Prolotherapy and LLLT

Anesth Pain Med. 2017 Oct 15;7(5):e14470. doi: 10.5812/aapm.14470. eCollection 2017 Oct.

Prolotherapy and Low Level Laser Therapy: A Synergistic Approach to Pain Management in Chronic Osteoarthritis.

Tieppo Francio V1,2,3, Dima RS4, Towery C1, Davani S1.

Author information

1
University of Science, Arts and Technology – USAT College of Medicine, Olveston, Montserrat, BWI.
2
Essential Integrative Health – Spine, Orthopaedics and Pain Management, Oklahoma City, OK, USA.
3
Variety Care – Community Health Center, Oklahoma City, OK, USA.
4
School of Interdisciplinary Sciences – McMaster University, Hamilton, ON, Canada.

Abstract

Regenerative injection therapy and low level laser therapy are alternative remedies known for their success in the treatment and symptomatic management of chronic musculoskeletal conditions. In response to the growing demand for alternative therapies in the face of the opioid epidemic, the authors conduct a literature review to investigate the potential for prolotherapy and LLLT to be used adjunctively to manage chronic osteoarthritis (OA). OA is a degenerative chronic musculoskeletal condition on the rise in North America, and is frequently treated with opioid medications. The regenerative action of prolotherapy and pain-modulating effects of LLLT may make these two therapies well-suited to synergistically provide improved outcomes for osteoarthritis patients without the side effects associated with opioid use. A narrative descriptive review through multiple medical databases (Google Scholar, PubMed, and MedLine) is conducted, restricted by the use of medical subject headings. 71 articles were selected for reading in full, and 40 articles were selected for use in the study after reading in full. A review of the literature revealed good clinical results in the use of prolotherapy and LLLT separately to manage chronic musculoskeletal pain due to osteoarthritis and other chronic conditions. It is also recognized in the literature that prolotherapy works most effectively when used adjunctively with other treatments. Downsides to the use of prolotherapy include mild side effects of pain, stiffness and bruising and potential adverse events as a result of injection. This study is limited by the lack of clinical trials available involving both LLLT and prolotherapy injections used adjunctively, and by the low number of high impact literature concerning the treatment of (specifically) osteoarthritis by alternative methods. The authors suggest that practicing health care providers consider utilizing LLLT and prolotherapy together as a supplementary method in the management of chronic pain due to osteoarthritis, to minimize the long-term prescription of opioids and emphasize a less invasive treatment for this debilitating condition.

Hand (N Y). 2014 Dec;9(4):419-46. doi: 10.1007/s11552-014-9642-x.

Non-surgical treatment of lateral epicondylitis: a systematic review of randomized controlled trials.

Sims SE1, Miller K2, Elfar JC1, Hammert WC1.

Author information

1
Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, 601 Elmwood Ave, Box 665, Rochester, NY 14642 USA.
2
University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 601, Rochester, NY 14642 USA.

Abstract

BACKGROUND:

Non-surgical approaches to treatment of lateral epicondylitis are numerous. The aim of this systematic review is to examine randomized, controlled trials of these treatments.

METHODS:

Numerous databases were systematically searched from earliest records to February 2013. Search terms included “lateral epicondylitis,” “lateral elbow pain,” “tennis elbow,” “lateral epicondylalgia,” and “elbow tendinopathy” combined with “randomized controlled trial.” Two reviewers examined the literature for eligibility via article abstract and full text.

RESULTS:

Fifty-eight articles met eligibility criteria: (1) a target population of patients with symptoms of lateral epicondylitis; (2) evaluation of treatment of lateral epicondylitis with the following non-surgical techniques: corticosteroid injection, injection technique, iontophoresis, botulinum toxin A injection, prolotherapy, platelet-rich plasma or autologous blood injection, bracing, physical therapy, shockwave therapy, or laser therapy; and (3) a randomized controlled trial design. Lateral epicondylitis is a condition that is usually self-limited. There may be a short-term pain relief advantage found with the application of corticosteroids, but no demonstrable long-term pain relief. Injection of botulinum toxin A and prolotherapy are superior to placebo but not to corticosteroids, and botulinum toxin A is likely to produce concomitant extensor weakness. Platelet-rich plasma or autologous blood injections have been found to be both more and less effective than corticosteroid injections. Non-invasive treatment methods such as bracing, physical therapy, and extracorporeal shockwave therapy do not appear to provide definitive benefit regarding pain relief. Some studies of lowlevel laser therapy show superiority to placebo whereas others do not.

CONCLUSIONS:

There are multiple randomized controlled trials for non-surgical management of lateral epicondylitis, but the existing literature does not provide conclusive evidence that there is one preferred method of non-surgical treatment for this condition. Lateral epicondylitis is a condition that is usually self-limited, resolving over a 12- to 18-month period without treatment.

LEVEL OF EVIDENCE:

Therapeutic Level II. See Instructions to Authors for a complete description of level of evidence.

KEYWORDS:

Elbow tendinopathy; Extensor tendinopathy; Lateral elbow pain; Lateral epicondylalgia; Lateral epicondylitis; Tennis elbow

Am Fam Physician. 2013 Apr 1;87(7):486-90.

Management of chronic tendon injuries.

Childress MA1, Beutler A.

Author information

1
Fort Belvoir Community Hospital, Fort Belvoir, VA 22060, USA.

Abstract

Chronic tendon injuries present unique management challenges. The assumption that these injuries result from ongoing inflammation has caused physicians to rely on treatments demonstrated to be ineffective in the long term. Nonsteroidal anti-inflammatory drugs should be limited in the treatment of these injuries. Corticosteroid injections should be considered for temporizing pain relief only for rotator cuff tendinopathy. For chronic Achilles tendinopathy (symptoms lasting longer than six weeks), an intense eccentric strengthening program of the gastrocnemius/ soleus complex improved pain and function between 60 and 90 percent in randomized trials. Evidence also supports eccentric exercise as a first-line option for chronic patellar tendon injuries. Other modalities such as prolotherapy, topical nitroglycerin, iontophoresis, phonophoresis, therapeutic ultrasound, extracorporeal shock wave therapy, and lowlevel laser therapy have less evidence of effectiveness but are reasonable second-line alternatives to surgery for patients who have persistent pain despite appropriate rehabilitative exercise.

Hemolasertherapy

Photomed Laser Surg. 2018 Apr;36(4):221-226. doi: 10.1089/pho.2017.4349.

Hemolasertherapy: A Novel Procedure for Gingival Papilla Regeneration-Case Report.

Zanin F1, Moreira MS2, Pedroni ACF3, Windlin M1, Brugnera AP4, Brugnera Júnior A5, Marques MM3.

Author information

1
1 Biophotonics Center at Institute Brugnera and Zanin-IBZ , São Paulo, Brazil .
2
2 Post Graduation Program, School of Dentistry, Ibirapuera University , Sao Paulo, Brazil .
3
3 Department of Restorative Dentistry of the School of Dentistry, University of Sao Paulo , Sao Paulo, Brazil .
4
4 PG student of Master Oral Laser Application, University of Liège , Liège, Belgium .
5
5 Associate Researcher of the National Institute of Science and Technology (INCT)-“Basic Optics and Applied to Life Sciences”-IFSC, University of Sao Paulo , Sao Paulo, Brazil .

Abstract

BACKGROUND:

Interdental papilla is of major importance to patients’ orofacial aesthetics, especially regarding anterior teeth as part of the smile’s harmony. Loss of gingival tissue, which constitutes interdental papilla, forms what in odontology is called black spaces. This loss, besides affecting the smile’s aesthetics, also provokes phonetic and functional damage.

OBJECTIVE:

The objective of the authors is to present the result of three clinical cases treated with an innovative technique called hemolasertherapy, which stimulates growth of gingival papilla and thus permanently fills in the black spaces.

METHODS:

The photobiomodulation therapy (PBMT) used a 660 nm diode laser (Laser Duo, MMO-São Carlos, SP, Brazil), punctual, contact mode in two steps: before the bleeding (first PBMT) and immediately after bleeding (second PBMT). Parameters used were power output: 100 mW, CW; diameter tip: 5 mm; spot area: 0.19 cm2; irradiation exposure time per point: 20 sec; 14 points per daily session; total of 2 sessions, with a 1-week interval; E: 2 J per point; E: per daily session, 28 J; irradiance per point: 0.52 W/cm2; fluence per point: 10.4 J/cm2. Total in two daily sessions: total energy: 56 J; total fluence: 294.75 J/cm, 560?sec total time. An in vitro preliminary study was simultaneously carried out to demonstrate what could happen at cellular level in hemotherapy clinical cases associated with PBMT laser application.

RESULTS:

This initial study demonstrated that the blood clot originated from the bleeding provoked in the gingival area is rich in mesenchymal stem cells. PBMT enables preservation, viability, and further differentiation, stimulating the return of gingival stem cells, which would support their survival and differentiation in the blood clot, thus favoring interdental papilla regeneration.

CONCLUSIONS:

Follow-up was done for a time span of 4-5 years and considered excellent with regard to papilla preservation.

KEYWORDS:

dentistry; periodontology; photobiomodulation; stem cells

J Bone Joint Surg Am. 1989 Mar;71(3):411-7.

Prevention of osteoporosis by pulsed electromagnetic fields.

Rubin CT, McLeod KJ, Lanyon LE.

Musculo-Skeletal Research Laboratory, Department of Orthopaedics, State University of New York, Stony Brook 11794.

Using an animal model, we examined the use of pulsed electromagnetic fields, induced at a physiological frequency and intensity, to prevent the osteoporosis that is concomitant with disuse. By protecting the left ulnae of turkeys from functional loading, we noted a loss of bone of 13.0 per cent compared with the intact contralateral control ulnae over an eight-week experimental period. Using a treatment regimen of one hour per day of pulsed electromagnetic fields, we observed an osteogenic dose-response to induced electrical power, with a maximum osteogenic effect between 0.01 and 0.04 tesla per second. Pulse power levels of more or less than these levels were less effective. The maximum osteogenic response was obtained by a decrease in the level of intracortical remodeling, inhibition of endosteal resorption, and stimulation of both periosteal and endosteal new-bone formation. These data suggest that short daily periods of exposure to appropriate electromagnetic fields can beneficially influence the behavior of the cell populations that are responsible for bone-remodeling, and that there is an effective window of induced electrical power in which bone mass can be controlled in the absence of mechanical loading.

Osteoporosis

J Bone Joint Surg Am. 1989 Mar;71(3):411-7.

Prevention of osteoporosis by pulsed electromagnetic fields.

Rubin CT, McLeod KJ, Lanyon LE.

Musculo-Skeletal Research Laboratory, Department of Orthopaedics, State University of New York, Stony Brook 11794.

Abstract

Using an animal model, we examined the use of pulsed electromagnetic fields, induced at a physiological frequency and intensity, to prevent the osteoporosis that is concomitant with disuse. By protecting the left ulnae of turkeys from functional loading, we noted a loss of bone of 13.0 per cent compared with the intact contralateral control ulnae over an eight-week experimental period. Using a treatment regimen of one hour per day of pulsed electromagnetic fields, we observed an osteogenic dose-response to induced electrical power, with a maximum osteogenic effect between 0.01 and 0.04 tesla per second. Pulse power levels of more or less than these levels were less effective. The maximum osteogenic response was obtained by a decrease in the level of intracortical remodeling, inhibition of endosteal resorption, and stimulation of both periosteal and endosteal new-bone formation. These data suggest that short daily periods of exposure to appropriate electromagnetic fields can beneficially influence the behavior of the cell populations that are responsible for bone-remodeling, and that there is an effective window of induced electrical power in which bone mass can be controlled in the absence of mechanical loading.

Tissue Engineered, Skin Substitutes

J Biomater Appl. 2018 Jan 1:885328218759385. doi: 10.1177/0885328218759385. [Epub ahead of print]

Red light accelerates the formation of a human dermal equivalent.

Oliveira AC1, Morais TF1, Bernal C2, Martins VC2, Plepis AM1,2, Menezes PF3, Perussi JR1,2.

Author information

1
1 Programa de Pós-Graduação Interunidades Bioengenharia – EESC/FMRP/IQSC, 67817 Universidade São Paulo-São Carlos-SP , Brazil.
2
2 Instituto de Química de São Carlos, 67817 Universidade de São Paulo-São Carlos-SP , Brazil.
3
3 Instituto de Física de São Carlos, Universidade de São Paulo-São Carlos-SP, Brazil.

Abstract

Development of biomaterials’ substitutes and/or equivalents to mimic normal tissue is a current challenge in tissue engineering. Thus, three-dimensional cell culture using type I collagen as a polymeric matrix cell support designed to promote cell proliferation and differentiation was employed to create a dermal equivalent in vitro, as well to evaluate the photobiomodulation using red light. Polymeric matrix cell support was prepared from porcine serous collagen (1.1%) hydrolyzed for 96 h. The biomaterial exhibited porosity of 95%, a median pore of 44 µm and channels with an average distance between the walls of 78 ± 14 µm. The absorption of culture medium was 95%, and the sponge showed no cytotoxicity to Vero cells, a non-tumor cell line. Additionally, it was observed that irradiation with light at 630 nm (fluency 30J/cm-2) leads to the cellular photobiomodulation in both monolayer and human dermal equivalent (three-dimensional cell culture system). It was also verified that the cells cultured in the presence of the polymeric matrix cell support, allows differentiation and extracellular matrix secretion. Therefore, the results showed that the collagen sponge used as polymeric matrix cell support and the photobiomodulation at 630 nm are efficient for the production of a reconstructed human dermal equivalent in vitro.

J Biomed Opt. 2009 May-Jun;14(3):034002. doi: 10.1117/1.3127201.

Effect of lowlevel laser treatment of tissue-engineered skin substitutes: contraction of collagen lattices.

Ho G1, Barbenel J, Grant MH.

Author information

1
Exploit Technologies, Biomedical Sciences Division, Agency of Science and Technology (A STAR), 30 Biopolis Street, Singapore 138671, Singapore.

Abstract

Fibroblast-populated collagen lattices (FPCL) are widely used in tissue-engineered artificial skin substitutes, but their main drawback is that interaction of fibroblasts and matrix causes contraction of the lattice, reducing it to about 20% of its original area. The effect of lowlevellaser treatment (LLLT) on the behavior of 3T3 fibroblasts seeded in collagen lattices containing 20% chondroitin-6-sulphate was investigated to determine whether LLLT could control the contraction of FPCL. A He-Ne laser was used at 632.8 nm to deliver a 5-mW continuous wave with fluences from 1 to 4 J/cm(2). Laser treatment at 3 J/cm(2) increased contraction of collagen lattices in the absence of cells but decreased contraction of cell seeded lattices over a 7-day period. The effect was energy dependent and was not observed at 1, 2, or 4 J/cm(2). There was no alteration in fibroblast viability, morphology, or mitochondrial membrane potential after any laser treatments, but the distribution of actin fibers within the cells and collagen fibers in the matrices was disturbed at 3 J/cm(2). These effects contribute to the decrease in contraction observed. LLLT may offer a means to control contraction of FPCL used as artificial skin substitutes.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Feb;20(2):172-6.

[Primary grafting research of tissue engineered oral mucosa lamina propria on skin full thickness wounds].

[Article in Chinese]
Wu Z1, Ding Y, Zhang L, Zhong S, Jiang T.

Author information

1
Department of Oral Surgery, 2nd Hospital of China Medical University, Shenyang Liaoning 110004, P R China. Lauwuzq@sina.com

Abstract

OBJECTIVE:

To study the allograft effect of two kinds of tissue engineered oral mucosa lamina propria on skin full-thickness wounds.

METHODS:

The cultured Wistar rat oral mucosa fibroblasts (OMF) were incorporated into collagen or chitosan-collagen to construct the tissue engineered oral mucosa lamina propria, and then the OMFs were labeled with BrdU. The full-thickness round skin defects were made with a round knife (diameter, 0.8 cm) on the backs of 36 Wistar rats (21-25 weeks old), which were divided into 2 experimental groups: the fibroblast-populated collagen lattices (FPCL) group (grafted by FPCLs) and the fibroblast-populated chitosan collagen lattices (FPCCL) group (grafted by FPCCLs), and the control group (only covered with gauges). All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively.

RESULTS:

There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups, which was replaced at 17 days. All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days, there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kind. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group (P < 0.01). Under the light microscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar phenomenon in the control group. Each skin wound in the three groups was only partly keratinocytes at 7 days postoperatively. The recipient wounds were wholly keratinocytes with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocytes with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustrated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defects without any allergic reaction during the period of the wound healing.

CONCLUSION:

The oral mucosa fibroblasts as the new seed cells can join in the repair of the skin defects effectively and feasible. The fibroblast-populated collagen lattices and the fibroblast-populated chitosan collagen lattices can repair skin defects effectively and feasible, too. And the quality of the new skins was better in the two experimental groups than in the control group.

Skin Grafts

Lasers Med Sci. 2018 Jan 24. doi: 10.1007/s10103-017-2430-4. [Epub ahead of print]

Effect of low-level laser therapy on the healing process of donor site in patients with grade 3 burn ulcer after skin graft surgery (a randomized clinical trial).

Vaghardoost R1, Momeni M2, Kazemikhoo N3, Mokmeli S4, Dahmardehei M1, Ansari F5, Nilforoushzadeh MA3, Sabr Joo P1, Mey Abadi S1, Naderi Gharagheshlagh S1, Sassani S6.

Author information

1
Burn Research center, Department of Plastic and Reconstructive Surgery, Iran University of Medical Sciences, Tehran, Iran.
2
Burn Research center, Department of Plastic and Reconstructive Surgery, Iran University of Medical Sciences, Tehran, Iran. mah_momeni@yahoo.com.
3
Skin and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran, Iran.
4
Canadian Optic and Laser Center, Victoria, BC, Canada.
5
Laser Application in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
6
Al Nasr Sport Club Medical Section, Dubai, United Arab Emirates.

Abstract

Skin graft is a standard therapeutic technique in patients with deep ulcers, but managing donor site after grafting is very important. Although several modern dressings are available to enhance the comfort of donor site, using techniques that accelerate wound healing may enhance patient satisfaction. Low-level laser therapy (LLLT) has been used in several medical fields, including healing of diabetic, surgical, and pressure ulcers, but there is not any report of using this method for healing of donor site in burn patients. The protocols and informed consent were reviewed according to Medical Ethics Board of Shahid Beheshti University of Medical Sciences (IR.SBMU.REC.1394.363) and Iranian Registry of Clinical Trials (IRCT2016020226069N2). Eighteen donor sites in 11 patients with grade 3 burn ulcer were selected. Donor areas were divided into 2 parts, for laser irradiation and control randomly. Laser area was irradiated by a red, 655-nm laser light, 150 mW, 2 J/cm2, on days 0 (immediately after surgery), 3, 5, and 7. Dressing and other therapeutic care for both sites were the same. The patients and the person who analyzed the results were blinded. The size of donor site reduced in both groups during the 7-day study period (P <0.01) and this reduction was significantly greater in the laser group (P =?0.01). In the present study, for the first time, we evaluate the effects of LLLT on the healing process of donor site in burn patients. The results showed that local irradiation of red laser accelerates wound healing process significantly.

KEYWORDS:

Low-level laser therapy; Skin graft; Wound healing

Lasers Med Sci. 2017 Apr;32(3):641-648. doi: 10.1007/s10103-017-2160-7. Epub 2017 Feb 2.

Photobiomodulation laser and pulsed electrical field increase the viability of the musculocutaneous flap in diabetic rats.

Leite GP1, das Neves LM1, Silva CA2, Guirro RR1, de Souza TR1, de Souza AK1, Garcia SB3, Guirro EC4,5.

Author information

1
Post-Graduate Program in Rehabilitation and Functional Performance, Ribeirão Preto Medical School-FMRP/USP, Ribeirão Preto, Brazil.
2
Post-Graduate Program in Human Movement Sciences, Methodist University of Piracicaba-UNIMEP, Piracicaba, Brazil.
3
Post-Graduate Program in Pathology, Ribeirao Preto Medical School-FMRP/USP, Ribeirão Preto, Brazil.
4
Post-Graduate Program in Rehabilitation and Functional Performance, Ribeirão Preto Medical School-FMRP/USP, Ribeirão Preto, Brazil. ecguirro@fmrp.usp.br.
5
Ribeirão Preto Medical School of the University of São Paulo (USP), Bandeirantes Avenue, 3900, Ribeirão Preto, SP, 14049-900, Brazil. ecguirro@fmrp.usp.br.

Abstract

The purpose of this study is to investigate the effect of pulsed electrical field (PEF) and photobiomodulation laser (PBM) on the viability of the TRAM flap in diabetic rats. Fifty Wistar rats were divided into five homogeneous groups: Group 1-control; Group 2-diabetics; Group 3-diabetics + PEF; Group 4-diabetic + laser 660 nm, 10 J/cm2, 0.27 J; Group 5-diabetic + laser 660 nm, 140 J/cm2, 3.9 J. The percentage of necrotic area was evaluated using software Image J®. The peripheral circulation of the flap was evaluated by infrared thermography FLIR T450sc (FLIR® Systems-Oregon USA). The thickness of the epidermis (haematoxylin-eosin), mast cell (toluidine blue), leukocytes, vascular endothelial growth factor, fibroblast and newly formed blood vessels were evaluated. For the statistical analysis, the Kruskal-Wallis test was applied followed by Dunn and ANOVA test followed by Tukey with critical level of 5% (p?<?0.05). The PEF reduced the area of necrosis, decreased the leukocytes, increased the mast cells, increased the thickness of epidermis and increased newly formed blood vessels when it was compared to the untreated diabetic group of animals. Laser 660 nm, fluence 140 J/cm2 (3.9 J) showed better results than the 10 J/cm2(0.27 J) related to reduction of the area of necrosis and the number of leukocytes, increased mast cells, increased thickness of the epidermis, increased vascular endothelial growth factor, increased fibroblast growth factor and increase of newly formed blood vessels in diabetic animals. The laser and pulsed electrical field increase the viability of the musculocutaneous flap in diabetic rats.

KEYWORDS:

Diabetes mellitus; Dosimetry; Fibroblast growth factor; Lowlevel laser therapy; Phototherapy; Physiotherapy; Vascular endothelial growth factor

Lasers Med Sci. 2017 Feb;32(2):335-341. doi: 10.1007/s10103-016-2118-1. Epub 2016 Dec 2.

Laser photobiomodulation (830 and 660 nm) in mast cells, VEGF, FGF, and CD34 of the musculocutaneous flap in rats submitted to nicotine.

das Neves LM1, Leite GP2, Marcolino AM2, Pinfildi CE3, Garcia SB4, de Araújo JE1, Guirro EC5.

Author information

1
Post-Graduate Program in Rehabilitation and Functional Performance of Ribeirão Preto Medical School of the University of São Paulo (FMRP/USP), Av. dos Bandeirantes, 3900, Ribeirão Preto, SP, 14049-900, Brazil.
2
Post-Graduate Program in Rehabilitation Sciences, Federal University of Santa Catarina – Campus Araranguá – UFSC/SC, Florianópolis, Brazil.
3
Post-Graduate Program in Interdisciplinary Health Sciences, Federal University of Sao Paulo – Santos Campus – UNIFESP/SP, Sao Paulo, Brazil.
4
Post-Graduate Program in Pathology, Ribeirão Preto Medical School – FMRP/USP, Sao Paulo, Brazil.
5
Post-Graduate Program in Rehabilitation and Functional Performance of Ribeirão Preto Medical School of the University of São Paulo (FMRP/USP), Av. dos Bandeirantes, 3900, Ribeirão Preto, SP, 14049-900, Brazil. ecguirro@fmrp.usp.br.

Abstract

The aim of this study was to investigate the effect of laser photobiomodulation (PBM) on the viability of the transverse rectus abdominis musculocutaneous (TRAM) flap in rats subjected to the action of nicotine. We evaluated 60 albino Wistar rats, divided into six groups of ten animals. Group 1 (saline) underwent the surgical technique to obtain a TRAM flap; group 2 (laser 830 nm) underwent the surgical technique and was irradiated with a laser 830 nm; group 3 (laser 660 nm) underwent the surgical technique and was irradiated with a laser 660 nm; group 4 was treated with nicotine subcutaneously (2 mg/kg/2×/day/4 weeks) and underwent surgery; group 5 (nicotine?+?laser 830 nm) was exposed to nicotine, underwent the surgical technique, and was irradiated with a laser 830 nm; group 6 (nicotine?+?laser 660 nm) was exposed to nicotine, underwent the surgical technique, and was irradiated with a laser 660 nm. The application of PBM occurred immediately after surgery and on the two following days. The percentage of necrosis was assessed using the AxioVision® software. The number of mast cells (toluidine blue staining) was evaluated, and immunohistochemistry was performed to detect vascular endothelial growth factor expression (anti-VEGF-A), fibroblasts (anti-basic FGF), and neoformed vessels (anti-CD34). PBM with a wavelength of 830 nm increased the viability of the TRAM flap, with a smaller area of necrosis, increased number of mast cells, and higher expression of VEGF and CD34. PBM increases the viability of musculocutaneous flaps treated with to nicotine.

Lasers Med Sci. 2016 Apr;31(3):497-502. doi: 10.1007/s10103-016-1896-9. Epub 2016 Feb 11.

Effects of low level laser therapy on the prognosis of split-thickness skin graft in type 3 burn of diabetic patients: a case series.

Dahmardehei M1, Kazemikhoo N2, Vaghardoost R1, Mokmeli S3, Momeni M1, Nilforoushzadeh MA4,5, Ansari F4, Amirkhani A4.

Author information

1
Burn Research Center, Iran University of Medical Sciences, Tehran, Iran.
2
Skin and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran, Iran. nooshakazemi@gmail.com.
3
Canadian Optic and Laser Center, Victoria, BC, Canada.
4
Skin and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran, Iran.
5
Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

Abstract

Significant populations in burn centers are diabetic burn patients. Healing process in these patients is more difficult due to diabetes complications. The gold standard treatment for patients with grade 3 burn ulcer is split-thickness skin grafting (STSG), but in the diabetic patients, the rate of graft failure and amputation is high due to impaired tissue perfusion. The technique of low level laser therapy (LLLT) improves tissue perfusion and fibroblast proliferation, increases collagen synthesis, and accelerates wound healing. The purpose of this case report is introducing a new therapeutic method for accelerating healing with better prognosis in these patients. The protocols and informed consent were reviewed according to the Medical Ethics, Board of Shahid Beheshti Medical Sciences (IR.SBMU.RAM.REC.13940.363). Diabetic type 2 patients with 13 grade 3 burn ulcers, candidate for amputation, were enrolled in the study. We used a 650-nm red laser light, 2 J/Cm for the bed of the ulcer and an 810-nm infrared laser light 6 J/Cm(2) for the margins along with intravenous laser therapy with a 660-nm red light, before and after STSG for treating grade 3 burn ulcers in 13 diabetic ulcers. The results of this study showed complete healing in the last 8 weeks for all patients who were candidates for amputation. In this case series, we present 13 cases of diabetic ulcer with type 3 burn wound, candidate for amputation, who healed completely using LLLT and STSG. This is the first time that these two techniques are combined for treatment of burn ulcer in diabetic patients. Using LLLT with STSG might be a promising treatment for burn victims especially diabetic patients.

KEYWORDS:

Burn wound; Low level laser therapy; Skin transplantation

Plast Surg (Oakv). 2015 Spring;23(1):35-9.

Inhibitory effects of low-level laser therapy on skin-flap survival in a rat model.

Baldan CS1, Masson IF1, Esteves Júnior I1, Baldan AM1, Machado AF1, Casaroto RA1, Liebano RE1.

Author information

1
Paulista University, São Paulo, Brazil.

Abstract

in English, French

BACKGROUND:

Although several studies have demonstrated the effects of low-level laser therapy (LLLT) on skin flap viability, the role of higher doses has been poorly investigated.

OBJECTIVE:

To investigate the inhibitory effect of the LLLT (?=670 nm) on the viability of random skin flaps in a rat model using an irradiation energy of 2.79 J at each point.

METHODS:

Sixteen Wistar rats were randomly assigned into two groups: sham laser irradiation (n=8); and active laser irradiation (n=8). Animals in the active laser irradiation group were irradiated with a 670 nm diode laser with an energy of 2.79 J/point, a power output 30 mW, a beam area of 0.028 cm(2), an energy density of 100 J/cm(2), an irradiance of 1.07 W/cm(2) for 93 s/point. Irradiation was performed in 12 points in the cranial skin flap portion. The total energy irradiated on the tissue was 33.48 J. The necrotic area was evaluated on postoperative day 7.

RESULTS:

The sham laser irradiation group presented a mean (± SD) necrotic area of 47.96±3.81%, whereas the active laser irradiation group presented 62.24±7.28%. There was a significant difference in skin-flap necrosis areas between groups (P=0.0002).

CONCLUSION:

LLLT (?=670 nm) increased the necrotic area of random skin flaps in rats when irradiated with an energy of 2.79 J (100 J/cm(2)).

KEYWORDS:

Low-level laser therapy; Necrosis; Rats; Surgical flaps

Minerva Stomatol. 2014 Mar;63(3):77-83.

Histological assessment of nonablative laser stimulation of tissue repair in acellular dermal grafts.

Silveira V1, Cenci R, Oliveira M, Moraes J, Etges A, Zerbinatti L.

Author information

1
Oral and maxillofacial Department Pontifícia Universidade do Rio Grande do Sul (PUCRS) Porto Alegre, RS, Brazil – gerhardtoliveira@gmail.com.

Abstract

AIM:

The objective of this study was to compare integration of AlloDerm® acellular dermal grafts in animals subjected to non-ablative laserirradiation and animals not exposed to this therapy.

METHODS:

Standardized AlloDerm® fragments measuring 5 mm² were grafted into the subcutaneous tissue overlying the calvaria in 32 Wistar rats. Laser therapy (685 nm), at a dose of 4 J/cm2 per session, was applied immediately after surgical intervention and every 48 hours thereafter for a total of four applications.

RESULTS:

Analysis of histology slides revealed significantly greater edema in the control group. There was no neutrophil infiltration in the laser-irradiated group at any point during the study period, whereas such infiltration was present in control animals at three of the four points of observation. In the laser therapy group, lymphocyte infiltration was observed from day 1, whereas in the control group, it was only apparent from day 3. Vascularization was substantially greater in the control group. In the experimental group, the AlloDerm® graft was completely replaced by fibrous tissue.

CONCLUSION:

These findings suggest that add-on non-ablative laser therapy is an effective stimulator of healing and graft integration after placement of AlloDerm® acellular dermal grafts.

Lasers Med Sci. 2013 May;28(3):755-61. doi: 10.1007/s10103-012-1130-3. Epub 2012 Jun 22.

What is better in TRAM flap survival: LLLT single or multi-irradiation?

Pinfildi CE1, Hochman BS, Nishioka MA, Sheliga TR, Neves MA, Liebano RE, Ferreira LM.

Author information

1
Department of Science of Human Movement, University Federal of São Paulo-UNIFESP, Campus Baixada Santista, Santos, São Paulo, Brazil. cepinfildi@hotmail.com

Abstract

Lowlevel laser therapy (LLLT) has been used with the aim of improving vascular perfusion of the skin and musculocutaneous flaps. This study evaluated the effect of LLLT on transverse rectus abdominis musculocutaneous flap (TRAM) viability, vascular angiogenesis, and VEGF release. Eighty-four Wistar rats were randomly divided into seven groups with 12 rats in each group. Group 1 received sham lasertreatment; group 2, 3 J/cm(2) at 1 point; group 3, 3 J/cm(2) at 24 points; group 4, 72 J/cm(2) at 1 point; group 5, 6 J/cm(2) at 1 point; group 6, 6 J/cm(2) at 24 points; and group 7, 144 J/cm(2) at 1 point. All experimental groups underwent LLLT immediately after the TRAM operation and on the following 2 days; thus, animals underwent 3 days of treatment. The percentage of skin flap necrosis area was calculated on the fourth postoperative day using the paper template method, and two skin samples were collected using a 1-cm(2) punch to evaluate alpha smooth muscle actin (1A4) and VEGF levels in blood vessels. Significant differences were found in necrosis percentage, and higher values were seen in group 1 than in the other groups. Statistically significant differences were not found among groups 3 to 7 (p<0.292). Groups 5 and 7 showed significantly higher VEGF levels compared to other groups. Groups 3 and 5 had an increase in levels of blood vessels compared to other groups. LLLT at energy densities of 6 to 144 J/cm(2) was efficient to increase angiogenesis and VEGF levels and promote viability in TRAM flaps in rats.

Rev Col Bras Cir. 2013 Jan-Feb;40(1):44-8.

Macro and microscopic analysis of island skin grafts after lowlevel laser therapy.

[Article in English, Portuguese]
da Silva EB1, Maniscalco CL, Ésper GV, Guerra RR, Kerppers II.

Author information

1
Department of Agricultural and Environmental Sciences, State University of Santa Cruz – UESC, Ilheus, Bahia State – BA, Brazil. elisangelavet@yahoo.com.br

Abstract

OBJECTIVE:

To observe the effects of low intensity laser therapy in inflammation, wound healing and epithelialization of island skin grafts.

METHODS:

Twenty rats were subjected to this grafting technique and divided subsequently into two equal groups, one treated with laser and the other control.

RESULTS:

there was less inflammation, faster healing, epithelialization and keratinization in the laser-treated animals when compared to the untreated.

CONCLUSION:

Low intensity laser therapy is helpful to island skin grafting.

Lasers Med Sci. 2012 Sep;27(5):1045-50. doi: 10.1007/s10103-011-1042-7. Epub 2011 Dec 30.

LED (660 nm) and laser (670 nm) use on skin flap viability: angiogenesis and mast cells on transition line.

Nishioka MA1, Pinfildi CE, Sheliga TR, Arias VE, Gomes HC, Ferreira LM.

Author information

1
Post Graduation Plastic Surgery, Federal University of São Paulo, R. Napoleão de Barros, 715, 4º andar, CEP 04024-900, São Paulo, SP, Brazil.

Abstract

Skin flap procedures are commonly used in plastic surgery. Failures can follow, leading to the necrosis of the flap. Therefore, many studies use LLLT to improve flap viability. Currently, the LED has been introduced as an alternative to LLLT. The objective of this study was to evaluate the effect of LLLT and LED on the viability of random skin flaps in rats. Forty-eight rats were divided into four groups, and a random skin flap (10?×?4 cm) was performed in all animals. Group 1 was the sham group; group 2 was submitted to LLLT 660 nm, 0.14 J; group 3 with LED 630 nm, 2.49 J, and group 4 with LLLT 660 nm, with 2.49 J. Irradiation was applied after surgery and repeated on the four subsequent days. On the 7th postoperative day, the percentage of flap necrosis was calculated and skin samples were collected from the viable area and from the transition line of the flap to evaluate blood vessels and mast cells. The percentage of necrosis was significantly lower in groups 3 and 4 compared to groups 1 and 2. Concerning blood vessels and mast cell numbers, only the animals in group 3 showed significant increase compared to group 1 in the skin sample of the transition line. LED and LLLT with the same total energies were effective in increasing viability of random skin flaps. LED was more effective in increasing the number of mast cells and blood vessels in the transition line of random skin flaps.

Acta Cir Bras. 2012 Feb;27(2):155-61.

The effects of different doses of 670 nm diode laser on skin flap survival in rats.

Baldan CS1, Marques AP, Schiavinato AM, Casarotto RA.

Author information

1
Physical Therapy Department, UNIP and Sao Paulo Metodista University, Brazil. cristianobaldan@yahoo.com.br

Abstract

PURPOSE:

To investigate the effects of different low-level laser therapy (LLLT) doses on random skin flap rats.

METHODS:

Forty Wistar rats were randomly divided in four groups. The control group (CG) was not irradiated. The experimental groups were irradiated with a diode laser 670 nm with different energies per point: group 2 (G2) with 0.06 J; group 3 (G3) 0.15 J and group 4 (G4) 0.57 J. The three groups were irradiated in 12 equally distributed points in the cranial skin flap portion. They were submitted to the irradiation during the immediate, first and second postoperative days. The necrosis area was evaluated in the seventh postoperative day.

RESULTS:

The CG shows 49.35% of necrosis area in the skin flap; G2, 39.14%; G3, 47.01% and G4, 29.17% respectively. There was a significantly difference when G4 was compared with CG`s skin flap necrosis area.

CONCLUSION:

The low-level laser therapy diode 670 nm with 0.57 J energy per point increases the survival in randomic skin flap rats.

Photomed Laser Surg. 2010 Aug;28(4):483-8. doi: 10.1089/pho.2009.2500.

Influence of the use of laser phototherapy (lambda660 or 790 nm) on the survival of cutaneous flaps on diabetic rats.

Santos NR1, dos Santos JN, dos Reis JA Jr, Oliveira PC, de Sousa AP, de Carvalho CM, Soares LG, Marques AM, Pinheiro AL.

Author information

1
School of Dentistry, Federal University of Bahia, Salvador, Bahia, Brazil.

Abstract

OBJECTIVE:

The aim of this study was to assess and compare the effects of laser phototherapy (LPT) on cutaneous flaps on diabetic rats.

BACKGROUND:

Diabetes mellitus is characterized by high blood glucose levels. Its main complications are delayed wound healing, an impaired blood supply, and a decrease in collagen production. Cutaneous flaps are routinely used in several surgical procedures, and most failures are related to poor blood supply. LPT has been studied using several healing models.

ANIMALS AND METHODS:

Twelve Wistar rats were randomized into three groups: group 1 (G1; diabetic animals without treatment), group 2 (G2; diabetic animals irradiated with lambda680 nm), and group 3 (G3; diabetic animals irradiated with lambda790 nm). Diabetes was induced with streptozotocin. A 2- x 8-cm cutaneous flap was raised on the dorsum of each animal, and a plastic sheet was introduced between the flap and the bed to cause poor blood supply. Nonirradiated animals acted as controls. The dose per session was 40 J/cm(2). Laser light was applied transcutaneously and fractioned on 16 contact points at the wound margins (16 x 2.5 J/cm(2)). Animal death occurred on day 8 after surgery. Specimens were taken, processed, cut, stained with eosin (HE) and sirius red, and underwent histological analysis.

RESULTS:

It is shown that accute inflammation was mostly discrete for G3. Chronic inflammation was more evident for G2. Fibroblast number was higher for G3. Angiogenesis was more evident for G3. Necrosis was more evident for G2. Statistical analysis among all groups showed significant differences (p = 0.04) on the level of acute inflammation between G1 and G3, tissue necrosis between G1 and G2 (p = 0.03), chronic inflammation between (p = 0.04), fibroblastic proliferation between G2 and G3 (p = 0.05), and neovascularization between G2 and G3 (p = 0.04).

CONCLUSION:

LPT was effective in increasing angiogenesis as seen on irradiated subjects and was more pronounced when IR laser light was used.

Photomed Laser Surg. 2010 Jun;28(3):379-84. doi: 10.1089/pho.2009.2535.

Effect of low-level laser therapy on malondialdehyde concentration in random cutaneous flap viability.

Prado R1, Neves L, Marcolino A, Ribeiro T, Pinfildi C, Ferreira L, Thomazini J, Piccinato C.

Author information

1
Department of Surgery and Anatomy, University of São Paulo-FMRP-USP, Ribeirão Preto, Brazil. paschoalrp@hotmail.com

Abstract

OBJECTIVE:

The aim of this study was to assess the effects of 830 and 670 nm laser on malondialdehyde (MDA) concentration in random skin-flap survival.

BACKGROUND DATA:

Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and activating superoxide-dismutase delivery, thus helping the inhibition of free-radical action and consequently reducing necrosis.

MATERIALS AND METHODS:

Thirty Wistar rats were used and divided into three groups, with 10 rats in each one. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group; group 2 received 830 nm laser radiation; and group 3 was submitted to 670 nm laser radiation. The animals underwent laser therapy with 36 J/cm(2) energy density immediately after surgery and on the 4 days subsequent to surgery. The application site of the laser radiation was 1 point, 2.5 cm from the flap’s cranial base. The percentage of the skin-flap necrosis area was calculated 7 days postoperative using the paper-template method, and a skin sample was collected immediately after as a way of determining the MDA concentration.

RESULTS:

Statistically significant differences were found between the necrosis percentages, with higher values seen in group 1 compared with groups 2 and 3. Groups 2 and 3 did not present statistically significant differences (p > 0.05). Group 3 had a lower concentration of MDA values compared to the control group (p < 0.05).

CONCLUSION:

LLLT was effective in increasing the random skin-flap viability in rats, and the 670 nm laser was efficient in reducing the MDA concentration.

Photomed Laser Surg. 2009 Jun;27(3):411-6. doi: 10.1089/pho.2008.2320.

Effect of application site of low-level laser therapy in random cutaneous flap viability in rats.

Prado RP1, Pinfildi CE, Liebano RE, Hochman BS, Ferreira LM.

Author information

1
Master of Basic Sciences in Plastic Surgery, São Paulo Federal University, São Paulo, SP, Brazil. paschoalrp@hotmail.com

Abstract

OBJECTIVE:

This study aimed to investigate the effect of diode laser (830 nm) irradiation on the viability of ischemic random skin flaps in rats, as well as to determine the most effective site for applying laser radiation to speed healing.

BACKGROUND DATA:

Low-level laser therapy (LLLT) has recently been used to improve the viability of ischemic random skin flaps in rats.

MATERIALS AND METHODS:

Seventy Wistar rats were used and divided into seven groups of 10 rats each: group 1, sham laser treatment; group 2, which received irradiation at 1 point 5 cm from the flap’s cranial base; group 3, which received irradiation at 2 points (5 and 7.5 cm from the flap’s base); group 4, which received irradiation at 3 points (2.5, 5 and 7.5 cm from the flap’s base); group 5, which received irradiation at 1 point 2.5 cm from the flap’s base; group 6, which received irradiation at 2 points (2.5 and 5 cm from the flap’s base); and group 7, which received irradiation at 1 point 7.5 cm from the flap’s base. The animals were subjected to laser therapy at an energy density of 36 J/cm(2) for 72 sec immediately after surgery, and one time on each of the four subsequent days. The percentage of necrotic skin flap area was calculated on the seventh postoperative day using a paper template.

RESULTS:

The results showed that the rats in group 5 had the highest increase in skin flap viability, with a statistically significant difference compared to the other groups. Statistically significant differences were not seen between any of the other groups.

CONCLUSION:

The diode laser was effective in increasing skin flap viability in rats, and laser irradiation of a point 2.5 cm from the cranial base flap was found to be the most effective.

Photomed Laser Surg. 2009 Apr;27(2):337-43. doi: 10.1089/pho.2008.2295.

Effect of low-level laser therapy on mast cells in viability of the transverse rectus abdominis musculocutaneous flap.

Pinfildi CE1, Liebano RE, Hochman BS, Enokihara MM, Lippert R, Gobbato RC, Ferreira LM.

Author information

1
Department of Plastic Surgery and IMES-FAFICA, São Paulo Federal University, São Paulo, SP, Brazil. cepinfildi@hotmail.com

Abstract

OBJECTIVE:

To assess the effect of low-level laser therapy (LLLT) on viability of mast cells of the transverse rectus abdominis musculocutaneous (TRAM) flap.

BACKGROUND DATA:

LLLT has been recently used on the TRAM flap to stimulate mast cells.

MATERIALS AND METHODS:

Eighty-four Wistar rats were randomly divided into seven groups of 12 rats in each: group 1 (sham laser therapy); group 2 received 3 J/cm(2) at one point; group 3 received 3 J/cm(2) at 24 points; group 4 received 72 J/cm(2) at 1 point; group 5 received 6 J/cm(2) at 1 point; group 6 received 6 J/cm(2) at 24 points; and group 7 received 144 J/cm(2) at 1 point. All experimental groups underwent LLLT immediately after TRAM surgery and on the next two following days, for three sessions in total. The percentage of the area of skin flap necrosis was calculated on the fourth postoperative day and two samples of skin were collected from each rat with a 1-cm(2) punch to perform mast cell evaluations with toluidine blue dye.

RESULTS:

Statistically significant differences were found in the percentage of necrosis, and higher values were seen in group 1 than in all other groups. Among groups 3-7 no statistically significant differences were found (p < 0.292). For mast cells, when group 1 was compared to groups 5 (6 J/cm(2) at 1 point) and 7 (144 J/cm(2) at 1 point), it had fewer mast cells.

CONCLUSION:

LLLT at a wavelength of 670 nm was effective at reducing the necrotic area, and we found that it can stimulate mast cells growth to increase vascular perfusion.

Ann Plast Surg. 1985 Mar;14(3):278-83.

Effects of low-power diode lasers on flap survival.

Kami T, Yoshimura Y, Nakajima T, Ohshiro T, Fujino T.

Abstract

We investigated the effect of low-power laser irradiation on the survival of experimental skin flaps in rats. A gallium-aluminum-arsenide diode laser that was developed by the Japan Medical Laser Laboratory was used. The laser power was 15 mW and the wavelength 830 nm. Irradiation was carried out, either before or after flap elevation, in two groups of 20 Wistar strain rats. A third group of 20 rats served as controls. A caudally based skin flap, 3 X 9 cm, was designed on the back of each rat. Laser irradiation therapy was performed for 5 consecutive days for 6 minutes per flap per day, preoperatively in one group and postoperatively in the other. Seven days postoperatively, the survival areas of the flaps were measured and compared. The survival area was increased significantly in both groups receiving laser therapy, probably due to the observed proliferation of blood vessels around the irradiated points and an increase in blood flow.

PMID:
3994272

Ischemia – Reperfusion Injury

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Link to Publisher's site
Biosci Rep. 2016 Dec; 36(6): e00420.
Published online 2016 Dec 5. Prepublished online 2016 Oct 25. doi:  10.1042/BSR20160082
PMCID: PMC5137536

Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats

Fenfen Ma,*,1 Wenwen Li,‡,1 Xinghui Li, Ba Hieu Tran,§ Rinkiko Suguro,§ Ruijuan Guan, Cuilan Hou, Huijuan Wang,? Aijie Zhang, Yichun Zhu, and YiZhun Zhu?¶,2
*Department of Pharmacy, Shanghai Pudong Hospital, Fudan University, Shanghai 201399, China
Shanghai Institute of Immunology & Department of Immunobiology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
Shanghai Key Laboratory of Bioactive Small Molecules and Research Center on Aging and Medicine, Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China
§Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China
?Longhua Hospital, Shanghai University of Tradition Chinese Medicine, Shanghai 201203,China
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore 119228, Singapore
1These authors contributed equally to the article.
2To whom correspondence should be addressed (email nc.ude.naduf@zyuhz).
Author information ? Article notes ? Copyright and License information ?
Received 2016 Mar 17; Revised 2016 Oct 11; Accepted 2016 Oct 17.
 .
Abstract

Extracorporeal pulsed electromagnetic field (PEMF) has shown the ability to regenerate tissue by promoting cell proliferation. In the present study, we investigated for the first time whether PEMF treatment could improve the myocardial ischaemia/reperfusion (I/R) injury and uncovered its underlying mechanisms.

In our study, we demonstrated for the first time that extracorporeal PEMF has a novel effect on myocardial I/R injury. The number and function of circulating endothelial progenitor cells (EPCs) were increased in PEMF treating rats. The in vivo results showed that per-treatment of PEMF could significantly improve the cardiac function in I/R injury group. In addition, PEMF treatment also reduced the apoptosis of myocardial cells by up-regulating the expression of anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and down-regulating the expression of pro-apoptosis protein (Bax). In vitro, the results showed that PEMF treatment could significantly reduce the apoptosis and reactive oxygen species (ROS) levels in primary neonatal rat cardiac ventricular myocytes (NRCMs) induced by hypoxia/reoxygenation (H/R). In particular, PEMF increased the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS), which might be closely related to attenuated cell apoptosis by increasing the releasing of nitric oxide (NO). Therefore, our data indicated that PEMF could be a potential candidate for I/R injury.

Keywords: apoptosis, Bax, B-cell lymphoma 2 (Bcl-2), ischaemia/reperfusion (I/R) injury, pulsed electromagnetic field (PEMF)

INTRODUCTION

Hypertension, arrhythmia, myocardial infarction (MI) and myocardial ischaemia/reperfusion (I/R) injury are all the most common cardiac diseases, which are the major causes of mortality in the world []. Among them, myocardial I/R injury is the most important cause of cardiac damage. Its pathological process is closely related to postoperative complications [,] caused by coronary artery vascular formation, coronary revascularization and heart transplantation. After myocardium suffered severe ischaemia, restoration of the blood flow is a prerequisite for myocardial salvage []. However, reperfusion may induce oxidative stress [], inflammatory cell infiltration and calcium dysregulation []. All these players contribute to the heart damage such as contraction and arrhythmias [], generally named myocardial I/R injury. Recently, more and more evolving therapies have been put into use for I/R injury.

Pulsed electromagnetic field (PEMF) is the most widely tested and investigated technique in the various forms of electromagnetic stimulations for wound healing [], alleviating traumatic pain and neuronal regeneration [,]. The rats were randomly divided into PEMF-treated (5 mT, 25 Hz, 1 h daily) and control groups. They hypothesized the possible mechanism that PEMF would increase the myofibroblast population, contributing to wound closure during diabetic wound healing. It is a non-invasive and non-pharmacological intervention therapy. Recent studies indicated that PEMF also stimulated angiogenesis in patients with diabetes [], and could improve arrhythmia, hypertension and MI []. The MI rats were exposed to active PEMF for 4 cycles per day (8 min/cycle, 30±3 Hz, 6 mT) after MI induction. In vitro, PEMF induced the degree of human umbilical venous endothelial cells tubulization and increased soluble pro-angiogenic factor secretion [VEGF and nitric oxide (NO)] []. However, the role of PEMF in ischaemia and reperfusion diseases remains largely unknown. Our study aimed to investigate the effects of PEMF preconditioning on myocardial I/R injury and to investigate the involved mechanisms.

In our study, we verified the cardioprotective effects of PEMF in myocardial I/R rats and the anti-apoptotic effects of PEMF in neonatal rat cardiac ventricular myocytes (NRCMs) subjected to hypoxia/reoxygenation (H/R). We hypothesized that PEMF treatment could alleviate myocardial I/R injury through elevating the protein expression of B-cell lymphoma 2 (Bcl-2), phosphorylation of protein kinase B (Akt). Meanwhile, it could decrease Bax. We emphatically made an effort to investigate the MI/R model and tried to uncover the underlying mechanisms.

MATERIALS AND METHODS

Animals

Male, 12-week-old Sprague Dawley (SD) rats (250–300 g) were purchased from Shanghai SLAC Laboratory Animal. Animals were housed in an environmentally controlled breeding room and given free access to food and water supplies. All animals were handled according to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH). Experimental procedures were managed according to the Institutional Aminal Care and Use Committee (IACUC), School of Pharmacy, Fudan University.

The measurement of blood pressure in SHR rats

At the end of 1 week treatment with PEMF, the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), the right common carotid artery (CCA) was cannulated with polyethylene tubing for recording of the left ventricle pressures (MFlab 200, AMP 20130830, Image analysis system of physiology and pathology of Fudan University, Shanghai, China).

Myocardial I/R injury rat model and measurement of infarct size

All the rats were divided into three groups: (1) Sham: The silk was put under the left anterior descending (LAD) without ligation; (2) I/R: Hearts were subjected to ischaemia for 45 min and then reperfusion for 4 h; (3) I/R + PEMF: PEMF device was provided by Biomobie Regenerative Medicine Technology. The I/R rats were pre-exposed to active PEMF for 2 cycles per day (8 min per cycle), whereas other two groups were housed with inactive PEMF generator. I/R was performed by temporary ligation of the LAD coronary artery for 45 min through an incision in the fourth intercostal space under anaesthesia []. Then, the ligature was removed after 45 min of ischaemia, and the myocardium was reperfused for 4 h. Ischaemia and reperfusion were confirmed and monitored by electrocardiogram (ECG) observation. The suture was then tightened again, and rats were intravenously injected with 2% Evans Blue (Sigma–Aldrich). After explantation of the hearts, the left ventricles were isolated, divided into 1 mm slices, and subsequently incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma–Aldrich) in 0.9% saline at 37°C for 25 min, to distinguish infarcted tissue from viable myocardium. These slices were flushed with saline and then fixed in 10% paraformaldehyde in PBS (pH 7.4) for 2 h. Next, the slices were placed on a glass slice and photographed by digital camera, the ImageJ software (NIH) was used in a blind fashion for analysis. Infarct size was expressed as a ratio of the infarct area and the area at risk [].

Pulsed electromagnetic field treatment

PEMF were generated by a commercially available healing device (length × width × height: 7 cm × 5cm × 3cm) purchased from Biomoble Regenerative Medicine Technology. The adapter input voltage parameter is approximately 100–240 V and output parameter is 5 V. Fields were asymmetric and consisted of 4.5 ms pulses at 30±3 Hz, with an adjustable magnetic field strength range (X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT). The I/R rats were housed in custom designed cages and exposed to active PEMF for 2 cycles per time (8 min for 1 cycle), whereas the I/R rats were housed in identical cages with inactive PEMF generator. For in vitro study, culture dishes were directly exposed to PEMF for 1–2 cycles as indicated (8 min for 1 cycle, 30 Hz, X-axis 0.22 mT, Y-axis 0.20 mT, Z-axis 0.06 mT) []. The background magnetic field in the room area of exposure animals/samples and controls is 0 mT.

Detection of myocardium apoptosis

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay was applied to analyse cardiomyocyte apoptosis. Heart samples were first fixed in 10% formalin and then paraffin embedded at day 14. Then, the hearts were cut into 5 ?m sections. TUNEL staining was carried out as described previously []. When apoptosis occurred, cells would look green.

Determination of myocardial enzymes in plasma

Blood samples were collected after haemodynamic measurement and centrifuged at 3000 g for 15 min to get the plasma. Creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase isoenzyme-MB (CKMB) and ?-hydroxybutyrate dehydrogenase (HBDH) were quantified by automatic biochemical analyzer (Cobas 6000, Roche). All procedures were performed according to the manufacturer’s protocols.

Myocardium cells morphology via TEM

At the end of the experiment, sections from myocardial samples of left ventricular were immediately fixed overnight in glutaraldehyde solution at 4°C and then incubated while protected from light in 1% osmium tetroxide for 2 h. After washing with distilled water for three times (5 min each), specimens were incubated in 2% uranyl acetate for 2 h at room temperature and then dehydrated in graded ethanol concentrations. Finally, sections were embedded in molds with fresh resin. The changes in morphology and ultrastructure of the myocardial tissues were observed and photographed under a TEM [].

Scal-1+/flk-1+ cells counting of endothelial progenitor cells

We applied antibodies to the stem cell antigen-1 (Sca-1) and fetal liver kinase-1 (flk-1) to sign endothelial progenitor cells (EPCs) as described before, and used the isotype specific conjugated anti-IgG as a negative control. The amount of Scal-1+/flk-1+ cells would be counted by flow cytometry technique [].

Measurement of nitric oxide concentration and Western blotting

Plasma concentrations of NO were measured with Griess assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. The expressions of Bax, Bcl-2, p-Akt, Akt, p-endothelial nitric oxide synthase (eNOS), eNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using Western blot as described recently []. Proteins were measured with Pierce BCA Protein Assay Kit (Thermo). Hippocampal protein lysates (50 mg/well) were separated using (SDS/PAGE) under reducing conditions. Following electrophoresis, the separated proteins were transferred to a PVDF membrane (Millipore). Subsequently, non-specific proteins were blocked using blocking buffer (5% skim milk or 5% BSA in T-TBS containing 0.05% Tween 20), followed by overnight incubation with primary rabbit anti-rat antibodies specific for target proteins as mentioned before (Cell Signaling Technology) at 4°C. Blots were rinsed three times (5 min each) with T-TBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Proteintech) for 2 h at room temperature. The blots were visualized by using enhanced chemiluminescence (ECL) method (Thermo). GAPDH was applied to be the internal control protein. Intensity of the tested protein bands was quantified by densitometry.

Cell culture

Primary neonatal rat cardiac ventricular myocytes (NRCMs) were collected as previously described []. Briefly, the ventricles of new born SD rats (1–3 days old) were minced and digested with 0.125% trypsin. Isolated cardiomyocytes were cultured in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life Technologies) supplemented with 10% (v/v) FBS (Life Technologies), 100 units/ml penicillin and 100 mg/ml streptomycin. The following experiments used spontaneously beating cardiomyocytes 48–72 hours after plating. (37°C with 5% CO2).

Cell treatment (hypoxia/reoxygenation)

NRCMs were prepared according to the methods recently described []. To establish the H/R model, the cells were cultured in DMEM/F-12 without glucose and serum. The cells were exposed to hypoxia (99% N2+5% CO2) for 8 h, followed by reoxygenation for 16 h. The cells were pretreated with PEMF for 30 min before the H/R procedure. The control group was cultured in DMEM/F-12 with low glucose (1000 mg/l) and 2% serum under normoxic air conditions for the corresponding times.

Cell viability assays

The viability of NRCMs cultured in 96-well plates was measured by using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies) according to the manufacturer’s instructions. The absorbance of CCK-8 was obtained with a microplate reader at 450 nm.

Measurement of intracellular reactive oxygen species levels

Reactive oxygen species (ROS) levels in NRVMs were determined by dihydroethidium (DHE, Sigma–Aldrich) fluorescence using confocal microscopy (Zeiss, LSM 710). After different treatments, cells were washed with D-PBS and incubated with DHE (10 ?mol/l) at 37°C for 30 min in the dark. Then, residual DHE was removed by PBS-washing. Fluorescent signals were observed (excitation, 488 nm; emission, 610 nm) under a laser confocal microscope (Zeiss).

Data analysis

All the data were presented as means ± S.E.M. Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05 was taken as statistically significant.

RESULTS

PEMF could lower blood pressure under treatment of certain PEMF intensity in SHR rat model (double-blind)

To determine whether PEMF has any effects on blood pressure of SHR rats, we treated SHR rats with different PEMF intensity 1–4 cycles per day for 7 days and measured the blood pressure changes via CCA. We observed that PEMF treatment could significantly lower the blood pressure in the Bioboosti WIN235 and WI215-stimulating groups than that in non-treated ones (Figures 1A and ?and1B).1B). But Bioboosti WIN221 and WC65 treating groups did not have any effects on the blood pressure in SHR rats, compared with the non-treated ones (Figures 1C and ?and1D).1D). Fields were asymmetric and consisted of 4.5 ms pulses at 30±3 Hz, with an adjustable magnetic field strength range (X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT). The I/R rats were housed in custom designed cages and exposed to active PEMF for 2 cycles per time (8 min for 1 cycle), whereas the I/R rats were housed in identical cages with inactive PEMF generator.

Figure 1

The effect of PEMF on SHR rats in vivo. PEMF could lower the blood pressure in SHR rats. At day 7 treatment with different intensity PEMF, blood pressure was recorded via CCA [1(A), 1(B), 1(C) and 1(D)]. Data were represented as the mean ± 

According to this result, we chose Bioboosti WIN235 as our needed PEMF to carry out the following experiments.

PEMF treatment could observably improve the abundance of EPCs

Amplifying EPCs abundance and function is an active focus of research on EPCs-mediated neovascularization after I/R. Thus, the number of circulating EPCs was identified by Sca-1/flk-1 dual positive cells as described. We determined that PEMF treatment could remarkably increase the number of Scal-1+/flk-1+ cells in peripheral blood at postoperative days 7 and 14 (Figure 2).

Figure 2

The effect of PEMF on the number of Scal-1+/flk-1+ cells after treating EPSc for 7 and 14 days. PEMF treatment notably increased the number of Scal-1+/flk-1+ cells after treating EPSc for 7 and 14 days. Data were represented as the mean 

Preliminary assessment of PEMF showed great protective effect against myocardial infarction/reperfusion injury (MI/RI) rat model

To examine the effect of PEMF on myocardial I/R, male SD rats were divided into three groups: Sham, I/R and I/R+ PEMF (2 cycles per day, 8 min per cycle) per day until 28 days. We observed that PEMF stimulation could significantly decrease four plasma myocardial enzymes (LDH, CK, CKMB and HBDH) in I/R rats (Figure 3A). Additionally, we found that pre-stimulating PEMF could improve the cardiac morphology via TEM, compared with I/R+ PEMF group. TEM revealed the rupture of muscular fibres, together with mitochondrial swelling, and intracellular oedema in Group I/R. The shape of nucleus was irregular, with evidence of mitochondrial overflow after cell death. Compared with Group I/R+ PEMF, less muscular fibres were ruptured, with mild swelling of mitochondria, mild intercellular oedema and less cell death. In Group Sham, the ruptured muscular fibres, mitochondrial or intracellular oedema and dead cells were not observed (Figure 3B). To further confirm protective effect of PEMF, we measured the MI size by applying TTC and Evans Blue staining in all three groups. The MI area in I/R+ PEMF group could be reduced, compared with the model rats in I/R group (Figure 3C).

Figure 3

Protective effect of PEMF on I/R rats in vivo. Plasma myocardial enzymes (LDH, CK, HBDH and CKMB) content was quantified by automatic biochemical analyzer (A) (n=18 in each group). Changes on cardiac cell morphology via TEM (B) (n=6 in 

In vivo, PEMF dramatically reduced cell apoptosis induced by I/R injury

As H/R of cardiomyocytes contributed to cell death, we also detected the effect on myocardial apoptosis by using TUNEL kit, as shown in Figure 4(A). We uncovered that PEMF pretreating could dramatically decrease apoptosis of myocardial cells in I/R + PEMF group, compared with I/R group. In addition, we also found that PEMF treatment could significantly increase the expression of anti-apoptosis protein Bcl-2, p-eNOS and p-Akt and down-regulated the expression of pro-apoptosis protein Bax in the heart tissue, as shown in Figure 4(B).

Figure 4

Apoptotic cardiomyocyte was identified by TUNEL analysis, apoptotic cardiomyocyte appears green whereas TUNEL-negative appears blue (A), photomicrographs were taken at ×200 magnification. Apoptosis-related protein Bcl-2, Bax, p-Akt level of different 

The effect of PEMF on cell viability in neonatal rat cardiac ventricular myocytes

To further investigate whether PEMF has the same effect in vitro, we simulated the I/R injury model in vitro. We applied NRCMs and hypoxia incubator to mimic myocardial I/R injury via H/R as described in the section ‘Materials and Methods’. We found that PEMF treatment (2 cycles) could remarkably improve cell viability, compared with the H/R group (Figure 5). For in vitro study, culture dishes were directly exposed to PEMF for 1–2 cycles as indicated (8 min for 1 cycle, 30±3 Hz, X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT).

Figure 5

NRCMs viability measured by CCK-8 assay at the end of the treatment for 72 h. PEMF treatment enhanced the cell viability of hypoxia NRCMs. Data were represented as the mean ± S.E.M.

Specific-density PEMF could decrease intracellular ROS levels of primary cardiomyocytes subjected to hypoxia/reperfusion

As shown in Figure 6(A), NRCMs that were subjected to H/R increased significantly the ROS level, whereas the ROS level had been decreased in PEMF group (2 cycles), in contrast with the H/R group. Representative images of the ROS level were displayed in Figure 6(B). At the same time, we identified the effect on NRCMs apoptosis after suffering H/R by using TUNEL kit. As shown in Figure 6(C), cell apoptosis in the H/R group was aggravated, whereas PEMF treatment could reduce the cell death. Representative images of TUNEL staining were shown in Figure 6(D).

Figure 6

PEMF protected Neonatal rat cardiac ventricular myocytes (NRCMs) from hypoxia/reoxygenation (H/R)-induced apoptosis via decreasing ROS levelat the end of the treatment for 72 h in vitro.

Effect of PEMF on NO releasing via Akt/eNOS pathway

Cultured NRCMs were treated with PEMF stimulation for 1 to 2 cycles and the supernatant and cell lysate were collected. When cells suffered H/R, intracellular levels of p-Akt, p-eNOS and Bcl-2 were decreased, whereas PEMF treatment could increase the phosphorylation of Akt, p-eNOS and Bcl-2 (Figures 7A–7C). The expression of Bax was increased when cells subjected to H/R whereas PEMF treatment reversed such increase (Figure 7C). Western blot analysis was shown in Figure 7(D) for p-Akt/Akt, Figure 7(E) for p-eNOS/eNOS, Figure 7(F) for Bcl-2 and Figure 7(G) for Bax.

Figure 7

The related protein expression about the effect of PEMF on apoptosis induced by hypoxia/reoxygenationat the end of the treatment for 72 h in vitro. PEMF increased the phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and the expression 

DISCUSSION

Our present study provides the first evidence that PEMF has novel functions as follows: (1) We treated SHR rats with different PEMF intensity (8 min for 1 cycle, 30±3 Hz, X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT) 1–4 cycles per day for 7 days. PEMF can lower blood pressure under treatment of certain PEMF intensity in SHR rat model (double-blind). (2) PEMF has a profound effect on improving cardiac function in I/R rat model. (3) PEMF plays a vital role in inhibiting cardiac apoptosis via Bcl-2 up-regulation and Bax down-regulation. (4) In vitro, PEMF treatment also has a good effect on reducing ROS levels by Akt/eNOS pathway to release NO and improving cell apoptosis in NRCMs subjected to hypoxia.

Many previous studies showed that extracorporeal PEMF-treated(5 mT, 25 Hz, 1 h daily) could enhance osteanagenesis, skin rapture healing and neuronal regeneration, suggesting its regenerative potency [,,]. And some researchers had found that PEMF therapy (8 min/cycle, 30±3 Hz, 6 mT) could improve the myocardial infarct by activating VEGF–Enos [] system and promoting EPCs mobilized to the ischaemic myocardium [,]. Consistent with the previous work, our present study demonstrated that PEMF therapy could significantly alleviate cardiac dysfunction in I/R rat model.

Recent evidence suggest that circulating EPCs can be mobilized endogenously in response to tissue ischaemia or exogenously by cytokine stimulation and the recruitment of EPCs contributes to the adult blood vessels formation [,,]. We hypothesized that PEMF could recruit more EPCs to the vessels. To confirm our hypothesis, we applied antibodies to the Sca-1 and flk-1 to sign EPC. The results indicated that PEMF could remarkably increase the number of EPCs in the PEMF group, compared with the I/R group.

Previous evidence indicated that when heart suffered I/R, cardiac apoptosis would be dramatically aggravated []. Myocardial apoptosis plays a significant role in the pathogenesis of myocardial I/R injury. We assumed that PEMF might play its role in improving cardiac function through inhibiting cell apoptosis. The Bcl-2 family is a group of important apoptosis-regulating proteins that is expressed on the mitochondrial outer membrane, endoplasmic reticulum membrane and nuclear membrane. Overexpression of Bcl-2 proteins blocks the pro-apoptosis signal transduction pathway, thereby preventing apoptosis caused by the caspase cascade []. The role Bax plays in autophagy is a debatable. Recently, new genetic and biochemical evidence suggest that Bcl-2/Bcl-xL may affect apoptosis through its inhibition of Bax []. Overexpression of Bax protein promotes the apoptosis signal pathway. In the present study, we applied TUNEL staining to find that PEMF has a perfect effect on cardiac cell apoptosis by regulating apoptosis-related proteins Bcl-2 and Bax [,,,].

To verify our findings in the rat model, we mimicked I/R condition in vitro by hypoxia exposure in NRCMs. Results showed that not only in vivo, hypoxia could induce cell apoptosis in vitro. And we also found that PEMF treatment could significantly alleviate cell apoptosis induced by hypoxia. At the basal level, ROS play an important role in mediating multiple cellular signalling cascades including cell growth and stress adaptation. Conversely, excess ROS can damage tissues by oxidizing important cellular components such as proteins, lipids and DNA, as well as activating proteolytic enzymes such as matrix metalloproteinases []. Previous studies showed that when cells were subjected to hypoxia, the intracellular ROS level would be sharply increased, and the overproduction of ROS would result in cell damage [,,]. In the present study, PEMF treatment could prominently down-regulate ROS levels. We also investigated how PEMF reduced the intracellular ROS level.

NO appears to mediate distinct pathways in response to oxidative stress via AKt–eNOS pathway [,]. NO is identified as gaseous transmitters. In vascular tissue, NO is synthesized from L-arginine by nitric oxide synthase (NOS) and it is considered to be the endothelium-derived relaxing factor. Evidence show that the NO generation in endothelium cells was damaged in hypertensive patients []. NO could also prevent platelet activation and promote vascular smooth muscle cells proliferation []. NO generation from eNOS is considered to be endothelium-derived relaxing and ROS-related factor [,]. Some researchers found that bradykinin limited MI induced by I/R injury via Akt/eNOS signalling pathway in mouse heart []. And bradykinin inhibited oxidative stress-induced cardiomyocytes senescence by acting through BK B2 receptor induced NO release []. Such evidence indicated that Akt phosphorylation could activate eNOS, which lead to NO releasing, and resulted in ROS reducing. In the present study, we found that PEMF decreased ROS via Akt/eNOS pathway.

In conclusion, this is the first study suggesting that PEMF treatment could improve cardiac dysfunction through inhibiting cell apoptosis. Furthermore, in vitro, we first clarified PEMF still plays a profound effect on improving cell death and removing excess ROS via regulating apoptosis-related proteins and Akt/eNOS pathway. All these findings highlight that PEMF would be applied as a potentially powerful therapy for I/R injury cure.

Acknowledgments

We thank all of the members of the Laboratory of Pharmacology of Chen Y., Ding Y.J. for their technical assistance.

Abbreviations

Akt protein kinase B
Bax Bcl-2 associated X protein
Bcl-2 B-cell lymphoma 2
CCA common carotid artery
CCK-8 Cell Counting Kit-8
CK creatine kinase
CKMB creatine kinase isoenzyme-MB
DAPI 4,6?-diamidino-2?-phenylindole
DHE dihydroethidium
DMEM/F12 Dulbecco’s modified Eagle’s medium/F-12
dUTP deoxyuridine triphosphate
eNOS endothelial nitric oxide synthase
EPCs endothelial progenitor cells
flk-1 fetal liver kinase-1
GAPDH glyceraldehyde-3-phosphate dehydrogenase
HBDH ?-hydroxybutyrate dehydrogenase
H/R hypoxia/reoxygenation
HRP horseradish peroxidase
I/R ischaemia/reperfusion
LAD left anterior descending
LDH lactate dehydrogenase
MI myocardial infarction
MI/R myocardial infarction/reperfusion
MI/RI myocardial infarction/reperfusion injury
NRCMs neonatal rat cardiac ventricular myocytes
PEMF pulsed electromagnetic field
ROS reactive oxygen species
Sca-1 stem cell antigen-1
SD Sprague Dawley
SHR spontaneously hypertensive rats
TTC 2,3,5-triphenyltetrazolium chloride
TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
VEGF vascular endothelial growth factor

AUTHOR CONTRIBUTION

Fenfen Ma designed and performed experiments on MI/RI rat model, histological stain and Western blot. Wenwen Li assisted the in vivo experiments, validated the effect in vitro experiments, analysed data and wrote the manuscript. Xinghui Li interpreted data and formatted manuscript. Rinkiko Suguro, Ruijuan Guan, Cuilan Hou, Huijuan Wang and Aijie Zhang interpreted data and edited manuscript. Yichun Zhu and YiZhun Zhu proposed the idea and supervised the project.

FUNDING

This work was supported by the key laboratory program of the Education Commission of Shanghai Municipality [grant number ZDSYS14005].

References

1. Hao C.N., Huang J.J., Shi Y.Q., Cheng X.W., Li H.Y., Zhou L., Guo X.G., Li R.L., Lu W., Zhu Y.Z., Duan J.L. Pulsed electromagnetic field improves cardiac function in response to myocardial infarction. Am. J. Transl. Res. 2014;6:281–290. [PMC free article] [PubMed]
2. Eltzschig H.K., Eckle T. Ischemia and reperfusion–from mechanism to translation. Nat. Med. 2011;17:1391–1401. doi: 10.1038/nm.2507. [PMC free article] [PubMed] [Cross Ref]
3. Thygesen K., Alpert J.S., Jaffe A.S., Simoons M.L., Chaitman B.R., White H.D. Third universal definition of myocardial infarction. Nat. Rev. Cardiol. 2012;9:620–633. doi: 10.1038/nrcardio.2012.122.[PubMed] [Cross Ref]
4. Nah D.Y., Rhee M.Y. The inflammatory response and cardiac repair after myocardial infarction. Korean Circ. J. 2009;39:393–398. doi: 10.4070/kcj.2009.39.10.393. [PMC free article] [PubMed] [Cross Ref]
5. Yellon D.M., Hausenloy D.J. Myocardial reperfusion injury. N. Engl. J. Med. 2007;357:1121–1135. doi: 10.1056/NEJMra071667. [PubMed] [Cross Ref]
6. Herron T.J., Milstein M.L., Anumonwo J., Priori S.G., Jalife J. Purkinje cell calcium dysregulation is the cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia. Heart Rhythm. 2010;7:1122–1128. doi: 10.1016/j.hrthm.2010.06.010. [PMC free article] [PubMed] [Cross Ref]
7. Kim S.S., Shin H.J., Eom D.W., Huh J.R., Woo Y., Kim H., Ryu S.H., Suh P.G., Kim M.J., Kim J.Y., et al. Enhanced expression of neuronal nitric oxide synthase and phospholipase C-gamma1 in regenerating murine neuronal cells by pulsed electromagnetic field. Exp. Mol. Med. 2002;34:53–59. doi: 10.1038/emm.2002.8. [PubMed] [Cross Ref]
8. Tepper O.M., Callaghan M.J., Chang E.I., Galiano R.D., Bhatt K.A., Baharestani S., Gan J., Simon B., Hopper R.A., Levine J.P., Gurtner G.C. Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2. FASEB J. 2004;18:1231–1233. [PubMed]
9. Weintraub M.I., Herrmann D.N., Smith A.G., Backonja M.M., Cole S.P. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch. Phys. Med. Rehabil. 2009;90:1102–1109. doi: 10.1016/j.apmr.2009.01.019. [PubMed] [Cross Ref]
10. Graak V., Chaudhary S., Bal B.S., Sandhu J.S. Evaluation of the efficacy of pulsed electromagnetic field in the management of patients with diabetic polyneuropathy. Int. J. Diab. Dev. Ctries. 2009;29:56–61. doi: 10.4103/0973-3930.53121. [PMC free article] [PubMed] [Cross Ref]
11. Kin H., Zhao Z.Q., Sun H.Y., Wang N.P., Corvera J.S., Halkos M.E., Kerendi F., Guyton R.A., Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc. Res. 2004;62:74–85. doi: 10.1016/j.cardiores.2004.01.006.[PubMed] [Cross Ref]
12. Yao L.L., Huang X.W., Wang Y.G., Cao Y.X., Zhang C.C., Zhu Y.C. Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP. Am. J. Physiol. Heart. Circ. Physiol. 2010;298:H1310–H1319. doi: 10.1152/ajpheart.00339.2009. [PubMed] [Cross Ref]
13. Zhikun G., Liping M., Kang G., Yaofeng W. Structural relationship between microlymphatic and microvascullar blood vessels in the rabbit ventricular myocardium. Lymphology. 2013;46:193–201.[PubMed]
14. Tsai S.H., Huang P.H., Chang W.C., Tsai H.Y., Lin C.P., Leu H.B., Wu T.C., Chen J.W., Lin S.J. Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells. PLoS ONE. 2012;7:e41065. doi: 10.1371/journal.pone.0041065.[PMC free article] [PubMed] [Cross Ref]
15. Jin S., Pu S.X., Hou C.L., Ma F.F., Li N., Li X.H., Tan B., Tao B.B., Wang M.J., Zhu Y.C. Cardiac H2S generation is reduced in ageing diabetic mice. Oxid. Med. Cell. Longev. 2015;2015:758358.[PMC free article] [PubMed]
16. Cheing G.L., Li X., Huang L., Kwan R.L., Cheung K.K. Pulsed electromagnetic fields (PEMF) promote early wound healing and myofibroblast proliferation in diabetic rats. Bioelectromagnetics. 2014;35:161–169. doi: 10.1002/bem.21832. [PubMed] [Cross Ref]
17. Weintraub M.I., Herrmann D.N., Smith A.G., Backonja M.M., Cole S.P. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch. Phys. Med. Rehabil. 2009;90:1102–1109. doi: 10.1016/j.apmr.2009.01.019. [PubMed] [Cross Ref]
18. Li J., Zhang Y., Li C., Xie J., Liu Y., Zhu W., Zhang X., Jiang S., Liu L., Ding Z. HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism. Cardiovasc. Res. 2013;99:674–684. doi: 10.1093/cvr/cvt139. [PubMed] [Cross Ref]
19. Goto T., Fujioka M., Ishida M., Kuribayashi M., Ueshima K., Kubo T. Noninvasive up-regulation of angiopoietin-2 and fibroblast growth factor-2 in bone marrow by pulsed electromagnetic field therapy. J. Orthop. Sci. 2010;15:661–665. doi: 10.1007/s00776-010-1510-0. [PubMed] [Cross Ref]
20. Asahara T., Masuda H., Takahashi T., Kalka C., Pastore C., Silver M., Kearne M., Magner M., Isner J.M. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ. Res. 1999;85:221–228. doi: 10.1161/01.RES.85.3.221. [PubMed] [Cross Ref]
21. Takahashi T., Kalka C., Masuda H., Chen D., Silver M., Kearney M., Magner M., Isner J.M., Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat. Med. 1999;5:434–438. doi: 10.1038/8462. [PubMed] [Cross Ref]
22. Freude B., Masters T.N., Robicsek F., Fokin A., Kostin S., Zimmermann R., Ullmann C., Lorenz-Meyer S., Schaper J. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J. Mol. Cell Cardiol. 2000;32:197–208. doi: 10.1006/jmcc.1999.1066. [PubMed] [Cross Ref]
23. Martindale J.J., Fernandez R., Thuerauf D., Whittaker R., Gude N., Sussman M.A., Glembotski C.C. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6. Circ. Res. 2006;98:1186–1193. doi: 10.1161/01.RES.0000220643.65941.8d. [PubMed] [Cross Ref]
24. Yu L., Lu M., Wang P., Chen X. Trichostatin A ameliorates myocardial ischemia/reperfusion injury through inhibition of endoplasmic reticulum stress-induced apoptosis. Arch. Med. Res. 2012;43:190–196. doi: 10.1016/j.arcmed.2012.04.007. [PubMed] [Cross Ref]
25. Maiuri M.C., Criollo A., Tasdemir E., Vicencio J.M., Tajeddine N., Hickman J.A., Geneste O., Kroemer G. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L) Autophagy. 2007;3:374–376. doi: 10.4161/auto.4237.[PubMed] [Cross Ref]
26. Lindqvist L.M., Heinlein M., Huang D.C., Vaux D.L. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc. Natl. Acad. Sci. U.S.A. 2014;111:8512–8517. doi: 10.1073/pnas.1406425111. [PMC free article] [PubMed] [Cross Ref]
27. Chandna S., Suman S., Chandna M., Pandey A., Singh V., Kumar A., Dwarakanath B.S., Seth R.K. Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of gamma-radiation. Int. J. Rad. Biol. 2013;89:1017–1027. doi: 10.3109/09553002.2013.825059. [PubMed][Cross Ref]
28. Xu M., Zhou B., Wang G., Wang G., Weng X., Cai J., Li P., Chen H., Jiang X., Zhang Y. miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells. Zhonghua Zhong Liu Za Zhi. 2014;36:897–902. [PubMed]
29. Zuo L., Best T.M., Roberts W.J., Diaz P.T., Wagner P.D. Characterization of reactive oxygen species in diaphragm. Acta Physiol. (Oxf.) 2015;213:700–710. doi: 10.1111/apha.12410. [PubMed] [Cross Ref]
30. Kalogeris T., Bao Y., Korthuis R.J. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2014;2:702–714. doi: 10.1016/j.redox.2014.05.006.[PMC free article] [PubMed] [Cross Ref]
31. Levraut J., Iwase H., Shao Z.H., Vanden H.T., Schumacker P.T. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am. J. Physiol. Heart Circ. Physiol. 2003;284:H549–H558. doi: 10.1152/ajpheart.00708.2002. [PubMed] [Cross Ref]
32. Dong R., Chen W., Feng W., Xia C., Hu D., Zhang Y., Yang Y., Wang D.W., Xu X., Tu L. Exogenous bradykinin inhibits tissue factor induction and deep vein thrombosis via activating the eNOS/phosphoinositide 3-kinase/Akt signaling pathway. Cell. Physiol. Biochem. 2015;37:1592–1606. doi: 10.1159/000438526. [PubMed] [Cross Ref]
33. Jin R.C., Loscalzo J. Vascular nitric oxide: formation and function. J. Blood Med. 2010;2010:147–162.[PMC free article] [PubMed]
34. Taddei S., Virdis A., Mattei P., Ghiadoni L., Sudano I., Salvetti A. Defective L-arginine-nitric oxide pathway in offspring of essential hypertensive patients. Circulation. 1996;94:1298–1303. doi: 10.1161/01.CIR.94.6.1298. [PubMed] [Cross Ref]
35. Tang E.H., Vanhoutte P.M. Endothelial dysfunction: a strategic target in the treatment of hypertension? Pflugers Arch. 2010;459:995–1004. doi: 10.1007/s00424-010-0786-4. [PubMed] [Cross Ref]
36. Beltowski J., Jamroz-Wisniewska A. Hydrogen sulfide and endothelium-dependent vasorelaxation. Molecules. 2014;19:21183–21199. doi: 10.3390/molecules191221183. [PubMed] [Cross Ref]
37. Wu D., Hu Q., Liu X., Pan L., Xiong Q., Zhu Y.Z. Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. Nitric Oxide. 2015;46:204–212. doi: 10.1016/j.niox.2014.11.006. [PubMed] [Cross Ref]
38. Li Y.D., Ye B.Q., Zheng S.X., Wang J.T., Wang J.G., Chen M., Liu J.G., Pei X.H., Wang L.J., Lin Z.X., et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J. Biol. Chem. 2009;284:4473–4483. doi: 10.1074/jbc.M806010200. [PMC free article] [PubMed] [Cross Ref]
39. Dong R., Xu X., Li G., Feng W., Zhao G., Zhao J., Wang D.W., Tu L. Bradykinin inhibits oxidative stress-induced cardiomyocytes senescence via regulating redox state. PLoS ONE. 2013;8:e77034. doi: 10.1371/journal.pone.0077034. [PMC free article] [PubMed] [Cross Ref]

Aging / Longevity

 2018 Sep;33(7):1513-1519. doi: 10.1007/s10103-018-2510-0. Epub 2018 Apr 26.

Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

Author information

1
Laboratório de Pesquisa em Células Tronco, Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, Vila Isabel, Rio de Janeiro, 20551-030, Brazil.
2
Laboratório de Biomorfologia e Patologia Experimental, Universidade Severino Sombra, Avenida Expedicionário Oswaldo de Almeida Ramos 280, Vassouras, Rio de Janeiro, 27700-000, Brazil.
3
Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, Vila Isabel, Rio de Janeiro, 20551-030, Brazil.
4
Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, Vila Isabel, Rio de Janeiro, 20551-030, Brazil. adnfonseca@yahoo.com.br.
5
Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, Rua Frei Caneca, 94, Rio de Janeiro, 20211-040, Brazil. adnfonseca@yahoo.com.br.

Abstract

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

KEYWORDS:

DNA; Laser; Muscle; Wistar rats

Photomed Laser Surg. 2018 Mar 23. doi: 10.1089/pho.2017.4393. [Epub ahead of print]

Aging Is a Sticky Business.

Sommer AP1.

Author information

1
Ulm, Germany .

Abstract

OBJECTIVE:

The objective of this work is to put forward a mechanism by which low-level light [red-to near infrared (NIR) laser or light emitting diodes (LED)] is instrumental in the process of accelerating the healing of wounds.

BACKGROUND DATA:

Interaction modalities of low-level light with oxidatively stressed cells and tissues are the focus of intense research efforts. Several models of the light/cell-interaction mechanism have been proposed. In the most popular model, cytochrome c oxidase is believed to play the role of the principal acceptor for red-to NIR photons.

METHODS:

Using as an illustrative example the successful LED treatment of an edematous limb ulcer, the results of recent in vitro tests and complementary laboratory experiments are presented and discussed.

RESULTS:

The most plausible mechanism of biostimulatory effect of red-to NIR light consists of its impact on the nanoscopic interfacial water layers in mitochondria and the extracellular matrix (ECM) where mitochondrial reactive oxygen species (ROS) induce an increase in the viscosity of the water layers bound to the predominantly hydrophilic surfaces in the intramitochondrial space as well as the ECM, where the process progressively propagates with age. The biostimulatory effect of red-to NIR light consists of counteracting the ROS-induced elevation of interfacial water viscosities, thereby instantly restoring the normal mitochondrial function, including the synthesis of adenosine triphosphate (ATP) by the rotary motor (ATP synthase).

CONCLUSIONS:

An understanding of the mechanism of interaction of red-to NIR light with mitochondria, cells, and tissues safeguards progress in the field of low-level light therapy (LLLT) and puts us in the position to design better therapies.

KEYWORDS:

ATP; LED; ROS; interfacial water viscosity; laser; mitochondria; wound

Neurobiol Aging. 2018 Feb 26;66:131-137. doi: 10.1016/j.neurobiolaging.2018.02.019. [Epub ahead of print]

Photobiomodulation reduces gliosis in the basal ganglia of aged mice.

El Massri N1, Weinrich TW2, Kam JH2, Jeffery G2, Mitrofanis J3.

Author information

1
Department of Anatomy F13, University of Sydney, Sydney, NSW, Australia.
2
Institute of Ophthalmology, University College London, London, England.
3
Department of Anatomy F13, University of Sydney, Sydney, NSW, Australia. Electronic address: john.mitrofanis@sydney.edu.au.

Abstract

This study explored the effects of long-term photobiomodulation (PBM) on the glial and neuronal organization in the striatum of aged mice. Mice aged 12 months were pretreated with PBM (670 nm) for 20 minutes per day, commencing at 5 months old and continued for 8 months. We had 2 control groups, young at 3 months and aged at 12 months old; these mice received no treatment. Brains were aldehyde-fixed and processed for immunohistochemistry with various glial and neuronal markers. We found a clear reduction in glial cell number, both astrocytes and microglia, in the striatum after PBM in aged mice. By contrast, the number of 2 types of striatal interneurons (parvalbumin+ and encephalopsin+), together with the density of striatal dopaminergic terminals (and their midbrain cell bodies), remained unchanged after such treatment. In summary, our results indicated that long-term PBM had beneficial effects on the aging striatum by reducing glial cell number; and furthermore, that this treatment did not have any deleterious effects on the neurons and terminations in this nucleus.

KEYWORDS:

Astrocytes; Caudate-putamen complex; Interneurons; Microglia; Substantia nigra

J Biophotonics. 2017 Dec 11. doi: 10.1002/jbio.201700282. [Epub ahead of print]

Aging of lymphoid organs: Can photobiomodulation reverse age-associated thymic involution via stimulation of extrapineal melatonin synthesis and bone marrow stem cells?

Odinokov D1, Hamblin MR2,3,4.

Author information

1
Department of Biomedical Engineering, Chinese University of Hong Kong, Hong Kong.
2
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, 02114, USA.
3
Department of Dermatology, Harvard Medical School, Boston, MA, 02115, USA.
4
Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA.

Abstract

Thymic atrophy and the subsequent reduction in T cell production are the most noticeable age-related changes affecting lymphoid organs in the immune system. In fact thymic involution has been described as “programmed aging”. New therapeutic approaches such as photobiomodulation (PBM) may reduce or reverse these changes. PBM (also known as low-level laser therapy or LLLT) involves the delivery of non-thermal levels of red or near-infrared light that are absorbed by mitochondrial chromophores, in order to prevent tissue death and stimulate healing and regeneration. PBM may reverse or prevent thymic involution due to its ability to induce extrapineal melatonin biosynthesis via cyclic AMP or NF-kB activation, or alternatively by stimulating bone marrow stem cells that can regenerate the thymus. This perspective puts forward a hypotheses that PBM can alter thymic involution, improve immune functioning in aged people, and even extend lifespan.

Neurobiol Aging. 2017 Oct;58:140-150. doi: 10.1016/j.neurobiolaging.2017.06.025. Epub 2017 Jul 6.

Transcranial low-level laser therapy improves brain mitochondrial function and cognitive impairment in D-galactose-induced aging mice.

Salehpour F1, Ahmadian N2, Rasta SH3, Farhoudi M2, Karimi P2, Sadigh-Eteghad S4.

Author information

1
Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Physics, Tabriz University of Medical Sciences, Tabriz, Iran.
2
Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran.
3
Department of Medical Physics, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medical Bioengineering, Tabriz University of Medical Sciences, Tabriz, Iran; School of Medical Sciences, University of Aberdeen, Aberdeen, UK.
4
Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: Saeed.sadigetegad@gmail.com.

Abstract

Mitochondrial function plays a key role in the aging-related cognitive impairment, and photoneuromodulation of mitochondria by transcranial low-level laser therapy (LLLT) may contribute to its improvement. This study focused on the transcranial LLLT effects on the D-galactose (DG)-induced mitochondrial dysfunction, apoptosis, and cognitive impairment in mice. For this purpose, red and near-infrared (NIR) laser wavelengths (660 and 810 nm) at 2 different fluencies (4 and 8 J/cm2) at 10-Hz pulsed wave mode were administrated transcranially 3 d/wk in DG-received (500 mg/kg/subcutaneous) mice model of aging for 6 weeks. Spatial and episodic-like memories were assessed by the Barnes maze and What-Where-Which (WWWhich) tasks. Brain tissues were analyzed for mitochondrial function including active mitochondria, adenosine triphosphate, and reactive oxygen species levels, as well as membrane potential and cytochrome c oxidase activity. Apoptosis-related biomarkers, namely, Bax, Bcl-2, and caspase-3 were evaluated by Western blotting method. Laser treatments at wavelengths of 660 and 810 nm at 8 J/cm2 attenuated DG-impaired spatial and episodic-like memories. Also, results showed an obvious improvement in the mitochondrial function aspects and modulatory effects on apoptotic markers in aged mice. However, same wavelengths at the fluency of 4 J/cm2 had poor effect on the behavioral and molecular indexes in aging model. This data indicates that transcranial LLLT at both of red and NIR wavelengths at the fluency of 8 J/cm2 has a potential to ameliorate aging-induced mitochondrial dysfunction, apoptosis, and cognitive impairment.

KEYWORDS:

Aging; Apoptosis; D-galactose; Episodic-like memory; Mitochondrial function; Spatial memory; Transcranial low-level laser therapy

Exp Brain Res. 2017 Oct;235(10):3081-3092. doi: 10.1007/s00221-017-5048-7. Epub 2017 Jul 25.

No evidence for toxicity after long-term photobiomodulation in normal non-human primates.

Moro C1, Torres N1, Arvanitakis K2, Cullen K2, Chabrol C1, Agay D1, Darlot F1, Benabid AL1, Mitrofanis J3.

Author information

1
University of Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus, 38000, Grenoble, France.
2
Department of Anatomy F13, University of Sydney, Camperdown, 2006, Australia.
3
Department of Anatomy F13, University of Sydney, Camperdown, 2006, Australia. john.mitrofanis@sydney.edu.au.

Abstract

In this study, we explored the effects of a longer term application, up to 12 weeks, of photobiomodulation in normal, naïve macaque monkeys. Monkeys (n = 5) were implanted intracranially with an optical fibre device delivering photobiomodulation (red light, 670 nm) to a midline midbrain region. Animals were then aldehyde-fixed and their brains were processed for immunohistochemistry. In general, our results showed that longer term intracranial application of photobiomodulation had no adverse effects on the surrounding brain parenchyma or on the nearby dopaminergic cell system. We found no evidence for photobiomodulation generating an inflammatory glial response or neuronal degeneration near the implant site; further, photobiomodulation did not induce an abnormal activation or mitochondrial stress in nearby cells, nor did it cause an abnormal arrangement of the surrounding vasculature (endothelial basement membrane). Finally, because of our interest in Parkinson’s disease, we noted that photobiomodulation had no impact on the number of midbrain dopaminergic cells and the density of their terminations in the striatum. In summary, we found no histological basis for any major biosafety concerns associated with photobiomodulation delivered by our intracranial approach and our findings set a key template for progress onto clinical trial on patients with Parkinson’s disease.

KEYWORDS:

670 nm; Behaviour; Macaque monkeys; Striatum; Substantia nigra; Tyrosine hydroxylase

Exp Brain Res. 2017 Oct;235(10):3081-3092. doi: 10.1007/s00221-017-5048-7. Epub 2017 Jul 25.

No evidence for toxicity after long-term photobiomodulation in normal non-human primates.

Moro C1, Torres N1, Arvanitakis K2, Cullen K2, Chabrol C1, Agay D1, Darlot F1, Benabid AL1, Mitrofanis J3.

Author information

1
University of Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus, 38000, Grenoble, France.
2
Department of Anatomy F13, University of Sydney, Camperdown, 2006, Australia.
3
Department of Anatomy F13, University of Sydney, Camperdown, 2006, Australia. john.mitrofanis@sydney.edu.au.

Abstract

In this study, we explored the effects of a longer term application, up to 12 weeks, of photobiomodulation in normal, naïve macaque monkeys. Monkeys (n = 5) were implanted intracranially with an optical fibre device delivering photobiomodulation (red light, 670 nm) to a midline midbrain region. Animals were then aldehyde-fixed and their brains were processed for immunohistochemistry. In general, our results showed that longer term intracranial application of photobiomodulation had no adverse effects on the surrounding brain parenchyma or on the nearby dopaminergic cell system. We found no evidence for photobiomodulation generating an inflammatory glial response or neuronal degeneration near the implant site; further, photobiomodulation did not induce an abnormal activation or mitochondrial stress in nearby cells, nor did it cause an abnormal arrangement of the surrounding vasculature (endothelial basement membrane). Finally, because of our interest in Parkinson’s disease, we noted that photobiomodulation had no impact on the number of midbrain dopaminergic cells and the density of their terminations in the striatum. In summary, we found no histological basis for any major biosafety concerns associated with photobiomodulation delivered by our intracranial approach and our findings set a key template for progress onto clinical trial on patients with Parkinson’s disease.

KEYWORDS:

670 nm; Behaviour; Macaque monkeys; Striatum; Substantia nigra; Tyrosine hydroxylase

Exp Brain Res. 2017 Jun;235(6):1861-1874. doi: 10.1007/s00221-017-4937-0. Epub 2017 Mar 15.

Photobiomodulation-induced changes in a monkey model of Parkinson’s disease: changes in tyrosine hydroxylase cells and GDNF expression in the striatum.

El Massri N1, Lemgruber AP1, Rowe IJ1, Moro C2, Torres N2, Reinhart F2, Chabrol C2, Benabid AL2, Mitrofanis J3.

Author information

1
Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia.
2
University of Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus, 38000, Grenoble, France.
3
Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia. john.mitrofanis@sydney.edu.au.

Abstract

Intracranial application of red to infrared light, known also as photobiomodulation (PBM), has been shown to improve locomotor activity and to neuroprotect midbrain dopaminergic cells in rodent and monkey models of Parkinson’s disease. In this study, we explored whether PBM has any influence on the number of tyrosine hydroxylase (TH)+cells and the expression of GDNF (glial-derived neurotrophic factor) in the striatum. Striatal sections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice and monkeys and 6-hydroxydopamine (6OHDA)-lesioned rats that had PBM optical fibres implanted intracranially (or not) were processed for immunohistochemistry (all species) or western blot analysis (monkeys). In our MPTP monkey model, which showed a clear loss in striatal dopaminergic terminations, PBM generated a striking increase in striatal TH+ cell number, 60% higher compared to MPTP monkeys not treated with PBM and 80% higher than controls. This increase was not evident in our MPTP mouse and 6OHDA rat models, both of which showed minimal loss in striatal terminations. In monkeys, the increase in striatal TH+ cell number in MPTP-PBM cases was accompanied by similar increases in GDNF expression, as determined from western blots, from MPTP and control cases. In summary, these results offer insights into the mechanisms by which PBM generates its beneficial effects, potentially with the use of trophic factors, such as GDNF.

KEYWORDS:

670 nm; 6OHDA; Caudate; MPTP; Near infrared light; Putamen

 2016 Aug;31(6):1161-7. doi: 10.1007/s10103-016-1956-1. Epub 2016 May 25.

Lowlevel infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

Author information

1
Laboratório de Pesquisa em Células Tronco, Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, Vila Isabel, Rio de Janeiro, 20551030, Brazil.
2
Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, 4° andar, Vila Isabel, Rio de Janeiro, 20551030, Brazil.
3
Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, 4° andar, Vila Isabel, Rio de Janeiro, 20551030, Brazil. adnfonseca@yahoo.com.br.
4
Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, Rua Frei Caneca, 94, Rio de Janeiro, 20211040, Brazil. adnfonseca@yahoo.com.br.

Abstract

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomerelength limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated lowlevel infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to lowlevel infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that lowlevelinfrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Lowlevel infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

KEYWORDS:

Low level laser; MyoD; Pax7; TRF1; TRF2

[Experimental study of effect of low power laser on telomere length of cells].

[Article in Chinese]

Author information

1
Department of Physiology, Guangxi Medical University, Nanning 530021, China.

Abstract

To investigate the effect of low power helium neon laser (He-Ne laser) on the telomere length of human fetal lung diploid fibroblast (2BS) cell, we used the laser (gamma = 632. 8 nm, P = 2 mW) to treat the young 2BS cells. Cell growth and proliferation was observed through MTT method after treating with low power laser. The relative telomere length of 2BS cells was detected by fluorescence real-time quantitative PCR (q-PCR). The results showed that the cells of the treated groups grew better than the untreated groups. The telomereDNA length of the old 2BS cells, treated by low power He-Ne laser when they were young, was longer than that of untreated group. The results of the present study indicated that the low power He-Ne laser might decrease shortening rate of telomere and delay the aging of cells. Therefore, this study provides the experimental basis for us to further investigate the effect of low power laser on cell aging at the gene level.

Vopr Kurortol Fizioter Lech Fiz Kult.  2011 Jul-Aug;(4):39-42.

The influence of pulsed infrared laser radiation on the hormone production in the thymus (an experimental study).

[Article in Russian]
[No authors listed]

Abstract

Local irradiation with pulsed (1500 Hz) low-energy infrared laser light of the thymus and thyroid gland region caused well-apparent stimulation of alpha-1-thymosin production in the healthy animals and normalized its level in the stressed ones. Similar stimulation of alpha-1-timosine biosynthesis was observed in an experiment with direct laser irradiation of the cultured HTSC epitheliocytes from the human thymus.

Adv Gerontol. 2010;23(4):547-53.

Induced thymus aging: radiation model and application perspective for low intensive laser radiation.

[Article in Russian]
Sevost’ianova NN, Trofimov AV, Lin’kova NS, Poliakova VO, Kvetno IM.

Abstract

The influence of gamma-radiation on morphofunctional state of thymus is rather like as natural thymus aging. However gamma-radiation model of thymus aging widely used to investigate geroprotectors has many shortcomings and limitations. Gamma-radiation can induce irreversible changes in thymus very often. These changes are more intensive in comparison with changes, which can be observed at natural thymus aging. Low intensive laser radiation can not destroy structure of thymus and its effects are rather like as natural thymus aging in comparison with gamma-radiation effects. There are many parameters of low intensive laser radiation, which can be changed to improve morphofunctional thymus characteristics in aging model. Using low intensive laser radiation in thymus aging model can be very perspective for investigations of aging immune system.

Biofizika. 2007 Jan-Feb;52(1):137-40.

Protective effect of low-power laser radiation in acute toxic stress.

[Article in Russian]
Novoselova EG, Glushkova OV, Khrenov MO, Chernenkov DA, Lunin SM, Novoselova TV, Chudnovski? VM, Iusupov VI, Fesenko EE.

Abstract

The effect of preliminary short-term irradiation with He-Ne laser light (632.8 nm, 0.2 mW/cm2) of the thymus zone projection of male NMRI mice subjected to acute toxic stress on the responses of immune cells was studied. Stress was modeled by lipopolysaccharide injection, 250 mg/100 g of body weight, which induced a significant increase in the production of several macrophage cytokines, IL-1alpha, IL-1beta, IL-6, IL-10 and TNF-alpha. A single irradiation with laser light did not provoke considerable variations in NO production in cells but induced an enhancement in the production of heat shock proteins Hsp25, Hsp70, and Hsp90. Nevertheless, when irradiation with red laser light was applied prior to toxic stress, considerable normalization of production of nearly all cytokines studied and nitric oxide was observed. Moreover, the normalization of production of heat shock proteins has been shown in these conditions. Thus, preliminary exposure of a small area of animal skin surface provoked a significant lowering in the toxic effect of lipopolysaccharide.

Izv Akad Nauk Ser Biol. 2006 Nov-Dec;(6):667-79.

Structure peculiarities of muscle regenerates and state of thymus under He-Ne laser therapy in different periods after muscle trauma.

[Article in Russian]
Buliakova NV, Azarova VS.

Abstract

We studied the gastrocnemius muscle regeneration and the reactive changes in thymus of rats under different regimens of He-Ne laser therapy of both operated legs (632.8 nm; 2.5-3.0 mW/cm(2) ). Laser radiation (10 exposures by 3 min within 1-15 days after muscle trauma, 4.5-5.4 J/cm(2) total dose per each leg) stimulated inflammatory reaction, muscle healing and favored preservation of muscle tissue in regenerates. The changes in thymus mass, its histological structure, size of cortex and thymocite mitotic index pointed to the increase of the functional load on thymus and delay of its recovery. The same dose of laser therapy of muscles within 16-30 days after trauma led to the increase of muscle tissue sclerotization in regenerates. The reactive changes in thymus were less pronounced. Threefold decrease of laser dose (10 exposures by 1 min for 1-15 days, 1.5-1.8 J/cm(2)) suppressed inflammatory reaction, impaired the muscle regeneration. The increase of functional activity in thymus was not observed.

Minim Invasive Ther Allied Technol. 2006;15(5):277-85.

Regeneration of skeletal muscles and state of thymus in gamma-irradiated rats under laser therapy of the area of muscle trauma.

Bulyakova NV, Azarova VS.

Source

A N Severtzov Institute of Ecology & Evolution, Russian Academy of Sciences, Moscow, Russia. admin@sevin.ru

Abstract

The gamma-irradiation of adult rats with a semi-lethal dose (6 Gy) suppressed the posttraumatic regeneration of skeletal muscles and brought about considerable destructive changes in the thymus. The effect of He-Ne laser radiation at a total dose 4.5-5.4 J/cm2 at each operated leg in irradiated rats stimulated the regenerative capacity of skeletal muscle tissue, the healing of skin-muscle wound, and the processes of postradiation recovery in thymus cells (a decrease of chromosome aberrations). The histological structure of regenerates had more muscle pattern. At the same time, the positive dynamics of regenerative processes in muscles was achieved by an increased functional load on the thymus. To stimulate the regeneration of irradiated muscles on the background of a more moderate load on the thymus, the prolonged period of laser therapy and fragmentary distribution of laser exposures during muscle regeneration were preferable. Wound healing improved visibly. Nor formation of chronic radiation ulcers on operated shins was observed.

Biofizika. 2006 Jan-Feb;51(1):123-35.

Effects of exposure of different skin areas to low-power laser light.

[Article in Russian]
Glushkov OV, Novoselova EG, Cherenkov DA, Novoselova TV, Khrenov MO, Lunin SM, Chudnovski? VM, Iusupov VI, Fesenko EE.

Abstract

The effect of helium-neon laser light of extremely low power of 0.2 mW/cm2 and wavelength 632.8 nm on the immune status of mice bearing solid tumors was studied. The evaluation of the status of tumor-bearing animals was provided by taking into account the number of immune cells, cytokine concentration (tumor necrosis factor, interleukin 2, production of nitric oxide, expression of heat shock proteins (Hsp70 and Hsp90), and activity of natural killers. The model of a solid tumor was formed by subcutaneous injection of Ehrlich carcinoma cells, and average life span of tumor-bearing mice achieved about 55 days. Different areas of the skin of tumor-bearing mice were subjected either to a single (1 min, dose 0.012 J/cm3) or repeated exposure to laser light (1 min, 48-h intervals, 30 days). Two different areas were irradiated: the thymus projection area or a hind limb with solid tumors. The results showed that chronic exposure of tumor-bearing mice in the thymus projection area, and especially, hind limb, reduced the resistance, which manifested itself in the acceleration of tumor growth and a tendency of mouse life span to decrease. On the contrary, a single exposure stimulated the antitumor immunity for several days after the exposure. The results show the expediency of further investigation of the immunomodulative effects of low-power laser light and the necessity of monitoring the immune system during laser therap

Nitric Oxide

Logo of oximed

Oxidative Medicine and Cellular Longevity
Oxid Med Cell Longev. 2017; 2017: 2181942.
Published online 2017 Sep 12. doi:  10.1155/2017/2181942
PMCID: PMC5613626

Benign Effect of Extremely Low-Frequency Electromagnetic Field on Brain Plasticity Assessed by Nitric Oxide Metabolism during Poststroke Rehabilitation

Natalia Cicho,corresponding author 1 Piotr Czarny, 2 Micha Bijak, 1 Elbieta Miller, 3 , 4 Tomasz liwiski, 5 Janusz Szemraj, 2 and Joanna Saluk-Bijak 1
1Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, Lodz, Poland
2Department of Medical Biochemistry, Medical University of Lodz, Mazowiecka 6/8, Lodz, Poland
3Department of Physical Medicine, Medical University of Lodz, Pl. Hallera 1, Lodz, Poland
4Neurorehabilitation Ward, III General Hospital in Lodz, Milionowa 14, Lodz, Poland
5Department of Molecular Genetics, Laboratory of Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Pomorska 141/143, Lodz, Poland
corresponding authorCorresponding author.
Academic Editor: Tanea T. Reed
Author information Article notes Copyright and License information
Received 2017 May 12; Revised 2017 Jul 2; Accepted 2017 Aug 14.

Abstract

Nitric oxide (NO) is one of the most important signal molecules, involved in both physiological and pathological processes. As a neurotransmitter in the central nervous system, NO regulates cerebral blood flow, neurogenesis, and synaptic plasticity. The aim of our study was to investigate the effect of the extremely low-frequency electromagnetic field (ELF-EMF) on generation and metabolism of NO, as a neurotransmitter, in the rehabilitation of poststroke patients. Forty-eight patients were divided into two groups: ELF-EMF and non-ELF-EMF. Both groups underwent the same 4-week rehabilitation program. Additionally, the ELF-EMF group was exposed to an extremely low-frequency electromagnetic field of 40Hz, 7mT, for 15min/day. Levels of 3-nitrotyrosine, nitrate/nitrite, and TNF? in plasma samples were measured, and NOS2 expression was determined in whole blood samples. Functional status was evaluated before and after a series of treatments, using the Activity Daily Living, Geriatric Depression Scale, and Mini-Mental State Examination. We observed that application of ELF-EMF significantly increased 3-nitrotyrosine and nitrate/nitrite levels, while expression of NOS2 was insignificantly decreased in both groups. The results also show that ELF-EMF treatments improved functional and mental status. We conclude that ELF-EMF therapy is capable of promoting recovery in poststroke patients.

1. Introduction

Cardiovascular diseases, including ischemic stroke (IS), are a serious problem of the modern age, killing 4 million people each year in Europe []. Stroke is caused by ischemia of brain tissue. Brain structure damage occurring during ischemia/reperfusion is due to the generation of significant amounts of reactive oxygen species and inflammatory mediators []. Damage to brain tissue as a result of a stroke cannot be undone. However, the most important part of poststroke therapy is immediate and long-term rehabilitation, considering the enormous plasticity of the brain []. Although extremely low-frequency electromagnetic field (ELF-EMF) therapy is not a standard treatment in the poststroke rehabilitation, some authors suggest its increased positive effect on patients []. ELF-EMF treatment is based on regeneration, osteogenesis, analgesics, and anti-inflammatory action. Its biological effect is related to processes of ion transport, cell proliferation, apoptosis, protein synthesis, and changes in the transmission of cellular signals []. The regenerative and cytoprotective effect of ELF-EMF is based on mechanism associated with nitric oxide induction, collateral blood flow, opioids, and heat shock proteins [].

Nitric oxide (NO) is an unstable, colourless, water-soluble gas with a short half-life (3–6sec). The compound has one unpaired electron, which makes it a highly reactive free radical. It is characterized by the multiplicity of action in the body, in both physiological and pathological conditions []. Synthesis of NO in the organism is catalysed by nitric oxide synthase (NOS), occurring in three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS), encoded by different genes whose expression is subject to varying regulation. The constituent isoforms of NOS are eNOS and nNOS, whose activity is associated with concentration of calcium ions and the level of calmodulin in a cell, as well as with hypoxia, physical activity, and the level of certain hormones, that is, oestrogens []. In contrast, because it is closely related with the calmodulin, iNOS does not require a high concentration of calcium ions but is regulated by various endogenous and exogenous proinflammatory factors [].

The two-stage synthesis of NO consists of the oxidation of L-arginine to N-hydroxy-L-arginine and, under the influence of NOS and oxygen, formation of L-citrulline and release of NO. All isoforms of NOS require the same cofactors: nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4), iron protoporphyrin IX (heme), and O2[].

Nitric oxide is one of the most important signal molecules, involved in both physiological and pathological processes. One of the major functions of NO is as a potent vasodilation, increasing the blood flow and regulation of blood pressure, which has been used in clinical practice for many years. Deficiency of this compound is observed in various disorders of many systems: cardiovascular, gastrointestinal, respiratory, and genitourinary []. The beneficial effects of NO lie in its platelet inhibition, macrophage cytotoxicity (antibacterial, antiviral, and antiparasitic), and protection of the mucosal lining of the digestive system. On the other hand, excessive expression of iNOS can be disadvantageous, for example, during sepsis. The adverse action of NO is associated with the production of superoxide anions and subsequent generation of peroxynitrite and hydroxyl radicals, which are highly toxic [].

In the central nervous system, NO as a neurotransmitter regulates cerebral blood flow, as well as neurogenesis and synaptic plasticity. Furthermore, neuronal death is caused by high concentrations of NO by caspase-dependent apoptosis process and promotion of inflammation. Elevated levels of nitric oxide promote necrosis by energy depletion. On the basis of these mechanisms, NO is involved in the etiology of many neurological diseases, such as major depression, schizophrenia, epilepsy, anxiety, and drug addiction [].

Our study was designed to investigate the effect of ELF-EMF on the metabolism of NO, as a signal molecule in the central nervous system, in the rehabilitation of acute poststroke patients.

2. Materials and Methods

2.1. Blood Sample Collection

Blood samples were collected into CPDA1-containing tubes (Sarstedt, Nümbrecht, Germany). Immediately upon doing so, a portion of the sample was frozen at 80°C and the rest of the samples centrifuged to isolate the plasma (15min, 1500g) at 25°C. Blood samples were collected twice, at an interval of 14 days before and after a standard 10 sessions of therapy. For additional analysis of 3-nitrotyrosine levels, the blood samples were collected three times, at an interval of 28 days: before treatment, after 10 treatments, and after 20 treatments. All blood samples were taken in the morning (between 7am and 9am) under patient fasting condition and stored using the same protocol.

2.2. Subject Presentation

Forty-eight poststroke patients were enrolled in the study. Participants were randomly divided into two groups: ELF-EMF (n = 25) and non-ELF-EMF (n = 23). Patients with metal and/or electronic implants (pacemakers, etc.) were excluded from the ELF-EMF group, for safety reasons. The ELF-EMF group had already undergone ELF-EMF therapy with specific parameters (40Hz frequency, magnetic induction of 5mT (B), rectangular and bipolar waveforms) (Figure 1), which was conducted using a Magnetronic MF10 generator (EiE Elektronika i Elektromedycyna, Otwock, Poland). The parameters were selected on the basis of the fact that low-intensity stimuli improve the vital functions of the body. In addition, rectangular pulses are more intense than sinusoidal and trapezoid, while bipolar pulses show more range of changes than unipolar pulses []. The ELF-EMF and non-ELF-EMF groups were treated for the same amount of time (15minutes). The non-ELF-EMF subjects were given only sham exposure. The pelvic girdle of the patients was exposed to the electromagnetic field, because exposure of the head to ELF-EMF can affect the activation of the epilepsy focus in the brain. The same therapeutic program was used for both subject groups. This consisted of aerobic exercise (30min), neurophysiological routines (60min), and psychological therapy (15min). Poststroke patients with moderate stroke severity according to NIHSS scores of 4.9 ±3.1 in the ELF-EMF group (aged 48.8 ±7.7) and 5.4 ±2.9 (aged 44.8 ±8.0) in the non-ELF-EMF group were enrolled in the study. Table 1 shows the clinical and demographic characteristics. Participants with haemorrhagic stroke, dementia, chronic or significant acute inflammatory factors, decreased consciousness, and/or neurological illness other than stroke in their medical prestroke history were excluded. The subjects had undergone neurorehabilitation for 4 weeks in Neurorehabilitation Ward III of the General Hospital in Lodz, Poland, as well as internal and neurological examinations. The Bioethics Committee of the Faculty of Biology and Environmental Protection of The University of Lodz, Poland, approved the protocol with resolution numbers 28/KBBN-U/II/2015 and 13/KBBN-U/II/2016. All participants provided written informed consent prior to participation. Depression was screened in both groups using the Geriatric Depression Scale (GDS). Cognitive status was estimated in a Mini-Mental State Examination (MMSE), and functional status using the Barthel Index of Activities of Daily Living (ADL). The GDS, ADL, and MMSE were administered either on the same day as the blood sampling or on the afternoon before.

Figure 1

ELF-EMF description. B=5mT; T = 1.3sec.

Table 1

Clinical demographic characteristics.

2.3. Magnetronic MF10 Devices

ELF-EMF therapy was performed by a Magnetronic MF10 generator as per accepted guidelines. This device is able to produce pulses in rectangular, trapezoid, and sinusoidal shapes. The pulses were applied using an AS-550 applicator (EiE, Otwock, Poland), which has the following properties: 550 mm in diameter, 270mm in length, and 5 layers of 187 turns of 1.45mm twin-parallel wires. Magnetic induction was set at 5mT. The electromagnetic field intensity was not uniformed; its distribution is vertical, while the induction coils are set horizontally. Induction of the electromagnetic field of 5mT is present at the geometric center of the applicator, and the value increases in the proximity to the surface about 7mT. Other factors that could affect EMF were eliminated (electronic measuring instruments occurring in rehabilitation room and other electronic equipment).

2.4. Immunodetection of 3-Nitrotyrosine by c-ELISA

Levels of 3-NT-containing proteins in plasma were determined using a modified c-ELISA method, as described by Khan et al. []. 96-well microtiter plates were coated with nitro-fibrinogen (nitro-Fg) (1mg/mL) and kept overnight at 4°C. Concentrations of nitrated proteins inhibiting the binding of anti-nitrotyrosine antibodies were assessed from the standard curve (10–100nM nitro-Fg equivalents) and expressed as nitro-Fg equivalents [].

2.5. Nitrate/Nitrite Estimation

Plasma samples were diluted twice before the measurement of nitrate/nitrite concentration using a Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Company, USA), based on the two-step Griess method. In the first step, the nitrate is converted to nitrite with nitrate reductase, while in the second step, after addition of the Griess reagent, the nitrite is converted to a deep purple azo compound. The absorbance measurement was performed at 540nm in a 96-well microplate reader (SPECTROstarNano, BMG Labtech, Ortenberg, Germany) [].

2.6. Determination of NOS2 Expression in Whole Blood Samples

RNA was isolated from the frozen whole blood samples (?80°C), in accordance with the manufacturer’s protocol using TRI Reagent® (Sigma-Aldrich, USA). The aqueous phase was purified in accordance with the manufacturer’s protocol using an InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Germany). The purity and quantity of isolated RNA were assessed using a Synergy HTX Multi-Mode Microplate Reader equipped with a Take3 Micro-Volume Plate and connected to a PC running Gen5 Software (BioTek Instruments Inc., Winooski, VT, USA). Isolated RNA (20ng/L) was transcribed onto cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). Quantitative assays were executed using a TaqMan Hs01075529_m1 probe for human NOS2 genes and an Hs02786624_g1 for endogenous control, which was GAPDH (Life Technologies). Reactions were carried out using a TaqMan Universal Master Mix II, without UNG (Life Technologies) in a BioRad CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA), all in accordance with the manufacturers’ protocols. Relative expression of NOS2 was obtained using the equation 2Ct, where Ct is the threshold cycle (Ct) value for the target gene minus Ct values obtained for the housekeeping gene GAPDH [].

2.7. Determination of TNF?

Measurements of human tumour necrosis factor alpha (TNF?) in plasma samples were made with a Human TNF? ELISA development kit (MABTECH, Cincinnati, OH, USA), in accordance with the manufacturer’s protocol. The combination of two coating antibodies (TNF3 and TNF4) were used for the analysis. The absorbance was measured at 450nm, and TNF?.

Oxidative Medicine and Cellular Longevity

2.8. Data Analysis

Biochemical and clinical data were expressed as mean ±SD. All measurements were executed in duplicate. Output value (100%) was determined for each measured parameter of each patient before treatment. Data from tests performed on the same study subjects after therapy constituted a percentage of the output value. Percentage values were presented as mean ± SD. Statistical analyses were performed using the Statistica 12 statistical software (StaftSoft Inc.). A Shapiro-Wilk test was used to analyse for normality. A paired Student t-test was used to the calculate differences between the values obtained for subjects before and after therapy, whereas unpaired Student t-test or Mann–Whitney U tests were used to determine differences between the ELF-EMF and non-ELF-EMF groups. p values of 0.05 were accepted as statistically significant for all analyses.

3. Results

Our comparative analysis demonstrated an increased level of 3-nitrotyrosine (3-NT) (p< 0.05) (Figure 2) and an elevated nitrate/nitrite concentration (p < 0.01) (Figure 3) in the plasma of patients from the ELF-EMF group. The gain in the 3-NT level was significantly higher with an increased amount of sessions (Figure 2). In the non-ELF-EMF group, we saw that the effect of rehabilitation on nitrative stress was largely weaker and not statistically significant (p > 0.05) (Figures (Figures22 and and3).3). The 3-NT level increased more in the ELF-EMF group than in the non-ELF-EMF after 10 treatments (68% versus 17%, p < 0.05) (Figure 2). The level of nitrate/nitrite in the non-ELF-EMF group even decreased after 10 treatments (although not statistically significantly) (Figure 3).

Figure 2

The comparison of 3-NT levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group. Statistical significance between the ELF-EMF and non-ELF-EMF groups: B versus D (p < 0.05).

Figure 3

The comparison of nitrate/nitrite levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group. Statistical significance between ELF-EMF and non-ELF-EMF groups: B versus D (p < 0.05).

In the next set of experiments, we determined the effect of magnetotherapy on gene expression in the whole blood samples of NOS2 mRNA. Its expression was unmeasurable in 35% of subjects from both the ELF-EMF and non-ELF-EMF groups. We observed a statistically insignificant decrease in the level of NOS2 mRNA expression after treatment in both the ELF-EMF and non-ELF-EMF groups (Figure 4).

Figure 4

The comparison of NOS2 mRNA expression obtained from the ELF-EMF group versus that from the non-ELF-EMF group.

Subsequently, we determined the concentration of proinflammatory cytokine TNF?. We found that the concentration of TNF? was comparable before treatment in both the ELF-EMF and non-ELF-EMF-groups. The cytokine level did not change in either groups after rehabilitation (Figure 5).

Figure 5

The comparison of TNF? levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group.

The ADL, MMSE, and GDS were used to evaluate the functional and mental status of poststroke patients undergoing rehabilitation. We demonstrated that treatment using ELF-EMF improves their clinical parameters, particularly in cognitive and psychosomatic functions.

Motor abilities estimated by ADL score changed at similar levels in both groups, with the observed improvement being statistically significant in all rehabilitated patients (p < 0.001) (Table 2).

Table 2

Clinical parameters: ADL, MMSE, and GDS measured in the ELF-EMF and non-ELF-EMF groups. Data presented as the delta of a clinimetric scale before and after the standard series of treatments ADL=the increase of ADL; MMSE= the 

The baseline MMSE values before treatment in both groups were comparable, but statistically different (p < 0.05) after rehabilitation. After 2 weeks of rehabilitation, MMSE parameters improved markedly in the ELF-EMF group (p = 0.002), while a small increase in the non-ELF-EMF group was not statistically significant (p = 0.2) (Table 2).

Depression syndrome expressed by GDS improved significantly in both groups after rehabilitation. However, the GDS value reached about a 60% lower result in the ELF-EMF group than in the non-ELF-EMF group (p = 0.018), starting from a similar base level in both groups (p > 0.05) (Table 2).

4. Discussion

In this study, we provide the evidence that application of extremely low-frequency electromagnetic field increases nitric oxide generation and its metabolism, as well as improving the effectiveness of poststroke ischemic patients’ treatments.

Ischemic stroke is one of the major causes of morbidity and mortality in the world’s population and is one of the main causes of long-term disability. The mechanisms of neurological function recovery after brain injury associated with neuroplasticity (cortical reorganization) are still insufficiently understood. Poststroke neurorehabilitation is designed to provide external stimuli, improving the effectiveness of compensatory plasticity [].

In the central nervous system, NO is both a pre- and postsynaptic signal molecule. The activity of NO is associated with a cGMP-mediated signalling cascade. The presynaptic excitatory action of NO is related to the phosphorylation of synaptophysin by the cGMP-dependent protein kinase G (PKG) pathway and the subsequent potentates of glutamatergic neurotransmission []. On the other hand, NO causes a neurotransmission inhibition through gamma-aminobutyric acid- (GABA-) ergic synaptic communication. It is associated with ion exchange and regulation of membrane excitation []. Moreover, NO as an important vasodilation factor mediates neurovascular coupling. The enlargement of vessel diameter is caused by increasing metabolic consumption as a result of neuronal activity. Neurovascular coupling maintains functional and structural brain integrity [].

This study was designed to investigate the impact of ELF-EMF on the metabolism of nitric oxide in the rehabilitation of acute poststroke patients.

In our study, we demonstrate that poststroke rehabilitation increases the level of 3-NT and nitrate/nitrite concentrations. Due to its vasodilating and proangiogenic effects, NO serves as a protective function during cerebral ischemia. Su et al. investigated the role of simvastatin-regulated TRPV1 receptors (transient receptor potential vanilloid type 1) in NO bioavailability, activation of eNOS, and angiogenesis in mice. They demonstrated that simvastatin causes an influx of calcium ions through the TRPV1-TRPA1 (transient receptor potential ankyrin 1) pathway, which then causes activation of CaMKII (Ca2+/calmodulin-dependent protein kinase II). This then enhances the formation of the TRPV1-eNOS complex, which also includes CaMKII, AMPK (5AMP-activated protein kinase), and Akt (protein kinase B), which leads to activation of eNOS, production of NO, and thus the promotion of endothelial angiogenesis []. There have been numerous reports of the protective effects of NO against inflammation and oxidative stress []. Transgenic eNOS-deficient mice demonstrated a more extensive infarct of the middle cerebral artery (MCA), compared to controls []. NO effects on the regulation of endothelial integrity, anti-inflammatory and anti-apoptotic effects, as well as maintenance of cerebral blood flow, inhibition of platelet aggregation, and reduction of leukocyte adhesion []. Khan et al. studied structurally different NO donors as agents of cerebrovascular protection in experimentally induced stroke in rats. They showed that NO donors promote cerebral blood flow through S-nitrosylation and may be an effective drug for acute stroke [].

Furthermore, Greco et al. proved the protective effect of nitroglycerin (donors of NO) on cerebral damage induced by MCA occlusion in Wistar rats. They observed a significant reduction in stroke volume in preinjected rats compared to their control group, which confirms the protective effect of nitroglycerin in vivo. They speculated that the mechanism of action is associated with the generation of a complex chain of phenomena, triggering activation of apoptosis and subsequent activation of antiapoptotic responses [].

The biological action of ELF-EMF is still being investigated. It is suggested that ELF-EMF has an impact on the physicochemical properties of water, the liquid crystal structure generated by cholesterol, and its derivatives []. Changes in ion balance caused by ELF-EMF appeal to the structure of tissue with piezoelectric and magnetostrictive properties, free radicals, diamagnetic molecules, and uncompensated magnetic spins of paramagnetic elements []. Therefore, ELF-EMF causes depolarization of cells having the ability to spontaneously depolarize, predominantly through Ca2+ influx []. In our previous study, we investigated the effect of ELF-EMF on oxidative stress in patients after ischemic stroke. We demonstrated that ELF-EMF causes activation of antioxidant enzymes [], which leads to reduction of the oxidative modification of plasma protein (this is detailed in an article published in Advances in Clinical and Experimental Medicine). As a highly reactive molecule, NO can also regulate the level of oxidative stress. Through the covalent interaction, NO influences the activity of various enzymes. Mechanisms of this modulation can be varied: NO reacts with coenzymes and active centers containing metal ions and interacts with cysteine residues of proteins [].

In the current study, we observed that in the ELF-EMF group, the level of plasma 3-NT was increased (Figure 2). The formation of 3-NT in protein molecules occurs in vivo by the action of nitrating agents on the polypeptide chain. The formation of 3-NT is mainly attributed to NO and superoxide anions (O2??), which react rapidly to form peroxynitrite (ONOO?). This is one of the major oxidizing and nitrating agents produced in vivo in acute and chronic inflammation, as well as in ischemia/reperfusion. Endothelial cells, macrophages, and neutrophils release large amounts of NO and O2?. Thus, increased amounts of NO contribute to the creation of 3-NT [].

To investigate the effect of ELF-EMF on NO metabolism, we determined nitrate/nitrite concentrations in plasma. We showed that in the ELF-EMF group, the level of nitrate/nitrite compounds in plasma increased after treatment (Figure 3), and these results correspond with the data presented by Chung et al. []. The authors investigated the effects of ELF-EMF (60Hz, 2mT) on the level of NO, biogenic amines, and amino acid neurotransmitters in the hippocampus, cortex, thalamus, cerebellum, and striatum in rats. They found a significant increase in NO concentration in the hippocampus, thalamus, and striatum. Moreover, ELF-EMF also caused a change in the level of biogenic amines and amino acid neurotransmitters in the brain. However, the observed effect and range were different, depending on the brain area. Balind et al. determined the effect of ELF-EMF (50Hz, 0.5mT) on oxidative stress in gerbils with induced cerebral ischemia. They measured the level of NO using the Griess reagent and showed an increased level of NO, provoked by electromagnetic fields. Moreover, ELF-EMF reduces oxidative stress generated during cerebral ischemia, thus leading to a decrease in the damaged brain tissue [].

NO is produced from L-arginine with the involvement of nitric oxide synthase. Three NOS isoforms are expressed in different tissues. Although, in the blood, only NOS2 is expressed, in 35% of the subjects in both the ELF-EMF and non-ELF-EMF groups, mRNA expression of NOS2 was under detection. In the remaining patients, the expression of NOS2 had not significantly changed after treatment. The NOS2 gene in fact encodes for iNOS, which is primarily activated during inflammation. In order to exclude deeper inflammation, we measured the concentration of TNF?, one of the main proinflammatory cytokines. TNF? is a pleiotropic cytokine that is involved in nearly all phenomena of inflammatory responses: initiating chemokine synthesis, promoting the expression of adhesion molecules, promoting the maturation of dendritic cells, and inducing the production of inflammatory mediators and other proinflammatory cytokines []. TNF? stimulates collagenase synthesis in synovial fibroblasts and synovial cartilage chondrocytes and activates osteoclasts, leading to joint cartilage damage, hypertrophy, bone resorption and erosion, and angiogenesis. It also activates monocytes and macrophages, enhancing their cytotoxicity and stimulating cytokine production. Chemokines and growth factors are responsible for T cell proliferation, proliferation and differentiation of B lymphocytes, and the release of inflammatory cytokines by the lymphocytes. Moreover, in the hypothalamus, TNF? stimulates prostaglandin E and IL-1 synthesis []. Pena-Philippides et al. investigated the effect of pulsed electromagnetic fields on injury size and neuroinflammation in mice after middle cerebral artery occlusion (MCAO). They found, using magnetic resonance imaging (MRI), that EMF reduced infarct size, as well as changed expression of genes encoding pro- and anti-inflammatory cytokines in the hemisphere with ischemic injury. After EMF exposure, genes encoding IL-1 and TNF superfamily were downregulated, while IL-10 expression was upregulated. Thus, the authors suggested that application of EMF to poststroke patients could have been beneficial through anti-inflammatory effect and reduction of injury size [].

On the basis of our results, we suggest that the observed increase in NO level is associated with nNOS and/or eNOS activities, but not with iNOS expression. Our research is consistent with evidence shown by Cho et al., who established that ELF-EMF (60Hz, 2mT) increased the expression and activation of nNOS in rat brains [].

The activities of nNOS and eNOS depend on calcium ions. There are many reports that the biological effect of ELF-EMF is related to the control of calcium channels []. In view of these findings, the observed mechanism of increased NO generation and metabolism may be associated with calcium-ion flux.

Additionally, we noticed that ELF-EMF treatment enhances the effectiveness of poststroke rehabilitation (Table 2). Some researchers suggest that electromagnetic fields have a beneficial effect on ischemic/reperfusion injury, and in some places, therapeutic programs using ELF-EMF are considered to be standard therapy for poststroke patients []. The beneficial effects of ELF-EMF include the following: improvement in the transport of cellular and mitochondrial membranes; normalization of blood rheological values; counteraction of tissue oxidation; intensification of regenerative processes; stimulation of axon growth in undamaged neurons; intensification of neuronal dissociation and differentiation; reduction of stress-induced emotional reactions and free radicals; acceleration of the return of fibre function in functional disorders; reduction of periapical scarring; and increase of the level of energetic substances in the brain tissue and erythrocytes []. Grant et al. estimated the impact of low-frequency pulsed electromagnetic field on cerebral ischemia in rabbit. They observed using MRI that exposure to electromagnetic field caused extenuation of cortical ischemia oedema and reduction of neuronal injury in cortical area [].

In conclusion, ELF-EMF therapy increases the metabolism and generation of NO, which has both neuroprotective and cytotoxic properties. An increase in NO level is probably associated with nNOS and/or eNOS activities, but not with iNOS expression, which increases mainly during inflammation. We suggested that in poststroke patients, NO demonstrated a protective effect due to significant improvement in patient functional status. Thus, our studies promote the validity of this method in poststroke rehabilitation therapy.

Acknowledgments

This study was supported by the Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz (no. 506/1136), and Laboratory of Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz (no. B161100000004601), and Grants for Young Scientists and PhD Students, Faculty of Biology and Environmental Protection, University of Lodz (B1611000001145.02).

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

References

1. Townsend N., Wilson L., Bhatnagar P., Wickramasinghe K., Rayner M., Nichols M. Cardiovascular disease in Europe: epidemiological update 2016. European Heart Journal2016;37(42):3232–3245. doi: 10.1093/eurheartj/ehw334. [PubMed][Cross Ref]
2. Li X., Su L., Zhang X., et al. Ulinastatin downregulates TLR4 and NF-kB expression and protects mouse brains against ischemia/reperfusion injury. Neurological Research2017;13:1–7. doi: 10.1080/01616412.2017.1286541. [PubMed] [Cross Ref]
3. Klarner T., Barss T. S., Sun Y., Kaupp C., Loadman P. M., Zehr E. P. Long-term plasticity in reflex excitability induced by five weeks of arm and leg cycling training after stroke. Brain Sciences2016;6(4) doi: 10.3390/brainsci6040054.[PMC free article] [PubMed] [Cross Ref]
4. Cheng Y., Dai Y., Zhu X., et al. Extremely low-frequency electromagnetic fields enhance the proliferation and differentiation of neural progenitor cells cultured from ischemic brains. Neuroreport2015;26(15):896–902. doi: 10.1097/WNR.0000000000000450. [PubMed] [Cross Ref]
5. Cichon N., Olejnik A. K., Miller E., Saluk J. The multipotent action of magnetic fields. Biologia2016;71(10):1103–1110.
6. Robertson J. A., Thomas A. W., Bureau Y., Prato F. S. The influence of extremely low frequency magnetic fields on cytoprotection and repair. Bioelectromagnetics2007;28(1):16–30. doi: 10.1002/bem.20258. [PubMed] [Cross Ref]
7. Kumar S., Singh R. K., Bhardwaj T. R. Therapeutic role of nitric oxide as emerging molecule. Biomedicine & Pharmacotherapy2017;85:182–201. doi: 10.1016/j.biopha.2016.11.125. [PubMed] [Cross Ref]
8. Alderton W. K., Cooper C. E., Knowles R. G. Nitric oxide synthases: structure, function and inhibition. The Biochemical Journal2001;357, Part 3:593–615.[PMC free article] [PubMed]
9. Li H., Poulos T. L. Structure-function studies on nitric oxide synthases. Journal of Inorganic Biochemistry2005;99:293–305. doi: 10.1016/j.jinorgbio.2004.10.016.[PubMed] [Cross Ref]
10. Lei J., Vodovotz Y., Tzeng E., Billiar T. R. Nitric oxide, a protective molecule in the cardiovascular system. Nitric Oxide2013;35:175–185. doi: 10.1016/j.niox.2013.09.004. [PubMed] [Cross Ref]
11. Rasool M., Ashraf M. A., Malik A., et al. Comparative study of extrapolative factors linked with oxidative injury and anti-inflammatory status in chronic kidney disease patients experiencing cardiovascular distress. PLoS One2017;12(2, article e0171561) doi: 10.1371/journal.pone.0171561. [PMC free article] [PubMed][Cross Ref]
12. Kozlov A. V., Bahrami S., Redl H., Szabo C. Alterations in nitric oxide homeostasis during traumatic brain injury. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease2017 doi: 10.1016/j.bbadis.2016.12.020. In press. [PubMed] [Cross Ref]
13. Mika T. In: Metodyka Magnetoterapii. Mika T., Kasprzak W., editors. Wydawnictwo Lekarskie PZWL. Warszawa: Fizykoterapia; 2013. pp. 337–339.
14. Khan J., Brennan D. M., Bradley N., Gao B., Bruckdorfer R., Jacobs M., Part 2 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. The Biochemical Journal1998;330:795–801. [PMC free article] [PubMed]
15. Kolodziejczyk J., Saluk-Juszczak J., Wachowicz B. L-Carnitine protects plasma components against oxidative alterations. Nutrition2011;27(6):693–699. doi: 10.1016/j.nut.2010.06.009. [PubMed] [Cross Ref]
16. Declèves A. É., Jadot I., Colombaro V., et al. Protective effect of nitric oxide in aristolochic acid-induced toxic acute kidney injury: an old friend with new assets. Experimental Physiology2016;101(1):193–206. doi: 10.1113/EP085333. [PubMed][Cross Ref]
17. Bobi?ska K., Szemraj J., Czarny P., Ga?ecki P. Expression and activity of metalloproteinases in depression. Medical Science Monitor2016;22:1334–1341.[PMC free article] [PubMed]
18. Désy O., Carignan D., Caruso M., de Campos-Lima P. O. Methanol induces a discrete transcriptional dysregulation that leads to cytokine overproduction in activated lymphocytes. Toxicological Sciences2010;117(2):303–313. doi: 10.1093/toxsci/kfq212. [PubMed] [Cross Ref]
19. Allman C., Amadi U., Winkler A. M., et al. Ipsilesional anodal tDCS enhances the functional benefits of rehabilitation in patients after stroke. Science Translational Medicine2016;8(330):p. 330re1. doi: 10.1126/scitranslmed.aad5651.[PMC free article] [PubMed] [Cross Ref]
20. Wang H. G., Lu F. M., Jin I., et al. Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins. Neuron2005;45:389–403. doi: 10.1016/j.neuron.2005.01.011. [PubMed] [Cross Ref]
21. Yang Y. R., Jung J. H., Kim S. J., et al. Forebrain-specific ablation of phospholipase C?1 causes manic-like behavior. Molecular Psychiatry2017 doi: 10.1038/mp.2016.261. [PubMed] [Cross Ref]
22. Fekete C. D., Goz R. U., Dinallo S., et al. In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex. The Journal of Comparative Neurology2017;525(5):1291–1311. doi: 10.1002/cne.24137. [PubMed] [Cross Ref]
23. Ungvari Z., Tarantini S., Hertelendy P., et al. Cerebromicrovascular dysfunction predicts cognitive decline and gait abnormalities in a mouse model of whole brain irradiation-induced accelerated brain senescence. Geroscience2017;39(1):33–42. doi: 10.1007/s11357-017-9964-z. [PMC free article] [PubMed] [Cross Ref]
24. Su K. H., Lin S. J., Wei J., et al. The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis. Acta Physiologica (Oxford, England) 2014;212(3):191–204. doi: 10.1111/apha.12378. [PubMed] [Cross Ref]
25. Cirino G., Fiorucci S., Sessa W. C. Endothelial nitric oxide synthase: the Cinderella of inflammation? Trends in Pharmacological Sciences2003;24:91–95. doi: 10.1016/S0165-6147(02)00049-4. [PubMed] [Cross Ref]
26. Huang Z., Huang P. L., Ma J., et al. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. Journal of Cerebral Blood Flow and Metabolism1996;16:981–987. doi: 10.1097/00004647-199609000-00023.[PubMed] [Cross Ref]
27. Li H., Forstermann U. Nitric oxide in the pathogenesis of vascular disease. The Journal of Pathology2000;190:244–254. doi: 10.1002/(SICI)1096-9896(200002)190:3<244::AID-PATH575>3.0.CO;2-8. [PubMed] [Cross Ref]
28. Khan M., Jatana M., Elango C., Paintlia A. S., Singh A. K., Singh I. Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke. Nitric Oxide2006;15(2):114–124. doi: 10.1016/j.niox.2006.01.008. [PubMed][Cross Ref]
29. Khan M., Sekhon B., Giri S., et al. S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke. Journal of Cerebral Blood Flow and Metabolism2005;25:177–192. doi: 10.1038/sj.jcbfm.9600012. [PubMed] [Cross Ref]
30. Greco R., Amantea D., Blandini F., et al. Neuroprotective effect of nitroglycerin in a rodent model of ischemic stroke: evaluation of Bcl-2 expression. International Review of Neurobiology2007;82:423–435. doi: 10.1016/S0074-7742(07)82024-1.[PubMed] [Cross Ref]
31. Sulpizio M., Falone S., Amicarelli F., et al. Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells. Journal of Cellular Biochemistry2011;112:3797–3806. doi: 10.1002/jcb.23310. [PubMed] [Cross Ref]
32. Yi G., Wang J., Wei X., et al. Effects of extremely low-frequency magnetic fields on the response of a conductance-based neuron model. International Journal of Neural Systems2014;24(1, article 1450007) doi: 10.1142/S0129065714500075. [PubMed][Cross Ref]
33. Brisdelli F., Bennato F., Bozzi A., Cinque B., Mancini F., Iorio R. ELF-MF attenuates quercetin-induced apoptosis in K562 cells through modulating the expression of Bcl-2 family proteins. Molecular and Cellular Biochemistry2014;397(1-2):33–43. doi: 10.1007/s11010-014-2169-1. [PubMed] [Cross Ref]
34. Morgado-Valle C., Verdugo-Díaz L., García D. E., Morales-Orozco C., Drucker-Colín R. The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation. Cell and Tissue Research1998;291(2):217–230. [PubMed]
35. Cichon N., Bijak M., Miller E., Saluk J. Extremely low-frequency electromagnetic field (ELF-EMF) reduces oxidative stress and improves functional and psychological status in ischemic stroke patients. Bioeletromagtetics2017;38(5):386–396. doi: 10.1002/bem.22055. [PubMed] [Cross Ref]
36. Wink D. A., Miranda K. M., Espey M. G., et al. Mechanisms of the antioxidant effects of nitric oxide. Antioxidants & Redox Signaling2001;3(2):203–213. doi: 10.1089/152308601300185179. [PubMed] [Cross Ref]
37. Ronson R. S., Nakamura M., Vinten-Johansen J. The cardiovascular effects and implications of peroxynitrite. Cardiovascular Research1999;44:47–59. [PubMed]
38. Chung Y. H., Lee Y. J., Lee H. S., et al. Extremely low frequency magnetic field modulates the level of neurotransmitters. The Korean Journal of Physiology and Pharmacology2015;19(1):15–20. doi: 10.4196/kjpp.2015.19.1.15. [PMC free article][PubMed] [Cross Ref]
39. Rauš B. S., Selakovi? V., Radenovi? L., Proli? Z., Jana? B. Extremely low frequency magnetic field (50 Hz, 0.5 mT) reduces oxidative stress in the brain of gerbils submitted to global cerebral ischemia. PLoS One2014;9(2, article e88921) doi: 10.1371/journal.pone.0088921. [PMC free article] [PubMed] [Cross Ref]
40. Wu P., Jia F., Zhang B., Zhang P. Risk of cardiovascular disease in inflammatory bowel disease. Experimental and Therapeutic Medicine2017;13(2):395–400. doi: 10.3892/etm.2016.3966. [PMC free article] [PubMed] [Cross Ref]
41. Godos J., Biondi A., Galvano F., et al. Markers of systemic inflammation and colorectal adenoma risk: meta-analysis of observational studies. World Journal of Gastroenterology2017;23(10):1909–1919. doi: 10.3748/wjg.v23.i10.1909.[PMC free article] [PubMed] [Cross Ref]
42. Pena-Philippides J. C., Yang Y., Bragina O., Hagberg S., Nemoto E., Roitbak T. Effect of pulsed electromagnetic field (PEMF) on infarct size and inflammation after cerebral ischemia in mice. Translational Stroke Research2014;5(4):491–500. doi: 10.1007/s12975-014-0334-1. [PubMed] [Cross Ref]
43. Cho S. I., Nam Y. S., Chu L. Y., et al. Extremely low-frequency magnetic fields modulate nitric oxide signaling in rat brain. Bioelectromagnetics2012;33(7):568–574. doi: 10.1002/bem.21715. [PubMed] [Cross Ref]
44. Walleczek J. Electromagnetic field effects on cells of the immune system: the role of calcium signaling. The FASEB Journal1992;6:3177–3185. [PubMed]
45. Grassi C., D’Ascenzo M., Torsello A., et al. Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium2004;35:307–315. doi: 10.1016/j.ceca.2003.09.001. [PubMed] [Cross Ref]
46. Piacentini R., Ripoli C., Mezzogori D., Azzena G. B., Grassi C. Extremely low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Cav1-channel activity. Journal of Cellular Physiology2008;215:129–139. doi: 10.1002/jcp.21293. [PubMed] [Cross Ref]
47. Gobba F., Malagoli D., Ottaviani E. Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: the role of calcium ion channels. Bioelectromagnetics2003;24:277–282. doi: 10.1002/bem.10102. [PubMed][Cross Ref]
48. Craviso G. L., Choe S., Chatterjee P., Chatterjee I., Vernier P. T. Nanosecond electric pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated Ca2+ channels. Cellular and Molecular Neurobiology2010;30:1259–1265. doi: 10.1007/s10571-010-9573-1. [PubMed] [Cross Ref]
49. Sieroñ A., Cieslar G. Use of magnetic fields in medicine – 15 years of personal experience. Wiadomo?ci Lekarskie2003;56:434–441. [PubMed]
50. Wolda?ska-Oko?ska M., Czernicki J. Effect of low frequency magnetic fields used in magnetotherapy and magnetostimulation on the rehabilitation results of patients after ischemic stroke. Przegla?d Lekarski2007;64(2):74–77. [PubMed]
51. Capone F., Dileone M., Profice P., et al. Does exposure to extremely low frequency magnetic fields produce functional changes in human brain? Journal of Neural Transmission (Vienna) 2009;116(3):257–265. doi: 10.1007/s00702-009-0184-2.[PubMed] [Cross Ref]
52. Miecznik A., Czernicki J., Krukowska J. Influence of magnetic field of different characteristics on blood pressure in patients with back pain syndromes and hypertensive disease. Acta Bio-Optica et Informatica Medica2001;7(1-2):9–13.
53. Di Lazzaro V., Capone F., Apollonio F., et al. A consensus panel review of central nervous system effects of the exposure to low-intensity extremely low-frequency magnetic fields. Brain Stimulation2013;6(4):469–476. doi: 10.1016/j.brs.2013.01.004.[PubMed] [Cross Ref]
54. Grant G., Cadossi R., Steinberg G. Protection against focal cerebral ischemia following exposure to a pulsed electromagnetic field. Bioelectromagnetics1994;15(3):205–216. [PubMed]

Measurements of human tumour necrosis factor alpha (TNF?) in plasma samples were made with a Human TNF? ELISA development kit (MABTECH, Cincinnati, OH, USA), in accordance with the manufacturer’s protocol. The combination of two coating antibodies (TNF3 and TNF4) were used for the analysis. The absorbance was measured at 450nm, and TNF? concentration was expressed as pg/mL [].