Alzheimer’s disease (AD) is a chronic, debilitating neurodegenerative disease with limited therapeutic options; at present there are no treatments that prevent the physical deterioration of the brain and the consequent cognitive deficits. Histopathologically, AD is characterised by neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein and amyloid-beta (A?) plaques [1,2]. The extent of these histopathological features is considered to vary with and to determine clinical disease severity . While the initiating pathogenic events underlying AD are still debated, there is strong evidence to suggest that oxidative stress and mitochondrial dysfunction have important roles in the neurodegenerative cascade [3–5]. Therefore, it has been proposed that targeting mitochondrial dysfunction could prove valuable for AD therapeutics .
One safe, simple yet effective approach to the repair of damaged mitochondria is photobiomodulation with near-infrared light (NIr). This treatment, which involves the irradiation of tissue with low intensity light in the red to near-infrared wavelength range (600 to 1000 nm), was originally pioneered for the healing of superficial wounds  but has been recently shown to have efficacy in protecting the central nervous system. While the mechanism of action remains to be elucidated, there is evidence that NIr preserves and restores cellular function by reversing dysfunctional mitochondrial cytochrome c oxidase (COX) activity, thereby mitigating the production of reactive oxygen species and restoring ATP production to normal levels [8,9].
To date, NIr treatment has yielded neuroprotective outcomes in animal models of retinal damage [9,10], traumatic brain injury [11,12], Parkinson’s disease [13–15] and AD [16,17]. Furthermore, NIr therapy has yielded beneficial outcomes in clinical trials of human patients with mild to moderate stroke  and depression . This treatment represents a promising alternative to drug therapy because it is safe, easy to apply and has no known side-effects at levels even higher than optimal doses .
The aim of this study was to assess the efficacy of NIr in mitigating the brain pathology and associated cellular damage that characterise AD. We utilised two mouse models, each manifesting distinct AD-related pathologies: the K3 tau transgenic model, which develops NFTs [21,22]; and the APP/PS1 transgenic model, which develops amyloid plaques . Here, we present histochemical evidence that NIr treatment over a period of 1 month reduces the severity of AD-related pathology and oxidative stress and restores mitochondrial function in brain regions susceptible to neurodegeneration in AD, specifically the neocortex and hippocampus. The findings extend our previous NIr work in models of acute neurodegeneration [13,14] to demonstrate that NIr is also effective in protecting the brain against chronic insults due to AD-related genetic aberrations, a pathogenic mechanism that is likely to more closely model the human neurodegenerative condition.
The K3 transgenic mouse model, originally generated as a model of frontotemporal dementia [21,22], harbours a human tau gene with the pathogenic K369I mutation; expression is driven by the neuron-specific mThy1.2 promoter. This model manifests high levels of hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We commenced our experiments on K3 mice and matched C57BL/6 wildtype (WT) controls at 5 months of age, when significant neuropathology is already present.
The APPswe/PSEN1dE9 (APP/PS1) transgenic mouse model, obtained from the Jackson Laboratory (Stock number 004462; Bar Harbor, ME, USA), harbours two human transgenes: the amyloid beta precursor protein gene (APP) containing the Swedish mutation; and the presenilin-1 gene (PS1) containing a deletion of exon 9 . The APP/PS1 mice exhibit increased A? and amyloid plaques by 4 months of age  and cognitive deficits by 6 months of age . We commenced our experiments on APP/PS1 mice and matched C57BL/6×C3H WT controls at 7 months of age, when numerous amyloid plaques and associated cognitive deficits are present.
Genotyping of mice was achieved by extracting DNA from tail tips through a modified version of the Hot Shot preparation method  and amplifying the transgene sequence by polymerase chain reaction. As reported previously, K3 mice were identified using the primers 5-GGGTGTCTCCAATGCCTGCTTCTTCAG-3 (forward) and 5-AAGTCACCCAGCAGGGAGGTGCTCAG-3 (reverse) [21,22] and APP/PS1 mice were genotyped using primers 5-AGGACTGACCACTCGACCAG-3 (forward) and 5-CGGGGGTCTAGTTCTGCAT-3 (reverse) .
For each series of experiments on K3 mice (aged 5 months) or APP/PS1 mice (aged 7 months) there were three experimental groups: untreated WT mice, untreated transgenic mice and NIr-treated transgenic mice (n=5 mice per experimental group for the K3 series, 15 mice in total; n=6 mice per experimental group for the APP/PS1 series, 18 mice in total). Our design did not include a WT control group exposed to NIr because NIr has no detectable impact on the survival and function of cells in normal healthy brain [13–15]. Given the consistency of the previous results, use of animals for this extra control group did not seem justified .
Mice in the NIr-treated groups were exposed to one 90-second cycle of NIr (670 nm) from a light-emitting device (LED) (WARP 10; Quantum Devices, Barneveld, WI, USA) for 5 days per week over 4 consecutive weeks. Light energy emitted from the LED during each 90-second treatment equates to 4 Joule/cm2; a total of 80 Joule/cm2 was delivered to the skull over the 4 weeks. Our measurements of NIr penetration across the fur and skull of a C57BL/6 mouse indicate that ~2.5% of transmitted light reaches the cortex.
For each treatment, the mouse was restrained by hand and the LED was held 1 to 2 cm above the head. The LED light generated no heat and reliable delivery of the radiation was achieved [13–15]. For the sham-treated WT, K3 and APP/PS1 groups, animals were restrained in the same way and the device was held over the head, but the light was not switched on. This treatment regime is similar to that used in previous studies where beneficial changes to neuropathology and behavioural signs were reported [13–15].
Experimental animals were housed two or more to a cage and kept in a 12-hour light (<5 lux)/dark cycle at 22°C; food pellets and water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the University of Sydney.
Histology and immunohistochemistry
At the end of the experimental period, mice were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg) and perfused transcardially with 4% buffered paraformaldehyde. Brains were post fixed for 3 hours, washed with phosphate-buffered saline and cryoprotected in 30% sucrose/phosphate-buffered saline. Tissue was embedded in OCT compound (ProSciTech, Thuringowa, QLD, Australia) and coronal sections of the neocortex and the hippocampus (between bregma ?1.8 and ?2.1) were cut at 20 ?m thickness on a Leica cryostat (Nussloch, Germany).
For most antibodies, antigen retrieval was achieved using sodium citrate buffer with 0.1% Triton. Sections were blocked in 10% normal goat serum and then incubated overnight at 4°C with a mouse monoclonal antibody – paired helical filaments-tau AT8, 1:500 (Innogenetics, Ghent, Belgium); 4-hydroxynonenal (4-HNE), 1:200 (JaICA, Fukuroi, Shizuoka, Japan); 8-hydroxy-2?-deoxyguanosine (8-OHDG), 1:200 (JaICA); COX, 1:200 (MitoSciences, Eugene, OR, USA) – and/or a rabbit polyclonal antibody (200 kDa neurofilament, 1:500; Sigma, St. Louis, MO, USA). Sections were then incubated for 3 hours at room temperature in Alexa Fluor-488 (green) and/or Alexa Fluor-594 (red) tagged secondary antibodies specific to host species of the primary antibodies (1:1,000; Molecular Probes, Carlsbad, CA, USA). Sections were then counterstained for nuclear DNA with bisbenzimide (Sigma).
Two different but complementary antibodies were used to label A? peptide: 6E10, which recognises residues 1 to 16; and 4G8, which recognises residues 17 to 24. We have previously used these two antibodies in combination to validate A? labelling, demonstrating identical labelling patterns in the rat neocortex and hippocampus . For double labelling using 6E10 antibodies (1:500; Covance, Princeton, NJ, USA) and anti-glial fibrillary acidic protein antibodies (1:1,000; DAKO, Glostrup, Denmark), antigen retrieval was achieved by incubation in 90% formic acid for 10 minutes, and primary antibody incubation was carried out overnight at room temperature. For labelling using the 4G8 (1:500; Covance) antibody, slides were treated with 3% H2O2 in 50% methanol, incubated in 90% formic acid and then washed several times in dH2O before the blocking step, as described previously . After blocking, sections were incubated overnight at room temperature with 4G8 antibody. Sections were then incubated in biotinylated goat anti-mouse IgG for 1 hour followed by ExtrAvidin peroxidase for 2.5 hours. The sections were then washed and developed with 3,3?-Diaminobenzidine.
Negative control sections were processed in the same fashion as described above except that primary antibodies were omitted. These control sections were immunonegative. Fluorescent images were taken using a Zeiss Apotome 2, Carl Zeiss, Oberkochen, Germany. Brightfield images were taken using a Nikon Eclipse E800, Nikon Instruments, Melville, NY, USA.
NFTs were assessed using the Bielschowsky silver staining method, as described previously [21,22]. Briefly, sections were placed in prewarmed 10% silver nitrate solution for 15 minutes, washed and then placed in ammonium silver nitrate solution at 40°C for a further 30 minutes. Sections were subsequently developed for 1 minute and then transferred to 1% ammonium hydroxide solution for 1 minute to stop the reaction. Sections were then washed in dH2O, placed in 5% sodium thiosulphate solution for 5 minutes, washed, cleared and mounted in dibutyl phathalate xylene.
As described previously , A? plaques were studied by staining with Congo red, a histological dye that binds preferentially to compacted amyloid with a ?-sheet secondary structure . Briefly, sections were treated with 2.9 M sodium chloride in 0.01 M NaOH for 20 minutes and were subsequently stained in filtered alkaline 0.2% Congo red solution for 1 hour.
Staining intensity and area measurements
To quantify the average intensity and area of antibody labelling within the neocortex and hippocampal regions, an integrated morphology analysis was undertaken using MetaMorph software. For each section, the level of nonspecific staining (using an adjacent region of unstained midbrain) was adjusted to a set level to ensure a standard background across different groups. Next, outlines of retrosplenial cortex area 29 and hippocampal CA1 region were traced and the average intensity and area of immunostaining were calculated by the program. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Statistical analyses were performed in Prism 5.0 (Graphpad, La Jolla, CA, USA) using one-way analysis of variance with Tukey’s multiple comparison post test. All values are given as mean±standard error of mean.
Amyloid-beta plaque measurements
Digital brightfield images of 4G8 staining in the neocortical and hippocampal regions (between bregma ?1.8 and ?2.1) were taken at 4× magnification and analysed with Metamorph, Molecular Devices LLC, Sunnyvale, CA, USA. The software was programmed to measure the number of plaques and the average size of plaques after thresholding for colour. The percentage of area covered by plaques (plaque burden) was calculated by multiplying the number of plaques by the average size of plaques, divided by the area of interest, as described previously . The average number of Congo red-positive plaques in the APP/PS1 brain regions was estimated using the optical fractionator method (StereoInvestigator; MBF Science, Williston, VT, USA), as outlined previously . Briefly, systematic random sampling of sites was undertaken using an unbiased counting frame (100 ?m×100 ?m). All plaques that came into focus within the frame were counted. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Plaque numbers and size were analysed using a two-tailed unpaired t test (when variances were equal) or Welch’s t test (when variances were unequal). All values are given as mean±standard error of mean. For all analyses, investigators were blinded to the experimental groups.
Evidence of NIr-induced neuroprotection is presented from the neocortex (retrosplenial area) and the hippocampus (CA1 and subiculum), two cortical regions affected in the early stages of human AD .
Near-infrared light mitigates the tau pathology of K3 cortex
Hyperphosphorylation of the neuronal microtubule stabilising protein tau and the resulting NFTs are much studied features of dementia pathology [2,31]. The K3 mouse model manifests hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We observe strong labelling for hyperphosphorylated tau in the neocortex and the hippocampus at 6 months of age; expression appears to plateau after this age, with similar labelling observed in 12-month-old mice (Figure 1A,B,C,D,E,F).
In the retrosplenial area of the neocortex there was a significant overall difference in AT8 immunolabelling for tau between the experimental groups, both when considering average intensity of labelling (P<0.01 by analysis of variance; Figure 2A) and labelled area (P<0.01; Figure 2B). Tukeypost hoc testing revealed significant differences between the untreated K3 group and the other two groups; labelling was much stronger and more widespread in K3 mice than WT controls (17-fold higher intensity, P<0.01), and this labelling was reduced by over 70% in NIr-treated mice (P<0.05). Interestingly, there was no significant difference between the WT and K3-NIr groups, suggesting that NIr treatment had reduced hyperphosphorylated tau to control levels in K3 mice. A similar trend was observed when considering the NFT pathology (Figure 2C,D,E). In contrast to WT brain, which showed no NFT-like lesions (Figure 2C), the K3 brain contained many ovoid shaped NFT-like lesions (that is, spheroids; Figure 2D). Such structures were less frequent in the K3-NIr brain (Figure 2E).
One should note that the large white matter pathways associated with the hippocampus were labelled intensely by silver staining in all three groups (Figure 3C,D,E). This labelling has been described previously and is not associated with any neuropathology .
Near-infrared light reduces oxidative stress in K3 cortex
Oxidative stress and damage are common features of neurodegenerative diseases such as AD, and may be a precursor to neuronal death [3–5]. We assessed two common markers of oxidative stress: 4-HNE, a toxic end-product of lipid peroxidation that may bind to proteins that then trigger mitochondrial dysfunction and cellular apoptosis in AD ; and 8-OHDG, a marker for nuclear and mitochondrial DNA oxidation, which is elevated in AD brains .
Overall, 4-HNE immunoreactivity in the neocortex was significantly different between the experimental groups (Figure 4), by both average labelling intensity (P<0.01) and labelled area (P<0.001). As with AT8 labelling above, the K3 group showed a much higher average 4-HNE labelling intensity and area than the WT group (fivefold and 20-fold, respectively) and this labelling was significantly reduced (by 50% and 80%, respectively) in the K3-NIr group. Again, these measures showed no significant differences between the WT and K3-NIr groups (P>0.05).
Similar patterns were observed for 8-OHDG immunoreactivity. Overall, there was a significant difference between the groups for 8-OHDG immunolabelling, by both average intensity (P<0.0001) and labelled area (P<0.0001). Again the K3 group showed significantly higher 8-OHDG labelling intensity and area than the WT group (sixfold and 17-fold, respectively), and the 8-OHDG labelling intensity and area were significantly reduced in the K3-NIr group relative to untreated K3 (65% and 85% reduction, respectively). The intensity and area of 8-OHDG labelling did not differ significantly between the WT and the K3-NIr groups (P>0.05), suggesting that NIr treatment reduces markers of oxidative stress to control levels. The representative photomicrographs of 8-OHDG immunoreactivity in the retrosplenial area (Figure 4H,I,J) reflect the quantitative data, with many 8-OHDG+ structures in the K3 group (Figure 4I) but not in the WT and K3-NIr groups (Figure 4H,J)
Near-infrared light mitigates mitochondrial dysfunction in K3 cortex
We assessed expression patterns of the mitochondrial enzyme COX in the neocortex and the hippocampus as a marker of mitochondrial function. Overall, there were statistically significant differences in the patterns of COX immunoreactivity between the different experimental groups, both in the neocortex and the hippocampus (both P<0.0001; Figure 5). Relative to WT mice, the COX labelling intensity and area were reduced in K3 mice in both the neocortex and the hippocampus (>70% and >75% reductions, respectively). The K3-NIr mice showed a significant recovery of COX immunoreactivity relative to untreated K3 mice in both the neocortex (>1.7-fold increase, P<0.05) and the hippocampus (>3.4-fold increase, P<0.001). However, recovery was not complete, with K3-NIr mice having significantly lower COX immunoreactivity than WT mice in the neocortex (~50%, P<0.001) and significantly lower COX labelling intensity (~20%, P<0.05) in the hippocampus. These two groups did not differ significantly in COX labelling area in the hippocampus (P>0.05).
Near-infrared mitigates amyloid pathology in APP/PS1 cortex
Along with NFTs, A? plaques are considered a primary pathological hallmark of AD and A? load is often used as a marker of AD severity [1,35]. We assessed the distribution of A? plaques and more immature forms of the A? peptide in the neocortex and hippocampus of APP/PS1 mice aged 7 months; this age is after the first signs of intracellular A? within cells (at 3 months; Figure 1G) and extracellular A? plaques (at 4.5 and 12 months; Figure 1H and ?and1I,1I, respectively).
Three quantitative measures of plaque pathology were used: percentage plaque burden, average plaque size and number of plaques. Immunohistochemical labelling with the anti-A? antibody 4G8 revealed a significant reduction in percentage plaque burden (Figure 6A,D), average plaque size (Figure 6B,E) and number of plaques (Figure 6C,F) in both the neocortex and the hippocampus of NIr-treated APP/PS1 mice relative to untreated APP/PS1 controls. Percentage plaque burden was reduced by over 40% in the neocortex (Figure 6A; P<0.001) and over 70% in the hippocampus (Figure 6D; P<0.01), average plaque size was reduced 25% in the neocortex (Figure 6B) and 30% in the hippocampus (Figure 6E), and the number of plaques was reduced by over 20% in the neocortex (Figure 6C) and by over 55% in the hippocampus (Figure 6F; all P<0.05).
The photomicrographs of the 4G8 immunoreactivity in Figure 6 reflect the quantitative data described earlier. The WT brain is free of plaques (Figure 6H,K); many 4G8+ plaques (arrows) are present in the neocortex (Figure 6I) and the hippocampus (Figure 6L) of untreated APP/PS1 mice, and fewer plaques are present in NIr-treated APP/PS1 mice (Figure 6J,M). Comparable immunolabelling was achieved using the 6E10 anti-A? antibody (data not shown).
A similar but less pronounced trend was observed when staining with Congo red (Figure 7), which stains only mature plaques. Mean counts of plaques in the neocortex (Figure 7A) and the hippocampus (Figure 7B) of NIr-treated APP/PS1 brains were lower than mean counts in untreated APP/PS1 brains (reductions >30%). However, the differences did not reach statistical significance; given the findings described above with the 4G8 and 6E10 anti-A? antibodies, this suggests that NIr may have greatest effect on recently formed A? deposits. The micrographs in Figure 7 show that mature plaques were absent from the WT brain (Figure 7C,D) but were present in the neocortex (Figure 7E) and hippocampus (Figure 7F) of untreated APP/PS1 brains. There appeared to be fewer plaques in the NIr-treated APP/PS1 brains (Figure 7G,H).
Using two mouse models with distinct AD-related pathologies (tau pathology in K3, amyloid pathology in APP/PS1), we report evidence that NIr treatment can mitigate the pathology characteristic of AD as well as reduce oxidative stress and restore mitochondrial function in brain regions affected early in the disease. Further, the extent of mitigation – to levels less than at the start of treatment – suggests that NIr can reverse some elements of AD-related pathology.
The present results add to our previous findings of NIr-induced neuroprotection in models of toxin-induced acute neurodegeneration (that is, MPTP-induced parkinsonism). When incorporated into the growing body of evidence that NIr can also protect against CNS damage in models of stroke, traumatic brain injury and retinal degeneration [9–12,36], the findings provide a basis for trialling NIr treatment as a strategy for protection against neurodegeneration from a range of causes. Present evidence is based on the use of multiple methods, immunohistochemical and histological, to demonstrate pathological features (for example, 4G8 antibody labelling and Congo red staining for amyloid plaques, AT8 antibody labelling and Bielschowsky silver staining for NFTs).
Relationship to previous studies
The present study focused on pathological features considered characteristic of AD, as well as on signs of cellular damage (for example, oxidative stress, mitochondrial dysfunction) that have been demonstrated in AD and in animal models [2–4]. Our observations in the K3 strain add to previous studies by providing the first evidence in this strain of extensive oxidative damage and mitochondrial dysfunction .
Our findings are consistent with previous reports of the effects of red to infrared light on AD pathology in animal models. De Taboada and colleagues assessed the capacity of 808 nm laser-sourced infrared radiation, delivered three times per week over 6 months, to reduce pathology in an APP transgenic model of A? amyloidosis . Treatment led to a reduction in plaque number, amyloid load and inflammatory markers, an increase in ATP levels and mitochondrial function, and mitigation of behavioural deficits. De Taboada and colleagues commenced treatment at 3 months of age, before the expected onset of amyloid pathology and cognitive effects. Similarly, Grillo and colleagues reported that 1,072 nm infrared light, applied 4 days per week for 5 months, reduces AD-related pathology in another APP/PS1 transgenic mouse model (TASTPM) . These investigators also initiated light treatment before the onset of pathology, at 2 months of age. Both studies thus provide evidence that infrared radiation can slow the progression of cerebral degeneration in these models. The present results confirm these observations, in two distinct transgenic strains; they also confirm that the wound-healing and neuroprotective effects of red-infrared length do not vary qualitatively with wavelength, over a wide range.
Evidence of reversal of pathology
Previous reports have described the natural history of the K3 [21,22] and APP/PS1 transgenic models [24,37]. Based on these previous reports and our own baseline data (Figure 1), significant brain pathology and functional deficits are present in both models at the ages when we commenced treatment. Our results therefore suggest that significant reversal of pathology has been induced by the NIr treatment. This has implications for clinical practice, where most patients are not diagnosed until pathogenic mechanisms have already been initiated and resultant neurologic symptoms manifest [15,27].
This evidence that AD-related neuropathology can be transient – appear then disappear – is not novel. Garcia-Alloza and colleagues described evidence of the transient deposition of A?, including the formation of plaque-like structures, in a transgenic model of A? deposition . Reversal of such pathology, by interventions such as NIr treatment, may therefore be possible. However our results suggest that reversal may also be limited to recently formed, immature plaques, as we observed a significant NIr-induced reduction in immunolabelling with the 4G8 and 6E10 antibodies but no significant difference in Congo red staining. Because the 4G8 and 6E10 antibodies recognise various forms of A?, while Congo red stains only mature, compacted plaques, a reasonable deduction is that NIr treatment reduces only the transient, recently formed A? deposits, with no substantial effect on mature plaques. As there is still no consensus as to the pathogenic roles of different forms of A?, it is unclear how this might impact on the therapeutic potential of NIr in a clinical setting.
The mechanisms underlying the neuroprotective actions of red to infrared light are not completely understood. There is considerable evidence that NIr photobiomodulation enhances mitochondrial function and ATP synthesis by activating photoacceptors such as COX and increasing electron transfer in the respiratory chain, while also reducing harmful reactive oxygen species [38–40]. NIr photobiomodulation could also upregulate protective factors such as nerve growth factor and vascular endothelial growth factor [41,42] and mesenchymal stem cells  that could target specific areas of degeneration.
The ability of NIr to reduce the expression of hyperphosphorylated tau, which in turn reduces oxidative stress , may be key to its neuroprotective effect. Oxidative stress and free radicals increase the severity of cerebrovascular lesions [45,46], mitochondrial dysfunction [4,47], oligomerisation of A? [5,48] and tauopathies and cell death [48,49] in AD. Considering the brain’s high consumption of oxygen and consequent susceptibility to oxidative stress, mitigating such stressors would probably have a pronounced protective effect .
Overall, our results in two transgenic mouse models with existing AD-related pathology suggest that low-energy NIr treatment can reduce characteristic pathology, oxidative stress and mitochondrial dysfunction in susceptible regions of the brain. These results, when taken together with those in other models of neurodegeneration, strengthen the notion that NIr is a viable neuroprotective treatment for a range of neurodegenerative conditions. We believe this growing body of work provides the impetus to begin trialling NIr treatment as a broad-based therapy for AD and other neurodegenerations.
A?: Amyloid-beta; AD: Alzheimer’s disease; APP: Amyloid beta precursor protein gene; COX: Cytochrome c oxidase; 4-HNE: 4-hydroxynonenal; LED: Light-emitting diode; NFT: Neurofibrillary tangle; NIr: Near-infrared light; 8-OHDG: 8-hydroxy-2?-deoxyguanosine; PS1: Presenilin 1; WT: Wildtype.
The authors declare that they have no competing interests.
SP undertook the bulk of the experimental work and analysis and wrote the manuscript. DMJ and JM were involved with the analysis of the data and the writing of the manuscript. CN was involved with genotyping and treating the animals. JS was involved in conceiving and designing the study and the writing of the manuscript. All authors read and approved the final manuscript.
The authors thank Tenix Corporation, Sir Zelman Cowen Universities Fund and Bluesand Foundation for funding. They are grateful to Prof. Lars Ittner for providing the breeding litter for K369I mice, and to Dr Louise Cole and the Bosch Advanced Microscopy facility for the help with MetaMorph. Sharon Spana was splendid for her technical help. DMJ is supported by a National Health and Medical Research Council of Australia (NHMRC) Early Career Fellowship.:
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Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders.
Irradiation in the red/near-infrared spectrum (R/NIR, 630-1000 nm) has been used to treat a wide range of clinical conditions, including disorders of the central nervous system (CNS), with several clinical trials currently underway for stroke and macular degeneration. However, R/NIR irradiation therapy (R/NIR-IT) has not been widely adopted in clinical practice for CNS injury or disease for a number of reasons, which include the following. The mechanism/s of action and implications of penetration have not been thoroughly addressed. The large range of treatment intensities, wavelengths and devices that have been assessed make comparisons difficult, and a consensus paradigm for treatment has not yet emerged. Furthermore, the lack of consistent positive outcomes in randomised controlled trials, perhaps due to sub-optimal treatment regimens, has contributed to scepticism. This review provides a balanced précis of outcomes described in the literature regarding treatment modalities and efficacy of R/NIR-IT for injury and disease in the CNS. We have addressed the important issues of specification of treatment parameters, penetration of R/NIR irradiation to CNS tissues and mechanism/s, and provided the necessary detail to demonstrate the potential of R/NIR-IT for the treatment of retinal degeneration, damage to white matter tracts of the CNS, stroke and Parkinson’s disease.
Parkinson’s disease is a major movement disorder characterised by the distinct signs of resting tremor, akinesia and/or lead pipe rigidity [1,2]. These arise after a substantial loss of dopaminergic cells, mainly within the substantia nigra pars compacta (SNc) of the midbrain [3,4]. The factors that generate this cell loss are not entirely clear, but there is evidence for mitochondrial dysfunction as a result of exposure to an environmental toxin (eg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine))  and/or the presence of a defective gene .
Many previous studies have shown that some substances, such as anti-oxidants like CoQ10 (coenzyme Q10)  and melatonin , help neuroprotect dopaminergic cells in the SNc against degeneration in animal models of Parkinson’s disease. These substances are thought to reduce mitochondrial dysfunction by lessening the oxidative stress caused by free radicals generated by defective mitochondria present in Parkinson’s disease. In addition to these substances, recent studies have reported on the neuroprotective properties of low intensity light therapy, known also as photobiomodulation or near infra-red light (NIr) treatment, after parkinsonian insult. For example, NIr treatment protects neural cells in vitro against parkinsonian toxins such as MPTP and rotenone [9,10]. Further, we have shown that NIr treatment offers in vivo protection for dopaminergic cells in the SNc in an acute  and chronic  MPTP mouse (Balb/c) model. There is also a brief report indicating that NIr treatment improves the locomotor activity of mice after MPTP insult . Although the mechanism of neuroprotection by NIr is not entirely clear, work on other systems indicate that NIr improves mitochondrial function and ATP synthesis in the damaged cells by increasing electron transfer in the respiratory chain and activating photoacceptors, such as cytochrome oxidase, within the mitochondria. Further, NIr has been shown to reduce the production of reactive oxygen species that are harmful to cells [14,15].
In this study, we sought to extend our earlier anatomical [11,12] and functional  studies by exploring the changes in locomotive behaviour of MPTP-treated mice after NIr treatment. Hitherto, this feature has not been reported extensively . We undertook this behavioural analysis, together with a stereological account of SNc cell number, in two strains of mice, Balb/c (albino) and C57BL/6 (pigmented). This was done because there are reports that MPTP has differential effects on behaviour and dopamine levels in the basal ganglia in different strains of mice [17,18], as well as rats . We wanted to determine whether there were mouse strain differences in the effect of NIr treatment after MPTP insult.
Male BALB/c (albino; n=40) and C57BL/6 mice (pigmented; n=40) mice were housed on a 12 hr light/dark cycle with unlimited access to food and water. Animals were 8–10 weeks old. All experiments were approved by the Animal Ethics Committee of the University of Sydney and COMETH (Grenoble).
We set up four experimental groups (see Figure 1). Mice received intraperitoneal injections of either MPTP or saline, combined with simultaneous NIr treatments or not. The different groups were; (1) Saline: saline injections with no NIr (2) Saline-NIr: saline injections with NIr (3) MPTP: MPTP injections with no NIr (4) MPTP-NIr: MPTP injections with NIr. Each experimental group comprised ten mice of each strain.
Following our previous work, we used an acute MPTP mouse model [11,16]. The acute model is a well-accepted model of the disease [20,21] and has revealed many aspects of the mechanisms of Parkinson’s disease over the years. Although it does not provide information on the chronic progressive nature of the disease, it does generate mitochondrial dysfunction, dopaminergic cell death and a reduction in locomotive activity [20,21]. The latter two issues were central in this study, making the acute model most appropriate for our use. Briefly, we made two MPTP (25 mg/kg injections; total of 50 mg/kg per mouse) or saline injections over a 24 hour period. Following each injection, mice in the MPTP-NIr and Saline-NIr groups were treated to one cycle of NIr (670 nm) of 90 seconds from a light-emitting device (LED; Quantum Devices WARP 10). This treatment equated to ~0.5 Joule/cm2 to the brain . Approximately 6 hours after each injection and first NIr treatment, mice in these groups received a second NIr treatment, but no MPTP or saline injection. Hence, each mouse in these groups received four NIr treatments, equalling ~2 joules/cm2 reaching the brain. This NIr treatment regime was similar to that used by previous studies, in particular, those reporting changes after trans-cranial irradiation [11,12,14–16]. For each treatment, the mouse was restrained by hand and the LED was held 1–2 cm above the head [11,12,16]. The LED generated no heat and reliable delivery of the radiation was achieved. For the Saline and MPTP groups, mice were held under the LED as described above, but the device was not turned on. After the last treatment, mice were allowed to survive for six days (Figure 1). This MPTP/NIr dose regime and survival period has been shown to furnish TH+ cell loss by MPTP and neuroprotection by NIr [8,11,16]. We also made some measurements of NIr penetration across the skin and fur of the two mouse strains. Skin was excised from the back of each mouse and positioned over a foil-coated vessel, with a calibrated light sensor at the bottom. NIr from the WARP-LED was then shone onto the skin and the penetration was recorded by the sensor (distance from WARP-LED to skin was ~4 cm and distance from skin to sensor was ~3 cm). For each strain, we compared the NIr penetration in cases where the fur was shaved from the skin to those that were unshaved. Each of the values obtained were compared to (and expressed as a percentage of) the values we recorded of NIr through the air, with no intervening skin.
Our experimental paradigm of simultaneous administration of parkinsonian insult and therapeutic application was similar to that of previous studies on animal models of Parkinson’s disease [8,11,12,16,22–24]. This paradigm is unlike the clinical reality where there is cell loss prior to therapeutic intervention. However, in our experimental study we hoped to determine the maximum effect of NIr neuroprotection.
Immunocytochemistry and cell analysis
Following the survival period, mice were anaesthetised with an intraperitoneal injection of chloral hydrate (4%; 1 ml/100 g). They were then perfused transcardially with 4% buffered paraformaldehyde. The brains were removed and post-fixed overnight in the same solution. Next, brains were placed in phosphate-buffered saline (PBS) with the addition of 30% sucrose until the block sank. The midbrain was then sectioned coronally and serially (at 50 ?m) using a freezing microtome. All sections were collected in PBS and then immersed in a solution of 1% Triton (Sigma) and 10% normal goat serum (Sigma) at room temperature for ~1 hour. Sections were then incubated in anti-tyrosine hydroxylase (Sigma; 1:1000) for 48 hours (at 4°C), followed by biotinylated anti-rabbit IgG (Bioscientific; 1:200) for three hours (at room temperature) and then streptavidin-peroxidase complex (Bioscientific; 1:200) for two hours (at room temperature). To visualise the bound antibody, sections were reacted in a 3,3?– diaminobenzidine tetrahydrochloride (Sigma) – PBS solution. Sections were mounted onto gelatinised slides, air dried overnight, dehydrated in ascending alcohols, cleared in Histoclear and coverslipped using DPX. Most of our immunostained sections were counterstained lightly with neutral red as well. In order to test the specificity of the primary antibody, some sections were processed as described above, except that there was no primary antibody used. These control sections were immunonegative.
In this study, we used TH immunocytochemistry to describe patterns of cell death and protection. As with many previous studies, we interpreted a change in TH+ cell number after experimental manipulation as an index of cell survival [8,11,12,22,23,25]. If cells lose TH expression, then they are likely to undergo death subsequently , which then leads to a reduction in Nissl-stained (and TH+) cell number [8,23]. Notwithstanding a small number of cells that may have transient loss of TH expression , a key aspect of our study was whether NIr treatment saved TH expression during a period when MPTP treatment alone would have abolished it [11,12]. In terms of analysis, the number of TH+ cells within the SNc was estimated using the optical fractionator method (StereoInvestigator, MBF Science), as outlined previously [8,11,12,23]. Briefly, systematic random sampling of sites – with an unbiased counting frame (100×100 ?m) – within defined boundaries of SNc was undertaken. Counts were made from every second section, and for consistency, the right hand side of the brain was counted in all cases. All cells (nucleated only) that came into focus within the frame were counted and at least five sites were sampled per section.
Digital images were constructed using Adobe Photoshop (brightness and contrast levels were adjusted on individual images in order to achieve consistency (eg, illumination) across the entire plate) and Microsoft PowerPoint programmes.
During the experimental period, we performed a standard open-field test . Mice were placed in white boxes (~20×20×20 cm) for C57BL/6 mice and black boxes for the Balb/c mice (this was important for software detection of contrast changes). Behavioural activity was measured and videotaped using a high definition camera (25000 images/sec) that detected changes in contrast and hence movement of mice. Mice were not acclimatised to the boxes prior to testing and boxes were cleaned thoroughly to avoid olfactory clues. Animal detection was made comparing a reference image that contained no subject with the live image containing the subject; the differences between the two were identified as subject pixel. Subject pixels changes were computed (Noldus, Ethovision, XT 8.5 version) to obtain different parameters of locomotor activity, for example velocity and mobility. Velocity was the mean speed of the mouse during trials (cm/sec) measured from the centre of gravity of the animal. To avoid “jittering”, a threshold of minimal distance moved of 0.3 cm was established. Mobility calculates the duration (in sec) during which the complete area detected as animal is changing even if the centre of gravity remains the same. High mobility refers to 10% or more of changes in percentage of body area detected between two samples, and immobility refers to less than 2% of changes. Each animal was tested at four time points (Figure 1); (T1) after first MPTP or saline injection and NIr (or no) treatment; (T2) after second NIr (or no) treatment; (T3) after second MPTP or saline injection and third NIr (or no) treatment; (T4) after fourth NIr (or no) treatment. Mice were tested for ~20 minutes at each time point. We tested locomotive activity at these points, particularly T1 and T3, because we wanted to explore the effects of NIr during a time when the MPTP was most effective (eg, immediately after injections), when the mice were most immobile and “sick” .
For comparisons between groups in the cell analysis, a one-way ANOVA test was performed, in conjunction with a Tukey-Kramer post-hoc multiple comparison test. For the behavioural analysis, groups were compared for time (T1,T2,T3,T4), drug (MPTP or not) and light (NIr or not) conditions using a three-way ANOVA test with a Bonferroni post-hoc test (using GraphPad Prism programme).
The results that follow will consider the cell and behavioural analyses for each strain separately.
Figure 2 shows the estimated number of TH+ cells in the SNc of the four groups in the Balb/c and C57BL/6 mice. Overall, the variations in number were significant for both Balb/c (ANOVA: F=4.9; p<0.001) and C57BL/6 (ANOVA: F=3.8; p<0.01) mice. For the Saline and Saline-NIr groups of both strains, the number of TH+ cells was similar; no significant differences were evident between these groups (Tukey test: p>0.05). For the MPTP groups, TH+ cell number was reduced compared to the saline control groups in both strains (~30%). These reductions were significant (Tukey test: p<0.05). In the MPTP-NIr groups, TH+ cell number was higher than in the MPTP groups of both strains, but more so in the Balb/c (~30%) compared to the C57BL/6 (~20%) mice. This increase reached statistical significance for the Balb/c group (Tukey test: p<0.05) but not the C57BL/6 group. Unlike the MPTP groups, the number of TH+ cells in the MPTP-NIr groups of both strains was not significantly different to the saline groups (Tukey test: p>0.05).
Figure 4 shows recorded values of locomotor activity in Balb/c (Figure 4A,B,C) and C57BL/6 (Figure 4A’,B’,C’) mice, in terms of velocity (Figure 4A,A’), high mobility (Figure 4B,B’) and immobility (Figure 4C,C’). Overall, there were significant interactions for time and drug conditions for velocity, high mobility and immobility in both Balb/c (ANOVA: F range=7.5-13.6; p<0.05) and C57BL/6 (ANOVA: F range=16.8-40.5; p<0.05) mice, while significant interactions for time, drug and light conditions were evident for these locomotive activities in Balb/c (ANOVA: F range=11.7-24.2; p<0.05), but not in C57BL/6 (ANOVA: F range=0.4-0.8; p>0.05) mice.
The patterns of locomotor activity in the Saline and Saline-NIr groups were similar in both strains of mice. There was no significant effect of the light in the different time conditions (T1-T4) in the saline-treated cases (Bonferroni test: p>0.05). Hence, for clarity, the values of these groups were pooled and are represented as a dotted line across each of the graphs. By contrast, distinct changes in locomotor activity were evident between the MPTP and MPTP-NIr groups; their values are hence represented as individual columns at each time point (Figure 4). The results for each locomotor activity in the two strains will be considered separately below.
For Balb/c mice, at T1 (after first MPTP injection and NIr treatment) and T2 (after second NIr treatment) the locomotor activities in the MPTP and MPTP-NIr groups were similar. There were no significant effects of the light in these two time conditions in the MPTP-treated cases (Bonferroni test: p>0.05; Figure 4A,B,C). The effects of MPTP were immediate; compared to the saline control groups, both groups showed less velocity (Figure 4A) and high mobility (Figure 4B) and greater immobility (Figure 4C) at T1. By T2, there was considerable recovery of each locomotor activity in both MPTP and MPTP-NIr groups, with their values returning to control levels (Figure 4A,B,C). At T3 (after second MPTP injection and third NIr treatment) and T4 (after fourth NIr treatment), unlike at T1 and T2, there were significant effects of the light in the MPTP-treated cases (Bonferroni test: p<0.05; Figure 4A,B,C). At T3 and T4, the MPTP-NIr group had greater velocity (Figure 4A) and high mobility (Figure 4B) and less immobility (Figure 4C) than the MPTP group. Compared to the saline control groups, the MPTP-NIr group had similar locomotor activities at T3 and in particular, at T4 (Figure 4A,B,C). By contrast, the MPTP group at both T3 and T4, still had considerably less velocity (Figure 4A) and high mobility (Figure 4B) and greater immobility (Figure 4C) than the saline controls.
For C57BL/6 mice, there were distinct differences in locomotor activity compared to Balb/c mice. First, in C57BL/6 mice, there were no significant effects of the light at all time conditions (T1-T4) in the MPTP-treated cases (Bonferroni test: p>0.05; Figure 4A’,B’,C’); for Balb/c mice, there was no effect of the light in the MPTP-treated cases at T1 and T2 only (Figure 4A,B,C). Second, the MPTP and MPTP-NIr groups had considerably less velocity (Figure 4A’) and high mobility (Figure 4B’) and greater immobility (Figure 4C’) than the saline controls at the majority of the time points. In contrast to Balb/c mice, there was no evidence of NIr-specific recovery of function at T3 and T4; instead MPTP-treated mice appeared to have some recovery after the second MPTP injection (T4; Figure 4A’,B’,C’) irrespective of whether or not they received NIr treatment. Finally, control C57BL/6 mice showed lower baseline velocity (Figure 4A’) and high mobility (Figure 4B’), but also less immobility (Figure 4C’), than Balb/c mice.
In order to explore whether these behavioural (and cellular) differences between the two strains was due to pigmentation, we compared the degree of NIr penetration across the skin and fur in the different strains. In the Balb/c mice, we found that NIr penetration in the unshaved cases was 16% while in the shaved cases, it was 28%. In the C57BL/6 mice, NIr penetration was less, being 19% in the shaved cases and, quite remarkably, only 0.2% in the unshaved cases. Hence, these measurements indicated that the pigmented fur of the C57BL/6 mice absorbed almost all the NIr, hence limiting severely its penetration through to the brain.
We have two main findings. First, the MPTP-NIr group of Balb/c mice had greater locomotor activity and, as shown previously (Shaw et al. 2010), more surviving dopaminergic cells than the MPTP group. Second, these differences in cell survival and locomotor activity between the two groups were not as clear in C57BL/6 mice. Overall, our results indicated that Balb/c mice were a better model for exploring the neuroprotective effects of NIr after MPTP treatment than C57BL/6 mice.
Comparison with previous studies
This study offers the first detailed description of changes in locomotor activity in MPTP-treated mice after NIr treatment. Whelan and colleagues  described briefly that NIr pre-treatment, but not post-treatment, improved locomotor activity in an acute MPTP mouse model (strain was not mentioned in that report). Our results in Balb/c mice confirms, at least in part, the results of that study.
There have been several previous reports on the behavioural and cellular changes in Balb/c and C57BL/6 mice after MPTP insult [17,18]. We confirm the findings of these reports in that there were fewer TH+ cells in the SNc of C57BL/6 mice than Balb/c mice (eg, saline controls) and that MPTP had a greater effect on locomotor activity in C57BL/6 than in Balb/c mice; further that Balb/c mice had some NIr-induced recovery of activity while C57BL/6 mice did not. Our results offered some differences to the previous studies, however. In particular, previous studies using non-stereological methods have reported a greater MPTP-induced cell loss in C57BL/6 compared to Balb/c mice [17,18]; our stereological analysis, by contrast, revealed a comparable loss in the two strains (~30%). The reason for these differences is not clear but they may reflect, for example, differences in our MPTP regimes (eg 50 mg/kg over 24 hrs vs. 60 mg/kg over 8 hrs) , methods of MPTP delivery (eg, intraperitoneal vs. intraventricular)  and methods of cell analysis (stereological vs. non-stereological) [17,18]. Finally, our control Balb/c mice had slightly better locomotor activity at baseline than the C57BL/6 mice, while Sedelis and colleagues  have reported the opposite. This discrepancy may reflect differences in the behavioural tests used and our measures of locomotor activity. For example, we measured velocity, high mobility and immobility using contrast changes, while the previous study recorded distance travelled with laser beam technology. Despite these differences in our studies, the key issue is that our MPTP regime was effective in generating TH+ cell loss and behavioural changes in the two strains, thereby allowing an assessment of neuroprotection by NIr treatment.
It should be noted that in this study, we did not undertake an analysis of the density of TH+ terminals in the striatum, nor of the locomotive activity of the mice after six days, the end of the experimental period. Previous studies have shown a complete recovery of TH+ terminal density in the striatum  and locomotive activity after six days in Balb/c mice using an acute model ; in C57BL/6 mice, although there are fewer TH+ terminals in the striatum of MPTP-treated animals compared to controls at this stage , the locomotive activity has been shown to return to control levels . Hence, from these data, there would have been no point for us to explore these issues, mainly because any impact of NIr treatment – the central issue considered in the present study – would not have been elucidated.
NIr treatment improved locomotor activity after MPTP insult in Balb/c mice
Our results showed that NIr treatment improved locomotor activity after MPTP insult in Balb/c mice, hence confirming the histological findings that there were more dopaminergic cells in MPTP-NIr than in MPTP groups [11,12]. The beneficial effect of NIr treatment was not immediate. It was only after the second MPTP injection (and subsequent NIr treatments; T3 and T4) that a clear difference in locomotor activity was recorded between the MPTP-NIr and MPTP groups. Before then (T1 and T2), no differences were evident between these two groups (with the MPTP effect being similar and immediate in both groups). Hence, it appears that it takes several doses of NIr treatment to elicit a beneficial outcome. The mitochondria of the dopaminergic cells, after the third and fourth NIr treatment, may have been stimulated further to increase ATP synthesis and reduce the production of reactive oxygen species [14,15], thereby being better prepared to protect against the second MPTP insult. It is noteworthy that Whelan and colleagues  reported improvement of locomotor activity in MPTP-treated mice after several NIr pre-treatments, but not after a single post-treatment. Indeed, previous studies reporting beneficial results in the majority of systems have used multiple NIr treatments of ~4 J/cm2[14,15]. There may well be a therapeutic window for NIr treatment and this may vary for different animals and systems .
Strain differences in the effectiveness of NIr treatment after MPTP insult
Somewhat surprisingly, the beneficial effects of NIr treatment after MPTP insult were not as clear in the C57BL/6 mice. When compared to the Balb/c mice, the C57BL/6 mice had a smaller increase in dopaminergic cell number (20% vs 30%) and no clear improvement in locomotor activity in the MPTP-NIr compared to the MPTP group, at least over the later part of the survival period used in this study. Future studies may explore if there is a linear correlation between cell pathology and behavioural decline (and recovery)  in different strains of MPTP-treated mice after NIr treatment in the long-term; further, it would be of interest to examine if the finer details of motor disturbances in mice after MPTP treatment are improved after NIr treatment in the different mouse strains .
The reason for this strain difference was likely to be due to the pigmented fur of the C57BL/6 mice absorbing the majority of the NIr, preventing penetration into the brain. Our measurements indicated that in unshaved C57BL/6 mice, unlike in the shaved C57BL/6 and Balb/c (shaved and unshaved), there was very little NIr penetration (>1%). Melanin is certainly capable of absorbing the 670 nm wavelength  and that seemed sufficient to limit neuroprotection in the C57BL/6 mice. It is of course possible that, in addition to these penetration issues, the albino and pigmented strains have distinct cellular enzyme differences also, responsible for the different responses to NIr-induced metabolic (and therefore therapeutic) changes.
In summary, although our results are in an animal model of the disease, a key point is that NIr appeared to have neuroprotective effects on structures deep in the brain. Our findings that NIr treatment reduced MPTP-induced degeneration among midbrain dopaminergic cells and improved locomotor activity in Balb/c mice, due to greater NIr penetration through skin and fur, form templates for future endeavour. It remains to be determined if NIr, when applied from an external device, is able to penetrate the thicker skull and meningeal layers, together with the greater mass of brain parenchyma to reach the SNc of humans.
CoQ10: Coenzyme Q10; ATP: Adenosine-5′-triphosphate; LED: Light emitting device; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NIr: Near-infrared light; PBS: Phosphate buffered saline; SNc: Substantia nigra pars compacta; SNr: Substantia nigra pars reticulata; TH: Tyrosine hydroxylase.
There was no conflict of interest for any of the authors: CM,NT, DR, DJ, JS, ALB and JM are full-time members of staff at their respective institutions, while CP and NEM are undergraduate students.
All authors contributed to the analysis of the data and the writing of the manuscript. CM, NT, NEM, DR and JM contributed to the experimental work. All authors read and approved the final manuscript.
We are forever grateful to Tenix corp, Salteri family, Sir Zelman Cowen Universities Fund, Fondation Philanthropique Edmond J Safra, France Parkinson and the French National Research Agency (ANR Carnot Institute) for funding this work. We thank Sharon Spana, Vincente Di Calogero, Christophe Gaude, Caroline Meunier and Leti-DTBS staff for excellent technical assistance. We thank Sarah-Jane Leigh and Kevin Keay for their invaluable assistance with the statistics.
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Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats
This study investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs in neuronal cells. MTT assays indicated no significant difference between the amount of cells with (LS+) and without (LS) laser treatment (P > 0.05). However, immunofluorescent staining and western blot analysis results indicated a significant increase in the neural stem-cell marker, nestin, following exposure to low-level laser irradiation (P < 0.05). Furthermore, stem cell implantation was applied to treat rats suffering from stroke. At 28 days posttreatment, the motor functions of the rats treated using i-ADSCs (LS+) did not differ greatly from those in the sham group and HE-stained brain tissue samples exhibited near-complete recovery with nearly no brain tissue damage. However, the motor functions of the rats treated using i-ADSCs (LS?) remained somewhat dysfunctional and tissue displayed necrotic scarring and voids. The western blot analysis also revealed significant expression of oligo-2 in the rats treated using i-ADSCs (LS+) as well as in the sham group (P < 0.05). The results demonstrated that low-level laser irradiation exerts a positive effect on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.
Stroke has become a common disease and has been shown to be associated with the consumption of high amounts of oil and salt. These dietary habits cause blood vessels to narrow and become prone to occlusion, which can lead to stroke. Strokes can be broadly classified into 2 categories: ischemic and hemorrhagic strokes. Differing treatment methods are used according to stroke type and lesion location. Therefore, at stroke onset, computed tomography or magnetic resonance imaging is often used for diagnosis to provide physicians with a basis of treatment . The treatment methods used for the 2 types of stroke are different. (1) Ischemic stroke is caused by thrombosis or blood clots in the brain vessels, which prevent blood flow into the brain tissues. Anticoagulants or antiplatelet medications must be administered to patients as soon as possible. (2) Hemorrhagic stroke is caused by the rupture or hemorrhaging of brain vessels caused by peripheral brain trauma (subarachnoid hemorrhage). The majority of cases of hemorrhagic stroke are caused by brain trauma (e.g., car and workplace accidents). Treating these patients often requires neurosurgical interventions . Ischemic strokes constitute the majority of stroke cases. When patients suffer from ischemic stroke, the brain tissues necrotize gradually because of the lack of nutrients if anticoagulants or antiplatelet medications are not administered within 3h of the stroke. After necrosis occurs, necrotic brain tissue cannot regenerate or recover its functions even if blood-vessel reperfusion occurs . In recent years, stem cell treatment has become an innovative therapy for brain tissues that cannot regenerate.
Body fat extraction (e.g., abdomen, thigh, or buttock fat) has been used for body shaping procedures in the past, and the extracted fat was discarded as medical waste. Presently, medical experts have confirmed that adipose tissues contain large amounts of mesenchymal stem cells that exhibit in vitro proliferation and multiple differentiation ability, characteristics that facilitate the repair and regeneration of damaged tissues or organs . In addition, adipose-derived stem cells (ADSCs) possess several characteristics: (1) they can be easily harvested in abundant quantities without invasiveness, (2) they can proliferate in in vitro cultures, and (3) they can be applied to a wide range of body tissue types because these cells migrate to lesion sites automatically to repair damage. Studies have shown that ADSCs can differentiate into many different cell types, such as adipose, bone, cartilage, smooth muscle, cardiac muscle, endothelial, blood, liver, and even neuronal cells . Because of these characteristics, ADSCs will likely be one of the major sources of autologous stem cells in the future.
The effectiveness of laser therapy on biological bodies has been confirmed, and laser therapy has been successfully applied in new technological applications such as microsurgery. Lasers can be classified as high or low power, depending on the energy levels used. High-power lasers involve using high energy levels to provoke blood coagulation, stop bleeding, cut tissues, and even damage cells, whereas low-level lasers involve using electromagnetic or photochemical processes to achieve therapeutic effects . A low-level laser is defined as a laser with extremely low power and energy too low to destroy the molecular bonding capacity (e.g., hydrogen bonds and van der Waals forces) of tissues. Therefore, low-level lasers do not cause molecular structural change, protein denaturation, or cell death. Irradiation from low-level lasers on tissue does not cause an obvious temperature increase at the treatment site (less than 0.1 to 0.5°C); thus, any physiological responses of the tissues are produced by the stimulation of the laser itself, and this mechanism is described by the theory of laser biostimulation . In addition, by using an appropriate energy level, low-level lasers are primarily used to stimulate biological cells to induce or strengthen physiological responses for facilitating local blood circulation, regulating cell functions, promoting immunological functions, and facilitating cell metabolism and proliferation. Using lasers to generate these physiological changes enables treatment goals such as anti-inflammation and wound-healing promotion to be met [8, 9]. Many studies have shown that low-level laser irradiation exerts beneficial biological effects on bone, neuronal, and skin healing [10–12]. However, the type, wavelength, power, and energy level of lasers used in previous studies have varied, and various effects have been observed in different cells when differing levels of laser energy were applied. Previous studies have shown that using a low-level laser with an 820–830nm wavelength can reduce neural damage, facilitate neuronal healing, and accelerate neural recovery after an osteotomy [13, 14]. Using a low-level laser with a 660nm wavelength has been demonstrated to exert healing effects on musculoskeletal injuries and inflammation . In addition, many studies have indicated that a low-level laser with a 660nm wavelength can effectively promote neural regeneration and accelerate the reinnervation of muscle fibers to promote the recovery of motor functions [16–18]. Recent evidence has suggested that protein aggregates such as ?-amyloid- (A?) associated neurotoxicity and dendrite atrophy might be a consequence of brain-derived neurotrophic-factor (BDNF) deficiency. Meng et al. observed that the upregulation of BDNF caused by using low-level laser therapy (LLLT) to activate the extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) pathway can ameliorate A?-induced neuron loss and dendritic atrophy, thus identifying a novel pathway through which LLLT protects against A?-induced neurotoxicity .
Currently, most low-level laser therapies in practical clinical applications emphasize treatment courses covering an extensive tissue area in a relatively short period. Multichannel-laser hair treatment, which is currently available for the physical treatment of alopecia, is one such example. Therefore, we used a large-area LLLT that differs from the irradiation methods previously reported in the literature (such as single-spot low-energy laser exposure or the scanning method) to increase the local area of exposure . In this study, stem cells were extracted from adipose tissues, and neural-stem-cell- (NSC-) differentiating agents were used to culture these ADSCs to transform them into induced adipose-derived stem cells (i-ADSCs), which were used as experimental cells. First, we investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs into neuronal cells. We then investigated whether i-ADSCs treated with laser irradiation and injected via an intravenous route could integrate and survive in various locations in rat brains. We investigated if this treatment could improve the neurological dysfunctions caused by ischemic brain damage in rats and if rats could produce BDNF, using this treatment. Studies using intravenous injection to transplant i-ADSC for the treatment of ischemic stroke with protocols similar to that used in the current study are rare. In this study, we further combined the biostimulation theory from large-area LLLT with transplantation of i-ADSCs to induce neural differentiation to investigate the effectiveness of ischemic stroke treatment. In a previous study , we established a transient ischemia-reperfusion stroke rodent model by using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. The effectiveness of the treatment was assessed by comparing HE-staining and western blot analysis, as well as the evaluation of motor skill indices by using the rotarod and grip-strength tests. Our protocol has the potential to be developed for application in the clinical treatment of patients with ischemic stroke.
2.1. Isolation and Culture of ADSCs
A flowchart illustrating the experimental design of the study is shown in Figure 1. Eight-week-old male Sprague-Dawley (SD) rats were used for isolating rat ADSCs. The ADSCs were harvested from the rats’ subcutaneous anterior abdominal wall. Inguinal fat pads were excised, washed sequentially in serial dilutions of betadine, and finely minced as tissues in phosphate-buffered saline (PBS). The tissues were digested with 0.3% of Type I collagenase (Sigma) at 37°C for 60min. The digested tissue/cell suspension was filtered through a 100-mesh filter to remove the debris, and the filtrate was centrifuged at 1000rpm for 10min. The cellular pellet was resuspended using DMEM/F12 (10% FBS, 1% P/S) and then cultured for 24h at 37°C in 5% CO2. Unattached cells and debris were then removed and the adherent cells were cultured using fresh medium. The cells were cultured to 80% confluency before being released with 0.05% trypsin and then subcultured.
2.2. ADSC Neuronal Predifferentiation
In this study, i-ADSCs obtained from culturing ADSCs by adding NSC-differentiating agents were used as experimental cells. ADSCs within 3–5 passages were detached and induced using NSC media supplementation. ADSCs were resuspended in a serum-free DMEM/F12 medium supplemented with an N2 supplement (Sigma), 20ng/mL of epidermal growth factor (Gibco, NY, USA), and fibroblast growth factor (Gibco, NY, USA).
2.3. Setup of a Low-Level Laser Application Method
The probe of the laser irradiation device was fixed vertically on a clean, open experimental bench. The distance between the probe and the cell culture dish was 30cm. Laser irradiation was applied in a 25°C environment by using an AlGaInP-diode laser (Konftec Co., Taipei, Taiwan) with a wavelength of 660nm at an output power of 50mW and frequency of 50Hz. In the control group, the cells that did not receive laser irradiation treatment, i-ADSCs (LS) (n = 10), were compared with the experimental cells, i-ADSCs (LS+) (n = 10), which were subjected to a laser irradiation treatment of 10min. The cells in the i-ADSCs (LS) group were cultured for 7 days, whereas the cells in the i-ADSCs (LS+) group were treated using low-level lasers on the following day for 10min and then cultured for 6 days. The cells receiving laser irradiation were collected at various times for analysis according to the purposes of the experimental protocols. After the completion of the cultures, an optical microscope was used for observing cell morphology.
2.4. MTT Assay
The principle of the MTT (3-[4,5-dimethylthiazol-2-y1]-2,4-diphenyltetrazolium bromide) assay is that the mitochondria of living cells can transform the yellow chemical substance MTT tetrazolium into the purple non-water-soluble substance MTT formazan through the effect of succinate dehydrogenase. DMSO can be used to dissolve the purple-colored products. No such response occurs in dead cells. An optical absorbance of 570nm was measured using an enzyme immunoassay analyzer. A higher absorbance value indicates a larger amount of cells. In this study, ADSCs neuronal predifferentiation was first distributed in a 96-well plate with approximately 10000 cells per well. The cells in the i-ADSCs (LS) group were then cultured in a 37°C environment and a 5% CO2 environment for 5 days and 7 days, respectively. The cells in the i-ADSCs (LS+) group were cultured in a 37°C environment and a 5% CO2 environment for 4 days and 6 days, respectively. After the completion of the cultures, the medium was removed by several rinses with PBS. An MTT solution of 100?L was added to each well of a 96-well plate in the dark (1mL of MTT reagent was added to 9mL of phenol-red-free, serum-free medium) and incubated in a 37°C, 5% CO2 environment for 2h. The MTT solution was then removed and the cells were dissolved using DMSO. An optical absorbance of 570nm was measured using an enzyme immunoassay analyzer to compare the values between the different groups.
2.5. Immunocytochemistry of i-ADSCs
After 7 days in culture, the subcultured neurospheres were washed using 0.1M PBS 3 times and fixed with 4% paraformaldehyde for 1h. Following the fixation, the cells were permeated with 0.1% of Triton X-100 for 10min and then blocked with 5% nonfat milk for 30min. The phenotypic expression of these neurospheres was examined by implementing immunocytochemical staining accompanied by antibodies against glial fibrillary acidic proteins (GFAPs) for astrocytes, mouse monoclonal antinestins for NSCs, and doublecortin (DCX), which has recently been used as a marker for neurogenesis. Briefly, the fixed cells were washed 3 times in cold PBS. After washing with PBS, the aforementioned primary antibodies were added and the slides were maintained at room temperature overnight. In the following day, the primary antibodies were removed by washing 3 times with PBS and the secondary antibodies were added before incubating the cells for 1h. After washing off the secondary antibodies, the cells were incubated with tertiary antibodies tagged with peroxidase-antiperoxidase for 1h. The tertiary antibodies were washed off using PBS. The cells were incubated with DAPI (Sigma, St. Louis, MO, USA) diluted with the cell culture medium for 10min. Finally, the cells were mounted with 90% glycerol and examined using fluorescent microscopy (Olympus IX-71, Inc., Trenton, NJ, USA).
2.6. Animals and Induction of the MCAO Model
In a previous study , we established a transient ischemia-reperfusion stroke rodent model, using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan. Thirty-two adult male SD rats were randomly allocated to 3 groups: the i-ADSCs (LS) therapy group (n = 12), the i-ADSCs (LS+) therapy group (n = 12), and a sham group (n = 8). The rats in all 3 groups were euthanized on the 28th day after MCAO was performed. For MCAO procedures, anesthesia was induced using 4% isoflurane (Baxter, USA) and maintained using 2% isoflurane. A midline cervical incision was made to isolate the right bilateral common carotid artery. A 25mm-long 3-0 nylon surgical thread was then inserted into the right carotid bifurcation. In this study, 2 rounds were used to provide a more complete blockage of blood flow in the artery. When the blunted distal end met resistance, the proximal end of the thread was tightened at the carotid bifurcation. The right common, internal, and external carotid arteries were carefully separated from the adjacent vagal nerve, and the distal portions of the external and common carotid arteries were ligated. A small incision was subsequently made at the proximal portion of the external carotid artery, and a 3-0 nylon monofilament suture was gently inserted (approximately 18mm) into the internal carotid artery. After 60min of MCAO, the nylon surgical thread was removed to allow complete reperfusion of the ischemic area. During ischemia, rectal temperature was monitored and maintained at approximately 37°C by using a heating pad and an overhead lamp. The anesthetized rats intravenously received i-ADSCs at a concentration of 2 × 107mL 1 via their femoral veins. The rats in the sham group underwent the same surgical procedures except that the right-sided middle cerebral artery was not occluded.
2.7. Rotarod Test
An accelerating rotarod test was performed for each rat before and on the 7th, 14th, 21st, and 28th day after cerebral ischemia-reperfusion was induced. Before the ischemia-reperfusion experiment was conducted, the animals were subjected to 3 training sessions per day for 3 days on the accelerating rotarod to obtain stable duration on the rotarod spindle. The diameter of the rotarod spindle was 7cm. The surface of the rotarod spindle was made of knurled Perspex to provide an adequate grip, which prevented animals from slipping off the spindle. The speed of the spindle was increased from 4 to 40rpm over a period of 5min and the duration that the animal stayed on the device was recorded. The rats that were capable of staying on the rotarod longer than 150s after 3 training sessions were selected for the experiments. On the testing days, the animals were tested twice, and the longest durations on the rotarod were recorded.
2.8. Grip Strength Test
Each rat was supported in a horizontal position approximately parallel to a grip bar (Model DPS-5R: range 0–5 kgf, Japan). The researcher set the rat’s forepaws on the grip bar and pulled the animal horizontally away from the bar by the base of its tail until the rat released its grip. The pulling motion was smooth and continuous. The researcher supported the rat by the abdomen when the grip was released. The reading on the strain gauge remained constant at the point of maximal value, which was recorded as the measure of forepaw grip strength. The researcher supported the rat body by both the chest and the base of the tail at an angle of ?45° down the tail. The rat was facing away from the grip bar. The rat was encouraged to grasp the bar by moving its hind paw to the bar. When the rat grasped the bar with both hind paws, establishing a “full” grip, the upper body of the rat was lowered so that the rat was in a nearly horizontal position. The rat was pulled horizontally by the base of the tail until it released its grip and was supported as previously described. The reading on the strain gauge remained constant at the point of maximal value (force was measured in grams), which was recorded as the measurement of forepaw grip strength. Three values were obtained in succession, and the median value was used as the daily score. The data were expressed as the percentage of the baseline (preischemic) value.
2.9. Hematoxylin-Eosin Staining of the Cerebellum
The SD rats were anesthetized using 10% chloral hydrate (4?L/kg), administered intraperitoneally, and were euthanized on the 28th day after the MCAO operation and sham treatment. For each rat, the left cerebellum was rapidly removed and postfixed in formalin for 24h. The postfixed tissues were embedded in paraffin wax and 6-?m-thick serial coronal sections were obtained and mounted on poly-L-lysine-coated glass slices. To assess the histological changes in the MCAO and sham groups, the paraffin-embedded left cerebellum sections were stained using hematoxylin-eosin (HE), according to standard protocol before the assay was performed.
2.10. Western Blot Analysis
Proteins were extracted from the rat brains by using a cold lysis buffer (10mM of tetra sodium pyrophosphate, 20mM of Hepes, 1% Triton X-100, 100mM NaCl, 2?g/mL of aprotinin, 2?g/mL of leupeptin, and 100?g/mL of phenylmethylsulfonyl fluoride). The protein concentrations from tissue extracts or ADSC-conditioned medium were determined using the Bradford protein assay. Equal amounts of protein were placed in a 2× sample buffer (0.125M Tris-HCl, pH 6.8, 2% glycerol, 0.2mg/mL of bromophenol blue dye, 2% SDS, and 10% ?-mercaptoethanol) and electrophoresed through 10% SDS-polyacrylamide gel. The proteins were then transferred onto a nitrocellulose membrane by using electroblotting. The membranes were blocked for 1h at room temperature in a Tris-buffered saline with Tween-20 (TBST) and 5% nonfat milk. The primary antibodies (1:1000) with appropriate dilutions were incubated for 1h at room temperature in TBST and 5% nonfat milk. The blots were then washed and incubated with a peroxidase-conjugated secondary antibody (1:2000) for 1h in TBST. The chemiluminescent substrate for the secondary antibody was developed using the ECL detection system (Amersham, UK). The blots were exposed to film for 3–5min and then developed.
2.11. Statistical Analysis
The data were expressed as the mean value ± standard error of the mean. The statistical significance of the differences between the groups was determined using a one-way analysis of variance followed by Tukey’s test. An alpha level of less than 0.05 (P < 0.05) was considered statistically significant.
3.1. Effects of the Low-Level Laser on Cell Morphology
The ADSCs were passaged 3–5 times after the initial plating of the primary culture. Rat ADSCs appeared to be a monolayer of large and flat cells (Figure 2(a)). Many cells in the i-ADSCs (LS?) and i-ADSCs (LS+) groups induced a neuronal phenotype and exhibited, among one another, bipolar and multipolar elongations of neuronally induced cell-forming networks. The results show that the stem cells in both i-ADSCs (LS?) and i-ADSCs (LS+) groups developed tentacles, indicating that ADSCs were facilitating the induction of differentiation into neuronal cells. Comparative optical micrographs revealed that some attached cells exhibited a spread-out shape with a spindle-like and fibroblastic phenotype in the i-ADSCs (LS?) group (Figure 2(b)). However, most of i-ADSCs-expressing neurites extended radially, connecting like bridges with those from adjacent cells in the i-ADSCs (LS+) group (Figure 2(c)).
3.2. Effects of the Low-Level Laser on Cell Proliferation and Differentiation
In this study, MTT assays were performed on Day 5 and Day 7 to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell proliferation. After analyzing the optical absorbance values, the results showed that, on Day 5, cell activity was slightly higher in the i-ADSCs (LS+) group compared with that in the i-ADSCs (LS) group. However, the difference was not statistically significant (P > 0.05). On Day 7, the cell amounts in both the i-ADSCs (LS+) and i-ADSCs (LS?) groups were larger than the amounts on Day 5. However, the cell proliferation rates were similar on Day 5 without major differences (P > 0.05) (Figure 3).
In this study, immunofluorescent staining and western blots were used to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell differentiation. Immunofluorescent staining was performed for the NSC marker, nestin, glial cell marker, GFAP antibody, and neuronal precursor-cell-marker protein, DCX. After the staining was completed, fluorescent microscopy was used to observe the amount of fluorescence expression of each antibody. The results showed that the fluorescence expression of the nestin was higher in the cells in the i-ADSCs (LS+) group than that in the cells in the i-ADSCs (LS?) group. These results indicated that ADSC differentiation into neuronal cells was facilitated after large-area low-level laser irradiation (Figure 4(a)). For GFAP, no difference was observed in the amount of fluorescence expression of GFAP between the i-ADSCs (LS?) and i-ADSCs (LS+) groups (Figure 4(b)). Furthermore, for DCX, no difference was observed between the 2 groups because the cells in both groups still exhibited stem cell morphology (Figure 4(c)).
Western blot is used to quantify the expression of marker proteins. Therefore, western blot analysis was used to compare the amount of nestin expression between the i-ADSCs (LS?) and i-ADSCs (LS+) groups in this study. The results showed that cells in the i-ADSCs (LS+) group exhibited a substantially higher nestin expression compared with the cells in the i-ADSCs (LS?) group (P < 0.05) (Figure 5). Regarding the results of GFAP and DCX, no difference was observed in the fluorescence expressions of the i-ADSCs (LS?) and i-ADSCs (LS+) groups. Therefore, western blot analyses were not shown for GFAP and DCX. This result is consistent with the findings obtained using immunofluorescent staining.
3.3. Evaluation of Behavior Recovery after Stroke in the Animals
In this study, treadmill and forepaw-grip tests were used to evaluate motor function recovery after stem-cell transplantation treatment in rats with ischemic stroke. The treadmill test was performed on Day 7 after stem-cell transplantation was performed on the rats with stroke. The rats from either the i-ADSCs (LS+) group or the i-ADSCs (LS) group were unable to run as quickly as the rats in the sham group. From Day 14, the rats in the i-ADSCs (LS+) group gradually recovered the ability to run. By contrast, it was observed that the rats in the i-ADSCs (LS?) group recovered slightly; however, the degree of recovery was lower than that in the i-ADSCs (LS+) group. On Day 21 after stem-cell transplantation, the recovery of running function was still more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS) group. On Day 28, the motor function of the rats in the i-ADSCs (LS+) group was approaching the level of the rats in the sham group, whereas the performance of the rats in the i-ADSCs (LS?) group was still considerably weaker than that of the sham group (Figure 6).
Grip-strength tests were performed on Day 7 after stem-cell transplantation. The findings were similar to the treadmill test results; the grip behavior of the rats in both the i-ADSCs (LS+) group and the i-ADSCs (LS) group was worse than that of the rats in the sham group. On Day 14, the grip strength of the rats in the i-ADSCs (LS+) group was considerably recovered. By contrast, although the rats in the i-ADSCs (LS) group showed some progress, the improvement was minimal. On Day 21, the rats in the i-ADSCs (LS+) group continued to recover, whereas it was observed that the animals in the i-ADSCs (LS?) group were not recovering as quickly. On Day 28, grip strength recovery in the i-ADSCs (LS+) group approached that of the sham group, whereas grip strength in the i-ADSCs (LS?) group was still lower (Figure 7).
3.4. Repair of Brain Tissues after Treatment in Animals
After euthanizing the rats, the brain specimens were treated with paraffin and then sliced. The HE-immunostaining method was used to observe the repair of brain tissues after stem-cell transplantation was performed. An upright microscope (10x) was used to macroscopically observe the brain tissue, and the results showed that brain tissue was completely repaired in the rats in the i-ADSCs (LS+) group with nearly no necrotic brain tissue. By contrast, obvious necrotic scars were observed at the ischemic sites of the brain tissue from the rats in the i-ADSCs (LS?) group (Figure 8(a)). Observed under a microscope and magnified 200 times, the results showed that the brain tissue from the rats in the i-ADSCs (LS+) group was as dense as normal brain tissues, whereas numerous cavities were observed in the brain tissue from rats in the i-ADSCs (LS?) group (Figure 8(b)).
Oligodendrocytes, which are glial cells found in normal brains, form myelin in the central nervous system. Oligodendrocytes substantially decrease after brain cells are damaged, leading to myelin collapse and the loss of neural-signal conduction. Therefore, a western blot was used to analyze the amount of expression of the oligodendrocyte cell protein, oligo-2, to confirm the repair of brain tissues after stem-cell transplantation was performed in rats with stroke. The results showed that the amount of oligo-2 protein response in the brain tissues of the i-ADSCs (LS+) group was as high as that in the sham group. By contrast, the oligo-2 protein response in the i-ADSCs (LS?) group was substantially lower (P < 0.05). These results indicated that stem-cell transplantation treatments can repair brain tissues damaged by ischemia (Figure 9).
Stem cells possess the ability to proliferate, regenerate, differentiate, and secrete cytokines. Previous studies have proven that stem-cell therapy substantially improves the damage caused by stroke. Stem cells are derived from various sites. Most previous studies have used bone marrow mesenchymal stem cells; however, we used adipose stem cells in the present study, which are easily accessible, and abundant and exhibit high differentiation and proliferation activity. Moreover, adipose stem cells do not trigger strong immune reactions (resulting in low exclusion) and rarely form teratomas. In the literature, it has been demonstrated that adipose stem cells are essential adult stem cells that differentiate into various mesoderm tissues similarly to bone marrow mesenchymal stem cells [21–23]. Furthermore, adipose stem cells are more easily accessible than mesenchymal stem cells; therefore, adipose stem cells can be used as a substitute for bone marrow mesenchymal stem cells for repairing damaged tissues in the future. Adipose stem cells are generally more practical than bone marrow mesenchymal stem cells for use in research [23–26].
In this study, we used large-area low-level laser irradiation to induce ADSCs to differentiate into neuronal cells. The MTT assay analysis showed that the cell activity of the i-ADSCs increased on Days 5 and 7 of culture after large-area low-level laser irradiation [27, 28]. However, although cell activity increased on both Days 5 and 7, the activity was also increased for the group that did not receive laser irradiation treatment. No significant difference was observed between the groups with or without laser treatment (P > 0.05). Previous studies have shown that, depending on irradiation parameters, various types of cell respond differently to laser irradiation . Although the mechanism underlying this phenomenon remains obscure, several hypotheses have been proposed to explain the mechanism of laser action . We speculate that the lack of significant effects might be attributable to the short duration of low-level laser irradiation, which might not provide sufficient energy to the ADSCs for them to facilitate proliferation. Alternatively, using low-level lasers on the ADSCs might not sufficiently enhance cell proliferation. Therefore, future studies should investigate the appropriate duration of low-level laser irradiation and determine how much energy is required to accelerate cell proliferation [31,32].
To understand if using large-area low-level lasers exerted a positive effect on cell differentiation, we used immunofluorescent staining and western blot analysis to evaluate whether these lasers were capable of accelerating the induction of cell differentiation . The immunofluorescent staining results showed that the nestin level in the group with i-ADSCs treated using large-area low-level laser irradiation increased substantially compared with that of the group that did not receive laser treatment. This result indicated that large-area low-level laser can accelerate the differentiation of ADSCs into neuronal cells [34, 35]. LLLT has been demonstrated to regulate neuronal function both in vitro and in vivo. Previous studies have reported that laser treatments accelerated nerve cell sprouting and cell migration, which begin within 24h of seeding. During the first week of cultivation, irradiated cultures contain a high number of neurons exhibiting large perikaria and branched neuronal fibers, which interconnect to form networks . The possible mechanism of LLLT at the cellular level has been attributed to the acceleration of electron transfer reactions, resulting in the increase of reactive oxygen species and Ca2+as versatile second messengers . Previous studies have shown that applying LLLT could influence cellular processes by altering DNA synthesis and protein expression , biomodulating cytoskeletal organization , and stimulating cellular proliferation . Such properties suggest that LLLT, or interventions with similar neurobiological effects, can be used to treat neurodegeneration, a phenomenon that underlies debilitating clinical conditions.
However, no obvious differences were observed for GFAP and DCX in the test results. This can be explained by the differentiation agent used in this study, which exerts its effects primarily by inducing ADSCs to differentiate into NSCs. Therefore, although the ADSCs were treated using a low-level laser, they still did not differentiate into neural glia cells. The DCX protein can only be discovered after neurons have been formed from NSCs, which demonstrates that DCX is a late-stage protein that cannot be expressed when cells still exhibit the morphology of NSCs. Therefore, the amount of DCX expression is not affected by low-level laser exposure. Western blot analysis was used to determine the amount of nestin expression after i-ADSCs were exposed to large-area low-level laser irradiation and culture for 7 days. The results showed that the amount of nestin was higher with laser treatment. This is similar to the findings from the immunofluorescent-staining method, indicating that laser irradiation can accelerate the differentiation of ADSCs into neuronal cells. These results indicated that ADSCs can be induced to differentiate into neuronal cells after treatment by large-area laser irradiation for 10min. Future studies should establish the precise duration of large-area LLLT required to achieve improved results.
In this study, treadmill and forepaws grip tests were used to evaluate motor function recovery after stem cell transplantation treatments in rats with stroke. The treadmill test results showed that the running function was weaker for the rats in the sham group on Day 7 after treatment. We speculate that this might have been because the brain-tissue lesion area from ischemia was too large; therefore, the transplanted stem cells did not have sufficient time to noticeably repair brain tissue. Therefore, the motor functions of the rats remained impaired on Day 7. When tested on Day 14, recoveries in motor functions were observed in both the i-ADSCs (LS?) and i-ADSCs (LS+) groups, with superior recovery in the i-ADSCs (LS+) group, indicating that the damaged brain tissue was repaired. On Day 21, the recovery of the rats’ running function was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group, indicating that the brain tissue repair capability was superior to that in the i-ADSCs (LS+) group. When tested on Day 28, the running function of the rats in the i-ADSCs (LS+) group was close to that of the rats in the sham group. However, the motor function of the i-ADSCs (LS?) group remained impaired, indicating that hind-paw motor function recovery was accelerated after i-ADSCs (LS+) treatment.
The grip test results were similar to the treadmill test results. On Day 7 after stem-cell transplantation treatment of the rats with stroke, grip strength was low for both the i-ADSCs (LS+) group and i-ADSCs (LS?) groups because the damaged brain tissues were just about to be repaired; therefore, the forepaws of the rats remained weak. When tested on Day 14, no major improvement was observed in the grip strength of the rats in the i-ADSCs (LS?) group. By contrast, the grip strength improved considerably in the i-ADSCs (LS+) group, indicating that the brain tissue repair capability was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Days 21 and 28, the tests showed that grip strength recovery was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the grip strength in the i-ADSCs (LS+) group recovered to a level close to that of the sham group, indicating that, after i-ADSCs (LS+) treatment, the damaged brain tissues of the rats with stroke were repaired quickly, enabling the recovery of forepaw-grip strength. Based on these results, we concluded that damaged brain tissues can be repaired faster and motor function can be recovered efficiently in rats with stroke after i-ADSCs (LS+) treatment.
The i-ADSCs could differentiate into neuronal cells after transplantation into the brain. As a result, they moved and repaired damaged cerebral tissue selectively and improved cerebral functions by enhancing angiogenesis, renewal of neurons, and proliferation of nerve cells [40, 41]. In this study, we used the HE-immunostaining method and western blot analysis to evaluate brain tissue repair on Day 28 after stem cell transplantation treatment was performed in rats with stroke. The results of immunostaining were observed using a microscope. The structures were magnified 200 times, which showed that the brain tissue in the stroke lesion was dense and similar to that of normal brain tissue in the i-ADSCs (LS+) group. By contrast, numerous cavities were observed in the ischemic lesions of the brain tissues of the rats in the i-ADSCs (LS?) group. These results indicated that necrotizing brain tissue after ischemia was quickly repaired when i-ADSCs (LS+) was used to treat the rats with stroke. We speculate that using i-ADSCs (LS+) treatment can accelerate the induction of ADSCs to differentiate into neuronal cells. Therefore, although the same number of stem cells was transplanted for treatment, a greater number of NSCs were observed in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. Furthermore, because NSCs can protect damaged brain tissues from continuous deterioration, they can also help brain tissues to repair. Therefore, transplanting a greater amount of NSCs would likely assist in repairing brain tissues more effectively.
Regarding the results of the western blot analysis, the oligo-2 amounts after stem-cell transplantation treatment were analyzed. Because oligodendrocytes form myelin in the central nervous system, myelin levels collapse when oligodendrocytes die. Nerve conduction is delayed or interrupted after the death of oligodendrocytes, leading to limb disabilities. Therefore, the evaluation of oligo-2 could be a crucial reference in assessing the degree of brain tissue recovery after stem-cell transplantation treatments in rats with stroke. In this study, using western blot analysis showed that the amount of expression of oligo-2 in brain tissues treated with i-ADSCs (LS+) was similar to that in normal brain tissues. By contrast, brain tissues treated with i-ADSCs (LS?) exhibited a lower oligo-2 expression, indicating that using i-ADSCs (LS+) treatment in rats with stroke can repair myelin in the central nervous system, leading to the recovery of neural-signal conduction and motor function. Based on these experimental results, we concluded that using i-ADSCs (LS+) treatment in rats with stroke cannot only accelerate the repair of damaged brain tissues for the partial recovery of motor functions, but also enable the central nervous system to recover the velocity of neural-signal conduction. These results confirm that the transplantation of i-ADSCs (LS+) can accelerate repairs in rats with ischemic stroke because i-ADSCs (LS+) can more efficiently differentiate into NSCs.
In this study, we used i-ADSCs treated with large-area low-level laser irradiation to evaluate the effects of a low-level laser on cell proliferation and differentiation. The results showed that although a low-level laser cannot facilitate cell proliferation, it can accelerate the induction of ADSCs differentiating into NSCs. In this study, we successfully created large-area cell and tissue damage in rat brains by using an embolic stroke animal model. Stem-cell transplantation with either i-ADSCs (LS+) or i-ADSCs (LS?) was performed to evaluate the degree of repair after stroke in the animals. Because large-area low-level laser irradiation can accelerate the differentiation of ADSCs into NSCs, and NSCs can protect damaged brain tissues to prevent continuous deterioration from damage and to help with repair, the motor function recovery was thus superior in the rats treated using i-ADSCs (LS+) compared with that in the rats treated using i-ADSCs (LS?). From the brain tissue slices from each group of rats, we discovered that i-ADSCs (LS+) treatment more effectively repaired necrotizing brain tissues after ischemia in rat brains. Furthermore, the western blot analysis also showed that the amount of oligo-2 increased in i-ADSCs (LS+)-treated rats with stroke, confirming the repair of myelin in cerebral neurons to further assist in the recovery of neural-signal conduction in the central nervous system.
Therefore, in the present study we demonstrated that using large-area low-level lasers exerts positive effects on inducing ADSCs differentiation, and it effectively treated ischemic stroke in rats, regarding motor function recovery. In future studies, the effects of large-area low-level laser irradiation time and the appropriate dosage for the proliferation and differentiation of ADSCs should be evaluated. If the optimal irradiation time and dosage for ADSC proliferation and differentiation can be discovered in animal experiments similar to those in this study, we believe that superior experimental results can be obtained. Furthermore, if primate or canine experimental animals can be used to conduct the experimental protocols described herein, the concerns associated with individual animal differences and errors associated with motor function assessments can be minimized to obtain more reliable experimental data. Therefore, the findings of this study contribute to the development of cell therapy, which can benefit patients with stroke.
There is no conflict of interests.
This work was supported by grants from the Taichung Veterans General Hospital and Central Taiwan University of Science and Technology (Grant. TCVGH-CTUST 1027701) and the National Science Council (Grant. NSC 102-2314-B-075A-019-MY2), Taiwan.
J Neuroinflammation. 2012 Sep 18;9(1):219. [Epub ahead of print]
Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases.
Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses.
To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell[trade mark sign] cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT.
Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway.
The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.
Therapeutic effect of near infrared (NIR) light on Parkinson’s disease models.
Department of Neurology, Medical College of Wisconsin, 8701 W. Watertown Plank Rd, Milwaukee, WI 53226, USA.
Parkinson’s disease (PD) is a neurodegenerative disorder that affects large numbers of people, particularly those of a more advanced age. Mitochondrial dysfunction plays a central role in PD, especially in the electron transport chain. This mitochondrial role allows the use of inhibitors of complex I and IV in PD models, and enhancers of complex IV activity, such as NIR light, to be used as possible therapy. PD models fall into two main categories; cell cultures and animal models. In cell cultures, primary neurons, mutant neuroblastoma cells, and cell cybrids have been studied in conjunction with NIR light. Primary neurons show protection or recovery of function and morphology by NIR light after toxic insult. Neuroblastoma cells, with a gene for mutant alpha-synuclein, show similar results. Cell cybrids, containing mtDNA from PD patients, show restoration of mitochondrial transport and complex I and IV assembly. Animal models include toxin-insulted mice, and alpha-synuclein transgenic mice. Functional recovery of the animals, chemical and histological evidence, and delayed disease progression show the potential of NIR light in treating Parkinson’s disease.
Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits.
Department of Zoology, Tel Aviv University, Faculty of Life Sciences, Tel Aviv 69978, Israel. email@example.com
Near-infrared transcranial laser therapy (TLT) has been found to modulate various biological processes including traumatic brain injury (TBI). Following TBI in mice, in this study we assessed the possibility of various near-infrared TLT modes (pulsed versus continuous) in producing a beneficial effect on the long-term neurobehavioral outcome and brain lesions of these mice. TBI was induced by a weight-drop device, and neurobehavioral function was assessed from 1 h to 56 days post-trauma using the Neurological Severity Score (NSS). The extent of recovery is expressed as the difference in NSS (dNSS), the difference between the initial score and that at any other later time point. An 808-nm Ga-Al-As diode laser was employed transcranially 4, 6, or 8 h post-trauma to illuminate the entire cortex of the brain. Mice were divided into several groups of 6-8 mice: one control group that received a sham treatment and experimental groups that received either TLT continuous wave (CW) or pulsed wave (PW) mode transcranially. MRI was taken prior to sacrifice at 56 days post-injury. From 5-28 days post-TBI, the NSS of the laser-treated mice were significantly lower (p<0.05) than those of the non-laser-treated control mice. The percentage of surviving mice that demonstrated full recovery at 56 days post-CHI (NSS=0, as in intact mice) was the highest (63%) in the group that had received TLT in the PW mode at 100 Hz. In addition, magnetic resonance imaging (MRI) analysis demonstrated significantly smaller infarct lesion volumes in laser-treated mice compared to controls. Our data suggest that non-invasive TLT of mice post-TBI provides a significant long-term functional neurological benefit, and that the pulsed laser mode at 100 Hz is the preferred mode for such treatment.
J Neurotrauma. 2012 Jan 20;29(2):408-17. doi: 10.1089/neu.2010.1745. Epub 2011 Sep 21.
Low-level laser light therapy improves cognitive deficits and inhibits microglial activation after controlled cortical impact in mice.
Neuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
Low-level laser light therapy (LLLT) exerts beneficial effects on motor and histopathological outcomes after experimental traumatic brain injury (TBI), and coherent near-infrared light has been reported to improve cognitive function in patients with chronic TBI. However, the effects of LLLT on cognitive recovery in experimental TBI are unknown. We hypothesized that LLLT administered after controlled cortical impact (CCI) would improve post-injury Morris water maze (MWM) performance. Low-level laser light (800 nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60-80 min after CCI. Injured mice treated with 60 J/cm² (500 mW/cm²×2 min) either transcranially or via an open craniotomy had modestly improved latency to the hidden platform (p<0.05 for group), and probe trial performance (p<0.01) compared to non-treated controls. The beneficial effects of LLLT in open craniotomy mice were associated with reduced microgliosis at 48 h (21.8±2.3 versus 39.2±4.2 IbA-1+ cells/200×field, p<0.05). Little or no effect of LLLT on post-injury cognitive function was observed using the other doses, a 4-h administration time point and 7-day administration of 60 J/cm². No effect of LLLT (60 J/cm² open craniotomy) was observed on post-injury motor function (days 1-7), brain edema (24 h), nitrosative stress (24 h), or lesion volume (14 days). Although further dose optimization and mechanism studies are needed, the data suggest that LLLT might be a therapeutic option to improve cognitive recovery and limit inflammation after TBI.
J Neurotrauma. 2011 Sep 21. [Epub ahead of print]
Low-Level Laser Light Therapy Improves Cognitive Deficits and Inhibits Microglial Activation after Controlled Cortical Impact in Mice.
1 Neuroscience Center, Massachusetts General Hospital , Harvard Medical School, Charlestown, Massachusetts.
Abstract Low-level laser light therapy (LLLT) exerts beneficial effects on motor and histopathological outcomes after experimental traumatic brain injury (TBI), and coherent near-infrared light has been reported to improve cognitive function in patients with chronic TBI. However, the effects of LLLT on cognitive recovery in experimental TBI are unknown. We hypothesized that LLLT administered after controlled cortical impact (CCI) would improve post-injury Morris water maze (MWM) performance. Low-level laser light (800?nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60-80?min after CCI. Injured mice treated with 60?J/cm(2) (500?mW/cm(2)×2?min) either transcranially or via an open craniotomy had modestly improved latency to the hidden platform (p<0.05 for group), and probe trial performance (p<0.01) compared to non-treated controls. The beneficial effects of LLLT in open craniotomy mice were associated with reduced microgliosis at 48?h (21.8±2.3 versus 39.2±4.2 IbA-1+ cells/200×field, p<0.05). Little or no effect of LLLT on post-injury cognitive function was observed using the other doses, a 4-h administration time point and 7-day administration of 60?J/cm(2). No effect of LLLT (60?J/cm(2) open craniotomy) was observed on post-injury motor function (days 1-7), brain edema (24?h), nitrosative stress (24?h), or lesion volume (14 days). Although further dose optimization and mechanism studies are needed, the data suggest that LLLT might be a therapeutic option to improve cognitive recovery and limit inflammation after TBI.
Improved cognitive function after transcranial, light-emitting diode treatments in chronic, traumatic brain injury: two case reports.
Two chronic, traumatic brain injury (TBI) cases, where cognition improved following treatment with red and near-infrared light-emitting diodes (LEDs), applied transcranially to forehead and scalp areas, are presented.
Significant benefits have been reported following application of transcranial, low-level laser therapy (LLLT) to humans with acute stroke and mice with acute TBI. These are the first case reports documenting improved cognitive function in chronic, TBI patients treated with transcranial LED.
Treatments were applied bilaterally and to midline sagittal areas using LED cluster heads [2.1? diameter, 61 diodes (9?×?633?nm, 52?×?870?nm); 12-15?mW per diode; total power: 500?mW; 22.2?mW/cm(2); 13.3?J/cm(2) at scalp (estimated 0.4?J/cm(2) to cortex)].
Seven years after closed-head TBI from a motor vehicle accident, Patient 1 began transcranial LED treatments. Pre-LED, her ability for sustained attention (computer work) lasted 20 min. After eight weekly LED treatments, her sustained attention time increased to 3 h. The patient performs nightly home treatments (5 years); if she stops treating for more than 2 weeks, she regresses. Patient 2 had a history of closed-head trauma (sports/military, and recent fall), and magnetic resonance imaging showed frontoparietal atrophy. Pre-LED, she was on medical disability for 5 months. After 4 months of nightly LED treatments at home, medical disability discontinued; she returned to working full-time as an executive consultant with an international technology consulting firm. Neuropsychological testing after 9 months of transcranial LED indicated significant improvement (+1, +2SD) in executive function (inhibition, inhibition accuracy) and memory, as well as reduction in post-traumatic stress disorder. If she stops treating for more than 1 week, she regresses. At the time of this report, both patients are continuing treatment.
Transcranial LED may improve cognition, reduce costs in TBI treatment, and be applied at home. Controlled studies are warranted
Postepy High Med Dosw (Online). 2011 Feb 17;65:73-92.
The role of biological sciences in understanding the genesis and a new therapeutic approach to Alzheimer’s disease.
T?gowska E, Wosi?ska A.
Zak?ad Toksykologii Zwierz?t, Wydzia? Biologii i Nauk o Ziemi, Uniwersytet Miko?aja Kopernika w Toruniu.
The paper contrasts the historical view on causal factors in Alzheimer’s disease (AD) with the modern concept of the symptoms’ origin. Biological sciences dealing with cell structure and physiology enabled comprehension of the role of mitochondrial defects in the processes of formation of neurofibrillary tangles and ?-amyloid, which in turn gives hope for developing a new, more effective therapeutic strategy for AD. It has been established that although mitochondria constantly generate free radicals, from which they are protected by their own defensive systems, in some situations these systems become deregulated, which leads to free radical-based mitochondrial defects. This causes an energetic deficit in neurons and a further increase in the free radical pool. As a result, due to compensation processes, formation of tangles and/or acceleration of ?-amyloid production takes place. The nature of these processes is initially a protective one, due to their anti-oxidative action, but as the amount of the formations increases, their beneficial effect wanes. They become a storage place for substances enhancing free radical processes, which makes them toxic themselves. It is such an approach to the primary causal factor for AD which lies at the roots of the new view on AD therapy, suggesting the use of methylene blue-based drugs, laser or intranasally applied insulin. A necessary condition, however, for these methods’ effectiveness is definitely an earlier diagnosis of the disease. Although there are numerous diagnostic methods for AD, their low specificity and high price, often accompanied by a considerable level of patient discomfort, make them unsuitable for early, prodromal screening. In this matter a promising method may be provided using an olfactory test, which is an inexpensive and non-invasive method and thus suitable for screening, although as a test of low specificity, it should be combined with other methods. Introducing new methods of AD treatment does not mean abandoning the traditional ones, based on enhancing cholinergic transmission. They are valuable as long as the therapy starts when abundant brain inclusions disturb the transmissions.
J Comp Neurol. 2010 Jan 1;518(1):25-40.
Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment.
Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J.
Discipline of Anatomy & Histology F13, University of Sydney, Australia.
This study explores whether near-infrared (NIr) light treatment neuroprotects dopaminergic cells in the substantia nigra pars compacta (SNc) and the zona incerta-hypothalamus (ZI-Hyp) from degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. BALB/c albino mice were divided into four groups: 1) Saline, 2) Saline-NIr, 3) MPTP, 4) MPTP-NIr. The injections were intraperitoneal and they were followed immediately by NIr light treatment (or not). Two doses of MPTP, mild (50 mg/kg) and strong (100 mg/kg), were used. Mice were perfused transcardially with aldehyde fixative 6 days after their MPTP treatment. Brains were processed for tyrosine hydroxylase (TH) immunochemistry. The number of TH(+) cells was estimated using the optical fractionator method. Our major finding was that in the SNc there were significantly more dopaminergic cells in the MPTP-NIr compared to the MPTP group (35%-45%). By contrast, in the ZI-Hyp there was no significant difference in the numbers of cells in these two groups. In addition, our results indicated that survival in the two regions after MPTP insult was dose-dependent. In the stronger MPTP regime, the magnitude of loss was similar in the two regions ( approximately 60%), while in the milder regime cell loss was greater in the SNc (45%) than ZI-Hyp ( approximately 30%). In summary, our results indicate that NIr light treatment offers neuroprotection against MPTP toxicity for dopaminergic cells in the SNc, but not in the ZI-Hyp.
J Photochem Photobiol B. 2009 Dec 2;97(3):145-51. Epub 2009 Sep 11.
Effect of phototherapy with low intensity laser on local and systemic immodulation following focal brain damage in rat.
Moreira MS, Velasco IT, Ferreira LS, Ariga SK, Barbeiro DF, Meneguzzo DT, Abatepaulo F, Marques MM.
LIM-51, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
Brain injury is responsible for significant morbidity and mortality in trauma patients, but controversy still exists over therapeutic management for these patients. The objective of this study was to analyze the effect of phototherapy with low intensity lasers on local and systemic immunomodulation following cryogenic brain injury. Laser phototherapy was applied (or not-controls) immediately after cryogenic brain injury performed in 51 adult male Wistar rats. The animals were irradiated twice (3 h interval), with continuous diode laser (gallium-aluminum-arsenide (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP), 660 nm) in two points and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). The experimental groups were: Control (non-irradiated), RL3 (visible red laser/ 3 J/cm(2)), RL5 (visible red laser/5 J/cm(2)), IRL3 (infrared laser/3 J/cm(2)), IRL5 (infrared laser/5 J/cm(2)). The production of interleukin-1IL-1beta (IL-1beta), interleukin6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) was analyzed by enzyme immunoassay technique (ELISA) test in brain and blood samples. The IL-1beta concentration in brain of the control group was significantly reduced in 24 h (p<0.01). This reduction was also observed in the RL5 and IRL3 groups. The TNF-alpha and IL-6 concentrations increased significantly (p<0.01 and p<0.05, respectively) in the blood of all groups, except by the IRL3 group. The IL-6 levels in RL3 group were significantly smaller than in control group in both experimental times. IL-10 concentration was maintained stable in all groups in brain and blood. Under the conditions of this study, it is possible to conclude that the laser phototherapy can affect TNF-alpha, IL-1beta and IL-6 levels in the brain and in circulation in the first 24 h following cryogenic brain injury.
Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Nov-Dec;(6):3-11.
Many-level polysensory stimulation of brain functions by physical therapeutic agents.
[Article in Russian]
Tyshkevich TG, Ponomarenko GN.
A combination of physiotherapeutic methods for neurorehabilitative treatment has been developed and applied to the treatment of 576 patients with neurosurgical problems including the loss of brain functions as a sequel to nervous system lesions of traumatic, vascular, and other origin. Methodologically, this complex is adapted to the level and extent of the lesion and the character of regeneration of the nervous tissues. It implies many-level stimulation of neuroregeneration by syndromically and pathogenetically substantiated application of physical factors in the early post-injury and postoperative periods. The proposed approach allows the brain function to be completely restored by virtue of persistent compensatory changes in the nervous system. A combination of many-level magnetic, electrical, and laser stimulation is recommended to manage lesions in the speech, motor, and visual analyzers. Combined laser and differential electrostimulation may be prescribed to patients with nerve lesions, extremely high frequency therapy to those with epileptic syndrome, combined microwave therapy to cases with impairment of consciousness, and a variant of systemic UV irradiation with underwater shower-massaging for the treatment of vegetative and asthenic disturbances. Selected physiological aspects of the action of the above physical factors are specified. This physiotherapeutic system is protected by 20 RF patents of invention.
Mol Neurodegener. 2009 Jun 17;4:26.
Reduced axonal transport in Parkinson’s disease cybrid neurites is restored by light therapy.
Trimmer PA, Schwartz KM, Borland MK, De Taboada L, Streeter J, Oron U.
University of Virginia, Morris K Udall Parkinson’s Research Center of Excellence and Department of Neurology, Charlottesville, Virginia, USA. firstname.lastname@example.org.
ABSTRACT: BACKGROUND: It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson’s disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid “cytoplasmic hybrid” neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined.
RESULTS: The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.
CONCLUSION: The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.
Lasers Surg Med. 2009 Apr;41(4):277-81.
Increase of neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy.
Rochkind S, El-Ani D, Nevo Z, Shahar A.
Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv 64239, Israel. email@example.com
BACKGROUND AND OBJECTIVES: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated.
MATERIALS AND METHODS: Whole brains were dissected from 16 days Sprague-Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4-14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVR-Gel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW.
RESULTS: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P<0.01), which formed dense branched interconnected networks of thick neuronal fibers.
CONCLUSIONS: 780 nm laser phototherapy of embryonic rat brain cultures embedded in hyaluronic acid-laminin gel and attached to positively charged cylindrical MCs, stimulated migration and fiber sprouting of neuronal cells aggregates, developed large size neurons with dense branched interconnected network of neuronal fibers and, therefore, can be considered as potential procedure for cell therapy of neuronal injury or disease.
Lasers Surg Med. 2009 Jan;41(1):52-9.
Light therapy and supplementary Riboflavin in the SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis (FALS).
Moges H, Vasconcelos OM, Campbell WW, Borke RC, McCoy JA, Kaczmarczyk L, Feng J, Anders JJ.
Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
BACKGROUND AND OBJECTIVE: Familial amyotrophic lateral sclerosis (FALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and death. Mitochondrial dysfunction and oxidative stress play an important role in motor neuron loss in ALS. Light therapy (LT) has biomodulatory effects on mitochondria. Riboflavin improves energy efficiency in mitochondria and reduces oxidative injury. The purpose of this study was to examine the synergistic effect of LT and riboflavin on the survival of motor neurons in a mouse model of FALS.
STUDY DESIGN/MATERIALS AND METHODS: G93A SOD1 transgenic mice were divided into four groups: Control, Riboflavin, Light, and Riboflavin+Light (combination). Mice were treated from 51 days of age until death. A single set of LT parameters was used: 810 nm diode laser, 140-mW output power, 1.4 cm(2) spot area, 120 seconds treatment duration, and 12 J/cm(2) energy density. Behavioral tests and weight monitoring were done weekly. At end stage of the disease, mice were euthanized, survival data was collected and immunohistochemistry and motor neuron counts were performed.
RESULTS: There was no difference in survival between groups. Motor function was not significantly improved with the exception of the rotarod test which showed significant improvement in the Light group in the early stage of the disease. Immunohistochemical expression of the astrocyte marker, glial fibrilary acidic protein, was significantly reduced in the cervical and lumbar enlargements of the spinal cord as a result of LT. There was no difference in the number of motor neurons in the anterior horn of the lumbar enlargement between groups.
CONCLUSIONS: The lack of significant improvement in survival and motor performance indicates study interventions were ineffective in altering disease progression in the G93A SOD1 mice. Our findings have potential implications for the conceptual use of light to treat other neurodegenerative diseases that have been linked to mitochondrial dysfunction.
Brain Res. 2008 Dec 3;1243:167-73. Epub 2008 Sep 30.
Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.
Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Parkinson’s disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.
Neuroscience. 2008 Jun 2;153(4):963-74. Epub 2008 Mar 26.
Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.
Liang HL, Whelan HT, Eells JT, Wong-Riley MT.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Parkinson’s disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson’s disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson’s disease by energizing the cells and increasing their viability.
J Neurotrauma. 2007 Apr;24(4):651-6.
Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits.
Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler V, Shohami E.
Department of Orthopedics, Assaf Harofeh Medical Center, Zerifin, Israel. firstname.lastname@example.org
Low-level laser therapy (LLLT) has been evaluated in this study as a potential therapy for traumatic brain injury (TBI). LLLT has been found to modulate various biological processes. Following TBI in mice, we assessed the hypothesis that LLLT might have a beneficial effect on their neurobehavioral and histological outcome. TBI was induced by a weight-drop device, and motor function was assessed 1 h post-trauma using a neurological severity score (NSS). Mice were then divided into three groups of eight mice each: one control group that received a sham LLLT procedure and was not irradiated; and two groups that received LLLT at two different doses (10 and 20 mW/cm(2) ) transcranially. An 808-nm Ga-As diode laser was employed transcranially 4 h post-trauma to illuminate the entire cortex of the brain. Motor function was assessed up to 4 weeks, and lesion volume was measured. There were no significant changes in NSS at 24 and 48 h between the laser-treated and non-treated mice. Yet, from 5 days and up to 28 days, the NSS of the laser-treated mice were significantly lower (p < 0.05) than the traumatized control mice that were not treated with the laser. The lesion volume of the laser treated mice was significantly lower (1.4%) than the non-treated group (12.1%). Our data suggest that a non-invasive transcranial application of LLLT given 4 h following TBI provides a significant long-term functional neurological benefit. Further confirmatory trials are warranted.
Photomed Laser Surg. 2006 Aug;24(4):458-66
Effects of power densities, continuous and pulse frequencies, and number of sessions of low-level laser therapy on intact rat brain.
Ilic S, Leichliter S, Streeter J, Oron A, DeTaboada L, Oron U.
Photothera Inc., Carlsbad, California, USA.
OBJECTIVE: The aim of the present study was to investigate the possible short- and long-term adverse neurological effects of low-level laser therapy (LLLT) given at different power densities, frequencies, and modalities on the intact rat brain.
BACKGROUND DATA: LLLT has been shown to modulate biological processes depending on power density, wavelength, and frequency. To date, few well-controlled safety studies on LLLT are available. METHODS: One hundred and eighteen rats were used in the study. Diode laser (808 nm, wavelength) was used to deliver power densities of 7.5, 75, and 750 mW/cm2 transcranially to the brain cortex of mature rats, in either continuous wave (CW) or pulse (Pu) modes. Multiple doses of 7.5 mW/cm2 were also applied. Standard neurological examination of the rats was performed during the follow-up periods after laser irradiation. Histology was performed at light and electron microscopy levels.
RESULTS: Both the scores from standard neurological tests and the histopathological examination indicated that there was no long-term difference between laser-treated and control groups up to 70 days post-treatment. The only rats showing an adverse neurological effect were those in the 750 mW/cm2 (about 100-fold optimal dose), CW mode group. In Pu mode, there was much less heating, and no tissue damage was noted. CONCLUSION: Long-term safety tests lasting 30 and 70 days at optimal 10x and 100x doses, as well as at multiple doses at the same power densities, indicate that the tested laser energy doses are safe under this treatment regime. Neurological deficits and histopathological damage to 750 mW/cm2 CW laser irradiation are attributed to thermal damage and not due to tissue-photon interactions.
Protective effect of low-level irradiation on acupuncture points combined with iontophoresis against focal cerebral ischemia-reperfusion injury in rats.
[Article in Chinese]
Dai JY, Ge LB, Zhou YL, Wang L.
Acupuncture Clinic, Institute of Qigong, Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China. email@example.com
OBJECTIVE: To investigate the effects of low-level laser irradiation on acupuncture points combined with iontophoresis against brain damage after middle cerebral artery occlusion (MCAO) in rats.
METHODS: Sixty-nine SD rats were randomly divided into five groups, including normal group, sham operation group, model group, electro-acupuncture group and low-level laser irradiation on acupuncture points combined with iontophoresis group (LLLI group). The cerebral ischemia-reperfusion (I/R) model was established by thread embolism of middle cerebral artery. The rats in the LLLI group, as well as the electro-acupuncture group were given treatment as soon as the occlusion finished (0 hour) and 12, 24 hours after the occlusion. We observed the changes of neurological deficit scores and the body weight of the rats at different time. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ratos brain tissue were tested.
RESULTS: The neurological deficit score of the LLLI group was significantly lower than that of the model group. The body weight and the activity of SOD of the rats decreased slightly, and the content of MDA decreased significantly after the treatment.
CONCLUSION: The low-level laser irradiation on acupuncture points combined with iontophoresis can prevent focal cerebral ischemia-reperfusion injury. One of its mechanisms may be increasing the activity of SOD and decreasing the damage of the oxidation products to the body.
Mitochondrion. 2004 Sep;4(5-6):559-67.
Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy.
Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.
Department of Health Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. firstname.lastname@example.org
Photobiomodulation by light in the red to near infrared range (630-1000 nm) using low energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing, improve recovery from ischemic injury in the heart and attenuate degeneration in the injured optic nerve. Recent evidence indicates that the therapeutic effects of red to near infrared light result, in part, from intracellular signaling mechanisms triggered by the interaction of NIR light with the mitochondrial photoacceptor molecule cytochrome c oxidase. We have demonstrated that NIR-LED photo-irradiation increases the production of cytochrome oxidase in cultured primary neurons and reverses the reduction of cytochrome oxidase activity produced by metabolic inhibitors. We have also shown that NIR-LED treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. Photobiomodulation improves wound healing in genetically diabetic mice by upregulating genes important in the promotion of wound healing. More recent studies have provided evidence for the therapeutic benefit of NIR-LED treatment in the survival and functional recovery of the retina and optic nerve in vivo after acute injury by the mitochondrial toxin, formic acid generated in the course of methanol intoxication. Gene discovery studies conducted using microarray technology documented a significant upregulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection. These findings provide a link between the actions of red to near infrared light on mitochondrial oxidative metabolism in vitro and cell injury in vivo. Based on these findings and the strong evidence that mitochondrial dysfunction is involved in the pathogenesis of numerous diseases processes, we propose that NIR-LED photobiomodulation represents an innovative and non-invasive therapeutic approach for the treatment of tissue injury and disease processes in which mitochondrial dysfunction is postulated to play a role including diabetic retinopathy, age-related macular degeneration, Leber’s hereditary optic neuropathy and Parkinson’s disease.
Biochemical and immunological indices of the blood in Parkinson’s disease and their correction with the help of laser therapy.
[Article in Russian]
Komel’kova LV, Vitreshchak TV, Zhirnova IG, Poleshchuk VV, Stvolinskii SL, Mikhailov VV, Gannushkina IV, Piradov MA.
The influence of laser therapy on the course of Parkinson’s disease (PD) was studied in 70 patients. This influence appeared adaptogenic both in the group with elevated and low MAO B and Cu/Zn SOD activity. Laser therapy resulted in reduction of neurological deficit, normalization of the activity of MAO B, Cu/Zn-SOD and immune indices. There was a correlation between humoral immunity and activity of the antioxidant enzymes (SOD, catalase). This justifies pathogenetically the use of laser therapy in PD.
Laser modification of the blood in vitro and in vivo in patients with Parkinson’s disease.
Vitreshchak TV, Mikhailov VV, Piradov MA, Poleshchuk VV, Stvolinskii SL, Boldyrev AA.
Institute of Neurology of the Russian Academy of Medical Sciences, Moscow.
The effect of He-Ne laser radiation on activity of MAO B, Cu/Zn-SOD, Mn-SOD, and catalase in blood cells from patients with Parkinson’s disease was studied in vivo and in vitro. The effects of intravenous in vivo irradiation (intravenous laser therapy) were more pronounced than those observed in similar in vitro experiments. It is concluded that generalized effect of laser therapy involves interaction between blood cells.
Proceedings of the SPIE, Volume 5229, pp. 97-103 (2003). Laser Technology VII: Applications of Lasers. DOI: 10.1117/12.520611
Laser biostimulation of patients suffering from multiple sclerosis in respect to the biological influence of laser light.
Peszynski-Drews, Cezary; Klimek, Andrzej; Sopinski, Marek; Obrzejta, Dominik
AA (Technical Univ. of Lodz (Poland)), AB (Copernicus Hospital (Poland)), AC(Technical Univ. of Lodz (Poland)), AD (Technical Univ. of Lodz (Poland))
The authors discuss the results, obtained so far during three years’ clinical examination, of laser therapy in the treatment of patients suffering from multiple sclerosis. They regard both the results of former laboratory experiments and so far discovered mechanisms of biological influence of laser light as an objective explanation of high effectiveness of laser therapy in the case of this so far incurable disease. They discuss wide range of biological mechanisms of laser therapy, examined so far on different levels (cell, tissue, organ), allowing the explanation of beneficial influence of laser light in pathogenetically different morbidities.
|Neurol Res. 2002 Jun;24(4):355-60.|
Transplantation of embryonal spinal cord nerve cells cultured on biodegradable microcarriers followed by low power laser irradiation for the treatment of traumatic paraplegia in rats.
Rochkind S, Shahar A, Amon M, Nevo Z.
Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Israel. email@example.com
This pilot study examined the effects of composite implants of cultured embryonal nerve cells and laser irradiation on the regeneration and repair of the completely transected spinal cord. Embryonal spinal cord nerve cells dissociated from rat fetuses and cultured on biodegradable microcarriers and embedded in hyaluronic acid were implanted in the completely transected spinal cords of 24 adult rats. For 14 consecutive post-operative days, 15 rats underwent low power laser irradiation (780 nm, 250 mW), 30 min daily. Eleven of the 15 (73%) showed different degrees of active leg movements and gait performance, compared to 4 (44%) of the 9 rats with implantation alone. In a controlgroup of seven rats with spinal cord transection and no transplantation or laser, six (86%) remained completely paralyzed. Three months after transection, implantation and laser irradiation, SSEPs were elicited in 69% of rats (p = 0.0237) compared to 37.5% in the nonirradiated group. The control group had no SSEPs response. Intensive axonal sprouting occurred in the group with implantation and laser. In the control group, the transected area contained proliferating fibroblasts and blood capillaries only. This suggests: 1. These in vitro composite implants are a regenerative and reparative source for reconstructing the transected spinal cord. 2. Post-operative low power laser irradiation enhances axonal sprouting and spinal cord repair.
|Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002 Jan;93(1):27-34.|
Low-level laser effect on neural regeneration in Gore-Tex tubes.
Miloro M, Halkias LE, Mallery S, Travers S, Rashid RG.
Department of Surgery, Division of Oral and Maxillofacial Surgery, University of Nebraska Medical Center, Omaha 68198-5180, USA.
PURPOSE: The purpose of this investigation was to determine the effects of low-level laser (LLL) irradiation on neural regeneration in surgically created defects in the rabbit inferior alveolar nerve.
STUDY DESIGN: Five adult female New Zealand White rabbits underwent bilateral exposure of the inferior alveolar nerve. A 6-mm segment of nerve was resected, and the nerve gap was repaired via entubulation by using a Gore-Tex conduit. The experimental side received 10 postoperative LLL treatments with a 70-mW gallium-aluminum-arsenide diode at 4 sites per treatment. At 15 weeks after surgery, the nerve segments were harvested bilaterally and prepared for light microscopy. Basic fuchsin and toluidine blue were used to highlight myelinated axons. The segments were examined histomorphometrically by using computer analysis to determine mean axonal diameter, total fascicular surface area, and axonal density along the repair sites.
RESULTS: Gross examination of all nerves showed intact neural bundles with variable degrees of osseous remodeling. Light microscopic evaluation revealed organized regenerated neural tissue in both groups with more intrafascicular perineural tissue in the control group. Histomorphometric evaluation revealed increased axonal density in the laser treated group as compared with the control.
CONCLUSIONS: LLL irradiation may be a useful noninvasive adjunct to promote neuronal wound healing in surgically created defects repaired with expanded polytetrafluoroethylene entubulation.
ACTA LASER BIOLOGY SINICA Vol. 8, No.2, 1999
Vascular Low Level Laser Irradiation Therapy in Treatment of Brain Injury
WANG Yu ZHU Jing, et al
(Department of Neurosurgery, Renji Hospital Affiliated to Shanghai Second Medical University, Shanghai Medical Centre for laser Research ,200001)
Abstract: To evaluate the effect and mechanism of Vascular Low Level Laser Irradiation Therapy on brain injury. In this study thirty-eight SpragueDawley rats received Feeney’s brain impact through a left lateral craniectomy under anesthesia. Control and treatment group are set up. According to the time exposed to laser and irradiating postinjury, the treatment group is divided in four subgroups by design. Semiconductor laser was used with a power of 5mW to irridate straightly Rat’s femur venous. The Y Water maze was used to assess cognitive performance. Superoxide dismutase(SOD) activity and the level of metabolic production of free radical MDA in Brain and erythrocyte were measured to determinate the level of free radical. We find Vascular Low Level Laser Irradiation Therapy can improve posttraumatic memory deficits. SOD activity is higher in treatment groups than the control group meanwhile the level of MDA is lower. These findings suggest that Vascular Low Level Laser Irradiation produced a significant reduction in free radical’s damage to the brain postinjury.
INFARED LASER RADIATION IN THE TREATMENT OF BRAIN INJURY CONSEQUENCES
E.L. Macheret, A.O. Korkushko, T.N. Kalishchuk, M.N. Matyash
Medical Academy of Post-Diploma Education, Kiev, Ukraine
The examination of 198 patients aged 16-47 has revealed a high frequency of progressive pathologic states in a form of asthenia, vegeto-vascular dystonia, hypertensive, somato-vegetative, vestibular syndroms. Taking into account the changes in cortico-undercortical interrelations and expansion of pathologic process in hypothalamic area during the head trauma, we have developed effective treating methods by means of laseropuncture. Laser rays influence on acupuncture points (AP) leads to a convergence of the afferent messages upon the neurones of spinal cord, reticular formation, thalamus, hypothalamus and brain cortex. As a result of that a dynamic balance between the inhibition and excitation processes in the structures of central nervous system leading vegetative function and endocrine secretion recovers. Use of infrared laser radiation is the most perspective. It docs not cause the direct photochemical reactions in biological tissues, but influences on physico-chemical structure of AP biomolecules. For laseropuncture we used an apparatus “BIOMED-01? with a wavelength of 0.89 nm. The work regime is impulsive-continuous with a modulation of frequency – from 0.1 to 1000 Hz. The middle power is up to 20 mW. The total time of the action for one sitting is till 20 min. The points selections was carried out on the grounds of the methods of acupuncture diagnosis, imagesking out the dominant clinical syndromes and including points of vascular, vegetotroimages, sedative orientation. Our clinical results, which were confirmed by paraclinical methods (EEG, dopplerography) and methods of acupuncture diagnosis have shown a high effectiveness of this therapy decreasing the drugs load and having no contradictions.
LASER-THERAPY AND ITS INFLUENCE ON HEMODYNAMICS WITH PATIENTS SUFFERED FROM GRAVE CRANIOCEREBRAL TRAUMA
Medical Academy, Dnepropetrovsk, Ukraine
Despite the maximal dosage of different medications taken for curing of grave craniocerebral trauma the resistance to the treatment carried out was observed. This fact stimulated the search of new methods and ways of therapy. One of the possible methods is a non-medicamental treatment based on blood irradiation with low-active helium-neon laser. The present paper presents some data concerning the laser-therapy influence in hemodynamics in the case of craniocerebral trauma. The total number of patients examined is 45. Laser-therapy was carried out through the subclavian vein (37 cases) or cubital vein (8 cases). For primary irradiation the preferable access was the central one. It was used in the acute period of craniocerebral trauma. The periferal access was used for irradiation in the posthospital period. The course of laser therapy for in-hospital patients consisted of 3-5 everyday procedures of 30 minutes each. To define the hemodynamic changes with the patients suffered from craniocerebral trauma both clinical observation and ultrasonic transcranial dopplerography were used. The last one gave the possibility to identify the type of blood flow speed disorders.
Paper received 10 May 1999; accepted after revision 23 August 1999.
Specific Effects of Laserpuncture on the Cerebral Circulation
G. Litscher (1), L. Wang (1), M. Wiesner-Zechmeister (2)(1)
Biomedical Engineering, Department of Anesthesiology and Critical Care, University of Graz, Graz, Austria(2) European Forum for Lasertherapy and Fractal Medicine
Abstract . Acupuncture is a form of traditional Chinese medicine that has developed over thousands of years. We studied the effects of laser puncture, needle acupuncture, and light stimulation on cerebral blood flow in 15 healthy volunteers (mean age 25.0±1.9 years, 5 female, 10 male) with non-invasive transcranial Doppler sonography. In addition 40-Hz stimulus-induced brain oscillations, heart rate, blood pressure, peripheral and cerebral oxygen saturation, and the bispectral index of the EEG were recorded. Stimulation with light significantly increased blood flow velocity in the posterior cerebral artery (p<0.01, ANOVA). Similar but less pronounced effects were seen after needle acupuncture (p< 0.05, ANOVA) and laserpuncture (n.s.) of vision-related acupuncture points. Furthermore both, laserpuncture and needle acupuncture, led to a significant increase in the amplitudes of 40-Hz cerebral oscillations. Stimulation of vision-related acupuncture points with laser light or needle acupuncture elicits specific effects in specific areas of the brain. The results indicate that the brain plays a key intermediate role in acupuncture. However, brain activity of itself does not explain anything about the healing power of acupuncture.
Keywords: Acupuncture; Brain; 40 Hz brain oscillations; Cerebral blood flow velocity; Laserpuncture; Light stimulation; Middle cerebral artery (MCA); Posterior cerebral artery (PCA); Transcranial Doppler sonography (TCD)
Light Therapy (LLLT) alters gene expression after acute spinal cord injury
K.R. Byrnes 1, R.W. Waynant 2, I.K. Ilev 2, B. Johnson 1, Pollard H. 1, Srivastava M. 1, Eidelman O. 1, Huang, W. 1, J.J. Anders1
1. Department of Anatomy, Physiology and Genetics, Uniformed Services University, Bethesda, MD, USA; 2. Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD, USA
Secondary injury in the spinal cord, which results in axonal degeneration, scar and cavity formation and cell death, occurs around the site of the initial trauma and is a primary cause for the lack of axonal regeneration observed after spinal cord injury (SCI). The immune response after SCI is under investigation as a potential mediator of secondary injury. Treatment of SCI with 810 nm light suppresses the immune response and improves axonal regeneration.
We hypothesize that these beneficial effects observed in the injured spinal cord are accompanied by alterations in gene expression within the spinal cord, particularly of those genes involved in secondary injury and the immune response. To test this hypothesis, a dorsal hemisection at vertebral level T9 was performed. The injured spinal cord from rat was then exposed to laser light (810nm, 150mW, 2,997 seconds, 0.3cm2 spot area, 1589 J/cm2) and spinal cord samples, including the injury site, were harvested at 6 and 48 hours and 4 days post-injury. Total RNA was extracted and purified from the lesioned spinal cord and cDNA copies were either labeled with [32P] for microarray analysis or amplified and analyzed with a polymerase chain reaction (PCR).
Microarray results revealed a suppression of genes involved in the immune response and excitotoxic cell death at 6 hours post-injury, as well as cell proliferation and scar formation at 48 hours post-injury in the light treated group. Analysis of the PCR products revealed that light treatment resulted in a significant suppression of expression of genes that normally peak between 6 and 24 hours post-injury, including the pro-inflammatory cytokine interleukin 6 (IL6), the chemokine monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS; p<0.05). Genes expressed earlier than 6 hours post-injury, such as IL1b, tumor necrosis factor a (TNFa) and macrophage inflammatory protein 1a (MIP-1a) were not affected by light treatment.
Although the precise role some of these genes play in axonal regeneration after spinal cord injury is currently unclear, these data demonstrate that light therapy has an anti-inflammatory effect on the injured spinal cord, and may reduce secondary injury, thus providing a possible mechanism by which light therapy may result in axonal regeneration.
Laser Therapy.1997; 9 (4): 151.
An innovative approach to induce regeneration and the repair of spinal cord injury.
Rochkind S, Shahar A. Nevo Z.
An Israeli research group has investigated an innovative method of repairing injured spinal cords. In a rat model the spinal cords were transected in 31 animals (between T7/T8). In vitro constructed composite implants were used in the transected area. These implants contained embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into the injured area. The whole lesion area was covered with a thin coagulated fibrin-based membrane. Control animals underwent the same laminectomy but did not receive any implant. In all animals the wound was closed normally. Laser therapy was started immediately after surgery. It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily. One group received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that received laser (A) showed different degrees of active movements in one or both legs, compared to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving no implant and no laser (C), 1 out of 7 showed some motor movements in one leg. Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months, and on one side in one animal in group B. Axon sprouting was observed as soon as three days post surgery, in group A only.
Laser Therapy.1997; 9 (4): 151
New hope for patients with spinal cord injuries.
Rochkind S, Shahar A. Nevo Z.
An Israeli research group has investigated an innovative method of repairing injured spinal cords. In a rat model the spinal cords were transected in 31 animals (between T7/T8). In vitro constructed composite implants were used in the transected area. These implants contained embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into the injured area. The whole lesion area was covered with a thin coagulated fibrin-based membrane. Control animals underwent the same laminectomy but did not receive any implant. In all animals the wound was closed normally. Laser therapy was started immediately after surgery. It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily. One group received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that received laser (A) showed different degrees of active movements in one or both legs, compared to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving no implant and no laser (C), 1 out of 7 showed some motor movements in one leg. Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months, and on one side in one animal in group B. Axon sprouting was observed as soon as three days post surgery, in group A only.
Spine (Phila Pa 1976). 1990 Jan;15(1):6-10.
Spinal cord response to laser treatment of injured peripheral nerve.
Rochkind S, Vogler I, Barr-Nea L.
Department of Neurosurgery, Ichilov Hospital, Tel-Aviv Medical Center, Israel.
The authors describe the changes occurring in the spinal cord of rats subjected to crush injury of the sciatic nerve followed by low-power laser irradiation of the injured nerve. Such laser treatment of the crushed peripheral nerve has been found to mitigate the degenerative changes in the corresponding neurons of the spinal cord and induce proliferation of neuroglia both in astrocytes and oligodendrocytes. This suggests a higher metabolism in neurons and a better ability for myelin production under the influence of laser treatment.
Lasers Surg Med. 1989;9(2):174-82.
Systemic effects of low-power laser irradiation on the peripheral and central nervous system, cutaneous wounds, and burns.
Rochkind S, Rousso M, Nissan M, Villarreal M, Barr-Nea L, Rees DG.
Department of Neurosurgery, Tel Aviv Medical Center, Ichilov Hospital, Israel.
In this paper, we direct attention to the systemic effect of low-power helium-neon (HeNe) laser irradiation on the recovery of the injured peripheral and central nervous system, as well as healing of cutaneous wounds and burns. Laser irradiation on only the right side in bilaterally inflicted cutaneous wounds enhanced recovery in both sides compared to the nonirradiated control group (P less than .01). Similar results were obtained in bilateral burns: irradiating one of the burned sites also caused accelerated healing in the nonirradiated site (P less than .01). However, in the nonirradiated control group, all rats suffered advanced necrosis of the feet and bilateral gangrene. Low-power HeNe laser irradiation applied to a crushed injured sciatic nerve in the right leg in a bilaterally inflicted crush injury, significantly increased the compound action potential in the left nonirradiated leg as well. The statistical analysis shows a highly significant difference between the laser-treated group and the control nonirradiated group (P less than .001). Finally, the systemic effect was found in the spinal cord segments corresponding to the crushed sciatic nerves. The bilateral retrograde degeneration of the motor neurons of the spinal cord expected after the bilateral crush injury of the peripheral nerves was greatly reduced in the laser treated group. The systemic effects reported here are relevant in terms of the clinical application of low-power laser irradiation as well as for basic research into the possible mechanisms involved.
Health Phys. 1989 May;56(5):687-90.
New biological phenomena associated with laser radiation.
Belkin M, Schwartz M.
Goldschleger Eye Research Institute, Tel-Aviv University, Sackler School of Medicine, Tel-Hashomer, Israel.
Low-energy laser irradiation produces significant bioeffects. These effects are manifested in biochemical, physiological and proliferative phenomena in various enzymes, cells, tissues, organs and organisms. Examples are given of the effect of He-Ne laser irradiation in preventing the post-traumatic degeneration of peripheral nerves and the postponement of degeneration of the central nervous system. The damage produced by similar radiant exposures to the corneal epithelium and endothelium is also described. It is suggested that the mechanism of laser/tissue interaction at these low levels of radiant exposure is photochemical in nature, explaining most of the characteristics of these effects. These low-energy laser bioeffects are of importance on a basic scientific level, from a laser safety aspect and as a medical therapeutic modality.