J Exp Neurosci. 2016 Feb 1;10:1-19. doi: 10.4137/JEN.S33444. eCollection 2016.
Neuroprotective Effects Against POCD by Photobiomodulation: Evidence from Assembly/Disassembly of the Cytoskeleton.
Liebert AD1, Chow RT2, Bicknell BT3, Varigos E4.
1University of Sydney, Sydney, NSW, Australia.
2Brain and Mind Institute, University of Sydney, Sydney, NSW, Australia.
3Australian Catholic University, Sydney, NSW, Australia.
4Olympic Park Clinic, Melbourne, VIC, Australia.
Postoperative cognitive dysfunction (POCD) is a decline in memory following anaesthesia and surgery in elderly patients. While often reversible, it consumes medical resources, compromises patient well-being, and possibly accelerates progression into Alzheimer’s disease. Anesthetics have been implicated in POCD, as has neuroinflammation, as indicated by cytokine inflammatory markers. Photobiomodulation (PBM) is an effective treatment for a number of conditions, including inflammation. PBM also has a direct effect on microtubule disassembly in neurons with the formation of small, reversible varicosities, which cause neural blockade and alleviation of pain symptoms. This mimics endogenously formed varicosities that are neuroprotective against damage, toxins, and the formation of larger, destructive varicosities and focal swellings. It is proposed that PBM may be effective as a preconditioning treatment against POCD; similar to the PBM treatment, protective and abscopal effects that have been demonstrated in experimental models of macular degeneration, neurological, and cardiac conditions.
Lasers Med Sci. 2015 May;30(4):1395-406. doi: 10.1007/s10103-014-1531-6. Epub 2014 Feb 12.
Benefits of laser phototherapy on nerve repair.
de Oliveira RF1, de Andrade Salgado DM, Trevelin LT, Lopes RM, da Cunha SR, Aranha AC, de Paula Eduardo C, de Freitas PM.
1Department of Restorative Dentistry, Special Laboratory of Lasers in Dentistry (LELO), School of Dentistry, University of São Paulo, São Paulo, Brazil.
Post-traumatic nerve repair represents a major challenge to health sciences. Although there have been great advances in the last few years, it is still necessary to find methods that can effectively enhance nerve regeneration. Laser therapy has been widely investigated as a potential method for nerve repair. Therefore, in this article, a review of the existing literature was undertaken with regard to the effects of low-power laser irradiation on the regeneration of traumatically/surgically injured nerves. The articles were selected using either electronic search engines or manual tracing of the references cited in key papers. In electronic searches, we used the key words as “paresthesia”, “laser therapy”, “low-power laser and nerve repair”, and “laser therapy and nerve repair”, considering case reports and clinical studies. According to the findings of the literature, laser therapy accelerates and improves the regeneration of the affected nerve tissues, but there are many conflicting results about laser therapy. This can be attributed to several variables such as wavelength, radiation dose, and type of radiation. All the early in vivo studies assessed in this research were effective in restoring sensitivity. Although these results indicate a potential benefit of the use of lasers on nerve repair, further double-blind controlled clinical trials should be conducted in order to standardize protocols for clinical application.
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).
These patterns are illustrated further in Figure 3 for both Balb/c (Figure 3A,C,E,G) and C57BL/6 (Figure 3B,D,F,H) in each of the Saline (Figure 3A,B), Saline-NIr (Figure 3C,D), MPTP (Figure 3E,F) and MPTP-NIr (Figure 3G,H) groups. Similar patterns of immunostaining were seen in both strains. Although there were fewer TH+ somata in the MPTP group (Figure 3E,F), those remaining were similar in overall appearance to those seen in the Saline (Figure 3A,B), Saline-NIr (Figure 3C,D) and MPTP-NIr (Figure 3G,H) groups. They had round or oval-shaped somata with one to two labelled dendrites.
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.
- Blandini F, Nappi G, Tassorelli C, Martignoni E. Functional changes of the basal ganglia circuitry in Parkinson’s disease. Prog Neurobiol. 2000;14:63–88. [PubMed]
- Bergman H, Deuschl G. Pathophysiology of Parkinson’s disease: from clinical neurology to basic neuroscience and back. Mov Disord. 2002;14:S28–S40. doi: 10.1002/mds.10140.[PubMed] [Cross Ref]
- Rinne JO. Nigral degeneration in Parkinson’s disease. Mov Disord 8 Suppl. 1993;14:S31–S35.[PubMed]
- McRitchie DA, Cartwright HR, Halliday GM. Specific A10 dopaminergic nuclei in the midbrain degenerate in Parkinson’s disease. Exp Neurol. 1997;14:202–213. doi: 10.1006/exnr.1997.6418.[PubMed] [Cross Ref]
- Langston JW. The etiology of Parkinson’s disease with emphasis on the MPTP story. Neurology.1996;14:S153–S160. doi: 10.1212/WNL.47.6_Suppl_3.153S. [PubMed] [Cross Ref]
- Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L, Riess O. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat Genet. 1998;14:106–118. doi: 10.1038/ng0298-106. [PubMed] [Cross Ref]
- LeWitt PA. Neuroprotection for Parkinson’s disease. J Neural Transm Suppl. 2006;14:113–122. doi: 10.1007/978-3-211-33328-0_13. [PubMed] [Cross Ref]
- Ma J, Shaw VE, Mitrofanis J. Does melatonin help save dopaminergic cells in MPTP-treated mice? Parkinsonism Relat Disord. 2009;14:307–314. doi: 10.1016/j.parkreldis.2008.07.008.[PubMed] [Cross Ref]
- Liang HL, Whelan HT, Eells JT, Wong-Riley MT. Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity. Neurosci. 2008;14:963–974. doi: 10.1016/j.neuroscience.2008.03.042.[PMC free article] [PubMed] [Cross Ref]
- Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT. Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone – and MPP+– induced neurotoxicity. Brain Res. 2008;14:167–173. [PMC free article] [PubMed]
- Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment. J Comp Neurol. 2010;14:25–40. [PubMed]
- Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson’s disease. Parkinsonism Relat Disord. 2012;14:469–476. doi: 10.1016/j.parkreldis.2012.01.005. [PubMed] [Cross Ref]
- Whelan HT, DeSmet KD, Buchmann E, Henry M, Wong-Riley M, Eells JT, Verhoeve J. Harnessing the cell’s own ability to repair and prevent neurodegenerative disease. SPIE Newsroom. 2008. pp. 1–3. [PMC free article] [PubMed] [Cross Ref]
- Desmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM, Buchmann EV, Connelly MP, Dovi JV, Liang HL, Henshel DS, Yeager RL, Millsap DS, Lim J, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT. Clinical and experimental applications of NIR-LED photobiomodulation. Photomed Laser Surg. 2006;14:121–128. doi: 10.1089/pho.2006.24.121.[PubMed] [Cross Ref]
- Hamblin MR, Demidova TN. In: Mechanisms for low-light therapy. Hamblin MR, Waynart RW, Anders J, editor. San Jose, CA, USA: Proc SPIE; 2006. Mechanisms of low level light therapy; p. 6140.
- Shaw VE, Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Patterns of cell activity in the subthalamic region associated with the neuroprotective action of near-infrared light treatment in MPTP-treated mice. Parkinson’s disease. 2012.[PMC free article] [PubMed]
- Sedelis M, Hofele K, Auburger GW, Morgan S, Huston JP, Schwarting RKW. MPTP Susceptibility in the Mouse: Behavioural, Neurochemical, and Histological Analysis of Gender and Strain Differences. Behav Gen. 2000;14:171–182. doi: 10.1023/A:1001958023096.[PubMed] [Cross Ref]
- Ito T, Suzuki K, Uchida K, Nakayama H. Different susceptibility to 1-methyl-4-phenylpyridium (MPP+)-induced nigro-striatal dopaminergic cell loss between C57BL/6 and BALB/c mice is not related to the difference of monoamine oxidase-B (MAO-B) Exp Toxic Path. 2011. EPub.[PubMed]
- Riachi NJ, Behmand RA, Harik SI. Correlation of MPTP neurotoxicity in vivo with oxidation of MPTP by the brain and blood–brain barrier in vitro in five rat strains. Brain Res. 1991;14:19–24. doi: 10.1016/0006-8993(91)90854-O. [PubMed] [Cross Ref]
- Schober A. Classic toxin-induced animal models of Parkinson’s disease: 6OHDA and MPTP. Cell Tissue Res. 2004;14:215–24. doi: 10.1007/s00441-004-0938-y. [PubMed] [Cross Ref]
- Bové J, Perier C. Neurotoxin-based models of Parkinson’s disease. Neurosci. 2012;14:51–76.[PubMed]
- Piallat B, Benazzouz A, Benabid AL. Subthalamic nucleus lesion in rats prevents dopaminergic nigral neuron degeneration after striatal 6-OHDA injection: behavioural and immunohistochemical studies. Eur J Neurosci. 1996;14:1408–1414. doi: 10.1111/j.1460-9568.1996.tb01603.x. [PubMed] [Cross Ref]
- Wallace BA, Ashkan K, Heise CE, Foote KD, Torres N, Mitrofanis J, Benabid AL. Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys. Brain. 2007;14:2129–2145. doi: 10.1093/brain/awm137. [PubMed][Cross Ref]
- Luquin N, Mitrofanis J. Does the cerebral cortex exacerbate dopamineric cell death in the substantia nigra of 6OHDA-lesioned rats? Parkinson Related Disord. 2008;14:213–223. doi: 10.1016/j.parkreldis.2007.08.010. [PubMed] [Cross Ref]
- Björklund A, Rosenblad C, Winkler C, Kirik D. Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of Parkinson’s disease. Neurobiol Dis. 1997;14:186–200. doi: 10.1006/nbdi.1997.0151. [PubMed] [Cross Ref]
- Huot P, Lévesque M, Parent A. The fate of striatal dopaminergic neurons in Parkinson’s disease and Huntington’s chorea. Brain. 2007;14:222–32. [PubMed]
- Paxinos G, Franklin BJ. The mouse brain in stereotaxic coordinates. 2. San Diego, CA, USA: Academic Press California USA; 2001.
- Bezard E, Dovero S, Bioulac B, Gross C. Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice. Exp Neurol. 1997;14:288–292. doi: 10.1006/exnr.1997.6648. [PubMed] [Cross Ref]
- Goldberg NR, Haack AK, Lim NS, Janson OK, Meshul CK. Dopaminergic and behavioural correlates of progressive lesioning of the nigrostriatal pathway with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurosci. 2011;14:256–271. [PubMed]
- Meredith P, Powell BJ, Riesz J, Nighswander-Rempel S, Pederson MR, Moore E. Towards structure–property-function relationships for eumelanin. Soft Matter. 2006;14:37.
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.
Low-level laser therapy improves repair following complete resection of the sciatic nerve in rats.
Department of Bioscience, Federal University of São Paulo (UNIFESP), Avenida Ana Costa 95, CEP 04021-001, Santos, SP, Brazil. firstname.lastname@example.org
The aim of this study is to analyze the effects of low-level laser therapy (LLLT) on the regeneration of the sciatic nerve in rats following a complete nerve resection. Male Wistar rats were divided into a control injury group, injury groups irradiated with a 660-nm laser at 10 or 50 J/cm(2), and injury groups irradiated with an 808-nm laser at 10 or 50 J/cm(2). Treatment began 24 h following nerve resection and continued for 15 days. Using the sciatic functional index (SFI), we show that the injured animals treated with 660 nm at 10 and 50 J/cm(2) had better SFI values compared with the control injury and the 808-nm groups. Animals irradiated with the 808-nm laser at 50 J/cm(2) show higher values for fiber density than do control animals. In addition, axon and fiber diameters were larger in animals irradiated with 660 nm at 50 J/cm(2) compared to the control group. These findings indicate that 660-nm LLLT is able to provide functional gait recovery and leads to increases in fiber diameter following sciatic nerve resection.
Functional and morphometric differences between the early and delayed use of phototherapy in crushed median nerves of rats.
Department of Physiotherapy, Federal University of Jequitinhonha and Mucuri Valleys, Campus JK – Rodovia MGT 367 – Km 583, nº 5000 – Alto da Jacuba CEP: 39100-000, Diamantina, MG, Brazil, email@example.com.
This study evaluated the functional and quantitative differences between the early and delayed use of phototherapy in crushed median nerves. After a crush injury, low-level laser therapy (GaAs) was applied transcutaneously at the injury site, 3 min daily, with a frequency of five treatments per week for 2 weeks. In the early group, the first laser treatment started immediately after surgery, and in the delayed group, after 7 days. The grasping test was used for functional evaluation of the median nerve, before, 10, and 21 days after surgery, when the rats were killed. Three segments of the median nerve were analyzed histomorphometrically by light microscopy and computer analysis. The following features were observed: myelinated fiber and axon diameters, myelin sheath area, g-ratio, density and number of myelinated fibers, and area and number of capillaries. In the proximal segment (site of crush), the nerves of animals submitted to early and delayed treatment showed myelinated fiber diameter and myelin sheath area significantly larger compared to the untreated group. In the distal segment, the myelin sheath area was significantly smaller in the untreated animals compared to the delayed group. The untreated, early, and delayed groups presented a 50, 57, and 81% degree of functional recovery, respectively, at 21 days after injury, with a significant difference between the untreated and delayed groups. The results suggest that the nerves irradiated with low-power laser exhibit myelinated fibers of greater diameter and a better recovery of function.
Lasers Med Sci. 2011 May 20. [Epub ahead of print]
The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts-an in vitro study.
Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran, firstname.lastname@example.org.
Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm(2) for BMSCs being induced to neurons, and 2 or 4 J/cm(2) for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm(2) in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm(2) dramatically facilitated the differentiation of BMSCs into neurons (p?<?0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p?<?0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2-6 J/cm(2), and at the same time increases BMSCs proliferation (except for 6 J/cm(2)). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.
Acta Cir Bras. 2011 Feb;26(1):12-18.
Histological analysis of low-intensity laser therapy effects in peripheral nerve regeneration in Wistar rats.
Câmara CN, Brito MV, Silveira EL, Silva DS, Simões VR, Pontes RW.
Department of Physiotherapy, UNAMA, Belem, PA, Brazil.
Purpose: Analyze the influence of low-intensity laser therapy in the sciatic nerve regeneration of rats submitted to controlled crush through histological analysis. Methods: Were used 20 Wistar rats, to analyze the influence of low-intensity laser therapy in the sciatic nerve regeneration, where the injury of the type axonotmesis was induced by a haemostatic clamp Crile (2nd level of the rack). The animals were randomly distributed in 2 groups. Control group (CG n = 10) and Laser group (LG n = 10). These were subdivided in 2 subgroups each, according to the euthanasia period: (CG14 _ n = 5 and CG21 _ n = 5) and (LG14 _ n = 5 and LG21 _ n = 5). At the end of treatment, the samples were removed and prepared for histological analysis, where were analyzed and quantified the following findings: Schwann cells, myelinic axons with large diameter and neurons. Results: In the groups submitted to low-intensity laser therapy, were observed an increase in the number of all analyzed aspects with significance level. Conclusion: The irradiation with low intensity laser (904nm) influenced positively the regeneration of the sciatic nerve in Wistar rats after being injured by crush (axonotmesis), becoming the nerve recovery more rapid and efficient.
Lasers Med Sci. 2010 May;25(3):423-30. Epub 2010 Feb 6.
Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion.
Barbosa RI, Marcolino AM, de Jesus Guirro RR, Mazzer N, Barbieri CH, de Cássia Registro Fonseca M.
Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, Medical School of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto 14049-900, SP, Brazil. email@example.com
Peripheral nerves are structures that, when damaged, can result in significant motor and sensory disabilities. Several studies have used therapeutic resources with the aim of promoting early nerve regeneration, such as the use of low-power laser. However, this laser therapy does not represent a consensus regarding the methodology, thus yielding controversial conclusions. The objective of our study was to investigate, by functional evaluation, the comparative effects of low-power laser (660 nm and 830 nm) on sciatic nerve regeneration following crushing injuries. Twenty-seven Wistar rats subjected to sciatic nerve injury were divided into three groups: group sham, consisting of rats undergoing simulated irradiation; a group consisting of rats subjected to gallium-aluminum-arsenide (GaAlAs) laser at 660 nm (10 J/cm(2), 30 mW and 0.06 cm(2) beam), and another one consisting of rats subjected to GaAlAs laser at 830 nm (10 J/cm(2), 30 mW and 0.116 cm(2)). Laser was applied to the lesion for 21 days. A sciatic functional index (SFI) was used for functional evaluation prior to surgery and on days 7, 14, and 21 after surgery. Differences in SFI were found between group 660 nm and the other ones at the 14th day. One can observe that laser application at 660 nm with the parameters and methods utilised was effective in promoting early functional recovery, as indicated by the SFI, over the period evaluated.
BMC Complement Altern Med. 2009 Apr 15;9:8.
Low infra red laser light irradiation on cultured neural cells: effects on mitochondria and cell viability after oxidative stress.
Giuliani A, Lorenzini L, Gallamini M, Massella A, Giardino L, Calzà L.
BioPharmaNet-DIMORFIPA, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia, Bologna, Italy. firstname.lastname@example.org
BACKGROUND: Considerable interest has been aroused in recent years by the well-known notion that biological systems are sensitive to visible light. With clinical applications of visible radiation in the far-red to near-infrared region of the spectrum in mind, we explored the effect of coherent red light irradiation with extremely low energy transfer on a neural cell line derived from rat pheochromocytoma. We focused on the effect of pulsed light laser irradiation vis-à-vis two distinct biological effects: neurite elongation under NGF stimulus on laminin-collagen substrate and cell viability during oxidative stress. METHODS: We used a 670 nm laser, with extremely low peak power output (3 mW/cm2) and at an extremely low dose (0.45 mJ/cm2). Neurite elongation was measured over three days in culture. The effect of coherent red light irradiation on cell reaction to oxidative stress was evaluated through live-recording of mitochondria membrane potential (MMP) using JC1 vital dye and laser-confocal microscopy, in the absence (photo bleaching) and in the presence (oxidative stress) of H2O2, and by means of the MTT cell viability assay. RESULTS: We found that laser irradiation stimulates NGF-induced neurite elongation on a laminin-collagen coated substrate and protects PC12 cells against oxidative stress. CONCLUSION: These data suggest that red light radiation protects the viability of cell culture in case of oxidative stress, as indicated by MMP measurement and MTT assay. It also stimulates neurite outgrowth, and this effect could also have positive implications for axonal protection.
Int Rev Neurobiol. 2009;87:445-64.
Chapter 25: Phototherapy in peripheral nerve injury: effects on muscle preservation and nerve regeneration.
Rochkind S, Geuna S, Shainberg A.
Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel.
Posttraumatic nerve repair and prevention of muscle atrophy represent a major challenge of restorative medicine. Considerable interest exists in the potential therapeutic value of laser phototherapy for restoring or temporarily preventing denervated muscle atrophy as well as enhancing regeneration of severely injured peripheral nerves. Low-power laser irradiation (laser phototherapy) was applied for treatment of rat denervated muscle in order to estimate biochemical transformation on cellular and tissue levels, as well as on rat sciatic nerve model after crush injury, direct or side-to-end anastomosis, and neurotube reconstruction. Nerve cells’ growth and axonal sprouting were investigated in embryonic rat brain cultures. The animal outcome allowed clinical double-blind, placebo-controlled randomized study that measured the effectiveness of 780-nm laser phototherapy on patients suffering from incomplete peripheral nerve injuries for 6 months up to several years. In denervated muscles, animal study suggests that the function of denervated muscles can be partially preserved by temporary prevention of denervation-induced biochemical changes. The function of denervated muscles can be restored, not completely but to a very substantial degree, by laser treatment initiated at the earliest possible stage post injury. In peripheral nerve injury, laser phototherapy has an immediate protective effect. It maintains functional activity of the injured nerve for a long period, decreases scar tissue formation at the injury site, decreases degeneration in corresponding motor neurons of the spinal cord, and significantly increases axonal growth and myelinization. In cell cultures, laser irradiation accelerates migration, nerve cell growth, and fiber sprouting. In a pilot, clinical, double-blind, placebo-controlled randomized study in patients with incomplete long-term peripheral nerve injury, 780-nm laser irradiation can progressively improve peripheral nerve function, which leads to significant functional recovery. A 780-nm laser phototherapy temporarily preserves the function of a denervated muscle, and accelerates and enhances axonal growth and regeneration after peripheral nerve injury or reconstructive procedures. Laser activation of nerve cells, their growth, and axonal sprouting can be considered as potential treatment for neural injury. Animal and clinical studies show the promoting action of phototherapy on peripheral nerve regeneration, which makes it possible to suggest that the time for broader clinical trials has come.
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.
Lasers Surg Med. 2009 Apr;41(4):277-81.
Increase in 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.
Neurosurg Focus. 2009;26(2):E8
Phototherapy in peripheral nerve regeneration: From basic science to clinical study.
Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel.
Object This review summarizes the continuous study of low-power laser radiation treatment of a severely injured peripheral nerve. Laser phototherapy was applied as a supportive factor for accelerating and enhancing axonal growth and regeneration after injury or a reconstructive peripheral nerve procedure. In nerve cell cultures, laser phototherapy was used to stimulate activation of nerve cells. Methods Low-power laser radiation was used for treatment of peripheral nerve injury using a rat sciatic nerve model after crush injury, neurorrhaphy, or neurotube reconstruction. Nerve cell growth and axonal sprouting were investigated using laser phototherapy on embryonic rat brain cultures. The outcome in animal studies facilitated a clinical double-blind, placebo-controlled, randomized study that measured the effectiveness of 780-nm laser phototherapy on patients suffering from incomplete peripheral nerve injuries for 6 months to several years. Results Animal studies showed that laser phototherapy has an immediate protective effect, maintains functional activity of the injured nerve, decreases scar tissue formation at the injury site, decreases degeneration in corresponding motor neurons of the spinal cord, and significantly increases axonal growth and myelinization. In cell cultures, laser irradiation accelerates migration, nerve cell growth, and fiber sprouting. A pilot clinical double-blind, placebocontrolled, randomized study showed that in patients with incomplete long-term peripheral nerve injury, 780-nm laser radiation can progressively improve peripheral nerve function, which leads to significant functional recovery. Conclusions Using 780-nm laser phototherapy accelerates and enhances axonal growth and regeneration after injury or a reconstructive peripheral nerve procedure. Laser activation of nerve cells, their growth, and axonal sprouting can be considered as potential treatment of neuronal injury. Animal and clinical studies show the promoting action of phototherapy on peripheral nerve regeneration, making it possible to suggest that the time for broader clinical trials has arrived.
Gen Dent. 2008 Nov-Dec;56(7):629-34.
Low level lasers in dentistry.
Ross G, Ross A.
Laser Light Canada.
Low level laser therapy (LLLT) uses light energy, in the form of adenosine triphosphate (ATP), to elicit biological responses in the body. The increased cellular energy and changes in the cell membrane permeability result in pain relief, wound healing, muscle relaxation, immune system modulation, and nerve regeneration. This article investigates the clinical effects of LLLT and explains how it can be applied in the dental field.
Photomed Laser Surg. 2007 Oct;25(5):436-42
Laser phototherapy (780 nm), a new modality in treatment of long-term incomplete peripheral nerve injury: a randomized double-blind placebo-controlled study.
Rochkind S, Drory V, Alon M, Nissan M, Ouaknine GE.
Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel. firstname.lastname@example.org
OBJECTIVE: The authors conducted this pilot study to prospectively investigate the effectiveness of low-power laser irradiation (780 nm) in the treatment of patients suffering from incomplete peripheral nerve and brachial plexus injuries for 6 months up to several years. BACKGROUND DATA: Injury of a major nerve trunk frequently results in considerable disability associated with loss of sensory and motor functions. Spontaneous recovery of long-term severe incomplete peripheral nerve injury is often unsatisfactory. METHODS: A randomized, double-blind, placebo-controlled trial was performed on 18 patients who were randomly assigned placebo (non-active light: diffused LED lamp) or low-power laser irradiation (wavelength, 780 nm; power, 250 mW). Twenty-one consecutive daily sessions of laser or placebo irradiation were applied transcutaneously for 3 h to the injured peripheral nerve (energy density, 450 J/mm(2)) and for 2 h to the corresponding segments of the spinal cord (energy density, 300 J/mm(2)). Clinical and electrophysiological assessments were done at baseline, at the end of the 21 days of treatment, and 3 and 6 months thereafter. RESULTS: The laser-irradiated and placebo groups were in clinically similar conditions at baseline. The analysis of motor function during the 6-month follow-up period compared to baseline showed statistically significant improvement (p = 0.0001) in the laser-treated group compared to the placebo group. No statistically significant difference was found in sensory function. Electrophysiological analysis also showed statistically significant improvement in recruitment of voluntary muscle activity in the laser-irradiated group (p = 0.006), compared to the placebo group. CONCLUSION: This pilot study suggests that in patients with long-term peripheral nerve injury noninvasive 780-nm laser phototherapy can progressively improve nerve function, which leads to significant functional recovery.
Photomed Laser Surg. 2007 Jun;25(3):137-43
Efficacy of 780-nm laser phototherapy on peripheral nerve regeneration after neurotube reconstruction procedure (double-blind randomized study).
Rochkind S, Leider-Trejo L, Nissan M, Shamir MH, Kharenko O, Alon M.
Division of Peripheral Nerve Reconstruction, Tel-Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel. email@example.com
OBJECTIVE: This pilot double-blind randomized study evaluated the efficacy of 780-nm laser phototherapy on the acceleration of axonal growth and regeneration after peripheral nerve reconstruction by polyglycolic acid (PGA) neurotube. BACKGROUND DATA: The use of a guiding tube for the reconstruction of segmental loss of injured peripheral nerve has some advantages over the regular nerve grafting procedure. Experimental studies have shown that laser phototherapy is effective in influencing nerve regeneration. METHODS: The right sciatic nerve was transected, and a 0.5-cm nerve segment was removed in 20 rats. A neurotube was placed between the proximal and the distal parts of the nerve for reconnection of nerve defect. Ten of 20 rats received post-operative, transcutaneous, 200-mW, 780-nm laser irradiation for 14 consecutive days to the corresponding segments of the spinal cord (15 min) and to the reconstructed nerve (15 min). RESULTS: At 3 months after surgery, positive somato-sensory evoked responses were found in 70% of the irradiated rats (p = 0.015), compared to 30% of the non-irradiated rats. The Sciatic Functional Index in the irradiated group was higher than in the non-irradiated group (p < 0.05). Morphologically, the nerves were completely reconnected in both groups, but the laser-treated group showed an increased total number of myelinated axons. CONCLUSION: The results of this study suggest that postoperative 780-nm laser phototherapy enhances the regenerative process of the peripheral nerve after reconnection of the nerve defect using a PGA neurotube.
Photomed Laser Surg. 2007 Apr;25(2):107-11
Promotion of regenerative processes in injured peripheral nerve induced by low-level laser therapy.
Mohammed IF, Al-Mustawfi N, Kaka LN.
Department of Anatomy, Al-Kindy Medical College, Baghdad University, Baghdad, Iraq. firstname.lastname@example.org
OBJECTIVE: This study aimed to assess in vitro the influence of low-level laser therapy (LLLT) on the regenerative processes of a peripheral nerve after trauma. BACKGROUND DATA: In peripheral nerve injury initiated after severing due to accident or by a surgeon during operation, photomodulation by light in the red to near-infrared range (530-1000 nm) using low-energy lasers has been shown to accelerate nerve regeneration. METHOD: Twenty-four New Zealand adult male rabbits were randomly assigned to two equal groups (control and laser-treated). General anesthesia was administered intramuscularly, and exploration of the peroneal nerve was done in the lateral aspect of the left leg. Complete section of the nerve was performed, which was followed by suturing of the neural sheath (epineurium). Irradiation was carried out directly after the operation and for 10 consecutive days. The laser used was diode with wavelength of 901 nm (impulsive) and power of 10 mW; it was a square-shaped window type (16 cm(2)), and its energy was applied by direct contact of the instrument’s window to the site of the operation. Three rabbits from each group were sacrificed at the end of weeks 2, 4, 6, and 8, and specimens were collected from the site of nerve suturing and sent for histopathological examination. RESULTS: Two important factors were examined via histopathology: diameter of the nerve fibers and individual internodal length. Compared to the control group, significant variations in regeneration were observed, including thicker nerve fibers, more regular myelin layers, clearer nodes of Ranvier with absence of short nodes in the treated group. Variations between the two groups for diameter were significant for the 2(nd) week (p < 0.05), highly significant for the 4(th) and 6(th) weeks, respectively (p < 0.01), and very highly significant for the 8(th) week (p < 0.001). Variations between the two groups for internodal length were highly significant for the 2(nd) and 4(th) weeks (p < 0.01), and very highly significant for the 6(th) and 8(th) weeks (p < 0.001). CONCLUSION: This experiment affirms the beneficial effect of LLLT on nerve regeneration, since LLLT produced a significant amount of structural and cellular change. The results of the present study suggest that laser therapy may be a viable approach for nerve regeneration, which may be of clinical relevance in scheduled surgery or microsurgery.
Neurol Res. 2004 Mar;26(2):161-6
Further development of reconstructivev and cell tissue-engineering technology for treatment of complete peripheral nerve injury in rats.
Rochkind S, Astachov L, el-Ani D, Hayon T, Graif M, Barsky L, Alon M, Odvak I, Nevo Z, Shahar A.
Department of Neurosurgery, Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel. email@example.com
In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve. In CONCLUSION: (1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.
Neurol Res. 2004 Mar;26(2):233-9
Phototherapy promotes regeneration and functional recovery of injured peripheral nerve.
Anders JJ, Geuna S, Rochkind S.
Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20854, USA. firstname.lastname@example.org
Numerous attempts have been made to enhance and/or accelerate the recovery of injured peripheral nerves. One of the methods studied is the use of phototherapy (low power laser or light irradiation) to enhance recovery of the injured peripheral nerve. A critical analysis of the literature on the employment of phototherapy for the enhancement of the regeneration process of the rat facial and sciatic nerve (after crush injury or transection followed by surgical reconstruction) is provided, together with the description of some of the most suitable basic biological mechanisms through which laser radiation exerts its action on peripheral nerve regeneration.
In Vivo. 2004 Jul-Aug;18(4):489-95.
Effect of Ga-as laser on the regeneration of injured sciatic nerves in the rat.
Bae CS, Lim SC, Kim KY, Song CH, Pak S, Kim SG, Jang CH.
College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University, Gwangju, Korea.
Laser irradiation is one of the therapeutic methods for the recovery of degenerated peripheral nerves. The aim of the present study was to determine if low-power laser treatment stimulates the regeneration process of damaged nerves. A standardized crush to the sciatic nerve was applied to cause extensive axonal degeneration. After this procedure, low-power infrared laser irradiation was administered transcutaneously to the injured sciatic nerve, 3 minutes daily to each of four treatment groups for 1, 3, 5 and 7 weeks, respectively. A nerve conduction study was done, and a morphological assessment was performed using both light and electron microscopy. With trauma of the nerve, both amplitude of compound motor action potential and nerve conduction velocity decreased significantly compared to the pre-trauma state. Morphologically, the numbers of myelinated axons and degenerated axons were decreased and increased, respectively, compared with the control. Typical aspects were of onion skin-type lamellation, fragmentation, edematous swelling and rarefaction in the myelin sheath. All these parameters recovered almost to the level of the pre-trauma state with laser irradiation, in direct proportion to the time spent for treatment. These results suggest that low-power infrared laser irradiation can relieve the mechanical damage of sciatic nerves and stimulate the regeneration of peripheral nerves.
LASER THERAPY – A NEW MODALITY IN THE TREATMENT OF PERIPHERAL NERVE INJURIES
(Twenty-five years experience from basic science to clinical studies)
S. Rochkind, MD Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Tel-Aviv University, Tel Aviv, Israel, E-mail: email@example.com
Since our first publication (Rochkind 1978), we have been studying and testing low power laser irradiation as a means to treat peripheral nerves, using both in vitro and in vivo methods. We have reached the clinical stage and are treating a variety of peripheral nerve injuries. This study is a review of my personal experience over the last twenty-five years in the use of laser therapy in treating these conditions.
I. Influence of Low Power Laser Irradiation on Nerve Cells
A study was done using direct 632.8nm HeNe laser irradiation to determine the effect of focused laser beams on aggregates of rat fetal brain cells and rat adult brain. The direct HeNe laser irradiation 3.6J/cm2 caused a significant amount of sprouting of cellular processes outgrowth in aggregates, compared to small amounts produced by non-irradiated controls. This observation suggests that low power laser irradiation applied to the area of an experimentally injured nerve may induce axonal processes sprouting, thereby improving nerve tissue recovery. The mechanism of low power laser on nerve tissue is not completely understood, but some studies partially explain the photochemical effect of laser irradiation on the biological system. Cytochromes are affected, thereby stimulating redox activity in the cellular respiratory chain, thereby causing increases in ATP production which activates Na+, K+ -ATPase and other ion carriers, thereby increasing cell activation.
II. Animal Studies – influence of laser therapy on the severely injured peripheral nerve
A radiation method for treating lesions in both the peripheral and central nervous systems was proposed in 1978 by Rochkind and modified over the years. The model used in this work was the rat sciatic nerve. Low power laser irradiation then was delivered to the crushed nerve either transcutaneously or directly. The effects of this laser therapy were measured both in the short-term, i.e. minutes and in the long-term, i.e. days and months. Short-term model: direct irradiation of the nerve was done through the open wound directly to the crushed injured nerve and the compound nerve action potential was measured. A variety of wavelengths and powers were applied and 540nm, 632.8nm and 780nm were found most effective (p=0.01). Long-term model: We found electrophysiolgical activity dropped as expected in the non-irradiated nerves following the crush injury, but the use of low power laser irradiation prevented or decreased this phenomenon (p=0.001), both immediately after the crush and in the long term. Furthermore, this investigation showed that when laser treatment was delivered to both the crushed nerve and the corresponding segments of the spinal cord, the recovery time and the quality of regeneration of the crushed sciatic nerve improved, compared to the application of irradiation to the nerve alone. Histological studies supported the electrophysiological findings: low power laser irradiation was found to prevent or decrease scar tissue formation in the injured area. Laser irradiation enhanced axonal sprouting in the crush-injured sciatic nerve, thus accelerating recovery of the severely injured peripheral nerve. In addition, a beneficial effect of low power laser irradiation was found not only in the laser-treated nerve, but in the corresponding segments of the spinal cord as well. Such laser treatment has been found to decrease significantly 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 to produce myelin under the influence of laser treatment. Also, low power laser irradiation exerts pronounced systemic effects on severely injured peripheral nerves and corresponding regions of the spinal cord.
III. Double-Blind Randomized Study Evaluating Regeneration of the Rat Sciatic Nerve after Suturing and Post-Operative Laser Therapy
The therapeutic effect of low power laser irradiation on peripheral nerve regeneration after complete transection and direct anastomosis of the rat sciatic nerve was studied recently. A 780nm laser wavelength was applied transcutaneously 30 minutes daily for 21 consecutive days to corresponding segments of the spinal cord and to the injured sciatic nerve immediately after closing the wound. Positive somato-sensory evoked responses were found in 55% of the irradiated rats and in 11% of the non-irradiated rats. Immuno-histochemical staining in the laser-treated group showed more intensive axonal growth and better quality of the regenerative process due to an increased number of large and medium diameter axons. IV. Clinical Pilot Studies The group of patients who were treated in the Department of Neurosurgery at Tel Aviv Sourasky Medical Center had been suffering from severe peripheral nerve and brachial plexus injuries for more than two years. Each of the 59 patients received laser treatment CW, 780nm, five hours daily for 21 consecutive days with the use of a laser system specially developed for our treatment method. Criterion for laser treatment in these cases was as follows: patients who suffered from partial motor and sensory disturbances and where surgery was not indicated. Fifty-six percent of the laser-treated patients showed good to excellent results in their motor function. V. Clinical Double-Blind Placebo-Controlled, Randomized Study of Low Power Laser in the Treatment of Peripheral Nerve Injures Since our previous pilot clinical results were positive, a final evaluation of the response to treatment was in order. Therefore, we performed a double-blind, placebo-controlled randomized study of patients who had been suffering from incomplete peripheral nerve and brachial plexus injuries from 6 months up to several years after injury. The protocol of this study was done with the permission of the Helsinki Committee of the Tel Aviv Sourasky Medical Center and with the approval of the Ministry of Health of Israel and by a grant from the Rehabilitation Department of the Ministry of Defence of Israel. The study evaluated the functional recovery of these patients after undergoing low power laser or placebo treatment. Recovery was classified by comparing each of the deficits present before and after surgery. The post-laser or post-placebo grade was determined by the change in strength compared to the pretreatment levels. In almost all cases, the level of motor function was minimal to poor pre-treatment. In the laser-treated group, statistically significant improvement was found in motor functional activity P=0.0001, compared to the placebo group). The electrophysiological findings also showed statistically significant improvement in the laser-treated group. Our twenty-five years of experience indicates that Laser Therapy is a low-cost, non-invasive method and will be recognized as standard additional treatment for improving the functional recovery of patients with peripheral nerve and brachial plexus injuries. According to our clinical experience, the main advantages of Laser Therapy are the enhancement and acceleration of the recovery of injured nerve tissue. The therapeutic results show that an objective progressive improvement appears in nerve function, leading to a significant and earlier recovery.
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. firstname.lastname@example.org
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.
J Reconstr Microsurg. 2001 Feb;17(2):133-7; discussion 138.
Double-blind randomized study evaluating regeneration of the rat transected sciatic nerve after suturing and postoperative low-power laser treatment.
Shamir MH, Rochkind S, Sandbank J, Alon M.
Koret School of Veterinary Medicine, Hebrew University of Jerusalem.
This double-blind randomized study evaluated the therapeutic effect of low-power laser irradiation (LPLI) on peripheral nerve regeneration, after complete transection and direct anastomosis of the rat sciatic nerve. After this procedure, 13 of 24 rats received postoperative LPLI, with a wavelength of 780 nm laser, applied transcutaneously, 30 min daily for 21 consecutive days, to corresponding segments of the spinal cord and to the injured sciatic nerve. Positive somatosensory evoked responses were found in 69.2 percent of the irradiated rats (p = 0.019), compared to 18.2 percent of the non-irradiated rats. Immunohistochemical staining in the laser-treated group showed an increased total number of axons (p = 0.026), and better quality of the regeneration process, due to an increased number of large-diameter axons (p = 0.021), compared to the non-irradiated control group. The study suggests that postoperative LPLI enhances the regenerative processes of peripheral nerves after complete transection and anastomosis.
Lasers Med Sci. 2003;18(2):83-8
No effect of GA-AS (904 nm) laser irradiation on the injured rat sciatic nerve.
Bagis S, Comelekoglu U, Coskun B, Milcan A, Buyukakilli B, Sahin G, Ozisik S, Erdogan C.
Mersin University, Adana, Turkey. email@example.com
We evaluated the electrophysiological and histopathological effects of low-energy gallium arsenide (904 nm) laser irradiation on the intact skin injured rat sciatic nerve. Twenty-four male Wistar rats were divided into three groups ( n=8 each). At the level of proximal third of the femur the sciatic nerve was crushed bilaterally with an aneurysm clip (Aesculap FE 751, Tuttingen, Germany) for half a second. A gallium arsenide laser (wavelength 904 nm, pulse duration 220 ns, peak power per pulse 27 W, spot size 0.28 cm2, pulse repetition rate 16, 128 and 1000 Hz; total applied energy density 0.31, 2.48 and 19 J/cm2) was applied to the right sciatic nerve for 15 min daily at the same time on 7 consecutive days. The same procedure was performed on the left sciatic nerve of same animal, but without radiation emission, and this was accepted as control. Compound muscle action potentials were recorded from right and left sides in all three groups before surgery, just at the end of injury, at the 24th hour and on the 14th and 21st days of injury in all rats using a BIOPAC MP 100 Acquisition System Version 3.5.7 (Santa Barbara, USA). BIOPAC Acknowledge Analysis Software (ACK 100 W) was used to measure CMAP amplitude, area, proximal and distal latency, total duration and conduction velocity. Twenty-one days after injury, the rats were sacrificed. The sciatic nerves of the operated parts were harvested from the right and left sides. Histopathological evaluation was performed by light microscopy. Statistical evaluation was done using analysis of variance for two factors (right and left sides) repeated-measures (CMAP variables within groups) and the Tukey-Kramer Honestly Significant Difference test (CMAP variables between laser groups). The significance was set at p < 0.05. No statistically significant difference (p > 0.05) was found regarding the amplitude, area, duration and conduction velocity of CMAP for each applied dose (0.31, 2.48 and 19 J/cm2) on the irradiated (right) side and the control (left) side, or between irradiated groups. Twenty-one days after injury there were no qualitative differences in the morphological pattern of the regenerated nerve fibres in either irradiated (0.31, 2.48 and 19 J/cm2) or control nerves when evaluated by light microscopy. This study showed that low-energy GaAs irradiation did not have any effect on the injured rat sciatic nerve.
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 innovative approach to induce regeneration and the repair of spinal cord injury. 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.
J. Käs . PNAS. 2002; 99: 16024-16028
Guiding neuronal growth with light
A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M. G. Raizen,
We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, we place a laser spot in front of a chosen area of the nerve’s leading edge, promoting growth into the beam focus. This allows us to guide neuronal turns as well as enhance growth. The power of our laser has been selected so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers, which uses large optical forces to manipulate entire structures. Our results therefore open a new avenue to controlling neuronal growth in vitro and in vivo with a simple, non-contact technique. Currently we have been using 800nm with continuous application of powers ranging from 20 to 130 mW over a circular area of 1 to 4 um in radius. Recently we’ve developed and active feedback mechanism to trace the contour of the growth cone and subsequently raster the beam image upon that, instead of the pure beam profile we had used previously.
(Abstract supplied by Allen Ehrlicher, main author)
|Neurosci Lett. 2003 Jun 26;344(2):71-4.|
Growth-associated protein-43 is elevated in injured rat sciatic nerve after low power laser irradiation.
Shin DH, Lee E, Hyun JK, Lee SJ, Chang YP, Kim JW, Choi YS, Kwon BS.
Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea.
Low power laser irradiation (LPLI) has been used in the treatment of peripheral nerve injury. In this study, we verified its therapeutic effect on neuronal regeneration by finding elevated immunoreactivities (IRs) of growth-associated protein-43 (GAP-43), which is up-regulated during neuronal regeneration. Twenty Sprague-Dawley rats received a standardized crush injury of the sciatic nerve, mimicking the clinical situations accompanying partial axonotmesis. The injured nerve received calculated LPLI therapy immediately after injury and for 4 consecutive days thereafter. The walking movements of the animals were scored using the sciatic functional index (SFI). In the laser treated rats, the SFI level was higher in the laser treated animals at 3-4 weeks while the SFIs of the laser treated and untreated rats reached normal levels at 5 weeks after surgery. In immunocytochemical study, although GAP-43 IRs increased both in the untreated control and the LPLI treated groups after injury, the number of GAP-43 IR nerve fibers was much more increased in the LPLI group than those in the control group. The elevated numbers of GAP-43 IR nerve fibers reached a peak 3 weeks after injury, and then declined in both the untreated control and the LPLI groups at 5 weeks, with no differences in the numbers of GAP-43 IR nerve fibers of the two groups at this stage. This immunocytochemical study using GAP-43 antibody study shows for the first time that LPLI has an effect on the early stages of the nerve recovery process following sciatic nerve injury.
|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.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996 Aug;82(2):132-8.
Effect of low-level laser treatment on neurosensory deficits subsequent to sagittal split ramus osteotomy.
Khullar SM, Emami B, Westermark A, Haanaes HR.
Department of Oral Surgery and Oral Medicine, University of Oslo, Norway.
OBJECTIVES: Low-level laser treatment has been advocated as a possible treatment for patients with paresthesia. An objectively verified improvement in sensory function is relevant if, at the same time, it is perceived as a subjective improvement by the patient. The aim of this double blind clinical study was to see if low-level laser treatment with a GaAlAs laser (820 nm, Rønvig, Denmark) resulted in objectively verified improvement in sensory function and whether this correlated with the patient’s subjective evaluation subsequent to treatment. STUDY DESIGN: The 13 patients in this study had all undergone saggittal split ramus osteotomy resulting in either compression or traction of the inferior alveolar nerve as reported by the surgery notes. The material was collected from a consecutive series of patients at the Karolinska Hospital, all of whom had shown reduced sensibility at their final 2-year postoperative checkup. The patients were randomly divided into two groups; one (eight subjects) group received real low-level laser treatment (4 x 6 J per treatment along the distribution of the inferior alveolar nerve, at the following points extraoral: lateral third of lower lip, intraoral; buccally to the apex of the second premolar tooth and the apex of the second molar tooth; lingually in the region of the mandibular foramen; for a total of 20 treatments). The other group received an equivalent placebo treatment. The study was conducted in a double blind fashion for both patient and doctor as the low-level laser equipment had two settings, A and B, one of which was an unknown void setting. The degree of mechanoceptor neurosensory deficit was assessed by Semmes Weinstein monofilaments (North Coast Medical, USA) and the degree of thermoceptor neurosensory deficit was assessed by a Thermotester (Somedic, Sweden). The degree of subjective neurosensory deficit was assessed by means of a visual analogue scale. Both variables and the degree of subjective injury were comparable between the two groups before starting treatment. RESULTS: The patients in the real low-level laser treatment group experienced a subjective improvement in both lip (p = 0.01) and chin (p = 0.02) after completion of the course of treatment. In addition, this group showed a significant decrease in the area of mechanoperception neurosensory deficit (p = 0.01) compared with no difference in the placebo group. The real low-level laser treatment group exhibited a strong tendency toward improvement in mechanoreceptor neurosensory deficit in the areas of most damage for both lip and chin. This improvement was especially pronounced in the lip region (p = 0.06). No similar tendency was demonstrated in the placebo group. Neither group showed any significant change or tendency to improvement in thermoception on completion of the course of treatment. CONCLUSION: In conclusion GaAlAs low-level laser treatment results in both a subjective and objective improvement in mechanical sensory perception in long-standing neurosensory deficit in the inferior alveolar nerve.
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.
Lasers Surg Med. 1987;7(5):441-3.
Response of peripheral nerve to He-Ne laser: experimental studies.
Rochkind S, Nissan M, Barr-Nea L, Razon N, Schwartz M, Bartal A.
Neurosurgery Department, Tel-Aviv Medical Center, Ichilov Hospital, Israel.
Low-energy He-Ne laser irradiation (LELI) was found to affect the electric activity and morphology in both intact and severely injured peripheral nerves in rats. Action potential (AP) in the healthy nerve increased by 33% following a single transcutaneous irradiation. Similar irradiation in crushed nerves caused AP to increase significantly over the AP of nonirradiated crushed nerve. Morphological observations revealed that a laser-irradiated injured nerve had diminished scar tissue as compared to an injured but not an irradiated nerve.