Exp Brain Res. 2016 Jul 5. [Epub ahead of print]
Near-infrared light treatment reduces astrogliosis in MPTP-treated monkeys.
El Massri N1, Moro C2, Torres N2, Darlot F2, Agay D2, Chabrol C2, Johnstone DM3, Stone J3, Benabid AL2, Mitrofanis J4.
- 1Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia.
- 2University Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus, 38000, Grenoble, France.
- 3Department of Physiology F13, University of Sydney, Sydney, 2006, Australia.
- 4Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia. email@example.com.
We have reported previously that intracranial application of near-infrared light (NIr) reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson’s disease. In this study, we explored whether NIr reduces the gliosis in this animal model. Sections of midbrain (containing the substantia nigra pars compacta; SNc) and striatum were processed for glial fibrillary acidic protein (to label astrocytes; GFAP) and ionised calcium-binding adaptor molecule 1 (to label microglia; IBA1) immunohistochemistry. Cell counts were undertaken using stereology, and cell body sizes were measured using ImageJ. Our results showed that NIr treatment reduced dramatically (~75 %) MPTP-induced astrogliosis in both the SNc and striatum. Among microglia, however, NIr had a more limited impact in both nuclei; although there was a reduction in overall cell size, there were no changes in the number of microglia in the MPTP-treated monkeys after NIr treatment. In summary, we showed that NIr treatment influenced the glial response, particularly that of the astrocytes, in our monkey MPTP model of Parkinson’s disease. Our findings raise the possibility of glial cells as a future therapeutic target using NIr.
Near-infrared light (670 nm) reduces MPTP-induced parkinsonism within a broad therapeutic time window.
1CLINATEC, EJ Safra Centre, CEA, LETI, University of Grenoble Alpes, 38000, Grenoble, France. firstname.lastname@example.org.
2Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia. email@example.com.
3Department of Physiology F13, University of Sydney, Sydney, 2006, Australia. firstname.lastname@example.org.
4Department of Physiology F13, University of Sydney, Sydney, 2006, Australia. email@example.com.
5Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia. firstname.lastname@example.org.
6CLINATEC, EJ Safra Centre, CEA, LETI, University of Grenoble Alpes, 38000, Grenoble, France. email@example.com.
7CLINATEC, EJ Safra Centre, CEA, LETI, University of Grenoble Alpes, 38000, Grenoble, France. firstname.lastname@example.org.
We have shown previously that near-infrared light (NIr), when applied at the same time as a parkinsonian insult (e.g. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPTP), reduces behavioural deficits and offers neuroprotection. Here, we explored whether the timing of NIr intervention-either before, at the same time or after the MPTP insult-was important. Mice received MPTP injections (total of 50 mg/kg) and, at various stages in relation to these injections, extracranial application of NIr. Locomotor activity was tested with an open-field test, and brains were processed for immunohistochemistry. Our results showed that regardless of when NIr was applied in relation to MPTP insult, behavioural impairment was reduced by a similar magnitude. The beneficial effect of NIr was fast-acting (within minutes) and long-lasting (for several days). There were more dopaminergic cells in the NIr-treated MPTP groups than in the MPTP group; there was no clear indication that a particular combination of NIr treatment and MPTP injection resulted in a higher cell number. In summary, irrespective of whether it was applied before, at the same time as or after MPTP insult, NIr reduced both behavioural and structural measures of damage by a similar magnitude. There was a broad therapeutic time window of NIr application in relation to the stage of toxic insult, and the NIr was fast-acting and long-lasting.
Turning On Lights to Stop Neurodegeneration: The Potential of Near Infrared Light Therapy in Alzheimer’s and Parkinson’s Disease.
2University Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus Grenoble, France.
Alzheimer’s and Parkinson’s disease are the two most common neurodegenerative disorders. They develop after a progressive death of many neurons in the brain. Although therapies are available to treat the signs and symptoms of both diseases, the progression of neuronal death remains relentless, and it has proved difficult to slow or stop. Hence, there is a need to develop neuroprotective or disease-modifying treatments that stabilize this degeneration. Red to infrared light therapy (? = 600-1070 nm), and in particular light in the near infrared (NIr) range, is emerging as a safe and effective therapy that is capable of arresting neuronal death. Previous studies have used NIr to treat tissue stressed by hypoxia, toxic insult, genetic mutation and mitochondrial dysfunction with much success. Here we propose NIr therapy as a neuroprotective or disease-modifying treatment for Alzheimer’s and Parkinson’s patients.
Intracranial application of near-infrared light in a hemi-parkinsonian rat model: the impact on behavior and cell survival.
1CEA, Leti, and Clinatec Departments, University Grenoble Alpes, Minatec Campus, Grenoble, France; and
2Departments of 2 Anatomy and.
3Physiology, University of Sydney, New South Wales, Australia.
Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in AAV-Based Rat Genetic Model of Parkinson’s Disease.
1Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Centre de Recherche du Centre Hospitalier de Québec, Axe Neuroscience et Département de Médecine Moléculaire de l’Université Laval, Québec, G1V4G2, Canada.
2Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Medos International Sàrl, a Johnson&Johnson company, Chemin Blanc 38, CH-2400, Le Locle, Switzerland.
3Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland.
4Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland.
5Medos International Sàrl, a Johnson&Johnson company, Chemin Blanc 38, CH-2400, Le Locle, Switzerland.
6Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, P.O. Box 5825, Doha, Qatar.
Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human ?-synuclein (?-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson’s disease (PD). In this model, daily exposure of both sides of the rat’s head to 808-nm near-infrared light for 28 consecutive days alleviated ?-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.
Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.
- 1CEA-Leti, Grenoble, France; and.
Previous experimental studies have documented the neuroprotection of damaged or diseased cells after applying, from outside the brain, near-infrared light (NIr) to the brain by using external light-emitting diodes (LEDs) or laser devices. In the present study, the authors describe an effective and reliable surgical method of applying to the brain, from inside the brain, NIr to the brain. They developed a novel internal surgical device that delivers the NIr to brain regions very close to target damaged or diseased cells. They suggest that this device will be useful in applying NIr within the large human brain, particularly if the target cells have a very deep location.
An optical fiber linked to an LED or laser device was surgically implanted into the lateral ventricle of BALB/c mice or Sprague-Dawley rats. The authors explored the feasibility of the internal device, measured the NIr signal through living tissue, looked for evidence of toxicity at doses higher than those required for neuroprotection, and confirmed the neuroprotective effect of NIr on dopaminergic cells in the substantia nigra pars compacta (SNc) in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson disease in mice.
The device was stable in freely moving animals, and the NIr filled the cranial cavity. Measurements showed that the NIr intensity declined as distance from the source increased across the brain (65% per mm) but was detectable up to 10 mm away. At neuroprotective (0.16 mW) and much higher (67 mW) intensities, the NIr caused no observable behavioral deficits, nor was there evidence of tissue necrosis at the fiber tip, where radiation was most intense. Finally, the intracranially delivered NIr protected SNc cells against MPTP insult; there were consistently more dopaminergic cells in MPTP-treated mice irradiated with NIr than in those that were not irradiated.
In summary, the authors showed that NIr can be applied intracranially, does not have toxic side effects, and is neuroprotective.
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.
Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains.
Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains
We have shown previously that near-infrared light (NIr) treatment or photobiomodulation neuroprotects dopaminergic cells in substantia nigra pars compacta (SNc) from degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Balb/c albino mice, a well-known model for Parkinson’s disease. The present study explores whether NIr treatment offers neuroprotection to these cells in C57BL/6 pigmented mice. In addition, we examine whether NIr influences behavioural activity in both strains after MPTP treatment. We tested for various locomotive parameters in an open-field test, namely velocity, high mobility and immobility.
Balb/c (albino) and C57BL/6 (pigmented) mice received injections of MPTP (total of 50 mg/kg) or saline and NIr treatments (or not) over 48 hours. After each injection and/or NIr treatment, the locomotor activity of the mice was tested. After six days survival, brains were processed for TH (tyrosine hydroxylase) immunochemistry and the number of TH+ cells in the substantia nigra pars compacta (SNc) was estimated using stereology. Results showed higher numbers of TH+ cells in the MPTP-NIr groups of both strains, compared to the MPTP groups, with the protection greater in the Balb/c mice (30% vs 20%). The behavioural tests revealed strain differences also. For Balb/c mice, the MPTP-NIr group showed greater preservation of locomotor activity than the MPTP group. Behavioural preservation was less evident in the C57BL/6 strain however, with little effect of NIr being recorded in the MPTP-treated cases of this strain. Finally, there were differences between the two strains in terms of NIr penetration across the skin and fur. Our measurements indicated that NIr penetration was considerably less in the pigmented C57BL/6, compared to the albino Balb/c mice.
In summary, our results revealed the neuroprotective benefits of NIr treatment after parkinsonian insult at both cellular and behavioural levels and suggest that Balb/c strain, due to greater penetration of NIr through skin and fur, provides a clearer model of protection than the C57BL/6 strain.
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.
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Therapeutic effect of near infrared (NIR) light on Parkinson’s disease models.
Department of Neurology, Medical College of Wisconsin, 8701 W. Watertown Plank Rd, Milwaukee, WI 53226, USA.
Parkinson’s disease (PD) is a neurodegenerative disorder that affects large numbers of people, particularly those of a more advanced age. Mitochondrial dysfunction plays a central role in PD, especially in the electron transport chain. This mitochondrial role allows the use of inhibitors of complex I and IV in PD models, and enhancers of complex IV activity, such as NIR light, to be used as possible therapy. PD models fall into two main categories; cell cultures and animal models. In cell cultures, primary neurons, mutant neuroblastoma cells, and cell cybrids have been studied in conjunction with NIR light. Primary neurons show protection or recovery of function and morphology by NIR light after toxic insult. Neuroblastoma cells, with a gene for mutant alpha-synuclein, show similar results. Cell cybrids, containing mtDNA from PD patients, show restoration of mitochondrial transport and complex I and IV assembly. Animal models include toxin-insulted mice, and alpha-synuclein transgenic mice. Functional recovery of the animals, chemical and histological evidence, and delayed disease progression show the potential of NIR light in treating Parkinson’s disease.
J Comp Neurol. 2010 Jan 1;518(1):25-40.
Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment.
Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J.
Discipline of Anatomy & Histology F13, University of Sydney, Australia.
This study explores whether near-infrared (NIr) light treatment neuroprotects dopaminergic cells in the substantia nigra pars compacta (SNc) and the zona incerta-hypothalamus (ZI-Hyp) from degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. BALB/c albino mice were divided into four groups: 1) Saline, 2) Saline-NIr, 3) MPTP, 4) MPTP-NIr. The injections were intraperitoneal and they were followed immediately by NIr light treatment (or not). Two doses of MPTP, mild (50 mg/kg) and strong (100 mg/kg), were used. Mice were perfused transcardially with aldehyde fixative 6 days after their MPTP treatment. Brains were processed for tyrosine hydroxylase (TH) immunochemistry. The number of TH(+) cells was estimated using the optical fractionator method. Our major finding was that in the SNc there were significantly more dopaminergic cells in the MPTP-NIr compared to the MPTP group (35%-45%). By contrast, in the ZI-Hyp there was no significant difference in the numbers of cells in these two groups. In addition, our results indicated that survival in the two regions after MPTP insult was dose-dependent. In the stronger MPTP regime, the magnitude of loss was similar in the two regions ( approximately 60%), while in the milder regime cell loss was greater in the SNc (45%) than ZI-Hyp ( approximately 30%). In summary, our results indicate that NIr light treatment offers neuroprotection against MPTP toxicity for dopaminergic cells in the SNc, but not in the ZI-Hyp.
Mol Neurodegener. 2009 Jun 17;4:26.
Reduced axonal transport in Parkinson’s disease cybrid neurites is restored by light therapy.
Trimmer PA, Schwartz KM, Borland MK, De Taboada L, Streeter J, Oron U.
University of Virginia, Morris K Udall Parkinson’s Research Center of Excellence and Department of Neurology, Charlottesville, Virginia, USA. email@example.com.
ABSTRACT: BACKGROUND: It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson’s disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid “cytoplasmic hybrid” neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined. RESULTS: The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT. CONCLUSION: The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.
Brain Res. 2008 Dec 3;1243:167-73. Epub 2008 Sep 30.
Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.
Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Parkinson’s disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.
Neuroscience. 2008 Jun 2;153(4):963-74. Epub 2008 Mar 26.
Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.
Liang HL, Whelan HT, Eells JT, Wong-Riley MT.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Parkinson’s disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson’s disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson’s disease by energizing the cells and increasing their viability.
Mitochondrion. 2004 Sep;4(5-6):559-67.
Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy.
Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.
Department of Health Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. firstname.lastname@example.org
Photobiomodulation by light in the red to near infrared range (630-1000 nm) using low energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing, improve recovery from ischemic injury in the heart and attenuate degeneration in the injured optic nerve. Recent evidence indicates that the therapeutic effects of red to near infrared light result, in part, from intracellular signaling mechanisms triggered by the interaction of NIR light with the mitochondrial photoacceptor molecule cytochrome c oxidase. We have demonstrated that NIR-LED photo-irradiation increases the production of cytochrome oxidase in cultured primary neurons and reverses the reduction of cytochrome oxidase activity produced by metabolic inhibitors. We have also shown that NIR-LED treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. Photobiomodulation improves wound healing in genetically diabetic mice by upregulating genes important in the promotion of wound healing. More recent studies have provided evidence for the therapeutic benefit of NIR-LED treatment in the survival and functional recovery of the retina and optic nerve in vivo after acute injury by the mitochondrial toxin, formic acid generated in the course of methanol intoxication. Gene discovery studies conducted using microarray technology documented a significant upregulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection. These findings provide a link between the actions of red to near infrared light on mitochondrial oxidative metabolism in vitro and cell injury in vivo. Based on these findings and the strong evidence that mitochondrial dysfunction is involved in the pathogenesis of numerous diseases processes, we propose that NIR-LED photobiomodulation represents an innovative and non-invasive therapeutic approach for the treatment of tissue injury and disease processes in which mitochondrial dysfunction is postulated to play a role including diabetic retinopathy, age-related macular degeneration, Leber’s hereditary optic neuropathy and Parkinson’s disease.
|Patol Fiziol Eksp Ter. 2004 Jan-Mar;(1):15-8.|
Biochemical and immunological indices of the blood in Parkinson’s disease and their correction with the help of laser therapy.
[Article in Russian]
Komel’kova LV, Vitreshchak TV, Zhirnova IG, Poleshchuk VV, Stvolinskii SL, Mikhailov VV, Gannushkina IV, Piradov MA.
The influence of laser therapy on the course of Parkinson’s disease (PD) was studied in 70 patients. This influence appeared adaptogenic both in the group with elevated and low MAO B and Cu/Zn SOD activity. Laser therapy resulted in reduction of neurological deficit, normalization of the activity of MAO B, Cu/Zn-SOD and immune indices. There was a correlation between humoral immunity and activity of the antioxidant enzymes (SOD, catalase). This justifies pathogenetically the use of laser therapy in PD.
|Bull Exp Biol Med. 2003 May;135(5):430-2.|
Laser modification of the blood in vitro and in vivo in patients with Parkinson’s disease.
Vitreshchak TV, Mikhailov VV, Piradov MA, Poleshchuk VV, Stvolinskii SL, Boldyrev AA.
Institute of Neurology of the Russian Academy of Medical Sciences, Moscow.
The effect of He-Ne laser radiation on activity of MAO B, Cu/Zn-SOD, Mn-SOD, and catalase in blood cells from patients with Parkinson’s disease was studied in vivo and in vitro. The effects of intravenous in vivo irradiation (intravenous laser therapy) were more pronounced than those observed in similar in vitro experiments. It is concluded that generalized effect of laser therapy involves interaction between blood cells.