We examined whether near-infrared light (NIr) treatment (photobiomodulation) saves dopaminergic amacrine cells of the retina in an acute and a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. For the acute model, BALB/c mice had MPTP (100 mg/kg) or saline injections over 30 hours, followed by a six-day-survival period. For the chronic model, mice had MPTP (200 mg/kg) or saline injections over five weeks, followed by a three-week-survival period. NIr treatment was applied either at the same time (simultaneous series) or well after (posttreatment series) the MPTP insult. There were four groups within each series: Saline, Saline-NIr, MPTP, and MPTP-NIr. Retinae were processed for tyrosine hydroxylase (TH) immunochemistry, and cell number was analysed. In the MPTP groups, there was a significant reduction in TH+ cell number compared to the saline controls; this reduction was greater in the acute (
Many previous studies have reported that mitochondrial dysfunction is a key component of the pathogenesis of Parkinson disease, a striking motor disorder that develops after a major loss of dopaminergic cells in the substantia nigra pars compacta (SNc) [
In this study, we sought to extend our previous findings on the SNc of MPTP-treated mice [
Male albino BALB/c mice (~20 g; ~8 weeks old;
An acute [
Outline of the different experimental groups used in this study, namely: Saline, Saline-NIr, MPTP, and MPTP-NIr, in either the simultaneous (a) or posttreatment (b) series of the acute and chronic models. Photomicrographs of TH+ amacrine cells in retinal wholemounts of the superior temporal region (c) and of the retinal edges ((d), (e)). The latter reveals the location of the TH+ somata and dendrites within the different layers. Most TH+ somata were located in the inner layers of the inner nuclear layer (c); very few were located in the ganglion cell layer (e). All images from Saline group of Ac-Sim case. Scale bars = 100
For the acute model, four (25 mg/kg injections; total of 100 mg/kg per mouse) MPTP or saline injections were made over a 30 hr period. After the last injection, mice were allowed to survive for six days. For the chronic model, mice had ten injections of MPTP (20 mg/kg per injection; total of 200 mg/kg per mouse) or saline combined with probenecid (250 mg/kg; decreases renal excretion of MPTP and hence maintains the effects of toxin during injection intervals), approximately three and a half days apart, over a five-week-period. After the last injection, mice were allowed to survive for three weeks. For both models, the dose regimes and survival periods were similar to those used by previous studies, including our own [
For the NIr treatment, mice in the MPTP-NIr and Saline-NIr groups of each model (acute and chronic) were treated with 670 nm light from a light-emitting device (Quantum Devices WARP 10) as described previously [
Following the survival periods, mice were anaesthetised with an intraperitoneal injection of sodium pentobarbital (60 mg/mL). They were then perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4), followed by 4% buffered formaldehyde. The retinae were removed and postfixed for ~20 mins in the same solution. Next, retinae were dissected free from other structures in the globe as wholemounts [
Each retina was scanned systematically under the fluorescence microscope and the number of TH+ cells and retinal area were calculated with the aid of a stereological programme (StereoInvestigator, MBF Science). Every TH+ cell was plotted and total number was recorded; for the areas, the boundaries of each retina were traced and the programme calculated the area (mm2). For comparisons between groups (using GraphPad Prism programme), a oneway ANOVA test (
The results that follow will be presented in four main parts: (i) morphology, (ii) retinal areas, (iii) number, and (iv) topography.
Confirming previous studies [
The graph in Figure
Graphs showing (a) the areas and (b) the number of TH+ amacrine cells in the retinae in the four experimental groups in the different cases (different shaded columns). Columns show the mean ± standard error of the total number of cells in each case. †Represents
The graph in Figure
For the Saline and Saline-NIr groups, TH+ cell number in the different cases were not significantly different (Tukey-Kramer test;
For the MPTP groups, TH+ cell number was reduced compared to the saline groups in all the cases. These reductions were significant (Tukey-Kramer test) in each of the Ac-Sim (
The number of TH+ cells in the substantia nigra pars compacta (SNc), from the same brains where the retinae were taken from, has been analysed too and full details of the results were published [
In the MPTP-NIr groups, TH+ cell number was higher than in the MPTP groups in all the cases (~30%). These increases were significant (Tukey-Kramer test) in the Ac-PT (
In summary, TH+ cell number in the MPTP groups was reduced from the saline groups, particularly in the acute cases. In the MPTP-NIr groups, there were more TH+ cells compared to the MPTP groups in all cases, although to a lesser extent in the Ac-Sim case.
We examined the distribution of TH+ cells in the different cases as to determine whether the MPTP or NIr treatment affected one retinal region more than another. Figure
Schematic diagrams of maps ((a), (c), (e), (g)) and photomicrographs ((b), (d), (f), (h)) of TH+ amacrine cells in the retinae of Saline ((a), (b)), Saline-NIr ((c), (d)), MPTP ((e), (f)), and MPTP-NIr ((g), (h)) groups of the Ac-Sim case (this case shown because it had the most change after MPTP treatment). The photomicrographs are of a midregion of superior temporal retina in each case. In the saline control groups ((a)–(d)), TH+ cells were distributed relatively uniformly across the retina, but with a slight concentration in superior and temporal retina. In the MPTP and MPTP-NIr groups ((e)–(h)), there was no particular region of retina that was affected particularly after MPTP (or NIr) treatment. Scale bar = 100
We had three main findings. First, there were fewer TH+ amacrine cells in the MPTP groups compared to the saline controls, particularly in the acute cases. Second, the magnitude of TH+ cell loss after MPTP insult was not as substantial as that seen in the SNc. Third, and importantly, there were more TH+ cells in the MPTP-NIr compared to the MPTP groups. Each of these issues will be discussed in this paper. First, a comparison with previous studies will be considered.
Previous studies have shown that NIr treatment offers
Although some of our TH+ cell loss may be due to transient TH expression after MPTP insult [
The magnitude of TH+ amacrine cell loss after MPTP insult was greater in the acute cases (~50%) than in the chronic ones (~30%). Our chronic model delivered double the dose of MPTP (200 versus 100 mg/kg), but it was not as damaging to the amacrine cells as the acute insult delivered over a much shorter time period, 30 hours as against 5 weeks. Such findings have been reported previously by many studies. In the SNc, for example, acute insults generate up to 70% cell loss, while the chronic insults generate only about 50% [
In the SNc, the reduction in TH+ cell number was ~15% greater than in the retina in both models [
There were more TH+ cells in the MPTP-NIr compared to the MPTP groups in all the cases, although to a lesser extent in the Ac-Sim case. There are three issues more to consider regarding this finding. First, despite the different types of intervening body tissue, whether transparent membranes of the globe or hair, skin, bone, and meninges, NIr treatment mitigated the MPTP insult just as effectively in the retina (~30%) and SNc (~35%) [
The precise mechanism(s) that saved the TH+ amacrine cells from degeneration is not known. Many authors have suggested that NIr triggers intrinsic trophic factors that enhance cell survival, for example, by increasing ATP production and reducing reactive oxygen species in the mitochondria (see Introduction). In addition, we suggest that NIr treatment stimulated the local release of melatonin, a powerful antioxidant and cell saving agent [
Acute-simultaneous
Acute-posttreatment
Adenosine-5′-triphosphate
Chronic-simultaneous
Chronic-posttreatment
Dopamine transporter molecule
Ganglion cell layer
Inner nuclear layer
Inner plexiform layer
Light emitting device
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Near-infrared light
Phosphate buffered saline
Substantia nigra pars compacta
Tyrosine hydroxylase.
There was no conflict of interests for any of the authors: G. Jeffery, J. Stone, G. E. Baker, and J. Mitrofanis were fulltime members of staff at their respective institutions, while C. Peoples and V. E. Show were fulltime postgraduate students.
The authors are forever grateful to Tenix Corp and Salteri family for their generous funding of the laboratory. The authors dedicate this work to their friend, colleague, and coauthor, Gary Baker, who past away during the final stages of manuscript preparation.