Neuroprotective Effect Of Transcorneal Electrical Stimulation On Ischemic Damage In The Rat Retina

Retinal ischemia plays an important role in the pathophysiology of various ocular diseases, such as glaucoma and diabetic retinopathy, and current therapies are often suboptimal and/or invasive. At the early stage of ischemia, increased glutamatergic stimulation is one essential element to trigger a self-reinforcing destructive cascade. On the other hand, glutamine synthetase (GS), a crucial component of the major mechanism for the clearance of extracellular glutamate, protects against glutamate excitotoxicity.Transcorneal electrical stimulation (TES), as a less invasive method, has shown encouraging neuroprotective effects for retinopathy and optic neuropathy in certain rodent models and scanty clinical case series studies. TES is drawing more attention as a new potential method to protect retinal neurons. However, the neuroprotective mechanism of TES needs further studies.The objective of this study was to test the hypothesis that TES also provides definite neuroprotection related to increasing GS levels in an ocular hypertension-induced retinal ischemia model. To evaluate the neuroprotective effects, we assessed the number of surviving retinal ganglion cells (RGCs), the retinal histology and electroretinagrams (ERG); and to reveal the neuroprotection mechanism of TES, the GS expression levels were measured by immunohistochemistry and Western blot analysis in the ischemic retinas with TES treatment or sham stimulation. PartⅠEvaluation of the Neuroprotective Effect of Transcorneal Electrical Stimulation on Ischemic Damage in the Rat RetinaPurpose. To investigate whether transcorneal electrical stimulation (TES) has a neuroprotective effect on retinal neurons after ischemic insults.Methods. Adult female Sprague-Dawley (SD) rats were selected and randomly divided into three groups:the normal group, the sham stimulation group (current intensity, OμA; stimulation duration,1 hour) the TES group (current intensity, OμA; stimulation duration,1 hour). Retinal ischemia was induced by elevating the intraocular pressure to 120 mmHg for 60 minutes and TES was commenced immediately after ischemic insults and thereafter applied for 1 hour every 2 days until day 14. Retinal ganglion cells (RGCs) were labeled retrogradely 7 days before ischemia and were quantified 7 and 14 days later. At the same time points, retinal function was assessed by scotopic electroretinography (ERG), combined with retinal histological analysis.Results. Quantification of RGCs showed that in normal eyes, the mean density of RGCs was 2365±60 cells/mm2. On day 7 after ischemia it was 1477±60 cells/mm2 in the sham stimulation group and 1785±59 cells/mm2 in TES group. Similarly, on day 14 the mean density of RGCs in the sham-stimulated eyes was 1147±56 cells/mm2, but it remained 1414±52 cells/mm2 in the TES group. The mean density of RGCs in TES group was significantly higher compared to retinas with sham stimulation on days 7 and 14 after ischemia (P<0.01). Histological analysis showed that in the normal retinas, the mean thickness of ILM-OLM, IPL, and ONL was 174.3±3.9 um,43.6±0.6μm,58.1±1.0 um, respectively. On day 7 after ischemia, the mean relative thickness (MRT) of ILM-OLM, compared with the normal group was:86.0% for the sham stimulation group,96.0% for the TES group; the MRT of IPL:80.9% for the sham stimulation group,93.1% for the TES group; and the MRT of ONL:84.4% for the sham stimulation group,97.3% for the TES group. And on day 14, the MRT of ILM-OLM, compared with the normal group was: 84.0% for the sham stimulation group,92.9% for the TES group; the MRT of IPL:77.9% for the sham stimulation group,89.1% for the TES group; and the MRT of ONL:82.4% for the sham stimulation group,93.6% for the TES group. Statistical analysis revealed that TES could significantly preserve the thickness of ILM-OLM (n=6; t-test, P<0.05), IPL (n=6; t-test, P<0.01) and ONL (n=6; t-test, P<0.01) compared with the sham stimulation group on both days 7 and 14. ERG studies indicated that by undergoing TES treatment, the b-wave amplitude was also significantly preserved on day 7 after ischemia and recovered robustly on day 14 (P<0.05).Conclusions. TES well preserved the morphology and function of retinas damaged by ischemic insults.PartⅡThe Effect of Transcorneal Electrical Stimulation on the Expression of Glutamine Synthetase after Retinal IschemiaPurpose. To investigate whether transcorneal electrical stimulation (TES) could upregulate GS levels in the retina, revealing the possible mechanism of TES neuroprotection.Methods. Adult female Sprague-Dawley (SD) rats were selected and randomly divided into three groups:the normal group, the sham stimulation group (current intensity, 0μA; stimulation duration,1 hour) the TES group (current intensity, 0μA; stimulation duration,1 hour). Retinal ischemia was induced by elevating the intraocular pressure to 120 mmHg for 60 minutes and TES was commenced immediately after ischemic insults and thereafter applied for 1 hour every 2 days until day 14. Six and 24 hours,3,7, and 14 days after ischemia, the glutamine synthetase (GS) immunoreactivity was compared between ischemic retinas with TES and those with sham stimulation under identical confocal laser microscope conditions. The immunohistochemical indications were confirmed by Western blot analysis.Results. Immunohistochemical and Western blot analysis both revealed that GS levels remarkably increased after TES and lasted for at least 7 days.Conclusions. TES could increase GS levels in the retina, which might attenuate glutamate-mediated excitotoxicity and thus protect retinal neurons against ischemic insults.