Ischemia-induced Retinal Damage Research Articles

Could Crocus sativus (saffron) be combined with conventional therapies for ocular diseases?

Dear Sir, Oxidative stress due the continuous contact of ocular tissues with radiation, atmospheric oxygen, environmental chemicals, and physical abrasion has been proposed to play a vital role in diseases such as glaucoma, cataract, uveitis, retrolental fibroplasias, age-related macular degeneration (AMD), and various forms of retinopathy. This offers a prospect for novel approach to their prevention and treatment. Unfortunately, there are no curative treatment options for AMD, and even palliative treatments are few. The utilized therapeutic modalities are associated with various adverse effects, are costly, and only manage the consequence without tackling the cause of the pathology. Hence, many novel options are being investigated. Conventional and newly promising management measures have not accomplished in reversing the outcome of ocular diseases to any radical extent. This has implied researchers to investigate alternative treatment options. Hence, the widespread collections of traditional medicinal knowledge systems from various parts of the world are re-investigated for their healing properties. Crocus sativus L., commonly known as saffron, is the raw material for one of the most expensive spices in the world, and it has been used in folk medicine for centuries. The major active constituents of saffron are crocin, crocetin, and safranal. There is increasing substantiation that the spice saffron, which contains powerful anti-oxidants, offers protection against neurodegenerative disorders, including AMD and Alzheimer's disease.[1] Ischemic retinopathy and AMD are the foremost ocular diseases that cause blindness. The etiology of these diseases is due in part to the reduction of blood flow in the retina and/or choroid. Crocin analogs isolated from C. sativus L. were found to significantly increase the blood flow in the retina and choroid and to facilitate retinal function recovery.[2] Experimental studies conducted by Ishizuka et al. have demonstrated that crocetin prevents ischemia-induced retinal damage through its inhibition of oxidative stress.[3] Results from the experiment by Yamauchi et al. suggest that crocetin has protective effects against retinal damage in vitro and in vivo, indicating that crocetin could suppress increase in caspase-3 and caspase-9 activities following retinal damage.[4] In a randomized clinical trial, it was demonstrated that short-term saffron supplementation improves retinal flicker sensitivity in early AMD.[5] Saffron extract prevented selenite-induced cataract formation in Wistar rats, probably due to the fortification of antioxidant status, diminution of the concentration of lipid peroxidation, shielding the sulfhydryl groups, and suppression of proteolysis of the lens water-soluble fraction. These findings highlight the anticataractogenic potential of saffron by virtue of its antioxidant property.[6] These attractive effects propose its presence as a therapeutic agent in future. Though saffron and its constituents have shown multiple useful effects, clinical evidence is still scarce in this regard, and more comprehensive studies with special focus on human clinical trials are required. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Anticancer Effect and Molecular Targets of Saffron Carotenoids.

Saffron carotenoids, crocin and crocetin, have shown anticancer activity in various animal models of cancer and against different cancerous cell lines. The radical scavenging property and activation of antioxidant defense system are two well-known characteristics of these compounds. However, the results of the studies indicated other mechanisms could also be involved in this function. Insights into various molecular mechanisms of action for crocin and crocetin have been obtained in recent years. The results indicated that despite the structural similarity of crocin and crocetin, their anticancer effects may exert through different mechanisms. Particular interest concerns the ROS-dependent signaling pathways of crocetin. Saffron compounds are safe and may provide inexpensive therapy for treating cancer. They also have protective potential in targeting other disorders including diabetes, Alzheimer's and cardiovascular disease, cognitive deficits, ischemia-induced retinal damage, and many other diseases.

Crocetin, a carotenoid derivative, inhibits retinal ischemic damage in mice

Crocetin, an aglycone of crocin, is found both in the saffron crocus (Crocus starus L.) and in gardenia fruit (Gardenia jasminoides Ellis). We evaluated the protective effects of crocetin against the retinal ischemia induced by 5h unilateral ligation of both the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. The effects of crocetin (20mg/kg, p.o.) on ischemia/reperfusion-induced retinal damage were examined by histological, electrophysiological, and anti-apoptotic analyses. Data for anti-apoptotic analysis was obtained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Using immunohistochemistry and immunoblotting, the protective mechanism mediating the effects of crocetin was evaluated by examining crocetin's effects on the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG; used as a marker of oxidative stress) and on phosphorylations of mitogen-activated protein kinases [MAPK; viz. extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK) and p38], and the redox-sensitive transcription factors nuclear factor-kappa B (NF-κB) and c-Jun. The histological analysis revealed that ischemia/reperfusion (I/R) decreased the cell number in the ganglion cell layer (GCL) and the thickness of inner nuclear layer (INL), and that crocetin inhibited GCL and INL. ERG measurements revealed that crocetin prevented the I/R-induced reductions in a- and b-wave amplitudes seen at 5 days after I/R. In addition, crocetin decreased the numbers of TUNEL-positive cells and 8-OHdG-positive cells, and the phosphorylation levels of p38, JNK, NF-κB, and c-Jun present in the retina after I/R. These findings indicate that crocetin prevented ischemia-induced retinal damage through its inhibition of oxidative stress.

Prothymosin-α plays a defensive role in retinal ischemia through necrosis and apoptosis inhibition

Prothymosin-alpha (ProTalpha) causes a switch in cell death mode from necrosis to neurotrophin-reversible apoptosis in primary cultured cortical neurons. In the present study, post-ischemic administration (3 or 24 h, intravenously) of recombinant mouse ProTalpha without neurotrophins completely prevented ischemia-induced retinal damage accompanying necrosis and apoptosis, as well as dysfunction assessed by electroretinogram. Treatments with anti-erythropoietin (EPO) or brain-derived neurotrophic factor (BDNF) immunoglobulin G (IgG) reversed ProTalpha-induced inhibition of apoptosis. ProTalpha upregulated retinal EPO and BDNF levels in the presence of ischemia. Moreover, intravitreous administration of anti-ProTalpha IgG or an antisense oligodeoxynucleotide for ProTalpha accelerated ischemia-induced retinal damage. We also observed that ischemia treatment caused a depletion of ProTalpha from retinal cells. Altogether, these results suggest that the systemic administration of ProTalpha switches ischemia-induced necrosis to apoptosis, which in turn is inhibited by neurotrophic factors upregulated by ProTalpha and ischemia. ProTalpha released upon ischemic stress was found to have a defensive role in retinal ischemia.

Gene deletion and pharmacological inhibition of aldose reductase protect against retinal ischemic injury

Retinal ischemic injury is common in patients with diabetes, atherosclerosis, hypertension, transient ischemia attack and amaurosis fugax. Previously, signs of ischemic stress, such as pericyte loss, blood–retinal barrier breakdown and neovascularization, which can lead to occlusion of retinal vessels, have been prevented in diabetic db/db mice with aldose reductase (AR) null mutation. To determine the role in retinal ischemic injury of AR and sorbitol dehydrogenase (SDH), the first and second enzymes in the polyol pathway, mice with deletion of AR (AR−/−) or SDH-mutation (SDH−/−), or C57BL/6N mice treated with AR or SDH inhibitors were subjected to transient retinal artery occlusion (2h of occlusion and 22h of reperfusion) by the intraluminal suture method. Neuronal loss and edema observed in wildtype (AR+/+) retinas after transient ischemia were prevented in the retinas of AR−/− mice or C57BL/6N mice treated with an AR inhibitor, Fidarestat. Fewer TUNEL-positive cells and smaller accumulations of nitrotyrosine and poly(ADP-ribose) were also observed in the retinas of AR−/− mice. However, SDH−/− mice and C57BL/6N mice treated with SDH inhibitor, CP-470,711, were not protected against ischemia-induced retinal damage. Taken together, AR contributes to retinal ischemic injury through increased edema and free radical accumulation. Therefore, AR inhibition should be considered for the treatment of retinal ischemic injury often observed in diabetic patients.

Neuroprotective effect of cilostazol against retinal ischemic damage via inhibition of leukocyte-endothelial cell interactions.

Cilostazol, a selective platelet phosphodiesterase inhibitor, has been shown to reduce neuronal injury after transient cerebral ischemia. Its neuroprotective effect is thought to result from an antiplatelet function. This study was designed to evaluate the inhibitory effects of cilostazol against retinal ischemic damage focusing on leukocyte-endothelial cell interactions. Retinal ischemia was induced for 60 min in male Sprague-Dawley rats (n = 144) by temporary ligation of the optic sheath. Cilostazol was administered just before ischemia induction. Leukocyte behavior in the retinal microcirculation was evaluated in vivo with scanning laser ophthalmoscopy and ex vivo with fluorescence microscopy. Retinal expression of P-selectin, intracellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor were evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction. Ischemia-induced retinal damage was evaluated histologically. Treatment with cilostazol significantly suppressed leukocyte-endothelial cell interactions; the maximal numbers of rolling leukocytes were reduced by 77.6% (P < 0.01) 12 h after ischemia. Twenty-four hours after ischemia, adherent and accumulated leukocytes were also suppressed by treatment with cilostazol (36.1% and 20.4% respectively, P < 0.01). The expressions of P-selectin and ICAM-1 mRNA were suppressed significantly in cilostazol-treated retinas (P < 0.05). The retinal histological examination demonstrated a significant protective effect of cilostazol against ischemia-induced retinal damage (P < 0.01). The present study demonstrates that cilostazol attenuates retinal injury after transient ischemia via inhibition of leukocyte-endothelial cell interactions. This inhibitory effect on postischemic leukocyte-endothelial cell interactions might partially contribute to its neuroprotective effects.

Intravitreous Injection of a Membrane Depolarization Agent Causes Retinal Degeneration Via Matrix Metalloproteinase-9

Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated. Normal adult CD-1 mice were treated with KCl by intravitreal injection. MMP activity in retinal protein extracts was determined by gelatin zymography. Tissue localization of MMP-9 in the retina was determined by immunohistochemistry. MMP-9, MMP-2, tissue inhibitor of MMP (TIMP)-1, TIMP-2, Bax, and BCl-2 proteins in retinal extracts were determined by Western blot analysis. Apoptotic cell death in the retina was determined by TUNEL assays. Retinal damage was assessed by immunolocalization studies with antibodies against neurofilament-light (NF-L) and calretinin. Depolarizing concentrations of KCl induced a dose- and time-related upregulation in MMP-9 activity and protein in the retina. KCl-mediated MMP-9 upregulation was associated with an increase in proapoptotic protein Bax and apoptotic death of cells in the ganglion cell (GCL) and inner nuclear layer (INL), and subsequent loss of NF-L-positive ganglion cells and calretinin-positive amacrine cells. Intravitreal injection of KCl along with an N-methyl-d-aspartate (NMDA)-type glutamate receptor antagonist, MK-801, and a non-NMDA-type glutamate receptor antagonist, NBQX, resulted in a reduction in KCl-mediated MMP-9 upregulation in the retina. Furthermore, a synthetic MMP inhibitor inhibited KCl-mediated MMP-9 upregulation, which led to a significant attenuation of KCl-induced retinal damage. These results suggest that upregulation of MMP-9, in part, plays a causative role in KCl-induced retinal damage.

Platelets adhering to the vascular wall mediate postischemic leukocyte-endothelial cell interactions in retinal microcirculation.

Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo. Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia-reperfusion injury. This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Argatroban attenuates leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia.

Argatroban, a direct thrombin inhibitor, has been shown to reduce neural injury after transient cerebral ischemia. It has also been reported that this neuroprotective effect results from an anticoagulant function. This study was designed to evaluate quantitatively the inhibitory effects of argatroban on leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. Retinal ischemia was induced for 60 minutes in male Long-Evans rats by temporary ligation of the optic sheath (n=342). Argatroban was administered just after induction of ischemia. Leukocyte and platelet behavior in the retinal microcirculation was then evaluated in vivo with scanning laser ophthalmoscopy. The expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) was evaluated by reverse transcription-polymerase chain reaction. After 10 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. Treatment with argatroban suppressed leukocyte-endothelial cell interactions; the maximum numbers of rolling and accumulated leukocytes were reduced by 90.1% (P<0.05) and 58.7% (P<0.05), respectively, at 12 hours after reperfusion. Treatment with argatroban also suppressed platelet-endothelial cell interactions; the maximum numbers of rolling and adhering platelets were reduced by 91.8% (P<0.01) and 78.9% (P<0.01), respectively, at 12 hours after reperfusion. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P<0.01). Histologic examination demonstrated the protective effect of argatroban on ischemia-induced retinal damage (P<0.01). Argatroban treatment suppressed leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. This inhibitory effect on postischemic blood cell-endothelial cell interactions might partially contribute to its neuroprotective effects.

Inhibitory Effects of Antithrombin III on Interactions between Blood Cells and Endothelial Cells during Retinal Ischemia–Reperfusion Injury

Infiltrating leukocytes have long been widely thought to be key mediators of ischemia-reperfusion injury. Recently, however, evidence suggests that platelets accumulating in postischemic tissues also contribute to ischemia-reperfusion injury because of their inflammatory properties and promotion of formation of thrombi. This study was designed to evaluate quantitatively the inhibitory effects of antithrombin (AT)-III on the interactions between blood cells and retinal endothelial cells in vivo after transient retinal ischemia. Transient retinal ischemia was induced for 60 minutes in male Long-Evans rats by ligation of the optic nerve. AT III (250 U/kg) was administered intravenously just after induction of ischemia. Leukocyte and platelet behavior in the retina was evaluated in vivo with a scanning laser ophthalmoscope. Expression of P-selectin and intracellular adhesion molecule (ICAM)-1 in the postischemic retina was investigated by reverse transcription-polymerase chain reaction and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. Administration of AT III significantly inhibited leukocyte rolling along the major retinal veins and subsequent accumulation of leukocytes in the postischemic retina. Furthermore, the maximum number of rolling and adherent platelets was reduced by 76% (P < 0.01) and 48% (P < 0.01), respectively, at 12 hours after reperfusion. Immunohistochemical studies also revealed the suppressive effect of AT III on expression of P-selectin and ICAM-1. Finally, histologic examination demonstrated the protective effects of AT III against retinal damage after transient retinal ischemia. This study demonstrates the inhibitory effects of AT III on leukocyte and platelet recruitment to the postischemic retina, which may account for the neuroprotective properties of this alpha-2 globulin against retinal ischemia-reperfusion injury.

Protective Action of Nipradilol against Ischemia-Induced Retinal Damage in Rats

The purpose of this study was to investigate whether nipradilol, a β-blocker having both vasodilating and α₁-blocking activities, can protect retinal cells from the injury induced by ischemia and reperfusion. Rats were anesthetized and, after an intravitreal injection of nipradilol, the intraocular pressure was raised for 45 min to induce retinal ischemia and reperfusion. Before, and 3 and 7 days after the ischemia, electroretinograms were recorded. After the ischemia, the mean amplitude of the b-waves in rats receiving 5 µl of 1.0 × 10^–6 M nipradilol was significantly larger than of controls (injected with phosphate-buffered saline). Histologically, the reduction in the number of retinal ganglion cells (1.0 × 10^–6 M), and the thickness of the inner and outer plexiform layers and the inner nuclear layer (1.0 × 10^–6, 10^–7 and 10^–8 M) was suppressed by nipradilol. These results indicate that nipradilol protected the retina against retinal ischemia and reperfusion and should be considered for therapeutic use in cases of transient retinal ischemia.

Protective Effects of Selectin Ligands/Inhibitor (SKK-60060) against Retinal Ischemia-Reperfusion Injury

A newly developed selectin ligand inhibitor (SKK-60060) has been shown to block P- and L-selectins in vitro. We examined its inhibition of leukocyte–endothelial interactions in vivo against retinal ischemia-reperfusion injury and protective effects on ischemia-induced retinal damage.Retinal ischemia was induced by temporary ligation of the optic sheath for 60min in anesthetized pigmented rats. SKK-60060 was administered 5min before reperfusion and 4, 12, 24 and 48hr thereafter, and leukocyte dynamics in the retinal microcirculation were evaluated using acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was also assessed histologically.SKK-60060 treatment suppressed leukocyte rolling during the reperfusion period; their numbers in the SKK-60060-treated rats were reduced by 67.0% (P<0.01) and 53.2% (P<0.01) at 12 and 24hr, respectively. The subsequent leukocyte accumulation was also inhibited in SKK-60060-treated rats; accumulated leukocytes in the SKK-60060-treated rats were reduced by 72.8% (P<0.01) and 53.4% (P<0.01) at 12 and 24hr, respectively. Retinal venous vasodilation in SKK-60060-treated rats were significantly suppressed at each time point (P<0.05). Histological examination demonstrated protective effects of SKK-60060 on ischemia-induced retinal damage, which were more substantial in the inner retina (P<0.01).SKK-60060 significantly inhibits the leukocyte rolling along the major retinal veins and their accumulation during the reperfusion period. These results suggest therapeutic potential of SKK-60060 for ischemia-reperfusion injury.

Tacrolimus (FK506) Attenuates Leukocyte Accumulation After Transient Retinal Ischemia

Tacrolimus, an immunosuppressant agent, has been shown to reduce tissue injury and leukocyte accumulation after transient ischemia. This study was designed to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo after transient retinal ischemia and the protective effects of tacrolimus on ischemia-induced neural damage. Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic sheath. Tacrolimus was administered at 10 minutes after ischemic induction. At 4, 12, 24, and 48 hours after reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. Treatment with tacrolimus suppressed leukocyte rolling; the maximum number of rolling leukocytes was reduced by 60.1% at 12 hours after reperfusion (P<0.05). In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P<0.01). The subsequent leukocyte accumulation was reduced by 61.6% at 24 hours after reperfusion (P<0.01). Histological examination demonstrated the protective effect of tacrolimus on ischemia-induced retinal damage, which was more substantial in the inner retina (P<0.01). The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation and the therapeutic potency to neural injury after transient retinal ischemia.

Techniques for evaluating neuronal death of the retina in vitro and in vivo

This review describes the techniques to evaluate retinal neurodegeneration induced by excitatory amino acids and transient ischemia. Glutamate-induced neurotoxicity was examined in cultured rat cortical cells. Cultures obtained from the retinas of fetal rats were incubated in Eagle's minimal essential medium supplemented with 10% fetal calf serum or 10% horse serum at 37 degrees C in a humidified 5% CO2 atmosphere for 10-14 days. The neurotoxicity induced by glutamate was quantified by trypan blue exclusion. The viability of cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 1 hr. N-methyl-D-aspartate (NMDA)-induced retinal damage was examined in adult rats. Transverse sections of the retinas through the optic disk were stained with hematoxylin and eosin. A single intravitreal injection of NMDA damaged the ganglion cell layer and the inner plexiform layer without affecting the other retinal layers 7 days after injection. Retinal ischemia was induced by elevating the intraocular pressure for 45 min through the needle which was placed in the anterior chamber. Ischemia-induced retinal damage was inhibited by MK-801. These results indicate that the techniques described in this review can be employed to develop new drugs possessing neuroprotective action against neurodegeneration that occurs during retinal ischemia.

Glutamate receptor antagonists protect against ischemia-induced retinal damage

The effects of intravitreal injections of excitatory amino acid receptor antagonists have been studied on the ischemic neuronal damage induced by photochemical occlusion of the retinal vessels. Rats were systemically injected with rose bengal fluorescein dye and one of their eyes was exposed to bright light. The activities of the enzymes, choline-acetyltransferase and glutamate decarboxylase, were measured as an index of neuronal loss in the lesioned tissue. Lesioned retinas had a 75 ± 5% reduction in choline-acetyltransferase activity and a 72 ± 8% reduction in glutamate-decarboxylase activity, suggesting that the lesion causes a massive loss of retinal neurons, which use acetylcholine or γ-aminobutyric acid (GABA) as neurotransmitter. A single intravitreal injection of excitatory amino acid receptor antagonists, performed immediately after the lesion, significantly reduced this loss. Both α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N- methyl- D- aspartate ) (NMDA) types of ionotropic glutamate receptor antagonists were active in a dose-dependent manner. Almost complete protection was also obtained with relatively large doses of thiokynurenic acid (400 nmol), a non-selective antagonist of both AMPA and NMDA glutamate receptors, while 7-Cl-thiokynurenic acid, a potent and selective glycine receptor antagonist, was not active up to 200 nmol. These results strongly suggest that excitotoxic mechanisms are involved in ischemia-induced neuronal death in the retina and that appropriate treatments with antagonists of both AMPA and NMDA receptor types may significantly reduce this damage.

Photochemically-induced lesion of the rat retina: a quantitative model for the evaluation of ischemia-induced retinal damage

The effects of ischemia-induced retinal damage were quantitatively evaluated in rats with the aim of obtaining a suitable model to study the pathogenesis of the loss of retinal neurons after ischemic episodes. Anaesthetized rats were injected with 80 mg/kg i.v. of the fluorescein rose bengal dye and one eye was exposed to cold light for different periods (from 5 to 30 min). The animals were sacrificed at different times (1 and 4 hr; 2 and 7 days) after the lesion and the photochemically-induced damage was evaluated. The damaged retinae appeared thicker, numerous neurons of the inner nuclear layers showed swelling of the perinuclear cytoplasm and the retinal vessels were enlarged. The activity of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), two marker enzymes of the GABAergic and cholinergic neurons, significantly decreased, indicating a degeneration of GABAergic and cholinergic amacrine cells.