Morphological and functional observations of a novel model of retinal ischemia injury induced by bilateral carotid artery stenosis in mice

AIM:To investigate the features of retinal ischemic injuries in a novel mouse model with bilateral carotid artery stenosis(BCAS).METHODS:BCAS was induced with microcoil implantation in 6-8-week-old C57BL6 mice.Cerebral blood flow was monitored at 2,7,and 28d postoperatively.Retinal morphological changes were evaluated by fundus photography and hematoxylin-eosin staining.Fluorescein fundus angiography(FFA)was performed to detect retinal vascular changes and circulation.The levels of apoptosis,activation of neurogliosis,and expression of hypoxiainducible factor(HIF)-1αin the retina were assessed by Western blotting and immunofluorescence staining,followed by retinal ganglion cell(RGC)density detection.Additionally,electrophysiological examinations including photopic negative response(PhNR)was also performed.RESULTS:The mice demonstrated an initial rapid decrease in cerebral blood flow,followed by a 4-week recovery period after BCAS.The ratio of retinal artery and vein was decreased under fundus photography and FFA.Compared with the sham mice,BCAS mice showed thinner retinal thickness on day 28.Additionally,apoptosis was increased and RGC density was decreased mainly in peripheral retinal region.Neurogliosis was mainly located in the inner retinal layers,with a stable increase in HIF-1αexpression.The dark-adapted electroretinogram showed a notable reduction in the a-,b-,and oscillatory potential(OP)wave amplitudes between days 2 and 7;this gradually recovered over the following 4wk.However,the b-and OPwave amplitudes were still significantly decreased on PhNR examination on day 28.CONCLUSION:BCAS can result in relatively mild retinal ischemia injuries in mice,mainly in the inner layer and peripheral region.Our study provides a novel animal model for investigating retinal ischemic diseases.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

Multimodal imaging in acute retinal ischemia: spectral domain OCT, OCT-angiography and fundus autofluorescence

AIM： To describe retinal findings of various imaging modalities in acute retinal ischemia. METHODS： Fluorescein angiography（FA）, spectral domain optical coherence tomography（SD-OCT）, OCTangiography（OCT-A） and fundus autofluorescence（FAF） images of 13 patients（mean age 64y, range 28-86y） with acute retinal ischemia were evaluated. Six suffered from branch arterial occlusion, 2 had a central retinal artery occlusion, 2 had a combined arteriovenous occlusions, 1 patient had a retrobulbar arterial compression by an orbital haemangioma and 2 patients showed an ocular ischemic syndrome.RESULTS： All patients showed increased reflectivity and thickening of the ischemic retinal tissue. In 10 out of 13 patients SD-OCT revealed an additional highly reflective band located within or above the outer plexiform layer. Morphological characteristics were a decreasing intensity with distance from the fovea, partially segmental occurrence and manifestation limited in time. OCT-A showed a loss of flow signal in the superficial and deep capillary plexus at the affected areas. Reduced flow signal was detected underneath the regions with retinal edema. FAF showed areas of altered signal intensity at the posterior pole. The regions of decreased FAF signal corresponded to peri-venous regions. CONCLUSION： Multimodal imaging modalities in retinal ischemia yield characteristic findings and valuable diagnostic information. Conventional OCT identifies hyperreflectivity and thickening and a mid-retinal hyperreflective band is frequently observed. OCT-A examination reveals demarcation of the ischemic retinal area on the vascular level. FAF shows decreased fluorescence signal in areas of retinal edema often corresponding to peri-venous regions.

Early activation of caspase-1 after retinal ischemia and reperfusion injury in mice

Background Caspases are important in the signaling pathway of cellular apoptosis Caspase-3 protein expression has been shown to increase and parallel to neuronal apoptosis in retinal ischemia injury This study was to determine whether caspase-1 is involved in neuronal cell death or in retinal ischemia and reperfusion injury Methods In twenty-one adult mice, ischemia was induced by increasing the intraocular pressure The animals were sacrificed at 1 hour, 3 hours, 6 hours, 1 day, 3 days and 7 days after reperfusion Frozen sections were used for caspase-1 immunostaining and TUNEL labeling Results In normal retina, no caspase-1 positive cells were seen One hour after ischemia, numerous positive cells were noted in the ganglion cell layer (GCL) and inner side of inner nuclear layer (INL) At 3 hours, caspase-1 positive cells continued to increase and peaked at 6 hours, then decreased significantly at 1 day TUNEL positive cells were detected at 3 hours and peaked at 1 day after ischemia Double labeling of caspase-1 and TUNEL only showed few cells with co-localization after ischemia Conclusion Caspase-1 immunoreactivity preceds to the TUNEL labeling in the GCL and INL after retinal ischemia and reperfusion injury and its early activation may play an important role in the initiation of neuronal apoptosis

Quantitative analysis of retinal intermediate and deep capillary plexus in patients with retinal deep vascular complex ischemia

AIM: To quantitatively analyze the retinal intermediate and deep capillary plexus(ICP and DCP) in patients with retinal deep vascular complex ischemia(RDVCI), using 3D projection artifacts removal(3D PAR) optical coherence tomography angiography(OCTA).METHODS: RDVCI patients and gender-and agematched healthy controls were assessed and underwent OCTA examinations. The parafoveal vessel density(PFVD) of retinal deep vascular complex(DVC), ICP, and DCP were analyzed, and the percentage of reduction(PR) of PFVD was calculated.RESULTS: Twenty-four eyes in 22 RDVCI patients(20 in acute phase and 4 in chronic phase) and 24 eyes of 22 healthy subjects were enrolled as the control group. Significant reduction of PFVD in DVC, ICP, and DCP was observed in comparison with the controls(DVC: acute: 43.59%±6.58% vs 49.92%±5.49%, PR=12.69%;chronic: 43.50%±3.33% vs 51.20%±3.80%, PR=15.04%. ICP: acute: 40.28%±7.91% vs 46.97%±7.14%, PR=14.23%;chronic: 41.48%±2.87% vs 46.43%±3.29%, PR=10.66%. DCP: acute: 45.44%±8.27% vs 51.51%±9.97%, PR=11.79%;chronic: 37.78%±3.48% vs 51.73%±5.17%, PR=26.97%;all P<0.05). No significant PR difference was found among DVC, ICP, and DCP of RDVCI in acute phase(P=0.812), but significant difference in chronic phase(P=0.006, DVC vs DCP, ICP vs DCP). No significant difference in PR between acute and chronic phases in the DVC(P=0.735) or ICP(P=0.681) was found, but significant difference in the DCP(P=0.041).CONCLUSION: The PFVD of DVC, ICP, and DCP in RDVCI is significantly decreased in both acute and chronic phases. ICP impairment is stabilized from acute to chronic phase in RDVCI, whereas subsequent DCP impairment is uncovered and can be explained by ischemia-reperfusion damage.

Retinal vessel diameter changes induced by transient high perfusion pressure

·AIM: To investigate the effects of transient high perfusion pressure on the retinal vessel diameter and retinal ganglion cells.·METHODS: The animals were divided into four groups according to different infusion pressure and infusion time(60 mm Hg-3min, 60 mm Hg-5min, 100 mm Hg-3min, 100 mm Hg-5min). Each group consisted of six rabbits. The left eye was used as the experimental eye and the right as a control. Retinal vascular diameters were evaluated before, during infusion, immediately after infusion, 5min, 10 min and 30 min after infusion based on the fundus photographs. Blood pressure was monitored during infusion. The eyes were removed after 24 h.Damage to retinal ganglion cell(RGC) was analyzed by histology.·RESULTS: Retina became whiten and papilla optic was pale during perfusion. Measurements showed significant decrease in retinal artery and vein diameter during perfusion in all of the four groups at the proximal of the edge of the optic disc. The changes were significant in the 100 mm Hg-3min group and 100 mm Hg-5min group compared with 60 mm Hg-3min group(P 1=0.025, P 2=0.000).The diameters in all the groups recovered completely after 30 min of reperfusion. The number of RGC)showed no significant changes at the IOP in 100 mm Hg with5 min compared with contralateral untreated eye(P 】0.05).·CONCLUSION: Transient fluctuations during infusion lead to temporal changes of retinal vessels, which could affect the retinal blood circulation. The RGCs were not affected by this transient fluctuation. Further studies are necessary to evaluate the effect of pressure during realtime phacoemusification on retinal blood circulation.

Protection of retinal ganglion cells against optic nerve injury by induction of ischemic preconditioning

AIM： To explore if ischemic preconditioning （IPC） can enhance the survival of retinal ganglion cells （RGCs） after optic nerve axotomy. METHODS： Twenty-four hours prior to retinal ischemia 60min or axotomy, IPC was applied for ten minutes in groups of （n=72） animals. The survival of RGCs, the cellular expression of heat shock protein 27 （HSP27） and heat shock protein 70 （HSP70） and the numbers of retinal microglia in the different groups were quantified at 7 and 14d post-injury. The cellular expression of HSP27 and HSP70 and changes in the numbers of retinal microglia were quantified to detect the possible mechanism of the protection of the IPC. RESULTS： Ten minutes of IPC promoted RGC survival in both the optic nerve injury （IPC-ONT） and the retinal ischemia 60min （IPC-IR60） groups, examined at 7d and 14d post-injury. Microglial proliferation showed little correlation with the extent of benefit effects of IPC on the rescue of RGCs. The number of HSP27-positive RGCs was significantly higher in the IPC-ONT group than in the sham IPC-ONT group, although the percentage of HSP27-positive RGCs did not significantly differ between groups. For the IPC-IR60 group, neither the number nor the percentage ofthe HSP27-positive RGCs differed significantly between the IPC and the sham-operated groups. The number of HSP70-positive RGCs was significantly higher for both the IPC-ONT and the IPC-IR60 experimental groups, but the percentages did not differ. CONCLUSION： The induction of IPC enhances the survival of RGCs against both axotomy and retinal ischemia.

Aqueous levels of erythropoietin in acute retinal vein occlusion with macular edema

AIM:To investigate the aqueous erythropoietin(EPO)levels and associated factors in patients with acute retinal vein occlusion(RVO).METHODS:The aqueous EPO level was measured in patients with macular edema(ME)secondary to acute branched retinal vein occlusion(BRVO)or central retinal vein occlusion(CRVO).Aqueous fluid from cataract patients served as the control.We also evaluated whether aqueous level of EPO was associated with factors such as serum EPO level,non-perfusion area,central macular thickness(CMT),and arterio-venous(AV)transit timeRESULTS:Twenty-seven RVO patients(16 BRVO,11CRVO)and 9 control subjects were enrolled in the study.The aqueous EPO level(mU/mL)was higher in RVO(68.2±54.3)than that in the control subjects(12.9±5.9).More specifically,the aqueous EPO level was higher in CRVO(118.9±52.1)than that in BRVO(33.3±10.8).However,no differences were found in serum EPO levels among three groups.CMT in RVO patients had a positive correlation with the aqueous EPO level(r=0.66).Also,in terms of non-perfusion area,the aqueous EPO levels were more elevated in the ischemic subgroup than in the non-ischemic subgroup in both BRVO and CRVO.CONCLUSION:Aqueous EPO levels are elevated in patients with macular edema secondary to recent onset RVO.Patients with CRVO have higher EPO levels than those with BRVO.The aqueous EPO level in RVO has a positive correlation with CMT and is associated with non-perfusion area.These results suggest that the aqueous EPO level could be associated with retinal ischemia and may be involved in the pathogenesis of macular edema secondary to RVO.

The novel chalcone analog L2H17 protects retinal ganglion cells from oxidative stress-induced apoptosis

Chalcone is a plant metabolite widely found in fruits,vegetables,spices and tea,and has anti-tumor,anti-inflammation,immunomodulation,antibacterial and anti-oxidation activities,as well as many other pharmacological and biological effects.Our team has shown that its analogs have antioxidant activity,and oxidative stress is a pathological hallmark of retinal ischemia/reperfusion injury that can lead to retinal damage and visual loss.This investigation aims to identify a chalcone that protects retinal ganglion cells in vitro from the effects of oxidative stress and examine its mechanism.Rat retinal ganglion cell-5 cells were pretreated with chalcones and then exposed to tert-butyl hydroperoxide that causes oxidative damage.Controls received dimethyl sulfoxide only or tert-butyl hydroperoxide in dimethyl sulfoxide.Only（E）-3,4-dihydroxy-2′-methylether ketone（L2 H17）,of the five chalcone analogs,markedly increased the survival rate of oxidatively injured RGC-5 cells.Thus,subsequent experiments only analyzed the results of the L2 H17 intervention.Cell viability and apoptosis were measured.Intracellular superoxide dismutase and reactive oxygen species levels were used to assess induced oxidative stress.The mechanism of action by L2 H17 was explored by measuring the ER stress/UPR pathway and the expression and localization of Nrf2.All results demonstrated that L2 H17 could reduce the apoptosis of oxidatively injured cells,inhibit caspase-3 activity,increase Bcl-2 expression,decrease Bad expression,increase the activity of superoxide dismutase,inhibit the production of reactive oxygen species,increase Nrf2 immunoreactivity,and reduce the activating transcription factor 4,phospho-eukaryotic initiation factor 2 and CHOP expression.L2 H17 protects retinal ganglion cells induced by oxidative stress by regulating Nrf2,which indicates that it has the potential to become a drug for retinal ischemia/reperfusion.

Connexins in neurons and glia: targets for intervention in disease and injury

Both neurons and glia throughout the central nervous system are organized into networks by gap junctions. Among glia, gap junctions facilitate metabolic homeostasis and intercellular communication. Among neurons, gap junctions form electrical synapses that function primarily for communication. However, in neurodegenerative states due to disease or injury gap junctions may be detrimental to survival. Electrical synapses may facilitate hyperactivity and bystander killing among neurons, while gap junction hemichannels in glia may facilitate inflammatory signaling and scar formation. Advances in understanding mechanisms of plasticity of electrical synapses and development of molecular therapeutics to target glial gap junctions and hemichannels offer new hope to pharmacologically limit neuronal degeneration and enhance recovery.