Selective over-expression of endothelin-1 in endothelial cells exacerbates inner retinal edema and neuronal death in ischemic retina

The level of endothelin-1 (ET-1), a potent vasoconstrictor, was associated with retinopathy under ischemia. The effects of endothelial endothelin-1 (ET-1) over-expression in a transgenic mouse model using Tie-1 promoter (TET-1 mice) on pathophysiological changes of retinal ischemia were investigated by intraluminal insertion of a microfilament up to middle cerebral artery (MCA) to transiently block the ophthalmic artery. Two-hour occlusion and twenty-two-hour reperfusion were performed in homozygous (Hm) TET-1 mice and their non-transgenic (NTg) littermates. Presence of pyknotic nuclei in ganglion cell layer (GCL) was investigated in paraffin sections of ipsilateral (ischemic) and contralateral (non-ischemic) retinae, followed by measurement of the thickness of inner retinal layer. Moreover, immunocytochemistry of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and aquaporin-4 (AQP4) peptides on retinal sections were performed to study glial cell reactivity, glutamate metabolism and water accumulation, respectively after retinal ischemia. Similar morphology was observed in the contralateral retinae of NTg and Hm TET-1 mice, whereas ipsilateral retina of NTg mice showed slight structural and cellular changes compared with the corresponding contralateral retina. Ipsilateral retinae of Hm TET-1 mice showed more significant changes when compared with ipsilateral retina of NTg mice, including more prominent cell death in GCL characterized by the presence of pyknotic nuclei, elevated GS immunoreactivity in Müller cell bodies and processes, increased AQP-4 immunoreactivity in Müller cell processes, and increased inner retinal thickness. Thus, over-expression of endothelial ET-1 in TET-1 mice may contribute to increased glutamate-induced neurotoxicity on neuronal cells and water accumulation in inner retina leading to edema. © 2011 Cheung et al.published_or_final_versio

Endothelin-1 over-expression leads to blood-brain barrier disruption resulting in increased infarct and edema after focal ischemia

Poster Session P13: Neurotoxicity, Neurodegeneration, Neuroprotection, Ischemia/Hypoxia and ApoptosisInduced endothelin-1 (ET-1) expression was observed in astrocytes after experimental ischemic stroke, suggesting a potential role of astrocytic ET-1 in ischemic brain injury. Previously, we showed that transgenic mice over-expressing ET-1 in astrocytes (GET mice) displayed increased cerebral infarct size and more severe neurological deficits upon focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Here, the expression profile of the endothelin system was examined in GET mouse brains after MCAO. Quantitative real-time PCR analyses showed that ET-1 mRNA levels in GET ipsilateral mouse brain after MCAO was further increased, while ET-3 levels stayed relatively constant when compared with sham-operated animals. There was an up-regulation of ETA receptor mRNA levels in GET ipsilateral brains while ETB receptor mRNA levels remained unchanged. In addition, Evans blue extravasation studies showed increased blood–brain barrier (BBB) breakdown in GET mice after MCAO, correlating with our findings that GET mice displayed increased brain swelling and brain water content. Moreover, linear regression analysis showed that severity of infarction and brain swelling correlated with the exacerbation of neurologic deficits. These results suggested that increased astrocytic ET-1 resulted in more BBB disruption leading to increased formation of brain edema, infarct and neurologic deficits after focal cerebral ischemia.link_to_OA_fulltex

Role of endothelial cell overexpressed endothelin-1 (ET-1) in mouse model of ischemic stroke

Endothelin-1 (ET-1) has been implicated in clinical stroke. We have demonstrated ET-1 mRNA expression is induced in astrocytes and endothelial cells after ischemic condition, suggesting that both of these cells synthesize ET-1 under this stress condition. ET-1 protected primary cultured astrocytes from ischemic stress. However, transgenic mice with over-expression of ET-1 in astrocytes displayed more severe neurological deficit and increased infract volume, suggesting that astrocytic ET-1 have neurotoxic effect on neurons. To further investigate the role of endothelial ET-1 in cerebral ischemic injury, several transgenic mouse lines (TET) were generated by microinjecting the construct, which include ET cDNA with SV40 poly A under tyrosine kinase receptor-specific for endothelial cell (Tie-1) promoter. TET mouse lines were further characterized for ET-1 over-expression. The RT-PCR analysis using the primers specific for tranagene ET-1 showed that transgene ET-1 is only expressed in the brain from TET mice. Total expression of ET-1 mRNA was also increased in brain of transgenic mice when compared to that of non-transgenic mice by semi-quantitative RT-PCR. In situ hybridization and immunocytochemical analyses showed that the increased ET-1 mRNA and peptide expressions are only in endothelial cells of cerebral vessels from the TET mice. ET-1 peptide level was also detected in the endothelial cells of cerebral vessel from TET mice. Under normal condition, no gross morphological change has been found in the brain. However, TET mice showed more severe neurological deficit, larger infarct size and volume after transient middle cerebral artery occlusion (MCAO), suggesting that overexpressing of ET-1 in endothelial cells is deleterious to neuronal survival under ischemic condition. Our present TET model will serve as an ideal model for studying the role of endothelial ET-1 in the pathogenesis of ischemic stroke.link_to_subscribed_fulltex

Endothelin-1 mediated signaling for increased blood-brain barrier breakdown and infarct after transient focal cerebral ischemia

Induced endothelin-1(ET-1) expression was observed in astrocytes after experi-mental ischemic stroke, suggesting a potential role of astrocytic ET-1 in ischemicbrain injury. Previously, we showed that transgenic mice over-expressing ET-1 inastrocytes (GET mice) displayed increased cerebral infarct size and more severeneurological deficits upon focal cerebral ischemia induced by middle cerebralartery occlusion (MCAO). However, the mechanism behind astrocytic ET-1 onischemia-induced brain injury was not clear. Here, expression profile of ET systemwas examined by real-time PCR analyses. In GET brain after MCAO ET-1 levelwas further increased while ET-3 level was lowered. ETA receptor level was up-regulated while ETB receptor level remained unchanged. After MCAO GET brainshowed lower occludin level and increased Evans blue extravasation, indicatingincreased blood–brain barrier (BBB) breakdown which correlated with our resultsthat GET mice displayed increased brain swelling and brain water content. Linearregression analysis also showed correlation of severity of infarction and brainswelling with neurological deficit exacerbation. There was significant p35 down-regulation after MCAO, suggesting involvement of p25/cdk5 cell death pathwayin increased infarct formation. Caspase9 expression was also increased. Theseresults suggested that increased astrocytic ET-1 resulted in BBB disruptionpossibly leading to increased formation of brain edema and swelling, infarct andneurological deficits mediated through cdk5 pathway and, therefore, imposedadverse effects on brain injury after focal cerebral ischemia. Further globalscreening of ET-1-mediated signaling after MCAO will be discussed.Acknowledgements: Supported by RGC and UGC of Hong Kong (AoE/B-15/01).link_to_OA_fulltex

Aldose reductase-deficient mice were protected from the neuro-retinal injury after carotid artery transient ischemia

Previously, we showed that AR deficiency prevents retinal abnormalities in 15 mos. diabetic db/db mice. Here, we explored the role of AR in ischemic retinal injury, since the retinal damage associated with diabetes is thought to be the consequence of ischemia. To induce retinal ischemia, we blocked the ophthalmic artery, which gives rise to central retinal artery, by using 2 hrs of internal carotid artery occlusion and 22 hrs of reperfusion by intraluminal insertion of filament. Here, we compared the ipsilateral retina of 8 wks old AR-wild type (AR+/+) to the similar aged AR−/− mice after transient ischemia. After the transient ischemia, there was a significant increase of total retinal thickness and neuronal cell loss in the ipsilateral side of AR+/+ retinas, but not of the AR−/− retinas when compared with the contralateral sides, respectively. Increase of neuro-retinal damage after the transient ischemia triggered the increased expression of glutamine synthetase, which was shown to protect against neuronal degeneration in injured retinal tissues, and markers of glial reactivation, such as glial acidic fibrillary protein (GFAP) and S-100 in Müller cells in the ipsilateral side of AR+/+, but not of the AR−/− retinas, when compared with the contralateral sides, respectively. This suggests that AR deficiency prevents neuro-retinal damage and glial reactivation induced by the carotid artery transient ischemia. This research was supported by Research Grants Council of Hong Kong Grant to Dr. S.K. Chung