Neuroinflammation in glaucoma：soluble tumor necrosis factor alpha and the connection with excitotoxic damage

Inflammation is a complex and highly regulated response that occurs early after infection or injury.This process is initiated by cells of the immune system to re-establish tissue homeostasis.When the injury is persistent,however,chronic inflammation leads to overproduction of noxious mediators that contribute to cell dysfunction and death.

AB015.Metabolic stress in glaucoma engages early activation of the energy biosensor adenosine monophosphate-activated protein kinase leading to neuronal dysfunction

Background:Metabolic stress has been proposed to contribute to neuronal damage in glaucoma,but the mechanism driving this response is not understood.The adenosine monophosphate-activated protein kinase(AMPK)is a master regulator of energy homeostasis that becomes active at the onset of energy stress.AMPK is a potent inhibitor of the mammalian target of rapamycin complex 1(mTORC1),which we showed is essential for the maintenance of retinal ganglion cell(RGC)dendrites,synapses,and survival.Here,we tested the hypothesis that AMPK is an early mediator of metabolic stress in glaucoma.Methods:Unilateral elevation of intraocular pressure was induced by injection of magnetic microbeads into the anterior chamber of mice expressing yellow fluorescent protein in RGCs.Inhibition of AMPK was achieved by administration of siRNA or compound C.RGC dendritic trees were 3D-reconstructed and analyzed with Imaris(Bitplane),and survival was assessed by counting Brn3a or RBPMS-labeled soma and axons in the optic nerve.RGC function was examined by quantification of anterograde axonal transport after intraocular administration of cholera toxinβ-subunit.Retinas from glaucoma patients were analyzed for expression of active AMPK.Results:Ocular hypertension triggered rapid upregulation of AMPK activity in RGCs concomitant with loss of mTORC1 function.AMPK inhibition with compound C or siRNA effectively restored mTORC1 activity and promoted an increase in total dendritic length,surface and complexity relative to control retinas.Attenuation of AMPK activity led to robust RGC soma and axon survival.For example,95%of RGCs(2,983±258 RGCs/mm2,mean±S.E.M.)survived with compound C compared to 77%in vehicle-treated eyes(2,430±233 RGCs/mm2)(ANOVA,P<0.001)at three weeks after glaucoma induction(n=8-10/group).Importantly,blockade of AMPK activity effectively restored anterograde axonal transport.Lastly,RGC-specific upregulation of AMPK activity was detected in human glaucomatous retinas relative to age-matched controls(n=10/group).Conclusions:Metabolic stress in glaucoma involves AMPK activation and mTORC1 inhibition promoting early RGC dendritic pathology,dysfunction and neurodegeneration.

AB018.Ocular hypertension promotes early mitochondrial fragmentation in retinal endothelial cells in a mouse model of glaucoma

Background:Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit.Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma.Here,we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier(BRB).Methods:Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice,a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells.Capillary density,mitochondrial volume,and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software.Dynamin-related protein(DRP-1),mitofusin-2(MFN-2)and optic atrophy-1(OPA-1)expression were assessed by western blot analysis of enriched endothelial cells.Mitochondrial structure was evaluated by transmission electron microscopy(TEM)and oxygen consumption rate was monitored by Seahorse analysis.The integrity of the BRB was evaluated by quantifying Evans blue leakage.Results:Our data demonstrate that two and three weeks after ocular hypertension induction,the total mitochondria volume in endothelial cells decreased from 0.140±0.002µm3 from non-injured retinas to 0.108±0.005 and 0.093±0.007µm3,respectively in glaucomatous eyes(mean±S.E.M,ANOVA,P<0.001;N=6/group).Frequency distribution showed a substantial increase of smaller mitochondria complexes(<0.5µm3)in endothelial cells from glaucomatous retinas.Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas,while MFN-2 and OPA-1 expression was not affected.Structural alteration in endothelial cell mitochondria was confirmed by TEM,which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas(n=3-6/group).In addition,this model did not trigger changes in the density of the vascular network,suggesting that mitochondrial fragmentation was not due to endothelial cell loss.Conclusions:This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria,contributing to vascular dysfunction in glaucoma.

AB011.Live imaging of retinal pericytes:evidence for early calcium uptake,capillary constriction and vascular dysregulation in ocular hypertension glaucoma

Background:Pericytes are contractile cells that wrap along the walls of capillaries.In the brain,pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand.The contribution of pericytes to microvascular deficits in glaucoma is currently unknown.To address this,we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.Methods:Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes(NG2-DsRed).Minimally invasive,multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one,two and three weeks after glaucoma induction.In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4.Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.Results:Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes.Calcium uptake correlated directly with the narrowing of capillaries in the superficial,inner,and outer vascular plexuses(capillary diameter:naïve control=4.7±0.1μm,glaucoma=4.0±0.1μm,n=5-6 mice/group,Student’s t-test P<0.05).Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries(≤3μm)and a decrease in larger-diameter microvessels(≥5-9μm)at three weeks after induction of ocular hypertension(n=5-6 mice/group,Student’s t-test P<0.05).Conclusions:Our data support two main conclusions.First,two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression.Second,ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction.This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.