Study On The Mechanism Of Retinal Degeneration In Rats Under Simulated Weightlessness

Background Mid-and-long-term spaceflight can cause ocular vision loss,optic disc edema,choroidal folds,thickening of nerve fiber layer,flat back of eye,and changes in distant vision and other symptoms,which are collectively referred to as Spaceflight-associated Neuro-ocular Syndrome(SANS for short).These eye changes can lead to decreased visual function,and some symptoms can last for years.SANS has become one of the most concerned issues for aerospace medicine and ophthalmology workers home and abroad.It is generally believed that SANS is mainly caused by head to head transfer of body fluids in microgravity environment,which causes venous congestion of head and neck,cerebrospinal fluid circulation disorder,intracranial pressure and optic nerve subarachnoid cerebrospinal fluid pressure increase,resulting in changes in the pressure gradient on the optic papilla,thus subsequently leading to changes in the anatomical structure of the optic papilla and corresponding ocular symptoms as well as visual dysfunction,which is also known as Visual impairment intracranial pressure(VIIP).We believe that SANS may be related to vein circulation disorder and may be the main initiating factor leading to eye injury.Objective In order to verify this hypothesis,this study intends to carry out this research by using tail suspension rat model to simulate weightlessness environment to observe the dynamic effects of simulated weightlessness on retinal function,eye structure and circulation,and to explore the molecular mechanism of choroidal vascular endothelial cell injury in microgravity environment,and to provide theoretical basis for clarifying the pathogenesis of SANS.Simultaneously,it provides intervention targets and strategies for combating SANS caused by long-term manned space flight.Methods1.The tail suspension model was used to simulate the effects of microgravity.SD rats were randomly divided into 10 groups: tail suspension group for 4 weeks(TS4W),tail suspension group for 5 weeks(TS5W),tail suspension group for 6 weeks(TS6W),tail suspension group for 7 weeks(TS7W)and tail suspension group for 8 weeks(TS8W),and corresponding control group was set up at each suspension time point.After the tail suspension simulated weightlessness for 4 weeks,the retinal function of rats was evaluated by Electroretinogram(ERG),the intraocular pressure was evaluated by intraocular pressure(IOP)technique,the changes of lens and ocular surface were evaluated by slit-lamp,and the changes of rat fundus were evaluated by small animal fundus camera in vivo.Optical coherence tomography angiography(OCTA)was used to evaluate choroidal vessel thickness,retinal thickness and choroidal capillary blood perfusion area respectively in each group.2.The tail suspension animal model was used to simulate the effect of microgravity.After the tail suspension simulated weightlessness for 4 weeks,HE staining was used to observe the pathological changes of retina.The morphology and structure of mitochondria in retinal layer were observed by transmission electron microscope.Mitochondrial DNA(mt-DNA)copy number was detected by RT-PCR.ROS and MDA kits were utilized to detect oxidative stress in retinal tissues.ATP content in retinal tissue was detected by ATP kit.Mitochondrial respiratory chain complex I and III kits were applied to detect mitochondrial respiratory chain enzyme activity in retinal tissue.Western blot was used to detect the expression of mitochondrial homeostasis and apoptosis related proteins.TUNE staining was used to detect retinal apoptosis.The molecular mechanism of the degeneration of the outer nuclear layer of retina was determined by proteomics.3.The tail suspension animal model was used to simulate the influence of microgravity.After the tail suspension simulated weightlessness for 4 weeks,the molecular protein expression related to Müller cell reverse differentiation,neural differentiation and gliosis was detected by immunofluorescence staining,and the molecular protein expression related to Müller cell neurotrophic factor was spotted by Western blot.4.The tail suspension model was used to simulate the effect of microgravity.After 4weeks of simulated weightlessness,the morphology and structure of chorioidal vascular endothelial cells and retinal pigment epithelial cells were observed by transmission electron microscopy.The expression of tight junction proteins Cx43,ZO-1 and occludin in the outer blood-retinal barrier were spotted by immunofluorescence staining and Western Blotting(WB)technique.The expression of Piezo1 in rat chorioidal vascular endothelial cells was observed by immunohistochemistry and WB techniques.The expression of Piezo1 in choroidal vascular endothelial cells was observed by applying different fluid shear forces(5,10,15 dyn/cm2,1 dyn/cm2=0.1Pa)to choroidal vascular cells with a fluid shear force device.In combination with Piezo1 agonist and inhibitor,the calcium content in chorioidal vascular endothelial cells,the calcium content in mitochondria,the changes of intracellular reactive oxygen species,mitochondrial membrane potential and the apoptosis rate of chorioidal vascular endothelial cells were observed by confocal laser microscopy and flow cytometry.Western blot technique was utilized to detect the expression of mitochondrial homeostasis related protein and endothelial cell compact junction protein.Small interfering RNA(si RNA)was applied to knock down the expression of Piezo1 gene in chorioidal vascular endothelial cells,and WB technology was used to detect the expression of apoptosis-related proteins.The molecular mechanism of the damage induced by Piezo1 to chorioidal vascular endothelial cells was determined by RNA-seq technique.Result1.The effect of medium and long-terms simulated weightlessness on eye structure and function in rats During the experiment,the weight of each group of rats increased gradually and the growth state was perfect.After the experiment,compared with the CON group,both the weight of rats after TS7 W and the soleus muscle mass of rats after TS4 W decreased significantly,suggesting that the tail suspension simulated weightlessness model was successfully constructed.It was observed by ERG that the light and dark vision 3.0 ERG response amplitude decreased drastically after TS5 W.By observing the fundus of the rats,it was found that the fundus of the rats in the TS group did not show obvious bleeding,papilledema and other changes,but the increased choroidal blood flow was observed in the fundus of the rats after TS4 W.The changes of anterior ganglia were observed by slit-lamp.Corneal epithelium in TS group was complete,transparent and the lens was not turbidity.After evaluating the IOP of rats,it was found that the tail suspension had a certain effect on the IOP of rats.The IOP of rats continued to rise within TS2 W,with statistical differences at some time points,but the fluctuations were all within the physiological range.It was witnessed by OCTA that the choroid thickened significantly after TS4 W,and the perfusion area of choroid capillaries decreased significantly after TS5 W.Additionally,the thickness of the outer nuclear layer of the retina was far thinner after TS6 W.2.Clarify the relevant mechanisms of retinal damage caused by medium to long-term simulated weightlessness HE staining showed that after TS6 W,the thickness of the outer nuclear layer of the retina was much thinner,the number of cell layers in the outer nuclear layer of the retina was dramatically reduced,and degenerative changes appeared.The retinal optic cell layer was observed by transmission electron microscopy,and it was found that more small vacuole and swollen mitochondria were visible in the inner segment of rod cells of rats after TS4W;the inner segment of rod cells of rats was dissolved and the membrane disc structure was dissolved after TS5W;most membrane disc structures of rod cells of rats were dissolved and most mitochondria of rod cells of rats were swollen after TS8 W.The ridge was broken,dissolved or even disappeared,and the remaining connective structure was visible between the rods and the outer nuclear layer cells.The gap between the outer nuclear layer cells was notably widened,and some of the outer nuclear layer cells had chromatin aggregation.Furthermore,we found that ROS and MDA were magnificently increased in the retinal tissues of rats after TS4 W,and the damage of mt DNA and the expression of DRP1 were drastically increased,while ATP,the activity of mitochondrial respiratory strepase,the expression of MFN1 and the homeostansis-related protein of mitochondrial fusion-division were drastically decreased.TUNEL staining showed that the number of TUNEL-positive cells in outer nuclear layer of retina increased significantly after TS5 W.Proteomic analysis was performed on the retinal tissues of rats after 8 weeks of simulated weightlessness by tail suspension and the retinal tissues of control rats.Over1.3 was utilized as a significantly up-regulated change threshold,and less than 1/1.3 was used as the significantly down-regulated change threshold.The results showed that 212 proteins were screened as differentially expressed proteins,among which 129 proteins were significantly up-regulated and 83 proteins were down-regulated.Among these up-regulated differential proteins,proteins GFAP,b FGF,and BFGF were found to be closely related to Müller cell activation.The protein MAP-1,which is closely related to neuronal repair,confirmed that Müller cells are involved in the regulation of retinal damage under simulated weightlessness.3.Study on the mechanism of Müller cells’ degeneration of the outer retina caused by simulated weightlessness.Compared with CON group,we found that the expression levels of retinal progenitor cell markers SOX2 and PAX6 in TS4 W group were dramatically increased,while the expression levels of SOX2 and PAX6 in retinal tissues of TS8 W group were decreased,SOX2 in particular.Moreover,we found that the expression levels of crx,which are related to the differentiation of retinal neurons,were drastically increased in TS4 W group,and decreased in TS8 W group.The expression of GFAP,a protein marker related to Müller cell gliosis,as well as GS and GLAST,which are related to glutamate metabolism,was changed.Compared with the control group,we found that the expression of GFAP was increased and the expression of GS and GLAST was decreased in retinal tissues of rats in tail TS4 W group.The expression of GFAP in TS8 W group was obviously increased,while the expression of GS and GLAST was notably decreased.Additionally,the expressions of GDNF,b FGF and MAP-1 in retinal tissue of TS group rats were significantly increased.4.The role and mechanism of Piezo1 in the damage of chorioidal vascular endothelial cells caused by simulated weightlessness The chorioidal vascular endothelial cells and the retinal pigment epithelial cellswere observed by transmission electron microscopy,and it was found that swollen mitochondria appeared in the chorioidal vascular endothelial cells of rats after TS4 W,erythrocyte stasis was observed in the chorioidal vascular cells of rats after TS6 W,apoptosis was observed in the chorioidal vascular endothelial cells of rats after TS7 W,and a small amount of autophagy was also observed in the chorioidal vascular endothelial cells of rats after TS8 W.Swelling mitochondria appeared in the retinal pigment epithelial cells after TS4 W,fat droplets,linear ridge breakage and dissolution,and a small amount of autophagy were discovered in the retinal pigment epithelial cells after TS5 W,and more pigment granules were found in the retinal pigment epithelial cells after TS7 W.The expression of Cx43,ZO-1 and occludin,which maintain the structure integrity of the outer blood-retinal barrier,decreased after TS4 W.After TS4 W,the expression of Piezo1 in chorioidal vascular endothelium was increased.Both shear stress and Yoda1 agonist dramatically increased Piezo1 expression in chorioidal vascular endothelial cells.Furthermore,Yoda1 agonists can drastically increase the concentration of calcium ions in the endothelial cells of choroidal vessels and in mitochondria,obviously decrease the mitochondrial membrane potential,mitochondrial homeostasia disorder,magnificently increase ROS,and increase the fine permeability of choroidal vascular endothelium.Using si RNA to knock down Piezo1 gene expression,calcium influx could not be activated by agonists and inhibited apoptosis of endothelial cells.Conclusion1.Simulated weightlessness leads to eye injury in rats,and its degree,signs and time are time-dependent.2.With the extension of simulated weightlessness time,the thickness of the outer core of the retina gradually thinned,the number of cell layers gradually decreased,the outer nuclear layer witnessed apoptosis,and the degeneration of the outer nuclear layer of the retina appeared.This is related to choroidal circulation disorders and retinal oxidative stress damage caused by simulated weightlessness.3.The simulated retinal degeneration changes in microgravity environment can activate Müller cells,induce Müller cells to express stem cell related molecules,neural differentiation and gliosis,and induce Müller cells to secrete neurotrophic factors to participate in the regulation of retinal damage in microgravity environment.4.Tail suspension simulated weightlessness increased the expression of Piezo1 in choroidal vascular endothelial cells,and Piezo1 played a significant role in regulating the damage of choroidal vascular endothelial cells.The elimination of Piezo1 could inhibit the damage of choroidal vascular endothelial cells under simulated weightlessness.