The Photosensitive Modification STIM1 Drives The Occurrence Of Stress Depression Induced By Retinal Injury And Its Related Mechanism

BackgroundDepressive disorder is a serious mental illness characterized by persistent sadness,loss of interest and social withdrawal.In severe cases,it can also lead to suicide.Depression is a multifactorial disorder that can manifest as a comorbidity with multiple diseases,and studies have shown a high rate（14-44%）of comorbidity between depression and visual impairment.However,there are few basic studies on the mechanism of the comorbidity of visual impairment and depression because no relevant animal model can be used for studying.Therefore,it will be of great significance to establish an animal model that can be used for exploring the correlation between retinal injury and stress induced depression and providing laboratory evidence for the subsequent development of precise prevention and treatment strategies.The rapid development of optogenetics provides us with new ideas for constructing animal models of retinal injury.It is reported that a novel optogenetics virus called AAV-Lovsoc-STIM1 can respond to blue light at wavelength of 420-560nm.Once this response occurs,stromal interaction molecule 1（STIM1）can activate the Ca²⁺release-activated Ca²⁺（CRAC）channels on the cell membrane and Ca²⁺in the extracellular will be driven to intracellular as a result.Continuous activation of this channel can cause intracellular calcium imbalance and cell damage.Based on this principle,we inject the photosensitive modified STIM1 virus into the mouse vitreous to infect the retinal tissue,CRAC channels in the retinal cell membrane can be effectively regulated in real time under the condition of 420-560nm blue light irritating.Continuous irradiation can keep CRAC channels open,leading to the retinal cell death due to calcium overload.Thus,this strategy can be applied to construct an animal model that the progressive damage of retinal tissue can be effectively and rapidly regulated.As an extension of the central nervous system,retinal dysfunction not only affects visual function,but also affects other brain function activities including emotion and cognition.Therefore,retinal damage may be related to the onset of stress depression.Accumulated evidence suggests that neuroinflammation may play a key role in the occurrence and development of depression.Various glial cells in the brain interact with each other correctly under physiological conditions.When the individual faced with different stress events,microglia,the innate immune cells in the central nervous system respond rapidly.The synthesis and secretion of proinflammatory mediators are induced and the maturation of oligodendrocytes inhibited,lead to myelination disorders.Consequently,the normal communication across different regions of the nervous system is blocked,the normal function of nervous system is destroyed.However,whether the inflammatory changes exist in the central nervous system during the occurrence of depression caused by retinal damage remains to be explored.Moreover,the hippocampus which is the main structure of the limbic system is a well-known brain region that regulates emotion.The hippocampus is extremely susceptible to dysfunction when neuroinflammation causes pathological changes in the brain.Therefore,we speculate that the occurrence of neuroinflammation in brain regions related to visual pathways in comorbid retinal injury and stress depression leads to demyelinating lesions in white matter and hippocampal dysfunction.MethodsThis study was divided into two sections.In section one,we established an animal model of stress-induced depression caused by photosensitive retinal injury.The first step was confirming the optimal mode of light to induce the retinal damage.AAV-Lovsoc-STIM1 virus was injected into the vitreous of mice.14 days after infection,the mice were exposed to blue light（460-465nm,5000lx）for 0,1,2 and 3h per day.After 7 days of continuous treatment,we compared the pathological damage of retinal cells to screen the best light conditions causing retinal damage.Next,the animals were randomly divided into three groups:photosensitive-group（Lovsoc-STIM1 virus injection without light exposure group）,control+group（Lovsoc-CON virus injection with light exposure group）,and photosensitive+group（Lovsoc-STIM1 virus injection with light exposure group）.The pathological changes of retina in the three groups were observed.Finally,the open field test,the sucrose preference test,the forced swimming test,and the tail suspension test were combined to evaluate whether the model mice had depressive-like symptoms,and the positive rate of depressive-like behaviors was calculated.In section two,we used the stress depressive mice model that caused by the photosensitive retinal injury to investigate the pathological changes in brain regions related to visual pathways.These changes included the inflammatory environment,the neuronal apoptosis,the myelin structure in the white matter and the neuroinflammation in the hippocampus.Immunofluorescence staining was applied to observe the morphological and quantitative changes of microglia in the visual cortex and dorsolateral geniculate nucleus of the three groups.RT-PCR was used to detect the m RNA expression of pro-inflammatory cytokine IL-6 and chemokine MIP-1αin the visual cortex of the three groups.By using Western-blot analysis,the expression of S100B protein,cleaved caspase-3 protein in the visual cortex and MBP protein in the white matter were tested.Immunofluorescence staining was used to observe the distribution and expression of MBP fluorescence signal in the corpus callosum（cc）and cingulate gyrus（cg）,and the changes of microglia and astrocytes in the hippocampus.ResultsThe results of the first part were as follows:1.14 days after AAV-Lovsoc-STIM1-m Cherry virus injection,red fluorescent signals were observed in all retinal layers,especially in ganglion cell layer and outer nuclear layer,suggesting that the retina had photosensitive properties.2.After AAV-Lovsoc-STIM1-m Cherry was expressed,the cell densities of the outer nuclear layer,inner nuclear layer and ganglion cell layer were significantly decreased in the 3h/d for 7 consecutive days group compared with those in the 0,1 and 2h/d groups（P<0.05）.It was suggested that the optimal mode of blue light exposure to induce retinal damage was 3hours per day for 7 consecutive days.3.Under the optimal light mode,the HE staining results revealed that the photosensitive+group showed had increased pyknotic cells compared with the photosensitive-group and the control+group（P<0.05）.Meanwhile,the retinal also showed disordered arrangement of cells in each layer and enlarged gaps between the cells,and the cell density was reduced in each layer（P<0.05）.This observation suggested that under the optimal blue light irradiation condition,the mice in the photosensitive+group had acute retinal damage.4.In the open field test,the mice in photosensitive+group showed less times and distance of entering the central area.The total distance of activity in the open field was also lower in the photosensitive+group and the depressive rate of the open field behavior test was65.12%.In the sucrose preference test,the sucrose preference index of the photosensitive+group was significantly lower than that of the photosensitive-group and the control+group,and the depressive rate of sucrose preference test was 60.47%.In forced swimming test and tail suspension test,the immobility time of the photosensitive+group were both longer than that of the photosensitive-group and the control+group,and the depressive rates of forced swimming test and tail suspension test were 72.09%and 76.74%,respectively.Venn diagram calculation showed that the positive depressive rate in all four behavioral tests was 23.26%.These results suggested that mice with retinal injury developed a stress-like depression phenotype.The results of the second part were as follows:1.Immunofluorescence staining showed that the Iba-1⁺microglial cells in the V2MM area of visual cortex and the dorsal lateral geniculate nucleus of mice in the photosensitive+group had increased cell size and dendritic branching,and these cells were activated form.Besides,the density of Iba-1⁺microglial cells in the V2MM area of visual cortex and the dorsal lateral geniculate nucleus in the photosensitive+group were higher than photosensitive-group and the control+group（P<0.05）.These results suggested the microglia cells were activated in the visual pathway.2.The results of RT-PCR showed that the m RNA expression of pro-inflammatory cytokine IL-6 and chemokine MIP-1αwere significantly up-regulated in the visual cortex of photosensitive+mice（P<0.0001）,suggesting the increased synthesis of pro-inflammatory mediators.Western-blot results showed that the expression of S100B protein and apoptosis-related protein Caspase-3 were significantly higher than those in the photosensitive-group and control+group in the visual cortex of mice in the photosensitive+group（P<0.001）,suggesting the occurrence of neuronal apoptosis.3.Western-blot analysis showed that the expression level of MBP protein in the white matter region of the brain of mice in the photosensitive+group was significantly lower than the photosensitive-and control+group（P<0.05）.The results of immunofluorescence staining showed that the fluorescence signals of MBP in the corpus callosum（cc）and cingulate gyrus（cg）of the white matter in the photosensitive+group were undistributed and absent to varying degrees.The relative fluorescence intensity was significantly weaker than that in the photosensitive-group and control+group（P<0.0001）indicating demyelinating lesions in the white matter,which were consistent with the WB results.4.The results of immunofluorescence staining showed that the density of IBa-1⁺cells and GFAP⁺cells in the photosensitive+group were both significantly higher than the other two groups（P<0.0001）.The cell sizes of both the microglial cells and astrocytes were increased,which were appeared to be activated.This observation indicated that the neuroinflammatory response occurred in the hippocampus.Conclusion1.After 14 days of AAV-Lovsoc-STIM1 virus injection,acute retinal injury occurred after 3 hours per day blue light irradiation for 7 consecutive days.We successfully established a photosensitive retinal injury animal model.2.The depressive symptoms including anxiety,anhedonia,and despair were occurred in23.26%of the acute retinal injury mice.This result indicated that the retinal injury could induce the occurrence of stress depression disorder.3.The microglial cells in the visual cortex V2MM and the dorsolateral geniculate nucleus were activated,the expression of inflammatory cytokines IL-6 and chemokine MIP-1αin the visual cortex were increased,and the apoptosis of nerve cells occurred in the visual cortex V2MM,the demyelinating lesions occurred in the corpus callosum and cingulate gyrus,the microglia and astrocytes were both enriched in the hippocampus in the stress depressive mice model induced by photosensitive retinal injury.In conclusion,the injection of photosensitive modification of STIM1 successfully induced a comorbidity model of retinal damage and stress depression.The occurrence of stress depression was related to the neuroinflammation in the brain visual pathway and hippocampus,the apoptosis of nerve cells in the visual cortex,and the demyelination of white matter.