The Underlying Mechanisms Of Polyunsaturated Fatty Acid Supplement On Alleviating Depression-incident Cognitive Dysfunction In Mice

Objective: Depression is one of the most prevalent neuropsychological diseases,apart from the mood symptoms of interest decrease,pessimism,despair,there are also incident cognitive dysfunction,such as retardation of thinking and memory deterioration.And this kind of cognitive dysfunction may be caused by brain metabolic waste accumulation(such as Aβ)and cerebrovascular dysfunction.Glymphatic system is a pathway for clearance of metabolic waste in central nervous system,it is constituted by vascular basement membrane and glial limitans,there is a kind of immune cell called perivascular macrophage(PVM)residing in perivascular space and playing a role in phagocytosis,immune regulation and vascular function maintenance.This research will explore the mechanisms of depression incident cognitive dysfunction and the therapeutic effects of polyunsaturated fatty acid from the aspects of glymphatic system and perivascular macrophages.Methods: The chronic unpredictable mild stress mouse model was used to mimic depression.After 8-week modeling,tail suspension test,open field test and sucrose preference test were used to examine the depressive symptoms of mice,while Morris water maze and novel object recognition were applied to assess the level of cognition.To understand the function of glymphatic system in CUMS mice,the fluorescent dextran was injected into cisterna magna,the distribution of dextran in brain was analyzed to define the influx function.Human Aβwas stereotactically injected in prefrontal cortex,and the brain residual content of Aβ was measured by ELISA to evaluate the efflux function.The expressions of inflammatory factors of TNF-α,IL-1β,IFN-γ were detected using Western blot.The expression and distribution of AQP4 in prefrontal cortex and cerebroventricular choroid plexus were examined by immunofluorescence and Western blot.To test the cortex vascular reactivity,laser speckle imaging was applied to detect the increase percentage of blood flow after dropping acetylcholine or adenosine solution on brain cortex.The type III collagen content was assessed by immunofluorescence to evaluate the degree of vascular sclerosis in CUMS mice.During modeling,PUFA or Es was administrated to mice,then the depressive symptoms,cognitive function,glymphatic function,expression of immune factors and AQP4,vascular reactivity and sclerosis were tested to evaluate the therapeutic effects of PUFA and Es on depression.The number of PVM,which is residing in glymphatic system,was detected using immunofluorescence and Western blot in CUMS mice to explore the mechanisms of vascular dysfunction.As aging is an important risk factor of vascular sclerosis,immunofluorescence,Western blot and flowcytometry were applied to examine the number of PVM in mice of different ages.After PVM depletion,q PCR and immunofluorescence were used to detect the markers of endothelial-to-mesenchymal transition,and laser speckle imaging was used to evaluate the vessel regulatory function.Results: After CUMS modeling,the mice manifested depressive symptoms,the latency of tail suspension was decreased,and the immobility extended,the movement distance of open field test was decreased and the sucrose percentage was reduced,PUFA and Es intervention could alleviate these depressive symptoms.The results of Morris water maze test and novel object recognition showed that in CUMS mice,the latency to platform extended,the number across platform decreased,the time staying on platform decreased,the time spending with novel object reduced,indicating cognitive deficits in CUMS mice,while PUFA could significantly improve cognitive function.After intracisternal injection of 40 k Da and 3 k Da fluorescent dextran for 30 min,the fluorescence mainly distributed in perivascular space of large arteries and veins,the area fraction of CUMS was significantly lower than Control and PUFA groups,and there was no difference between CUMS and Es group.After human Aβinjection in prefrontal cortex,the brain residual Aβ content tested by ELISA showed significantly higher than other groups.All of these results implicate that PUFA can alleviate glymphatic function impaired by CUMS modeling.Besides,the results of Western blot showed that the expressions of TNF-α,IL-1β,IFN-γ were increased in prefrontal cortex of CUMS mice,PUFA diet could reduce the content of these inflammatory factors.The expression and distribution of AQP4 in prefrontal cortex and ventricular choroid plexus were analyzed using immunofluorescence and Western blot,AQP4 showed significant reduction and depolarization in CUMS mice,and PUFA diet ameliorated it.Laser speckle imaging was applied to test the level of blood flow increase after acetylcholine or adenosine administration,the increase rate of blood flow in CUMS mice was higher than the Control,while PUFA group was higher than the CUMS.The expression of vascular type III collagen in CUMS mice was increased than Control and PUFA groups,indicating that PUFA can alleviate vascular sclerosis.Brain perivascular macrophage was analyzed by immunofluorescence and flowcytometry,it showed PVM specifically expressed CD206 and Lyve1,without expressing microglia specific marker TMEM119.PVM cell number in CUMS mouse prefrontal cortex was lower than the Control group,and the number of PVM was higher in PUFA group compared with CUMS.In 2 weeks,3 months and 18 months old mice,as the age increased,the number of PVM reduced.After specific deletion of PVM,the results of immunofluorescence and q PCR showed endothelial-to mesenchymal transition,laser speckle imaging showed a reduction in vascular reactivity.Conclusion: Polyunsaturated fatty acid can reduce inflammatory response,alleviate prefrontal cortex AQP4 expression and depolarization in CUMS mice,ameliorate brain vascular regulatory dysfunction through increasing the number of perivascular macrophages,and improve glymphatic clearance capacity,further alleviate depression incident cognitive dysfunction.