| Objective: This study aims to delve deeply into the neuroprotective effects of Exendin-4 on mice with traumatic brain injury(TBI),with a specific focus on elucidating the critical role and mechanism of the glymphatic system in this context.Methods:1.First Part: Firstly,a mouse TBI model was established using a Fluid Percussion Injury(FPI)device.The glymphatic system’s lymphatic influx function was assessed by injecting a fluorescent tracer into the cisterna magna.Subsequently,the glymphatic system’s lymphatic clearance function was evaluated by injecting a fluorescent tracer into the brain parenchyma of the hippocampus.Finally,the function of solute diffusion in the perivascular spaces was assessed using in vivo two-photon microscopy imaging.2.Second Part: First,a mouse TBI model was created using the FPI device.The potential side effects of Exendin-4 treatment on TBI were evaluated by monitoring blood glucose levels and body weight.This was followed by an assessment of the effect of Exendin-4 treatment on the blood-brain barrier(BBB)post-TBI using Evans Blue(EB)leakage assay,Collagen IV and CD31 immunofluorescence staining,and Western Blot analysis.Subsequently,the impact of Exendin-4 treatment on axonal injury and neuronal apoptosis post-TBI was evaluated using immunohistochemical staining for Amyloid Precursor Protein(APP),Hematoxylin and Eosin staining,Nissl staining,TUNEL fluorescence staining,and Western Blot analysis.3.Third Part: Firstly,the impact of TGN-020 on the glymphatic system’s fluid transport function was assessed by injecting fluorescent tracers into the cisterna magna and the brain parenchyma of the hippocampus.Subsequently,a mouse TBI model was created using the FPI device.The effect of Exendin-4 treatment on cognitive function in TBI mice was further evaluated using the Morris Water Maze(MWM)test and the Novel Object Recognition(NOR)test.4.Fourth Part: First,a mouse TBI model was created using the FPI device,and the effect of Exendin-4 treatment on cerebral vascular pulsation function post-TBI in mice was assessed using in vivo two-photon microscopy imaging.Subsequently,the impact of Exendin-4 treatment on cardiac function in mice post-TBI was evaluated using echocardiography.This was followed by an assessment of the effect of Exendin-4 treatment on reactive astrocyte proliferation in the injured hemisphere of mice post-TBI,using GFAP and Aquaporin 4(AQP4)immunofluorescence staining.Finally,the impact of Exendin-4treatment on AQP4 polarization in the injured hemisphere post-TBI was evaluated using AQP4 and CD31 immunofluorescence staining.Results:1.First Part: After TBI,the lymphatic influx function of the mouse glymphatic system was significantly reduced.Treatment with Exendin-4 substantially improved this lymphatic influx function.However,if a competitive antagonist of GLP-1R,Exendin-(9-39),was administered 20 minutes prior to Exendin-4,no improvement in lymphatic influx function was observed.Similarly,the lymphatic clearance function of the glymphatic system was notably diminished after TBI,but saw significant improvement following Exendin-4treatment.Yet,this improvement in lymphatic clearance was not observed when Exendin-(9-39)was administered 20 minutes before Exendin-4.Furthermore,the function of perivascular space solute diffusion in the glymphatic system was significantly reduced after TBI.Treatment with Exendin-4 markedly enhanced this function,but pre-treatment with Exendin-(9-39)followed by Exendin-4 did not lead to any observed improvement in the diffusion of solutes in the perivascular space.2.Second Part: Treatment with Exendin-4 in mice post-TBI did not result in a noticeable decrease in blood glucose and body weight.After TBI,there was a significant disruption of the blood-brain barrier(BBB)in mice.Treatment with Exendin-4 ameliorated Evans Blue(EB)leakage in the brain tissue on the injury side,protected the microvascular basement membrane,and alleviated the reduction of tight junction proteins.However,if a competitive antagonist of GLP-1R,Exendin-(9-39),was administered 20 minutes prior to Exendin-4,no mitigation of BBB disruption caused by Exendin-4 was observed.Moreover,significant axonal injury and neuronal apoptosis occurred on the injured side in mice post-TBI.Exendin-4 treatment reduced the TBI-induced axonal damage and neuronal apoptosis,eased the decrease in Bcl2 expression,and reduced the elevation of Cleaved caspase-3.However,the improvement in neuronal apoptosis was not observed when Exendin-(9-39)was administered 20 minutes before Exendin-4.3.Third Part: TGN-020 effectively inhibited the lymphatic influx and clearance functions of the glymphatic system.After TBI,mice exhibited a significant decline in cognitive function.Treatment with Exendin-4 alleviated the deterioration of cognitive function in mice post-TBI.However,when the AQP4 inhibitor TGN-020 was administered15 minutes prior to Exendin-4,no alleviation of the cognitive decline by Exendin-4 was observed.4.Fourth Part: After TBI,there was a significant decline in the pulsatile function of the intracerebral surface arteries and penetrating arteries in mice.Treatment with Exendin-4mitigated the reduction in pulsatile function of these arteries post-TBI.However,no improvement in arterial pulsation was observed when a competitive antagonist of GLP-1R,Exendin-(9-39),was administered 20 minutes prior to Exendin-4.Post-TBI,cardiac function in mice was notably impaired,with a significant decrease in Left Ventricular Ejection Fraction(LVEF)and Fractional Shortening(FS).Treatment with Exendin-4 alleviated the reduction in LVEF and FS in mice post-TBI,but no improvement in cardiac function was observed when Exendin-(9-39)was administered 20 minutes before Exendin-4.Furthermore,after TBI,significant reactive astrocyte proliferation and loss of AQP4 polarization were observed in the injured hemisphere of the brain in mice.Treatment with Exendin-4 relieved the post-TBI reactive astrocyte proliferation and loss of AQP4 polarization.However,when Exendin-(9-39)was given 20 minutes before Exendin-4,no improvement was observed in the reactive astrocyte proliferation and AQP4 polarization.Conclusion:1.Exendin-4 significantly improves the fluid transport function of the glymphatic system.2.Exendin-4 alleviates BBB disruption and neuronal apoptosis in mice post-TBI.3.Exendin-4 ameliorates cognitive impairments post-TBI through the glymphatic system.4.The mechanism of Exendin-4’s regulation of the glymphatic system’s function in TBI mice involves improved arterial pulsatility,alleviated cardiac dysfunction,inhibited reactive astrocyte proliferation,and restored AQP4 polarization. |
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