| Objectives:1)To establish a mouse model of PND in male C57/BL6 wild-type mice by using a unilateral tibial fracture with internal fixation using an intramedullary nail,to use a fear conditioning test as an index to evaluate the short-term learning and memory abilities of the mice,To explore whether the surgery caused the disorder of mice’s contextual memory,and to evaluate the effect of intraperitoneal injection of Gal on mice’s contextual memory;2)To investigate the characteristics of the molecular biological and electrophysiological changes of different cells,receptors and synaptic functions in the hippocampal region of mice in the surgically induced PND model by examining the inflammatory cytokines,the activation state of main immune cells,the electrophysiology of immune and neuronal cells and the state of cholinergic nerve functions in the hippocampal region;3)To evaluate the effects of Gal on the molecular biology and electrophysiology of different cells in the hippocampus of the surgical mice by intraperitoneal injection of Gal or by using Gal to infusioning in the acute brain slices.Methods:1)The male C57/BL6 wild-type mice of 10-12 months old were selected and raised according to the document standards of the Chinese Regulations on the Administration of Laboratory Animal Affairs.Animal experiment design is conducted according to the "3R principle" of experimental animals in the Technical Principles of Humanitarian Experiment.Before the surgery,the mice completed the fear conditioning trainingin the fear-conditioning chamber.After the unilateral tibial fracture and intramedullary nail fixation was anesthetized in the surgical group,while the sham group was anesthetized only.Gal(4mg/kg)or physiological saline was injected intraperitoneally once a day in the two groups of mice,and the contextual memory was evaluated on the 3rd and 7th days after surgery.At the same time,open field test and pole climbing test were used to evaluate the motor ability of mice between the surgical mice and the sham group to determine whether the difference in motor ability were caused by leg surgery.Graphs were plotted and statistically analyzed using SigmaPlot 11.0 or GraphPad Prism 7.0,and data were expressed as mean±standard error.For comparisons between two groups of data,a two-tailed Student’s t was used,and comparisons between multiple groups were performed by one-way ANOVA or two-way ANOVA.P<0.05 was considered statistically significant;2)Mice were randomly divided into two groups according to the sham and surgical groups.Hippocampal tissues of mice were dissected according to the experimental operation specification at 3rd days after surgery,and microglia(Iba-1 staining)and astrocytes(GFAP staining)of the sections were observed and imaged and counted by using confocal microscope.Pro-inflammatory cytokines(IL-1β,TNF-α and IL-6)and anti-inflammatory cytokines(IL-4 and IL-10)were measured by ELISA in the hippocampus.After the mice were anesthetized by intraperitoneal injection of pentobarbital sodium(40 mg/kg),the mice were fixed with the mouse head stereotactic framework and injected with the viral vector carrying ACh sensor(AAV-hSyn-GACh3.0)into the dorsal hippocampus at a rate of 50 nl/min.Three weeks later,acute 300-μm-thick hippocampal sections were prepared for experiments,and data were collected by imaging GACh in the CA1 region of the hippocampus by fluorescence microscopy on electrical stimulation from O/A region.In carbogenated ACSF,astrocytes in hippocampal slices were stained with sulfohodamine 101 and Fluo-4 AM.Astrocytes labeled with sulfohodamine 101 were observed with red fluorescent lighting,and calcium signals displayed by Fluo-4 AM were observed with green fluorescent lighting.It was Observed and recorded the change(ΔF)of fluorescence intensity caused by the change of calcium concentration in astrocytes in CA1 region induced by electrical stimulation from O/A region.Electrophysiological signals from neurons,astrocytes and fEPSPs in the hippocampal CA1 region were recorded by using a whole-cell membrane clamp technique,in which stimulating electrode at a distance of 400-500 μM from record electrode in acute hippocampal brain slices.3)Gal(4mg/kg)or physiological saline was injected intraperitoneally in mice once a day in the sham and surgical groups,and hippocampal tissues of mice were dissected according to the experimental operation specification at 3rd days after surgery.Microglia(Iba-1 staining)and astrocytes(GFAP staining)of the sections were observed and imaged and counted by using confocal microscope.Pro-inflammatory cytokines(IL-1β,TNF-α and IL-6)and anti-inflammatory cytokines(IL-4 and IL-10)were measured by ELISA in the hippocampus.Hippocampal acute brain slices were labeled with red fluorescent agent SR101 and green fluorescent agent Fluo-4,and were perfused Gal of 10μM concentration,observed the change(ΔF)of fluorescence intensity of calcium signal induced by electrical stimulation.The acute hippocampal brain slices from mice with dorsal hippocampus containing an ACh sensor viral vector(AAV-hSyn-GACh3.0)were perfused with Gal and the signal of GACh induced by electrical stimulation in the CA1 region of the hippocampus were collected in fluorescence microscopy imaging.The stimulation electrode was placed on the Schaffer collateral branch and 400-500 away from the recording electrode after infusion of Gal(10μM)or FC(100μM).The electrophysiological signals from astrocytes,hippocampal neurons and fEPSPs in CA1 region were recorded by patch clamp technique.Results:1)There was no significant difference in the percentage of postural freezing time between sham and surgical mice prior to fear training.At 3rd and 7th days after training.Compared with baseline levels,freezing time increased in sham group and surgical group(P<0.01)and was significantly less in surgical group than in sham group(P<0.05),with no statistically significant differences in locomotor performance between the two groups after the open field test and pole climbing test and Gal intervention(P>0.05).Mice in the surgical group had a significantly longer percentage of freezing time after Gal intervention compared with the saline surgical group(P<0.05).2)The number of microglia in the hippocampus of the two groups of mice was detected by immunohistochemistry.The number of microglia in the surgical group was about 2 times higher than that in the sham surgical group(P<0.01),and there was no regional specificity,including in the density of astrocytes and total GFAP immunoreactive area.Pro-inflammatory cytokine IL-1β(P<0.05),TNF-α(P<0.01)and IL-6(P<0.05)in hippocampus of surgical mice increased.There was no statistical difference in the content of anti-inflammatory cytokines(IL-4 and IL-10)in the hippocampus of the two groups(P>0.05).Compared with the sham group,resting membrane potential and conductance(P<0.01)and capacitance(P<0.05)were increased in hippocampal astrocytes of surgical group.In the surgical group,AMPA receptor-mediated EPSCs in pyramidal neurons of mice and fEPSPs decreased significantly(P<0.01),but the rectification index had no statistical difference.In the acute hippocampal slices,the peak response of released acetylcholine and the peak response of astrocytes induced by electrical stimulation in the surgical group were significantly lower than those in the sham group(P<0.01).Scop(5μM),non selective muscarinic receptor antagonist,inhibited significantly the increase of calcium induced by electrical stimulation,which was 73.75%±7.07%and 76.25±3.85%in the two groups of mice,respectively.3)On the 3rd day after surgery,the number of microglia activation in the sham+saline group was smaller than in the surgery+Gal group(P<0.05)and that the surgery+saline group was higher than surgery+Gal(P<0.05).The hippocampal IL-1β,TNF-α,and IL-6 in the surgery+Gal group showed a statistically significant decrease compared to in surgery+saline group.There was no significant difference between the IL-4 and IL-10 in each groups.In the hippocampal acute slices on the 3rd day after surgery,the magnitude of AMPA receptor mediated EPSCs in the surgical group were greater than those of the sham group(P<0.05).The degree of change in electrical stimulation-evoked fluorescence intensity in astrocytes in the CA1 region of the hippocampus was greater in the surgical group than in the sham group(P<0.01).After Gal or FC infusion,the fEPSP amplitude of the surgical group and the sham group increased significantly compared with the baseline level(P<0.01).The fEPSP amplitude in the surgical group of mice reached the same level as in the sham group without Gal infusion(P>0.05).Conclusions:1)Gal intervention can improve the situation memory disorder of mice caused by surgery;2)The operation itself can lead to the decrease of cholinergic nerve function in the hippocampus of mice,the activation of immune cells,and the increase of inflammatory cytokines in the central hippocampus of mice,which is manifested in the dysfunction of immune cells and neurons in the hippocampus and the decrease of synaptic plasticity;3)Through the intervention of intraperitoneal injection of Gal,it can reduce the inflammatory reaction in the hippocampus of surgical mice and inhibit the activation of immune cells.The abnormal function of immune cells and neurons in the hippocampus caused by surgery can be restored by the infusion of Gal in the hippocampal slices,and the decrease of synaptic plasticity caused by surgery in hippocampus can be restored by the perfusion of Gal and FC in hippocampus slices.To sum up,strengthening central cholinergic function and/or reducing inflammatory reaction in mice may be potential targets and strategies for improving PND symptoms. |
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