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Objective:To explore the effect of NOD-like receptor thermal protein 3 ( NLRP3) knockout in γ-aminobutyric acid (GABA)-ergic neurons in the hippocampal CA1 area on improving cognitive dysfunction in mice after traumatic brain injury (TBI). Methods:Forty-eight healthy male NLRP3 flox/flox mice weighing 25-28 g were randomly divided into 4 groups ( n=12): sham-operated+control virus group (SV group), sham-operated+ NLRP3 specific knockout group (SG group), TBI+control virus group (TV group), TBI+ NLRP3 specific knockout group (TG group). TBI in the TV and TG groups was established by free-fall method, while surgical procedures such as scalp incision and cranial window opening without impact were given to the SV and SG groups. Adenovirus was injected into the hippocampal CA1 area of SG and TG groups 21 d before TBI to induce NLRP3 specific knockout in GABA-ergic neurons in the hippocampal CA1 area; empty virus was injected into the CA1 area of SV and TV groups. Cognitive function was evaluated using novel object recognition test 30 and 31 d after TBI, and learning and memory functions were assessed using Morris water maze test 32-36 d after TBI. Field potentials in the hippocampal CA1 area were recorded during novel object recognition 31 d after TBI. After behavioral tests, these mice were sacrificed. Immunofluorescent staining was used to detect the fluorescent intensity of microtubule-associated protein2 (MAP2), glutamic acid decarboxylase 67 (GAD67), and postsynaptic density protein 95 (PSD95) in the hippocampal CA1 area, as well as percentage of pyroptosis-associated inflammatory factor interleukin-18 (IL-18)/GAD67 double-positive neurons in total GAD67 positive neurons. Results:Compared with the SV and SG groups, the TV and TG groups had decreased novel object recognition index, decreased number of platform crossings during the experimental period, increased escape latency on day 3 and day 4 of the training period in Morris water maze test, decreased θ and γ oscillation power in the hippocampal CA1 area during novel object recognition, decreased fluorescent intensity of MAP2, GAD67, and PSD95 in the hippocampal CA1 area, increased percentage of IL-18/GAD67 double-positive neurons, with significant differences ( P<0.05). Compared with the TV group, the TG group had increased novel object recognition index, increased number of platform crossings in Morris water maze test, decreased escape latency during the training period, increased θ and γ oscillation power in the hippocampal CA1 area during novel object recognition, increased fluorescence intensity of MAP2, GAD67, and PSD95 in the hippocampal CA1 area, decreased percentage of IL-18/GAD67 double-positive neurons, with significant differences ( P<0.05). Conclusion:Specific inhibition of NLRP3 expression in GABA-ergic neurons in the hippocampal CA1 area can improve cognitive dysfunction in mice after TBI, whose mechanism may be related to inhibited GABA-ergic neuronal pyroptosis in the hippocampal CA1 area.
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Objective:To evaluate the role of astrocytic NOD-like receptor protein 3 (NLRP3) in the lateral hypothalamus (LHA) in anxiety-like behaviors after hemorrhagic shock and resuscitation in mice.Methods:Forty-eight clean-grade male C57BL/6 mice, aged 10 weeks, weighing 25-30 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group C), hemorrhagic shock and resuscitation group (group H), hemorrhagic shock and resuscitation + adeno-associated virus group (group HI), and hemorrhagic shock and resuscitation + control virus group (group HIV). The model of hemorrhagic shock and resuscitation was developed by bleeding and re-transfusion through the femoral vein in H, HI and HIV groups. At 21 days before developing the model, AAV-GfaABC1D-EGFP-Cre was injected into bilateral LHA in group HI, and AAV-GfaABC1D-EGFP was administered as a control in group HIV. Anxiety-like behaviors were evaluated by EPM-maze and bead-burying tests at 14 days after resuscitation. Mice were immediately sacrificed at the end of behavioral tests, and LHA-containing brain tissues were obtained for determination of co-localization of NLRP3 with glial fibrillary acidic protein (GFAP), the fluorescence intensity of Wisteria floribunda agglutinin was measured using immunofluorescent staining to reflect the expression of extracellular matrix in the LHA, and the percentage of cleaved caspase-1/GFAP and IL-18/GFAP positive cells in total cells was calculated. Results:Compared with group C, the number of buried beads and percentage of time of staying at the open arm were significantly decreased, the expression of extracellular matrix in the LHA was down-regulated, and the percentage of cleaved caspase-1/GFAP and IL-18/GFAP positive cells was increased in H, HI and HIV groups, and the co-localization coefficient of NLRP3 and GFAP was significantly decreased in group HI ( P<0.01). Compared with group H, the number of buried particles and percentage of time of staying at the open arm were significantly decreased, the expression of extracellular matrix in the LHA was up-regulated, the co-location coefficient of NLRP3 and GFAP was decreased, the percentage of cleaved caspase-1/GFAP and IL-18/GFAP positive cells was decreased ( P<0.01), and no significant change was found in the parameters mentioned above in group HIV ( P>0.05). Conclusions:Anxiety-like behaviors after hemorrhagic shock and resuscitation is associated with astrocytic NLRP3-induced pyroptosis in the LHA and reduction of extracellular matrix in mice.
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Objective:To investigate the effect of activation of microglia in prefrontal cortex on long-term spatial memory in post-stroke depression mice.Methods:Forty-eight male C57BL/6 mice were divided into sham operation group, stroke group, post-stroke depression group and depression group according to the random number table method with 12 in each group, and 36 mice were divided into solvent group, enrofloxacin group and minocycline group according to the random number table method with 12 in each group.Middle cerebral artery occlusion (MCAO) was use to establish the stroke model, and forced swimming was used to establish the depression model.The post-stroke depression model mice were received MCAO first and then received forced swimming on the 4th day after stroke to establish the model.Mice in enrofloxacin group and minocycline group were treated with enrofloxacin and minocycline injection once a clay for 14 days from the 5th day after stroke, respectively.Forced swimming test and sugar water preference test were used to evaluate the depression of mice in each group, Morris water maze test was used to detect the spatial memory function of mice in each group, and Nissl staining and immunofluorescence staining were used to detect the neuronal function and the number and type of microglia activation.The expression of inflammatory cytokines IL-6 and IL-1β were detected by Western blot.GraphPad Prism 8.0.1 statistical software was used for statistical analysis.The single factor variance analysis was used to compare the difference among multiple groups, and pairwise comparison was performed with SNK- q test. Results:(1) There were statistically significant differences in depression, learning and memory, neuron damage, activation of microglia, inflammatory factors and other indicators in sham operation group, stroke group, post-stroke depression group and depression group ( F=43.58-255.70, all P<0.05). Compared with stroke group, post-stroke depression group had longer floating immobility time ((222.70±29.12) s, (79.25±46.78) s, P<0.05), the preference rate of sugar water was significantly lower ( (49.44±6.19) %, (84.49±4.73) %, P<0.05), and the average value of platform approach after correction was higher((125.00±9.95) mm, (96.79±12.57) mm, P<0.05), Nissl bodies expression was lower ((53.50±15.78) cells /mm 2, (85.67±17.52) cells /mm 2, P<0.05), NeuN positive expression rate was lower ((29.78±3.70) %, (45.73±4.51) %, P<0.05), the percent of M1 microglia expression was significantly higher ((75.55±8.84) %, (58.19±5.69) %, P<0.05), the percent of M2 microglia expression was lower ((43.46±5.11)%, (57.14±5.40)%, P<0.05), and the expression levels of IL-6 ((1.14±0.03), (0.94±0.05), P<0.05) and IL-1β((1.17±0.03), (0.56±0.04), P<0.05) were significantly higher.(2) Depression, learning and memory, neuron injury, activation of microglia, inflammatory factors and other indicators of mice in solvent group, enrofloxacin group and minocycline group were significantly different ( F=7.13-94.35, all P<0.05). Compared with enrofloxacin group, mice in minocycline group had shorter floating immobility time ((169.30±13.04) s, (224.30±22.60) s, P<0.05) and higher sugar water preference rate ((62.81±7.75) %, (47.71±8.11) %, P<0.05), the mean value of platform approach estimation after water maze correction was lower ((97.66±14.56) mm, (120.20±12.08) mm, P<0.05), and the expression level of Nissl bodies was higher ((80.17±10.55) cells /mm 2, (52.00±8.94) cells /mm 2, P<0.05), NeuN expression rate was high ((45.04±3.62) %, (28.88±4.50) %, P<0.05), Iba-1 expression was lower ((97.33±10.67) cells/mm 2, (112.50±6.54)cells/mm 2, P<0.05), the percent of M1 microglia expression was lower ((54.43±5.22) %, (73.82±6.88) %, P<0.05), and the percent of M2 microglia expression was significantly higher ((51.86±6.22) %, (36.30±5.72) %, P<0.05). The expression levels of IL-6 ((0.75±0.06), (1.21±0.07), P<0.05) and IL-1β ((0.61±0.06) (1.09±0.09), P<0.05) were lower. Conclusion:The long-term spatial memory impairment of post-stroke depression mice is aggravated, which is related to the neuron damage caused by increased activation of M1 microglia in PFC area.Inhibition of M1 microglia by minocycline can effectively improve the spatial memory ability of mice.
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Objective:To investigate the effect of Spautin-1 (an inhibitor of autophagy) on improving anxiety-like behaviors and its mechanism in mice after traumatic brain injury (TBI).Methods:Thirty-six C57BL/6 mice were randomly divided into sham-operated group, TBI group, and TBI+Spautin-1 group ( n=12); TBI models in the latter two groups were established by modified Feeney free fall epidural impingement method. Mice in TBI+Spautin-1 group were administered with Spautin-1 (2 μL, 10 mmol/L) into the lateral ventricle 10 min after modeling, but mice in the other two groups were only injected with same volume of solvent. Neuropathy Symptom Score (NSS) was used to evaluate the functions of motor, sensory and reflexes of mice on 1 st, 7 th and 14 th d of modeling. On 15 th and 16 th d of modeling, open field test and elevated plus maze test were used to evaluate the anxiety-like behaviors in mice. The number of Nissl bodies in the amygdala of mice was calculated by Nissl staining 16 d after modeling. The numbers of neuron specific nucleoprotein (NeuN) positive cells, interleukin (IL)-18 and IL-1β positive astrocytes in the amygdala were detected by immunofluorescent staining. Western blotting was used to detect the autophagy-and pyrotopic-associated protein expressions in the amygdala region of mice. Results:(1) As compared with the sham-operated group, the TBI group and TBI+Spautin-1 group had significantly increased NSS scores on 1 st and 7 th d of modeling ( P<0.05). (2) Open field test showed that as compared with the sham-operated group, the TBI group and TBI+Spautin-1 group had significantly smaller number of crossing grids, significantly decreased percentage of time spending in the central zone ([central area residence time/total time] × 100%), significantly decreased percentage of frequencies entering into opening arm (OE) (OE/[OE+frequencies of entering closing arm]×100%) and opening arm time (OT) percentage (OT/[OT+time of closing arm]×100%); as compared with the TBI group, TBI+Spautin-1 group had significantly larger number of crossing grids, and significantly increased time percentage spending in the central zone, OE percentage, and OT percentage ( P<0.05). (3) As compared with sham-operated group, the TBI group and TBI+Spautin-1 group had significantly smaller numbers of Nissl bodies and NeuN positive cells in the amygdala of mice ( P<0.05); as compared with TBI group, TBI+Spautin-1 group had significantly larger numbers of Nissl bodies and NeuN positive cells in the amygdala of mice ( P<0.05). (4) As compared with the sham-operated group, the TBI group and TBI+Spautin-1 group had significantly increased percentages of IL-1β and IL-18 positive astrocytes in amygdala of mice ( P<0.05); as compared with the TBI group, the TBI+Spautin-1 group had significantly decreased percentages of IL-1β and IL-18 positive astrocytes in amygdala of mice ( P<0.05). (5) As compared with sham-operated group, the TBI group and TBI+Spautin-1 group had significantly higher protein expressions of NOD-like receptor family protein 3 (NLRP3), activated cysteine aspartate protease-1 (Caspase-1), pore-forming protein D-N terminal fragment (GSDMD-N), ubiquitin specific peptidase (USP) 13 and B-lymphocytoma-2 interacting protein (Beclin1), and statistically higher ratio of microtubule-associated protein 1 light chain (LC)3 II/LC3 I ( P<0.05); as compared with TBI group, the TBI+Spautin-1 group had significantly decreased protein expressions of NLRP3, activated Caspase-1, GSDMD-N, USP13 and Beclin1 in the amygdala, and statistically lower ratio of LC3 II/LC3 I ( P<0.05). Conclusion:Spautin-1 improves the anxiety-like behaviors in mice after TBI, whose mechanism may be associated with the inhibition of astrocytic pyroptosis in the amygdala.