Objective:To evaluate the changes in cortical electroencephalogram (EEG) burst suppression rate (BSR) during sevoflurane anesthesia in septic mice and the correlation with cerebral blood perfusion.Methods:Forty SPF male C57BL/6J mice, aged 8-10 weeks, weighing 22-25 g, were divided into 2 groups ( n=20 each) by the random number table method: sham operation group (Sham group) and cecal ligation perforation group (CLP group). The sepsis model was established by cecal ligation and puncture in anesthetized animals. Mice in both groups inhaled 2% sevoflurane for 2 h. During sevoflurane anesthesia, BSR (30 min as an epoch) on electroencephalogram was recorded, and the cortical cerebral blood perfusion was recorded using the laser speckle flow imaging at 30, 60, 90 and 120 min of anesthesia. Results:Compared with Sham group, the cortical EEG BSR was significantly increased, and the cortical cerebral blood perfusion was decreased during sevoflurane anesthesia in CLP group ( P<0.05). Cortical EEG BSR was negatively correlated with cortical cerebral blood perfusion ( P<0.05). Conclusions:Cortical EEG BSR increases during sevoflurane anesthesia in septic mice, which may be related to decreased cortical cerebral blood perfusion.

Objective:To evaluate the role of ZIP7 in sepsis-induced cardiomyopathy in mice.Methods:Ninety wild-type and 90 cardiomyocyte-specific knockout ZIP7 (ZIP7 cKO) male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups using a random number table method: wild-type sham operation group (Sham group) and wild-type sepsis group (Sep group), ZIP7 cKO sham operation group (cKO + Sham group) and ZIP7 cKO sepsis group (cKO + Sep group), with 45 mice in each group. The sepsis-induced cardiomyopathy model was developed using the cecal ligation and puncture in anesthetized mice. Twenty mice were randomly selected to record the survival for 10 days postoperatively. At 18 h after surgery, the left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were measured by echocardiography, and serum concentrations of cardiac troponin T (cTnT), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay. The contents of hydroxyl radical (·OH) and peroxynitrite anion (ONOO -) were determined using a colorimetric assay, the morphology of myocardial mitochondria was observed with a transmission electron microscope, and the expression of dynamin-related protein 1 (Drp1), mitofusin-2 (Mfn2), and optic atrophy 1 (Opa1) in myocardial tissues was detected using Western blot. Results:Compared to Sham group, the survival rate, LVEF and LVFS were significantly decreased, serum concentrations of cTnT, TNF-α and IL-6 were increased, the contents of ·OH and ONOO - in myocardial tissues were increased, the expression of Drp1 was up-regulated, the expression of Mfn2 and Opa1 was down-regulated ( P<0.05), myocardial cells exhibited mitochondrial swelling, and marked destruction of mitochondrial cristae was observed in Sep group, and no significant differences were found in the aforementioned parameters in cKO+ Sham group ( P>0.05). Compared to Sep group, the survival rate, LVEF and LVFS were significantly increased, serum concentrations of cTnT, TNF-α and IL-6 were decreased, the contents of ·OH and ONOO - in myocardial tissues were decreased, the expression of Drp1 was down-regulated, the expression of Mfn2 and Opa1 was up-regulated ( P<0.05), and mitochondrial swelling in myocardial cells was mild, with less dissolution and destruction of mitochondrial cristae in cKO+ Sep group. Conclusions:Myocardial ZIP7 can promote mitochondrial fusion and inhibit mitochondrial fission, potentially contributing to the mechanism of sepsis-induced cardiomyopathy in mice.

Objective:To evaluate the role of cannabinoid receptor 1 (CB1) in the spinal membrane in electroacupuncture (EA)-induced alleviation of morphine-triggered opioid-induced hyperalgesia (OIH) and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups ( n=12 each) using a random number table method: normal saline group (NS group), morphine group (M group), morphine+ EA group (ME group), and morphine+ EA+ CB1 antagonist group (MEA group). The OIH model was established by intrathecal injection of morphine 15 μg (10 μl) twice a day for 7 consecutive days. The equal volume of normal saline 10 μl was given instead in NS group. EA of the " Yanglingquan" (GB34) and " Zusanli" (ST36) acupoints lasting 30 min was performed after the first administration of medication each day, with a current intensity of 2 mA and frequency of 2 Hz in ME group. In MEA group, morphine (15 μg) and CB1 antagonist AM251 30 μg (10 μl) were intrathecally injected twice a day for 7 consecutive days, with other treatments similar to those previously described in ME group. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before administration (T 0) and on days 1, 3, 5 and 7 after administration (T 1-4). Rats were deeply anesthetized and sacrificed at T 3, 4 after administration, and the L 4-6 spinal cord tissues were collected for determination of the expression of membrane CB1, ERK1/2 and p-ERK1/2 by Western blot. Results:Compared with NS group, the MWT was significantly decreased and the TWL was shortened at T 3, 4 in M, ME and MEA groups, the expression of spinal membrane CB1 and p-ERK1/2 was significantly up-regulated at T 4 after administration in M group, and the expression of spinal membrane CB1 was significantly up-regulated at T 3, 4 after administration in ME and MEA groups ( P<0.05). Compared with M group, the MWT was significantly increased and the TWL was prolonged at T 3, 4, and the expression of p-ERK1/2 was down-regulated at T 4 after administration in ME group ( P<0.05), no significant change was found in MWT or TWL at T 3, 4 in MEA group ( P>0.05), and the expression of spinal membrane CB1 was significantly up-regulated at T 3, 4 after administration in ME and MEA groups ( P<0.05). Compared with ME group, the MWT was significantly decreased and the TWL was shortened at T 3, 4, and the expression of spinal membrane CB1 and p-ERK1/2 was up-regulated at T 4 after administration in MEA group ( P<0.05). Conclusions:Up-regulation of spinal membrane CB1 expression is involved in EA-mediated alleviation of morphine-induced OIH, which is associated with the inhibition of ERK1/2 phosphorylation in rats.

Objective:To explore the expression changes of nerve growth factor (NGF)/tropomyosin receptor kinase A (TrkA) signaling pathway of dorsal root ganglia (DRG) in incisional rat remifentanil-induced hyperalgesia and its effect on the expression of membrane delta opioid receptor (DOR).Methods:A total of 48 SPF male SD rats were randomly divided into 6 groups based on body weight matching, with 8 in each group, which were control group (infusion of 0.9% NaCl solution via the tail vein), incision pain group (incision pain model established using the Brennan method), remifentanil group (infusion of remifentanil via the tail vein), incision pain+ remifentanil model group (incision pain model established using the Brennan method, followed by infusion of remifentanil via the tail vein), NGF group and TrkA inhibitor group(established incision pain+ remifentanil model after intrathecal injection of NGF (0.06 μg/g) or K252a (0.3 μg/g, TrkA inhibitor)). Mechanical paw withdrawal threshold (PWT) was used to assess pain sensitivity in rats. Western blot was employed to measure the expression of NGF, TrkA, and the total DOR(tDOR) and the membrane DOR(mDOR) in DRG tissues. Immunoelectron microscopy was used to detect subcellular DOR expression in DRG. Data were processed using SPSS 24.0 software. Multiple comparisons among groups were conducted by repeated measures ANOVA or one-way ANOVA, and post-hoc comparisons were performed using the Bonferroni test.Results:(1) The results of pain behavior showed that there was a significant interaction effect between time and group in the comparison of PWT among the six groups of rats before and after intervention ( F=345.817, P<0.001). At each time point after intervention, the PWTs of the incision pain+ remifentanil group were lower than those of the incision pain group and remifentanil group, the PWTs of the NGF group were lower than those of the incision pain+ remifentanil group, and the PWTs of the TrkA inhibitor group were higher than those of the incision pain+ remifentanil group and NGF group (all P<0.05). (2)The Western blot results showed that there were statistically significant differences in the relative levels of NGF, TrkA, and mDOR in the DRG tissues of the six groups of rats ( F=156.2, 163.8, 421.2, all P<0.001). The levels of NGF, TrkA, and mDOR proteins in the incision pain+ remifentanil group (1.45±0.07, 1.46±0.04, 3.01±0.20) were higher than those in the incision pain group (1.25±0.05, 1.24±0.04, 1.84±0.05) and remifentanil group (1.24±0.04, 1.26±0.03, 1.84±0.04) (all P<0.05). The levels of NGF, TrkA, and mDOR in the NGF group (1.57±0.03, 1.58±0.07, 3.74±0.25) were higher than those in the incision pain+ remifentanil group (all P<0.05). The relative expression levels of TrkA, and mDOR in the TrkA inhibitor group (1.25±0.04, 1.68±0.07) were lower than those in the incision pain+ remifentanil group and the NGF group (all P<0.05). (3)The results of immunoelectron microscopy showed that there were statistically significant differences in the localization of DOR in the cell membrane, subcellular sites of synthesis pathways, and subcellular localization of degradation pathways among the six groups of rat DRG tissues ( F=140.3, 60.63, 60.28, all P<0.01). The DOR of the synthesis pathway of incision pain+ remifentanil group was higher than that of the incision pain group and remifentanil group, while the DOR of the synthesis pathway of NGF was higher than that of the incision pain+ remifentanil group.The DOR of the synthesis pathway of TrkA inhibitor group was lower than that of the incision pain+ remifentanil group and NGF group (both P<0.05). The DOR of the degradation pathway in the incision pain+ remifentanil group was lower than that in the incision pain group and remifentanil group, the DOR of the degradation pathway in the NGF group was lower than that in the incision pain+ remifentanil group, and the DOR of the degradation pathway in the TrkA inhibitor group was higher than that in the incision pain+ remifentanil group and NGF group (both P<0.05). Conclusion:The NGF/TrkA signaling pathway is involved in rat incisional pain-remifentanil hyperalgesia by upregulating the delta opioid receptor of the dorsal root ganglia.

Objective To investigate the effects of regenerating islet-derived protein 3β(Reg3β)on intestinal function and glycolysis in septic mice,as well as its role in promoting lactylation.Methods ① In vivo experiments:a total of 36 adult male C57BL/6 mice,including wild-type(WT)and Reg3β knockout(KO)mice,were randomly divided into six groups using a random number table:WT sham group,WT cecal ligation and puncture(CLP)-induced sepsis group(WT CLP group),WT sham+Reg3β intervention group(WT sham group),WT CLP+Reg3β intervention group(WT CLP+Reg3β group),KO sham group,and KO CLP group(n=6 per group).Blood glucose levels were measured at 24 hours and 48 hours after modeling;At 48 hours after modeling,ileum tissues were collected for hematoxylin-eosin(HE)staining to observe histopathological changes,immunofluorescence staining was performed to assess the positive expression levels of lactylated proteins,Western blotting was used to detect the expression levels of lactylated proteins in ileum tissues.② In vitro experiments:Cultured RAW264.7 cells were randomly divided into four groups using a random number table:blank control group,lipopolysaccharide(LPS)-induced sepsis model group(LPS group),Reg3β group,and LPS+Reg3β group.After 24 hours of drug induction,cells were collected,and Western blotting was performed to measure the levels of lactylated proteins,the culture medium was collected to determine lactylation levels.Results ① Histopathological observations showed that compared with the WT CLP group,the WT CLP+Reg3β group exhibited milder villus breakage and inflammatory cell infiltration.The KO CLP group showed more severe damage,with significantly shortened intestinal villi and separation of the epithelial layer from the lamina propria.Compared with the WT CLP group,blood glucose levels were significantly higher in the KO CLP group(mmol/L:6.83±1.15 vs.4.78±1.37,P<0.05).Both Western blotting and immunofluorescence staining results indicated that,compared with the WT CLP group,lactylation levels were significantly decreased in the KO CLP group[lactylated protein expression(lactylated protein/β-actin):0.48±0.20 vs.0.78±0.09;positive lactylated protein expression(mean fluorescence intensity):59.84±6.02 vs.100.00±5.26,both P<0.01].② Western blotting results of RAW264.7 cells cultured for 24 hours showed that compared with the LPS group,the LPS+Reg3β group exhibited significantly increased lactylated protein expression levels(lactylated protein expression/β-actin:3.67±0.48 vs.1.64±0.49,P<0.01).Compared with the blank control group,the lactate levels in the culture medium of the LPS group were significantly increased(mmol/L:4.95±0.20 vs.3.82±0.09,P<0.01).Compared with the LPS group,the lactate levels in the culture medium of the LPS+Reg3β group were also significantly increased(mmol/L:6.03±0.32 vs.4.95±0.20,P<0.01).Conclusion Reg3β promotes intestinal protein lactylation and exerts a protective effect on the intestine in sepsis,suggesting that Reg3β may serve as a novel therapeutic target for sepsis.

Objective:To evaluate the role of tubulin tyrosin ligase like-6 (TTLL6)-mediated microtubule polyglutamylation and Spastin(a microtubule cleaving protein)-induced excessive microtubule cleavage in the developmental impairment of dendritic spines in neonatal mice following repeated sevoflurane anesthesia, by utilizing TTLL6 conditional knockout mice.Methods:Fifty SPF female TTLL6 brain tissue-specific knockout (TTLL6 CKO: Camk2-Cre + ; TTLL6 f/f) and fifty control (TTLL6 CON: TTLL6 f/f) mice with C57BL/6J background, aged 6 days old were selected.TTLL6 CKO mice were divided into TTLL6 CON control group and TTLL6 CON sevoflurane group, and TTLL6 CKO mice were divided into TTLL6 CKO control group and TTLL6 CKO sevoflurane group, with 25 mice in each group by random block method.Mice in the sevoflurane groups were exposed to 3% sevoflurane with 60% O 2 for 2 hours daily on postnatal days 6, 8, and 10.The mice in control groups received only 60% O 2 under the same condition.The polyGlu-Tubulin and postsynaptic density 95(PSD95) protein expression were detected using Western blot. The expressions of TTLL6, Spastin, and α-Tubulin were assessed via immunofluorescence.Golgi staining and electron microscopy were employed to observe the density of hippocampal dendritic spines and synaptic conditions. The Morris water maze test was used to evaluate spatial memory capabilities. Statistical analysis was performed using GraphPad Prism 8.0 software. Results:(1) Behavioral results showed significant time and group interactions among the four groups in terms of latency to find the platform ( F=8.22, P<0.001).Mice in the TTLL6 CON sevoflurane group had a significantly longer escape latency on days 3-7 compared with the TTLL6 CON control group (all P<0.05), while there was no significant difference between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group (all P>0.05). The number of platform crossings differed significantly among the four groups ( H=11.95, P=0.007).The TTLL6 CON sevoflurane group had significantly fewer crossing times than the TTLL6 CON control group ( P<0.05), but no significant difference was observed between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group ( P>0.05). (2) Golgi staining and electron microscopy results revealed significant differences in dendritic spine density and synapse number among the four groups( F=29.00, 41.94, both P<0.001). The dendritic spine density ((5.83±0.40)/10 μm) and the number of synapses ((3.67±0.58)/10 μm) in the TTLL6 CON sevoflurane group were both significantly lower than those in the TTLL6 CON control group ((12.87±1.70)/10 μm, (9.33±0.57)/10 μm)(both P<0.05). In contrast, there was no statistically significant difference between the TTLL6 CKO sevoflurane group and TTLL6 CKO control group (both P>0.05). (3) Immunofluorescence results showed significant differences in the percentage of TTLL6 and Spastin and α-Tubulin co-expressed positive cells in the CA3 region of the hippocampus among the four groups of mice ( F=215.20, 26.08, both P<0.001). The percentage of TTLL6 and Spastin and α-Tubulin co-expressed positive cells in the TTLL6 CON sevoflurane group ((16.75±1.81) %, (47.98±8.42) %) were significantly higher than those in the TTLL6 CON control group ((2.44±0.58) %, (20.07±4.54) %)(both P<0.05), while there was no statistically significant difference between the TTLL6 CKO sevoflurane group and TTLL6 CKO control group ( P>0.05). (4) Western blot results indicated significant differences in the expression of polyGlu-Tubulin and PSD95 proteins in the hippocampal tissue among the four groups of mice ( F=19.66, 8.57, both P<0.001). The TTLL6 CON sevoflurane group had higher polyGlu-Tubulin expression (0.86±0.19) and lower PSD95 expression (0.61±0.13) compared to the TTLL6 CON control group (0.51±0.11, 1.01±0.07) (both P<0.05).However, there was no significant difference between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group ( P>0.05). Conclusion:The mechanism underlying long-term cognitive impairment in developing brain of neonatal mice caused by repeated sevoflurane anesthesia may relate to the upregulation of TTLL6-induced microtubule polyglutamylation and accelerated Spastin-mediated microtubule severing, which ultimately leads to abnormal dendritic spine development.

Objective:To evaluate the changes in cortical electroencephalogram (EEG) burst suppression rate (BSR) during sevoflurane anesthesia in septic mice and the correlation with cerebral blood perfusion.Methods:Forty SPF male C57BL/6J mice, aged 8-10 weeks, weighing 22-25 g, were divided into 2 groups ( n=20 each) by the random number table method: sham operation group (Sham group) and cecal ligation perforation group (CLP group). The sepsis model was established by cecal ligation and puncture in anesthetized animals. Mice in both groups inhaled 2% sevoflurane for 2 h. During sevoflurane anesthesia, BSR (30 min as an epoch) on electroencephalogram was recorded, and the cortical cerebral blood perfusion was recorded using the laser speckle flow imaging at 30, 60, 90 and 120 min of anesthesia. Results:Compared with Sham group, the cortical EEG BSR was significantly increased, and the cortical cerebral blood perfusion was decreased during sevoflurane anesthesia in CLP group ( P<0.05). Cortical EEG BSR was negatively correlated with cortical cerebral blood perfusion ( P<0.05). Conclusions:Cortical EEG BSR increases during sevoflurane anesthesia in septic mice, which may be related to decreased cortical cerebral blood perfusion.

Objective:To evaluate the role of ZIP7 in sepsis-induced cardiomyopathy in mice.Methods:Ninety wild-type and 90 cardiomyocyte-specific knockout ZIP7 (ZIP7 cKO) male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups using a random number table method: wild-type sham operation group (Sham group) and wild-type sepsis group (Sep group), ZIP7 cKO sham operation group (cKO + Sham group) and ZIP7 cKO sepsis group (cKO + Sep group), with 45 mice in each group. The sepsis-induced cardiomyopathy model was developed using the cecal ligation and puncture in anesthetized mice. Twenty mice were randomly selected to record the survival for 10 days postoperatively. At 18 h after surgery, the left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were measured by echocardiography, and serum concentrations of cardiac troponin T (cTnT), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay. The contents of hydroxyl radical (·OH) and peroxynitrite anion (ONOO -) were determined using a colorimetric assay, the morphology of myocardial mitochondria was observed with a transmission electron microscope, and the expression of dynamin-related protein 1 (Drp1), mitofusin-2 (Mfn2), and optic atrophy 1 (Opa1) in myocardial tissues was detected using Western blot. Results:Compared to Sham group, the survival rate, LVEF and LVFS were significantly decreased, serum concentrations of cTnT, TNF-α and IL-6 were increased, the contents of ·OH and ONOO - in myocardial tissues were increased, the expression of Drp1 was up-regulated, the expression of Mfn2 and Opa1 was down-regulated ( P<0.05), myocardial cells exhibited mitochondrial swelling, and marked destruction of mitochondrial cristae was observed in Sep group, and no significant differences were found in the aforementioned parameters in cKO+ Sham group ( P>0.05). Compared to Sep group, the survival rate, LVEF and LVFS were significantly increased, serum concentrations of cTnT, TNF-α and IL-6 were decreased, the contents of ·OH and ONOO - in myocardial tissues were decreased, the expression of Drp1 was down-regulated, the expression of Mfn2 and Opa1 was up-regulated ( P<0.05), and mitochondrial swelling in myocardial cells was mild, with less dissolution and destruction of mitochondrial cristae in cKO+ Sep group. Conclusions:Myocardial ZIP7 can promote mitochondrial fusion and inhibit mitochondrial fission, potentially contributing to the mechanism of sepsis-induced cardiomyopathy in mice.

Objective:To evaluate the role of cannabinoid receptor 1 (CB1) in the spinal membrane in electroacupuncture (EA)-induced alleviation of morphine-triggered opioid-induced hyperalgesia (OIH) and the relationship with phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups ( n=12 each) using a random number table method: normal saline group (NS group), morphine group (M group), morphine+ EA group (ME group), and morphine+ EA+ CB1 antagonist group (MEA group). The OIH model was established by intrathecal injection of morphine 15 μg (10 μl) twice a day for 7 consecutive days. The equal volume of normal saline 10 μl was given instead in NS group. EA of the " Yanglingquan" (GB34) and " Zusanli" (ST36) acupoints lasting 30 min was performed after the first administration of medication each day, with a current intensity of 2 mA and frequency of 2 Hz in ME group. In MEA group, morphine (15 μg) and CB1 antagonist AM251 30 μg (10 μl) were intrathecally injected twice a day for 7 consecutive days, with other treatments similar to those previously described in ME group. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before administration (T 0) and on days 1, 3, 5 and 7 after administration (T 1-4). Rats were deeply anesthetized and sacrificed at T 3, 4 after administration, and the L 4-6 spinal cord tissues were collected for determination of the expression of membrane CB1, ERK1/2 and p-ERK1/2 by Western blot. Results:Compared with NS group, the MWT was significantly decreased and the TWL was shortened at T 3, 4 in M, ME and MEA groups, the expression of spinal membrane CB1 and p-ERK1/2 was significantly up-regulated at T 4 after administration in M group, and the expression of spinal membrane CB1 was significantly up-regulated at T 3, 4 after administration in ME and MEA groups ( P<0.05). Compared with M group, the MWT was significantly increased and the TWL was prolonged at T 3, 4, and the expression of p-ERK1/2 was down-regulated at T 4 after administration in ME group ( P<0.05), no significant change was found in MWT or TWL at T 3, 4 in MEA group ( P>0.05), and the expression of spinal membrane CB1 was significantly up-regulated at T 3, 4 after administration in ME and MEA groups ( P<0.05). Compared with ME group, the MWT was significantly decreased and the TWL was shortened at T 3, 4, and the expression of spinal membrane CB1 and p-ERK1/2 was up-regulated at T 4 after administration in MEA group ( P<0.05). Conclusions:Up-regulation of spinal membrane CB1 expression is involved in EA-mediated alleviation of morphine-induced OIH, which is associated with the inhibition of ERK1/2 phosphorylation in rats.

Objective:To evaluate the role of tubulin tyrosin ligase like-6 (TTLL6)-mediated microtubule polyglutamylation and Spastin(a microtubule cleaving protein)-induced excessive microtubule cleavage in the developmental impairment of dendritic spines in neonatal mice following repeated sevoflurane anesthesia, by utilizing TTLL6 conditional knockout mice.Methods:Fifty SPF female TTLL6 brain tissue-specific knockout (TTLL6 CKO: Camk2-Cre + ; TTLL6 f/f) and fifty control (TTLL6 CON: TTLL6 f/f) mice with C57BL/6J background, aged 6 days old were selected.TTLL6 CKO mice were divided into TTLL6 CON control group and TTLL6 CON sevoflurane group, and TTLL6 CKO mice were divided into TTLL6 CKO control group and TTLL6 CKO sevoflurane group, with 25 mice in each group by random block method.Mice in the sevoflurane groups were exposed to 3% sevoflurane with 60% O 2 for 2 hours daily on postnatal days 6, 8, and 10.The mice in control groups received only 60% O 2 under the same condition.The polyGlu-Tubulin and postsynaptic density 95(PSD95) protein expression were detected using Western blot. The expressions of TTLL6, Spastin, and α-Tubulin were assessed via immunofluorescence.Golgi staining and electron microscopy were employed to observe the density of hippocampal dendritic spines and synaptic conditions. The Morris water maze test was used to evaluate spatial memory capabilities. Statistical analysis was performed using GraphPad Prism 8.0 software. Results:(1) Behavioral results showed significant time and group interactions among the four groups in terms of latency to find the platform ( F=8.22, P<0.001).Mice in the TTLL6 CON sevoflurane group had a significantly longer escape latency on days 3-7 compared with the TTLL6 CON control group (all P<0.05), while there was no significant difference between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group (all P>0.05). The number of platform crossings differed significantly among the four groups ( H=11.95, P=0.007).The TTLL6 CON sevoflurane group had significantly fewer crossing times than the TTLL6 CON control group ( P<0.05), but no significant difference was observed between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group ( P>0.05). (2) Golgi staining and electron microscopy results revealed significant differences in dendritic spine density and synapse number among the four groups( F=29.00, 41.94, both P<0.001). The dendritic spine density ((5.83±0.40)/10 μm) and the number of synapses ((3.67±0.58)/10 μm) in the TTLL6 CON sevoflurane group were both significantly lower than those in the TTLL6 CON control group ((12.87±1.70)/10 μm, (9.33±0.57)/10 μm)(both P<0.05). In contrast, there was no statistically significant difference between the TTLL6 CKO sevoflurane group and TTLL6 CKO control group (both P>0.05). (3) Immunofluorescence results showed significant differences in the percentage of TTLL6 and Spastin and α-Tubulin co-expressed positive cells in the CA3 region of the hippocampus among the four groups of mice ( F=215.20, 26.08, both P<0.001). The percentage of TTLL6 and Spastin and α-Tubulin co-expressed positive cells in the TTLL6 CON sevoflurane group ((16.75±1.81) %, (47.98±8.42) %) were significantly higher than those in the TTLL6 CON control group ((2.44±0.58) %, (20.07±4.54) %)(both P<0.05), while there was no statistically significant difference between the TTLL6 CKO sevoflurane group and TTLL6 CKO control group ( P>0.05). (4) Western blot results indicated significant differences in the expression of polyGlu-Tubulin and PSD95 proteins in the hippocampal tissue among the four groups of mice ( F=19.66, 8.57, both P<0.001). The TTLL6 CON sevoflurane group had higher polyGlu-Tubulin expression (0.86±0.19) and lower PSD95 expression (0.61±0.13) compared to the TTLL6 CON control group (0.51±0.11, 1.01±0.07) (both P<0.05).However, there was no significant difference between the TTLL6 CKO sevoflurane group and the TTLL6 CKO control group ( P>0.05). Conclusion:The mechanism underlying long-term cognitive impairment in developing brain of neonatal mice caused by repeated sevoflurane anesthesia may relate to the upregulation of TTLL6-induced microtubule polyglutamylation and accelerated Spastin-mediated microtubule severing, which ultimately leads to abnormal dendritic spine development.