Objective To observe the growth situation of Blastocystis hominis in vitro and select the optimal method for culti?vation of B. hominis in different media. Methods Ten positive stools with B. hominis were inoculated in three different media for cultivating,namely 1640,Jone’s medium and vitro medium. And the stools with good growth status and high quantities of B. hominis were chosen to inoculate in the three media with equal amount after subcultivation,and the number of B. hominis was counted every 24 h for ten days,and the morphological changes and growth status were also observed. Results The densities of B. hominis in the 1640 and Jone’s medium were higher than that in the vitro medium 48 h after the inoculation. The same stool sample was inoculated to the three different media and observed for ten days,and the results indicated that the growth of B. homi?nis presented regular changes in the three media,the growth peaks were on the third,sixth and ninth day post inoculation;and the density of B. hominis was the highest in the Jone’s medium. The morphology of B. hominis was the clearest and most dynamic in the vitro medium,while various reproductive forms were observed in the Jone’s medium. Conclusion Jone’s medium is suitable for the growth of B. hominis and can be the first choice for the cultivation of B. hominis in vitro,and vitro medium is the best medium for observing the growth situation of B. hominis.

Objective:To evaluate the role of reactive oxygen species (ROS)-mediated mitochondrial pathway of apoptosis in long-term cognitive impairment induced by multiple exposures to sevoflurane in the neonatal rats.Methods:Sixty SPF healthy neonatal Sprague-Dawley rats, weighing 12-20 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple exposures to sevoflurane for anesthesia group (group S) and ROS inhibitor group (group A). Group S and group A inhaled 3% sevoflurane for 2 h starting from 6, 7 and 8 days after birth, while group C inhaled air.In group A, ROS inhibitor N-acetylcysteine (NAC) 150 mg/kg was intraperitoneally injected before each anesthesia with sevoflurane.The spontaneous activity was evaluated by open field test on day 35 after birth.The cognitive function was determined by Morris water maze test on day 36 after birth.The rats were sacrificed after the end of Morris water maze test, and the hippocampal tissues were obtained for determination of the apoptosis rate of hippocampal neurons, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) (by flow cytometry) and levels of Cyt c and cleaved caspase-9 and caspase-3 (by Western blot). The expression of Bcl-2 and Bax mRNA was detected by real-time polymerase chain reaction.The ultrastructure of mitochondria in hippocampal neurons was observed with a transmission electron microscope. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal neurons and levels of ROS and MMP were increased, the expression of Cyt c, cleaved caspase-9, cleaved caspase-3 and Bax mRNA was up-regulated, the expression of Bcl-2 mRNA was down-regulated, the ratio of Bax/Bcl-2 was increased ( P<0.05), mitochondria were swollen, and mitochondrial cristae structure was broken in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, the apoptosis rate of hippocampal neurons and levels of ROS and MMP were decreased, the expression of Cyt c, cleaved caspase-9, cleaved caspase-3 and Bax mRNA was down-regulated, the expression of Bcl-2 mRNA was up-regulated, the ratio of Bax/Bcl-2 was decreased ( P<0.05), and the mitochondrial swelling and rupture of cristae structure were improved in group A. Conclusion:The mechanism by which multiple exposures to sevoflurane induce long-term cognitive impairment may be related to activating the ROS-mediated mitochondrial pathway of apoptosis in neonatal rats.

Objective:To evaluate the role of RhoA/ROCK2 signaling pathway in multiple exposures to sevoflurane-induced long-term cognitive impairment in neonatal rats.Methods:Sixty SPF healthy neonatal Sprague-Dawley rats of either sex, aged 6 days, weighing 12-20 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple exposures to sevoflurane group (group S) and RhoA/ROCK2 signaling pathway inhibitor Y-27632 group (group Y). Group S and group Y inhaled 3% sevoflurane for 2 h at days 6, 7 and 8 after birth.In group Y, Y-27632 5 mg/kg was intraperitoneally injected before sevoflurane anesthesia.The spontaneous activity was evaluated by open field test on day 35 after birth.The cognitive function was detected by Morris water maze test at day 36 after birth.The rats were sacrificed after Morris water maze test, and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons and cytoplasmic calcium concentration ([Ca 2+ ] i) (by flow cytometry) and expression of phosphorylated RhoA (p-RhoA), ROCK2 and cleaved-caspase-3 (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:There was no significant difference in movement speed, distance and time of stay in the open field center in the open field test among the three groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal neurons and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological injury to hippocampal neurons was found in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, the apoptosis rate of hippocampal neurons and [Ca 2+ ] i were decreased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was down-regulated ( P<0.05), and the pathological injury to hippocampal neurons was attenuated in group Y. Conclusions:The mechanism by which multiple exposures to sevoflurane induces long-term cognitive impairment is related to activation of RhoA/Rock2 signaling pathway and induction of apoptosis rate of hippocampal neurons in neonatal rats.

Objective:To evaluate the effect of esketamine on hippocampal neuronal necroptosis in aged rats with postoperative cognitive dysfunction.Methods:One hundred and twenty SPF-grade healthy male Sprague-Dawley rats, aged 22 months, weighing 550-600 g, were divided into 4 groups ( n=30 each) using a random number table method: control group (group C), postoperative cognitive dysfunction group (group P), postoperative cognitive dysfunction+ esketamine group (group PE), and esketamine group (group CE). Rats received exploratory laparotomy under sevoflurane anesthesia, and esketamine 10 mg/kg and the equal volume of 0.9% sodium chloride were intraperitoneally injected at the end of surgery once a day for 6 consecutive days in group P and group PE, respectively. Rats received no anesthesia and surgery, and esketamine 10 mg/kg and the equal volume of 0.9% sodium chloride were intraperitoneally injected at the end of surgery once a day for 6 consecutive days in group CE and group C, respectively. Morris water maze test was performed at 7th day after surgery. The escape latency, times of crossing the original platform and time spent in the original platform quadrant were recorded. The rats were sacrificed at the end of Morris water maze test, and the hippocampal tissues were collected for determination of the rate of necroptosis and cytosolic Ca 2+ concentrations (by flow cytometry) and expression of mixed lineage kinase domain-like protein (MLKL), phosphorylated MLKL (p-MLKL), receptor-interacting protein kinase-3 (RIPK3), phosphorylated RIPK3 (p-RIPK3), receptor-interacting protein kinase-1 (RIPK1) and phosphorylated RIPK1 (p-RIPK1) (by Western blot). Results:Compared with group C, the escape latency was significantly prolonged, the times of crossing the original platform were decreased, the time spent in the original platform quadrant was shortened, the necroptosis rate of hippocampal neurons and cytosolic Ca 2+ concentrations were increased, and the expression of MLKL, p-MLKL, RIPK3, p-RIPK3, RIPK1 and p-RIPK1 was up-regulated in group P and group PE ( P<0.05). Compared with group P, the escape latency was significantly shortened, the times of crossing the original platform were increased, the time spent in the original platform quadrant was prolonged, the necroptosis rate of hippocampal neurons and cytosolic Ca 2+ concentrations were decreased, and the expression of MLKL, p-MLKL, RIPK3, p-RIPK3, RIPK1 and p-RIPK1 was down-regulated in group PE ( P<0.05). Conclusions:The mechanism by which esketamine attenuates postoperative cognitive dysfunction may be related to inhibition of necroptosis in hippocampal neurons of aged rats.

Objective:To evaluate the effect of esketamine on the efficacy of postoperative patient-controlled intravenous analgesia (PCIA) in the patients with moderate central sensitization undergoing high tibial osteotomy.Methods:Fifty-four patients of both sexes with moderate central sensitization, aged 45-64 yr, with body mass index of 18.0-32.5 kg/m 2, of American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ, undergoing elective high tibial osteotomy, were divided into 2 groups ( n=27 each) using a random number table method: control group (group C) and esketamine group (group ES). Ultrasound-guided femoral nerve block was performed with 0.5% ropivacaine 30 ml on the operated side at 30 min before induction of anesthesia.In C and ES groups, midazolam 0.1 mg/kg, sufentanil 0.2 μg/kg, propofol 1.5 mg/kg, and cisatracurium besilate 0.15 mg/kg were intravenously injected in turn during induction of anesthesia, and in addition esketamine hydrochloride 0.5 mg/kg was injected in ES group, and the equal volume of 0.9% sodium chloride was injected in C group, and then a laryngeal mask airway was placed.Anesthesia was maintained with intravenous infusion of remifentanil 0.1-0.3 μg·kg -1·min -1 and propofol 4-6 mg·kg -1·h -1.Esketamine hydrochloride 0.2 mg/kg was intravenously injected once every 20 min until 30 min before the end of operation in ES group, the equal volume of 0.9% sodium chloride was injected according to the amount of esketamine hydrochloride injected for the same body weight at the same time point in C group, and additional cisatracurium besilate was administered intermittently according to the degree of muscle relaxation.Intraoperative BIS values were maintained at 40-60.Postoperative PCIA was performed, and the patient was admitted to the post-anesthesia care unit.When the efficacy of PCIA was not good, ketorolac tromethamine 30 mg was intravenously injected for rescue analgesia.The intraoperative consumption of remifentanil and propofol and emergence time in the anesthesia recovery room were recorded.The pressing times of PCA and the number of rescue analgesia in each group were recorded within 2 days after operation.The Chinese Richards-Campbell Sleep Questionnaire was used to assess the nighttime sleep quality on the night of surgery and 1 and 2 days after surgery.The Chinese Quality of Recovery was used to assess the early recovery quality at 1 and 2 days after surgery.The first postoperative off-bed time and first walked distance were recorded.The adverse reactions were recorded. Results:Compared with group C, the consumption of remifentanil and propofol was significantly reduced, the emergence time in the anesthesia recovery room was prolonged, the pressing times of PCA and the number of rescue analgesia were decreased within 2 days after operation, the quality of nighttime sleep was improved on the night of surgery and 1 and 2 days after operation, the quality of early recovery on 1 and 2 days after operation was increased, the first postoperative off-bed time was shortened, the first walked distance was prolonged, and the incidence of postoperative adverse effects was decreased in group ES ( P<0.05). Conclusions:Esketamine can enhance the efficacy of postoperative PCIA in the patients with moderate central sensitization undergoing high tibial osteotomy.

Objective:To evaluate the role of 1, 4, 5-inositol triphosphate receptor (IP3R) in necroptosis of hippocampal neurons induced by sevoflurane anesthesia in aged rats.Methods:Sixty healthy male Sprague-Dawley rats, aged 18 months, weighing 500-600 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), sevoflurane anesthesia group (group S) and sevoflurane anesthesia + IP3R antagonist group (group S+ I). S and S+ I groups inhaled 2% sevoflurane for 5 h. In group S+ I, IP3 receptor antagonist 2-APB 3 mg/kg was intraperitoneally injected at 10 min before sevoflurane inhalation, and the equal volume of dimethyl sulfoxide was intraperitoneally injected in group C and group S. Morris water maze test was used to test the cognitive function on the day after the end of sevoflurane anesthesia.Then the animals were sacrificed and the brain tissues were obtained for microscopic examination of the pathological changes after HE staining and Nissl staining (with a light microscope) and for determination of the free calcium concentration ([Ca 2+ ] i) and rate of necroptosis of hippocampal neurons (by flow cytometry) and expression of IP3R, receptor-interacting protein kinase-1 (RIPK1), receptor-interacting protein kinase-3 (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) (by Western blot). Results:Compared with group C, the escape latency was significantly prolonged, the times of crossing the platform were reduced, the time of staying at the target quadrant was shortened, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were increased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was up-regulated in group S and group S+ I ( P<0.05). Compared with group S, the escape latency was significantly shortened, the times of crossing the platform were increased, the time of staying at the target quadrant was prolonged, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were decreased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was down-regulated in group S+ I ( P<0.05). Conclusions:The mechanism by which sevoflurane induces cognitive dysfunction may be related to the imbalance of calcium homeostasis caused by activation of IP3R and thus inducing programmed necrosis in aged rats.

Objective:To evaluate the effect of pre-injection of young rat plasma on cognitive dysfunction after cerebral ischemia-reperfusion (I/R) in aged rats and the role of phosphatidylinositol 3-kinase/serine threonine protein kinase (PI3K/Akt) signaling pathway.Methods:Seventy-two SPF-grade healthy male Sprague-Dawley rats, aged 18 months, weighing 600-650 g, were divided into 4 groups ( n=18 each) by the random number table method: control group (group C), cerebral I/R group (group IR), pre-injection of young rat plasma group (group P) and PI3K inhibitor LY294002 group (group LY). In group P and group LY, young rat plasma 100 μl/time was injected via the tail vein. In group C and group IR, the equal volume of normal saline was injected via the the tail vein, 2 times a week for 4 weeks. Then the model of cerebral I/R injury was developed under sevoflurane anesthesia in IR, P and LY groups. LY294002 0.3 mg/kg was injected through the tail vein at 1 h before anesthesia in LY group. The neurological deficit score (Longa score) was performed at 24 h after reperfusion, and then 6 rats were randomly sacrificed, and brain tissues were obtained to determine the cerebral infarct volume. Spontaneous mobility and anxiety-like behavior were assessed by the open field test at day 29 of reperfusion, and cognitive function was assessed by the novel object recognition test at day 30 of reperfusion. At the end of the behavioral test, rats were sacrificed, hippocampal tissues were isolated for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), postsynaptic dense protein-95 (PSD-95) and synaptic vesicle protein (SYN) (by Western blot), and the dendritic length and dendritic spine density of neurons in the hippocampal CA1 region. Results:There was no significant difference in motor speed, distance traveled, and time of staying at the center of the open field among the four groups ( P>0.05). Compared with group C, the Longa score and cerebral infarct volume were significantly increased, the percentage of novel object exploration and discrimination index were decreased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was down-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were decreased in IR, P and LY groups ( P<0.05). Compared with group IR, Longa score and cerebral infarct volume were significantly decreased, the percentage of novel object exploration and discrimination index were increased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was up-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were increased in group P ( P<0.05), and no significant change was found in the parameters mentioned above in group LY ( P>0.05). Compared with group P, Longa score and cerebral infarct volume were significantly increased, the percentage of novel object exploration and discrimination index were decreased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was down-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were decreased in group LY ( P<0.05). Conclusions:Pre-injection of young rat plasma can attenuate cognitive dysfunction after cerebral I/R in aged rats, and the mechanism is related to activation of hippocampal PI3K/Akt signaling pathway and improvement in synaptic plasticity.

Objective:To evaluate the role of N-methyl-D-aspartate receptors (NMDA receptors) in sevoflurane anesthesia-caused necroptosis in hippocampal neurons of aged mice.Methods:Ninety clean-grade healthy male C57BL/6 mice, aged 18 months, weighing 27-30 g, were divided into 3 groups ( n=30 each) using a random number table method: control group (group C), sevoflurane anesthesia group (group S) and sevoflurane anesthesia plus NMDA receptor antagonist memantine hydrochloride group (group S+ M). Mice inhaled 3% sevoflurane for 2 h for 3 consecutive days in S group and S+ M group, and memantine hydrochloride 20 mg/kg was intraperitoneally injected at 1 h before each inhalation of sevoflurane in S+ M group.Mice only inhaled pure oxygen for 2 h in group C. Ten mice of each group were selected on 1 day before anesthesia and 3 and 7 days after anesthesia to perform Morris water maze test.The mice were sacrificed immediately after Morris water maze test, and hippocampus was removed for microscopic examination of pathological changes (with a light microscope) and for determination of the necroptosis rate of neurons and cytoplasmic free calcium concentration([Ca 2+ ] i)(by flow cytometry), and expression of NMDA receptor subtypes GluN2A, GluN2B and receptor-interacting protein kinase 1 (RIP1) (by Western blot). Results:Compared with group C, the escape latency was significantly prolonged, and the frequency of crossing the original platform was decreased, and the [Ca 2+ ] i and neuronal necroptosis rate in the hippocampus were increased at each time point after anesthesia, and the expression of GluN2A, GluN2B and RIP1 was up-regulated( P<0.05), and the pathologic changes were accentuated in S group and S+ M group.Compared with group S, the escape latency was significantly shortened, and the frequency of crossing the original platform was increased, and the [Ca 2+ ] i and neuronal necroptosis rate in the hippocampus were decreased at each time point after anesthesia, and the expression of GluN2A, GluN2B and RIP1 was down-regulated ( P<0.05), and the pathologic changes were attenuated in group S+ M. Conclusions:NMDA receptors are involved in the process of cognitive dysfunction induced by sevoflurane anesthesia in aged mice, and the mechanism may be related to the promotion of necrptosis in hippocampal neurons.

Objective:To evaluate the effect of pre-infusion of young rat plasma on postoperative cognitive function in aged rats and role of phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway.Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 18 months, weighing 550-650 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), operation group (group O), young rat plasma group (group P) and PI3K inhibitor LY294002 group (group LY). The young rat plasma 100 μl/time was injected via the caudal vein twice a week for 4 consecutive weeks in group P and group LY, while the equal volume of normal saline was given instead in group C and group O. Rats received internal fixation for unilateral tibial fracture under sevoflurane anesthesia in O, P and LY groups.Rats received no treatment in group C. PI3K inhibitor LY294002 0.3 mg/kg was injected through the caudal vein before anesthesia in group LY.The ability of spontaneous activity was evaluated by open field test at 3 days after surgery, and then the cognitive function was assessed by Morris water maze test.The rats were sacrificed after the end of behavioral testing, and the hippocampal tissues were isolated for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), synapsin, synaptophysin I and synaptic vesicle protein (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). The number of synapses was recorded. Results:There was no significant difference in the movement speed and length and time spent in the central zone among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was down-regulated, and the number of synapses was reduced in O and LY groups ( P<0.05), and no significant change was found in the parameters mentioned above in group P ( P>0.05). Compared with group O, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was up-regulated, and the number of synapses was increased in group P ( P<0.05), and no significant change was found in the parameters mentioned above in group LY ( P>0.05). Compared with group P, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was down-regulated, and the number of synapses was reduced in group LY ( P<0.05). Conclusion:Pre-infusion of young rat plasma can improve postoperative cognitive function in aged rats, and the mechanism is related to activation of PI3K/Akt pathway and improvement of synaptic plasticity.

Objective:To evaluate the role of brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) signaling pathway in pre-injection of young rat plasma-induced reduction of sevoflurane-caused cognitive dysfunction in aged rats.Methods:Eighty SPF healthy male Sprague-Dawley rats, aged 18 months, weighing 550-650 g, were divided into 4 groups ( n=20 each) using a random number table method: control group (group C), sevoflurane anesthesia group (group S), young rat plasma group (group Y) and BDNF/TrkB signaling pathway inhibitor K252a group (group K). The plasma 100 μl obtained from 3-month-old young rats was injected via the tail vein in group Y and group K, while the equal volume of normal saline was given via the tail vein in group C and group S, twice a week, for 4 weeks.In S, Y and K groups, 3% sevoflurane was inhaled for 3 h starting from the end of treatment, and BDNF/TrkB signaling pathway inhibitor K252a was injected via the tail vein before anesthesia in group K. The open field test and Morris water maze test were performed at 3 days after anesthesia to assess the spontaneous motor ability and cognitive function.Then the rats were sacrificed, and the hippocampal tissues were isolated for determination of the expression of BDNF, phosphorylated TrkB (p-TrkB), postsynaptic dense protein-95 (PSD-95) and synaptic vesicle protein (SYN) (by Western blot), dendritic length and dendritic ridge density of neurons in hippocampal CA1 area (by Golgi staining), and the number of synapses and length of synaptic active area (with a transmission electron microscope). Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-TrkB, BDNF, PSD-95 and SYN was down-regulated, and the dendritic length, dendritic ridge density, the number of synapses and length of synaptic active area were decreased in group S ( P<0.05). Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-TrkB, BDNF, PSD-95 and SYN was up-regulated, and the dendritic length, dendritic ridge density, the number of synapses and length of synaptic active area were increased in group Y ( P<0.05). Compared with group Y, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-TrkB, BDNF, PSD-95 and SYN was down-regulated, and the dendritic length, dendritic ridge density, the number of synapses and length of synaptic active area were decreased in group K ( P<0.05). Conclusions:The mechanism by which pre-injection of young rat plasma reduces sevoflurane-induced cognitive dysfunction is related to activation of BDNF/TrkB signaling pathway and improvement in synaptic plasticity in the hippocampus of aged rats.