Objective To explore the possible mechanism for the neuroprotective effect of ifenprodil by investigating its effects on inducible nitric oxide synthase (iNOS) expression and activity and apoptosis in the ischemic penumbra following focal cerebral ischemia-reperfusion (I/R) in rats.Methods Fifty-four adult male SD rats weighing 280-320 g were randomly divided into 3 groups ( n = 18 each) : I sham operation group (group S) ; II focal cerebral I/R group (group I/R) and Ⅲ ifenpradil preconditioning group (group IF) received intraperitoneal ifenprodil 10 mg/kg before focal cerebral I/R. Focal cerebral I/R was induced by middle cerebral artery occlusion (MCAO) . A 3-0 nylon thread with rounded tip was inserted into right internal jugular vein and threaded cranially until resistance was met. MCAO was maintained for 2 h. At 48 h after reperfusion, the animals were assessed for neurological function which was scored (0 = no functional deficit, 4 = unable to crawl, unconscious) and then decapitated. The brains were immediately removed for microscopic examination and determination of iNOS protein expression and activity, NO content and apoptosis in the ischemic core (IC) and penumbra (IP). Results Ifenprodil pretreatment significantly decreased the cerebral infarct size and neurological scores in group IF as compared with group I/R. In group I/R the iNOS activity was increased compared with group S.The iNOS activity and NO content were significantly lower in IP than in IC in group IR and IF. The TUNEL-positive cells were also mainly confined to IP. Compared with group I/R, in group IF the iNOS protein expression was significantly down-regulated in IC and IP and the iNOS activity and NO content in IC and IP were suppressed and TUNEL-positive cells were significantly reduced in IP. Conclusion Ifenprodil pretreatment has protective effect against cerebral I/R injury by inhibiting iNOS protein expression in IP, suppressing iNOS activity and NO content and reducing apoptosis.

Objective To evaluate the effect of hypothermia on the expression of dynamin?related protein 1 ( Drp1) in brain tissues during global cerebral ischemia?reperfusion ( I∕R) in rats. Methods Thirty?six healthy male Sprague?Dawley rats, weighing 280-320 g, were divided into 3 groups ( n=12 each) using a random number table: sham operation group ( group Sham ) , global cerebral I∕R group ( group I∕R) and hypothermia group ( group H) . Cardiac arrest was induced by transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the global cerebral I∕R model in anesthetized rats in I∕R and H groups. In group H, the body temperature ( rectal temperature) was cooled down to 32-34 ℃ within 15 min starting from the beginning of reperfusion, and maintained at this level for 6 h. At 72 h of reperfusion, neurological deficit was scored, and the rats were sacrificed, and the whole brain was removed for examination of the pathological changes in hippocampal CA1 region and for determination of nor?mal pyramidal cell count and neuronal apoptosis in hippocampal CA1 region and expression of Drp1 and cy?tochrome c (Cyt c) in hippocampal tissues (by Western blot). The apoptosis rate was calculated. Re?sults Compared with group S, the neurological deficit score and apoptosis rate were significantly in?creased, and the number of normal pyramidal cells was decreased in I∕R and H groups, the expression of Drp1 and Cyt c in hippocampal tissues was significantly up?regulated in group I∕R ( P<0.05) , and no sig?nificant change was found in the expression of Drp1 and Cyt c in hippocampal tissues in group H ( P>0.05) . Compared with group I∕R, the neurological deficit score and apoptosis rate were significantly de?creased, the number of normal pyramidal cells was increased, and the expression of Drp1 and Cyt c in hip?pocampal tissues was down?regulated in group H ( P<0.05) . Conclusion The mechanism by which hypo?thermia inhibits cell apoptosis during global cerebral I∕R may be related to down?regulation of Drp1 expres?sion in rats.

Objective To investigate the effect of mild hypothermia combined with mitochondrial divison inhibitor 1 in mitochondrial after cerebral ischemia-reperfusion (IR).Methods Fourty male healthy Sprague-Dawley (SD) rats, weighing 280-320 g, were randomly divided into 5 groups (n=8 each): group Sham, group IR, hypothermia group (group H), Mdivi-1 group (group M) and hypothermia+Mdivi-1 group (group HM).Animal models of global cerebral IR were established by transoesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation (ischemia 4 min and reperfusion 6 h).The group Sham was similarly treated to group IR except the cardiac arrest and cardiopulmonary resuscitation.In groups H and HM, the core temperature was cooled down to 32-34℃ within 15 min starting from the beginning of reperfusion, and maintained for 6 h.In the other groups, the core temperature was maintained at the normal temperature.In groups M and HM, the animals were given Mdivi-1 (1.2 mg/kg) intravenously at the beginning of the reperfusion and the other groups were given the same Volume of dimethylsnlfone (DMSO).After 6 h of reperfusion, the rats were sacrificed, and bilateral hippocampi were immediately removed for determination the protein level of dynamin-related proten 1 (Drp1) and cytochrome C (Cyt-C) expression by Western blot and obsevation of the mitochondrial structure of pyramidal cell in hippocampal CA1 under electronic microscope.Results Compared with group Sham, the expression of Drp1 and Cyt-C was up-regulated in groups IR, H, M and HM (P＜0.05).Compared with group IR, the expression of Drp1 and Cyt-C was down-regulated in groups H, M and HM (P＜0.05).Compared with groups H and M, the expression of Drp1 and Cyt-C was down-regulated in group HM (P＜0.05).There was no significant difference in the expression of Drp1 and Cyt-C between groups H and M.The mitochondria were rod-shaped with clear and sound structure in group Sham, while mitochondria showed various degree of fission, swollen structures, matrix deposit, vacuoles formation and cristae collapse in other groups.The changes of group HM were relatively slight.Conclusion Mild hypothermia combined with mitochondrial divison inhibitor 1 alleviate mitochondrial damage after global cerebral IR of rats.The combined effect is better than that of any individual application.

Objective To investigate the effect of hydrogen-rich saline combined with mild hypothermia on cerebral ischemia-reperfusion (IR) injury in rats. Methods Fifty male SD rats, aged 9-10 weeks, weiging 250-300 g, were randomly divided into 5 groups (n= 10 each): sham operation group (group S), group IR, hygrogen-rich saline group (group H), mild hypothermia group (group M) and hydrogen-rich saline + mild hypothermia group (group HM). In group IR, H, M and HM cerebral IR was induced by 15 min ligation of bilateral carotid artery followed by 6 h reperfusion. In group H and HM intraperitoneal hydrogen-rich saline 5 ml/kg was injected immediately after reperfusion, while the equal volume of normal saline was injected instead of hydrogen-rich saline in the other three groups. At the same time, the rectal temperature was maintained at 37-38 ℃ in group S,IR and M, while it was reduced to 32-34 ℃ by physical method within 15 min, lasting for 6 h, in group M and HM. The animals were sacrificed after 6 h of reperfusion, and then the hippocampus was removed for microscopic examination. The expression of HO-1 and content of MDA and TNF-α were determined by Western blot. Results The cerebral IR injury was attenuated in group H, M and HM compared with group IR, with the slightest injury in group HM. The expression of HO-1 and content of MDA and TNF-α were significantly higher in group IR, H, M and HM than in group S. The expression of HO-1 was significantly higher, while the content of MDA and TNF-α were lower in group H, M and HM than in group IR, and in group HM than in group H and M. There was no significant difference in the expression of HO-1 and content of MDA and TNF-α between group H and M. Conclusion Hydrogen-rich saline combined with mild hypothermia can attenuate cerebral I/R injury in rats via up-regulating the expression of HO-1 and decreasing the content of MDA and TNF-α in hippocampus.

Objective To investigate the role of mitochondrial permeability transition pore (mPTP) of hippocampal neurons in process of hydrogen-rich saline attenuating global cerebral ischemia-reperfusion (I/R) injury in rats.Methods Seventy-two male Sprague Dawley rats,weighing 250-300 g,were randomly divided into six groups ( n =12 each):sham operation group (group S),cerebral ischemia-reperfusion group (group IR),normal saline group (group NS),hydrogen-rich saline group (group H),atractyloside group (group A) and hydrogen-rich saline + atractyloside group (group HA).Global cerebral I/R injury was produced by four-vessel occlusion method.Bilateral vertebral arteries were cauterized.Then bilateral common carotid arteries were occluded for 15min and followed by reperfusion.In groups H and HA,hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after reperfusion,while equal volume of normal saline was injected in the other four groups.The rats in groups A and HA received intracerebroventricular injection of atractyloside 15 μl 10 min before reperfusion,while groups NS and H received intracerebroventricular injection of equal volume of normal saline.After the neurological behavior was evaluated at 24 h of reperfusion,8 rats in each group were sacrificed and the hippocampi were immediately isolated and homogenized followed by density gradient centrifugation.The opening degree of mPTP was assayed with spectrophotometry and the mitochondrial membrane potential (MMP) was detected with Rhodamine 123 method.Four rats in each group were killed at 72 h of reperfusion and the brains were removed for microscopic examination of the area CA1 of the hippocampus and determination of the number of normal pyramidal neurons.Results Compared with group S,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in the other five groups ( P ＜ 0.05).The neurological behavior was better,MMP was increased and mPTP opening degree was decreased in groups H and HA as compared with group IR ( P ＜ 0.05).Compared with group H,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in group HA ( P ＜ 0.05).Compared with group IR,the number of normal pyramidal neurons at 72 h of reperfusion in the CA1 region of the hippocampus was higher in group HA ( P ＜0.05).The injury of the CA1 region of the hippocampus at 72 h of reperfusion was attenuated in group H as compared with groups IR,NS,A and HA.Conclusion Hydrogen-rich saline can attenuate global cerebral I/R injury throngh inhibiting the mPTP opening and reducing the dissipation of MMP,thus maintaining the mitochondrial function.

BACKGROUND:This review explores the current research status and frontier hot spots of functional magnetic resonance imaging(fMRI)in the field of traditional Chinese medicine(TCM)for the treatment of ischemic stroke,and attempts to grasp future research trends,with a view to providing a reference for subsequent research in this field. OBJECTIVE:To visualize and analyze the hotspots and frontiers in the TCM treatment of ischemic stroke based on fMRI using CiteSpace knowledge mapping combined with binary logistic regression equations,in order to grasp the future research trends and further explore the distribution of brain regions with abnormal neural activity related to the types of post-stroke dysfunction. METHODS:CNKI,WanFang,VIP,Chinese Biomedical Literature Database and Web of Science core set database were searched.CiteSpace was used to plot keyword co-occurrence,keyword clustering timeline,burst term detection,co-cited literature mapping to analyze hotpots and frontiers in this field.Binary logistic regression analysis fitted the distribution of brain regions with abnormal neural activity associated with different dysfunction after ischemic stroke. RESULTS AND CONCLUSION:A total of 354 articles were included for CiteSpace knowledge mapping analysis.The number of annual publications showed that the research popularity has been raised from 2000 to 2022 with a good development prospect,but the core strength is mainly concentrated in China.Keywords co-occurrence and clustering time line analysis showed that aphasia,hemiplegia and cognitive impairment are the hot poststroke dysfunction types.Electroacupuncture,acupuncture and head acupuncture are hotspot intervention measures.Functional connectivity is a hotspot analysis method,and resting fMRI is a hotspot scanning technology.The time span of each research hotspot is long,indicating that it has a certain research value and the relevant research is gradually deepening,promoting the research progress in this field.However,acupuncture is the main intervention measure,and there is a lack of research on traditional Chinese medicine,Chinese patent medicine,acupuncture and medicine combination and other TCM therapy.Burst term detection results showed that functional connectivity,graph theory,degree centrality,default mode network,randomized controlled trials have great influence and strong explosive power.They are the current and future frontier hot spots in this field,suggesting that future research should focus on the brain network information integration and strengthen the scientific and rigorous clinical trial design.The results of co-cited literature analysis showed that the epidemiological investigation of ischemic stroke,the safety and effectiveness of acupuncture in the treatment of stroke,the brain activation patterns under different tasks,and the neuropathological mechanism of brain network dysfunction after stroke are the theoretical basis of this field.Future research direction in this field is to explore TCM-targeted brain regions and neural networks to reveal the brain effect mechanism of TCM promoting neural remodeling after stroke.A total of 255 articles were included for binary Logistic regression analysis.The results showed that sensorimotor cortex and premotor area dysfunction are positively correlated with the incidence of motor dysfunction after stroke;hippocampus,cerebellum posterior lobe,precuneus,inferior temporal gyrus and anterior cingulate nerve dysfunction are positively correlated with the incidence of cognitive impairment after stroke;cuneus,angular gyrus and prefrontal lobe neural dysfunction were positively correlated with the incidence of affective disorder after stroke;anterior cingulate,cerebellum posterior lobe neural dysfunction are positively correlated with the incidence of swallowing disorder after stroke.The above brain regions are the core brain regions of the sensorimotor network,default mode network and reward loop,suggesting that functional abnormalities within or between brain networks related to dysfunction may be potential target areas for TCM intervention,but the specific changes in neural activity activation or inhibition still need to be refined.

Objective:To investigate the efficacy of a SARS-CoV-2 recombinant protein vaccine as a booster dose.Methods:A new immunogen, namely RBD-sc-trimer, was designed by tandem repeating of single receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein to mimic the trimeric form of RBD presented by the virus. The RBD-sc-trimer protein was expressed as a His-tagged fusion protein using a baculovirus expression system and purified by nickel affinity column. The purified protein was identified by Western blot. Its in vitro binding activity to human angiotensin converting enzyme 2 (hACE2) was analyzed by ELISA. The immunogenicity of RBD-sc-trimer as well as RBD proteins of other forms including RBD dimer (RBD-Fc), RBD monomer (RBD) and S protein trimer (S trimer) as a booster dose was evaluated in BALB/c mice. Results:In terms of both binding and neutralizing antibodies against SARS-CoV-2, RBD-sc-trimer showed an immunogenicity that was superior to that of RBD-Fc and RBD and close to the level of S trimer. The antibody response induced by RBD-sc-trimer was characterized as Th1-biased. Moreover, it displayed a stronger cross-neutralization activity against SARS-CoV-2 Beta, Delta and Omicron variants. The titer of neutralizing antibody against Omicron induced by RBD-sc-trimer only decreased by 9.1 folds relative to the prototype strain, while the antibody response induced by RBD-Fc and S trimer decreased by 68.4 and 70.8 folds, respectively.Conclusions:The recombinant protein, RBD-sc-trimer, which was capable of eliciting stronger humoral response in mice as a booster dose and showed the superiority in raising cross-reactive antibodies against SARS-CoV-2 variants over non-trimeric RBD forms, should be considered as an optimal immunogen for the development of more effective SARS-CoV-2 vaccines.

Objective:To reduce the immunogenicity of vaccinia virus vector by replacing the D8L region, which is a neutralizing antibody epitope in vaccinia virus, with an exogenous gene.Methods:A gene fragment encoding influenza virus hemagglutinin (HA) was inserted into the D8L region to replace it using homologous recombination technique. Then, a recombinant vaccinia virus influenza vaccine was constricted. A recombinant vaccinia virus vaccine with the TK region expressing HA was used as a control. The expression of HA was validated by Western blot. BALB/c mice were immunized with the vaccines and the serum antibody titers two weeks after each immunization were evaluated by ELISA and hemagglutination inhibition assay. The protective efficacy of the recombinant vaccinia virus was assessed through a challenge experiment.Results:Western blot confirmed the successful expression of HAD8L protein in the constructed recombinant vaccines. ELISA and hemagglutination inhibition assay showed that after the primary immunization, the anti-HA antibody titer induced by the recombinant vaccinia virus with D8L region mutation was slightly higher than that induced by the vaccine with TK region mutation, and the difference was statistically significant with the increase of immunization times ( P<0.05). The recombinant vaccinia virus with D8L region mutation showed significantly lower immunogenicity than the recombinant virus with TK region mutation after the primary immunization, but there was no significant difference between them with the increase of immunization times ( P>0.05). After H1N1pdm challenge, no virus was detected in the mice immunized with the recombinant vaccinia virus with D8L region mutation and the mice showed mild lung inflammation and less tissue damage. Conclusions:This study indicated that inserting exogenous genes into the D8L region of the neutralizing antibody epitope in the vaccinia virus vector could help to reduce the immunogenicity of the vector itself and enhance the immunogenicity of the exogenous genes. This provided a reference for the use of the vaccinia virus vector as a delivery tool in the field of vaccines or gene therapy.

OBJECTIVETo determine the safety and effectiveness of autotransfusion of shed mediastinal blood after open heart surgery.

METHODSSixty patients undergoing coronary artery bypass grafting (CABG) were selected randomly to receive either nonwashed shed mediastinal blood (Group 1, n = 30) or banked blood (Group 2, n = 30). Drainage and transfusion volume were determined after the operation. Hb, RBC, HCT and PLT were detected immediately before and after the operation, as well as 24 hours and 7 days after the operation. Data were analyzed using Fisher's exact test. A P < 0.05 was considered significant.

RESULTSThere were no significant differences in Hb, HCT, PLT or length of cardiopulmonary bypass (CPB) (P > 0.05). In the two groups, no significant difference in the mean blood loss was observed during 24 hours after the operation (660 +/- 300 ml in Group 1 and 655 +/- 280 ml in Group 2, P > 0.05). In Group 1, the mean volume autotransfused was 280 +/- 160 ml, and the patients required 360 +/- 80 ml banked blood compared with 660 +/- 120 ml in Group 2. In other words, the banked blood requirement in Group 1 was 40% lower.

CONCLUSIONSAutotransfusion of shed mediastinal blood after an open heart operation is safe and effective.

Adult ; Aged ; Blood Banks ; Blood Transfusion, Autologous ; Cardiopulmonary Bypass ; Coronary Artery Bypass ; Drainage ; Female ; Humans ; Male ; Mediastinum ; Middle Aged

Objective:To evaluate the effect of hydrogen on the expression of hippocampal cold-inducible RNA-binding protein (CIRP) after cardiac arrest-resuscitation in rats.Methods:Ninety clean-grade healthy male Sprague-Dawley rats, weighing 280-320 g, were randomly divided into 3 groups: sham group (group Sham, n=20), cardiac arrest-cardiopulmonary resuscitation group (group CPR, n=35), and hydrogen-rich saline group (group H 2, n=35). Cardiac arrest was induced by transoesophageal cardiac pacing followed by CPR in group CPR.Only femoral arteriovenous puncture and tracheal intubation were performed in group Sham.Hydrogen-rich saline 5 ml/kg was intraperitoneally injected immediately after recovery of spontaneous circulation (ROSC) and at 6 and 12 h after ROSC in group H 2 , while the equal volume of normal saline was given instead in the other two groups.Neuro-functional deficit was assessed using neurologic deficit scores (NDS) at 1 and 3 days after ROSC.The animals were sacrificed immediately after intubation in group Sham and at 6 h and 1, 2 and 3 days after ROSC in CPR and H 2 groups, and the hippocampal tissues were obtained to detect the expression of nuclear and cytoplasmic CIRP by Western blot. Results:Compared with group Sham, NDS was significantly decreased at each time point after ROSC in group CPR and group H 2, the expression of nuclear CIRP was significantly down-regulated at 1, 2 and 3 days after ROSC, and the expression of cytoplasmic CIRP was up-regulated at 1 and 2 days after ROSC in group CPR, and the expression of nuclear CIRP was significantly down-regulated at each time point after ROSC, and the expression of cytoplasmic CIRP was down-regulated at 2 and 3 days after ROSC in group H 2 ( P<0.05). Compared with group CPR, NDS was significantly increased at each time point after ROSC, the expression of nuclear CIRP was down-regulated at 6 h after ROSC, and the expression of cytoplasmic CIRP was down-regulated at 1 and 2 days after ROSC in group H 2 ( P<0.05). Conclusion:The nechanism by which hydrogen reduces brain injury after cardiac arrest-resuscitation may be related to down-regulating hippocampal CIRP expression in rats.