Brain injury remains a major problem in patients suffering cardiac arrest (CA). Disruption of the blood-brain barrier (BBB) is an important factor leading to brain injury. Therapeutic hypothermia is widely accepted to limit neurological impairment. However, the efficacy is incomplete. Hydrogen sulfide (H2S), a signaling gas molecule, has protective effects after cerebral ischemia reperfusion injury. This study showed that combination of hypothermia and H2S after resuscitation was more beneficial for attenuated BBB disruption and brain edema than that of hypothermia or H2S treatment alone. CA was induced by ventricular fibrillation for 4 min. Hypothermia was performed by applying alcohol and ice bags to the body surface under anesthesia. We used sodium hydrosulphide (NaHS) as the H2S donor. We found that global brain ischemia induced by CA and cardiopulmonary resuscitation (CPR) resulted in brain edema and BBB disruption; Hypothermia or H2S treatment diminished brain edema, decreased the permeability and preserved the structure of BBB during the early period of CA and resuscitation, and more importantly, improved the neurologic function, increased the 7-day survival rate after resuscitation; the combination of hypothermia and H2S treatment was more beneficial than that of hypothermia or H2S treatment alone. The beneficial effects were associated with the inhibition of matrix metalloproteinase-9 expression, attenuated the degradation of the tight junction protein occludin, and subsequently protected the structure of BBB. These findings suggest that combined use of therapeutic hypothermia and hydrogen sulfide treatment during resuscitation of CA patients could be a potential strategy to improve clinical outcomes and survival rate.

Therapeutic hypothermia is effective to attenuate brain ischemia/reperfusion (I/R) injury after cardiac arrest, and multiple mechanisms have been proposed. Dynamin-related protein 1 (Drp1), a large GTPases of dynamin superfamily, predominantly controls mitochondrial fission and is related to IR-induced Cyt C release and apoptosis. However, the effect of therapeutic hypothermia on Drp1 and mitochondrial fission after cardiac arrest remains still unclear. In this study, non-cardiac arrest and post-cardiac arrest rats received 6-h normothermia (37-38°C) or therapeutic hypothermia (32-34°C), and the hippocampus was harvested at 6h and 72h after cardiac arrest. Results showed the expression of Drp1 and Cyt C increased after cardiac arrest, but therapeutic hypothermia partially reversed this increase at 6h after cardiac arrest. Transmission electron microscopy (TEM) also showed a change in morphology following therapeutic hypothermia after cardiac arrest. Moreover, therapeutic hypothermia could decrease the histopathological damage, inhibit the apoptosis of CA1 neurons and improve the survival and neurological outcomes at 72h after cardiac arrest. Taken together, our study demonstrates that therapeutic hypothermia is neuroprotective against global cerebral I/R injury, which is, at least partially, ascribed to the inhibition Drp1 and Cyt C expression and the protection of mitochondrial structure.

Background RNA binding motif protein 3 (RBM3) is a cold shock protein,produced upon hypothermia,in various cell types,including neurons and glial cells.RBM3 involves in many processes that protect neurons from a variety of injury.Objective To review structure,function and protective effects of RBM3.Content RBM3 contains a consensus seqnence-RNA-binding domain,which binds DNAs or RNAs to regulate their transcription or translation in a plethora of species,tissues and cell types.In the nervous system,RBM3 is implicated in maintaining synthesis of proteins implicated in synaptic plasticity.The upregulation of RBM3 is triggered by hypothemia,spinal cord injury,endoplasmic reticulum stress,and plays important roles in preventing neuronal apoptosis.Trend RBM3 is a promising target for the treatment of neurodegenerative diseases because it prevents neuronal apoptosis and improves impaired synaptic plasticity.

Objective To evaluate the effect of hydrogen sulfide on hippocampal endoplasmic reticulum stress during global cerebral ischemia-reperfusion (I/R) in rats.Methods Seventy-two pathogen-free healthy male Sprague-Dawley rats,weighing 280-320 g,aged 8-10 weeks,were divided into 3 groups (n=24 each) using a random number table:sham operation group (group Sham),global cerebral I/R group (group I/R) and global cerebral I/R plus sodium hydrosulfide group (group I/R+NaHS).Cardiac arrest was induced with transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the global cerebral I/R model.Immediately after recovery of spontaneous circulation,sodium hydrosulfide 2.5 mg/kg was intraperitoneally injected in group I/R+NaHS,and normal saline 5 ml/kg was given in group I/R.The hippocampi were immediately removed at 24 h of reperfusion for determination of the expression of glucose-regulated protein 78 (GRP78),C/EBP-homologous protein (CHOP) and caspase-12 in hippocampal tissues (by Western blot).At 1,3 and 7 days of reperfusion,the hippocampal tissues were obtained and stained with haematoxylin and eosin for examination of the pathological changes in hippocampal CA1 region (under a light microscope) and for determination of apoptosis in hippocampal cells (using TUNEL staining),and the apoptosis rate was calculated.Results Compared with group Sham,the apoptosis rate of hippocampal tissues at 1,3 and 7 days of reperfusion in group I/R and at 3 and 7 days of reperfusion in group I/R+NaHS were significantly increased,and the expression of GRP78,CHOP and caspase-12 in hippocampal tissues was significantly up-regulated in I/R and I/R+NaHS groups (P＜0.05).Compared with group I/R,the apoptosis rate of hippocampal tissues was significantly decreased,and the expression of GRP78,CHOP and caspase-12 was down-regulated at 1,3 and 7 days of reperfusion (P＜0.05),and the pathological changes were significantly attenuated in group I/R+NaHS.Conclusion The mechanism by which hydrogen sulfide reduces apoptosis in hippocampal cells is related to inhibition of endoplasmic reticulum stress during global cerebral I/R in rats.

Objective To investigate whether the nuclear factor E2 related factor/heme synthase-1 (Nrf2/HO-1) pathway is involved in the mechanism of hydrogen sulfide-induced reduction of blood brain barrier(BBB) injury in rats with cardiac arrest and the possible mechanism involved in the effect.Methods Cardiac arrest was induced with transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the cardiac arrest model and the time of ischemia was 4 min.140 adult male SD rats were randomly divided into five groups:sham operation group (Sham group,n=20),cardiac arrest model group (CPR group,n=30),sodium hydrogen sulfide (NaHS) group (n=30),NaHS+solvent group [NaHS+dimethyl sulfoxide (DMSO) group,n=30],NaHS+Nrf2 inhibitor retinoic acid(RA) group(NaHS+RA group,n=30).In Sham group,only anesthesia was performed,and the trachea was inserted through the mouth,and the catheters were performed via femoral artery and vein.The other four groups were all induced cardiac arrest by electrical stimulation.NaHS group and NaHS+RA group,rats received NaHS (0.5 mg/kg) at the start of CPR intravenously,followed by a continuous infusion of NaHS (1.5 mg ·kg-1 ·h-1) for 3 h.NaHS+RA group,RA was given i.p.daily at 10 mg/kg for 1 week before experiment and immediately after recovery.NaHS+DMSO group was given the same amount of solvent daily for 1 week before experiment and immediately after recovery.CPR group was given the same amount of normal saline at the start of CPR intravenously.After recovery,7 days were observed continuously and the survival condition was recorded,and neurological function were evaluated at 1,3 d,and 7 d after recovery,brain water content,BBB permeability and Nrf2,HO-1,BBB tight junction protein occludin expression was detected at 24 h after resuscitation,and the expression of matrix metalloproteinase-9(MMP-9) was detected by immunohistochemical staining.Results The survival rates,neurologic deficit scores (NDS),Nrf2 and HO-1 expression,and occludin expression in NaHS group were higher than CPR group (P＜0.05),while the NaHS+RA group was lower than the NaHS group(P＜0.05).The brain water content and evans blue(EB) content,and MMP-9 positive cells number in NaHS group were significantly lower than that in CPR group(P＜0.05),and the content of brain water content and EB content,and MMP-9 positive cells number in NaHS+RA group were higher than that in NaHS group (P＜0.05).There was no significant difference between the NaHS group and the NaHS+DMSO group (P＞0.05).Conclusions Hydrogen sulfide could reduce the BBB damage in rats with cardiac arrest,which may be related to the activation of Nrf2/HO-1 pathway,inhibiting the expression of MMP-9 and decreasing the loss of occludin protein.

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 volumn 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.

Therapeutic hypothermia is well known for its protective effect against brain injury after cardiac arrest, but the exact mechanism remains unclear. Cold-inducible RNA-binding protein (CIRP), a member of cold shock protein, enables mammalian cells to withstand decreased temperature by regulating gene translation. However, the role of CIRP in global cerebral ischemia after therapeutic hypothermia has not been clearly elucidated. In the present study, rats resuscitated from 4 min of cardiac arrest were separately treated with therapeutic hypothermia (immediately after return of spontaneous circulation (ROSC); targeted temperature at 33 °C) and therapeutic normothermia (targeted temperature at 36.8 °C) for 6 h. The hippocampus was harvested at 0 h (baseline), 6 h, 12 h, 1 day, 3 days, and 7 days after ROSC. The expression of CIRP messenger RNA (mRNA) was assessed by real-time PCR. CIRP and mitochondrial apoptosis-associated proteins were tested by Western blot. The histological changes and neurological function were respectively evaluated by hematoxylin-eosin staining and neurological deficit score (NDS). Compared with baseline, rats resuscitated from cardiac arrest showed increased expression of CIRP, Bax, Caspase 9, and Caspase 3 and decreased expression of Bcl-2 in hippocampus (P < 0.05). However, therapeutic hypothermia after ROSC alleviated the alterations of Bax, Caspase 9, Caspase 3, and Bcl-2, while further increased the hippocampal expression of CIRP mRNA and protein, when compared with the normothermia rats (P < 0.05). In addition, compared with the therapeutic normothermia rats, histopathological damage in CA1 zone and NDS were respectively decreased and increased in the hypothermia rats (P < 0.05). Our findings suggest that 32 °C therapeutic hypothermia exerts an important neuroprotective effects by up-regulating CIRP expression and inhibiting mitochondrial apoptosis factor production in the cardiac arrest rat model.

Objective To evaluate the effect of hypothermia on the expression of cold-inducible RNA-binding protein (CIRP) after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest.Methods Ninety pathogen-free male Sprague-Dawley rats,weighing 280-320 g,were randomly divided into 3 groups (n =30 each) using a random number table:sham operation group (group Sham),cardiac arrest-CPR group (group CPR),and hypothermia group (group H).Group Sham underwent only cannulation of the artery and vein and tracheal intubation.In group CPR,cardiac arrest was induced by electric stimulation via the esophagus,and then CPR was performed.In group H,the rectal temperature was cooled down to 32-34 ℃ within 15 min by putting rats on the ice bags immediately after recovery of spontaneous circulation (ROSC),and maintained at this level for 6 h,while the rectal temperature was maintained at 36-37 ℃ in the other groups.Neurological deficit score (NDS) was evaluated at 24 and 72 h after ROSC,and the rats were then sacrificed.The hippocampus was removed for examination of the pathological changes of pyramidal cells in the hippocampal CA1 region (using hematoxylin and eosin staining),and for determination of the expression of CIRP mRNA and protein (using fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction or Western blot).Results Compared with group Sham,NDS score was significantly decreased,and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in CPR and H groups (P＜0.05).Compared with group CPR,NDS score was significantly increased at 72 h after ROSC,and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in group H (P＜0.05).Conclusion Hypothermia can effectively reduce cerebral ischemia-reperfusion injury and improve neurological function after CPR,and the mechanism is probably related to up-regulation of CIRP expression in a rat model of cardiac arrest.

Objective To investigate the efficacy of ulinastatin for improvement of cerebral resuscitation using therapeutic hypothermia (TH) in rats with cardiac arrest (CA).Methods Sixty pathogenfree healthy male Sprague-Dawley rats,weighing 280-320 g,were divided into 5 groups (n =12 each) using a random number table:sham operation group (group Sham);CA/cardiopulmonary resuscitation (CPR) group (group CA/CPR);group TH;ulinastatin group (group U);ulinastatin + TH group (group U+TH).CA was induced through transesophageal cardiac pacing,and CPR was performed at 4 min of untreated arrest to establish CA/CPR model.In group TH,the rectal temperature was cooled down to 32-34 ℃ by rubbing the body surface with ethanol within 15 min starting from the time point immediately after restoration of spontaneous circulation (ROSC),and maintained at this level for 6 h.In group U,ulinastatin 100 000 U/kg was injected via the femoral vein immediately after ROSC.In group U+TH,ulinastatin 100 000 U/kg was intravenously injected immediately after ROSC,and rectal cooling was then started.The neurological function was evaluated and scored at 24 and 72 h after ROSC.The rats were sacrificed after the neurological function was scored at 72 h after ROSC,and the hippocampi were removed for microscopic examination of pathological changes in hippocampal CA1 region and for determination of caspase-3 expression (by immunohistochemical staining and Western blot) and Bax and Bcl-2 expression (by Western blot) in hippocampal tissues.Bax/Bcl-2 ratio was calculated.Results Compared with group Sham,the neurological function score was significantly decreased,the expression of caspase-3 and Bax was up-regulated,the expression of Bcl-2 was down-regulated,and Bax/Bcl-2 ratio was increased after ROSC in group CA/CPR (P＜0.05).Compared with group CA/CPR,the neurological function score was significantly increased at 24 and 72 h after ROSC in TH and U+TH groups and at 24 h after ROSC in group U,caspase3 expression was significantly down-regulated,Bcl-2 expression was up-regulated,and Bax/Bcl-2 ratio was decreased at 72 h after ROSC in TH,U and U+TH groups,and the expression of Bax was significantly down-regulated in U and U+TH groups (P＜0.05).Compared with group TH or group U,the neurological function score was significantly increased at 24 h after ROSC,and the expression of caspase-3 was downregulated and Bax/Bcl-2 ratio was decreased at 72 h after ROSC in group U+TH (P＜0.05).The pathological changes of hippocampal tissues were significantly mitigated in group U+TH when compared with group TH or group U.Conclusion Ulinastatin can improve the efficacy of TH for cerebral resuscitation in rats with cardiac arrest,and the mechanism may be related to inhibition of neuronal apoptosis.

Objective To evaluate the efficacy of sodium hydrosulfide for improvement of cerebral protection provided by mild hypothermia in a rat model of cardiac arrest and resuscitation.Methods One hundred and forty pathogen-free healthy male Sprague-Dawley rats,weighing 280-320 g,aged 7-8 weeks,were randomly divided into 5 groups using a random number table:sham operation group (group S,n =20),cardiac arrest and resuscitation group (group CAR,n=30),sodium hydrosulfide group (group SH,n=30),mild hypothermia group (group MH,n=30) and sodium hydrosulfide plus mild hypothermia group (group SH+MH,n=30).Cardiac arrest was induced by transesophageal cardiac pacing,and cardiopulmonary resuscitation was started after 4 min untreated arrest.The administration regimen for sodium hydrosulfide was as follows:sodium hydrosulfide was intravenously injected as a bolus of 0.5 mg/kg immediately after restoration of spontaneous circulation followed by an infusion of 1.5 mg · kg-1,h-1 for 3 h.Mild hypothermia was performed as follows:the rectal temperature was cooled down to 32-34 ℃ by rubbing the body surface with ethanol within 15 min starting from the time point immediately after restoration of spontaneous circulation and maintained at this level for 4 h,and then increased to 38-39 ℃ within 2 h.The animals were observed for 7 consecutive days after resuscitation,the survival rate was recorded,and the neurological function was evaluated by the tape removal test.At 24 h after resuscitation,brain tissues were harvested for determination of brain water content,permeability of blood brain barrier,expression of occludin in hippocampi (by Western blot),and expression of matrix metalloproteinase-9 (MMP-9) in hippocampi (by immunohistochemical staining).Results Compared with group S,the survival rate was significantly decreased,the tape removal time was significantly prolonged,the brain water content and permeability of blood brain barrier were significantly increased,the expression of occludin was significantly downregulated,and the expression of MMP-9 was significantly up-regulated in CAR,SH,MH and SH+MH groups (P＜0.05).Compared with group CAR,the survival rate was significantly increased,the tape removal time was significantly shortened,the brain water content and permeability of blood brain barrier were significantly decreased,the expression of occludin was significantly up-regulated,and the expression of MMP-9 was significantly down-regulated in SH,MH and SH+MH groups,and the changes in the parameters mentioned above were more obvious in group SH+MH (P＜0.05).Conclusion Sodium hydrosulfide significantly improves cerebral protection provided by mild hypothermia in a rat model of cardiac arrest and resuscitation,and the mechanism is related to reduction of occludin degradation,down-regulation of MMP-9 expression and decrease in the permeability of blood brain barrier.

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 evaluate the changes in the expression of cold-inducible RNA binding protein (CIRP) in the hippocampus after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest. Methods Eighty-five male Sprague-Dawley rats, weighing 280–320 g, aged 3 months, were randomly divided into 3 groups using a random number table: control (group C, n=15), sham operation group (group S, n=15) and CPR group (n=55). Cardiac arrest was induced through ventricular fibrillation caused by transoesophageal cardiac pacing, and then CPR was performed in group CPR.At 6 h, 12 h, and 1, 3 and 7 days after CPR, and the hippocampi were removed for detection of the expression of CIRP mRNA (using real-time reverse transcriptase polymerase chain reaction) and CIRP protein (using Western blot). The brain tissues were also obtained at 3 days after CPR for detection of CIRP expression in the hippocampal CA1 region by immunohistochemistry. Results Compared with group C, the expression of CIRP mRNA was significantly up-regulated at 1, 3, and 7 days after CPR, and the expression of CIRP was up-regulated at 1 and 3 days after CPR in group CRP (P 0.05). Compared with the values at 3 days after CPB, the expression of CIRP mRNA was significantly down-regulated at each time point after CPR, and the expression of CIRP was down-regulated at 6 h, 12 h, and 7 days after CPR in group CRP (P<0.05). CIRP was mainly expressed in the nucleus of pyramidal cells in the hippocampal CA1 region. Conclusion Up-regulated expression of hippocampal CIRP is one of the endogenous mechanisms that inhibit cell apoptosis in the brain tissues of rats. Key words: Heart arrest, induced; Cardiopulmonary resuscitation; RNA-binding proteins; Hippocampus