| BackgroundCardiac arrest is one of the leading cause of death. According to the official statistics, approximately 100,000 people are suffered from out-of-hospital cardiac arrest (OHCA) in the United States each year. However, it is estimated that the real number of sudden death is two to three times higher. At present, although there is not any large-scale statistical data in our country, but a survey study on Beijing, Guangzhou, Karamay and Yu County reveals that the annual incidence of CA is about 41.8/100,000 people. In recent years, along with the unceasing enhancement of the modern cardiopulmonary resuscitation (CPR) technology and technical levels of emergency medical personnel, more CA patients are able to receive timely and effective treatment and the proportion of return of spontaneous circulation (ROSC) has been improved, but no significant improvement in the outcome of post-CA patients is observed. According to statistics, less than 10% of patients admitted to the hospital after successfully resuscitated from OHCA will leave the hospital without major neurological impairments.The exacerbation of post-CA patients could be related to the prolonged, complete whole body ischemia and the reperfusion injury caused by successful resuscitation. This disease state is called "post cardiac arrest syndrome (PCAS)", which includes three main contents:(1) post-CA brain injury; (2) post-CA cardiac dysfunction; (3) systematic ischemia/reperfusion reaction. In addition, the persisting pathological state that leading to CA may be the fourth factor that influencing the outcome. Among them, post-CA brain injury is the main cause of death and disability in those who achieved ROSC. In a survey study on patients who survived from CA and admitted to ICU but later died in hospital,68% of OHCA patients and 23% of in-hospital CA patients died of brain damage. The mechanisms that lead to brain damage after CA/CPR is very complicated, including neuronal exitotoxicity, calcium imbalance, generation of free radicals, pathological protease cascades, and the activation of several cell death signals. Histologically, selectively vulnerable neuron subpopulations in the hippocampus, cortex, cerebellum, corpus striatum, and thalamus degenerate over a period of hours to days. Both neuronal necrosis and apoptosis have been reported after cardiac arrest. The relative contribution of each cell-death pathway remains controversial, however, and is depend in part on patient age and the neuronal subpopulation under examination. The relatively protracted duration of injury cascades and histological change suggests a broad therapeutic window for neuroprotective strategies after cardiac arrest.To date, target temperature management (TTM), also known as therapeutic mild hypothermia, is the only approach proven to improve outcome for patients with OHCA. Therapeutic mild hypothermia or TTM, refers to the course of reducing the core temperature of patients to 32～34℃ (32～36℃) in a short period, maintaining it for 12～24 hours and then slow rewarming to normothermia. The direct evidences of mild hypothermia as a recommendation for routine usage of post-CA treatment came from two landmark studies published in 2002. In one study,273 OHCA-patients with presumed cause of ventricular fibrillation were randomized to mild hypothermia (32～34℃, maintained for 24 hours) or normothermia group. It is found that mild hypothermia improves neurological outcome and survival as compared to nomorthermia control. Another study also divided patients into mild hypothermia (33℃, maintained for 12 hours) and normothermia group, showing similar results with higher survival rate and proportion of good outcome in mild hypothermia group. However, an international multicenter study leaded by Nielsen et al. randomly divided subjects into 33℃ and 36℃ groups, but found that target temperature at 33℃ did not showed benefit in survival and neurological outcome as compared to 36℃, making best target temperature being disputed. Currently, the newest guideline from the American Heart Association recommends that post-CA patients should be rapidly cooled to 32～36℃, and maintained for 24 hours or longer when reaching target temperature. Meanwhile, TTM as a paradigm aiming to strict control of target temperature seems to be more acceptable.Despite prominent advantages found in post-CA patients with ventricular fibrillation or pulseless ventricular tachycardia cause, the neuroprotection of TTM in post-CA patients with un-shockable cause still lacks enough evidence. In addition, the side effects brought by TTM and the increasing labor and costs are also issues of concern. Overall, TTM is still underutilized. Therefore, seeking alternative or additional approaches are in urgent need for treating PC AS.Glibenclamide is a sulfonylurea that has been used as an oral antidiabetic for decades. All sulfonylureas work through inhibiting the sulfonylurea receptor 1. In treating type 2 diabetes, glibenclamide blocks the potassium ATP channel (SUR1-Kir6.2) in the pancreas β cell and facilitates the release of insulin. In recent years, glibenclamide receives great concerns for its pluripotential neuroprotection in acute neurological injury through blocking the SURl-transient receptor potential M4 (SURl-TRPM4) channel. Researches show that glibenclamide inhibits SUR1 activity and exerts neuroprotection in several animal models of central nervous system injury, including ischemic and hemorrhagic stroke, traumatic brain injury, spinal injury, neonatal ischemic-hypoxic encephalopathy, encephalopathy of prematurity and brain metastatic tumor. Moreover, in two retrospective studies, patients with acute ischemic stroke but long taking sulfonylureas to treat diabetes and continued to receive such drugs after stroke onset were associated with less neurologic deficit and lower risk of hemorrhagic transformation, compared with those whose treatment regimen did not include sulfonylureas. The promising results from animal studies and retrospective clinical studies have led to two prospective clinical trials testing the efficacy of glibenclamide on traumatic brain injury as well as malignant edema and stroke. However, whether glibenclamide is neuroprotective in brain injury caused by CA/CPR is still unknown.This study aims to:(1) evaluate the neuroprotection of glibenclamide in a rat model of asphyxial cardiac arrest/cardiopulmonary resuscitation (ACA/CPR) and the involvement of SUR1-TRPM4 channel in this animal model; (2) compare the efficacy of glibenclamide with TTM in neuroprotection in this animal model; (3) find out whether combination of glibenclamide and TTM exerts synergistic effect.MethodsTo achieve these research purposes, we conducted the study from four parts. Specific methods and contents are as follows:1. The neuroprotection of glibenclamide in a rat model of 8-min ACA/CPROne hundred and twenty-six male SD rats underwent 8-min ACA/CPR (n=111) or sham operation (n=15). In 84 rats ROSC were achieved,8 of them died before pre-defined time points were exclude from the following study. The rest 76 rats suffered from ACA/CPR were randomized to vehicle control (Vehicle group) or glibenclamide treatment (GBC group) at 10 minutes after ROSC. Rats in the GBC group were intraperitoneally administered glibenclamide with a loading dose of 10μg/kg, followed by four maintenance dose of 1.2μg every 6 hours, while rats in the Vehicle group received equivalent volume of vehicle (5% DMSO in saline). Sham-operated rats underwent orotracheal intubation and cannula of femoral artery and vein, but not asphyxia cardiac arrest and cardiopulmonary resuscitation.In the first section, rats in the Vehicle group (n=22), GBC group (n=22) and Sham group (n=5) were followed up for 7 days and survival, neurological function and histological injury were evaluated. Neurological function was assessed using a validated neurological deficit scale (NDS,0=normal,100=brain death). Nissl staining was performed to observe the number of viable neuron in the hippocampal CA1 region. In the second section, rats were euthanized at 24 hours after ROSC, and the brains were harvested for following examinations. (1) Propidium iodide (PI) staining for detection of neuronal necrosis; (2) TdT-mediated dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunohistochemistry for detection of neuronal apoptosis; (3) RNA were extracted from brain tissue and quantitative PCR were used to detect the mRNA expression of several cytokines, including tumor necrosis factor a (TNFa), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β) and interlrukin-6 (IL-6); (4) RNA were extracted from brain tissues, separated as cerebral cortex and hippocampus, and quantitative PCR was performed to check the mRNA expression of Abcc8 (encoding SUR1) and Trpm4; (5) Total protein were extracted from brain tissues separated as cerebral cortex and hippocampus and Western blotting was performed to assess the expression of SUR1 and TRPM4 before and after ACA/CPR.2. Comparison of glibenclamide and TTM in neuroprotection in the 8-min ACA/CPR modelFifty-five male SD rats underwent 8-min ACA/CPR (n=48) or sham-operation (n=7). Forth rats achieved ROSC were randomly divided into four groups (n=10 in each group):normothermia and vehicle (NT), normothermia and glibenclamide (GBC), target temperature management and vehicle (TTM), TTM and GBC (TTM+GBC). Glibenclamide and vehicle were given as described above. Rats in the TTM and TTM+GBC groups were rapidly cooled to 33℃ by using ethyl alcohol spray and an electric fan, maintained at 33℃ for 2 hours and then rewarmed to normothermia at a rate of 0.5℃/h. Rats in the nomothermic groups were maintained at 37±0.5℃. All rats were followed up for 7 days and survival, neurological outcome, Morris water maze and histological injury were evaluated. Neurological function were assessed used the NDS score as described above. The Morris water maze performance included two parts, the acquiring phase observed during the 8th to 11th days after ROSC and the exploring trial conducted at the 12th day after ROSC. Time required for finding the platform (latency), time stayed in the target quarter and the frequency crossing the location of platform were recorded. Nissl staining was used for histological injury evaluation.3. The establishment of 10-min ACA/CPR model and the determination of duration for target temperature managementForty-three male SD rats underwent 10-min ACA/CPR (n=40) or sham-operation (n=3). Different from the 8-min ACA/CPR model used above, the 10-min ACA/CPR utilized mechanical compression and delayed administration of epinephrine to prolong the CPR time. ROSC was achieved in 30 rats and they were randomized into three groups at 15 minutes after ROSC (n=10 for each group): normothermia control (NT), target temperature management of 4-hour duration (TTM-4h) and target temperature management of 12-hour duration (TTM-12h). All rats were followed up for 7 days and survival, neurological outcome and histological injury were evaluated. Due to the higher incidence of seizure in the 10-min ACA/CPR model than the 8-min ACA/CPR model, and seizure was considered to be a predictor of poor outcome, another version of NDS score (0=brain death,80=normal) were used for assessment of neurological function. Nissl staining was used for examination of histopathological injury.4. Comparison of glibenclamide and target temperature management in neuroprotection in the 10-min ACA/CPR modelTwo hundred and ten rats underwent 10-min ACA/CPR (n=195) or sham operation (n=15). ROSC was achieved in 152 rats but 12 of them died before pre-defined time points and were excluded from the following study. The rest 140 post-CA rats were randomly divided into four groups at 15 minutes after ROSC: normothermia and vehicle (NT), normothermia and glibenclamide (GBC); TTM and vehicle; TTM+GBC. Glibenclamide and vehicle were given as described above. Rats in the TTM and TTM+GBC were rapidly cooled to 33℃ at 15 minutes after ROSC, maintained at 33℃ for 4 hours and then rewarmed to normothermia at a rate of 0.5℃/h. This study included three sections. In the first section, rats in the NT (n=32), GBC (n=20), TTM (n=20) and TTM+GBC (n=20) were followed up for 7 days and survival, neurological function and histological injury were evaluated. Neurological outcome was assessed used the 80-point version of NDS score. Histological injury was determined by immunohistochemistry, using specific marker of neuron (neuronal specific neucleoprotein, NeuN), neuronal dendrite (microtubule associated protein, MAP2), microglia (ionized calcium binding adaptor molecule-1, Iba-1) and astrocyte (glial fibrillary acidic protein, GFAP). NeuN-staining cells were counted automatically, while MAP2, Iba-1 and GFAP staining was reported as the relative intensity of staining (percent). In the second section, the expression of SUR1 and TRPM4 at different time points after 10-min ACA/CPR were checked. Rats were euthanized at 3,6, and 24 hours after ROSC and brains were harvested for extraction of RNA and total protein. The mRNA levels of Abcc8 (encoding SUR1) and Trpm4 were detected by using quantitative PCR and their protein levels were examined by Western blotting. Two subset of rats were euthanized at either 24 hours (n=5) or 72 hours (n=5) after ROSC. Brains were harvested and cryo-section were obtained. Immunofluorescence was conducted for detection of SUR1 and TRPM4-positve cells in the brain cortex and the hippocampal CA1 region. Triple immunofluorescent labeling was used to illustrate the cellular location of SUR1 and TRPM4, using cell marker of NeuN, Von Willebrand Factor (vwf), Iba-1 AND GFAP. In the third section, the expression of SUR1 and TRPM4 under different treatment were determined using Western blotting.Results1. Glibenclamide improved 7-day survival and neurological outcome after 8-min ACA/CPR in ratsThe 7-day survival rate in the Vehicle group was 31.8%(7/22) while in the GBC group was 68.2%(15/22). There was significant difference between these two groups (x2=4.559, P=0.033). Neurological deficit was remarkable at 24 hours after ROSC in ACA/CPR-treated rats, but recovered gradually. Compared with the Vehicle group, rats in the GBC group showed less NDS scores at 24,48, and 72 hours after ROSC (all P<0.05). Results from Nissl staining showed that there were significantly more viable neurons in the sham-operated group (102±11) than in the ACA/CPR groups, while the number of viable neurons in the GBC group (25±9) was more than that in the Vehicle group (15±4) (both P<0.05). In acute phase, rare PI-positive cells were observed in the brain cortex, hippocampus and putamen in sham-operated rats, but significantly more PI-positive number was observed in these three regions after ACA/CPR, especially in the hippocampus. There were less PI-positive cells in the GBC group than the Vehicle group in these zones (all P< 0.05), indicating that glibenclamide was effective in suppressing cellular necrosis. In addition, more TUNEL-positive and cleaved caspase 3-positive cells were detected in the Vehicle group, implying the occurrence of cellular apoptosis after ACA/CPR. As compared to the Vehicle group, less TUNEL- and Cleaved caspase-3 cells were observed in the GBC group, which suggested that glibenclamide inhibited cellular apoptosis induced by ACA/CPR. Moreover, the mRNA levels of cytokines including TNFa, MCP-1, IL-1β and IL-6 were found elevated after ACA/CPR, and the expressions of TNFa and MCP-1 were much lower in the GBC-treated animals. Finally, we found that both the mRNA and protein levels of SUR1 and TRPM4 were upregulated at 24 hours after ACA/CPR and not inhibited by GBC-treatment, indicating that glibenclamide works through mediating the channel activity rather than the expression of the SUR1-TRPM4 channel.2. Glibenclamide was comparable to target temperature management in improving cognitive function after 8-min ACA/CPRIn survival study, the 7-day cumulative survival rate was 70%(7/10) in the NT group,90% in the GBC group,80%(9/10) in the TTM group and 90%(9/10) in the TTM+GBC group. No significance was found among these four groups(x2=2.052, P=0.152). At 24 hours after ROSC, significant difference in NDS was found among the NT, GBC, TTM and TTM+GBC groups, with rats in the GBC, TTM and TTM+GBC groups all showed less neurological deficit than the NT group as speculated from their median values. Repeated measurement of data from acquiring phase of Morris water maze showed that significant differences in latency existed among each time points (F=10.419, P=0.000). Lesser time was required with time elapse. There were also significant in latency among each groups, with the shortest latency in the Sham group, followed by the GBC, TTM and TTM+GBC groups, and the longest latency in the NT group. No interaction was found between time points and treatments (F=0.786, P=0.664). Analysis in a specific time points revealed that there was no significant difference among groups at the first training day (8 days after ROSC). However, in the third and fourth day of acquiring training, shorted time in latencies were observed in rats from the GBC, TTM and TTM+GBC groups as compared to rats in the NT groups. In exploring trial, the time of staying in the target quarter and the frequency of crossing the location of platform were much lower in the NT group as compared to the Sham group. Time stay in target quarter seemed to be slightly longer in the GBC, TTM, and TTM+GBC groups, but no statistical significance was found. However, the frequency in crossing the location of platform was much more in the GBC, TTM and TTM+GBC groups. Results from Nissl staining demonstrated less viable neurons in the ACA/CPR group as compared to the Sham group (74±6). Compared with the NT group (20±8), the number of viable neurons were much more in the GBC (43±10), TTM (37±6) and TTM+GBC (48±21) groups. Although the number appeared to be slightly higher in the TTM+GBC group as compared to the TTM or GBC group, no statistical significance was found.3. TTM-4h was comparable to TTM-12h in improving 7-day survival and neurological outcome after 10-min ACA/CPRAfter global cerebral ischemia, rectal temperature may not accurately reflect the change of brain temperature. Therefore, we synchronously monitored brain temperature and rectal temperature in a subset group of rats in the NT and TTM-4h groups. It was found that during asphyxia and cardiac arrest, brain temperature dropped faster than rectal temperature. However, brain temperature raised faster than rectal temperature after ROSC and these two temperatures showed linear trend gradually, indicating that rectal temperature could reflect brain temperature when re-circulation was achieved. In survival analysis, the 7-day survival rate in the NT group was 30%(6/10), and in both the TTM-4h and TTM-12h groups were 60% (6/10). Compared with the NT group, higher survival rate seemed to be obtained in these two TTM groups, but no statistical significance was found (x2=2.133, P= 0.145). Neurological function evaluation represented as NDS scores demonstrate different degrees of neurological deficit in rats after 10-min ACA/CPR. Compared with the NT group, the NDS scores in the TTM-4h and TTM-12h groups at 24 and 48 hours after ROSC were both much higher (both P<0.05). Results from Nissl staining showed that compared with sham-operated rats (81±4), rats in the NT group that underwent 10-min ACA/CPR (9±6) revealed prominent neuronal loss in the hippocampal CA1 region (P<0.05). However, neuronal loss was partly preserved in both the TTM-4h (29±6) and TTM-12h (36±12) groups. There was no significant difference between the TTM-12h group and the TTM-4h group.4. GBC was comparable to TTM in improving 7-day survival and neurological outcome after 10-min ACA/CPRThe 7-day survival rate was 34.4%(11/32) in the NT group,65%(13/20) in the GBC group,50%(10/20) in the TTM group and 70%(14/20) in the TTM+GBC group. Survival rate in the GBC (x2= 4.580, P=0.032) and TTM+GBC (x2=5.474, P=0.019) groups were both higher than that in the NT group, while no significant difference was found between the TTM and NT group (x2= 1.771, P= 0.183). As compared to the NT group, rats in the GBC, TTM, and TTM+GBC groups all revealed higher NDS scores at 24,48,72 hours and 7 days after ROSC, but no significant difference was found among the former three groups. Histopathologically, less viable neurons were detected in rats underwent ACA/CPR than rats with sham-operation. TTM or GBC alone significantly reduced neuronal loss compared to the control group treated with NT and vehicle, while their combination resulted in a further reduction of neuronal loss compared to each treatment alone (P<0.05 versus GBC or TTM). In addition, ACA/CPR caused massive neuronal dendritic injury, as revealed by decreased density of MAP2 immuno-staining. Both GBC and TTM reduced the dendrite injury, with combined treatment of them showed better effects. Furthermore, prominent activation of microglia and astrocyte in the hippocampal CA1 region were detected after ACA/CPR, presented as increased intensity of Iba-1 and GFAP immune-staining. GBC and TTM showed similar efficacy in suppressing the activation of microglia and astrocytes, with TTM+GBC revealed better effect in inhibiting activation of microglia than GBC alone. Both the mRNA and protein levels of SUR1 and TRPM4 levels were found significantly upregulated at 6 hours and further increased at 24 hours after ROSC, by using quantitative PCR and Western blotting analysis. Immunofluorescence results also demonstrated more SUR1-and TRPM4-positive cell in the brain cortex and hippocampus. SUR1 was illustrated to be expressed in neuron, endothelial cell, astrocyte and microglia. Moreover, SUR1 co-localized closely with TRPM4. Finally, we found that TTM significantly inhibited the expression of SUR1 and TRPM4, indicating that combination of TTM and GBC might synchronously inhibited the channel activity and structural expression of the SUR1-TRPM4 channel and exert synergistic or additional effects.Conclusions1. Glibenclamide improves 7-day survival and neurological outcome as well as alleviating neuronal loss in the hippocampal CA1 region in a rat model of 8-min ACA/CPR, without causing significant hypoglycemia. The neuroprotection of glibenclamide may be exerted via mediating the channel activity of SUR1-TRPM4 channel and thereby reducing the cellular necrosis and apoptosis as well as suppressing the expression of cytokines.2. Since TTM is the only approach proven to provide neuroprotection after CA/CPR, we thus compare the efficacy of GBC and TTM in neuroprotection after 8-min ACA/CPR. We find that GBC and TTM is comparable in improving neurological and cognitive function after 8-min ACA/CPR. In addition, the neuroprotective effect of GBC and TTM for neuronal loss in the hippocampal CA1 region is not significantly different.3. TTM-4h and TTM-12h are comparable in improving neurological outcome and histopathological injury.4. In a rat model of 10-min ACA/CPR, GBC is comparable to TTM in improving 7-day survival and neurological outcome, as well as in attenuating neuronal loss and glia activation in the hippocampal CA1 region, with combination of them seems to show better effects in ameliorating histopathological injury. The potential therapeutic target of GBC, the subunits of the SUR1-TRPM4 channel, were upregulated after ACA/CPR, with the upregulated SUR1 illustrated to express in neuron, endothelial cell, astrocyte and microglia. These findings indicate that the SUR1-TRPM4 may be involved in the post-ACA/CPR brain injury, and GBC may mediate the activity of this channel and provide salutary effects. TTM is found to inhibit the upregulation of SUR1 and TRPM4 induced by ACA/CPR, which might be a potential mechanism of TTM-mediated neuroprotection. Combination of GBC and TTM might target the SUR1-TRPM4 by synchronously inhibiting the channel activity and structural expression and provide additive or synergistic effects. |
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