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Objective:To explore the role and mechanism of tubastatin A (TubA) in alleviating brain injury after cardiac arrest and cardiopulmonary resuscitation (CA-CPR) by inhibiting endoplasmic reticulum stress-mediated cell apoptosis in swine.Methods:Twenty-three conventional male white swine, weighing 33-40 kg, aged 4 to 6 months, were divided into 3 groups by random number table method: sham group ( n=6), CA-CPR group ( n=9), and TubA group ( n=8). The CA-CPR swine model was established by 9 min of electrically induced CA through pacing catheter in the right ventricle and then 6 min of CPR in the CA-CPR group. The CA-CPR swine model was established by the same method, and then a dose of 4.5 mg/kg of TubA at 5 min after resuscitation was intravenously infused in the TubA group. The serum concentrations of neuron specific enolase (NSE) and S100β protein (S100β) were measured using ELISA before modeling and at 1, 2, 4 and 24 h after resuscitation. Neurological deficit score (NDS) was evaluated at 24 h after resuscitation. Thereafter, the animals were euthanized, and brain cortex tissues were harvested, and the expression levels of caspase-12 and caspase-3 were measured using immunohistochemistry. Cell apoptosis index was detected by TUNEL assay. The variables among the three groups were compared with one-way analysis of variance and the Bonferroni hoc test using SPSS software. Results:Twenty-four h after resuscitation, the serum concentrations of NSE and S100β were significantly increased, and NDS was markedly elevated in the CA-CPR and TubA groups compared with the sham group (all P<0.05). Compared with the CA-CPR group, serum concentration of NSE starting 2 h after resuscitation and serum concentration of S100β starting 1 h after resuscitation were significantly decreased in the TubA group [NSE (ng/mL): (23.1±2.0) vs. (20.2±2.0) at 2 h, (28.4±2.3) vs. (23.7±1.9) at 4 h, (32.1±2.7) vs. (26.6±2.0) at 24 h; S100β (pg/mL): (2239±193) vs. (1923±101) at 1 h, (2817±157) vs. (2360±141) at 2 h, (3384±250) vs. (2691±210) at 4 h, (3965±303) vs. (3119±260) at 24 h, all P<0.05], and NDS was markedly reduced (240±30 vs. 63±44, P<0.05). At 24 h after resuscitation, brain cortex tissue detection showed that the expression levels of caspase-12 and caspase-3 were significantly increased, and cell apoptosis index was markedly elevated in the CA-CPR and TubA groups compared with the sham group (all P<0.05). However, the expression levels of caspase-12 and caspase-3 were significantly decreased [caspase-12:(7.1±0.7) vs. (4.2±0.4); caspase-3: (13.3±1.6) vs. (7.7±0.8), all P<0.05], and cell apoptosis index was markedly reduced in the TubA group compared to the CA-CPR group [(31.1±8.6) vs. (17.3±2.2), P<0.05]. Conclusions:TubA alleviates brain injury and neurological dysfunction after CA-CPR in swine, which may be related to the inhibition of cell apoptosis mediated by endoplasmic reticulum stress.
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OBJECTIVES@#Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms.@*METHODS@#Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery.@*RESULTS@#The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) .@*CONCLUSIONS@#HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.