Glibenclamide Improves Survival and Neurologic Outcome After Cardiac Arrest in Rats* Kaibin Huang, MD; Yong Gu, PhD; Yafang Hu, PhD; Zhong Ji, MD, PhD; Shengnan Wang, MD; Zhenzhou Lin, MD; Xing Li, MD; Zuoshan Xie, MD; Suyue Pan, MD, PhD
Objectives: Glibenclamide confers neuroprotection in animal models as well as in retrospective clinical studies. This study determines whether glibenclamide improves outcome after cardiac arrest in rats. Design: Prospective randomized laboratory study. Setting: University research laboratory. Subjects: Male Sprague-Dawley rats (n = 126). Interventions: Rats successfully resuscitated from 8-minute asphyxial cardiac arrest were randomized to glibenclamide or vehicle group. Rats in the glibenclamide group were intraperitoneally administered glibenclamide with a loading dose of 10 μg/ kg at 10 minutes and a maintenance dose of 1.2 μg at 6, 12, 18, and 24 hours after return of spontaneous circulation, whereas rats in the vehicle group received equivalent volume of vehicle solution. Measurements and Main Results: Survival was recorded every day, and neurologic deficit scores were assessed at 24, 48, and 72 hours and 7 days after return of spontaneous circulation (n = 22 in each group). Results showed that glibenclamide treatment increased 7-day survival rate, reduced neurologic deficit scores, and prevented neuronal loss in the hippocampal cornu ammonis 1 region. To investigate the neuroprotective effects of glibenclamide in acute phase, we observed neuronal injury at 24 hours after return of spontaneous circulation and found that glibenclamide significantly decreased the rate of neuronal necrosis and apoptosis. In addition, glibenclamide reduced the messenger RNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 in the cortex after return of spontaneous circulation. Furthermore, the sulfonylurea receptor 1 and transient *See also p. 2040. All authors: Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China. This work is supported, in part, by the National Natural Science Foundation of China (grant No. 81271521, 81471339), the Science and Technology Planning Project of Guangdong Province of China (grant No. 2012A030400011), and the Doctoral Fund of Ministry of Education of China (grant No. 20124433110017). For information regarding this article, E-mail: [email protected] Copyright © 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. DOI: 10.1097/CCM.0000000000001093
Critical Care Medicine
receptor potential M4 heteromers, the putative therapeutic targets of glibenclamide, were up-regulated after cardiac arrest and cardiopulmonary resuscitation, indicating that they might be involved in neuroprotective effect of glibenclamide. Conclusions: Glibenclamide treatment substantially improved survival and neurologic outcome throughout a 7-day period after return of spontaneous circulation. The salutary effects of glibenclamide were associated with suppression of neuronal necrosis and apoptosis, as well as inflammation in the brain. (Crit Care Med 2015; 43:e341–e349) Key Words: apoptosis; cardiac arrest; cytokines; glibenclamide; necrosis
C
ardiac arrest (CA) is a leading cause of death worldwide (1). Despite advances in cardiopulmonary resuscitation (CPR) methods, up to 67% of adults successfully resuscitated from out-of-hospital CA (OHCA) die in hospital, and only 68% of survivors have a good outcome (2). Most of the post-CA mortality and morbidity are caused by post-CA syndrome, including neurologic damage, myocardial dysfunction, and systemic inflammation (2). Although therapeutic hypothermia confers significant neuroprotection after ventricular fibrillation–induced OHCA in adults, it has been shown to benefit, at most, 20% of victims in whom return of spontaneous circulation (ROSC) is achieved (3, 4). The benefit of therapeutic hypothermia on those who experienced nonventricular fibrillation–induced OHCA is even less (5, 6). Alternative approaches should be developed to favor post-CA patients. Sulfonylurea receptor 1 (SUR1) is a member of the large superfamily of adenosine triphosphate (ATP)-binding cassette proteins and for decades used as the target of sulfonylurea drugs to treat type-2 diabetes mellitus (7). Transient receptor potential M4 (TRPM4) is a pore-forming protein that exclusively and nonselectively conduct monovalent cations (8). Coexpression of SUR1 and TRPM4 yields SUR1-TRPM4 heteromers and exhibits pharmacological properties of SUR1 and biophysical properties of TRPM4 (9). SUR1-TRPM4 complex is involved in neurologic injury in several disease models such as cerebral www.ccmjournal.org
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injury as well as malignant edema and stroke are now under testing in two clinical trials (ClinicalTrials.gov Identifier: NCT01454154 and NCT01794182) (17). However, the role of GBC in CA and followed CPR (CA/CPR) has not been elucidated. In this study, we aimed to determine whether GBC was salutary in a rat model of asphyxial CA/CPR. We found that low-dosage GBC treatment significantly improved 7-day survival and neurologic outcome and ameliorated neuronal injury and inflammatory cytokine expression after CA/CPR in rats.
MATERIALS AND METHODS
Figure 1. A, Experimental procedure and measurements during baseline, asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR), and return of spontaneous circulation (ROSC). Blood analysis includes arterial blood gases, electrolytes, pH, base excess, hematocrit, hemoglobin, blood glucose, and lactate concentration. B, Flow diagram of the experimental groups. DMSO = dimethyl sulfoxide, ECG = electrocardiogram, GBC = glibenclamide, i.p. = intraperitoneal injection, HR = heart rate, MAP = mean arterial pressure, RT-PCR = real-time polymerase chain reaction, TUNEL = terminal deoxynucleotide transferase–mediated dUTP-biotin nick-end labeling.
infraction, traumatic brain injury, subarachnoid hemorrhage, hemorrhagic encephalopathy of prematurity, and spinal cord injury (10–14). Blockage of SUR1-TRPM4 with low-dose glibenclamide (GBC) alleviates brain damage without causing obvious hypoglycemia (15). In a clinical setting, patients who experienced ischemic stroke but long taking sulfonylureas such as GBC to treat diabetes were associated with reduced symptomatic hemorrhagic transformation compared with those whose treatment regimen did not include sulfonylureas (16). More inspiringly, the applications of GBC on traumatic brain e342
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Animal Preparation This study was approved by the Animal Care and Use Committee of the Nanfang Hospital, Southern Medical University and followed the National Guidelines for Animal Experimentation. Male Sprague-Dawley rats weighing between 300 and 350 g were obtained from the Experimental Animal Center of Southern Medical University and housed on a 12-hour light and dark cycle with free access to water and food.
CA Model The 8-minute asphyxial CA model was established with a slight modification of the model used in the study by Gao et al (18) (Fig. 1A). In brief, rats were anesthetized, orotracheally intubated with a 14G cannula (BD, Suzhou, China), and connected to a ventilator (RWD, Shenzhen, China). Intravascular catheters (24G; BD) were inserted into the right femoral artery and vein for dynamic artery blood pressure monitoring and drug administration. After 10-minute stabilization, the rats were chemically paralyzed by IV vecuronium (2 mg/kg), and the ventilator was disconnected for 8 minutes to induce CA, which September 2015 • Volume 43 • Number 9
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TABLE 1.
Physiological Parameters Comparison Among the Three Treatment Groups
Parameters
Mean arterial pressure (mm Hg)
Heart rate (beats/min)
Rectal temperature (°C)
PH
Paco2 (mm Hg)
Lactic acid (mmol/L)
Glucose (mmol/L)
Time Points
Sham
Vehicle
Glibenclamide
Baseline
104 ± 2
114 ± 16
108 ± 8
10 min
107 ± 2
124 ± 28
117 ± 17
30 min
107 ± 4
87 ± 9a
81 ± 12a
60 min
112 ± 2
86 ± 12a
82 ± 14a
Baseline
408 ± 14
403 ± 19
400 ± 33
10 min
415 ± 14
410 ± 37
413 ± 35
30 min
407 ± 27
382 ± 52
397 ± 58
60 min
431 ± 18
399 ± 36
404 ± 55
Baseline
37.1 ± 0.3
37.1 ± 0.3
36.8 ± 0.3
10 min
37.2 ± 0.2
36.8 ± 0.3
36.6 ± 0.1a
30 min
37.2 ± 0.2
37.0 ± 0.2
36.9 ± 0.3
60 min
37.2 ± 0.3
37.1 ± 0.2
37.0 ± 0.3
Baseline
7.35 ± 0.01
7.33 ± 0.02
7.35 ± 0.01
10 min
7.38 ± 0.03
a
7.05 ± 0.05
7.10 ± 0.06a
30 min
7.40 ± 0.03
7.13 ± 0.07a
7.20 ± 0.03a
60 min
7.39 ± 0.03
7.29 ± 0.06a
7.28 ± 0.01a
a
Baseline
49.67 ± 4.39
43.65 ± 5.04
46.88 ± 3.28
10 min
49.20 ± 4.98
87.63 ± 10.58
30 min
47.90 ± 3.10
82.1 ± 10.34a
62.55 ± 9.19a
60 min
48.33 ± 3.46
59.85 ± 14.21
55.95 ± 6.59
Baseline
1.23 ± 0.45
1.05 ± 0.13
1.07 ± 0.10
10 min
1.20 ± 0.46
a
6.78 ± 2.43
4.85 ± 2.88a
30 min
0.97 ± 0.38
4.18 ± 0.81a
2.83 ± 1.16a
60 min
1.03 ± 0.15
2.20 ± 0.81
1.87 ± 0.69
Baseline
7.1 ± 0.5
6.9 ± 0.8
7.5 ± 0.6
10 min
8.7 ± 1.8
10.4 ± 1.3a
11.5 ± 1.3a
30 min
7.5 ± 0.8
7.0 ± 2.0
6.0 ± 0.8
60 min
6.4 ± 1.6
5.4 ± 1.2
5.7 ± 0.8
90 min
6.8 ± 1.2
5.2 ± 1.0
5.0 ± 0.5
120 min
7.3 ± 0.8
5.5 ± 0.8
6.4 ± 1.0
a
70.18 ± 10.74a,b
p < 0.05 versus sham group. b p < 0.05 versus vehicle group. Physiological variables were measured at baseline and at 10, 30, and 60 min after return of spontaneous circulation or sham operation. The values are expressed as mean ± sd. a
was arbitrarily defined as mean arterial pressure (MAP) dropped to 25 mm Hg below. Typically, CA occurred within 4 minutes asphyxia, leading to 4–5 minutes pulselessness. At the end of the 8-minute asphyxia, CPR was initiated by reconnecting the ventilator, injection of epinephrine (0.01 mg/kg) and sodium bicarbonate (1 mEq/kg), and performing thoracic compressions (200 compressions per minute). ROSC was characterized by an increase Critical Care Medicine
of MAP beyond 60 mm Hg for 10 minutes. Rats failed to ROSC within 2 minutes were excluded from the continuing experiments. Ventilation parameters were adjusted according to the values of blood gas analysis. Blood glucose level was intermittently detected using a glucometer (Optium, Abbott, CA). After spontaneous breath started, the animals were weaned from ventilator and extubated. To avoid spontaneous hypothermia, www.ccmjournal.org
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to cages. No additional fluids were administered to the rats during the whole procedure. Study Protocol At 10 minutes after ROSC, rats were randomized to GBC or vehicle group, by using random number table (Fig. 1A). GBC (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline. Rats in the GBC group were intraperitoneally administered GBC with a loading dose of 10 μg/kg at 10 minutes and a maintenance dose of 1.2 μg at 6, 12, 18, and 24 hours after ROSC, whereas rats in the vehicle group received equivalent volume of DMSO and saline. The present study included two parts (Fig. 1B). In the first part, 7-day effects of GBC in the CA/CPR model were assessed. Forty-four rats that successfully resuscitated (n = 22 for each group) were followed up for 7 days, and survival rate, neurologic outcome, and neuronal degeneration were evaluated. In the second part, the protective effect of GBC in acute phase (24 hr) was investigated. Rats survived for 24 hours after ROSC (n = 16 for each group) were selected for the detection of in situ cellular necrosis and apoptosis, as well as messenger RNA (mRNA) and protein expression of relevant genes in the brain. Rats underwent all procedures except asphyxial CA and CPR were used as sham control (n = 5 in part 1; n = 10 in part 2). Figure 2. Glibenclamide (GBC) improved survival and neurologic outcome after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A, Survival rate of rats during 7-day follow-up after CA/CPR. Solid line, vehicle group; dashed line, GBC group. B, Neurologic deficit scores of survived rats at 24, 48, and 72 hr and at 7 days after CA/CPR. #p < 0.05 versus vehicle group.
rectal temperatures were monitored and maintained at 37.0°C ± 0.5°C with a temperature feedback system (RWD) during the surgery and for another 6 hours before rats were returned
Survival Study and Neurologic Function Evaluation CA/CPR rats were followed up to 7 days, and survival rate was recorded every day. A previously validated scale of neurologic deficit score (NDS) was used to assess the neurologic outcome at 24, 48, and 72 hours and 7 days after ROSC, which was conducted by two investigators who were unaware of animal grouping (19). The total NDS was consisted of five components: consciousness and respiration, cranial nerve function, motor function, sensory function, and coordination. An NDS of 0% was considered as normal, whereas 100% was brain death.
Figure 3. Glibenclamide (GBC) prevented neuronal loss in hippocampal cornu ammonis 1 (CA1) region after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A, Representative photomicrographs (Nissl staining, purple; magnification ×400) showing the surviving neurons in the hippocampal CA1 region at 7 days after CA/CPR. Bars indicate 40 μm. B, Quantification of viable neurons in hippocampal CA1 region. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group.
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Histological Examination Seven days after ROSC or sham surgery, rats were deeply anesthetized and transcardially perfused with saline and followed by 4% paraformaldehyde for brain fixation. Brains were removed and immersed at 4°C in 15% and 30% sucrose for cryoprotection. Coronal brain sections located at 3.5 mm posterior to bregma were obtained (Leica CM1800, Heidelberg, Germany), stained with cresyl violet (Beyotime, Hangzhou, China), and observed under microscope (Olympus, Tokyo, Japan). Viable September 2015 • Volume 43 • Number 9
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neurons in the hippocampal CA1 region were those with visible nucleus and intact cytoplasm with discernable and rich Nissl staining. Neurons with shrunken cell bodies surrounding by empty spaces were excluded (20). Cellular Necrosis and Apoptosis To label necrotic cells, propidium iodide (PI; 1 mg/mL; Sigma) was administered (1 mg/kg, intraperitoneal injection) at 1 hour prior to killing (21). Brain sections were obtained, fixed in 100% ethanol, and observed with excitation/emission filters 568/585. From each rat brain, five fields in cortex, hippocampal CA1, and putamen from eight brain sections were selected for analysis. The mean counts of PI-positive cells per field were automatically calculated using Image-Pro Plus version 6.0 (Media Cybernetics, Warrendale, PA). For detection of apoptosis, cryosections were stained with either terminal deoxynucleotide transferase–mediated
dUTP-biotin nick-end labeling (TUNEL) or cleaved caspase 3 antibody. TUNEL was performed using an in situ cell apoptosis detection kit (TMR Red; Roche, Mannheim, Germany) according to the manufacturer’s recommendations. Immunohistochemistry was applied to detect cleaved caspase 3 in coronal cryosections. After blocking, sections were incubated overnight with primary antibody against cleaved caspase 3 (1:500; Cell Signaling, Beverly, MA), followed by goat anti-rabbit secondary antibody conjugated with biotin (ZSGB-BIO, Beijing, China). After rinsing in phosphate-buffer saline, the sections were incubated with streptavidin peroxidase and observed with a microscope. Western Blotting Denatured protein samples from cortex and hippocampus of each rat were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA). After blocking, membrane was incubated overnight at 4°C with antibodies including mouse anti-SUR1 (1:500; Abcam, Cambridge, United Kingdom), rabbit anti-TRPM4 (1: 1,000; Abcam), and mouse anti-βactin (1:10,000; CWBIO, Beijing, China). Bound primary antibodies were detected with the antimouse or anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:5,000; CWBIO). All signals were detected by the enhanced chemiluminescence detection method (Millipore). The densities of protein blots were quantified by using Image J (National Institutes of Health, Bethesda, MD) and normalized to β-actin levels.
Figure 4. Glibenclamide (GBC) reduced neuronal necrosis in brain cortex, hippocampal CA1, and putamen at 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Representative photomicrographs of propidium iodide (PI) staining. Bars indicate 100 μm. B, Quantification of PI-positive cells. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group.
Critical Care Medicine
Measurement of Gene Expression Total RNA was isolated using a TRIzol kit (Takara, Dalian, Japan) and reverse-transcribed to complementary DNA (cDNA) using PrimeScript RT Master Mix Kit (Takara). cDNA samples were amplified by quantitative real-time polymerase chain reaction (RT-PCR) using SYBR Green RT-PCR Master Mix (Roche) to determine mRNA level of tumor necrosis factor-α (TNFα), monocyte chemoattractant protein www.ccmjournal.org
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s) and time required for ROSC (51.4 ± 24.5 vs 43.5 ± 17.6 s) were comparative between the vehicle and GBC groups. Eight rats that resuscitated from CA but failed to survive for 24 hours were excluded from this study. The rest 78 CA/CPR rats were subjected to either 7-day (part 1, n = 44) or 24-hour (part 2, n = 32) investigation. There was no significant difference in physiologic variables among three groups at baseline, whereas prominent acidosis was observed in both CA/CPR groups (Table 1). Notably, no hypoglycemia was detected in the GBC group during the 2-hour monitoring after ROSC (Table 1). Results in the 7-day survival assay showed that 31.8% rats (7 of 22) in the vehicle group were survived at day 7, which was much lower than that in the GBC group (68.2%, 15 of 22) (p < 0.05) (Fig. 2A). We also determined the NDSs, which represent the neuFigure 5. Glibenclamide (GBC) inhibited neuronal apoptosis in the hippocampal cornu ammonis 1 (CA1) rologic deficiency, and found that region at 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Representative photomicrographs of the NDSs at 24, 48, and 72 hours terminal deoxynucleotide transferase–mediated dUTP-biotin nick-end labeling (TUNEL) staining (red). after ROSC were lower in the GBC B, Representative photomicrographs of immunohistochemistry using cleaved caspase 3 antibody (brown granules). C and D, Quantification of TUNEL-positive and cleaved caspase 3–positive cells. Bars indicate group than those in the vehicle 40 μm. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group. group with statistical significance (p < 0.05) (Fig. 2B). No significant difference was found at 7 days although an improved trend was (MCP)-1, interleukin (IL)-1β, IL-6, ABCC8, TRPM4, and observed in the GBC group (Fig. 2B). Together, these results sugglyceraldehyde phosphate dehydrogenase (GAPDH). Changes gest that low-dosage GBC treatment within 24 hours improves in the relative mRNA expression were normalized to levels of 7-day survival and neurologic outcome after CA/CPR. GAPDH. Statistical Analysis All data were presented as means ± sds. Continuous data were analyzed with unpaired t tests or one-way analysis of variance followed by post hoc multiple comparison tests (least significant difference test). Difference in survival rate was analyzed by the log-rank test. SPSS 20.0 (IBM, Armonk, NY) and GraphPad Prism 5.0 (GraphPad, La Jolla, CA) were used for statistical analyses. p value less than 0.05 was considered statistically significant.
RESULTS GBC Treatment Improved Survival and Neurologic Outcome After CA/CPR A total of 126 rats were prepared for the study (Fig. 1B). Among them, 111 underwent asphyxial CA/CPR, whereas the rest 15 rats received sham operation. For animals that successfully resuscitated (n = 84), asphyxial time to CA (211.8 ± 27.7 vs 218.1 ± 23.9 e346
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GBC Attenuated Cell Death in the Brain After CA/CPR To further evaluate neurologic injury at 7 days after ROSC, brains in each group were removed for neuronal degeneration assay with Nissl staining. Results in Figure 3 demonstrated that CA/CPR induced neuronal degeneration in the hippocampal CA1 region, which was partly preserved by GBC (Fig. 3). We also examined, in acute stage (at 24 hr after ROSC), the effect of GBC on cellular necrosis and apoptosis in the brain after CA/CPR. To detect cell necrosis, rats were administered with PI before killing. Results in Figure 4 showed that PI-positive cells were observed in the cortex, hippocampal CA1, and putamen in the CA/CPR rats treated with vehicle solution, which was significantly reduced by GBC. Meanwhile, TUNEL and cleaved caspase 3 staining were performed to evaluate cell apoptosis. A large number of TUNEL-positive and cleaved caspase 3–positive cells were observed in the hippocampal CA1 region at 24 September 2015 • Volume 43 • Number 9
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were up-regulated after CA/CPR (p < 0.05). Consistently, SUR1 and TRPM4 protein levels were also increased in both vehicle and GBC groups (p < 0.05). Given that inhibiting the channel conductance rather than reducing the expression of SUR1-TRPM4 contributes the neuroprotective role of GBC, it is reasonable that the expression of SUR1 and TRPM4 was not affected after GBC treatment. Thus, it is possible that elevated expression of SUR1 and TRPM4 after CA/CPR contribute to the post-CA injury; through inhibiting the channel conductance, GBC prevents brain injury.
DISCUSSION In the present study, we found that low-dosage GBC treatment after successful resuscitation Figure 6. Messenger RNA (mRNA) expression of tumor necrosis factor-α (TNFα), monocyte chemoattractant from CA markedly improved protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 in the brain cortex at 24 hr in rats received sham operation (sham) and cardiac arrest and cardiopulmonary resuscitation treated with vehicle solution or glibenclamide 7-day survival and neurologic (GBC). *p < 0.05 versus sham group. #p < 0.05 versus vehicle group. outcome in rats. The neuroprotective role of GBC was associated with the attenuation of the CA/CPR-induced neuronal necrosis hours after CA/CPR (Fig. 5). Furthermore, GBC treatment decreased the number of TUNEL-positive and cleaved caspase and apoptosis at 24 hours after ROSC. The salutary impact of GBC on the outcome of CA/CPR was also related to the inhibi3–positive cells (Fig. 5). These results, when taken together, suggest that GBC treatment prevents neuronal loss by suppressing tion of cytokine expression and the activity of SUR1-TRPM4. Asphyxia is the most common cause of CA in patients both necrosis and apoptosis in the brain after CA/CPR. who experience severe head injury, drowning, foreign body obstruction, or intoxication (23). The 8-minute asphyxial CA/ GBC Treatment Inhibited Cytokine Expression in CPR model used in this study was reported to reproduce the Cortex After CA/CPR pathophysiology of hypoxic-ischemic encephalopathy and After CA/CPR, inflammatory cytokines are accumulated in induce histological injury similar to humans with asphyxia the brain cortex and may bring detrimental effects to neu(24, 25). Furthermore, the damage of this model is still amenable rons (22). We next investigated whether GBC could inhibit to interventions including neuroprotectant and mild hypotherthe expression of cytokines in the brain cortex after CA/CPR. mia (26, 27). Consistent with previous studies, rats underwent Results showed that mRNA level of TNFα, MCP-1, IL-1β, and asphyxial CA/CPR in the present study displayed neurologic IL-6 was markedly up-regulated in the brain cortex of rats subdeficiency in acute phase, neuronal loss in delayed stage jected to CA/CPR (Fig. 6). Compared with vehicle treatment, (19, 28), and similar 7-day survival rate (29). GBC prevented mRNA expression of TNFα and MCP-1, but GBC is an FDA-approved drug for the treatment of type-2 not IL-1β and IL-6, in the brain cortex. These results indicate diabetes. Since the elegant work by Simard et al (10) in 2006, that TNFα and MCP-1 might be associated with the neuroprothe role of GBC in neuroprotection was paid attention by neutective effect of GBC in the brain after CA/CPR. rologic researchers. Low-dose GBC was reported to significantly reduce cerebral edema, infarct volume, and mortality by CA/CPR Induced the Expression of SUR1 and TRPM4 50% without causing obvious hypoglycemia (10). Later, GBC in Brain Cortex and Hippocampus was found to be effective in the treatment of traumatic brain SUR1-TRPM4 complex is the putative therapeutic target of GBC. injury (30) and malignant stroke (31). The relevant clinical To further study the therapeutic targets of GBC, RT-PCR and trials are ongoing (17). Other studies on global brain injury Western blot were performed to detect the mRNA and protein (13, 32), subarachnoid hemorrhage (12, 33), and spinal cord level of SUR1 and TRPM4 at 24 hours after CA/CPR. As shown in injury (34) add more supportive evidences to the benefits of GBC. Here, we stepped forward to demonstrate the role of GBC Figure 7, both ABCC8 (encoding SUR1) and TRPM4 mRNA levels Critical Care Medicine
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depletion and calcium overload (2), which can activate TRPM4 channel (36). Through interfering with SUR1 unit, GBC blocks the activity of TRPM4 channel and decreases neuronal necrosis and apoptosis in the model of subarachnoid hemorrhage and spinal cord injury (12, 37). Therefore, we logically proposed that blocking SUR1-TRPM4 channel was the mechanism for the salutary effect of GBC in CA/CPR model. Although the opening frequency of SUR1-TRPM4 was not determined due to technique issues, we found both SUR1 and TRPM4 were up-regulated after CA/CPR, indicating that SUR1-TRPM4 complex is involved in post-CA injury, through which, GBC attenuates brain injury, and thereby improving neurologic outcome and survival.
CONCLUSIONS Our study demonstrated that low-dosage GBC treatment markedly improved 7-day survival and neurologic outcome, reduced cellular necrosis and apoptosis, and decreased TNFα and MCP-1 mRNA expression in the brain. Because clinical trials of GBC are underway, the findings in the present study may favor its clinical application.
ACKNOWLEDGMENT We thank Xiaoyong Zhao at the Department of Anesthesiology, Tangdu Hospital, Fourth Millitary Medical University, for technical assistance.
REFERENCES
Figure 7. Sulfonylurea receptor 1 (SUR1) and transient receptor potential M4 (TRPM4) were up-regulated in brain cortex and hippocampus after cardiac arrest and cardiopulmonary resuscitation. A, Quantitative polymerase chain reaction results of messenger RNA (mRNA) expression of ABCC8 (encoding SUR1) and TRPM4. B, Representative Western blot pictures of SUR1 and TRPM4. C, Quantification of the blot densities of SUR1 and TRPM4. *p < 0.05 versus sham group. GBC = glibenclamide.
in the model of CA/CPR. We found that low dosage of GBC was enough to improve survival and neurologic function, reduce cellular necrosis and apoptosis, and decrease mRNA expression of TNFα and MCP-1 in the brain after CA/CPR in rats. As the dosage of GBC required for neuroprotection is far lower than that for hypoglycemic effect (15), neither our study nor others, including the pilot study of GBC in patients with a large infarction of ischemic stroke, observed hypoglycemia (10, 17, 35). Opening of SUR1-TRPM4 channel leads to complete depolarization, cell blebbing, and cell death (36). CA/CPR induces ATP e348
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1. Field JM, Hazinski MF, Sayre MR, et al: Part 1: Executive summary: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2010; 122:S640–S656 2. Neumar RW, Nolan JP, Adrie C, et al: Post-cardiac arrest syndrome: Epidemiology, pathophysiology, treatment, and prognostication. A consensus statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian and New Zealand Council on Resuscitation, European Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, Resuscitation Council of Asia, and the Resuscitation Council of Southern Africa); the American Heart Association Emergency Cardiovascular Care Committee; the Council on Cardiovascular Surgery and Anesthesia; the Council on Cardiopulmonary, Perioperative, and Critical Care; the Council on Clinical Cardiology; and the Stroke Council. Circulation 2008; 118:2452–2483 3. Bernard SA, Gray TW, Buist MD, et al: Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002; 346:557–563 4. Hypothermia after Cardiac Arrest Study Group: Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med 2002; 346:549–556 5. Lundbye JB, Rai M, Ramu B, et al: Therapeutic hypothermia is associated with improved neurologic outcome and survival in cardiac arrest survivors of non-shockable rhythms. Resuscitation 2012; 83:202–207 6. Testori C, Sterz F, Behringer W, et al: Mild therapeutic hypothermia is associated with favourable outcome in patients after cardiac arrest with non-shockable rhythms. Resuscitation 2011; 82:1162–1167 7. Aittoniemi J, Fotinou C, Craig TJ, et al: Review. SUR1: A unique ATPbinding cassette protein that functions as an ion channel regulator. Philos Trans R Soc Lond B Biol Sci 2009; 364:257–267 8. Vennekens R, Nilius B: Insights into TRPM4 function, regulation and physiological role. Handb Exp Pharmacol 2007; 0:269–285 September 2015 • Volume 43 • Number 9
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Online Laboratory Investigations 9. Woo SK, Kwon MS, Ivanov A, et al: The sulfonylurea receptor 1 (Sur1)-transient receptor potential melastatin 4 (Trpm4) channel. J Biol Chem 2013; 288:3655–3667 10. Simard JM, Chen M, Tarasov KV, et al: Newly expressed SUR1regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke. Nat Med 2006; 12:433–440 11. Zweckberger K, Hackenberg K, Jung CS, et al: Glibenclamide reduces secondary brain damage after experimental traumatic brain injury. Neuroscience 2014; 272:199–206 12. Simard JM, Geng Z, Woo SK, et al: Glibenclamide reduces inflammation, vasogenic edema, and caspase-3 activation after subarachnoid hemorrhage. J Cereb Blood Flow Metab 2009; 29:317–330 13. Tosun C, Koltz MT, Kurland DB, et al: The protective effect of glibenclamide in a model of hemorrhagic encephalopathy of prematurity. Brain Sci 2013; 3:215–238 14. Gerzanich V, Woo SK, Vennekens R, et al: De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury. Nat Med 2009; 15:185–191 15. Simard JM, Yurovsky V, Tsymbalyuk N, et al: Protective effect of delayed treatment with low-dose glibenclamide in three models of ischemic stroke. Stroke 2009; 40:604–609 16. Kunte H, Busch MA, Trostdorf K, et al: Hemorrhagic transformation of ischemic stroke in diabetics on sulfonylureas. Ann Neurol 2012; 72:799–806 17. Sheth KN, Kimberly WT, Elm JJ, et al: Pilot study of intravenous glyburide in patients with a large ischemic stroke. Stroke 2014; 45:281–283 18. Gao CJ, Li JP, Wang W, et al: Effects of intracerebroventricular application of the delta opioid receptor agonist [D-Ala2, D-Leu5] enkephalin on neurological recovery following asphyxial cardiac arrest in rats. Neuroscience 2010; 168:531–542 19. Katz L, Ebmeyer U, Safar P, et al: Outcome model of asphyxial cardiac arrest in rats. J Cereb Blood Flow Metab 1995; 15:1032–1039 20. Gao CJ, Niu L, Ren PC, et al: Hypoxic preconditioning attenuates global cerebral ischemic injury following asphyxial cardiac arrest through regulation of delta opioid receptor system. Neuroscience 2012; 202:352–362 21. You Z, Savitz SI, Yang J, et al: Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab 2008; 28:1564–1573 22. Wang J, Fujiyoshi T, Kosaka Y, et al: Inhibition of soluble epoxide hydrolase after cardiac arrest/cardiopulmonary resuscitation induces a neuroprotective phenotype in activated microglia and improves neuronal survival. J Cereb Blood Flow Metab 2013; 33:1574–1581
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23. Hickey RW, Ferimer H, Alexander HL, et al: Delayed, spontaneous hypothermia reduces neuronal damage after asphyxial cardiac arrest in rats. Crit Care Med 2000; 28:3511–3516 24. Xiao F, Pardue S, Arnold T, et al: Effect of ifenprodil, a polyamine site NMDA receptor antagonist, on brain edema formation following asphyxial cardiac arrest in rats. Resuscitation 2004; 61:209–219 25. Katz LM, Young A, Frank JE, et al: Neurotensin-induced hypothermia improves neurologic outcome after hypoxic-ischemia. Crit Care Med 2004; 32:806–810 26. Callaway CW, Rittenberger JC, Logue ES, et al: Hypothermia after cardiac arrest does not alter serum inflammatory markers. Crit Care Med 2008; 36:2607–2612 27. Yang L, Zhao X, Sun M, et al: Delta opioid receptor agonist BW373U86 attenuates post-resuscitation brain injury in a rat model of asphyxial cardiac arrest. Resuscitation 2014; 85:299–305 28. Ebmeyer U, Esser T, Keilhoff G: Low-dose nitroglycerine improves outcome after cardiac arrest in rats. Resuscitation 2014; 85:276–283 29. Huo TT, Zeng Y, Liu XN, et al: Hydrogen-rich saline improves survival and neurological outcome after cardiac arrest and cardiopulmonary resuscitation in rats. Anesth Analg 2014; 119:368–380 30. Patel AD, Gerzanich V, Geng Z, et al: Glibenclamide reduces hippocampal injury and preserves rapid spatial learning in a model of traumatic brain injury. J Neuropathol Exp Neurol 2010; 69:1177–1190 31. Simard JM, Tsymbalyuk N, Tsymbalyuk O, et al: Glibenclamide is superior to decompressive craniectomy in a rat model of malignant stroke. Stroke 2010; 41:531–537 32. Zhou Y, Fathali N, Lekic T, et al: Glibenclamide improves neurological function in neonatal hypoxia-ischemia in rats. Brain Res 2009; 1270:131–139 33. Tosun C, Kurland DB, Mehta R, et al: Inhibition of the Sur1-Trpm4 channel reduces neuroinflammation and cognitive impairment in subarachnoid hemorrhage. Stroke 2013; 44:3522–3528 34. Simard JM, Popovich PG, Tsymbalyuk O, et al: MRI evidence that glibenclamide reduces acute lesion expansion in a rat model of spinal cord injury. Spinal Cord 2013; 51:823–827 35. Ortega FJ, Jolkkonen J, Mahy N, et al: Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia. J Cereb Blood Flow Metab 2013; 33:356–364 36. Simard JM, Woo SK, Gerzanich V: Transient receptor potential melastatin 4 and cell death. Pflugers Arch 2012; 464:573–582 37. Simard JM, Woo SK, Norenberg MD, et al: Brief suppression of Abcc8 prevents autodestruction of spinal cord after trauma. Sci Transl Med 2010; 2:28ra29
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Objectives: Glibenclamide confers neuroprotection in animal models as well as in retrospective clinical studies. This study determines whether glibenclamide improves outcome after cardiac arrest in rats. Design: Prospective randomized laboratory study. Setting: University research laboratory. Subjects: Male Sprague-Dawley rats (n = 126). Interventions: Rats successfully resuscitated from 8-minute asphyxial cardiac arrest were randomized to glibenclamide or vehicle group. Rats in the glibenclamide group were intraperitoneally administered glibenclamide with a loading dose of 10 μg/ kg at 10 minutes and a maintenance dose of 1.2 μg at 6, 12, 18, and 24 hours after return of spontaneous circulation, whereas rats in the vehicle group received equivalent volume of vehicle solution. Measurements and Main Results: Survival was recorded every day, and neurologic deficit scores were assessed at 24, 48, and 72 hours and 7 days after return of spontaneous circulation (n = 22 in each group). Results showed that glibenclamide treatment increased 7-day survival rate, reduced neurologic deficit scores, and prevented neuronal loss in the hippocampal cornu ammonis 1 region. To investigate the neuroprotective effects of glibenclamide in acute phase, we observed neuronal injury at 24 hours after return of spontaneous circulation and found that glibenclamide significantly decreased the rate of neuronal necrosis and apoptosis. In addition, glibenclamide reduced the messenger RNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 in the cortex after return of spontaneous circulation. Furthermore, the sulfonylurea receptor 1 and transient *See also p. 2040. All authors: Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China. This work is supported, in part, by the National Natural Science Foundation of China (grant No. 81271521, 81471339), the Science and Technology Planning Project of Guangdong Province of China (grant No. 2012A030400011), and the Doctoral Fund of Ministry of Education of China (grant No. 20124433110017). For information regarding this article, E-mail: [email protected] Copyright © 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. DOI: 10.1097/CCM.0000000000001093
Critical Care Medicine
receptor potential M4 heteromers, the putative therapeutic targets of glibenclamide, were up-regulated after cardiac arrest and cardiopulmonary resuscitation, indicating that they might be involved in neuroprotective effect of glibenclamide. Conclusions: Glibenclamide treatment substantially improved survival and neurologic outcome throughout a 7-day period after return of spontaneous circulation. The salutary effects of glibenclamide were associated with suppression of neuronal necrosis and apoptosis, as well as inflammation in the brain. (Crit Care Med 2015; 43:e341–e349) Key Words: apoptosis; cardiac arrest; cytokines; glibenclamide; necrosis
C
ardiac arrest (CA) is a leading cause of death worldwide (1). Despite advances in cardiopulmonary resuscitation (CPR) methods, up to 67% of adults successfully resuscitated from out-of-hospital CA (OHCA) die in hospital, and only 68% of survivors have a good outcome (2). Most of the post-CA mortality and morbidity are caused by post-CA syndrome, including neurologic damage, myocardial dysfunction, and systemic inflammation (2). Although therapeutic hypothermia confers significant neuroprotection after ventricular fibrillation–induced OHCA in adults, it has been shown to benefit, at most, 20% of victims in whom return of spontaneous circulation (ROSC) is achieved (3, 4). The benefit of therapeutic hypothermia on those who experienced nonventricular fibrillation–induced OHCA is even less (5, 6). Alternative approaches should be developed to favor post-CA patients. Sulfonylurea receptor 1 (SUR1) is a member of the large superfamily of adenosine triphosphate (ATP)-binding cassette proteins and for decades used as the target of sulfonylurea drugs to treat type-2 diabetes mellitus (7). Transient receptor potential M4 (TRPM4) is a pore-forming protein that exclusively and nonselectively conduct monovalent cations (8). Coexpression of SUR1 and TRPM4 yields SUR1-TRPM4 heteromers and exhibits pharmacological properties of SUR1 and biophysical properties of TRPM4 (9). SUR1-TRPM4 complex is involved in neurologic injury in several disease models such as cerebral www.ccmjournal.org
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injury as well as malignant edema and stroke are now under testing in two clinical trials (ClinicalTrials.gov Identifier: NCT01454154 and NCT01794182) (17). However, the role of GBC in CA and followed CPR (CA/CPR) has not been elucidated. In this study, we aimed to determine whether GBC was salutary in a rat model of asphyxial CA/CPR. We found that low-dosage GBC treatment significantly improved 7-day survival and neurologic outcome and ameliorated neuronal injury and inflammatory cytokine expression after CA/CPR in rats.
MATERIALS AND METHODS
Figure 1. A, Experimental procedure and measurements during baseline, asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR), and return of spontaneous circulation (ROSC). Blood analysis includes arterial blood gases, electrolytes, pH, base excess, hematocrit, hemoglobin, blood glucose, and lactate concentration. B, Flow diagram of the experimental groups. DMSO = dimethyl sulfoxide, ECG = electrocardiogram, GBC = glibenclamide, i.p. = intraperitoneal injection, HR = heart rate, MAP = mean arterial pressure, RT-PCR = real-time polymerase chain reaction, TUNEL = terminal deoxynucleotide transferase–mediated dUTP-biotin nick-end labeling.
infraction, traumatic brain injury, subarachnoid hemorrhage, hemorrhagic encephalopathy of prematurity, and spinal cord injury (10–14). Blockage of SUR1-TRPM4 with low-dose glibenclamide (GBC) alleviates brain damage without causing obvious hypoglycemia (15). In a clinical setting, patients who experienced ischemic stroke but long taking sulfonylureas such as GBC to treat diabetes were associated with reduced symptomatic hemorrhagic transformation compared with those whose treatment regimen did not include sulfonylureas (16). More inspiringly, the applications of GBC on traumatic brain e342
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Animal Preparation This study was approved by the Animal Care and Use Committee of the Nanfang Hospital, Southern Medical University and followed the National Guidelines for Animal Experimentation. Male Sprague-Dawley rats weighing between 300 and 350 g were obtained from the Experimental Animal Center of Southern Medical University and housed on a 12-hour light and dark cycle with free access to water and food.
CA Model The 8-minute asphyxial CA model was established with a slight modification of the model used in the study by Gao et al (18) (Fig. 1A). In brief, rats were anesthetized, orotracheally intubated with a 14G cannula (BD, Suzhou, China), and connected to a ventilator (RWD, Shenzhen, China). Intravascular catheters (24G; BD) were inserted into the right femoral artery and vein for dynamic artery blood pressure monitoring and drug administration. After 10-minute stabilization, the rats were chemically paralyzed by IV vecuronium (2 mg/kg), and the ventilator was disconnected for 8 minutes to induce CA, which September 2015 • Volume 43 • Number 9
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Online Laboratory Investigations
TABLE 1.
Physiological Parameters Comparison Among the Three Treatment Groups
Parameters
Mean arterial pressure (mm Hg)
Heart rate (beats/min)
Rectal temperature (°C)
PH
Paco2 (mm Hg)
Lactic acid (mmol/L)
Glucose (mmol/L)
Time Points
Sham
Vehicle
Glibenclamide
Baseline
104 ± 2
114 ± 16
108 ± 8
10 min
107 ± 2
124 ± 28
117 ± 17
30 min
107 ± 4
87 ± 9a
81 ± 12a
60 min
112 ± 2
86 ± 12a
82 ± 14a
Baseline
408 ± 14
403 ± 19
400 ± 33
10 min
415 ± 14
410 ± 37
413 ± 35
30 min
407 ± 27
382 ± 52
397 ± 58
60 min
431 ± 18
399 ± 36
404 ± 55
Baseline
37.1 ± 0.3
37.1 ± 0.3
36.8 ± 0.3
10 min
37.2 ± 0.2
36.8 ± 0.3
36.6 ± 0.1a
30 min
37.2 ± 0.2
37.0 ± 0.2
36.9 ± 0.3
60 min
37.2 ± 0.3
37.1 ± 0.2
37.0 ± 0.3
Baseline
7.35 ± 0.01
7.33 ± 0.02
7.35 ± 0.01
10 min
7.38 ± 0.03
a
7.05 ± 0.05
7.10 ± 0.06a
30 min
7.40 ± 0.03
7.13 ± 0.07a
7.20 ± 0.03a
60 min
7.39 ± 0.03
7.29 ± 0.06a
7.28 ± 0.01a
a
Baseline
49.67 ± 4.39
43.65 ± 5.04
46.88 ± 3.28
10 min
49.20 ± 4.98
87.63 ± 10.58
30 min
47.90 ± 3.10
82.1 ± 10.34a
62.55 ± 9.19a
60 min
48.33 ± 3.46
59.85 ± 14.21
55.95 ± 6.59
Baseline
1.23 ± 0.45
1.05 ± 0.13
1.07 ± 0.10
10 min
1.20 ± 0.46
a
6.78 ± 2.43
4.85 ± 2.88a
30 min
0.97 ± 0.38
4.18 ± 0.81a
2.83 ± 1.16a
60 min
1.03 ± 0.15
2.20 ± 0.81
1.87 ± 0.69
Baseline
7.1 ± 0.5
6.9 ± 0.8
7.5 ± 0.6
10 min
8.7 ± 1.8
10.4 ± 1.3a
11.5 ± 1.3a
30 min
7.5 ± 0.8
7.0 ± 2.0
6.0 ± 0.8
60 min
6.4 ± 1.6
5.4 ± 1.2
5.7 ± 0.8
90 min
6.8 ± 1.2
5.2 ± 1.0
5.0 ± 0.5
120 min
7.3 ± 0.8
5.5 ± 0.8
6.4 ± 1.0
a
70.18 ± 10.74a,b
p < 0.05 versus sham group. b p < 0.05 versus vehicle group. Physiological variables were measured at baseline and at 10, 30, and 60 min after return of spontaneous circulation or sham operation. The values are expressed as mean ± sd. a
was arbitrarily defined as mean arterial pressure (MAP) dropped to 25 mm Hg below. Typically, CA occurred within 4 minutes asphyxia, leading to 4–5 minutes pulselessness. At the end of the 8-minute asphyxia, CPR was initiated by reconnecting the ventilator, injection of epinephrine (0.01 mg/kg) and sodium bicarbonate (1 mEq/kg), and performing thoracic compressions (200 compressions per minute). ROSC was characterized by an increase Critical Care Medicine
of MAP beyond 60 mm Hg for 10 minutes. Rats failed to ROSC within 2 minutes were excluded from the continuing experiments. Ventilation parameters were adjusted according to the values of blood gas analysis. Blood glucose level was intermittently detected using a glucometer (Optium, Abbott, CA). After spontaneous breath started, the animals were weaned from ventilator and extubated. To avoid spontaneous hypothermia, www.ccmjournal.org
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Huang et al
to cages. No additional fluids were administered to the rats during the whole procedure. Study Protocol At 10 minutes after ROSC, rats were randomized to GBC or vehicle group, by using random number table (Fig. 1A). GBC (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline. Rats in the GBC group were intraperitoneally administered GBC with a loading dose of 10 μg/kg at 10 minutes and a maintenance dose of 1.2 μg at 6, 12, 18, and 24 hours after ROSC, whereas rats in the vehicle group received equivalent volume of DMSO and saline. The present study included two parts (Fig. 1B). In the first part, 7-day effects of GBC in the CA/CPR model were assessed. Forty-four rats that successfully resuscitated (n = 22 for each group) were followed up for 7 days, and survival rate, neurologic outcome, and neuronal degeneration were evaluated. In the second part, the protective effect of GBC in acute phase (24 hr) was investigated. Rats survived for 24 hours after ROSC (n = 16 for each group) were selected for the detection of in situ cellular necrosis and apoptosis, as well as messenger RNA (mRNA) and protein expression of relevant genes in the brain. Rats underwent all procedures except asphyxial CA and CPR were used as sham control (n = 5 in part 1; n = 10 in part 2). Figure 2. Glibenclamide (GBC) improved survival and neurologic outcome after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A, Survival rate of rats during 7-day follow-up after CA/CPR. Solid line, vehicle group; dashed line, GBC group. B, Neurologic deficit scores of survived rats at 24, 48, and 72 hr and at 7 days after CA/CPR. #p < 0.05 versus vehicle group.
rectal temperatures were monitored and maintained at 37.0°C ± 0.5°C with a temperature feedback system (RWD) during the surgery and for another 6 hours before rats were returned
Survival Study and Neurologic Function Evaluation CA/CPR rats were followed up to 7 days, and survival rate was recorded every day. A previously validated scale of neurologic deficit score (NDS) was used to assess the neurologic outcome at 24, 48, and 72 hours and 7 days after ROSC, which was conducted by two investigators who were unaware of animal grouping (19). The total NDS was consisted of five components: consciousness and respiration, cranial nerve function, motor function, sensory function, and coordination. An NDS of 0% was considered as normal, whereas 100% was brain death.
Figure 3. Glibenclamide (GBC) prevented neuronal loss in hippocampal cornu ammonis 1 (CA1) region after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A, Representative photomicrographs (Nissl staining, purple; magnification ×400) showing the surviving neurons in the hippocampal CA1 region at 7 days after CA/CPR. Bars indicate 40 μm. B, Quantification of viable neurons in hippocampal CA1 region. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group.
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Histological Examination Seven days after ROSC or sham surgery, rats were deeply anesthetized and transcardially perfused with saline and followed by 4% paraformaldehyde for brain fixation. Brains were removed and immersed at 4°C in 15% and 30% sucrose for cryoprotection. Coronal brain sections located at 3.5 mm posterior to bregma were obtained (Leica CM1800, Heidelberg, Germany), stained with cresyl violet (Beyotime, Hangzhou, China), and observed under microscope (Olympus, Tokyo, Japan). Viable September 2015 • Volume 43 • Number 9
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neurons in the hippocampal CA1 region were those with visible nucleus and intact cytoplasm with discernable and rich Nissl staining. Neurons with shrunken cell bodies surrounding by empty spaces were excluded (20). Cellular Necrosis and Apoptosis To label necrotic cells, propidium iodide (PI; 1 mg/mL; Sigma) was administered (1 mg/kg, intraperitoneal injection) at 1 hour prior to killing (21). Brain sections were obtained, fixed in 100% ethanol, and observed with excitation/emission filters 568/585. From each rat brain, five fields in cortex, hippocampal CA1, and putamen from eight brain sections were selected for analysis. The mean counts of PI-positive cells per field were automatically calculated using Image-Pro Plus version 6.0 (Media Cybernetics, Warrendale, PA). For detection of apoptosis, cryosections were stained with either terminal deoxynucleotide transferase–mediated
dUTP-biotin nick-end labeling (TUNEL) or cleaved caspase 3 antibody. TUNEL was performed using an in situ cell apoptosis detection kit (TMR Red; Roche, Mannheim, Germany) according to the manufacturer’s recommendations. Immunohistochemistry was applied to detect cleaved caspase 3 in coronal cryosections. After blocking, sections were incubated overnight with primary antibody against cleaved caspase 3 (1:500; Cell Signaling, Beverly, MA), followed by goat anti-rabbit secondary antibody conjugated with biotin (ZSGB-BIO, Beijing, China). After rinsing in phosphate-buffer saline, the sections were incubated with streptavidin peroxidase and observed with a microscope. Western Blotting Denatured protein samples from cortex and hippocampus of each rat were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA). After blocking, membrane was incubated overnight at 4°C with antibodies including mouse anti-SUR1 (1:500; Abcam, Cambridge, United Kingdom), rabbit anti-TRPM4 (1: 1,000; Abcam), and mouse anti-βactin (1:10,000; CWBIO, Beijing, China). Bound primary antibodies were detected with the antimouse or anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:5,000; CWBIO). All signals were detected by the enhanced chemiluminescence detection method (Millipore). The densities of protein blots were quantified by using Image J (National Institutes of Health, Bethesda, MD) and normalized to β-actin levels.
Figure 4. Glibenclamide (GBC) reduced neuronal necrosis in brain cortex, hippocampal CA1, and putamen at 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Representative photomicrographs of propidium iodide (PI) staining. Bars indicate 100 μm. B, Quantification of PI-positive cells. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group.
Critical Care Medicine
Measurement of Gene Expression Total RNA was isolated using a TRIzol kit (Takara, Dalian, Japan) and reverse-transcribed to complementary DNA (cDNA) using PrimeScript RT Master Mix Kit (Takara). cDNA samples were amplified by quantitative real-time polymerase chain reaction (RT-PCR) using SYBR Green RT-PCR Master Mix (Roche) to determine mRNA level of tumor necrosis factor-α (TNFα), monocyte chemoattractant protein www.ccmjournal.org
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s) and time required for ROSC (51.4 ± 24.5 vs 43.5 ± 17.6 s) were comparative between the vehicle and GBC groups. Eight rats that resuscitated from CA but failed to survive for 24 hours were excluded from this study. The rest 78 CA/CPR rats were subjected to either 7-day (part 1, n = 44) or 24-hour (part 2, n = 32) investigation. There was no significant difference in physiologic variables among three groups at baseline, whereas prominent acidosis was observed in both CA/CPR groups (Table 1). Notably, no hypoglycemia was detected in the GBC group during the 2-hour monitoring after ROSC (Table 1). Results in the 7-day survival assay showed that 31.8% rats (7 of 22) in the vehicle group were survived at day 7, which was much lower than that in the GBC group (68.2%, 15 of 22) (p < 0.05) (Fig. 2A). We also determined the NDSs, which represent the neuFigure 5. Glibenclamide (GBC) inhibited neuronal apoptosis in the hippocampal cornu ammonis 1 (CA1) rologic deficiency, and found that region at 24 hr after cardiac arrest and cardiopulmonary resuscitation. A, Representative photomicrographs of the NDSs at 24, 48, and 72 hours terminal deoxynucleotide transferase–mediated dUTP-biotin nick-end labeling (TUNEL) staining (red). after ROSC were lower in the GBC B, Representative photomicrographs of immunohistochemistry using cleaved caspase 3 antibody (brown granules). C and D, Quantification of TUNEL-positive and cleaved caspase 3–positive cells. Bars indicate group than those in the vehicle 40 μm. *p < 0.05 versus sham group. #p < 0.05 versus vehicle group. group with statistical significance (p < 0.05) (Fig. 2B). No significant difference was found at 7 days although an improved trend was (MCP)-1, interleukin (IL)-1β, IL-6, ABCC8, TRPM4, and observed in the GBC group (Fig. 2B). Together, these results sugglyceraldehyde phosphate dehydrogenase (GAPDH). Changes gest that low-dosage GBC treatment within 24 hours improves in the relative mRNA expression were normalized to levels of 7-day survival and neurologic outcome after CA/CPR. GAPDH. Statistical Analysis All data were presented as means ± sds. Continuous data were analyzed with unpaired t tests or one-way analysis of variance followed by post hoc multiple comparison tests (least significant difference test). Difference in survival rate was analyzed by the log-rank test. SPSS 20.0 (IBM, Armonk, NY) and GraphPad Prism 5.0 (GraphPad, La Jolla, CA) were used for statistical analyses. p value less than 0.05 was considered statistically significant.
RESULTS GBC Treatment Improved Survival and Neurologic Outcome After CA/CPR A total of 126 rats were prepared for the study (Fig. 1B). Among them, 111 underwent asphyxial CA/CPR, whereas the rest 15 rats received sham operation. For animals that successfully resuscitated (n = 84), asphyxial time to CA (211.8 ± 27.7 vs 218.1 ± 23.9 e346
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GBC Attenuated Cell Death in the Brain After CA/CPR To further evaluate neurologic injury at 7 days after ROSC, brains in each group were removed for neuronal degeneration assay with Nissl staining. Results in Figure 3 demonstrated that CA/CPR induced neuronal degeneration in the hippocampal CA1 region, which was partly preserved by GBC (Fig. 3). We also examined, in acute stage (at 24 hr after ROSC), the effect of GBC on cellular necrosis and apoptosis in the brain after CA/CPR. To detect cell necrosis, rats were administered with PI before killing. Results in Figure 4 showed that PI-positive cells were observed in the cortex, hippocampal CA1, and putamen in the CA/CPR rats treated with vehicle solution, which was significantly reduced by GBC. Meanwhile, TUNEL and cleaved caspase 3 staining were performed to evaluate cell apoptosis. A large number of TUNEL-positive and cleaved caspase 3–positive cells were observed in the hippocampal CA1 region at 24 September 2015 • Volume 43 • Number 9
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were up-regulated after CA/CPR (p < 0.05). Consistently, SUR1 and TRPM4 protein levels were also increased in both vehicle and GBC groups (p < 0.05). Given that inhibiting the channel conductance rather than reducing the expression of SUR1-TRPM4 contributes the neuroprotective role of GBC, it is reasonable that the expression of SUR1 and TRPM4 was not affected after GBC treatment. Thus, it is possible that elevated expression of SUR1 and TRPM4 after CA/CPR contribute to the post-CA injury; through inhibiting the channel conductance, GBC prevents brain injury.
DISCUSSION In the present study, we found that low-dosage GBC treatment after successful resuscitation Figure 6. Messenger RNA (mRNA) expression of tumor necrosis factor-α (TNFα), monocyte chemoattractant from CA markedly improved protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 in the brain cortex at 24 hr in rats received sham operation (sham) and cardiac arrest and cardiopulmonary resuscitation treated with vehicle solution or glibenclamide 7-day survival and neurologic (GBC). *p < 0.05 versus sham group. #p < 0.05 versus vehicle group. outcome in rats. The neuroprotective role of GBC was associated with the attenuation of the CA/CPR-induced neuronal necrosis hours after CA/CPR (Fig. 5). Furthermore, GBC treatment decreased the number of TUNEL-positive and cleaved caspase and apoptosis at 24 hours after ROSC. The salutary impact of GBC on the outcome of CA/CPR was also related to the inhibi3–positive cells (Fig. 5). These results, when taken together, suggest that GBC treatment prevents neuronal loss by suppressing tion of cytokine expression and the activity of SUR1-TRPM4. Asphyxia is the most common cause of CA in patients both necrosis and apoptosis in the brain after CA/CPR. who experience severe head injury, drowning, foreign body obstruction, or intoxication (23). The 8-minute asphyxial CA/ GBC Treatment Inhibited Cytokine Expression in CPR model used in this study was reported to reproduce the Cortex After CA/CPR pathophysiology of hypoxic-ischemic encephalopathy and After CA/CPR, inflammatory cytokines are accumulated in induce histological injury similar to humans with asphyxia the brain cortex and may bring detrimental effects to neu(24, 25). Furthermore, the damage of this model is still amenable rons (22). We next investigated whether GBC could inhibit to interventions including neuroprotectant and mild hypotherthe expression of cytokines in the brain cortex after CA/CPR. mia (26, 27). Consistent with previous studies, rats underwent Results showed that mRNA level of TNFα, MCP-1, IL-1β, and asphyxial CA/CPR in the present study displayed neurologic IL-6 was markedly up-regulated in the brain cortex of rats subdeficiency in acute phase, neuronal loss in delayed stage jected to CA/CPR (Fig. 6). Compared with vehicle treatment, (19, 28), and similar 7-day survival rate (29). GBC prevented mRNA expression of TNFα and MCP-1, but GBC is an FDA-approved drug for the treatment of type-2 not IL-1β and IL-6, in the brain cortex. These results indicate diabetes. Since the elegant work by Simard et al (10) in 2006, that TNFα and MCP-1 might be associated with the neuroprothe role of GBC in neuroprotection was paid attention by neutective effect of GBC in the brain after CA/CPR. rologic researchers. Low-dose GBC was reported to significantly reduce cerebral edema, infarct volume, and mortality by CA/CPR Induced the Expression of SUR1 and TRPM4 50% without causing obvious hypoglycemia (10). Later, GBC in Brain Cortex and Hippocampus was found to be effective in the treatment of traumatic brain SUR1-TRPM4 complex is the putative therapeutic target of GBC. injury (30) and malignant stroke (31). The relevant clinical To further study the therapeutic targets of GBC, RT-PCR and trials are ongoing (17). Other studies on global brain injury Western blot were performed to detect the mRNA and protein (13, 32), subarachnoid hemorrhage (12, 33), and spinal cord level of SUR1 and TRPM4 at 24 hours after CA/CPR. As shown in injury (34) add more supportive evidences to the benefits of GBC. Here, we stepped forward to demonstrate the role of GBC Figure 7, both ABCC8 (encoding SUR1) and TRPM4 mRNA levels Critical Care Medicine
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Huang et al
depletion and calcium overload (2), which can activate TRPM4 channel (36). Through interfering with SUR1 unit, GBC blocks the activity of TRPM4 channel and decreases neuronal necrosis and apoptosis in the model of subarachnoid hemorrhage and spinal cord injury (12, 37). Therefore, we logically proposed that blocking SUR1-TRPM4 channel was the mechanism for the salutary effect of GBC in CA/CPR model. Although the opening frequency of SUR1-TRPM4 was not determined due to technique issues, we found both SUR1 and TRPM4 were up-regulated after CA/CPR, indicating that SUR1-TRPM4 complex is involved in post-CA injury, through which, GBC attenuates brain injury, and thereby improving neurologic outcome and survival.
CONCLUSIONS Our study demonstrated that low-dosage GBC treatment markedly improved 7-day survival and neurologic outcome, reduced cellular necrosis and apoptosis, and decreased TNFα and MCP-1 mRNA expression in the brain. Because clinical trials of GBC are underway, the findings in the present study may favor its clinical application.
ACKNOWLEDGMENT We thank Xiaoyong Zhao at the Department of Anesthesiology, Tangdu Hospital, Fourth Millitary Medical University, for technical assistance.
REFERENCES
Figure 7. Sulfonylurea receptor 1 (SUR1) and transient receptor potential M4 (TRPM4) were up-regulated in brain cortex and hippocampus after cardiac arrest and cardiopulmonary resuscitation. A, Quantitative polymerase chain reaction results of messenger RNA (mRNA) expression of ABCC8 (encoding SUR1) and TRPM4. B, Representative Western blot pictures of SUR1 and TRPM4. C, Quantification of the blot densities of SUR1 and TRPM4. *p < 0.05 versus sham group. GBC = glibenclamide.
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