Differential Effects of Targeted Temperature Management on Sex-Dependent Outcomes After Experimental Asphyxial Cardiac Arrest.

Asphyxial cardiac arrest (ACA) survivors face lasting neurological disability from hypoxic ischemic brain injury. Sex differences in long-term outcomes after cardiac arrest (CA) are grossly understudied and underreported. We used rigorous targeted temperature management (TTM) to understand its influence on survival and lasting sex-specific neurological and neuropathological outcomes in a rodent ACA model. Adult male and female rats underwent either sham or 5-minute no-flow ACA with 18 hours TTM at either ∼37°C (normothermia) or ∼36°C (mild hypothermia). Survival, temperature, and body weight (BW) were recorded over the 14-day study duration. All rats underwent neurological deficit score (NDS) assessment on days 1-3 and day 14. Hippocampal pathology was assessed for cell death, degenerating neurons, and microglia on day 14. Although ACA females were less likely to achieve return of spontaneous circulation (ROSC), post-ROSC physiology and biochemical profiles were similar between sexes. ACA females had significantly greater 14-day survival, NDS, and BW recovery than ACA males at normothermia (56% vs. 29%). TTM at 36°C versus 37°C improved 14-day survival in males, producing similar survival in male (63%) versus female (50%). There were no sex or temperature effects on CA1 histopathology. We conclude that at normothermic conditions, sex differences favoring females were observed after ACA in survival, NDS, and BW recovery. We achieved a clinically relevant ACA model using TTM at 36°C to improve long-term survival. This model can be used to more fully characterize sex differences in long-term outcomes and test novel acute and chronic therapies.

Therapeutic Administration of Oxcarbazepine Saves Cerebellar Purkinje Cells from Ischemia and Reperfusion Injury Induced by Cardiac Arrest through Attenuation of Oxidative Stress.

Research reports using animal models of ischemic insults have demonstrated that oxcarbazepine (a carbamazepine analog: one of the anticonvulsant compounds) extends neuroprotective effects against cerebral or forebrain injury induced by ischemia and reperfusion. However, research on protective effects against ischemia and reperfusion cerebellar injury induced by cardiac arrest (CA) and the return of spontaneous circulation has been poor. Rats were assigned to four groups as follows: (Groups 1 and 2) sham asphyxial CA and vehicle- or oxcarbazepine-treated, and (Groups 3 and 4) CA and vehicle- or oxcarbazepine-treated. Vehicle (0.3% dimethyl sulfoxide/saline) or oxcarbazepine (200 mg/kg) was administered intravenously ten minutes after the return of spontaneous circulation. In this study, CA was induced by asphyxia using vecuronium bromide (2 mg/kg). We conducted immunohistochemistry for calbindin D-28kDa and Fluoro-Jade B histofluorescence to examine Purkinje cell death induced by CA. In addition, immunohistochemistry for 4-hydroxy-2-nonenal (4HNE) was carried out to investigate CA-induced oxidative stress, and immunohistochemistry for Cu, Zn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) was performed to examine changes in endogenous antioxidant enzymes. Oxcarbazepine treatment after CA significantly increased the survival rate and improved neurological deficit when compared with vehicle-treated rats with CA (survival rates ≥ 63.6 versus 6.5%), showing that oxcarbazepine treatment dramatically protected cerebellar Purkinje cells from ischemia and reperfusion injury induced by CA. The salvation of the Purkinje cells from ischemic injury by oxcarbazepine treatment paralleled a dramatic reduction in 4HNE (an end-product of lipid peroxidation) and increased or maintained the endogenous antioxidant enzymes (SOD1 and SOD2). In brief, this study shows that therapeutic treatment with oxcarbazepine after CA apparently saved cerebellar neurons (Purkinje cells) from CA-induced neuronal death by attenuating oxidative stress and suggests that oxcarbazepine can be utilized as a therapeutic medicine for ischemia and reperfusion brain (cerebellar) injury induced by CA.

Electroacupuncture attenuates brain injury through α7 nicotinic acetylcholine receptor-mediated suppression of neuroinflammation in a rat model of asphyxial cardiac arrest.

We determined whether electroacupuncture (EA) ameliorated brain injury following asphyxial cardiac arrest (CA) and evaluated the role of the α7 nicotinic acetylcholine receptor (α7nAChR)-mediated anti-inflammatory pathway. In CA-induced rats, EA reduced brain injury and promoted behavioral recovery. Morris water maze escape latency time reduced after Baihui (GV20) and Shuigou (DU26) stimulation. EA reduced α7nAChR downregulation after cardiopulmonary resuscitation (CPR), reducing tumor necrosis factor alpha, interleukin-1, and interleukin-6 expression and ionized calcium binding adapter molecule 1 production. The α7nAChR antagonist methyllycaconitine reversed EA effect. EA stimulation of acupuncture points alleviated brain damage after CPR and reduced the inflammatory response via α7nAChR activation.

Effect of therapeutic hypothermia against renal injury in a rat model of asphyxial cardiac arrest: Α focus on the survival rate, pathophysiology and antioxidant enzymes.

Although multi­organ dysfunction is associated with the survival rate following cardiac arrest (CA), the majority of studies to date have focused on hearts and brains, and few studies have considered renal failure. The objective of the present study, therefore, was to examine the effects of therapeutic hypothermia on the survival rate, pathophysiology and antioxidant enzymes in rat kidneys following asphyxial CA. Rats were sacrificed one day following CA. The survival rate, which was estimated using Kaplan­Meier analysis, was 42.9% one day following CA. However, hypothermia, which was induced following CA, significantly increased the survival rate (71.4%). In normothermia rats with CA, the serum blood urea nitrogen level was significantly increased one day post­CA. In addition, the serum creatinine level was significantly increased one day post­CA. However, in CA rats exposed to hypothermia, the levels of urea nitrogen and creatinine significantly decreased following CA. Histochemical staining revealed a significant temporal increase in renal injury after the normothermia group was subjected to CA. However, renal injury was significantly decreased in the hypothermia group. Immunohistochemical analysis of the kidney revealed a significant decrease in antioxidant enzymes (copper­zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase and catalase) with time in the normothermia group. However, in the hypothermia group, these enzymes were significantly elevated following CA. Collectively, the results revealed that renal dysfunction following asphyxial CA was strongly associated with the early survival rate and therapeutic hypothermia reduced renal injury via effective antioxidant mechanisms.

Hypothermic treatment reduces matrix metalloproteinase-9 expression and damage in the liver following asphyxial cardiac arrest in rats.

BACKGROUND: Hypothermic treatment is known to protect organs against cardiac arrest (CA) and improves survival rate. However, few studies have evaluated the effects of hypothermia on CA-induced liver damages. This study was designed to analyzed the possible protective effects of hypothermia on the liver after asphyxial CA (ACA). Rats were randomly subjected to 5 min of ACA followed by return of spontaneous circulation (ROSC). Body temperature was controlled at 37 ± 0.5 °C (normothermia group) or 33 ± 0.5 °C (hypothermia group) for 4 h after ROSC. Liver tissues were extracted and examined at 6 h, 12 h, 1 day, and 2 days after ROSC. RESULTS: The expression of infiltrated neutrophil marker CD11b and matrix metallopeptidase-9 (MMP9) was investigated via immunohistochemistry. Morphological damage was assessed via hematoxylin and eosin (H & E) staining. Hypothermic treatment improved the survival rate at 6 h, 12 h, 1 day, and 2 days after ACA. Based on immunohistochemical analysis, the expression of CD11b and MMP9 was significantly increased from 6 h after ACA in the normothermia group. However, the expressions of CD11b and MMP9 was significantly decreased in the hypothermia group compared with that of the normothermia group. In addition, in the results of H & E, sinusoidal dilatation and vacuolization were apparent after ACA; however, these ACA-induced structural changes were reduced by the 4 h-long hypothermia. CONCLUSIONS: In conclusion, hypothermic treatment for 4 h inhibited the increases in CD11b and MMP9 expression and reduced the morphological damages in the liver following ACA in rats. This study suggests that hypothermic treatment after ACA reduces liver damages by regulating the expression of CD11b and MMP9.

Effects of hypothermia on inflammatory cytokine expression in rat liver following asphyxial cardiac arrest.

Hypothermic treatment is known to protect against cardiac arrest (CA) and improve survival rate. However, few studies have evaluated the CA-induced liver damage and the effects of hypothermia on this damage. Therefore, the aim of the present study was to determine possible protective effects of hypothermia on the liver after asphyxial CA. Rats were subjected to a 5-min asphyxial CA followed by return of spontaneous circulation (ROSC). The body temperature was controlled at 37±0.5˚C (normothermia group) or 33±0.5˚C (hypothermia group) for 4 h after ROSC. Livers were examined at 6, 12 h, 1 and 2 days after ROSC. Histopathological examination was performed by H&E staining. Alterations in the expression levels of pro-inflammatory (TNF-α and interleukin IL-2) and anti-inflammatory cytokines (IL-4 and IL-13) were investigated by immunohistochemistry. Sinusoidal dilatation and vacuolization were observed after asphyxial CA by histopathological examination. However, these CA-induced structural alterations were prevented by hypothermia. In immunohistochemical examination, the expression levels of pro-inflammatory cytokines were reduced in the hypothermia group compared with those in the normothermia group while the expression levels of anti-inflammatory cytokines were increased in the hypothermia group compared with those in the normothermia group. In conclusion, hypothermic treatment for 4 h following asphyxial CA in rats inhibited the increase of pro-inflammatory cytokines and stimulated the expression of anti-inflammatory cytokines compared with the normothermic group. The results of the present study suggested that hypothermic treatment after asphyxial CA reduced liver damage via the regulation of inflammation.

Continuous chest compressions with asynchronous ventilation improve survival in a neonatal swine model of asphyxial cardiac arrest.

BACKGROUND: Guidelines for neonatal resuscitation recommend a 3:1 compression to ventilation ratio. However, this recommendation is based on expert opinion and consensus rather than strong scientific evidence. Our primary aim was to assess whether continuous chest compressions with asynchronous ventilations would increase return of spontaneous circulation (ROSC) rate and survival compared to the 3:1 chest compression to ventilation ratio. METHODS: This was a prospective, randomized, laboratory study. Twenty male Landrace-Large White pigs, aged 1-4 days with an average weight 1.650 ± 228.3 g were asphyxiated and left untreated until heart rate was less than 60 bpm or mean arterial pressure was below 15 mmHg. Animals were then randomly assigned to receive either continuous chest compressions with asynchronous ventilations (n = 10), or standard (3:1) chest compression to ventilation ratio (n = 10). Heart rate and arterial pressure were assessed every 30 s during cardiopulmonary resuscitation (CPR) until ROSC or asystole. All animals with ROSC were monitored for 4 h. RESULTS: Coronary perfusion pressure (CPP) at 30 s of CPR was significantly higher in the experimental group (45.7 ± 16.9 vs. 21.8 ± 6 mmHg, p < 0.001) and remained significantly elevated throughout the experiment. End-tidal carbon dioxide (ETCO2) was also significantly higher in the experimental group throughout the experiment (23.4 ± 5.6 vs. 14.7 ± 5.9 mmHg, p < 0.001). ROSC was observed in six (60%) animals treated with 3:1 compression to ventilation ratio and nine (90%) animals treated with continuous chest compressions and asynchronous ventilation (p = 0.30). Time to ROSC was significantly lower in the experimental group (30 (30-30) vs. 60 (60-60) sec, p = 0.021). Of note, 7 (77.8%) animals in the experimental group and 1 (16.7%) animal in the control group achieved ROSC after 30 s (0.02). At 4 h, 2 (20%) animals survived in the control group compared to 7 (70%) animals in the experimental group (p = 0.022). CONCLUSION: Continuous chest compressions with asynchronous ventilations significantly improved CPP, ETCO2, time to ROSC, ROSC at 30 s and survival in a porcine model of neonatal resuscitation.

Repetitive anodal transcranial direct current stimulation improves neurological recovery by preserving the neuroplasticity in an asphyxial rat model of cardiac arrest.

BACKGROUND: Non-shockable rhythms present an increasing proportion of out-of-hospital cardiac arrest (CA) patients, but are associated with poor prognosis and received limited therapeutic effect of targeted temperature management (TTM). Previous study showed repetitive anodal transcranial direct current stimulation (tDCS) improved neurological outcomes in animals with ventricular fibrillation. Here, we examine the effectiveness of tDCS on neurological recovery and the potential mechanisms in a rat model of asphyxial CA. METHOD: Cardiopulmonary resuscitation was initiated after 5 min of untreated asphyxial CA. Animals were randomized to three experimental groups immediately after successful resuscitation (n = 12/group, 6 males): no-treatment control (NTC) group, TTM group, and tDCS group. Post resuscitation hemodynamics, quantitative electroencephalogram (EEG), neurological deficit score, and 96-h survival were evaluated. Brain tissues of additional animals undergoing same experimental procedure was harvested for enzyme-linked immunoassay-based quantification assays of neuroplasticity-related biomarkers and compared with the sham-operated rats (n = 6/group). RESULTS: We observed that after resuscitation tDCS-treated animals exhibited significantly higher mean arterial pressure and left ventricular ejection fraction than NTC group and showed greatly improved EEG characteristics including weighted-permutation entropy and gamma band power, and neurologic deficit scores and 96-h survival rates compared to NTC and TTM groups. Furthermore, neuroplastic biomarkers including microtubule-associated protein 2, growth-associated protein 43, postsynaptic density protein 95 and synaptophysin, were significantly higher in tDCS group when compared with NTC and TTM groups. CONCLUSION: In this rat model of asphyxial CA, repetitive anodal tDCS commenced after resuscitation improved neurological recovery, and it may exert a neuroprotective effect by preserving the neuroplasticity.

Palmitic acid methyl ester inhibits cardiac arrest-induced neuroinflammation and mitochondrial dysfunction.

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator released from the sympathetic ganglion with vasoactive properties. Post-treatment with PAME can enhance cortical cerebral blood flow and functional learning and memory, while inhibiting neuronal cell death in the CA1 region of the hippocampus under pathological conditions (i.e. cerebral ischemia). Since mechanisms underlying PAME-mediated neuroprotection remain unclear, we investigated the possible neuroprotective mechanisms of PAME after 6 min of asphyxial cardiac arrest (ACA, an animal model of global cerebral ischemia). Our results from capillary-based immunoassay (for the detection of proteins) and cytokine array suggest that PAME (0.02 mg/kg) can decrease neuroinflammatory markers, such as ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) and inflammatory cytokines after cardiopulmonary resuscitation. Additionally, the mitochondrial oxygen consumption rate (OCR) and respiratory function in the hippocampal slices were restored following ACA (via Seahorse XF24 Extracellular Flux Analyzer) suggesting that PAME can ameliorate mitochondrial dysfunction. Finally, hippocampal protein arginine methyltransferase 1 (PRMT1) and PRMT8 are enhanced in the presence of PAME to suggest a possible pathway of methylated fatty acids to modulate arginine-based enzymatic methylation. Altogether, our findings suggest that PAME can provide neuroprotection in the presence of ACA to alleviate neuroinflammation and ameliorate mitochondrial dysfunction.

The Spinal Cord Damage in a Rat Asphyxial Cardiac Arrest/Resuscitation Model.

BACKGROUND: After cardiac arrest/resuscitation (CA/R), animals often had massive functional restrictions including spastic paralysis of hind legs, disturbed balance and reflex abnormalities. Patients who have survived CA also develop movement restrictions/disorders. A successful therapy requires detailed knowledge of the intrinsic damage pattern and the respective mechanisms. Beside neurodegenerations in the cerebellum and cortex, neuronal loss in the spinal cord could be a further origin of such movement artifacts. METHODS: Thus, we aimed to evaluate the CA/R-induced degeneration pattern of the lumbar medulla spinalis by immunocytochemical expression of SMI 311 (marker of neuronal perikarya and dendrites), IBA1 (microglia marker), GFAP (marker of astroglia), calbindin D28k (marker of the cellular neuroprotective calcium-buffering system), MnSOD (neuroprotective antioxidant), the transcription factor PPARγ and the mitochondrial marker protein PDH after survival times of 7 and 21 days. The CA/R specimens were compared with those from sham-operated and completely naïve rats. RESULTS & CONCLUSION: The main ACA/R-mediated results were: (1) degeneration of lumbar spinal cord motor neurons, characterized by neuronal pyknotization and peri-neuronal tissue artifacts; (2) attendant activation of microglia in the short-term group; (3) attendant activation of astroglia in the long-term group; (4) degenerative pattern in the intermediate gray matter; (5) activation of the endogenous anti-oxidative defense systems calbindin D28k and MnSOD; (6) activation of the transcription factor PPARγ, especially in glial cells of the gray matter penumbra; and (7) activation of mitochondria. Moreover, marginal signs of anesthesia-induced cell stress were already evident in sham animals when compared with completely naïve spinal cords. A correlation between the NDS and the motor neuronal loss could not be verified. Thus, the NDS appears to be unsuitable as prognostic tool.

Protective effects of therapeutic hypothermia on renal injury in an asphyxial cardiac arrest rat model.

Cardiac arrest (CA) is a leading cause of mortality worldwide. Most of post-resuscitation related deaths are due to post-cardiac arrest syndrome (PCAS). After cardiopulmonary resuscitation (CPR), return of spontaneous circulation (ROSC) leads to renal ischemia-reperfusion injury, also known as PCAS. Many studies have focused on brain and heart injuries after ROSC, but renal failure has largely been ignored. Therefore, we investigated the protective effects of therapeutic hypothermia (TH) on asphyxial CA-induced renal injury in rats. Thirty rats were randomly divided into five groups: 1) the control group (sham); 2) the normothermic CA (nor.); 3) a normothermic CA group that received TH immediately within 2 h after CPR (Hypo. 2 hrs); 4) a normothermic CA group that received TH within 4 h after CPR (Hypo. 4 hrs); and 5) a normothermia CA group that received TH within 6 h after CPR (Hypo. 6 h). One day after CPR, all rats were sacrificed. Compared with the normothermic CA group, the TH groups demonstrated significantly increased survival rate (P < 0.05); decreased serum blood urea nitrogen, creatinine, and lactate dehydrogenase levels; and lower histological damage degree and malondialdehyde concentration in their renal tissue. Terminal deoxynucleotidyl transferase dUTP nick end labeling stain revealed that the number of apoptotic cells significantly decreased after 4 h and 6 h of TH compared to the results seen in the normothermic CA group. Moreover, TH downregulated the expression of cyclooxygenase-2 in the renal cortex compared to the normothermic CA group one day after CPR. These results suggest that TH exerts anti-apoptotic, anti-inflammatory, and anti-oxidative effects immediately after ROSC that protect against renal injury.

Stearic acid methyl ester affords neuroprotection and improves functional outcomes after cardiac arrest.

Cardiac arrest causes neuronal damage and functional impairments that can result in learning/memory dysfunction after ischemia. We previously identified a saturated fatty acid (stearic acid methyl ester, SAME) that was released from the superior cervical ganglion (sympathetic ganglion). The function of stearic acid methyl ester is currently unknown. Here, we show that SAME can inhibit the detrimental effects of global cerebral ischemia (i.e. cardiac arrest). Treatment with SAME in the presence of asphyxial cardiac arrest (ACA) revived learning and working memory deficits. Similarly, SAME-treated hippocampal slices after oxygen-glucose deprivation inhibited neuronal cell death. Moreover, SAME afforded neuroprotection against ACA in the CA1 region of the hippocampus, reduced ionized calcium-binding adapter molecule 1 expression and inflammatory cytokines/chemokines, with restoration in mitochondria respiration. Altogether, we describe a unique and uncharted role of saturated fatty acids in the brain that may have important implications against cerebral ischemia.

Therapeutic Hypothermia Improves Hind Limb Motor Outcome and Attenuates Oxidative Stress and Neuronal Damage in the Lumbar Spinal Cord Following Cardiac Arrest.

: Hypothermia enhances outcomes of patients after resuscitation after cardiac arrest (CA). However, the underlying mechanism is not fully understood. In this study, we investigated effects of hypothermic therapy on neuronal damage/death, microglial activation, and changes of endogenous antioxidants in the anterior horn in the lumbar spinal cord in a rat model of asphyxial CA (ACA). A total of 77 adult male Sprague-Dawley rats were randomized into five groups: normal, sham ACA plus (+) normothermia, ACA + normothermia, sham ACA + hypothermia, and ACA + hypothermia. ACA was induced for 5 min by injecting vecuronium bromide. Therapeutic hypothermia was applied after return of spontaneous circulation (ROSC) via rapid cooling with isopropyl alcohol wipes, which was maintained at 33 ± 0.5 °C for 4 h. Normothermia groups were maintained at 37 ± 0.2 °C for 4 h. Neuronal protection, microgliosis, oxidative stress, and changes of endogenous antioxidants were evaluated at 12 h, 1 day, and 2 days after ROSC following ACA. ACA resulted in neuronal damage from 12 h after ROSC and evoked obvious degeneration/loss of spinal neurons in the ventral horn at 1 day after ACA, showing motor deficit of the hind limb. In addition, ACA resulted in a gradual increase in microgliosis with time after ACA. Therapeutic hypothermia significantly reduced neuronal loss and attenuated hind limb dysfunction, showing that hypothermia significantly attenuated microgliosis. Furthermore, hypothermia significantly suppressed ACA-induced increases of superoxide anion production and 8-hydroxyguanine expression, and significantly increased superoxide dismutase 1 (SOD1), SOD2, catalase, and glutathione peroxidase. Taken together, hypothermic therapy was found to have a substantial impact on changes in ACA-induced microglia activation, oxidative stress factors, and antioxidant enzymes in the ventral horn of the lumbar spinal cord, which closely correlate with neuronal protection and neurological performance after ACA.

Effects of regional body temperature variation during asphyxial cardiac arrest on mortality and brain damage in a rat model.

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.

The influences of morphine or ketamine pre-treatment on hemodynamic, acid-base status, biochemical markers of brain damage and early survival in rats after asphyxial cardiac arrest.

BACKGROUND: In different models of hypoxia, blockade of opioid or N-methyl-D-aspartate (NMDA) receptors shows cardio- and neuroprotective effects with a consequent increase in animal survival. The aim of the study was to investigate effects of pre-treatment with Morphine or Ketamine on hemodynamic, acid-base status, early survival, and biochemical markers of brain damage in a rat model of asphyxial cardiac arrest (ACA). METHODS: Under anaesthesia with Thiopental Sodium 60 mg/kg, i.p., Wistar rats (n = 42) were tracheostomized and catheters were inserted in a femoral vein and artery. After randomization, the rats were pre-treated with: Morphine 5 mg/kg i.v. (n = 14); Ketamine 40 mg/kg i.v. (n = 14); or equal volume of i.v. NaCl 0.9% as a Control (n = 14). ACA was induced by corking of the tracheal tube for 8 min, and defined as a mean arterial pressure (MAP) < 20 mmHg. Resuscitation was started at 5 min after cardiac arrest (CA). Invasive MAP was recorded during experiments. Arterial pH and blood gases were sampled at baseline (BL) and 10 min after CA. At the end of experiments, all surviving rats were euthanised, brain and blood samples for measurement of Neuron Specific Enolase (NSE), s100 calcium binding protein B (s100B) and Caspase-3 (CS-3) were retrieved. RESULTS: At BL no differences between groups were found in hemodynamic or acid-base status. After 3 min of asphyxia, all animals had cardiac arrest (CA). Return of spontaneous circulation (MAP > 60 mmHg) was achieved in all animals within 3 min after CA. At the end of the experiment, the Ketamine pre-treated group had increased survival (13 of 14; 93%) compared to the Control (7 of 14; 50%) and Morphine (10 of 14; 72%) groups (p = 0.035). Biochemical analysis of plasma concentration of NSE and s100B as well as an analysis of CS-3 levels in the brain tissue did not reveal any differences between the study groups. CONCLUSION: In rats after ACA, pre-treatment with Morphine or Ketamine did not have any significant influence on hemodynamic and biochemical markers of brain damage. However, significantly better pH level and increased early survival were found in the Ketamine pre-treated group.

Therapeutic hypothermia attenuates paraplegia and neuronal damage in the lumbar spinal cord in a rat model of asphyxial cardiac arrest.

Spinal cord ischemia can result from cardiac arrest. It is an important cause of severe spinal cord injury that can lead to serious spinal cord disorders such as paraplegia. Hypothermia is widely acknowledged as an effective neuroprotective intervention following cardiac arrest injury. However, studies on effects of hypothermia on spinal cord injury following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) are insufficient. The objective of this study was to examine effects of hypothermia on motor deficit of hind limbs of rats and vulnerability of their spinal cords following asphyxial CA/CPR. Experimental groups included a sham group, a group subjected to CA/CPR, and a therapeutic hypothermia group. Severe motor deficit of hind limbs was observed in the control group at 1 day after asphyxial CA/CPR. In the hypothermia group, motor deficit of hind limbs was significantly attenuated compared to that in the control group. Damage/death of motor neurons in the lumbar spinal cord was detected in the ventral horn at 1 day after asphyxial CA/CPR. Neuronal damage was significantly attenuated in the hypothermia group compared to that in the control group. These results indicated that therapeutic hypothermia after asphyxial CA/CPR significantly reduced hind limb motor dysfunction and motoneuronal damage/death in the ventral horn of the lumbar spinal cord following asphyxial CA/CPR. Thus, hypothermia might be a therapeutic strategy to decrease motor dysfunction by attenuating damage/death of spinal motor neurons following asphyxial CA/CPR.

Melatonin alleviates asphyxial cardiac arrest-induced cerebellar Purkinje cell death by attenuation of oxidative stress.

Although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and activation of autophagy in Purkinje cells following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) remains unclear. Rats used in this study were randomly assigned to 6 groups as follows; vehicle-treated sham operated group, vehicle-treated asphyxial CA/CPR operated group, melatonin-treated sham operated group, melatonin-treated asphyxial CA/CPR operated group, PDOT (a MT2 melatonin receptor antagonist) plus (+) melatonin-treated sham operated group and PDOT+melatonin-treated asphyxial CA/CPR operated group. Melatonin (20 mg/kg, i.p., 4 times before CA and 3 times after CA) treatment significantly improved survival rate and neurological deficit compared with the vehicle-treated asphyxial CA/CPR rats (survival rates ≥40% vs 10%), showing that melatonin treatment exhibited protective effect against asphyxial CA/CPR-induced Purkinje cell death. The protective effect of melatonin against CA/CPR-induced Purkinje cell death paralleled a remarkable attenuation of autophagy-like processes (Beclin-1, Atg7 and LC3), as well as a dramatic reduction in superoxide anion radical (O2·-), intense enhancements of CuZn superoxide dismutase (SOD1) and MnSOD (SOD2) expressions. Furthermore, the protective effect was notably reversed by treatment with PDOT, which is a selective MT2 antagonist. In brief, melatonin conferred neuroprotection against asphyxial CA/CPR-induced Purkinje cell death via inhibiting autophagic activation by reducing expressions of O2·- and increasing expressions of antioxidant enzymes, and suggests that MT2 is involved in neuroprotective effect of melatonin against Purkinje cell death caused by asphyxial CA/CPR.

The relationship between low survival and acute increase of tumor necrosis factor α expression in the lung in a rat model of asphyxial cardiac arrest.

Cardiac arrest (CA) is sudden loss of heart function and abrupt stop in effective blood flow to the body. The patients who initially achieve return of spontaneous circulation (RoSC) after CA have low survival rate. It has been known that multiorgan dysfunctions after RoSC are associated with high morbidity and mortality. Most previous studies have focused on the heart and brain in RoSC after CA. Therefore, the aim of this research was to perform serological, physiological, and histopathology study in the lung and to determine whether or how pulmonary dysfunction is associated with low survival rate after CA. Experimental animals were divided into sham-operated group (n=14 at each point in time), which was not subjected to CA operation, and CA-operated group (n=14 at each point in time), which was subjected to CA. The rats in each group were sacrificed at 6 hours, 12 hours, 24 hours, and 2 days, respectively, after RoSC. Then, pathological changes of the lungs were analyzed by hematoxylin and eosin staining, Western blot and immunohistochemistry for tumor necrosis factor α (TNF-α). The survival rate after CA was decreased with time past. We found that histopathological score and TNF-α immunoreactivity were significantly increased in the lung after CA. These results indicate that inflammation triggered by ischemia-reperfusion damage after CA leads to pulmonary injury/dysfunctions and contributes to low survival rate. In addition, the finding of increase in TNF-α via inflammation in the lung after CA would be able to utilize therapeutic or diagnostic measures in the future.

Gynostemma pentaphyllum is neuroprotective in a rat model of cardiopulmonary resuscitation.

Asphyxial cardiac arrest (ACA)-induced ischemia results in acute and delayed neuronal cell death. The early reperfusion phase is critical for the outcome. Intervention strategies directed to this period are promising to reduce ACA/resuscitation-dependent impairments. This study focused on the evaluation of the protective potential of an extract from Gynostemma pentaphyllum (GP), a plant used in traditional medicine with antioxidative, glucose lowering and neuroprotective activities, in an ACA rat model. We tested the following parameters: i) Basic systemic parameters such as pCO2 and blood glucose value within the first 30 min post-ACA; ii) mitochondrial response by determining activities of citrate synthase, respiratory chain complexes I + III and II + III, and the composition of cardiolipin 6 and 24 h post-ACA; iii) neuronal vitality of the CA1 hippocampal region by immunohistochemistry 24 h and 7 days post-ACA; and iv) cognitive function by a novel object recognition test 7 days post-ACA. GP, administered after reaching spontaneous circulation, counteracted the following: i) ACA-mediated increases in arterial CO2 tension and blood glucose values; ii) transient increase in the activity of the respiratory chain complexes II + III; iii) elevation in cardiolipin content; iv) hippocampal CA1 neurodegeneration, and v) loss of normal novelty-object seeking. The protective effects of GP were accompanied by side effects of the vehicle DMSO, such as the stimulation of citrate synthase activity in control animals, inhibition of cardiolipin synthesis in ACA animals and complex II + III activity in both control and ACA animals. The results emphasize the importance of the early post-resuscitation phase for the neurological outcome after ACA/resuscitation, and demonstrated the power of GP substitution as neuroprotective intervention. Moreover, the results underline the need of a careful handling of the popular vehicle DMSO.

Changes in histopathology and tumor necrosis factor-α levels in the hearts of rats following asphyxial cardiac arrest.

OBJECTIVE: Post cardiac arrest (CA) syndrome is associated with a low survival rate in patients who initially have return of spontaneous circulation (ROSC) after CA. The aim of this study was to examine the histopathology and inflammatory response in the heart during the post CA syndrome. METHODS: We induced asphyxial CA in male Sprague-Dawley rats and determined the survival rate of these rats during the post resuscitation phase. RESULTS: Survival of the rats decreased after CA: 66.7% at 6 hours, 36.7% at 1 day, and 6.7% at 2 days after ROSC following CA. The rats were sacrificed at 6 hours, 12 hours, 1 day, and 2 days after ROSC, and their heart tissues were examined. Histopathological scores increased at 12 hours post CA and afterwards, histopathological changes were not significant. In addition, levels of tumor necrosis factor-α immunoreactivity gradually increased after CA. CONCLUSION: The survival rate of rats 2 days post CA was very low, even though histopathological and inflammatory changes in the heart were not pronounced in the early stage following CA.