Normothermic Research Articles

Experimental early‐life febrile seizures induce changes in GABAAR‐mediated neurotransmission in the dentate gyrus

Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ-aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA(A) R function in dentate granule cells. We applied an immature rat model of hyperthermia (HT)-induced FS. GABA(A) R-mediated neurotransmission was studied using whole-cell patch-clamp recordings from dentate granule neurons in hippocampal slices within 6-9 days post-HT. Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole-cell GABA responses revealed a doubled GABA(A) R sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole-cell GABA responses showed similar kinetics in postsynaptic GABA(A) Rs of HT and NT rats. quantitative real-time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA(A) R subunit expression, which was most pronounced for the α3 subunit. The data support the hypothesis that FS persistently alter neuronal excitability.

Adenosine and Opioid Receptors Do Not Trigger the Cardioprotective Effect of Mild Hypothermia

Mild hypothermia (32°C-34°C) exerts a potent cardioprotection in animal models of myocardial infarction. Recently, it has been proposed that this beneficial effect is related to survival signaling. We, therefore, hypothesized that the well-known cardioprotective pathways dependent on adenosine and/or opioid receptors could be the trigger of hypothermia-induced salvage. Open-chest rabbits were accordingly exposed to 30 minutes of coronary artery occlusion (CAO) under normothermic (NT) or hypothermic ([HT] 32°C) conditions. In the latter, hypothermia was induced by total liquid ventilation with temperature-controlled perfluorocarbons in order to effect ultrafast cooling and to accurately control cardiac temperature. After 4 hours of reperfusion, infarct and no-reflow zone sizes were assessed and quantified as a percentage of the risk zone. In animals experiencing HT ischemia, the infarct size was dramatically reduced as compared to NT animals (9% ± 3% vs 55% ± 2% of the risk zone, respectively). Importantly, administration of opioid and adenosine receptor antagonists (naloxone [6 mg/kg iv] and 8-(p-sulfophenyl) theophylline [20 mg/kg iv], respectively) did not alter the infarct size or affect the cardioprotective effect of hypothermia. Doses of these 2 antagonists were appropriately chosen since they blunted infarct size reduction induced by selective opioid or adenosine receptor stimulation with morphine (0.3 mg/kg iv) or N (6)-cyclopentyladenosine ([CPA] 100 μg/kg iv), respectively. Therefore, the cardioprotective effect of mild hypothermia is not triggered by either opioid or adenosine receptor activation, suggesting the involvement of other cardioprotective pathways.

The effect of moderate hypothermia on the electrographic seizure burden in neonatal hypoxic ischaemic encephalopathy

BackgroundModerate therapeutic hypothermia improves outcome in babies with hypoxic ischaemic encephalopathy by reducing neuronal damage during the reperfusion stage at which time seizures often commence. Some evidence suggests seizures may...

Effect of Delayed Mild Brain Hypothermia on Edema Formation After Intracerebral Hemorrhage in Rats

Secondary consequences of intracerebral hemorrhage (ICH) including inflammation, edema, and oxidative damage all contribute to cell death after ICH. Brain hypothermia (BH) has been used as an effective neuroprotective treatment in experimental brain ischemia and traumatic brain injury. In this study, we first attempted to evaluate the effect of delayed mild BH (35 degrees C) on brain edema formation 48 hours after ICH. BH was started 3, 6, 12, and 24 hours after the induction of 100 muL of autologous blood into the basal ganglia (hypothermic [HT]; HT3: n = 4, HT6: n = 6, HT12: n = 11, HT24: n = 6) in rats. To examine the protective mechanism of BH, blood-brain barrier (BBB) permeability to Evans blue, accumulation of polymorphonuclear leukocyte, and oxidative DNA damage in the lesion were compared between normothermic (NT) (37 degrees C) and HT6 rats 48 hours after ICH. Finally, neurologic recovery was assessed using behavioral tests in NT and HT6 rats 48 hours after ICH. Brain water content in the ispilateral basal ganglia was significantly reduced with delayed BT compared with NT (n = 7, 81.8 +/- 0.7% v HT3: 78.9 +/- 0.8%, P < .01; HT6: 78.7 +/- 0.6%, P < .01; HT12: 79.4 +/- 1.1%, P < .01; HT24: 80.3 +/- 0.6%, P < .01). The BBB disruption to Evans blue was significantly reduced with BH (HT6: n = 6) compared with NT (n = 6) rats in the ipsilateral basal ganglia (23.0 +/- 5.2 v 42.3 +/- 4.0 ng/g wet tissue, P < .05). HT6 treatment (n = 6) significantly inhibited the accumulation of polymorphonuclear leukocyte compared with NT treatment (n = 6) (0.43 +/- 0.22 v 1.49 +/- 0.61 DeltaAbs/mg tissue, P < .05). HT6 treatment (n = 3) also significantly reduced oxidative DNA damage determined with 8-hydroxyl-2'-deoxyguanosine compared with NT treatment (n = 3) (92 +/- 18 v 40 +/- 7 pg 8-hydroxyl-2'-deoxyguanosine/mug DNA, P < .05). Furthermore, HT6 treatment (n = 5) significantly improved neurologic recovery assessed with forelimb placing score compared with NT treatment (42.0 +/- 5.8 v 12.0 +/- 3.7, P < .05). In conclusion, mild BH significantly reduces the brain edema formation after ICH, even when the BH is applied 24 hours after hematoma induction in rats. Several neuroprotective mechanisms, including reduced BBB disruption, inflammation and oxidative damage, are suggested in this study.

Dynamic cerebral autoregulation during passive heat stress

This study tested the hypothesis that passive heating impairs cerebral autoregulation. Transfer function analysis of resting arterial blood pressure (BP) and middle cerebral artery blood velocity (MCAVmean) were conducted on 9 healthy subjects, dressed in a water‐perfused suit, under normothermic (NT) and passive heat stress (HS, Δ core temperature 1.1 ± 0.2 °C, P&lt;0.001) conditions. Passive heating reduced MCAVmean (8 ± 8 cm•s−1, P=0.01), whilst mean arterial BP was maintained (2 ± 5 mm Hg, P=0.36). In the very low frequency range (&lt;0.07 Hz) coherence was lower during HS compared to NT conditions (0.26 ± 0.10 vs 0.57 ± 0.13, respectively, P&lt;0.001). Coherence was &gt;0.5 and similar between NT and HS in the low (0.07–0.20 Hz, 0.64 ± 0.19 vs 0.59 ± 0.10, respectively, P=0.40) and high (0.20–0.35 Hz, 0.63 ± 0.19 vs 0.72 ± 0.14, respectively, P=0.12) frequency ranges. Phase was higher during HS in the low frequency range (NT: 0.55 ± 0.31 vs HS: 0.82 ± 0.17°, P=0.01), whereas it was unchanged by HS in the high frequency range (NT: −0.10 ± 0.20 vs HS: 0.12 ± 0.71°, P=0.14). Transfer gain was higher during HS in the low (NT: 1.53 ± 0.41 vs HS: 1.73 ± 0.38 cm•s −1•mmHg−1, P=0.03) and high (NT: 1.55 ± 0.55 vs HS: 2.06 ± 0.37 cm•s−1•mmHg−1, P=0.03) frequency ranges. Higher transfer function estimates of gain suggest that dynamic cerebral autoregulation is impaired during HS.Supported by NIH Grant HL‐61388 &amp; HL‐84072

Differential Neuroprotective Properties of Endogenous and Exogenous Erythropoietin in a Mouse Model of Traumatic Brain Injury

Both heat acclimation (HA) and post-injury treatment with recombinant human erythropoietin (Epo, rhEpo, exogenous Epo) are neuroprotective against traumatic brain injury (TBI). Our previous data demonstrated that HA-induced neuroprotection includes improved functional recovery and reduced cerebral edema formation. Additionally, in earlier Western-blot analyses, we found that HA mice display increased expression of the specific erythropoietin receptor (EpoR) and of hypoxia-inducible factor-1 alpha (HIF-1 alpha), the inducible subunit of the transcription factor, which regulates Epo gene expression, but not of Epo itself. In light of this, the aim of the current study was threefold: (1) to assess Epo expression in the trauma area and hippocampus following HA, rhEpo administration, or combined HA-rhEpo treatment, using immunohistochemical methods that offer enhanced anatomical resolution; (2) to examine the effects of endogenous and exogenous Epo on edema formation in normothermic (NT) mice; and (3) to evaluate the effects of exogenous Epo administration on neuroprotective outcome measures in HA animals. HA induced enhanced expression of endogenous Epo in the trauma area and the hippocampus. Treatment with anti-Epo antibody given to NT mice increased edema formation, whereas rhEpo induced no beneficial effect. Cognitive performance testing and immunohistochemical findings reinforced HA and rhEpo as separate protective interventions but showed no advantage to combining the two strategies. We therefore suggest that HA-induced neuroprotection is shaped by pre-existing mediators but cannot be modified by post-injury treatment aimed at increasing the levels of neuroprotective agents.

Altered cytokine expression and sustained hypothermia following traumatic brain injury in heat acclimated mice

Long-term exposure to moderate ambient heat (heat acclimation, HA, 30 days at 34 ± 1 °C) provides protection toward a variety of stressors including traumatic brain injury. As previous studies suggested an anti-inflammatory effect of HA and given the ability of augmented pre-injury anti-inflammatory cytokine expression to harbor neuroprotection and to attenuate early post-injury expression of pro-inflammatory mediators, we hypothesized that HA-induced neuroprotection may involve enhanced pre-injury expression of anti-inflammatory mediators or a reduction in post-injury TNF alpha (TNFα) expression. Since the attenuation of inflammatory-associated entities has also been linked to mild hypothermia, an established neuroprotective paradigm, the effect of HA on post-injury body temperature was also studied. HA mice and normothermic (NT) counterparts were examined using a closed head injury model. Cytokines were measured within the ipsilateral cortex. Pre-injury protein levels of anti-inflammatory interleukins 10 and 4 (IL-10, IL-4) were quantified by enzyme-linked immunosorbent assays (ELISA). mRNA and protein levels of TNFα were quantified during the initial 2 h post-injury using semi-quantitative and real-time polymerase chain reaction (sqRT-PCR and qRT-PCR) or ELISA, respectively. Rectal temperatures were measured. HA induced augmented pre-injury IL-10 expression and a post-injury reduction in TNFα mRNA levels, as well as altered expression dynamics of TNFα protein. TNFα protein levels decreased relative to the sham state in HA mice only. HA mice displayed sustained post-injury hypothermia, namely significantly lower body temperature at 4 h post-injury. Given the evidence on the neuroprotective nature of hypothermia and anti-inflammatory cytokines, we suggest that these changes may contribute to HA-induced neuroprotection.

Hypothermia Affects Translocation of Numerous Cytoplasmic Proteins Following Global Cerebral Ischemia

Using a decapitation ischemia model, we studied translocation of proteins to and from the cytosol in normothermic (NT) and hypothermic (HT) rat brains. 2D gel analysis identified 74 proteins whose cytosolic level changed significantly after 15 min of ischemia. HT preserved the cytosolic levels of several glycolytic enzymes, as well as many plasticity related proteins, otherwise decreased following NT ischemia. The levels of redox-related proteins was lower in HT than in NT. Our results indicate that translocation of proteins to and from the cytosol is an important issue during ischemia.

Recombinant activated factor VII efficacy and safety in a model of bleeding and thrombosis in hypothermic rabbits: a blind study.

Recombinant activated factor VII (rFVIIa) is increasingly used to secure hemostasis in hemorrhagic situations in trauma and surgical patients. Hypothermia is often observed in these clinical settings. To study the efficacy and safety of rFVIIa in hypothermia in a rabbit model of bleeding and thrombosis. Sixty-nine rabbits were anesthetized, ventilated and monitored for blood pressure, temperature and carotid flow. The Folts model was used: a stenosis (75%) and an injury were carried out on the carotid artery, inducing thrombosis. Blood flow decreased as thrombus size increased until the pressure gradient was such that the thrombus was released and local arterial blood flow was suddenly restored. This is known as a cyclic flow reduction (CFR). After counting baseline CFRs during a 20-min period (P1), rabbits were randomized blindly to one of four groups: normothermic (NT) placebo or rFVIIa (150 microg kg(-1)), hypothermic (HT) (34 degrees C) placebo or rFVIIa. Then CFRs were recorded over a second period (P2). At the end of the experiment, a hepato-splenic section was performed and the amount of blood loss was recorded. After each period, the following were measured: ear immersion bleeding time (BT), hemoglobin, platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen. Hypothermia increased BT and blood loss. These effects were reversed by rFVIIa. In NT rabbits, rFVIIa shortened BT but did not reduce blood loss. rFVIIa-treated rabbits bled similarly regardless of temperature. The incidence of CFRs was higher in treated than placebo animals regardless of temperature. rFVIIa decreased PT and aPTT without modifying platelet count or fibrinogen level. Hemostatic efficacy of rFVIIa was maintained in hypothermia. However, the number of CFRs was higher in the rFVIIa-treated group than in the placebo groups, whether for NT or HT rabbits.

The central chemoreflex ventilatory response to carbon dioxide following acclimation to a hot environment

Studies in non-human mammals have shown that the chemosensitive areas on the ventral surface of the medulla oblongata are temperature sensitive. We asked in this study if human chemosensitivity adapts to heat acclimation (HA) as do human thermoregulatory responses. We hypothesized HA would alter the central chemoreflex ventilatory response to CO2. Six male participants performed a hyperoxic modified Read re-breathing test at a normothermic (NT) and hyperthermic (HT) esophageal temperature (TES) prior to and following a 10 day passive heat acclimation. Acclimation was confirmed by a decrease in the TES threshold for eccrine sweating (ESW) and temple cutaneaous blood velocity (CBVTEMPLE) (ESW: 37.62°C ± 0.22 versus 37.22°C ± 0.25; p=0.01 and CBVTEMPLE: 37.43°C ± 0.16 versus 37.16°C ± 0.17; p=0.02). The pre-acclimation PETCO2 ventilatory recruitment thresholds (NT: 46.50 mm Hg ± 1.90 and HT: 47.68 mm Hg ± 3.69) were not different than that observed post-acclimation (NT: 47.08 mm Hg ± 1.89; p=0.310 and HT: 47.00 ± 2.86; p=0.259). Additionally, pre-acclimation sensitivities of the central chemoreflex ventilatory response to CO2 (NT: 3.18 L·min−1·mm Hg−1 ± 2.43 and HT: 4.03 L·min−1·mm Hg−1 ± 1.75) were unaffected (NT: 3.45 L·min−1·mm Hg−1 ± 2.01; p=0.267 and HT: 4.33 L·min−1·mm Hg−1 ± 2.00; p=0.225). In conclusion, the central chemoreflex ventilatory response to CO2 did not appear to be modified by passive acclimation to a hot environment. Acknowledgements: This study was supported by grants from Natural Sciences and Engineering Research Council of Canada, Canadian Institutes for Health Research (SafetyNet) and Canadian Foundation for Innovation.

Is Hypothermia Simply a Marker of Shock and Injury Severity or an Independent Risk Factor for Mortality in Trauma Patients? Analysis of a Large National Trauma Registry

Successful application of induced hypothermia (HT) after stroke and cardiac arrest has led to a resurgence of interest in its potential protective effects in trauma patients. Although clinical studies demonstrate an adverse effect, they consist of small studies from single institutions. We tested the hypothesis that HT is associated with improved survival in trauma patients by analyzing a large, national trauma database. The study included all patients in the National Trauma Data Bank (1994-2002) aged 18 to 55 years old with temperature at presentation to the emergency department and base deficit (BD) recorded. Hypothermic (temperature, <35 degrees C; n = 3267) patients and patients with normothermia (temperature, > or =35 degrees C; n = 35,283) patients were compared. Two analyses were conducted to control for the association between HT and injury severity. First, hypothermic and normothermic (NT) patients were stratified by injury severity score (ISS) and shock (blood pressure < or = 90 mm Hg). Next, logistic regression was used to control for effects of age, sex, mechanism, ISS, head, chest, and abdominal injuries, BP, Glasgow Coma Scale score, and BD, with survival as the dependent and HT as the predictor variable. Although HT was more common in more seriously injured patients, stratified analysis revealed that hypothermic patients have significantly higher mortality than patients with the same severity of injury who remain NT. Even after controlling for injury severity and the other potential confounders listed above, HT remained a strong, independent predictor of mortality (odds ratio, 1.19; 95% confidence interval, 1.05-1.35). There is no apparent protective effect of HT in trauma patients. HT itself, not just its association with shock, injury severity, and other cofactors lead to a significant increase in mortality in injured patients. Continued, aggressive efforts to prevent and treat HT are warranted.

Application of heart rate variability to determine physiologic changes in hypothermic rats

Time domain and power spectral density (PSD) analyses of ECG were used as indices of physiologic change in telemetry-equipped hypothermic rats. Anesthetized rats were either cooled (Hypo, Tc=20°C) and then rewarmed or maintained normothermic (NC). Heart rate, blood pressure, interbeat interval (IBI), normalized standard deviation (NSD) and PSD were determined. NSD in Hypo rats was significantly elevated at Tc=30°C which was coincident with the transition from increasing to decreasing HR and BP during cooling. With no change in PSD at Tc=30°C, the increased NSD suggests physiological events lacking an autonomic nervous system origin.

Application of heart rate variability to determine physiologic changes in hypothermic rats

Time domain and power spectral density (PSD) analyses of ECG were used as indices of physiologic change in telemetry-equipped hypothermic rats. Anesthetized rats were either cooled (Hypo, Tc=20°C) and then rewarmed or maintained normothermic (NC). Heart rate, blood pressure, interbeat interval (IBI), normalized standard deviation (NSD) and PSD were determined. NSD in Hypo rats was significantly elevated at Tc=30°C which was coincident with the transition from increasing to decreasing HR and BP during cooling. With no change in PSD at Tc=30°C, the increased NSD suggests physiological events lacking an autonomic nervous system origin.

Delayed onset of prolonged hypothermia improves outcome after intracerebral hemorrhage in rats.

Prolonged hypothermia reduces ischemic brain injury, but its efficacy after intracerebral hemorrhagic (ICH) stroke is unresolved. Rats were implanted with core temperature telemetry probes and subsequently subjected to an ICH, which was produced by infusing bacterial collagenase into the striatum. Animals were kept normothermic (NORMO), or were made mildly hypothermic (33-35 degrees C) for over 2 days starting 1 hour (HYP-1), 6 hours (HYP-6), or 12 hours (HYP-12) after collagenase infusion. Others were cooled for 7 hours beginning 1 hour after infusion (BRIEF). Skilled reaching, walking, and spontaneous forelimb use were assessed. Normothermic ICH rats sustained, on average, a 36.9-mm3 loss of tissue at 1 month. Only the HYP-12 group had a significantly smaller lesion (25.5 mm3). Some functional improvements were found with this and other hypothermia treatments. Cerebral edema was observed in NORMO rats, and was not lessened significantly by hypothermia (HYP-12). Blood pressure measurements, as determined by telemetry, in BRIEF rats showed that hypothermia increased blood pressure. This BRIEF treatment also resulted in significantly more bleeding at 12 hours after ICH (79.2 microL) versus NORMO-treated rats (58.4 microL) as determined by a spectrophotometric hemoglobin assay. Accordingly, these findings suggest that early hypothermia may fail to lessen lesion size owing to complications, such as elevated blood pressure, whereas much-delayed hypothermia is beneficial after ICH. Future experiments should assess whether counteracting the side effects of early hypothermia enhances protection.

Effect of intra-ischemic hypothermia on the expression of c-Fos and c-Jun, and DNA binding activity of AP-1 after focal cerebral ischemia in rat brain

It is unknown whether immediate early gene (IEG) induction and subsequent late gene regulation after ischemia is beneficial or deleterious. The aim of this study was to examine the effect of hypothermia on expression of c-Fos and c-Jun, and AP-1 DNA binding activity, after transient focal cerebral ischemia in rat brain, and clarify the role of IEGs and AP-1 after insults. Male Wistar rats underwent right middle cerebral artery occlusion for 1 h with the intraluminal suture method. During ischemia, animals were assigned to either normothermic (NT) or hypothermic (HT) groups. In the NT group, brain temperature was observed to spontaneously increase to 40 °C during ischemia. In the HT group, brain temperature decreased to 30 °C. Infarct volume in cortex was decreased in the HT group, compared with that in the NT group ( P<0.001). Increased c-Fos immunoreactivity in the cortex was observed at 3 h after reperfusion in the HT, but not the NT group, while c-Jun expression was not affected by HT treatment. There was also a significant increase in AP-1 DNA binding activity at 3 h in the HT group when compared to the NT group ( P<0.01). In conclusion, hypothermia decreased cerebral infarction in association with early increases in c-Fos expression and AP-1 DNA binding activity in peri-infarct cortex. It remains to be established whether such responses are a cause or consequence of cell survival, but these results clearly establish that altered transcription is a key feature of tissue spared following hypothermic focal ischemia.

Delayed transient ischemic attacks kill some CA1 neurons previously salvaged with postischemic hypothermia: neuroprotection undone

Delayed hypothermia reduces ischemic hippocampal CA1 injury. However, there are residual structural and functional abnormalities. Therefore, we studied whether these apparently vulnerable rescued neurons are susceptible to secondary insults. All gerbils were subjected to normothermic forebrain ischemia (ISC, 5 min) or SHAM operation. Gerbils were treated with mild hypothermia (HYPO; 33 °C for 24 h+35 °C for 24 h) beginning 12 h after surgery, or they remained normothermic (NORMO). Then 5 and 6 days following ISC/SHAM operation gerbils received sublethal transient ischemic attacks (TIA, 1.5 min) or sham (SH) surgeries. Behavioral testing was done and animals survived for 30 days for quantification of medial, middle and lateral CA1 sector cell death. The SHAM groups were not significantly different. The ISC+NORMO+SH group lost 87.3% (of SHAM) of medial CA1 neurons, which was not significantly exacerbated in the ISC+NORMO+TIA group (91.1%, P=0.633). However, the ISC+HYPO+TIA group (58.8% loss) had significantly more cell death than the ISC+HYPO+SH group (42.8%; P=0.035), although CA1 protection was still better than in ISC+NORMO groups ( P<0.001). Trends were similar in middle and lateral CA1, but the deleterious effects of TIAs were not statistically significant. Behavioral testing did not distinguish groups with or without TIA, but did reveal deficits in ISC+NORMO groups and protection in ISC+HYPO groups. These data, like previous ultrastructural findings, show that while most hypothermia-rescued CA1 neurons are healthy, some are susceptible. Perhaps other neuroprotectants, especially weaker ones, might be undone by delayed insults (e.g. TIA, fever).

Effect of mild hypothermia on energy state recovery following transient forebrain ischemia in the gerbil.

A transient (lasting for 15 min) bilateral carotid artery occlusion model was created by using male Mongolian gerbils ( n=20, weight 50-60 g). The animals were divided into a group with mild hypothermia (34 degrees C, n=10) and a normothermic group (37 degrees C, n=10). High-energy phosphate metabolism (ATP, PCr, Pi) and intracellular pH were sequentially measured using (31)P-MRS during ischemia and after reperfusion for 1 week. The same animals were also subjected to a histopathological evaluation. During ischemia, there were no statistically significant differences between the two groups in the quantities of the metabolites. However, after reperfusion the rate of metabolic recovery by the mildly hypothermic (MH) group was significantly higher (by 10-20%) than the normothermic (NT) group. The intracellular pH decreased about 0.4 in both groups after ischemia; and after reperfusion the intracellular pH of the MH group returned to baseline levels faster than in the NT group. One week after ischemia, energy metabolism gradually decreased about 10-20% in both groups. In the histopathological evaluation, pyramidal cell damage in the hippocampus was 33% on average in the MH group and 79% in the NT group. The neuronal damage to the cerebral cortex was 26% in the MH group and 61% in the NT group. Astrocyte reactivity in the hippocampus and cerebral cortex was 2.9% and 1.1% in the MH group and 9.7% and 5.2% in the NT group. The results of this experiment indicate that the protective effect of mild hypothermia is due to the high recovery rate of ATP and PCr and the prevention of a secondary decline in high phosphate energy.

Mild hypothermia mitigates post‐ischemic neuronal death following focal cerebral ischemia in rat brain: Immunohistochemical study of Fas, caspase‐3 and TUNEL

Mild hypothermia is considered to have a protective effect during ischemic neuronal cell death. The present study provides experimental evidence for this beneficial role of mild hypothermia using reversible middle cerebral artery occlusion (MCAo) in a Sprague–Dawley (SD) rat model. MCAo was induced in rats for 1 h followed by reperfusion at different periods. Hematoxylin–eosin (HE) staining in normothermic (NT) 37°C and hypothermic (HT) 33°C groups of rats confirmed cerebral infarcts. The mean per cent infarct area was significantly reduced in the HT group of rats. Immunohistochemical analysis was done using anti‐Fas and caspase‐3 antibodies. The immunohistochemical expression of Fas and caspase‐3 was demonstrable as early as 5 h after reperfusion, but the expression pattern maximized at 24 h after reperfusion. The expression of Fas and caspase‐3 proteins showed a clear decrease in the HT group over the NT group. In situ detection of DNA fragmentation was done using the terminal deoxy‐nucleotidyl transferase‐mediated dUTP‐biotin nick end‐ labeling method (TUNEL). TUNEL‐positive cells were first observed at 5 h after reperfusion and progressively increased by 24 h. A higher number of TUNEL‐positive cells was found in the NT group, but they were significantly decreased in the HT group. Further, DNA fragmentation was confirmed by size fractionation in agarose gel. These findings demonstrate a positive relation between the expression of Fas, caspase‐3 and TUNEL‐positive cells. Mild expression of Fas and caspase‐3 proteins and a reduced number of TUNEL‐positive cells in the HT group is clear evidence for the protective role of hypothermia in ischemia‐induced cell death.

Mild hypothermia mitigates post-ischemic neuronal death following focal cerebral ischemia in rat brain: immunohistochemical study of Fas, caspase-3 and TUNEL.

Mild hypothermia is considered to have a protective effect during ischemic neuronal cell death. The present study provides experimental evidence for this beneficial role of mild hypothermia using reversible middle cerebral artery occlusion (MCAo) in a Sprague-Dawley (SD) rat model. MCAo was induced in rats for 1 h followed by reperfusion at different periods. Hematoxylin-eosin (HE) staining in normothermic (NT) 37 degrees C and hypothermic (HT) 33 degrees C groups of rats confirmed cerebral infarcts. The mean per cent infarct area was significantly reduced in the HT group of rats. Immunohistochemical analysis was done using anti-Fas and caspase-3 antibodies. The immunohistochemical expression of Fas and caspase-3 was demonstrable as early as 5 h after reperfusion, but the expression pattern maximized at 24 h after reperfusion. The expression of Fas and caspase-3 proteins showed a clear decrease in the HT group over the NT group. In situ detection of DNA fragmentation was done using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method (TUNEL). TUNEL-positive cells were first observed at 5h after reperfusion and progressively increased by 24h. A higher number of TUNEL-positive cells was found in the NT group, but they were significantly decreased in the HT group. Further, DNA fragmentation was confirmed by size fractionation in agarose gel. These findings demonstrate a positive relation between the expression of Fas, caspase-3 and TUNEL-positive cells.

Immunohistochemical expression of Bcl-2, Bax and cytochrome c following focal cerebral ischemia and effect of hypothermia in rat

Recent studies suggest that mild hypothermia significantly alleviate damage following cerebral ischemia though the precise mechanism is poorly defined. In the present study, middle cerebral artery occlusion (MCAo) was induced in Sprague–Dawley (SD) rats for 1 h followed by varying periods of reperfusion. Cerebral infarcts identified by hematoxylin & eosin (H&E) staining revealed extensive lesion in normothermic (NT) 37°C and small lesion in hypothermic (HT) 33°C group of rats. Immunohistochemical analysis revealed Bcl-2 was induced in many neurons of HT group, while Bax and cytochrome c was induced in few neurons. In situ detection of DNA fragmentation using 3′-OH end labeling method (terminal dUTP nick-end labelling (TUNEL)) indicated, higher number of TUNEL-positive cells in NT group, but significantly decreased in HT group. The expression pattern revealed many neurons at the penumbra region could survive in HT group whereas, many neurons are committed to die in NT group. Our results suggest that hypothermia is selectively interfering at more than one place and providing protection.