Postischemic Hypothermia Research Articles

Hypothermia After Cardiac Arrest in Large Animals (HACA-LA): Study protocol of a randomized controlled experimental trial

BackgroundInduced hypothermia post-cardiac arrest is neuroprotective in animal experiments, but few high-quality studies have been performed in larger animals with human-like brains. The neuroprotective effect of postischemic hypothermia has recently been questioned in human trials. Our aim is to investigate whether hypothermia post-cardiac arrest confers a benefit compared to normothermia in large adult animals. Our hypothesis is that induced hypothermia post cardiac arrest is neuroprotective and that the effect diminishes when delayed two hours. MethodsAdult female pigs were anesthetized, mechanically ventilated and kept at baseline parameters including normothermia (38 °C). All animals were subjected to ten minutes of cardiac arrest (no-flow) by induced ventricular fibrillation, followed by four minutes of cardiopulmonary resuscitation with mechanical compressions, prior to the first countershock. Animals with sustained return of spontaneous circulation (systolic blood pressure >60 mmHg for ten minutes) within fifteen minutes from start of life support were included and randomized to three groups; immediate or delayed (2 h) intravenous cooling, both targeting 33 °C, or intravenously controlled normothermia (38 °C). Temperature control was applied for thirty hours including cooling time, temperature at target and controlled rewarming (0.5 °C/h). Animals were extubated and kept alive for seven days. The primary outcome measure is histological brain injury on day seven. Secondary outcomes include neurological and neurocognitive recovery, and the trajectory of biomarkers of brain injury. ConclusionHigh-quality animal experiments in clinically relevant large animal models are necessary to close the gap of knowledge regarding neuroprotective effects of induced hypothermia after cardiac arrest.Trial registration:Preclinicaltrials.eu (PCTE0000272), published 2021-11-03.

Hypothermia decreased the expression of heat shock proteins in neonatal rat model of hypoxic ischemic encephalopathy.

Hypothermia (HT) is a well-established neuroprotective strategy against neonatal hypoxic ischemic encephalopathy (HIE). The overexpression of heat shock proteins (HSP) has been shown to provide neuroprotection in animal models of stroke. We aimed to investigate the effect of HT on HSP70 and HSP27 expression in a neonatal rat model of HIE. Seven-day-old rat pups were exposed to hypoxia for 90min to establish the Rice-Vannucci model and were assigned to the following four groups: hypoxic injury (HI)-normothermia (NT, 36°C), HI-HT (30°C), sham-NT, and sham-HT. After temperature intervention for 24h, the mRNA and protein expression of HSP70 and HSP27 were measured. The association between HSP expression and brain injury severity was also evaluated. The brain infarct size was significantly smaller in the HI-HT group than in the HI-NT group. The mRNA and protein expression of both HSPs were significantly greater in the two HI groups, compared to those in the two sham groups. Moreover, among the rat pups subjected to HI, HT significantly reduced the mRNA and protein expression of both HSPs. The mRNA expression level of the HSPs was proportional to the brain injury severity. Post-ischemic HT, i.e., a cold shock attenuated the expression of HSP70 and HSP27 in a neonatal rat model of HIE. Our study suggests that neither HSP70 nor HSP27 expression is involved in the neuroprotective mechanism through which prolonged HT protects against neonatal HIE.

Abstract W P97: Post-ischemic Trans-arterial Regional Hypothermia Therapy Attenuates Microvascular Narrowing due to Astrocyte End-feet Swelling and Inhibits Subsequent Inflammatory Reactions in Ischemia Reperfusion Injury

Background and Purpose: We previously presented the robust neuroprotective effects of post-ischemic trans-arterial regional hypothermia therapy in I/R injury model rats (ISC 2014, San Diego). This treatment showed a significant suppression of infarct volume and inflammatory reaction at 24hrs after reperfusion. To characterize the neuroprotective effects in terms of microvascular integrity, we investigated microvascular morphological changes and inflammatory reactions by a time course analysis within 24hrs. Methods: At reperfusion onset, 2hr-MCAO rats were divided into cold saline group (10°C, 20ml/kg of trans-arterial infusion from the recanalized artery for 15mins), warm saline group (37°C, in the same manner as cold saline group), and control group (recanalization only). Microvascular morphological changes were examined by vWF Immunohistochemistry (IHC) and transmission electron microscopy (TEM), and inflammatory reactions were examined by Western blotting and IHC at the time point of 0hr, 2hrs, 6hrs, and 24hrs of reperfusion. Results: IHC analysis and TEM observation revealed that astrocyte end-feet swelling occurred at 2hrs and progressed to 6hrs, and microvascular narrowing due to the swelling occurred at 6hrs in control group. This microvascular morphological change was attenuated in cold saline group. While endothelial ICAM1 overexpression started at 6hrs in control group, MPO, MMP9, and Iba-1 overexpression occurred only at 24hrs in the same group. These inflammatory reactions were also inhibited in cold saline group. Conclusions: Post-ischemic trans-arterial regional hypothermia therapy attenuated astrocyte end-feet swelling from 2hrs and the swelling induced microvascular narrowing at 6hrs. The treatment also inhibited endothelial ICAM-1 overexpression at 6hrs, and subsequently inhibited inflammatory reactions at 24hrs. The maintenance of microvascular morphological and functional integrity may be the primary cause of the neuroprotective effects by the regional hypothermia.

Regional and time-dependent neuroprotective effect of hypothermia following oxygen-glucose deprivation.

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6- to 8-day-old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty-five-minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia.

AMP-Dependent Hypothermia Affords Protection from Ischemic Brain Injury

In light of the relevance of therapeutic hypothermia to stroke treatment, we investigated whether 5'-adenosine monophosphate (AMP)-dependent cooling affords protection from ischemic brain injury. We show that hypothermia by AMP is because of adenosine A1 receptor (A1R) activation and is not invariantly associated with hypotension. Inhibition of ecto-5'-nucleotidase-dependent constitutive degradation of brain extracellular AMP by methylene-ADP (AMPCP) also suffices to prompt A1R-dependent hypothermia without hypotension. Both intraischemic and postischemic hypothermia by AMP or AMPCP reduce infarct volumes and mortality of mice subjected to transient middle cerebral artery occlusion. Data disclose that AMP-dependent hypothermia is of therapeutic relevance to treatment of brain ischemia.

Post-ischemic hypothermia promotes generation of neural cells and reduces apoptosis by Bcl-2 in the striatum of neonatal rat brain

Hypothermia is a potential therapy for cerebral hypoxic ischemic injury in adults and neonates. However, the mechanism of hypothermia neuroprotection after hypoxic-ischemia (HI) on the developing rat brain remains unclear. In this research, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2 h. They were divided into hypothermia (rectal temperature, 32–33 °C for 24 h) and normothermia (36–37 °C for 24 h) groups immediately after hypoxia-ischemia. All rats were given 50 mg/kg/day 5-bromodeoxyuridine (BrdU) intraperitoneally at 4–6 days and sacrificed at 1 or 2 weeks after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The numbers of nestin-labeled cells did not change greatly, but β-tubulin III (Tuj-1) immuno-positive cells increased significantly in the striatum at 1 and 2 weeks after HI in the hypothermia compared to normothermia group. Neurogenesis was assessed by double immunohistochemical/immunofluorescent labeling of BrdU with nestin, Tuj-1 or microtubule-associated protein 2 (Map-2). Newborn neural progenitors (BrdU +-nestin +) did not change dramatically, but newborn immature (BrdU +-Tuj-1 +) and mature (BrdU +-Map-2 +) neurons increased significantly in the hypothermia compared with normothermia group. Meanwhile, the apoptosis rate of neural precursors, immature and mature neurons, assessed by double labeling of active Casp-3 with nestin/Tuj-1/Map-2, decreased noticeably in the hypothermia compared with normothermia group. We also found that hypothermia significantly increased expression of Bcl-2, which coexisted with nestin/Tuj-1/Map-2. Inhibition of Bcl-2 expression reversed the decreased apoptosis rate of neural precursors and neurons in hypothermia animal striatum of neonatal rat brain. These results suggest that neuroprotection effects of hypothermia on injured developing rat brain may associate with enhanced generation of neuronal cells and Bcl-2-mediated reduction of apoptosis of these cells. These observations are noteworthy regarding clinical hypothermia therapy following cerebral HI injury during the perinatal period.

Long-Term Neuroprotective Effect of Postischemic Hypothermia in a Neonatal Rat Model of Severe Hypoxic Ischemic Encephalopathy: A Comparative Study on the Duration and Depth of Hypothermia

It is not known whether deeper or longer hypothermia (HT) can achieve better neuroprotection against hypoxic ischemic encephalopathy (HIE) in neonates. To compare the neuroprotective effects of different durations and temperatures of postischemic HT in neonatal rats with severe HIE, 7-d-old rats were subjected to the Rice-Vannucci model for 150 min hypoxia. Only the rats with identified brain lesions in diffusion-weighted MRI were assigned to normothermia (NT, 36° C/48 h) or four HT (HT-30° C/48 h; HT-30° C/24 h; HT-33° C/48 h; and HT-33° C/24 h) groups. H-magnetic resonance spectroscopy (H-MRS) and T2-weighted MRI were obtained serially, and functional studies were performed. HT groups showed significantly greater residual hemispheric volume and better rotarod and cylinder tests than did the NT group at 5 wk postischemia. HT groups also showed lower lactate-plus-lipid level in H-MRS than did the NT group at 7 d postischemia. All of these outcome variables, however, did not differ among the 4 HT subgroups, despite a trend toward greater residual brain volume in the 48-h HT versus 24-h HT subgroups. In conclusion, neither reducing the target temperature from 33 to 30° C nor prolonging the duration from 24 to 48 h produced further improvements in neurologic outcomes in neonatal rat with HIE.

Mild Hypothermia Has No Long-Term Impact on Postischemic Neurogenesis in Rats

Postischemic improvement of functional outcome by therapeutic hypothermia may be related to cerebral regeneration by postischemic neurogenesis. We investigated whether mild peri-ischemic hypothermia leads to a long-term increase in postischemic neurogenesis. Seventy male sevoflurane-anesthetized Sprague Dawley rats were randomly assigned to the following treatment groups: normothermic ischemia, intraischemic hypothermia, and postischemic hypothermia with corresponding sham-operated controls. Fifteen naïve rats were investigated as reference for natural neurogenesis. Forebrain ischemia was induced by bilateral common carotid artery occlusion and hemorrhagic hypotension. In normothermic groups, the pericranial temperature was maintained at 37.5 degrees C. Animals in the hypothermic groups were cooled to a pericranial temperature of 33 degrees C for 45 min. All animals received 5-bromo-2-deoxyuridine for 7 days. Histopathological damage and 5-bromo-2-deoxyuridine-positive neurons of the hippocampus were analyzed after 28 days. Hypothermia had no impact on natural neurogenesis. Cerebral ischemia increased the number of new neurons regardless of pericranial temperature. Forty-five minutes of hypothermia beginning before ischemia diminished hippocampal injury to <10% in the CA1 and CA3 regions, whereas 45 min of postischemic hypothermia beginning after reperfusion did not reduce neuronal injury compared with normothermia. Neither intraischemic nor postischemic hypothermia affected the ischemia-induced increase in endogenous neurogenesis. Intraischemic hypothermia reduced hippocampal damage, whereas postischemic hypothermia as applied here did not prevent formation of histopathological injury. This indicates that, 28 days after cerebral ischemia, postischemic neurogenesis is not significantly increased by mild peri-ischemic hypothermia and not affected by the severity of histopathological damage.

Therapeutic Window of Selective Profound Cerebral Hypothermia for Resuscitation of Severe Cerebral Ischemia in Primates

It is well recognized that brain death starts to occur just 4-6 min after cardiac arrest, and few attempts at resuscitation succeed after 10 min of severe cerebral ischemia and anoxia. We sought to determine the therapeutic window of selective cerebral profound hypothermia of primates following severe cerebral ischemia in primates. Fourteen rhesus monkeys with severe cerebral ischemia were divided into four groups: normothermia (n = 3); profound hypothermia I (n = 4), with cooling initiated 10 min after ischemia; profound hypothermia II (n = 4), with cooling initiated 15 min after ischemia; and profound hypothermia III (n = 3), with cooling initiated 20 min after ischemia. Severe cerebral ischemia was induced by clamping both the internal and external carotid arteries, as well as the internal and external jugular veins. Profound cerebral hypothermia (15.8 degrees +/- 0.9 degrees C) was achieved and maintained for 60 min, and the animals were then re-warmed gradually. All four animals in hypothermia group I survived without any neurological deficits. Only 1 animal survived and 3 animals died in hypothermia group II. All 4 animals died in both hypothermia group III and the normothermia group. Neurological functions were normal in all surviving animals, and MRI scans showed no cerebral infarction in these animals. Microscopic examination showed no injured neurons in the hippocampus and cerebral cortex of the surviving animals, and showed that the heart, lung, liver, and kidneys were normal in these animals. Our data indicate that post-ischemic profound cerebral hypothermia provided significant cerebral protection with no systemic complications, and that the effective therapeutic window is more than 10 min, but less than 15 min, after severe cerebral ischemia.

Importance of normothermia control in investigating delayed neuronal injury in a mouse global ischemia model

This study aims to establish a mouse global cerebral ischemia model in which the physiological parameter measurements and neuronal injury evaluations are conducted in the same group of animals and to identify the effect of post-ischemic core temperature (CT) on the outcome of neuronal injury. Global ischemia was induced by 12-min bilateral common carotid artery occlusion followed by 7 days of reperfusion in C57BL/6 mice. Immediately after occlusion, mice were randomly assigned to be kept in environments of different temperatures [25 °C (room temperature, group 1), 33–34 °C for 2 h (group 2), and 33–34 °C for 24 h (group 3)] before being returned to their home cages. We found that in group 1, CT declined to ∼32 °C after ischemia and then recovered at 24 h post-ischemia; in group 2, CT remained at the pre-ischemia level during the first 2 h, declined after the mice were returned to room temperature, and recovered at 24 h post-ischemia; and in group 3, CT remained constant at the pre-ischemia level throughout the reperfusion period. The number of surviving neurons in a sector of the hippocampal CA1 region was significantly lower in all ischemic groups than in the sham controls, but the number was significantly higher in group 1 than that in groups 2 or 3 ( P < 0.05). We observed that CT declines initially but recovers spontaneously at 24 h post-ischemia. Early post-ischemic hypothermia impacts the delayed neuronal injury, suggesting that tight temperature control immediately following ischemia is important to obtain the most reproducible neuronal damage in mouse models of cerebral global ischemia.

Hypothermia enhances heat-shock protein 70 production in ischemic brains

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Post-ischemic hypothermia for 24 h in P7 rats rescues hippocampal neuron: Association with decreased astrocyte activation and inflammatory cytokine expression

Hypothermia is an effective method for reducing the neuronal damage induced by hypoxia–ischemia (HI) but the underlying mechanism remains unclear. To investigate the effects of post-HI hypothermia on the developing brain, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2 h. They were divided into a hypothermia group (rectal temperature 32–33 °C for 24 h) and a normothermia group (36–37 °C for 24 h) immediately after hypoxia–ischemia. Animals were sacrificed at 12, 24 and 72 h for gene analysis and 0, 1, 3 and 7 days for protein analysis after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The hypothermia group had more neuronal nuclei (NeuN) positive neurons and lower levels of glial fibrillary acidic protein (GFAP) mRNA and immunoreactivity in the hippocampus CA1 region than the normothermia group. Real-time PCR showed no significant difference in glial cell line-derived neurotrophic factor (GDNF) mRNA expression in the hippocampus in the two groups at various time points after HI. However, GDNF protein level was significantly increased in the hypothermia group. On the other hand, mRNA and protein levels of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were dramatically decreased in the hypothermia compared with the normothermia group. The present findings highlight an apparent association between inhibition of hippocampal neuron loss by hypothermia and decreased astrocytosis and inflammatory cytokine release after hypoxia–ischemia in the developing brain.

Initiation time of post-ischemic hypothermia on the therapeutic effect in cerebral ischemic injury

Objective: To study the efficacy of mild brain hypothermia beginning at different time intervals on cerebral ischemic injury.Methods: Male Sprague–Dawley rats were divided into sham-operated group, normothermia (37–38°C) and mild hypothermia (31–32°C) ischemia groups. The last group was subdivided into four groups: 240 minute hypothermia, 30 minute normothermia plus 210 minute hypothermia, 60 minute normothermia plus 180 minute hypothermia, and 90 minute normothermia plus 150 minute hypothermia (n=8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model for 20 minutes and mild hypothermia was applied after 20 minutes of ischemia. Brain tissue was collected following 20 minute cerebral ischemia and 240 minute reperfusion, and used to measure the levels of malondialdehyde (MDA), lactate, water content and the amounts of electrolytes, such as sodium (Na+), potassium (K+) and calcium (Ca++).Results: Mild hypothermia beginning at 0–60 minutes decreased the levels of malondialdehyde and lactate (p<0.05 or p<0.01), decreased water content, Na+ and Ca++, and increased the amount of K+ (p<0.05 or p<0.01) in ischemic tissue, except the amounts of Na+, K+ and Ca++ in mild hypothermia beginning at 60 minute ischemia group (p>0.05). Mild hypothermia beginning at 90 minutes had little effect on the levels of targeted molecules, water content and amounts of electrolytes of Na+, K+ and Ca++ in ischemic tissue (p>0.05).Discussion: Post-ischemic mild brain hypothermia can decrease the accumulation of lactate and lipid peroxidation in ischemic tissue, and delay the development of brain edema following ischemic reperfusion. The best neuroprotection of mild hypothermia to attenuate ischemic injury was begun within 60 minutes.

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood–brain barrier disruption after global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague–Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42–45 mm Hg). In a separate group of animals, a mild (32 °C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from ∼ 500 brain vessels (20–100 μm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using 3H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p < 0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for 3H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.

Treatment of the Term Newborn With Brain Injury: Simplicity As the Mother of Invention

Neonatal brain injury remains a common cause of developmental disability, despite tremendously enhanced obstetrical and neonatal care. The timing of brain injury occurs throughout gestation, labor, and delivery, providing an evolving form of brain injury and a moving target for therapeutic intervention. Nonetheless, markedly improved methods are available to identify those infants injured at birth, via clinical presentation with neonatal encephalopathy and neuroimaging techniques. Postischemic hypothermia has been shown to be of tremendous clinical promise in several completed and ongoing trials. As part of this approach to the treatment of the newborn, other parameters of physiologic homeostasis can and should be attended to, with strong animal and clinical evidence that their correction will have dramatic influence on the outcome of the newborn infant. This review addresses aspects of newborn care to which we can direct our attention currently, and which should result in a safe and efficacious improvement in the prognosis of the newborn with neonatal encephalopathy.

Postischemic mild hypothermia alleviates hearing loss because of transient ischemia

The effect of postischemic mild hypothermia on the inner ear has not been clarified. In this study, we investigated whether hypothermia after transient ischemia could prevent cochlear damage and its therapeutic time window. Mongolian gerbils were divided into six groups: a sham-operation group, a normothermia group, and four hypothermia groups in which hypothermia was induced 1-7, 1-4, 3-6, and 6-9 h after reperfusion. Animals subjected to postischemic mild hypothermia within 3 h after reperfusion had attenuated hearing loss and inner hair cell loss. The protective effect was greater when hypothermia was induced earlier and had a longer duration. This implies that mild hypothermia after ischemia could have therapeutic effects for inner ear ischemic damage.

Therapeutic effect of post-ischemic hypothermia duration on cerebral ischemic injury

Objective: To study the efficacy of post-ischemic mild brain hypothermia lasting for different time intervals on cerebral ischemic reperfusion injury.Method: Male Sprague–Dawley rats were divided into a sham-operated group, normothermia (37–38° C) ischemia group and mild hypothermia (31–32° C) group. The last group was subdivided into four groups: 30 minute hypothermia plus 210 minute normothermia, 60 minute hypothermia plus 180 minute nomothermia,120 minute hypothermia plus 120 minute normothermia, and 240 minute hypothermia (n=8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model. Brain tissue was collected following a 20 minute cerebral ischemia and 240 minute reperfusion, and was used to measure the levels of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin(5-HT) and hydroxyindoleacetic acid (5-HIAA), nitrite (NO2), endothelin-1 (ET1), tumor necrosis factor alpha(TNFα) and interleukin-1beta (IL-1β). Serum was collected to measure the levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK) and its brain band isoenzyme (CK-BB).Results: Hypothermia lasting for 60–240 minutes delayed the decrease in these amino acids, postponed the decrease in DA, NE and 5-HT and increase in hydroxyindoleacetic acid (5-HIAA), and decreased the levels of IL-1β, TNFα, ET1 and NO2 in brain tissue. Hypothermia also decreased the levels of LDH, AST, CK and CK-BB in serum as compared to normothermia group (p<0.05 or p<0.01). Hypothermia lasting for 30 minutes delayed the decreases in these amino acids and 5-HT and increase in 5-HIAA in brain tissue (p<0.05), but failed to influence the levels of IL-1β, TNFα, ET1 and NO2 in brain tissue and the amounts of LDH, AST, CK and CK-BB in serum as compared to normothermia ischemia group (p>0.05).Conclusions: Post-ischemic mild brain hypothermia can significantly suppress the excessive release of amino acids, monoamine neurotransmitters and inflammation response in ischemic tissue. It can also stabilize the function of the cell membrane, which is associated with the mechanism of cerebral protection by mild hypothermia. These results suggest that mild hypothermia should be applied immediately after ischemia and last for more than 60 minutes in order to obtain neuroprotective effects.

General versus Specific Actions of Mild-Moderate Hypothermia in Attenuating Cerebral Ischemic Damage

Mild or moderate hypothermia is generally thought to block all changes in signaling events that are detrimental to ischemic brain, including ATP depletion, glutamate release, Ca(2+) mobilization, anoxic depolarization, free radical generation, inflammation, blood-brain barrier permeability, necrotic, and apoptotic pathways. However, the effects and mechanisms of hypothermia are, in fact, variable. We emphasize that, even in the laboratory, hypothermic protection is limited. In certain models of permanent focal ischemia, hypothermia may not protect at all. In cases where hypothermia reduces infarct, some studies have overemphasized its ability to maintain cerebral blood flow and ATP levels, and to prevent anoxic depolarization, glutamate release during ischemia. Instead, hypothermia may protect against ischemia by regulating cascades that occur after reperfusion, including blood-brain barrier permeability and the changes in gene and protein expressions associated with necrotic and apoptotic pathways. Hypothermia not only blocks multiple damaging cascades after stroke, but also selectively upregulates some protective genes. However, most of these mechanisms are addressed in models with intraischemic hypothermia; much less information is available in models with postischemic hypothermia. Moreover, although it has been confirmed that mild hypothermia is clinically feasible for acute focal stroke treatment, no definite beneficial effect has been reported yet. This lack of clinical protection may result from suboptimal criteria for patient entrance into clinical trials. To facilitate clinical translation, future efforts in the laboratory should focus more on the protective mechanisms of postischemic hypothermia, as well as on the effects of sex, age and rewarming during reperfusion on hypothermic protection.

Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia

Hypothermia is the only neuroprotective therapy proven to be clinically effective. Identifying the molecules that play important roles in the efficacy of hypothermia, we developed a multi-channel computer-controlled system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 °C, and examined the time window necessary to exert its significant neuroprotective effects. Eight-week-old SD rats were subjected to a 2 h middle cerebral artery occlusion (MCAO) with an intraluminal filament, and post-ischemic hypothermia was introduced at 0, 2, 4, or 6 h after reperfusion until the rats were killed 2 days after MCAO. Since a significant protection was observed when hypothermia was started within 4 h after reperfusion, it was concluded that the therapeutic time window of mild hypothermia lasts for 4 h after reperfusion in our model. On the basis of the window, comprehensive gene expression analyses using oligonucleotide microarrays were conducted and identified potential genes related to the efficacy of hypothermia, which included inflammatory genes like osteopontin, early growth response-1, or macrophage inflammatory protein-3α. Therefore, the neuroprotective effects of post-ischemic mild hypothermia were strongly suggested to be mainly associated with the reduction of neuronal inflammation.

Post-ischemic hypothermia reduced IL-18 expression and suppressed microglial activation in the immature brain

Inflammation is an important factor for hypoxia–ischemia (HI) brain injury. Interleukin (IL)-18 is a proinflammatory cytokine which may be a contributor to injury in the immature brain after HI. To investigate the effects of post-HI hypothermia on IL-18 in the developing brain, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 60 min and divided into a hypothermia group (rectal temperature 32 °C for 24 h) and a normothermia group (36 °C for 24 h). The IL-18 mRNA was analyzed with real-time RT-PCR, and the protein level was analyzed by Western blot, and the location and source of IL-18 were assessed by immunohistochemistry. The significant increase of the IL-18 mRNA was observed in the ipsilateral hemispheres of the normothermia group at 24 h and 72 h after HI compared with controls, but the level in the ipsilateral hemispheres of the hypothermia group was significantly reduced at both time points, compared with the normothermia group, respectively. The IL-18 protein level in the ipsilateral hemispheres of the normothermia group significantly increased at 72 h after HI compared with controls, however, the protein level of the hypothermia group was significantly decreased, compared with the normothermia group. IL-18-positive cells were observed throughout the entire cortex, corpus callosum (CC) and striatum in the ipsilateral hemispheres of normothermia group at 72 h after HI, however, little positive cells were observed in the hypothermia group. Double labeling immunostaining found that most of the IL-18-positive cells were colocalized with lectin, which is a marker of microglia. The number of ameboid microglia (AM) in the normothermia group was significantly increased in cortex and CC, compared with the number in controls, but there were very few ramified microglia (RM) in these areas. In contrast, the number of AM in the hypothermia group was significantly decreased in cortex and CC, compared with the number in the normothermia group, and there were no significant differences in the number of AM and RM between the hypothermia group and controls. In conclusion, we found that IL-18 mRNA and the protein level were attenuated by post-HI hypothermia and that post-HI hypothermia may decrease microglia activation in the developing brain.