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1.  The protective effect of early hypothermia on PTEN phosphorylation correlates with free radical inhibition in rat stroke 
We recently showed that intraischemic moderate hypothermia (30°C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and δ-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (3 h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (3 h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-SOD (superoxide dismutase) and phosphorylated Akt and blocked δ-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.
PMCID: PMC3221613  PMID: 19553907
focal ischemia; hypothermia; neuroprotection; stroke
2.  When hypothermia meets hypotension and hyperglycemia: the diverse effects of adenosine 5′-monophosphate on cerebral ischemia in rats 
Mild hypothermia renders potent neuroprotection against acute brain injury. Recent reports show that adenosine 5′-monophosphate (AMP) plays a role in thermoregulation and induces hypothermia in mice. Therefore, this study sought to determine whether AMP induces hypothermia in rats and to study its collective effects on cerebral ischemia induced by 2-h middle cerebral artery occlusion. An intraperitoneal injection of AMP induced hypothermia dose-dependently. At the dose of 4 mmol/kg, AMP induced promising mild hypothermia for 2.5 h. Unexpectedly, the AMP-induced hypothermia failed to reduce infarct volume after brain ischemia; instead, it exaggerated the ischemic damage, indicated by an increased infarct volume, as well as increased incidences of hemorrhagic transformation, seizure, and animal death. Physiologic parameter monitoring revealed that AMP causes profound hypotension, leading to cerebral hypoperfusion. Furthermore, AMP administration resulted in severe hyperglycemia, metabolic acidosis, and hypocalcemia. In addition, western blots showed early dephosphorylation and degradation of AMP-activated kinase in the ischemic cortex in AMP-treated rats. Taken together, our findings suggest that AMP induces hypothermia in rats, probably by limiting cellular access to glucose. However, the potential neuroprotection of AMP-mediated hypothermia against ischemia was overwhelmed by the detrimental effects of hypotension and hyperglycemia, thus making AMP an unlikely agent for inducing hypothermia to protect the brain against ischemic injury.
PMCID: PMC2726621  PMID: 19319149
acidosis; AMPK; Compound C; hibernation; hypocalcemia; insulin
3.  Limitations of Mild, Moderate, and Profound Hypothermia in Protecting Developing Hippocampal Neurons After Simulated Ischemia 
Mild hypothermia (33°C–34°C) after cerebral ischemia in intact animals or ischemia-like conditions in vitro reduces neuron death. However, it is now clear that more profound hypothermia or delayed hypothermia may not provide significant protection. To further define the limitations of hypothermia after cerebral ischemia, we used hippocampal slice cultures to examine the effects of various degrees, durations, and delays of hypothermia on neuron death after an ischemia-like insult. Organotypic cultures of the hippocampus from 7- to 8 day-old rat pups were cooled to 32°C, 23°C, 17°C, or 4°C immediately or after a 2–4 hour delay from an injurious insult of oxygen and glucose deprivation (OGD). Cell death in CA1, CA3 and dentate regions of the cultures was assessed 24 hours later with SYTOX® or propidium iodide, both of which are fluorescent markers labeling damaged cells. OGD caused extensive cell death in CA1, CA3, and dentate regions of the hippocampal cultures. Hypothermia (32°C, 23°C and 17°C) for 4–6 hours immediately after OGD was protective at 24 hours, but when hypothermia was applied for longer periods or delayed after OGD, no protection or increased death was seen. Ultra-profound hypothermia (4°C) increased cell death in all cell areas of the hippocampus even when after a milder insult of only hypoxia. In an in vitro model of recovery after an ischemia-like insult, mild to profound hypothermia is protective only when applied without delay and for limited periods of time (6–8 hours). Longer durations of hypothermia, or delayed application of the hypothermia can increase neuron death. These findings may have implications for clinical uses of therapeutic hypothermia after hypoxic or ischemic insults, and suggest that further work is needed to elucidate the limitations of hypothermia as a protective treatment after ischemic stress.
PMCID: PMC3868300  PMID: 24380031
4.  Moderate Hypothermia Inhibits Brain Inflammation and Attenuates Stroke-induced Immunodepression in Rats 
CNS neuroscience & therapeutics  2013;20(1):10.1111/cns.12160.
Stroke causes both brain inflammation and immunodepression. Mild to moderate hypothermia is known to attenuate brain inflammation but its role in stroke-induced immunodepression (SIID) of the peripheral immune system remains unknown. This study investigated the effects in rats of moderate intra-ischemic hypothermia on SIID and brain inflammation.
Stroke was induced in rats by permanent distal MCA occlusion combined with transient bilateral CCA occlusion while body temperature was reduced to 30°C. Real-time PCR, flow cytometry, in vitro T cell proliferation assays and confocal microscopy were used to study SIID and brain inflammation.
Brief Intra-Ischemic hypothermia helped maintain certain leukocytes in the peripheral blood and spleen, and enhanced T cell proliferation in vitro and delayed-type hypersensitivity in vivo, suggesting that hypothermia reduces SIID. In contrast, in the brain, brief intra-Ischemic hypothermia inhibited mRNA expression of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines INF-γ, TNF-α, IL-2, IL-1β and MIP-2. Brief intra-Ischemic hypothermia also attenuated the infiltration of lymphocytes, neutrophils (MPO+ cells) and macrophages (CD68+ cells) into the ischemic brain, suggesting that hypothermia inhibited brain inflammation.
Brief intra-ischemic hypothermia attenuated SIID and protected against acute brain inflammation.
PMCID: PMC3867545  PMID: 23981596
focal cerebral ischemia; hypothermia; inflammation; immunodepression; leukocytes
5.  Time-dependent effects of hypothermia on microglial activation and migration 
Therapeutic hypothermia is one of the neuroprotective strategies that improve neurological outcomes after brain damage in ischemic stroke and traumatic brain injury. Microglial cells become activated following brain injury and play an important role in neuroinflammation and subsequent brain damage. The aim of this study was to determine the time-dependent effects of hypothermia on microglial cell activation and migration, which are accompanied by neuroinflammation.
Microglial cells in culture were subjected to mild (33 °C) or moderate (29 °C) hypothermic conditions before, during, or after lipopolysaccharide (LPS) or hypoxic stimulation, and the production of nitric oxide (NO), proinflammatory cytokines, reactive oxygen species, and neurotoxicity was evaluated. Effects of hypothermia on microglial migration were also determined in in vitro as well as in vivo settings.
Early-, co-, and delayed-hypothermic treatments inhibited microglial production of inflammatory mediators to varying degrees: early treatment was the most efficient, and delayed treatment showed time-dependent effects. Delayed hypothermia also suppressed the mRNA levels of proinflammatory cytokines and iNOS, and attenuated microglial neurotoxicity in microglia-neuron co-cultures. Furthermore, delayed hypothermia reduced microglial migration in the Boyden chamber assay and wound healing assay. In a stab injury model, delayed local hypothermia reduced migration of microglia toward the injury site in the rat brain.
Taken together, our results indicate that delayed hypothermia is sufficient to attenuate microglial activation and migration, and provide the basis of determining the optimal time window for therapeutic hypothermia. Delayed hypothermia may be neuroprotective by inhibiting microglia-mediated neuroinflammation, indicating the therapeutic potential of post-injury hypothermia for patients with brain damages exhibiting some of the inflammatory components.
PMCID: PMC3470995  PMID: 22776061
Hypothermia; Microglia; Cell migration; Neuroinflammation; Neuroprotection
6.  FasL shedding is reduced by hypothermia in experimental stroke 
Journal of neurochemistry  2008;106(2):541-550.
Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor mediated pathways, such as that triggered by Fas/FasL. Male Sprague Dawley rats subjected to 2h middle cerebral artery occlusion with 2h intraischemic mild hypothermia (33C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by ~50–60% at 6 and 24h post insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by ~50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.
PMCID: PMC2735469  PMID: 18410517
apoptosis; hypothermia; cerebral ischemia; matrix metalloproteinases; Fas/FasL; stroke
7.  ATP induces mild hypothermia in rats but has a strikingly detrimental impact on focal cerebral ischemia 
Ischemic stroke is a devastating condition lacking effective therapies. A promising approach to attenuate ischemic injury is mild hypothermia. Recent studies show that adenosine nucleotides can induce hypothermia in mice. The purpose of the present study was to test the hypothesis that adenosine 5′-triphosphate (ATP) induces mild hypothermia in rats and reduces ischemic brain injury. We found that intraperitoneal injections of ATP decreased core body temperature in a dose-dependent manner; the dose appropriate for mild hypothermia was 2 g/kg. When ATP-induced hypothermia was applied to stroke induced by middle cerebral artery occlusion, however, a neuroprotective effect was not observed. Instead, the infarct volume grew even larger in ATP-treated rats. This was accompanied by an increased rate of seizure events, hemorrhagic transformation, and higher mortality. Continuous monitoring of physiologic parameters revealed that ATP reduced heartbeat rate and blood pressure. ATP also increased blood glucose, accompanied by severe acidosis and hypocalcemia. Western blotting showed that ATP decreased levels of both phospho-Akt and total-Akt in the cortex. Our results reveal that, despite inducing hypothermia, ATP is not appropriate for protecting the brain against stroke. Instead, we show for the first time that ATP treatment is associated with exaggerated ischemic outcomes and dangerous systemic side effects.
PMCID: PMC3597371  PMID: 23072747
acidosis; ATP; brain ischemia; hyperglycemia; hypothermia
8.  Mild hypothermia alone or in combination with anesthetic post-conditioning reduces expression of inflammatory cytokines in the cerebral cortex of pigs after cardiopulmonary resuscitation 
Critical Care  2010;14(1):R21.
Hypothermia improves survival and neurological recovery after cardiac arrest. Pro-inflammatory cytokines have been implicated in focal cerebral ischemia/reperfusion injury. It is unknown whether cardiac arrest also triggers the release of cerebral inflammatory molecules, and whether therapeutic hypothermia alters this inflammatory response. This study sought to examine whether hypothermia or the combination of hypothermia with anesthetic post-conditioning with sevoflurane affect cerebral inflammatory response after cardiopulmonary resuscitation.
Thirty pigs (28 to 34 kg) were subjected to cardiac arrest following temporary coronary artery occlusion. After seven minutes of ventricular fibrillation and two minutes of basic life support, advanced cardiac life support was started according to the current American Heart Association guidelines. Return of spontaneous circulation was achieved in 21 animals who were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group: n = 7) for 24 hours. The effects of hypothermia and the combination of hypothermia with sevoflurane on cerebral inflammatory response after cardiopulmonary resuscitation were studied using tissue samples from the cerebral cortex of pigs euthanized after 24 hours and employing quantitative RT-PCR and ELISA techniques.
Global cerebral ischemia following resuscitation resulted in significant upregulation of cerebral tissue inflammatory cytokine mRNA expression (mean ± SD; interleukin (IL)-1β 8.7 ± 4.0, IL-6 4.3 ± 2.6, IL-10 2.5 ± 1.6, tumor necrosis factor (TNF)α 2.8 ± 1.8, intercellular adhesion molecule-1 (ICAM-1) 4.0 ± 1.9-fold compared with sham control) and IL-1β protein concentration (1.9 ± 0.6-fold compared with sham control). Hypothermia was associated with a significant (P < 0.05 versus normothermia) reduction in cerebral inflammatory cytokine mRNA expression (IL-1β 1.7 ± 1.0, IL-6 2.2 ± 1.1, IL-10 0.8 ± 0.4, TNFα 1.1 ± 0.6, ICAM-1 1.9 ± 0.7-fold compared with sham control). These results were also confirmed for IL-1β on protein level. Experimental settings employing hypothermia in combination with sevoflurane showed that the volatile anesthetic did not confer additional anti-inflammatory effects compared with hypothermia alone.
Mild therapeutic hypothermia resulted in decreased expression of typical cerebral inflammatory mediators after cardiopulmonary resuscitation. This may confer, at least in part, neuroprotection following global cerebral ischemia and resuscitation.
PMCID: PMC2875536  PMID: 20158893
9.  Hypothermia and Postconditioning after Cardiopulmonary Resuscitation Reduce Cardiac Dysfunction by Modulating Inflammation, Apoptosis and Remodeling 
PLoS ONE  2009;4(10):e7588.
Mild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia.
Methodology/Principal Findings
Thirty pigs (28–34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34±7 versus 57±12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1β and IL-6 mRNA up-regulation, and (iv) IL-1β protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached.
Hypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression.
PMCID: PMC2764338  PMID: 19855846
10.  Mild hypothermia of 34°C reduces side effects of rt-PA treatment after thromboembolic stroke in rats 
Hypothermia is neuroprotective in experimental stroke and may extend the so far limited therapeutic time window for thrombolysis. Therefore, hypothermia of 34°C and its effects on delayed thrombolysis including reperfusion-associated injury were investigated in a model of thromboembolic stroke (TE).
Male Wistar rats (n = 48) were subjected to TE. The following treatment groups were investigated: control group - normothermia (37°C); thrombolysis group - rt-PA 90 min after TE; hypothermia by 34°C applied 1.5 to 5 hours after TE; combination therapy- hypothermia and rt-PA. After 24 hours infarct size, brain edema and neuroscore were assessed. Protein markers for inflammation and adhesion, gelatinase activity, and blood brain barrier (BBB) disruption were determined. MRI-measurements investigated infarct evolution and blood flow parameters.
The infarct volume and brain swelling were smaller in the hypothermia group compared to the other groups (p < 0.05 to p < 0.01). Thrombolysis resulted in larger infarct and brain swelling than all others. Hypothermia in combination with thrombolysis reduced these parameters compared to thrombolysis (p < 0.05). Moreover, the neuroscore improved in the hypothermia group compared to control and thrombolysis. Animals of the combination therapy performed better than after thrombolysis alone (p < 0.05). Lower serum concentration of sICAM-1, and TIMP-1 were shown for hypothermia and combination therapy. Gelatinase activity was decreased by hypothermia in both groups.
Therapeutic hypothermia reduced side-effects of rt-PA associated treatment and reperfusion in our model of TE.
PMCID: PMC3320523  PMID: 22397464
focal ischemia; stroke; thrombolysis; hypothermia; reperfusion; MRI; thromboembolic model; rat
11.  Combination Therapy with Hypothermia for Treatment of Cerebral Ischemia 
Journal of neurotrauma  2009;26(3):325-331.
Mild hypothermia is an established neuroprotectant in the laboratory, showing remarkable and consistent effects across multiple laboratories and models of brain injury. At the clinical level, mild hypothermia has shown benefits in patients who have suffered cardiac arrest and in some pediatric populations suffering hypoxic brain insults. However, a review of the literature has demonstrated that in order to appreciate the maximum benefits of hypothermia, brain cooling needs to begin soon after the insult, maintained for relatively long period periods of time, and, in the case of ischemic stroke, should be applied in conjunction with the re-establishment of cerebral perfusion. Translating this to the clinical arena can be challenging, especially rapid cooling and the reestablishment of perfusion. The addition of a second neuroprotectant could potentially (1) enhance overall protection, (2) prolong the temporal therapeutic window for hypothermia, or (3) provide protection where hypothermic treatment is only transient. Combination therapies resulting in recanalization following ischemic stroke would improve the likelihood of a good outcome, as the experimental literature suggests more consistent neuroprotection against ischemia with reperfusion, than ischemia without. Since recombinant tissue plasiminogen activator (rt-PA) is the only FDA approved treatment for acute ischemic stroke, and acts to recanalize occluded vessels, it is an obvious initial strategy to combine with hypothermia. However, the effects of thrombolytics are also temperature dependent, and the risk of hemorrhage is significant. The experimental data nevertheless seem to favor a combinatorial approach. Thus, in order to apply hypothermia to a broader range of patients, combination strategies should be further investigated.
PMCID: PMC2752358  PMID: 19216635
hypothermia; neuroprotection; stroke; tissue plasminogen activator
12.  Combination Therapy with Hypothermia for Treatment of Cerebral Ischemia 
Journal of Neurotrauma  2009;26(3):325-331.
Mild hypothermia is an established neuroprotectant in the laboratory, showing remarkable and consistent effects across multiple laboratories and models of brain injury. At the clinical level, mild hypothermia has shown benefits in patients who have suffered cardiac arrest and in some pediatric populations suffering hypoxic brain insults. However, a review of the literature has demonstrated that in order to appreciate the maximum benefits of hypothermia, brain cooling needs to begin soon after the insult, maintained for relatively long period periods of time, and, in the case of ischemic stroke, should be applied in conjunction with the re-establishment of cerebral perfusion. Translating this to the clinical arena can be challenging, especially rapid cooling and the re-establishment of perfusion. The addition of a second neuroprotectant could potentially (1) enhance overall protection, (2) prolong the temporal therapeutic window for hypothermia, or (3) provide protection where hypothermic treatment is only transient. Combination therapies resulting in recanalization following ischemic stroke would improve the likelihood of a good outcome, as the experimental literature suggests more consistent neuroprotection against ischemia with reperfusion, than ischemia without. Since recombinant tissue plasiminogen activator (rt-PA) is the only FDA approved treatment for acute ischemic stroke, and acts to recanalize occluded vessels, it is an obvious initial strategy to combine with hypothermia. However, the effects of thrombolytics are also temperature dependent, and the risk of hemorrhage is significant. The experimental data nevertheless seem to favor a combinatorial approach. Thus, in order to apply hypothermia to a broader range of patients, combination strategies should be further investigated.
PMCID: PMC2752358  PMID: 19216635
hypothermia; neuroprotection; stroke; tissue plasminogen activator
13.  Acute and Delayed Protective Effects of Pharmacologically Induced Hypothermia in an Intracerebral Hemorrhage Stroke Model of Mice 
Neuroscience  2013;252:489-500.
Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2 mg/kg bolus injection followed by 2 injections of 1 mg/kg, i.p.) were initiated at 1 or 24 hrs after ICH. HPI-201 induced mild hypothermia within 30 min and maintained body and brain temperatures at 32.7±0.4°C for at least 6 hrs without causing observable shivering. With the 1 hr delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24 hrs after the onset of ICH, it still significantly attenuated brain edema, cell death and blood brain barrier breakdown. HPI-201 significantly decreased the expression of MMP-9, reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-hr delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 hrs after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.
PMCID: PMC3961766  PMID: 23912033
Intracerebral hemorrhage; pharmacological hypothermia; PIH; neurotensin receptor; ABS-201; HPI-201
14.  Post-Stroke Hypothermia Provides Neuroprotection through Inhibition of AMP-Activated Protein Kinase 
Journal of Neurotrauma  2011;28(7):1281-1288.
Hypothermia is robustly protective in pre-clinical models of both global and focal ischemia, as well as in patients after cardiac arrest. Although the mechanism for hypothermic neuroprotection remains unknown, reducing metabolic drive may play a role. Capitalizing on the beneficial effects of hypothermia while avoiding detrimental effects such as infection will be the key to moving this therapy forward as a treatment for stroke. AMPK is a master energy sensor that monitors levels of key energy metabolites. AMPK is activated via phosphorylation (pAMPK) when cellular energy levels are low, such as that seen during ischemia. AMPK activation appears to be detrimental in experimental stroke, likely via exacerbating ischemia-induced metabolic failure. We tested the hypothesis that hypothermia reduces AMPK activation. First, it was found that hypothermia reduced infarct after middle cerebral artery occlusion. Second, induced hypothermia reduced brain pAMPK in both sham control and stroke mice. Third, hypothermic neuroprotection was ameliorated after administration of compound C, an AMPK inhibitor. Finally, deletion of one of the catalytic isoforms of AMPK completely reversed the effect of hypothermia on stroke outcome after both acute and chronic survival. These effects were mediated by a reduction in AMPK activation rather than a reduction in LKB1, an upstream AMPK kinase. In summary, these studies provide evidence that hypothermia exerts its protective effect in part by inhibiting AMPK activation in experimental focal stroke. This suggests that AMPK represents a potentially important biological target for stroke treatment.
PMCID: PMC3136745  PMID: 21446786
AMPK; hypothermia; middle cerebral artery occlusion; stroke
15.  Hypothermia and operative mortality during on-pump coronary artery bypass grafting 
Controversy surrounds the effect of hypothermia on operative mortality during cardiac surgery. The present study accessed a large clinical database of coronary artery bypass graft operations to address the issue.
A retrospective review of the Society of Thoracic Surgeons Adult Cardiac Surgery Database identified patients treated with isolated, nonemergency, on-pump coronary artery bypass grafting from July 2011 to December 2012. The patients were divided into 3 groups according to their lowest core temperature during the procedure: moderate hypothermia (≤34°C), mild hypothermia (>34°C but ≤36°C), and normothermia (>36°C). The primary endpoint of the study was operative mortality, defined according to the Database criteria.
During the study period, 142,541 patients were available for analysis; 94,777 (66.5%) received moderate hypothermia, 42,750 (30.3%) mild hypothermia, and 5014 (3.5%) normothermia. Operative mortality occurred in 1394 patients (1.5%) in the moderate hypothermia, 534 (1.3%) in the mild hypothermia, and 105 (2.1%) in the normothermia group. Multivariate analysis identified hypothermia (both mild [odds ratio, 0.66; 95% confidence interval, 0.54–0.81; P<.0001] and moderate [odds ratio, 0.73; 95% confidence interval, 0.60–0.89; P =.0015]) was protective against operative mortality compared with normothermia. No incremental benefit was noted between the different hypothermia grades (P = .0827).
Most patients receive hypothermia during on-pump coronary artery bypass grafting. Hypothermia is protective against operative mortality compared with normothermia in such patients. Moderate hypothermia does not provide additional survival benefit.
PMCID: PMC4336164  PMID: 25125205
16.  Hypothermia Blocks β-catenin Degradation after Focal Ischemia in Rats 
Brain research  2008;1198:182-187.
Dephosphorylated and activated glycogen synthase kinase (GSK) 3β hyperphophorylates β-catenin, leading to its ubiquitin-proteosome-mediated degradation. β-catenin-knockdown increases while β-catenin overexpression prevents neuronal death in vitro; in addition, protein levels of β-catenin are reduced in the brain of Alzheimer’s patients. However, whether β-catenin degradation is involved in stroke-induced brain injury is unknown. Here we studied activities of GSK3 β and β-catenin, and the protective effect of moderate hypothermia (30 °C) on these activities after focal ischemia in rats. The results of Western blot showed that GSK3 β was dephosphorylated at 5 and 24 hours after stroke in the normothermic (37 °C) brain; hypothermia augmented GSK3β dephosphorylation. Because hypothermia reduces infarction, these results contradict with previous studies showing that GSK3β dephosphorylation worsens neuronal death. Nevertheless, hypothermia blocked degradation of total GSK3β protein. Corresponding to GSK3β activity in normothermic rats, β-catenin phosphorylation transiently increased at 5 hours in both the ischemic penumbra and core, and the total protein level of β-catenin degraded after normothermic stroke. Hypothermia did not inhibit β-catenin phosphorylation, but it blocked β-catenin degradation in the ischemic penumbra. In conclusion, moderate hypothermia can stabilize β-catenin, which may contribute to the protective effect of moderate hypothermia.
PMCID: PMC2350209  PMID: 18241848
Focal ischemia; hypothermia; GSK-3β; β-catenin
17.  The neuroprotective effect of post ischemic brief mild hypothermic treatment correlates with apoptosis, but not with gliosis in endothelin-1 treated rats 
BMC Neuroscience  2012;13:105.
Stroke remains one of the most common diseases with a serious impact on quality of life but few effective treatments exist. Mild hypothermia (33°C) is a promising neuroprotective therapy in stroke management. This study investigated whether a delayed short mild hypothermic treatment is still beneficial as neuroprotective strategy in the endothelin-1 (Et-1) rat model for a transient focal cerebral ischemia. Two hours of mild hypothermia (33°C) was induced 20, 60 or 120 minutes after Et-1 infusion. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry in the striatum, which represents the core of the infarct. Functional outcome and infarct volume were assessed 24 hours after the insult. In this sub-acute phase following stroke induction, the effects of the hypothermic treatment on apoptosis, phagocytosis and astrogliosis were assessed as well. Apoptosis was determined using caspase-3 immunohistochemistry, phagocytic cells were visualized by CD-68 expression and astrogliosis was studied by glial fibrillary acidic protein (GFAP) staining.
Cooling could be postponed up to 1 hour after the onset of the insult without losing its positive effects on neurological deficit and infarct volume. These results correlated with the caspase-3 staining. In contrast, the increased CD-68 expression post-stroke was reduced in the core of the insult with all treatment protocols. Hypothermia also reduced the increased levels of GFAP staining, even when it was delayed up to 2 hours after the insult. The study confirmed that the induction of the hypothermia treatment in the Et-1 model does not affect the CBF.
These data indicate that in the Et-1 rat model, a short mild hypothermic treatment delayed for 1 hour is still neuroprotective and correlates with apoptosis. At the same time, hypothermia also establishes a lasting inhibitory effect on the activation of astrogliosis.
PMCID: PMC3502503  PMID: 22920191
Hypothermia; Cerebral ischemia; Endothelin-1; Caspase-3; Gliosis; Phagocytosis
18.  Endothelin-1 Is Increased in Cerebrospinal Fluid and Associated with Unfavorable Outcomes in Children after Severe Traumatic Brain Injury 
Journal of Neurotrauma  2010;27(10):1819-1825.
Severe pediatric traumatic brain injury (TBI) is associated with unfavorable outcomes secondary to injury from activation of the inflammatory cascade, the release of excitotoxic neurotransmitters, and changes in the reactivity of cerebral vessels, causing ischemia. Hypoperfusion of injured brain tissues after TBI is also associated with unfavorable outcomes. Therapeutic hypothermia is an investigational treatment strategy for use in patients with severe TBI that has shown differential effects on various cerebrospinal fluid (CSF) mediators in pediatric patients. Endothelin-1 (ET-1) is a powerful vasoconstrictor that exerts its effects on the cerebrovascular endothelium for sustained periods after TBI. The purpose of this study was to determine if CSF concentrations of ET-1 are increased after severe TBI in children, and if they are associated with demographics and outcomes that are affected by therapeutic hypothermia. This was an ancillary study to a prospective, randomized-controlled trial of early hypothermia in a tertiary care pediatric intensive care unit. Children (n = 34, age 3 months–15 years) suffering from severe TBI were randomized to hypothermia (n = 19) and normothermia (n = 15) as part of the efficacy study. Children undergoing diagnostic lumbar puncture (n = 11) to rule out infection were used as controls. Patients received either mild to moderate hypothermia (32–33°C) or normothermia as part of their treatment protocol. CSF was serially collected during the first 5 days after TBI. ET-1 concentrations were quantitated in patient and control CSF samples by a validated ELISA in duplicate with a limit of quantification of 0.195 pg/mL. CSF ET-1 concentrations were increased by two- to threefold in children after TBI compared to controls, and the increase was sustained for up to 5 days post-TBI. This relationship was not affected by hypothermia, and there were no differences in ET-1 response between children with inflicted and accidental TBI. Group-based trajectory analysis revealed two distinct groups with similar ET-1 levels over time. Univariate analysis showed a significant association between ET-1 levels and Glasgow Outcome Scale (GOS) scores, for which higher ET-1 levels over time were associated with unfavorable outcomes. ET-1 is increased in children with severe TBI and is associated with unfavorable outcomes. This increase in ET-1 may mediate the hypoperfusion or cerebrovascular dysfunction accompanying severe TBI in children. Importantly, hypothermia does not affect the brain's ET-1 response as measured in the CSF.
PMCID: PMC2953926  PMID: 20684675
abusive head trauma; cerebral blood flow; endothelin; hypoperfusion; inflicted childhood neurotrauma; pediatric brain injury; vasospasm
19.  Delayed Treatment With Hypothermia Protects Against the No‐Reflow Phenomenon Despite Failure to Reduce Infarct Size 
Many studies have shown that when hypothermia is started after coronary artery reperfusion (CAR), it is ineffective at reducing necrosis. However, some suggest that hypothermia may preferentially reduce no‐reflow. Our aim was to test the effects of hypothermia on no‐reflow when initiated close to reperfusion and 30 minutes after reperfusion, times not associated with a protective effect on myocardial infarct size.
Methods and Results
Rabbits received 30 minutes coronary artery occlusion/3 hours CAR. In protocol 1, hearts were treated for 1 hour with topical hypothermia (myocardial temperature ≈32°C) initiated at 5 minutes before or 5 minutes after CAR, and the results were compared with a normothermic group. In protocol 2, hypothermia was delayed until 30 minutes after CAR and control hearts remained normothermic. In protocol 1, risk zones were similar and infarct size was not significantly reduced by hypothermia initiated close to CAR. However, the no‐reflow defect was significantly reduced by 43% (5 minutes before CAR) and 38% (5 minutes after CAR) in hypothermic compared with normothermic hearts (P=0.004, ANOVA, P=ns between the 2 treated groups). In protocol 2, risk zones and infarct sizes were similar, but delayed hypothermia significantly reduced no‐reflow in hypothermic hearts by 30% (55±6% of the necrotic region in hypothermia group versus 79±6% with normothermia, P=0.008).
These studies suggest that treatment with hypothermia reduces no‐reflow even when initiated too late to reduce infarct size and that the microvasculature is especially receptive to the protective properties of hypothermia and confirm that microvascular damage is in large part a form of true reperfusion injury.
PMCID: PMC3603258  PMID: 23525431
ischemia; myocardial infarction; reperfusion
20.  Whole body hypothermia broadens the therapeutic window of intranasally administered IGF-1 in a neonatal rat model of cerebral hypoxia-ischemia 
Brain research  2011;1385:246-256.
To investigate whether whole body hypothermia after neonatal cerebral hypoxia-ischemia (HI) could broaden the therapeutic window of intranasal treatment of IGF-1 (iN-IGF-1), postnatal day 7 rat pups were subjected to right common carotid artery ligation, followed by 8% oxygen inhalation for 2 h. After HI, one group of pups were returned to their dams and kept at room temperature (24.5±0.2°C). A second group of pups were subjected to whole body hypothermia in a cool environment (21.5±0.3°C) for 2 or 4 h before being returned to their dams. Two doses of 50 μg recombinant human IGF-1 were administered intranasally at a 1 h interval starting at 0, 2 or 4 h after hypothermia. Hypothermia decreased the rectal temperature of pups by 4.5°C as compared to those kept at room temperature. While hypothermia or iN-IGF-1 administered 2 h after HI alone did not provide neuroprotection, the combined treatment of hypothermia with iN-IGF-1 significantly protected the neonatal rat brain from HI injury. Hypothermia treatment extended the therapeutic window of IGF-1 to 6 h after HI. The extended IGF-1 therapeutic window by hypothermia was associated with decreases in infiltration of polymorphonuclear leukocytes and activation of microglia/macrophages and with attenuation of NF-κB activation in the ipsilateral hemisphere following HI.
PMCID: PMC3065539  PMID: 21316352
hypothermia; IGF-1; intranasal administration; neonatal hypoxia-ischemia; polymorphonuclear cell infiltration; NF-κB
21.  Delayed minocycline but not delayed mild hypothermia protects against embolic stroke 
BMC Neurology  2002;2:2.
Inflammatory reactions occurring in the brain after ischemia may contribute to secondary damage. In the present study, effects of minocycline, an anti-inflammatory agent, alone or in combination with mild hypothermia on focal embolic cerebral ischemia have been examined.
Focal ischemic injury was induced by embolizing a preformed clot into the middle cerebral artery (MCA). Infarction volume was measured at 48 h after the injury. Mortality was also recorded.
Delayed administration of minocycline alone or delayed minocycline plus delayed mild hypothermia reduced the infarction volume significantly. However, delayed mild hypothermia alone was not protective and delayed mild hypothermia in combination with minocycline did not show any additive effect.
These results suggest that minocycline is beneficial in focal ischemic brain injury, and the lack of the enhanced neuroprotection may be due to the brief exposure to hypothermia.
PMCID: PMC107740  PMID: 11960560
22.  Molecular Mechanisms Underlying Hypothermia-Induced Neuroprotection 
Stroke Research and Treatment  2010;2011:809874.
Stroke is a dynamic event in the brain involving heterogeneous cells. There is now compelling clinical evidence that prolonged, moderate cerebral hypothermia initiated within a few hours after severe ischemia can reduce subsequent neuronal death and improve behavioral recovery. The neuroprotective role of hypothermia is also well established in experimental animals. However, the mechanism of hypothermic neuroprotection remains unclear, although, presumably involves the ability of hypothermia to suppress a broad range of injurious factors. In this paper, we addressed this issue by utilizing comprehensive gene and protein expression analyses of ischemic rat brains. To predict precise target molecules, we took advantage of the therapeutic time window and duration of hypothermia necessary to exert neuroprotective effects. We proposed that hypothermia contributes to protect neuroinflammation, and identified candidate molecules such as MIP-3α and Hsp70 that warrant further investigation as targets for therapeutic drugs acting as “hypothermia-like neuroprotectants.”
PMCID: PMC2995905  PMID: 21151700
Mesenteric ischemia-reperfusion injury (IRI) leads to systemic inflammation and multiple organ failure in both clinical and laboratory settings. We investigated the lung structural, functional, and genomic response to mesenteric IRI with and without regional intraischemic hypothermia (RIH) in rodents, and hypothesized that RIH would protect the lung and preferentially modulate the distant organ transcriptome under these conditions.
Sprague-Dawley rats underwent sham laparotomy or superior mesenteric artery occlusion (SMAO) for 60 minutes with or without RIH. Gut temperature was maintained at 15–20°C during SMAO, and systemic normothermia (37°C) was maintained throughout the study period. At 6 or 24 hours, lung tissue was collected for 1) histology, 2) MPO activity 3) bronchoalveolar lavage (BAL) fluid protein concentrations, 4) lung wet-dry ratios, and 5) total RNA isolation and hybridization to Illumina's Sentrix BeadChips (>22,000 probes) for gene expression profiling. Significantly affected genes (false discovery rate <5% and fold change ≤1.5%) were linked to gene ontology (GO) terms using MAPPFinder and hypothermia suppressed genes were further analyzed with Pubmatrix.
Mesenteric IRI induced lung injury as evidenced by leukocyte trafficking, alveolar hemorrhage, increased BAL protein and wet-dry ratios, and activated a pro-inflammatory lung transcriptome when compared to sham. In contrast, rats treated with RIH exhibited lung histology, BAL protein, and wet-dry ratios similar to sham. At six hours, GO analysis indentified 232 hypothermia-suppressed genes related to inflammation, innate immune response and cell adhesion, and 33 hypothermia-activated genes related to lipid and amine metabolism and defense response. Quantitative RT-PCR validated select array changes in top hypothermia-suppressed genes lipocalin-2 (lcn-2) and chemokine ligand 1 (CXCL-1); prominent genes associated with neutrophil activation and trafficking.
Therapeutic hypothermia during SMAO provides distant organ protection and preferentially modulates the IRI-activated transcriptome in the rat lung. This study identifies potential novel diagnostic and therapeutic targets of mesenteric IRI, and provides a platform for further mechanistic study of hypothermic protection at the cellular and sub-cellular level.
PMCID: PMC2949511  PMID: 20678877
24.  The Combination of Either Tempol or FK506 with Delayed Hypothermia: Implications for Traumatically Induced Microvascular and Axonal Protection 
Journal of Neurotrauma  2011;28(7):1209-1218.
Following traumatic brain injury (TBI), inhibition of reactive oxygen species and/or calcineurin can exert axonal and vascular protection. This protection proves optimal when these strategies are used early post-injury. Recent work has shown that the combination of delayed drug administration and delayed hypothermia extends this protection. Here we revisit this issue in TBI using the nitroxide antioxidant Tempol, or the immunophilin ligand FK506, together with delayed hypothermia, to determine their effects upon cerebral vascular reactivity and axonal damage. Animals were subjected to TBI and treated with Tempol at 30 or 90 min post-injury, or 90 min post-injury with concomitant mild hypothermia (33°C). Another group of animals were treated in the same fashion with the exception that they received FK506. Cranial windows were placed to assess vascular reactivity over 6 h post-injury, when the animals were assessed for traumatically induced axonal damage. Vasoreactivity was preserved by early Tempol administration; however, this benefit declined with time. The coupling of hypothermia and delayed Tempol, however, exerted significant vascular protection. The use of early and delayed FK506 provided significant vascular protection which was not augmented by hypothermia. The early administration of Tempol provided dramatic axonal protection that was not enhanced with hypothermia. Early and delayed FK506 provided significant axonal protection, although this protection was not enhanced by delayed hypothermia. The current investigation supports the premise that Tempol coupled with hypothermia extends its benefits. While FK506 proved efficacious with early and delayed administration, it did not provide either increased vascular or axonal benefit with hypothermia. These studies illustrate the potential benefits of Tempol coupled to delayed hypothermia. However, these findings do not transfer to the use of FK506, which in previous studies proved beneficial when coupled with hypothermia. These divergent results may be a reflection of the different animal models used and/or their associated injury severity.
PMCID: PMC3136741  PMID: 21521034
axonal injury; pial vessels; vascular reactivity; traumatic brain injury
25.  Mild hypothermia causes differential, time-dependent changes in cytokine expression and gliosis following endothelin-1-induced transient focal cerebral ischemia 
Stroke is an important cause of morbidity and mortality and few therapies exist thus far. Mild hypothermia (33°C) is a promising neuroprotective strategy to improve outcome after ischemic stroke. However, its complete mechanism of action has not yet been fully elaborated. This study is the first to investigate whether this neuroprotection occurs through modulation of the neuroinflammatory response after stroke in a time-dependent manner.
The Endothelin-1 (Et-1) model was used to elicit a transient focal cerebral ischemia in male Wistar rats. In this model, the core and penumbra of the insult are represented by the striatum and the cortex respectively. We assessed the effects of 2 hours of hypothermia, started 20 minutes after Et-1 injection on neurological outcome and infarct volume. Furthermore, pro- and anti-inflammatory cytokine expression was determined using ELISA. Microgliosis and astrogliosis were investigated using CD-68 and GFAP staining respectively. All parameters were determined 8, 24, 72 hours and 1 week after the administration of Et-1.
Et-1 infusion caused neurological deficit and a reproducible infarct size which increased up to 3 days after the insult. Both parameters were significantly reduced by hypothermia. The strongest reduction in infarct volume with hypothermia, at 3 days, corresponded with increased microglial activation. Reducing the brain temperature affected the stroke induced increase in interleukin-1β and tumor necrosis factor α in the striatum, 8 hours after its induction, but not at later time points. Transforming growth factor β increased as a function of time after the Et-1-induced insult and was not influenced by cooling. Hypothermia reduced astrogliosis at 1 and 3 days after stroke onset.
The beneficial effects of hypothermia after stroke on infarct volume and functional outcome coincide with a time-dependent modulation of the cytokine expression and gliosis.
PMCID: PMC3127770  PMID: 21627837
Stroke; Hypothermia; Neuroinflammation; Cytokines; Gliosis

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