Although many studies have shown the great potential of induced hypothermia in stroke treatment, we recognize that there are limitations to the protective effects of hypothermia even in the laboratory. Here, we review our experiments on the protective effects of mild-to-moderate hypothermia in rats. Focal ischemia was induced by bilateral common carotid artery (CCA) occlusion for 1 to 2 hours combined with permanent or transient middle cerebral artery (MCA) occlusion. We compared the effects of mild (33°C) and moderate (30°C) hypothermia, evaluated therapeutic time windows, and studied the underlying mechanisms. On review, our findings revealed that the protective effects of induced mild hypothermia (33°C) were limited, and the therapeutic time window of even moderate hypothermia (30°C) was very short in our specific models, although this limitation might be due to the relatively brief periods of hypothermia used. In addition, we found that hypothermia reduced brain injury by preserving Akt activity, PTEN phosphorylation and εPKC activity, while inhibiting ROS production, and δPKC activity.
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.”
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.
AMPK; hypothermia; middle cerebral artery occlusion; stroke
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
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.
acidosis; AMPK; Compound C; hibernation; hypocalcemia; insulin
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.
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a dual-specificity lipid and protein phosphatase. The loss of PTEN was originally discovered in numerous human cancers. PTEN inhibition by bisperoxovanadium (bpV) reduces neurological damage after ischemic brain injury. The purpose of this study was to identify the optimal neuroprotective dose of bpV when administrated after focal ischemia/reperfusion (I/R) injury in rats. Focal I/R injury was induced using the middle cerebral artery occlusion method. bpV at doses of 0.25, 0.50 and 1.0 mg/kg were injected intraperitoneally just after reperfusion, with saline serving as a vehicle control. A maximal reduction in brain injury was observed with 1.0 mg/kg bpV. This dose of bpV also significantly blocked apoptosis in the penumbral cortex of rats. This beneficial effect was associated with the increasing levels of Akt phosphorylation in the penumbral cortex. These results demonstrate that the pharmacological inhibition of PTEN protects against I/R injury in a dose-dependent manner and the protective effect might be induced through upregulation of the phosphoinositide-3 kinase/Akt pro-survival pathway, suggesting a new therapeutic strategy to combat ischemic brain injury.
bisperoxovanadium; phosphatase and tensin homologue deleted on chromosome 10; cerebral ischemia; neuroprotection; Akt
Neuroinflammation is a key element in the ischemic cascade after cerebral ischemia that results in cell damage and death in the subacute phase. However, anti-inflammatory drugs do not improve outcome in clinical settings suggesting that the neuroinflammatory response after an ischemic stroke is not entirely detrimental. This review describes the different key players in neuroinflammation and their possible detrimental and protective effects in stroke. Because of its inhibitory influence on several pathways of the ischemic cascade, hypothermia has been introduced as a promising neuroprotective strategy. This review also discusses the influence of hypothermia on the neuroinflammatory response. We conclude that hypothermia exerts both stimulating and inhibiting effects on different aspects of neuroinflammation and hypothesize that these effects are key to neuroprotection.
The proapoptotic function of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphatase has been linked to its capacity to antagonize the phosphatidylinositol-3-kinase–Akt signaling pathway. Previous studies have shown that the Forkhead transcriptional factor (FOXO3a) is a critical effector of the PTEN-mediated tumor suppressor. However, whether the PTEN–Akt– FOXO3a pathway is involved in neuronal apoptosis in developing rat brain after hypoxia–ischemia (HI) is unclear. In this study, we generated an HI model using postnatal day 10 rats. Immunohistochemistry and western blot were used to detect the expression of total and phosphorylated PTEN, Akt, and FOXO3a, as well as its target gene Bim. We found that dephosphorylation of PTEN was accompanied by dephosphorylation of Akt and FOXO3a, which induced FOXO3a translocation into the nucleus and upregulated the expression of Bim. Furthermore, we found that PTEN inhibition by bisperoxovanadium significantly increased the phosphorylation of Akt and FOXO3a, decreased the nuclear translocation of FOXO3a, and inhibited Bim expression after HI. Moreover, the downregulation of Bim caused by PTEN inhibition attenuated cellular apoptosis in developing rat brain. Our findings suggest that the PTEN–Akt–FOXO3a pathway is involved in neuronal apoptosis in neonatal rat brain after HI. Agents targeting PTEN may offer a promise to rescue neurons from HI brain damage.
Akt; Bim; FOXO3a; hypoxia-ischemia; neuronal apoptosis; PTEN
In experimentally induced myocardial infarction, mild hypothermia (33–35°C) is beneficial if applied prior to ischemia or reperfusion. Hypothermia, when applied after reperfusion seems to confer little or no benefit. The mechanism by which hypothermia exerts its cell-protective effect during cardiac ischemia remains unclear. It has been hypothesized that hypothermia reduces the reperfusion damage; the additional damage incurred upon the myocardium during reperfusion. Reperfusion results in a massive increase in blood flow, reactive hyperemia, which may contribute to reperfusion damage. We postulated that hypothermia could attenuate the post-ischemic reactive hyperemia.
Sixteen 25–30 kg pigs, in a closed chest model, were anesthetized and temperature was established in all pigs at 37°C using an intravascular cooling catheter. The 16 pigs were then randomized to hypothermia (34°C) or control (37°C). The left main coronary artery was then catheterized with a PCI guiding catheter. A Doppler flow wire was placed in the mid part of the LAD and a PCI balloon was then positioned proximal to the Doppler wire but distal to the first diagonal branch. The LAD was then occluded for ten minutes in all pigs. Coronary blood flow was measured before, during and after ischemia/reperfusion.
The peak flow seen during post-ischemic reactive hyperemia (during the first minutes of reperfusion) was significantly reduced by 43 % (p < 0.01) in hypothermic pigs compared to controls.
Mild hypothermia significantly reduces post-ischemic hyperemia in a closed chest pig model. The reduction of reactive hyperemia during reperfusion may have an impact on cardiac reperfusion injury.
Intercellular adhesion molecule-1 (ICAM-1) in cerebral vascular endothelium induced by ischemic insult triggers leukocyte infiltration and inflammatory reaction. We investigated the mechanism of hypothermic suppression of ICAM-1 in a model of focal cerebral ischemia. Rats underwent 2 hours of middle cerebral artery occlusion and were kept at 37°C or 33°C during occlusion and rewarmed to normal temperature immediately after reperfusion. Under hypothermic condition, robust activation of extracellular signal-regulated kinase-1/2 (ERK1/2) was observed in vascular endothelium of ischemic brain. Hypothermic suppression of ICAM-1 was reversed by ERK1/2 inhibition. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) in ischemic vessel was attenuated by hypothermia. STAT3 inhibitor suppressed ICAM-1 production induced by stroke. ERK1/2 inhibition enhanced phosphorylation and DNA binding activity of STAT3 in hypothermic condition. In this study, we demonstrated that hypothermic suppression of ICAM-1 induction is mediated by enhanced ERK1/2 activation and subsequent attenuation of STAT3 action.
The basal ganglia of newborns are extremely vulnerable to hypoxic ischemia (HI). Striatal neurons undergo prominent necrosis after HI. The mechanisms for this degeneration are not well understood. Post-asphyxic hypothermia ameliorates the striatal necrosis, but the mechanisms of hypothermia-induced neuroprotection are not known. We used a newborn piglet model of hypoxic-asphyxic cardiac arrest to test the hypotheses that N-methyl-D-aspartate receptor activation and free radical damage coexist, prior to neurodegeneration, early after resuscitation, and that these changes are attenuated with hypothermia. Piglets were subjected to 30 minutes of hypoxia followed by 7 minutes of airway occlusion, causing asphyxia cardiac arrest, and then were resuscitated and survived normothermically for 5 minutes, 3 hours, or 6 hours, or hypothermically for 3 hours. By 6 hours of normothermic recovery, 50% of neurons in putamen showed ischemic cytopathology. Striatal tissue was fractionated into membrane or soluble proteins and was assayed by immunoblotting for carbonyl modification, phosphorylation of the N-methyl-D-aspartate receptor subunit NR1, and neuronal nitric oxide synthase. Significant accumulation of soluble protein carbonyls was present at 3 hours (196% of control) and 6 hours (142% of control). Phosphorylation of serine-897 of NR1 was increased significantly at 5 minutes (161% of control) and 3 hours (226% of control) after HI. Phosphorylation of serine-890 of NR1 was also increased after HI. Membrane associated neuronal nitric oxide synthase was increased by 35% at 5 minutes. Hypothermia attenuated the oxidative damage and the NR1 phosphorylation in striatum. We conclude that neuronal death signaling in newborn striatum after HI is engaged rapidly through N-methyl-D-aspartate receptor activation, neuronal nitric oxide synthase recruitment, and oxidative stress. Postasphyxic, mild whole body hypothermia provides neuroprotection by suppressing N-methyl-D-aspartate receptor phosphorylation and protein oxidation.
asphyxia; cardiac arrest; neonatal brain ischemia; pediatric brain damage; protein carbonyl; nitric oxide
Hypothermia has long been known to be a potent neuroprotectant. In this mini review, we highlighted clinical experience that hypothermia protects the brain from cerebral injury. We discussed the clinical practice of hypothermia in ischemic stroke. Multiple factors play a significant role in the mechanisms. Clinical application drew first from two clinical trials with comatose patients after cardiac arrest is attractive. The Australian and European study have led to renewed interest in these patients. More and more evidence bring insight into its effects on cerebral ischemia. The type of cooling technique to be used, the duration of cooling and speed of rewarming appear to be key factors in determining whether hypothermia is effective in preventing or mitigating neurological injury. Although until now, there are no clear therapeutic standards of the parameters in therapeutic hypothermia, it is well accepted that cooling should be initiated as soon as possible. By combining hypothermia with other neuroprotectants, it may be possible to enhance protective effects, reduce side effects and lengthen the maximum time. In addition to its neuroprotective properties hypothermia may extend the therapeutic window for other neuroprotective treatment. Thus, combination therapies with neuroprotective, anti-inflammatory and thrombolytic agents are likely to be investigated in the clinical setting in the future.
Although the protective mechanisms of delayed ischemic preconditioning have received extensive studies, few have addressed the mechanisms associated with rapid ischemic postconditioning. We investigated whether ischemic tolerance induced by rapid preconditioning is regulated by the Akt survival signaling pathway. Stroke was generated by permanent occlusion of the left distal middle cerebral artery (MCA) plus 30 min or 1 h occlusion of the bilateral common carotid artery (CCA) in male rats. Rapid preconditioning performed 1h before stroke onset reduced infarct size by 69% in rats with 30 min CCA occlusion, but by only 19% with 1 h occlusion. After control ischemia with 30 min CCA occlusion, Western Blot showed that P-Akt was transiently increased while Akt kinase assay showed that Akt activity was decreased. Although preconditioning did not change P-Akt levels at 1h and 5h compared with control ischemia, it attenuated reduction in Akt activity at 5h in the penumbra. However, preconditioning did not change the levels of P-PDK1, P-PTEN, and P-GSK3β in the Akt pathway, all of which were decreased after stroke. At last, the PI3K kinase inhibitor, LY294002, completely reversed the protection from ischemic preconditioning. In conclusion, Akt contributes to the protection of rapid preconditionin against stroke.
rapid preconditioning; ischemic tolerance; cerebral ischemia; focal ischemia; neuroprotection; Akt
Changes in the dynamic interactions of macromolecules in cell membranes appear to underlie the robust neuroprotective effect of hypothermia against selective neuronal degeneration in the CA1 region of the rat hippocampus after transient cerebral ischemia, but the detailed mechanisms are still elusive. Using the two-vessel occlusion model of transient normothermic cerebral ischemia of 15 min duration, we investigated the tyrosine phosphorylation of synaptic proteins in general and that of the NMDA receptor subunits in particular, at different times of recirculation. Specifically, the effect of intra-ischemic hypothermia (33°C), which provides neuroprotection to the CA1 region of the hippocampus, was studied. Phosphorylation of tyrosine residues on the NMDA receptor (NR) 2, but not of the NR1 or the AMPA receptor subunit 1 (GluR1) proteins, was markedly enhanced following cerebral ischemia. Protein tyrosine phosphorylation was persistently increased in the postsynaptic densities of the vulnerable CA1 region, but was transient in the CA3/dentate gyrus (DG) neurons where cell death was not evident. The phospho-tyrosine phosphatase activity decreased during reperfusion in the CA1 region but not in CA3/DG. Importantly, decreasing body temperature to 33°C during ischemia modified the dynamics of the protein tyrosine phosphorylation of NR2 in the CA1 region, which was transient and similar in time course to that seen in the CA3/DG region after normothermic ischemia. We conclude that the protracted tyrosine phosphorylation of the NR2 subunit in the hippocampus CA1 region following normothermic ischemia is attenuated by hypothermia and therefore constitutes an important target for hypothermic neuroprotection.
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.
Hypothermia; Microglia; Cell migration; Neuroinflammation; Neuroprotection
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.
apoptosis; hypothermia; cerebral ischemia; matrix metalloproteinases; Fas/FasL; stroke
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.
Focal ischemia; hypothermia; GSK-3β; β-catenin
Seizures are associated with adverse outcome in infants with hypoxic-ischemic encephalopathy. We hypothesized that early administration of the anticonvulsant phenobarbital after cerebral hypoxia-ischemia could enhance the neuroprotective efficacy of delayed-onset hypothermia. We tested this hypothesis in a neonatal rodent model. Seven-day-old rats (n=104) underwent right carotid ligation, followed by 90 min 8%O2 exposure; 15 min later, they received injections of phenobarbital (40 mg/kg) or saline. One or 3h later, all were treated with hypothermia (30°C, 3h). Function and neuropathology were evaluated after 7 days (“early outcomes”) or 1 month (“late outcomes”). Early outcome assessment demonstrated better sensorimotor performance and less cortical damage in phenobarbital-treated groups; there were no differences between groups in which the hypothermia delay was shortened from 3h to 1h. Late outcome assessment confirmed sustained benefits of phenobarbital+hypothermia treatment; sensorimotor performance was better (persistent attenuation of contralateral forepaw placing deficits and absence of contralateral forepaw neglect); neuropathology scores were lower (medians, phenobarbital 2, saline 8.5, p<0.05), and less ipsilateral cerebral hemisphere %Damage (mean±SD, 11±17 vs. 28±22, p<0.05). These results suggest that early post-hypoxia-ischemia administration of phenobarbital may augment the neuroprotective efficacy of therapeutic hypothermia.
Although treatment of stroke patients with mild hypothermia is a promising therapeutic approach, chemicals inducing prompt and safe reduction of body temperature are an unmet need. We measured the effects of the transient receptor potential vanilloid-1 (TRPV1) agonist rinvanil on thermoregulation and ischemic brain injury in mice. Intraperitoneal or intracerebroventricular injection of rinvanil induces mild hypothermia that is prevented by the receptor antagonist capsazepine. Both intraischemic and postischemic treatments provide permanent neuroprotection in animals subjected to transient middle cerebral artery occlusion (MCAo), an effect lost in mice artificially kept normothermic. Data indicate that TRPV1 receptor agonists are promising candidates for hypothermic treatment of stroke.
hypothermia; neuroprotectants; TRPV1 receptor
Endoplasmic reticulum (ER) stress has been implicated in the pathology of cerebral ischemia. Apoptotic cell death occurs during prolonged period of stress or when the adaptive response fails. Hypothermia blocked the TNF or Fas-mediated extrinsic apoptosis pathway and the mitochondria pathway of apoptosis, however, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study aimed to elucidate whether hypothermia attenuates brain cerebral ischemia/reperfusion (I/R) damage by suppressing ER stress-induced apoptosis. A 15 min global cerebral ischemia rat model was used in this study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in hippocampus CA1 were assessed after reperfusion of the brain. The expressions of C/EBP-homolo gous protein (CHOP) and glucose-regulated protein 78 (GRP78) in ischemic hippocampus CA1 were measured at 6, 12, 24 and 48 h after reperfusion. The results showed that hypothermia significantly attenuated brain I/R injury, as shown by reduction in cell apoptosis, CHOP expression, and increase in GRP78 expression. These results suggest that hypothermia could protect brain from I/R injury by suppressing ER stress-induced apoptosis.
Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.
Neuroprotection is a major health care priority, given the enormous burden of human suffering and financial cost caused by perinatal brain damage. With the advent of hypothermia as therapy for term hypoxic–ischemic encephalopathy, there is hope for repair and protection of the brain after a profound neonatal insult. However, it is clear from the published clinical trials and animal studies that hypothermia alone will not provide complete protection or stimulate the repair that is necessary for normal neurodevelopmental outcome. This review critically discusses drugs used to treat seizures after hypoxia–ischemia in the neonate with attention to evidence of possible synergies for therapy. In addition, other agents such as xenon, N-acetylcysteine, erythropoietin, melatonin and cannabinoids are discussed as future potential therapeutic agents that might augment protection from hypothermia. Finally, compounds that might damage the developing brain or counteract the neuroprotective effects of hypothermia are discussed.
Anticonvulsants; Hypoxia; Ischemia; Neuroprotection; Repair; Seizures
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.
focal ischemia; stroke; thrombolysis; hypothermia; reperfusion; MRI; thromboembolic model; rat
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.
Stroke; Hypothermia; Neuroinflammation; Cytokines; Gliosis