Sage Journals: Discover world-class research

Abstract

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, Ca²⁺ 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.

Keywords

apoptosis cerebral ischemia hypothermia necrosis neuroprotection stroke

Introduction

Neuroprotection provided by induced hypothermia has been documented for several decades (Colbourne et al, 1997; Maher and Hachinski, 1993). Deep hypothermia (less than 20°C) is effective in reducing cerebral ischemic damage in patients after cardiac arrest; however, it can produce deleterious complications (Krieger et al, 2005). Since Busto et al, (1987) reported in 1987 that even a 1 to 2°C temperature reduction profoundly protects against experimental stroke in rats, the protection of mild (33 to 36°C) to moderate (28 to 32°C) hypothermia against central nervous system injury and its potential mechanisms have received extensive study in the past 20 years (Corbett and Thornhill, 2000; Krieger and Yenari, 2004).

Cerebral ischemia results from a reduction or complete loss of cerebral blood flow (CBF), followed by depletion of ATP and occurrence of anoxic depolarization (AD) and/or spreading depression (SD)-like depolarization (Colbourne et al, 1997; Nedergaard and Hansen, 1993; Obrenovitch and Urenjak, 1997; Ohta et al, 1997; Figure 1). A large amount of glutamate is released from the intracellular space into the extracellular space (Colbourne et al, 1997; Obrenovitch and Urenjak, 1997; Zhao et al, 1997), which stimulates N-methyl-d-aspartate receptors and leads to increased intracellular calcium levels (Choi and Rothman, 1990). Ischemia/reperfusion generates products of reactive oxygen species in large quantities (Chan, 2001). These acute cascades lead to necrotic neuronal death by causing mitochondrial disruption. In addition, proteins misfold after stroke, causing enzyme dysfunction (Hu et al, 2001). blood—brain barrier (BBB) disruption also contributes to ischemic necrosis (Dietrich et al, 1990). Furthermore, inflammatory factors such as matrix metalloproteinases (Rosenberg, 2002), nuclear transcription factor κB (Zheng and Yenari, 2004), and stress-activated mitogen-activated protein kinase are activated after cerebral ischemia and exacerbate necrosis by releasing free radicals (Kaminska, 2005). Apoptosis, or programmed cell death, also occurs after stroke. Antiapoptotic proteins of the Bcl-2 family, Bcl-2 and Bcl-xL, are selectively upregulated in surviving neurons. Conversely, proapoptotic proteins of the Bcl-2 family, Bax, Bid, and Bad, are highly expressed in dying cells after stroke (Graham and Chen, 2001). After ischemia, cytochrome c is released from the mitochondria into the cytosol causing caspase-9 and-3 activity, leading to apoptosis; apoptosis-inducing factor translocates from the mitochondria into the nuclei, generating large fragments of DNA (Cao et al, 2003; Colbourne et al, 1997; Graham and Chen, 2001; Zhao et al, 2004). However, other studies did not detect the typical apoptotic morphology of neuronal death in focal ischemia (van Lookeren Campagne and Gill, 1996) and global ischemia (Colbourne et al, 1999), and little convincing evidence for the protective effect of a caspase inhibitor has been reported. Thus, apoptosis and necrosis probably represent overlapping ends of a cell death continuum; these two types of cell death can occur in the same tissues after cerebral ischemia (MacManus and Buchan, 2000). Activation of nuclear transcription factor κB, p38 mitogen-activated protein kinase, matrix metalloproteinases, which contribute to necrosis and the inflammatory response, may also contribute to apoptosis. Furthermore, the phosphoinositide 3-kinase (PI3K)/Akt kinase (protein kinase B) survival signaling pathways support neuronal survival in experimental stroke (Colbourne et al, 1997; Zhao et al, 2006, 2005a).

Figure 1

A diagram showing the major cascades that occur after stroke reviewed in this article. AD, anoxic depolarization; AIF, apoptosis-inducing factor; BBB, blood—brain barrier; Cas, caspase; CBF, cerebral blood flow; cyto c, cytochrome c; Fas L, Fas ligand; FKHR, forkhead homologue in rhabdomyosarcoma; Glu, glutamate; GSK3β, glycogen synthase kinase 3β; MMP, matrix metalloprotease; NOS, nitric oxide synthesis; NO, nitric oxide; ONOO⁻, peroxynitrite; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidyliositol-4,5-bisphosphate; PIP3, phosphatidyliositol-3,4,5-bisphosphate;PKC, protein kinase C; P-Akt, phosphorylated Akt; PTEN, phosphatase and tensin homologue deleted on chromosome 10; P-PDK1, phosphorylated phosphoinositide-dependent protein kinase-1; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; VDCC, voltage-dependent calcium channel.

Understanding the mechanisms by which hypothermia protects brain tissue may lead to advances in stroke treatment. We review the effects of mild to moderate hypothermia on various events according to the ischemic cascade depicted in Figure 1. Unless otherwise specified, the term hypothermia in this review refers to mild to moderate hypothermia.

The Ostensible Pan-Inhibitive Effects of Hypothermia

We summarized some studies on the ‘pan-inhibiting’ effects of mild to moderate hypothermia on detrimental cascades caused by stroke in Table 1. Intraischemic hypothermia delays or attenuates both ATP depletion (Ibayashi et al, 2000; Sutton et al, 1991; Welsh et al, 1990) and anoxic depolarization (Bart et al, 1998; Nakashima and Todd, 1996; Takeda et al, 2003), it also blocks glutamate release (Busto et al, 1989b; Patel et al, 1994; Winfree et al, 1996), suppresses inflammation (Kawai et al, 2000; Wang et al, 2002), maintains the integrity of the BBB (Dietrich et al, 1990; Huang et al, 1999; Kawanishi, 2003), reduces free radical production (Maier et al, 2002), inhibits protein kinase C translocation (Cardell et al, 1991; Shimohata et al, 2007a, b ; Tohyama et al, 1998), inhibits matrix metalloproteinase expression (Hamann et al, 2004), and blocks both necrosis and apoptosis. Intraischemic hypothermia also preserves the base-excision repair pathway, which repairs oxidative damage (Luo et al, 2007). In addition to those cascades directly associated with neuronal injury, hypothermia further blocks astrocyte activity and inhibits white matter injury (Colbourne et al, 1997; Dempsey et al, 1987; Kimura et al, 2002). Similarly, postischemic hypothermia blocks free radical generation (Horiguchi et al, 2003), attenuates inflammation (Horstmann et al, 2003; Ohta et al, 2007), prevents BBB permeability (Preston and Webster, 2004), and suppresses caspase activities (Van Hemelrijck et al, 2005). Indeed, a browse through the literature gives an overwhelming impression that hypothermia seems to block every damaging event associated with necrosis or apoptosis. One reason for this impression of pan-inhibition may lie in the causality of ischemic damage. For example, is the inflammatory response the cause of tissue damage or is it induced by brain injury? If it is the latter, then since hypothermia prevents tissue damage, it certainly also prevents the inflammatory response.

Table 1

Hypothermia blocks changes in many detrimental cascades occurring after stroke

Reference	Model	S	T (°C)	Factors
Takeda et al (2003)	Global	G	31 and 34	Anoxic depolarization
Busto et al (1989b)	Global	R	30 and 33	Glutamate
Dietrich et al (1990)	Global	R	30 and 33	BBB
Kawanishi (2003)	Hemorrhage	R	35	Edema; BBB; PMNL
Kawai et al (2000)	Focal	R	33	ICAM-1 mRNA; PMNL
Wang et al (2002)	Focal	R	30	ICAM-1; neutrophil and monocyte; microglia
Hamann et al (2004)	Focal	R	32 and 34	MMP-2; MMP-9; μ-PA; t-PA
Maier et al (2002)	Focal	R	33	O^2-
Karibe et al (1994a)	Focal	R	33	Ascorbate; glutathione
Kader et al (1994)	Focal	R	33	NOS; nitrite
Toyoda et al (1996)	Focal	R	30	Neutrophil
Chopp et al (1992)	Global	R	30	HSP-70
Mancuso et al (2000)	Focal	R	33	HSP-70; C-fos
Tohyama et al (1998)	Focal	R	30	PKC
Shimohata et al (2007a)	Focal	R	30	ɛPKC
Shimohata et al (2007b)	Focal	R	30	δPKC
Harada et al (2002)	Global	R	32	CaM kinase II; PKC-α,β,γ synaptosome
Tsuchiya et al (2002)	Global	M	33	Zn²⁺
Phanithi et al (2000)	Focal	R	33	Fas; caspase-3
Zhao et al (2007)	Focal	R	33	Cytochrome c and AIF
Karabiyikoglu et al (2003)	Focal	R	33 intra or	iNOS; nNOS
			post
Wagner et al (2003)	Focal	R	33 post	BBB; MMP-9
Inamasu et al (2000)	Focal	R	34.5 post	Neutrophil infiltration; microglia
Horstmann et al (2003)	Stroke	Hu	33 post	MMP-9
Horiguchi et al (2003)	Global	R	32 post	Hydroxyl radical
Han et al (2003)	Focal	R	33 post	NF-κB; iNOS; TNF-α
Van Hemelrijck et al (2005)	Focal	R	34 post	Caspase-3; nNOS
Inamasu et al (2000)	Focal	R	34.5 post	Bax
Friedman et al (2001)	Global	R	30 intra/post	GluR1A; GluR2B; GluR3C; NMDAR1
Ohta et al (2007)	Focal	R	35 post	Inflammatory genes: osteopontin, early growth response-1, and macrophage inflammatory protein-3α
Luo et al (2007)	Focal	R	33 post	Base-excision repair pathway
Preston and Webster (2004)	Global	R	32 post	BBB
Liebetrau et al (2004)	Focal	R	32 post	Calpain

AIF, apoptosis-inducing factor; BBB, blood—brain barrier; G, gerbil; Hu, human; HSP-70, heat-shock protein-70; iNOS, inducible nitric oxide synthase; intra, intraischemic hypothermia; MMP-9, matrix metalloprotease-9; M, mouse; NF-κB, nuclear transcription factor κB; NOS, nitric oxide synthesis; nNOS, neuronal nitric oxide synthase; PKC, protein kinase C; PMNL, polymorphonuclear leukocytes; post, postischemic hypothermia; R, rat; S, species; T(°C), intraischemic temperature, unless specified; TNF-α, tumor necrosis factor-α.

The ‘pan-inhibiting’ effect of hypothermia seems to apply not only to detrimental cascades, but also to some protective genes as well. For instance, overexpression of heat-shock protein-70 (HSP-70) protects neurons (Kelly et al, 2002), but hypothermia blocks HSP-70 expression after stroke; therefore, it was believed that protection by hypothermia is independent of HSP-70 (Chopp et al, 1992; Mancuso et al, 2000). However, the truth may be more complex. It is possible that neurons saved by hypothermia do not require HSP-70 expression; HSP-70 may only be upregulated in neurons that are injured. Furthermore, neurons may not overexpress HSP-70 even if they need it, because hypothermia generally inhibits protein translation. Nerve growth factors also improve neuron survival after cerebral ischemia; however, hypothermia does not enhance expression of nerve growth factors, brain-derived neurotrophic factor, neurotrophin 3, and tyrosine kinase receptor B in global ischemia, suggesting that the protective effect of hypothermia is not achieved this way (Boris-Moller et al, 1998). The argument that applies to HSP-70 expression may also apply here.

Findings such as these ‘pan-inhibiting’ effects may lead one to assume that hypothermia simply blocks, inhibits, or reverses any ischemia-induced damaging cascade, even including some beneficial cascades that occur, thereby preventing cell damage. By extension, hypothermia's actions may be so broad that it may provide no real clues for understanding the mechanisms of cell death due to ischemia. Nevertheless, such an assumption is oversimplified and misleading. Although it is true that hypothermia has multiple effects on various cascades after ischemia, and this is almost certain why hypothermia has such profound protective effects, hypothermia does not simply block every detrimental downstream reaction after stroke.

In fact, the mechanisms underlying hypothermic protection are controversial, and a bias appears to favor results showing that hypothermia blocks all damaging cascades after ischemia, whereas results to the contrary have often been under appreciated. We focus on the more controversial issues surrounding hypothermia's effects on CBF, glutamate release, and survival signaling pathways, and do not dwell on hypothermia's effects on BBB permeability and inflammation, because there is somewhat more of a consensus in the literature on these issues. However, first, we summarize the protection of mild to moderate hypothermia in experimental stroke models.

Hypothermia Does Not Always Attenuate Stroke Even in the Laboratory

To translate hypothermia to the clinic, it is important to recognize that its neuroprotective effects are limited in the laboratory as well. Intraischemic hypothermia is most effective in transient ischemia or ‘reperfusion’ models (Busto et al, 1989a; Carroll and Beek, 1992; Colbourne and Corbett, 1995); the effects of intraischemic hypothermia in permanent ischemia models are far less uniform (Baker et al, 1991; Kader et al, 1992; Kozlowski et al, 1997; Lo et al, 1993; Morikawa et al, 1992; Moyer et al, 1992; Ridenour et al, 1992; Zhao et al, 2007). In addition, it will be most meaningful clinically if hypothermia protects when applied postischemically; however, therapeutic time windows for postischemic hypothermia are limited. For example, mild hypothermia applied 15 mins after reperfusion failed to improve neurologic function in global ischemia, whereas it protected when applied immediately after reperfusion (Kuboyama et al, 1993). This therapeutic time window might be similar to what occurs in the penumbra after focal ischemia. In addition to the onset time of hypothermia, duration and depth of hypothermia and animal species/strains also determine the protective effects of postischemic hypothermia (Busto et al, 1989a; Chopp et al, 1991; Coimbra and Wieloch, 1992, 1994; Colbourne and Corbett, 1994, 1995; Colbourne et al, 1997; Doerfler et al, 2001; Huh et al, 2000; Maier et al, 2001; Ren et al, 2004; Yanamoto et al, 1996). Such limitations suggest that hypothermia cannot alter some events that inevitably lead to ischemic damage.

The Effects of Hypothermia on Cerebral Blood Flow and Metabolism

Cerebral Blood Flow

Hypothermia reduces CBF in anesthetized animals and human patients in the absence of cerebral ischemia, as reviewed recently by Erecinska et al (2003). Hypothermia does not affect CBF during complete global ischemia (Busto et al, 1987; Jiang et al, 1994; Kil et al, 1996; Sonn et al, 2002; Sugimura et al, 1998); global ischemia reduces CBF to less than 5% of normal (Busto et al, 1987), making it unlikely that hypothermia would have a further significant effect on CBF. However, there are a wide range of findings on CBF after reperfusion in global ischemia: hypothermia increases (Jenkins et al, 2001; Jiang et al, 1994; Karibe et al, 2000), decreases (Huang et al, 1999; Mori et al, 1998), or has no effect on CBF (Baldwin et al, 1991; Hoffman and Thomas, 1996; Horiguchi et al, 2003; Kil et al, 1996; Sonn et al, 2002; Sugimura et al, 1998).

Focal ischemia reduces CBF in the penumbra to various levels (e.g., 50 to 60% of baseline), and one would expect that hypothermia might further reduce CBF in the penumbra. However, most reports suggest that mild to moderate hypothermia has no effect on the CBF level during ischemia in the penumbra (Kawai et al, 2000; Morikawa et al, 1992; Yanamoto et al, 2001). Nevertheless, one study reported that moderate hypothermia (30°C) significantly reduced CBF using a diffusion-weighted imaging technique (Jiang et al, 1994). Some studies suggest that hypothermia does not affect CBF after reperfusion in focal ischemia (Kawai et al, 2000; Morikawa et al, 1992), whereas others suggest that hypothermia blocks the postischemic hyperperfusion and the delayed, sustained hypoperfusion (Huang et al, 1998; Jiang et al, 1994; Karibe et al, 1994b).

Although the discrepancies may be due to animal species, anesthesia, temperature controls, or other variables, we speculate that the techniques used by most of these studies to measure CBF are the key source of variation. Laser Doppler flowmetry only reflects the relative changes in CBF levels, and it is unreliable to compare such levels across different temperature groups using this technique. Other potential factors affecting CBF results are differences in cooling techniques, the rewarming process, and shivering. Rapid rewarming not only exacerbates axonal injury (Suehiro and Povlishock, 2001), but it also causes dysfunction of blood vessels after brain trauma. Furthermore, shivering during rewarming, especially when anesthesia is withdrawn, is a common phenomenon that significantly affects CBF and ATP recovery (Ueda et al, 2004). However, previous studies usually ignored this issue and so the degree to which rewarming impacts CBF after ischemia/reperfusion is not known. More stringently designed experiments are needed to clarify the effect of hypothermia on CBF in cerebral ischemia.

Hypothermia Promotes Energy Recovery after Reperfusion

After stroke onset, ATP depletes immediately after the reduction of CBF, and the extent of ATP depletion should be proportional to the extent of CBF reduction at a fixed temperature. ATP production and cerebral metabolism closely associate with brain temperature. A 10° decrease in temperature reduces ATP consumption and the cerebral metabolic rate of glucose, oxygen, and lactate two- to four-fold (Erecinska et al, 2003). Hypothermia may not significantly improve energy levels during global ischemia, as the CBF level is already significantly reduced (Busto et al, 1987).

Whether mild hypothermia protects against ischemia in adult animal models by maintaining ATP levels is controversial. One report showed that mild hypothermia attenuated phosphate depletion and lactate accumulation during global ischemia, and attributed the neuroprotective effect of hypothermia to preserved ATP levels (Ibayashi et al, 2000). However, other studies disagree with this conclusion (Busto et al, 1987; Kimura et al, 2002; Kozlowski et al, 1997; Shimizu et al, 1997; Sutton et al, 1991). At the end of 20 mins of global ischemia, # Busto et al (1987) reported that phosphocreatine, glucose, and pyruvate all decreased to a similar degree among groups treated with different temperatures from 30 to 36°C. Similarly, during 15 mins of global ischemia, the hypothermic effect (34°C) did not differ from that of normothermia (37°C) on the amount of high-energy phosphate metabolites including ATP, phosphocreatine, and inorganic phosphate (Kimura et al, 2002). Taken together, it is reasonable to assume that under severe ischemia in which CBF is almost or completely cut off, energy would be depleted within several minutes even with hypothermia, but mild hypothermia would still significantly reduce ischemic damage. Thus, hypothermia's effect on energy depletion during ischemia is unlikely to play a significant role in its neuroprotective effects.

Almost all the studies mentioned above agree that after severe ischemia, hypothermia accelerates ATP recovery during reperfusion (Haraldseth et al, 1992; Kimura et al, 2002; Kozlowski et al, 1997; Shimizu et al, 1997; Sutton et al, 1991). For example, mild hypothermia (34°C) increased the rate of metabolic recovery relative to normothermia by 10 to 20% for the first 10 to 25 mins of reperfusion after 15 mins of transient global ischemia in gerbils (Kimura et al, 2002); metabolism recovered more at 20 to 60 mins after the onset of reperfusion in hypothermic rats subjected to 60 mins of global ischemia (Shimizu et al, 1997); in hippocampal slices of adult guinea pigs, mild hypothermia improved ATP recovery at 12 h after oxygen-glucose deprivation, although mild hypothermia had no evident effect at 2 h after oxygen-glucose deprivation (Berger et al, 1998).

In conclusion, mild to moderate hypothermia may not maintain ATP levels during ischemia. Rather, a more likely mechanism of neuroprotection by hypothermia in adult animal models is the acceleration of ATP recovery after reperfusion.

Hypothermia's Effect on Anoxic Depolarization and Glutamate Release

Anoxic Depolarization

Almost all studies agree that hypothermia merely delays the occurrence of AD in global ischema. These reports consistently show a brief delay in the onset of AD after ischemia in mild to moderate hypothermic animals (e.g., 1 to 2 mins at 33 to 34°C; Table 2). However, few have studied the effects of hypothermia on AD in focal ischemia. Unlike in global ischemia, both AD- and SD-like depolarization occur in the ischemic core and penumbra, respectively, in focal ischemia. In addition, AD- or SD-like depolarization occurs along with redistribution of ions such as Ca²⁺, K⁺, and Na⁺. Changes in these ions' extracellular or intracellular concentrations may actually reflect changes in AD- or SD-like depolarization. Sick et al (1999) showed that hypothermia did not change the onset time of the extracellular potassium level ([K⁺]_o) shift and the concentration of [K⁺]_o in the ischemic core. Furthermore, hypothermia did not affect the duration and frequency of SD-like depolarization and [K⁺]_o in the penumbra, although hypothermia promoted [K⁺]_o recovery after reperfusion. In contrast, Chen et al (1993) showed that hypothermia reduced the number of SD-like depolarizations; differences between the core and the penumbra were not addressed in this study. Moreover, intracellular Ca²⁺ levels play a critical role in neurotoxicity. A few reports have convincingly showed that hypothermia merely delays Ca²⁺ mobilization without altering its concentration from normothermic levels (Kristian et al, 1992; Mitani et al, 1991). Again, these studies were performed in the context of global ischemia. Whether hypothermia has similar effects in focal ischemia is not known.

Table 2

Hypothermia delays onset time of AD in global ischemia or inhibits frequency of SD in focal ischemia

Author	Model	S	T(°C)	Onset time of AD (mins) unless specified
Takeda et al (2003)	Global	G	31	2.4 ± 0.7
			34	1.6 ± 0.4
			37	1.3 ± 0.2
Kaminogo et al (1999)	Global	R	33	5.1 ± 0.3
			37.2	2.2 ± 0.5
Bart et al (1998)	Global	R	31	5.4 ± 0.2
			33	∼3.3
			35	∼2.5
			37	1.8 ± 0.3
Nakashima and Todd (1996)	Global	R	25	4.9 ± 0.6
			31	2.8 ± 0.4
			34	1.7 ± 0.2
			38	1.3 ± 0.2
Mitani et al (1991)	OGD	G	31	4.6
			33	3.9
			35	3.0
			37	2.2
Mori et al (2002)	Global	R		[K⁺]_e
			31.5	1.33 ± 0.4
			37	0.55 ± 0.
				[Na⁺]_e
			31.5	2.87 ± 1.2
			37	1.47 ± 0.5
Sick et al (1999)	Focal	R		Onset time of [K⁺]_o in core
			31.5–32	0.81 ± 0.12
			35–36	0.96 ± 0.12
				Frequency of [K⁺]_o in penumbra
			31.5–32	2.89 ± 1.2 times
			35–36	2.17 ± 1.0 times
Chen et al (1993)	Focal	R	30	Single AD
			37	Multiple
			40	Multiple
Kristian et al (1992)	Cardiac arrest	R		[Ca²⁺]_e
			33	1.52–1.58
			37	1
Mitani et al (1991)	Hypoxia	G		[Ca²⁺]_i
			31	4.6
			33	3.86
			35	3.03
			37	2.17

AD, anoxic depolarization; [Ca²⁺]_i, intracellular concentration of Ca²⁺; D, dog; G, gerbil; [K⁺]_e,[Na⁺]_e, and [Ca²⁺]_e, extracellular concentration of K⁺, Na⁺, and Ca²⁺; [K⁺]_o, onset time of the extracellular potassium level; OGD, oxygen-glucose deprivation; R, rat; S, species; SD, spreading depression. All original data were changed from seconds to minutes.

Glutamate Release

Unlike the consistent effects of hypothermia on anoxic depolarization in global ischemia, the effects of hypothermia on extracellular glutamate concentration ([Glu]_e) are controversial (Table 3). It has been reported that mild hypothermia completely blocks (Busto et al, 1989b; Li et al, 1999; Martinez-Tica and Zornow, 2000; Patel et al, 1994), partially blocks (Mitani and Kataoka, 1991; Winfree et al, 1996; Yamamoto et al, 1999), merely delays (Asai et al, 2000, 1998), or does not inhibit an increase in [Glu]_e after cerebral ischemia (Berger et al, 1998; Zhao et al, 1997). Furthermore, the effect of hypothermia on [Glu]_e depends on the brain area, ischemic severity, and animal age (Berger et al, 2002; Boris-Moller and Wieloch, 1998; Lo et al, 1993; Van Hemelrijck et al, 2003).

Table 3

The effects of hypothermia on glutamate release

Author	Model	S	T (°C)	Glu release
Busto et al (1989b)	Global	R	30 and 33	Completely inhibited Glu release
Koinig et al (2001b)	Global	Rbt	30	Inhibited Glu to baseline
Martinez-Tica and Zornow (2000)	Global	Rbt	30	Inhibited to baseline
Li et al (1999)	Global	R	30	Completely blocked Glu release
Patel et al (1994)	Global	R	34	No Glu increase
Yamamoto et al (1999)	Global	G	31	Inhibited Glu release to 19%
Mitani et al (1991)	Global	G	33 and35	Glu at 35 and 33°C is 50.25% of at 37°C
Patel et al (1994)	Global	R	30 and 34	34 and 30°C inhibited Glu release
Nakashima and Todd (1996)	Cardiac	R	25, 31, and 34	Delayed the onset and rate of Glu release, Glu even significantly
	arrest		34	increased at 25°C
Koinig et al (2001a)	Global	Rbt	33 and 34.5	33°C attenuated Glu release but 34.5°C did not inhibit
	ischemia
Zhao et al (1998)	Global	R	32	Reduced CBF threshold for Glu release
Zhao et al (1997)	Global	R	32	Did not block Glu release but promoted its uptake
Asai et al (1998)	Global	R	28 and 32	Merely delayed onset of Glu release
Asai et al (2000)	Global	R	32	Did not block Glu release but promoted uptake after ischemia
Boris-Moller et al (1998)	Global	R	33	Straitum: inhibited to ∼50% cortex: no inhibition
Berger et al (2002)	Stroke	Hu	33	Inhibited Glu to 38% in penumbra but not in the core
Lo et al (1993)	Focal	Rbt	33	Inhibited Glu in the central regions not in the peripheral region
Winfree et al (1996)	Focal	R	33	33°C inhibited Glu release in penumbra to 34%
Van Hemelrijck et al (2003)	Focal	R	34	Inhibited Glu release in the striatum but not in the cortex

CBF, cerebral blood flow; G, gerbil; Glu, glutamate; Hu, human; P, pig; R, rat; Rbt, rabbit; S, species; T, hypothermic temperature.

It is not clear why there is such variation across studies concerning [Glu]_e. It may reflect species variation, ischemia models, and differences in systems used for monitoring glutamate. We postulate that the different methodology used for detecting glutamate release may be largely responsible for such variations as seen in CBF detection. As we discussed, various studies have shown strikingly consistent results about the effect of hypothermia on AD in global ischemia, probably because the way of detecting AD using direct current potential electrodes is simple and straightforward. However, for glutamate release detection, samples are usually collected using microdialysis probes and are then measured by high-performance liquid chromatography (Li et al, 1999; Phillis et al, 2000; Rossi et al, 2000; Zhao et al, 1999). Extracellular glutamate enters the probe through a semipermeable membrane and is washed out by a continuous flow of solution. The type of probe, the speed of flow, the sample-collecting period, and the high-performance liquid chromatography detection systems could each affect the results.

The mechanisms of glutamate release after cerebral ischemia are not completely understood. [Glu]_e reflects a balance of release and uptake. Glutamate starts to release almost at the same time that AD occurs. After reperfusion and ATP recovery, released glutamate is taken back into the intracellular space when AD starts to recover.

As occurrence of AD reflects ATP depletion that leads to glutamate release, AD usually synchronizes glutamate release under both normothermia and hypothermia after stroke (Asai et al, 2000, 1998; Obrenovitch and Urenjak, 1997; Zhao et al, 1997). Thereafter, released glutamate stimulates N-methyl-d-aspartate receptors leading to intracellular Ca²⁺ accumulation, which is believed a major source for the increased intracellular Ca²⁺ after stroke (Choi and Rothman, 1990). As we have discussed, mild and moderate hypothermia merely delays the onset of AD and intracellular Ca²⁺ accumulation in global ischemia; it is unlikely that the hypothermia would completely inhibit glutamate release when AD occurs and intracellular Ca²⁺ increases. Rather, hypothermia may only delay the onset of glutamate release or attenuate the total amount of glutamate released. Therefore, it may have been overstated in some studies that hypothermia blocks ischemic damage by completely inhibiting glutamate release.

In summary, intraischemic mild hypothermia merely delays ATP depletion, the occurrence of AD, glutamate release, and Ca²⁺ mobilization during severe cerebral ischemia. This delay is not sufficient to explain the strong protection of mild or moderate hypothermia (Bart et al, 1998; Takeda et al, 2003), but it might contribute to a reduction in ischemic damage, especially when combined with the myriad other beneficial effects. Nevertheless, in the case of mild to moderate hypothermia, neuroprotection cannot be solely attributed to its effect on intraischemic events; its effect on subsequent cascades also plays a critical role, which will be discussed in the following sections.

Hypothermia Does Not Simply Block Cell Signaling Pathways of Apoptosis or Necrosis

Although hypothermia generally blocks protein synthesis in the absence of an insult (Sayegh et al, 1992), it actually improves protein synthesis compared with normothermia after ischemia (Wu et al, 1995; Yamashita et al, 1991). Sparing of tissue may contribute to the improved protein synthesis; however, hypothermia selectively upregulates some proteins after ischemia. For example, expression of the mineralocorticoid receptor increased under hypothermia after stroke, which may promote neuronal survival in the hippocampal dentate gyrus (Macleod et al, 2003). Hypothermia also enhanced the glucose-regulated protein-78, of which overexpression via adenovirus gene therapy rescued hippocampal neurons (Aoki et al, 2001). In addition, mild hypothermia improved the protein synthesis rate after 12 h of recovery in hippocampal slices from immature or mature fetuses subjected to oxygen-glucose deprivation from 10 to 40 mins, and improved protein synthesis rate in slices from adult animals exposed to 20 to 30 mins, but not 40 mins, oxygen-glucose deprivation. The neuroprotective effect of mild hypothermia on protein synthesis depends on the animal's age and the severity of the ischemia (Berger et al, 1998). We summarize such specific effects of hypothermia in Table 4.

Table 4

Hypothermia specifically regulates protein levels or mRNA of genes or BBB

Reference	Model	S	T (°C)	Genes	Results
Yamashita et al (1991)	Global	G	< 33	Ubiquitin	Hypothermia promoted ubiquitin synthesis in CA1 at 24 and 48 h after ischemia
Wu et al (1995)	Global	G	30	β-Actin	Hypothermia preserved β-actin mRNA levels
Aoki et al (2001)	Global	G	34	GRP 78	Hypothermia maintained (glucose-regulated protein) GRP78 levels
Macleod et al (2003)	Global	R	33	MR	Hypothermia increased mineralocorticoid receptor (MR) expression
Kumar et al (1996)	Global	G	30	c-fos and fos-B	Hypothermia promoted c-fos and fox-B expression as early as 1 h in vulnerable area
Kamme et al (1995)	Global	R	33	c-fos, fos-B, fra-1, fra-2 c-jun, and jun-B	Biphasic induction: 1–2 h after reperfusion in the DG, 12-36h after reperfusion in CA1 and CA3. Hypothermia made the second phase earlier compared with normothermia
Akaji et al (2003)	Focal	R	30	C-Fos	Hypothermia inhibited the early increase in c-Fos expression but did not affect c-Jun at 3 h after reperfusion
Hicks et al (2000)	Global	R	33 post	Erk/2, JNK, P38	Hypothermia increased ERK and JNK phosphorylation
Tomasevic et al (1999)	Global	R	33	P53, p21WAF1/Cip1	Hypothermia increased p53 and p21WAF1/Cip1 expression in the CA1
Eberspacher et al (2003)	Global	R	34	Bax, Bcl-2, P53, Mdm-2	Hypothermia inhibits Bax expression at 4 h after ischemia, but does not affect other proteins
Yenari et al (2002)	Focal	R	33	Cytochrome c, caspase-3, Bcl-2, and Bax	Hypothermia transiently blocked cytochrome c release, but did not alter Bcl-2, Bax expression; no caspase-3 activity is detected
Zhang et al (2001)	Global	R	33	Bax and Bcl-2	Hypothermia upregulated Bcl-2 expression, no influence on Bax
Huang et al (1999)	Focal	R	28.5 32.5 post	BBB	Biphasic BBB opening: first: immediate after reperfusion then closed at 1–4 h; second time: 22–46 h. Hypothermia inhibited only the late opening
Zhao et al (2005b)	Global	R	33	Cytochrome c, caspase-9 and −3	Hypothermia blocked caspase activity and the second large phase of cytochrome c release; However, it did not block the early phase of cytochrome c release
Zhao et al (2005a)	Focal	R	30	Akt, PTEN, PDK1, GSK3β, and β-catenin	Hypothermia blocked changes in phosphorylated Akt, PTEN, PDK1, FKHR and inhibited β-catenin translocation, but it did not attenuate dephosphorylation of GSK3β

BBB, blood-brain barrier; G, gerbil; GSK3β, glycogen synthase kinase 3β; JNK, Jun N-terminal kinase; post, postischemic hypothermia; PTEN, phosphatase and tensin homologue deleted on chromosome 10 R, rat; S, species; T, temperature.

The Jun N-Terminal Kinase/Early Genes and the Erk Pathway

Jun N-terminal kinase (JNK) and Erk1/2 are two well-characterized members of the mitogen-activated protein kinase family; they are implicated in both apoptosis and neuroprotection after stroke, thus their exact roles remain elusive. In addition, JNK functions by regulating the early genes, c-jun and jun-B, which are upregulated immediately after cerebral ischemia (Akins et al, 1996). As with JNK and Erk1/2, it is not known whether such early gene expression is beneficial or detrimental. Hicks et al (2000) showed that phosphorylated/activated JNK increased at 6, 12, and 24 h after cardiac arrest in rats, and that mild hypothermia further increased phosphorylated JNK at 24 h, but not at 6 and 12 h compared with normothermia; moreover, phosphorylated JNK also increased in the ischemia-resistant regions of the dentate gyrus and of the CA3. The hypothermia-induced increase in phosphorylated JNK coincides with an increase in downstream early gene expression of JNK (Kamme et al, 1995). Kamme et al (1995) showed that the early gene expression patterns of c-jun and jun-B, as well as c-fos and fos-b, is biphasic after global ischemia: the early phase occurred at 1 to 2 h after reperfusion in the dentate gyrus and the late phase appeared at 12 to 36 h in CA-1 and −3. Mild hypothermia accelerated the occurrence of the second phase of gene expression earlier in CA1. Furthermore, Kumar et al (1996) showed that both c-fos and fos-B were induced in the hippocampal dentate gyrus as early as 1 h after global ischemia, and expression extended to vulnerable areas at 4 to 6 h. Hypothermia accelerated their expression to 1 h in the vulnerable regions and temperature did not change expression of the two genes at 6 h, 1 and 2 days. Although another experiment showed that hypothermia decreased expression of c-fos, this occurred in the penumbra of a focal ischemia, and hypothermia did not inhibit c-jun expression at 3 h after cerebral ischemia (Akaji et al, 2003). The conclusion that upregulation of immediate early genes is detrimental is at odds with the fact that these genes are generally induced in hypothermia (Akins et al, 1996).

As with JNK, the function of the kinase Erk1/2 after ischemia is not understood. Whereas some studies state that Erk1/2 expression contributes to ischemic damage (Sugawara et al, 2002), others showed that Erk1/2 activity promoted neuronal survival after ischemia (Kilic et al, 2005). Consistent with the latter, Hicks et al (2000) showed that hypothermia promotes Erk1/2 expression after global ischemia, and Erk1/2 overexpression correlates with selective upregulation of brain-derived neurotrophic factor, an upstream element of the Erk1/2 pathway (D'Cruz et al, 2002).

Hypothermia Unexpectedly Enhances the p53 Pathway

Striking findings have emerged concerning the tumor suppressor p53, a protein that causes cell death. p53 expression after stroke has been associated with neuronal damage, and p53 induction is generally believed to contribute to apoptosis after cerebral ischemia (Halterman and Federoff, 1999). Therefore, induction of p53 is used as a marker for neuronal death, but how it causes neuronal death is not clear. Surprisingly, Tomasevic et al (1999) found that p53 induction might be neuroprotective through their research in hypothermia. They investigated the expression of both p53 and one of its effector genes, p21^WAF/Cip1, which is upregulated in p53-dependent cell cycle arrest and apoptosis. Both p53 and p21^WAF/Cip1 mRNA are upregulated in the resistant area of the hippocampus after transient global ischemia in normothermic and hypothermic rat brains. p53 protein levels are increased in CA3 neurons after ischemia in normothermic rats. But hypothermia increases p53 expression in the protected CA1 neurons, where it translocates into the nuclei, suggesting that its induction is not a marker for neuronal death (Tomasevic et al, 1999). Actually, p53 and p21^WAF/Cip1 are also involved in DNA repair, and lack of their activity leads to failure of repair (Tomasevic et al, 1999). Therefore, the authors argue that p53 induction correlates with neuronal survival, which is supported by a recent report using p53 knockout mice (Maeda et al, 2001). Maeda et al induced a 1 h transient focal ischemia in wild type (p53 ⁺ ^/+), heterozygous (p53 ⁺ ^{/ −}), and homozygous (p53^−/−) mice for p53 deficiency. Infarct size increased inversely to the p53 gene dosage, suggesting that p53 prevents rather than worsens ischemic damage. Taken together, hypothermia may enhance p53 expression promoting repair after stroke (Tomasevic et al, 1999).

Caspase and Cytochrome c-Mediated Apoptotic Pathways

Hypothermia blocks apoptosis after experimental stroke. Mild hypothermia inhibits Fas and caspase-3 expression after 1 h of focal ischemia (Phanithi et al, 2000), it also inhibits Bax overexpression 4 h after 30 mins of incomplete cerebral ischemia but does not affect Bcl-2, p53, and Mdm-2 expression (Eberspacher et al, 2003). In addition, Yenari et al (2002) showed that hypothermia transiently attenuated cytochrome c release but did not alter Bcl-2 and Bax expression in a focal ischemia model. Caspase activity was not observed, however, which suggests that ischemic damage is caspase-independent (Yenari et al, 2002). In contrast, they found that hypothermia specifically increased Bcl-2 protein expression after global ischemia (Zhang et al, 2001).

Recently, we observed a biphasic cytochrome c release after global ischemia: a small peak of cytochrome c release occurred at 5 h, and a larger peak occurred at 48 h after ischemia onset (Zhao et al, 2005b). Moreover, caspase-9 and −3 activity significantly increased at 12 and 24 h after the first phase of cytochrome c release, suggesting that the small amount of cytochrome c might be responsible for the subsequent caspase activity. To our surprise, mild hypothermia did not block the first phase of cytochrome c release, but it did significantly block caspase activity and the second phase of cytochrome c release. These results suggest that low levels of cytochrome c release might be reversible after global ischemia, and may define a time window for intervention for stroke treatment.

Hypothermia Does Not Indiscriminately Upregulate all Signals in the Akt/protein kinase B Pathway

The PI3K/Akt kinase pathways promote neuron survival after ischemia (reviewed by Zhao et al, 2006; Figure 1). Akt activity is believed to be regulated by phosphorylation at Ser-473 and Thr-308 via some upstream molecules, such as phosphoinositide-dependent protein kinase-1 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Whereas activated phosphoinositide-dependent protein kinase-1 phosphorylates Akt, activated PTEN dephosphorylates Akt through degrading phosphatidyliositol-3,4,5-bisphosphate to phosphatidyliositol-4,5-bisphosphate. Activated Akt then blocks caspase/cytochrome c-mediated apoptosis by phosphorylating Akt substrates, such as forkhead homologue in rhabdomyosarcoma and glycogen synthase kinase 3β (GSK3β).

The level of phosphorylated Akt at Ser-473 (P-Akt) has been used to represent Akt activity, and most reports indicate that P-Akt is transiently increased within hours after stroke, but then decreased after 24 h or longer (Zhao et al, 2006). Protection by some agents such as corticosteroids, brain-derived neurotrophic factor, preconditioning, and copper—zinc superoxide dismutase overexpression maintains P-Akt after stroke (Zhao et al, 2006).

We recently showed that PI3K/Akt pathway indeed plays critical roles in neuroprotection by moderate hypothermia (Zhao et al, 2005a). Phosphorylated Akt levels are transiently increased after stroke, but phosphorylation levels of PTEN, phosphoinositide-dependent protein kinase-1, GSK3β, and forkhead homologue in rhabdomyosarcoma are decreased (Zhao et al, 2005a). Intriguingly, in vitro Akt kinase assays showed that the true Akt activity decreased when P-Akt levels reached a peak, suggesting that P-Akt does not represent the true Akt activity. Consistent with this observation, the true Akt activity and phosphorylation levels of PTEN, phosphoinositide-dependent protein kinase-1, GSK3β, and forkhead homologue in rhabdomyosarcoma are decreased at early time points in the penumbra after ischemia, and preceded degradation of MAP-2 and cytochrome c release. Thus, disruption of the Akt pathway might mediate ischemic damage (Zhao et al, 2005a).

Hypothermia serves as a useful tool for understanding the roles of the Akt pathway in neuroprotection after stroke. To our surprise, hypothermia blocks the increases in P-Akt after stroke, but it maintains the true Akt activity, further suggesting that P-Akt does not represent true Akt activity (Zhao et al, 2005a). A functional role for this hypothermia-maintained activity is supported by the finding that the PI3K/Akt inhibitor, LY294002, enlarged infarct size in hypothermic animals, suggesting that the PI3K/Akt pathways play critical roles in neuroprotection (Zhao et al, 2005a).

Further evidence for the role of Akt activity in hypothermic protection comes from the study of a negative regulator of the Akt pathway, PTEN. The activity of PTEN is downregulated by phosphorylation at Ser-380 (P-PTEN) (Torres and Pulido, 2001; Vazquez et al, 2000). Dephosphorylated (activated) PTEN results in dephosphorylation of phosphatidyliositol-3,4,5-bisphosphate to phosphatidyliositol-4,5-bisphosphate and prevents recruitment of Akt to the membrane for phosphorylation (Zhao et al, 2006). Our data indicate that hypothermia blocks ischemic damage by attenuating a decrease in P-PTEN after stroke onset. In fact, the level of P-PTEN is markedly and consistently preserved by hypothermia at various time points from 0.5 to 48 h after stroke, compared with P-Akt, phosphorylated phosphoinositide-dependent protein kinase-1, P-GSK3β, and phosphorylated forkhead homologue in rhabdomyosarcoma. Therefore, hypothermia may exert its neuroprotective action partly through its effects on P-PTEN.

However, hypothermia does not indiscriminately attenuate every aspect of the Akt pathway. Glycogen synthase kinase 3β, a kinase downstream of Akt, contributes to ischemic neurotoxicity (Kelly et al, 2004). Glycogen synthase kinase 3β activity is downregulated when phosphorylated at Ser-9 by Akt. Although hypothermia maintains many steps in the Akt pathway after stroke, it does not elevate GSK3β (Ser-9) phosphorylation (Zhao et al, 2005a). It is a puzzle that the protein level of the active form of nonphosphorylated GSK3β was increased after hypothermic stroke, which is supposed to induce cell death, but no ischemic damage was detected under hypothermia. We therefore explored if hypothermia affects the downstream target of GSK3β, β-catenin, a critical transcription factor. Glycogen synthase kinase 3β phosphorylase β-catenin leading to its degradation and apoptosis (Zhao et al, 2005a). Despite protein levels of phosphorylated β-catenin being unknown, we have found that β-catenin translocates into nuclei in the penumbra after stroke, and that hypothermia blocks such translocation. This result suggests that hypothermia may block ischemic damage by blocking events downstream of GSK3β (Zhao et al, 2005a).

In sum, hypothermia provides multiple protective effects against damaging cascades after stroke. However, contrary to the popular belief that hypothermia simply blocks all apoptotic or necrotic pathways, it actually specifically upregulates or downregulates expression of certain beneficial or detrimental genes. Nevertheless, one must be cautious not to over extrapolate these findings to draw conclusions concerning the roles of such genes in pro- or anti ischemic damage, since hypothermia may provide overall protection in several pathways, but does not always promote expression of all beneficial genes nor does it inhibit every detrimental event.

Clinical Translation and Future Directions

Hypothermic studies done in the laboratory have led to clinical investigations for cerebral ischemia. Four recent landmark prospective randomized-controlled studies showed that induced mild to moderate hypothermia improved neurologic function in patients suffering cardiac arrest from ventricular fibrillation (Hypothermia after Cardiac Arrest Study Group, 2002; Bernard et al, 2002) and reduced risk of death or disability in neonates hypoxic—ischemic encephalopathy (Gluckman et al, 2005; Shankaran et al, 2005). In the case of acute stroke treatment, temperature on admission correlates with neurologic outcome: hyperthermia is associated with poor prognosis (den Hertog et al, 2007; Krieger et al, 2005). Therefore, hyperthermia prevention for acute stroke patients is warranted (Ginsberg and Busto, 1998) by using antipyretics to inhibit hyperthermia (e.g., den Hertog et al, 2007).

Several preliminary clinical trials have been completed to test the protection of induced mild to moderate hypothermia against focal stroke (den Hertog et al, 2007; Krieger et al, 2005) and three trials are still ongoing (http://www.strokecenter.org). In these studies, cooling methods from surface cooling to endovascular cooling have been used. Mild to moderate hypothermia was instituted from 6 h to 1 day after stroke onset, and target temperatures were achieved from 1 to 6 h after cooling application (den Hertog et al, 2007; Krieger et al, 2005). These studies have confirmed the feasibility and safety of hypothermia application, but no definite beneficial effect was reported.

Although we cannot directly extrapolate settings in the laboratory into clinical trials, a comparison might provide insights for future clinical trials. In the laboratory, hypothermia must be initiated at very early hours after reperfusion in transient focal ischemia to achieve neuroprotection, and it has minimal protective effects in permanent ischemic models. However, in most clinical studies, mild to moderate hypothermia was initiated as late as 5 to 6 h after focal stroke, and it took one to several hours to reach target temperature (den Hertog et al, 2007; Krieger et al, 2005). In addition, patients might not have reperfusion or reperfusion occurred in a very late stage. Although laboratory studies have provided strong rational for clinical application of hypothermia for acute stroke treatment, we must emphasize that the protective effect of hypothermia is limited even in the laboratory. In the laboratory, a number of crucial variables need to be considered, including the onset time of hypothermia, its depth, and whether the models used include reperfusion. Compared with experimental stroke in the laboratory, it is not surprising that beneficial effects were not found in the past clinical trials. Therefore, entrance criteria for clinical trials must be carefully re-considered. For patients with focal stroke, early reperfusion and rapid hypothermia initiation should be used to achieve protection.

Understanding the protective mechanisms involved in hypothermia provides insights for clinical trials. To date, the intraischemic hypothermia studies suggest that the effects of hypothermia on acute events during ischemia are not the sole decisive events underlying its neuroprotection. Overemphasizing the effects of hypothermia on ATP depletion, anoxic depolarization, glutamate release, and Ca²⁺ mobilization during ischemia may be misleading. Instead, studies of the effects of hypothermia on the expression of various genes after reperfusion have enhanced our understanding of the mechanisms of ischemic neuronal damage. Hypothermia not only blocks multiple damaging cascades after stroke, it also selectively upregulates some protective genes. In fact, hypothermia provides a unique opportunity for us to understand the function of some genes that have puzzled us.

Unfortunately, despite the great potential clinical relevance of postischemic hypothermia, few have studied its protective mechanisms; future projects will concentrate on cellular and molecular mechanisms of postischemic hypothermia. In addition, how gender, age, and rewarming factor into protection by hypothermia need to be studied in the laboratory. Finally, the literature shows that the protective effects of hypothermia, especially when applied after stroke, are highly variable; additional well-designed experiments are needed to resolve these outstanding issues.

Footnotes

Acknowledgements

We thank Dr David Schaal, Dr Bruce Schaar, and Ms Felicia F Beppu for manuscript assistance and Ms Elizabeth Hoyte for figure preparation.

References

Akaji

Suga

Fujino

Mayanagi

Inamasu

Horiguchi

Sato

Kawase

(2003) Effect of intraischemic hypothermia on the expression of c-Fos and c-Jun, and DNA binding activity of AP-1 after focal cerebral ischemia in rat brain. Brain Res 975:149–57

Akins

Liu

Hsu

(1996) Immediate early gene expression in response to cerebral ischemia. Friend or foe? Stroke 27:1682–7

Aoki

Tamatani

Taniguchi

Yamaguchi

Bando

Kasai

Miyoshi

Nakamura

Vitek

Tohyama

Tanaka

Sugimoto

(2001) Hypothermic treatment restores glucose regulated protein 78 (GRP78) expression in ischemic brain. Brain Res Mol Brain Res 95:117–28

Asai

Zhao

Kohno

Takahashi

Nagata

Ishikawa

(2000) Quantitative evaluation of extracellular glutamate concentration in postischemic glutamate re-uptake, dependent on brain temperature, in the rat following severe global brain ischemia. Brain Res 864:60–8

Asai

Zhao

Takahashi

Nagata

Kohno

Ishikawa

(1998) Minimal effect of brain temperature changes on glutamate release in rat following severe global brain ischemia: a dialysis electrode study. Neuroreport 9:3863–8

Baker

Onesti

Barth

Prestigiacomo

Solomon

(1991) Hypothermic protection following middle cerebral artery occlusion in the rat. Surg Neurol 36:175–80

Baldwin

Kirsch

Hurn

Toung

Traystman

(1991) Hypothermic cerebral reperfusion and recovery from ischemia. Am J Physiol 261:H774–81

Bart

Takaoka

Pearlstein

Dexter

Warner

(1998) Interactions between hypothermia and the latency to ischemic depolarization: implications for neuroprotection. Anesthesiology 88:1266–73

Berger

Schabitz

Georgiadis

Steiner

Aschoff

Schwab

(2002) Effects of hypothermia on excitatory amino acids and metabolism in stroke patients: a microdialysis study. Stroke 33:519–24

10.

Berger

Jensen

Hossmann

Paschen

(1998) Effect of mild hypothermia during and after transient in vitro ischemia on metabolic disturbances in hippocampal slices at different stages of development. Brain Res Dev Brain Res 105:67–77

11.

Bernard

Gray

Buist

Jones

Silvester

Gutteridge

Smith

(2002) Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. New Engl J Med 346:557–63

12.

Boris-Moller

Kamme

Wieloch

(1998) The effect of hypothermia on the expression of neurotrophin mRNA in the hippocampus following transient cerebral ischemia in the rat. Brain Res Mol Brain Res 63:163–73

13.

Boris-Moller

Wieloch

(1998) Changes in the extracellular levels of glutamate and aspartate during ischemia and hypoglycemia. Effects of hypothermia. Exp Brain Res 121:277–84

14.

Busto

Dietrich

Globus

Ginsberg

(1989a) Postischemic moderate hypothermia inhibits CA1 hippocampal ischemic neuronal injury. Neurosci Lett 101:299–304

15.

Busto

Dietrich

Globus

Valdes

Scheinberg

Ginsberg

(1987) Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 7:729–38

16.

Busto

Globus

Dietrich

Martinez

Valdes

Ginsberg

(1989b) Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke 20:904–10

17.

Cao

Clark

Pei

Yin

Zhang

Sun

Graham

Chen

(2003) Translocation of apoptosis-inducing factor in vulnerable neurons after transient cerebral ischemia and in neuronal cultures after oxygen-glucose deprivation. J Cereb Blood Flow Metab 23:1137–50

18.

Cardell

Boris-Moller

Wieloch

(1991) Hypothermia prevents the ischemia-induced translocation and inhibition of protein kinase C in the rat striatum. J Neurochem 57:1814–7

19.

Carroll

Beek

(1992) Protection against hippocampal CA1 cell loss by post-ischemic hypothermia is dependent on delay of initiation and duration. Metab Brain Dis 7:45–50

20.

Chan

(2001) Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 21:2–14

21.

Chen

Chopp

Bodzin

Chen

(1993) Temperature modulation of cerebral depolarization during focal cerebral ischemia in rats: correlation with ischemic injury. J Cereb Blood Flow Metab 13:389–94

22.

Choi

Rothman

(1990) The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci 13:171–82

23.

Chopp

Chen

Dereski

Garcia

(1991) Mild hypothermic intervention after graded ischemic stress in rats. Stroke 22:37–43

24.

Chopp

Dereski

Levine

Yoshida

Garcia

(1992) Hypothermia reduces 72-kDa heat-shock protein induction in rat brain after transient forebrain ischemia. Stroke 23:104–7

25.

Coimbra

Wieloch

(1992) Hypothermia ameliorates neuronal survival when induced 2 hours after ischaemia in the rat. Acta Physiol Scand 146:543–4

26.

Coimbra

Wieloch

(1994) Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia. Acta Neuropathol (Berl) 87:325–31

27.

Colbourne

Corbett

(1994) Delayed and prolonged post-ischemic hypothermia is neuroprotective in the gerbil. Brain Res 654:265–72

28.

Colbourne

Corbett

(1995) Delayed postischemic hypothermia: a six month survival study using behavioral and histological assessments of neuroprotection. J Neurosci 15:7250–60

29.

Colbourne

Sutherland

Corbett

(1997) Postischemic hypothermia. A critical appraisal with implications for clinical treatment. Mol Neurobiol 14:171–201

30.

Colbourne

Sutherland

Auer

(1999) Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia. J Neurosci 19:4200–10

31.

Corbett

Thornhill

(2000) Temperature modulation (hypothermic and hyperthermic conditions) and its influence on histological and behavioral outcomes following cerebral ischemia. Brain Pathol 10:145–52

32.

D'Cruz

Fertig

Filiano

Hicks

DeFranco

Callaway

(2002) Hypothermic reperfusion after cardiac arrest augments brain-derived neurotrophic factor activation. J Cereb Blood Flow Metab 22: 843–851

33.

Dempsey

Combs

Maley

Cowen

Roy

Donaldson

(1987) Moderate hypothermia reduces postischemic edema development and leukotriene production. Neurosurgery 21:177–81

34.

den Hertog

van der Worp

van Gemert

Dippel

(2007) Therapeutic hypothermia in acute ischemic stroke. Expert Rev Neurother 7:155–64

35.

Dietrich

Busto

Halley

Valdes

(1990) The importance of brain temperature in alterations of the blood—brain barrier following cerebral ischemia. J Neuropathol Exp Neurol 49:486–97

36.

Doerfler

Schwab

Hoffmann

Engelhorn

Forsting

(2001) Combination of decompressive craniectomy and mild hypothermia ameliorates infarction volume after permanent focal ischemia in rats. Stroke 32:2675–81

37.

Eberspacher

Werner

Engelhard

Pape

Gelb

Hutzler

Henke

Kochs

(2003) The effect of hypothermia on the expression of the apoptosis-regulating protein Bax after incomplete cerebral ischemia and reperfusion in rats. J Neurosurg Anesthesiol 15:200–8

38.

Erecinska

Thoresen

Silver

(2003) Effects of hypothermia on energy metabolism in mammalian central nervous system. J Cereb Blood Flow Metab 23:513–30

39.

Friedman

Ginsberg

Belayev

Busto

Alonso

Lin

Globus

(2001) Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia. Brain Res Mol Brain Res 86:34–47

40.

Ginsberg

Busto

(1998) Combating hyperthermia in acute stroke: a significant clinical concern. Stroke 29:529–34

41.

Gluckman

Wyatt

Azzopardi

Ballard

Edwards

Ferriero

Polin

Robertson

Thoresen

Whitelaw

Gunn

(2005) Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet 365:663–70

42.

Graham

Chen

(2001) Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 21:99–109

43.

Halterman

Federoff

(1999) HIF-1alpha and p53 promote hypoxia-induced delayed neuronal death in models of CNS ischemia. Exp Neurol 159:65–72

44.

Hamann

Burggraf

Martens

Liebetrau

Jager

Wunderlich

DeGeorgia

Krieger

(2004) Mild to moderate hypothermia prevents microvascular basal lamina antigen loss in experimental focal cerebral ischemia. Stroke 35:764–9

45.

Han

Karabiyikoglu

Kelly

Sobel

Yenari

(2003) Mild hypothermia inhibits nuclear factor-kappaB translocation in experimental stroke. J Cereb Blood Flow Metab 23:589–98

46.

Harada

Maekawa

Tsuruta

Kaneko

Sadamitsu

Yamashima

Yoshida Ki

(2002) Hypothermia inhibits translocation of CaM kinase II and PKC-alpha, beta, gamma isoforms and fodrin proteolysis in rat brain synaptosome during ischemia—reperfusion. J Neurosci Res 67:664–9

47.

Haraldseth

Gronas

Southon

Thommessen

Borchgrevink

Jynge

Gisvold

Unsgard

(1992) The effects of brain temperature on temporary global ischaemia in rat brain. A 31-phosphorous NMR spectroscopy study. Acta Anaesthesiol Scand 36:393–9

48.

Hicks

Parmele

DeFranco

Klann

Callaway

(2000) Hypothermia differentially increases extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest. Neuroscience 98:677–85

49.

Hoffman

Thomas

(1996) Effects of graded hypothermia on outcome from brain ischemia. Neurol Res 18:185–9

50.

Horiguchi

Shimizu

Ogino

Suga

Inamasu

Kawase

(2003) Postischemic hypothermia inhibits the generation of hydroxyl radical following transient forebrain ischemia in rats. J Neurotrauma 20:511–20

51.

Horstmann

Kalb

Koziol

Gardner

Wagner

(2003) Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients: influence of different therapies. Stroke 34:2165–70

52.

Janelidze

Ginsberg

Busto

Perez-Pinzon

Sick

Siesjo

Liu

(2001) Protein aggregation after focal brain ischemia and reperfusion. J Cereb Blood Flow Metab 21:865–75

53.

Huang

Zhou

Yang

(1998) The effect of extending mild hypothermia on focal cerebral ischemia and reperfusion in the rat. Neurol Res 20:57–62

54.

Huang

Xue

Preston

Karbalai

Buchan

(1999) Biphasic opening of the blood—brain barrier following transient focal ischemia: effects of hypothermia. Can J Neurol Sci 26:298–304

55.

Huh

Belayev

Zhao

Koch

Busto

Ginsberg

(2000) Comparative neuroprotective efficacy of prolonged moderate intraischemic and postischemic hypothermia in focal cerebral ischemia. J Neurosurg 92:91–9

56.

Hypothermia after Cardiac Arrest Study Group (2002) Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. New Engl J Med 346:549–56

57.

Ibayashi

Takano

Ooboshi

Kitazono

Sadoshima

Fujishima

(2000) Effect of selective brain hypothermia on regional cerebral blood flow and tissue metabolism using brain thermo-regulator in spontaneously hypertensive rats. Neurochem Res 25:369–75

58.

Inamasu

Suga

Sato

Horiguchi

Akaji

Mayanagi

Kawase

(2000) Postischemic hypothermia attenuates apoptotic cell death in transient focal ischemia in rats. Acta Neurochir Suppl 76:525–7

59.

Jenkins

DeWitt

Johnston

Davis

Prough

(2001) Intraischemic mild hypothermia increases hippocampal CA1 blood flow during forebrain ischemia. Brain Res 890:1–10

60.

Jiang

Chopp

Zhang

Helpern

Ordidge

Ewing

Jiang

Marchese

(1994) The effect of hypothermia on transient focal ischemia in rat brain evaluated by diffusion- and perfusion-weighted NMR imaging. J Cereb Blood Flow Metab 14:732–41

61.

Kader

Brisman

Maraire

Huh

Solomon

(1992) The effect of mild hypothermia on permanent focal ischemia in the rat. Neurosurgery 31:1056–60; discussion 1060-1061

62.

Kader

Frazzini

Baker

Solomon

Trifiletti

(1994) Effect of mild hypothermia on nitric oxide synthesis during focal cerebral ischemia. Neurosurgery 35:272–7; discussion 277

63.

Kaminogo

Ichikura

Onizuka

Takahata

Matsuo

Kitagawa

Shibata

(1999) Mild hypothermia on anoxic depolarization and subsequent cortical injury following transient ischemia. Neurol Res 21:670–6

64.

Kaminska

(2005) MAPK signalling pathways as molecular targets for anti-inflammatory therapy—from molecular mechanisms to therapeutic benefits. Biochim Biophys Acta 1754:253–62

65.

Kamme

Campbell

Wieloch

(1995) Biphasic expression of the fos and jun families of transcription factors following transient forebrain ischaemia in the rat. Effect of hypothermia. Eur J Neurosci 7:2007–16

66.

Karabiyikoglu

Han

Yenari

Steinberg

(2003) Attenuation of nitric oxide synthase isoform expression by mild hypothermia after focal cerebral ischemia: variations depending on timing of cooling. J Neurosurg 98:1271–6

67.

Karibe

Chen

Zarow

Gafni

Graham

Chan

Weinstein

(1994a) Mild intraischemic hypothermia suppresses consumption of endogenous anti-oxidants after temporary focal ischemia in rats. Brain Res 649:12–8

68.

Karibe

Sato

Shimizu

Tominaga

Koshu

Yoshimoto

(2000) Intraoperative mild hypothermia ameliorates postoperative cerebral blood flow impairment in patients with aneurysmal subarachnoid hemorrhage. Neurosurgery 47:594–9; discussion 599-601

69.

Karibe

Zarow

Graham

Weinstein

(1994b) Mild intraischemic hypothermia reduces postischemic hyperperfusion, delayed postischemic hypoperfusion, blood—brain barrier disruption, brain edema, and neuronal damage volume after temporary focal cerebral ischemia in rats. J Cereb Blood Flow Metab 14:620–7

70.

Kawai

Okauchi

Morisaki

Nagao

(2000) Effects of delayed intraischemic and postischemic hypothermia on a focal model of transient cerebral ischemia in rats. Stroke 31:1982–9; discussion 1989

71.

Kawanishi

(2003) Effect of hypothermia on brain edema formation following intracerebral hemorrhage in rats. Acta Neurochir Suppl 86:453–6

72.

Kelly

Zhang

Zhao

Giffard

Sapolsky

Yenari

Steinberg

(2002) Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2. Ann Neurol 52:160–7

73.

Kelly

Zhao

Hua Sun

Cheng

Qiao

Luo

Martin

Steinberg

Harrison

Yenari

(2004) Glycogen synthase kinase 3beta inhibitor Chir025 reduces neuronal death resulting from oxygen-glucose deprivation, glutamate excitotoxicity, and cerebral ischemia. Exp Neurol 188:378–86

74.

Kil

Zhang

Piantadosi

(1996) Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats. J Cereb Blood Flow Metab 16:100–6

75.

Kilic

Soliz

Bassetti

Gassmann

Hermann

(2005) Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J 19:2026–8

76.

Kimura

Sako

Tanaka

Kusakabe

Tanaka

Nakada

(2002) Effect of mild hypothermia on energy state recovery following transient forebrain ischemia in the gerbil. Exp Brain Res 145:83–90

77.

Koinig

Morimoto

Zornow

(2001a) The combination of lamotrigine and mild hypothermia prevents ischemia-induced increase in hippocampal glutamate. J Neurosurg Anesthesiol 13:106–12

78.

Koinig

Vornik

Rueda

Zornow

(2001b) Lubeluzole inhibits accumulation of extracellular glutamate in the hippocampus during transient global cerebral ischemia. Brain Res 898:297–302

79.

Kozlowski

Buchan

Tuor

Xue

Huang

Chaundy

Saunders

(1997) Effect of temperature in focal ischemia of rat brain studied by 31P and 1H spectroscopic imaging. Magn Reson Med 37:346–54

80.

Krieger

Schwab

Kammersgard

(2005) Focal cerebral ischemia: clinical studies. In: Tisherman

Sterz

(eds), Therapautic hypothermia 1st edn. Springer, pp 43–61

81.

Krieger

Yenari

(2004) Therapeutic hypothermia for acute ischemic stroke: what do laboratory studies teach us? Stroke 35:1482–9

82.

Kristian

Katsura

Siesjo

(1992) The influence of moderate hypothermia on cellular calcium uptake in complete ischaemia: implications for the excitotoxic hypothesis. Acta Physiol Scand 146:531–2

83.

Kuboyama

Safar

Radovsky

Tisherman

Stezoski

Alexander

(1993) Delay in cooling negates the beneficial effect of mild resuscitative cerebral hypothermia after cardiac arrest in dogs: a prospective, randomized study. Crit Care Med 21:1348–1358

84.

Kumar

Evans

(1996) Expression of c-fos and fos-B proteins following transient forebrain ischemia: effect of hypothermia. Brain Res Mol Brain Res 42:337–43

85.

Miyashita

Howllet

Siesjo

Shuaib

(1999) Hypothermia ameliorates ischemic brain damage and suppresses the release of extracellular amino acids in both normo- and hyperglycemic subjects. Exp Neurol 158:242–53

86.

Liebetrau

Burggraf

Martens

Pichler

Hamann

(2004) Delayed moderate hypothermia reduces calpain activity and breakdown of its substrate in experimental focal cerebral ischemia in rats. Neurosci Lett 357:17–20

87.

Steinberg

Panahian

Maidment

Newcomb

(1993) Profiles of extracellular amino acid changes in focal cerebral ischaemia: effects of mild hypothermia. Neurol Res 15:281–7

88.

Luo

Ling

Zhang

Cao

Chen

(2007) Impaired DNA repair via the base-excision repair pathway after focal ischemic brain injury: a protein phosphorylation-dependent mechanism reversed by hypothermic neuroprotection. Front Biosci 12:1852–62

89.

Macleod

Johansson

Soderstrom

Lai

Gido

Wieloch

Seckl

Olsson

(2003) Mineralocorticoid receptor expression and increased survival following neuronal injury. Eur J Neurosci 17:1549–55

90.

MacManus

Buchan

(2000) Apoptosis after experimental stroke: fact or fashion? J Neurotrauma 17:899–914

91.

Maeda

Hata

Gillardon

Hossmann

(2001) Aggravation of brain injury after transient focal ischemia in p53-deficient mice. Brain Res Mol Brain Res 88:54–61

92.

Maher

Hachinski

(1993) Hypothermia as a potential treatment for cerebral ischemia. Cerebrovasc Brain Metab Rev 5:277–300

93.

Maier

Sun

Cheng

Yenari

Chan

Steinberg

(2002) Effects of mild hypothermia on superoxide anion production, superoxide dismutase expression, and activity following transient focal cerebral ischemia. Neurobiol Dis 11:28–42

94.

Maier

Sun

Kunis

Yenari

Steinberg

(2001) Delayed induction and long-term effects of mild hypothermia in a focal model of transient cerebral ischemia: neurological outcome and infarct size. J Neurosurg 94:90–6

95.

Mancuso

Derugin

Hara

Sharp

Weinstein

(2000) Mild hypothermia decreases the incidence of transient ADC reduction detected with diffusion MRI and expression of c-fos and hsp70 mRNA during acute focal ischemia in rats. Brain Res 887:34–45

96.

Martinez-Tica

Zornow

(2000) Effects of adenosine agonists and an antagonist on excitatory transmitter release from the ischemic rabbit hippocampus. Brain Res 872:110–5

97.

Mitani

Kadoya

Kataoka

(1991) Temperature dependence of hypoxia-induced calcium accumulation in gerbil hippocampal slices. Brain Res 562:159–63

98.

Mitani

Kataoka

(1991) Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia: brain microdialysis study. Neuroscience 42:661–70

99.

Mori

Miyazaki

Iwase

Maeda

(2002) Temporal profile of changes in brain tissue extracellular space and extracellular ion (Na(+), K(+)) concentrations after cerebral ischemia and the effects of mild cerebral hypothermia. J Neurotrauma 19:1261–70

100.

Morikawa

Ginsberg

Dietrich

Duncan

Kraydieh

Globus

Busto

(1992) The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12:380–9

101.

Moyer

Welsh

Zager

(1992) Spontaneous cerebral hypothermia diminishes focal infarction in rat brain. Stroke 23:1812–6

102.

Nakashima

Todd

(1996) Effects of hypothermia, pentobarbital, and isoflurane on postdepolarization amino acid release during complete global cerebral ischemia. Anesthesiology 85:161–8

103.

Nedergaard

Hansen

(1993) Characterization of cortical depolarizations evoked in focal cerebral ischemia. J Cereb Blood Flow Metab 13:568–74

104.

Obrenovitch

Urenjak

(1997) Altered glutamatergic transmission in neurological disorders: from high extracellular glutamate to excessive synaptic efficacy. Prog Neurobiol 51:39–87

105.

Ohta

Terao

Shintani

Kiyota

(2007) Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia. Neurosci Res 57:424–33

106.

Ohta

Graf

Rosner

Heiss

(1997) Profiles of cortical tissue depolarization in cat focal cerebral ischemia in relation to calcium ion homeostasis and nitric oxide production. J Cereb Blood Flow Metab 17:1170–81

107.

Patel

Drummond

Cole

Yaksh

(1994) Differential temperature sensitivity of ischemia-induced glutamate release and eicosanoid production in rats. Brain Res 650:205–11

108.

Phanithi

Yoshida

Santana

Kawamura

Yasui

(2000) Mild hypothermia mitigates post-ischemic neuronal death following focal cerebral ischemia in rat brain: immunohistochemical study of Fas, caspase-3 and TUNEL. Neuropathology 20:273–82

109.

Phillis

Ren

O'Regan

(2000) Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate. Brain Res 868:105–12

110.

Preston

Webster

(2004) A two-hour window for hypothermic modulation of early events that impact delayed opening of the rat blood—brain barrier after ischemia. Acta Neuropathol (Berl) 108:406–12

111.

Ren

Hashimoto

Pulsinelli

Nowak

Jr (2004) Hypothermic protection in rat focal ischemia models: strain differences and relevance to ‘reperfusion injury’. J Cereb Blood Flow Metab 24:42–53

112.

Ridenour

Warner

Todd

McAllister

(1992) Mild hypothermia reduces infarct size resulting from temporary but not permanent focal ischemia in rats. Stroke 23:733–8

113.

Rosenberg

(2002) Matrix metalloproteinases in neuroinflammation. Glia 39:279–91

114.

Rossi

Oshima

Attwell

(2000) Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 403:316–21

115.

Sayegh

Sershen

Lajtha

(1992) Different effects of hypothermia on amino acid incorporation and on amino acid uptake in the brain in vivo. Neurochem Res 17:553–7

116.

Shankaran

Laptook

Ehrenkranz

Tyson

McDonald

Donovan

Fanaroff

Poole

Wright

Higgins

Finer

Carlo

Duara

Cotten

Stevenson

Stoll

Lemons

Guillet

Jobe

(2005) Whole-body hypothermia for neonates with hypoxic—ischemic encephalopathy. New Engl J Med 353:1574–84

117.

Shimizu

Chang

Litt

Zarow

Weinstein

(1997) Effect of brain, body, and magnet bore temperatures on energy metabolism during global cerebral ischemia and reperfusion monitored by magnetic resonance spectroscopy in rats. Magn Reson Med 37:833–9

118.

Shimohata

Zhao

Steinberg

(2007a) Epsilon PKC may contribute to the protective effect of hypothermia in a rat focal cerebral ischemia model. Stroke 38:375–80

119.

Shimohata

Zhao

Sung

Sun

Mochly-Rosen

Steinberg

(2007b) Suppression of deltaPKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia. J Cereb Blood Flow Metab, doi:10.1038/sj.jcbfm.9600450

120.

Sick

Perez-Pinzon

(1999) Mild hypothermia improves recovery of cortical extracellular potassium ion activity and excitability after middle cerebral artery occlusion in the rat. Stroke 30:2416–21; discussion 2422

121.

Sonn

Granot

Etziony

Mayevsky

(2002) Effect of hypothermia on brain multi-parametric activities in normoxic and partially ischemic rats. Comp Biochem Physiol A Mol Integr Physiol 132:239–46

122.

Suehiro

Povlishock

(2001) Exacerbation of traumatically induced axonal injury by rapid posthypothermic rewarming and attenuation of axonal change by cyclosporin A. J Neurosurg 94:493–8

123.

Sugawara

Noshita

Lewen

Gasche

Ferrand-Drake

Fujimura

Morita-Fujimura

Chan

(2002) Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation. J Neurosci 22:209–17

124.

Sugimura

Sako

Tohyama

Yonemasu

(1998) Consecutive in vivo measurement of nitric oxide in transient forebrain ischemic rat under normothermia and hypothermia. Brain Res 808:313–6

125.

Sutton

Clark

Norwood

Woodford

Welsh

(1991) Global cerebral ischemia in piglets under conditions of mild and deep hypothermia. Stroke 22:1567–73

126.

Takeda

Namba

Higuchi

Hagioka

Takata

Hirakawa

Morita

(2003) Quantitative evaluation of the neuroprotective effects of hypothermia ranging from 34 degrees C to 31 degrees C on brain ischemia in gerbils and determination of the mechanism of neuroprotection. Crit Care Med 31:255–60

127.

Tohyama

Sako

Yonemasu

(1998) Hypothermia attenuates the activation of protein kinase C in focal ischemic rat brain: dual autoradiographic study of [3H]phorbol 12, 13-dibutyrate and iodo[14C]antipyrine. Brain Res 782:348–51

128.

Tomasevic

Kamme

Stubberod

Wieloch

(1999) The tumor suppressor p53 and its response gene p21WAF1/Cip1 are not markers of neuronal death following transient global cerebral ischemia. Neuroscience 90:781–92

129.

Torres

Pulido

(2001) The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J Biol Chem 276:993–998

130.

Toyoda

Suzuki

Kassell

Lee

(1996) Intraischemic hypothermia attenuates neutrophil infiltration in the rat neocortex after focal ischemia—reperfusion injury. Neurosurgery 39:1200–5

131.

Tsuchiya

Hong

Suh

Kayama

Panter

Weinstein

(2002) Mild hypothermia reduces zinc translocation, neuronal cell death, and mortality after transient global ischemia in mice. J Cereb Blood Flow Metab 22:1231–8

132.

Ueda

Suehiro

Wei

Kontos

Povlishock

(2004) Uncomplicated rapid posthypothermic rewarming alters cerebrovascular responsiveness. Stroke 35:601–6

133.

Van Hemelrijck

Hachimi-Idrissi

Sarre

Ebinger

Michotte

(2005) Post-ischaemic mild hypothermia inhibits apoptosis in the penumbral region by reducing neuronal nitric oxide synthase activity and thereby preventing endothelin-1-induced hydroxyl radical formation. Eur J Neurosci 22:1327–37

134.

Van Hemelrijck

Vermijlen

Hachimi-Idrissi

Sarre

Ebinger

Michotte

(2003) Effect of resuscitative mild hypothermia on glutamate and dopamine release, apoptosis and ischaemic brain damage in the endothelin-1 rat model for focal cerebral ischaemia. J Neurochem 87:66–75

135.

van Lookeren Campagne

Gill

(1996) Ultrastructural morphological changes are not characteristic of apoptotic cell death following focal cerebral ischaemia in the rat. Neurosci Lett 213:111–4

136.

Vazquez

Ramaswamy

Nakamura

Sellers

(2000) Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell Biol 20:5010–8

137.

Wagner

Nagel

Kluge

Schwab

Heiland

Koziol

Gardner

Hacke

(2003) Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia. Brain Res 984:63–75

138.

Wang

Deng

Maier

Sun

Yenari

(2002) Mild hypothermia reduces ICAM-1 expression, neutrophil infiltration and microglia/monocyte accumulation following experimental stroke. Neuroscience 114:1081–1090

139.

Welsh

Sims

Harris

(1990) Mild hypothermia prevents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab 10:557–63

140.

Winfree

Baker

Connolly

Jr Fiore

Solomon

(1996) Mild hypothermia reduces penumbral glutamate levels in the rat permanent focal cerebral ischemia model. Neurosurgery 38:1216–22

141.

Evans

Kumar

(1995) Hypothermia preserves expression of beta-actin mRNA in ischemic brain. Neuroreport 7:302–4

142.

Yamamoto

Mitani

Cui

Takechi

Irita

Suga

Arai

Kataoka

(1999) Neuroprotective effect of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia. Neuroscience 91:501–9

143.

Yamashita

Eguchi

Kajiwara

Ito

(1991) Mild hypothermia ameliorates ubiquitin synthesis and prevents delayed neuronal death in the gerbil hippocampus. Stroke 22:1574–81

144.

Yanamoto

Hong

Soleau

Kassell

Lee

(1996) Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex. Brain Res 718:207–11

145.

Yanamoto

Nagata

Niitsu

Zhang

Xue

Sakai

Kikuchi

(2001) Prolonged mild hypothermia therapy protects the brain against permanent focal ischemia. Stroke 32:232–9

146.

Yenari

Iwayama

Cheng

Sun

Fujimura

Morita-Fujimura

Chan

Steinberg

(2002) Mild hypothermia attenuates cytochrome c release but does not alter Bcl-2 expression or caspase activation after experimental stroke. J Cereb Blood Flow Metab 22:29–38

147.

Zhang

Sobel

Cheng

Steinberg

Yenari

(2001) Mild hypothermia increases Bcl-2 protein expression following global cerebral ischemia. Brain Res Mol Brain Res 95:75–85

148.

Zhao

Asai

Kohno

Ishikawa

(1998) Effects of brain temperature on CBF thresholds for extracellular glutamate release and reuptake in the striatum in a rat model of graded global ischemia. Neuroreport 9:3183–8

149.

Zhao

Asai

Ishikawa

(1999) Neither L-NAME nor L-arginine changes extracellular glutamate elevation and anoxic depolarization during global ischemia and reperfusion in rat. Neuroreport 10:313–8

150.

Zhao

Asai

Kanematsu

Kunimatsu

Kohno

Ishikawa

(1997) Real-time monitoring of the effects of normothermia and hypothermia on extracellular glutamate re-uptake in the rat following global brain ischemia. Neuroreport 8:2389–93

151.

Zhao

Sapolsky

Steinberg

(2006) PI3K/Akt survival signal pathways are implicated in neuronal survival after stroke. Mol Neurobiol 234:249–70

152.

Zhao

Shimohata

Wang

Sun

Schaal

Sapolsky

Steinberg

(2005a) Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats. J Neurosci 25:9794–806

153.

Zhao

Wang

Shimohata

Sun

Yenari

Sapolsky

Steinberg

(2007) Conditions of protection by hypothermia and effects on apoptotic pathways in a model of permanent middle cerebral artery occlusion. J Neurosurg, in press

154.

Zhao

Yenari

Cheng

Barreto-Chang

Sapolsky

Steinberg

(2004) Bcl-2 transfection via herpes simplex virus blocks apoptosis inducing factor translocation after focal ischemia in rat. J Cereb Blood Flow Metab 24:681–92

155.

Zhao

Yenari

Cheng

Sapolsky

Steinberg

(2005b) Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia. J Cereb Blood Flow Metab 25:1119–29

156.

Zheng

Yenari

(2004) Post-ischemic inflammation: molecular mechanisms and therapeutic implications. Neurol Res 26:884–92

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

Abstract

Keywords

Introduction

The Ostensible Pan-Inhibitive Effects of Hypothermia

Hypothermia Does Not Always Attenuate Stroke Even in the Laboratory

The Effects of Hypothermia on Cerebral Blood Flow and Metabolism

Cerebral Blood Flow

Hypothermia Promotes Energy Recovery after Reperfusion

Hypothermia's Effect on Anoxic Depolarization and Glutamate Release

Anoxic Depolarization

Glutamate Release

Hypothermia Does Not Simply Block Cell Signaling Pathways of Apoptosis or Necrosis

The Jun N-Terminal Kinase/Early Genes and the Erk Pathway

Hypothermia Unexpectedly Enhances the p53 Pathway

Caspase and Cytochrome c-Mediated Apoptotic Pathways

Hypothermia Does Not Indiscriminately Upregulate all Signals in the Akt/protein kinase B Pathway

Clinical Translation and Future Directions

Footnotes

Acknowledgements

References