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(Stroke. 1996;27:1578-1585.)
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Articles

Long-lasting Neuroprotective Effect of Postischemic Hypothermia and Treatment With an Anti-inflammatory/Antipyretic Drug

Evidence for Chronic Encephalopathic Processes Following Ischemia

Cicero Coimbra, MD, PhD; Mikael Drake, MD; Fredrik Boris-Moller, MD Tadeusz Wieloch, MD, PhD

the Laboratory for Experimental Brain Research, Lund (Sweden) University, and Laboratory for Experimental Neurology, Escola Paulista de Medicina, Sao Paulo, Brazil (C.C.)

Correspondence to Dr Cicero Coimbra, Laboratory for Experimental Neurology, Escola Paulista de Medicina, R Botucatu 862, 04023-900, Sao Paulo, Brazil.

	Abstract

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Background and Purpose It has been recognized that postischemic pharmacological interventions may delay the evolution of neuronal damage rather than provide long-lasting neuroprotection. Also, fever complicates recovery after stroke in humans. Here we report the effects of late postischemic treatment with hypothermia and an antipyretic/anti-inflammatory drug, dipyrone, on cell damage at 1 week and 2 months of survival.

Methods Rats were subjected to 10 minutes of forebrain ischemia. Hypothermia (33°C) was induced at 2 hours of recovery and maintained for 7 hours. Dipyrone (100 mg·kg^-1IP) was given every 3 hours from 14 to 72 hours of recovery. Temperature was measured every 6 hours for 60 days. Neuronal damage was assessed at 7 days and 2 months of recovery.

Results From 17 to 72 hours of recovery, a period of hyperthermia was observed, which dipyrone abolished but postischemic hypothermia treatment did not. Dipyrone treatment diminished neuronal damage by 43% at 7 days, and at 2 months of survival, a minor (16%) protection was seen. Postischemic hypothermia treatment alone delayed neuronal damage. In contrast, combined treatment of hypothermia followed by dipyrone markedly diminished neuronal damage by more than 50% at both 7 days and 2 months of recovery.

Conclusions Neuronal degeneration may be ongoing for months after a transient ischemic insult, and prolonged protective measures need to be instituted for long-lasting neuroprotective effects. Hyperthermia during recovery worsens ischemic damage, and processes associated with inflammation may contribute to the development of neuronal damage. An early and extended period of postischemic hypothermia provides a powerful and long-lasting protection if followed by treatment with anti-inflammatory/antipyretic drugs.

Key Words: temperature • neuronal damage • rats • hypothermia • anti-inflammatory agents

	Introduction

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Recent studies have demonstrated that body temperature is a major determinant of neuronal survival long into the recovery phase after cerebral ischemia. Provided that moderate hypothermia is properly long,^{1 2} it may reduce or delay neuronal death when induced several hours into the recovery phase. Moderate hypothermia (33°C) from 2 to 7 hours of recovery largely reduced the number of degenerating neurons in most rat forebrain structures at 7 days of recovery.² This hypothermic treatment is capable of augmenting the number of surviving neurons in the CA1 region even when induced from 12 hours of recovery but is ineffective if initiated 24 hours after the ischemic insult.² Recently, it has been reported that postischemic hypothermia to a large degree delays neuronal necrosis rather than persistently prevents damage. Three hours of immediate hypothermia (30°C) following a 10-minute normothermic ischemic episode in the rat provided a marked protection at 7 days but not at 2 months of recovery.³ Similarly, in the gerbil, 12 hours of hypothermia did not persistently protect neurons against damage unless the ischemic period was short (3 minutes).⁴ More recently, it was demonstrated that prolonged mild hypothermia (32°C) for 24 hours diminished neuronal damage in the hippocampus by 70%, a protection that persisted for at least 6 months.⁵

Intraischemic hyperthermia is known to aggravate neuronal damage.⁶ However, the effect of postischemic hyperthermia has not been addressed experimentally in much detail. This is of clinical relevance because several reports have indicated that fever worsens outcome after ischemia.⁷ We recently observed a sustained period of spontaneous hyperthermia from the end of the 1st day to the end of the 3rd day of recovery in our model of rat forebrain ischemia.⁸ Maintaining normothermic levels during this period by effective antipyretic therapy or cooling decreased neuronal loss observed at 7 days of recovery in the hippocampus and cerebral cortex. This suggested that vulnerable neurons may succumb because of hyperthermic stress late during recovery.

In this study, we investigated whether postischemic hypothermia of moderate duration or antipyretic treatment provides a long-lasting (2 months) neuroprotective effect. Also, since the two treatments provide a substantial neuroprotection at 7 days of recovery and are instituted at different phases of recovery, we investigated the effect of sequential treatment with hypothermia followed by antipyresis.

	Materials and Methods

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Induction of Ischemia
The model of ischemia used in this study has been described in detail elsewhere.⁹ All animal experiments were approved by the ethics committee at Lund University. Male Wistar rats (Moellegaard Avlslaboratorium, Copenhagen, Denmark) weighing between 330 and 380 g were used. Anesthesia was induced by 3.5% halothane in N₂O/O₂ (70:30) in rats fasted overnight. During ischemia, halothane was maintained at 0.3% in N₂O/O₂ (70:30). Muscle relaxation was achieved with vecurone bromide (Norcuron, Organon Teknika BV, with a loading dose of 1.2 mg·kg^-1 followed by 6.0 mg·kg^-1·h^-1). Ischemia was induced by combining the clamping of both common carotid arteries with hypotension (mean arterial pressure, 50 mm Hg) achieved by the withdrawal of venous blood from the systemic circulation. After 10 minutes the blood was reinfused, the clamps were removed, and 75.0 mg·kg^-1 NaHCO₃ was administered. Halothane was discontinued at recirculation, but ventilatory assistance was maintained for the first 30 minutes of recovery. Arterial blood gases and pH were monitored and kept within the following preischemic ranges: PaO₂, 95 to 110 mm Hg; and PaCO₂, 35 to 40 mm Hg, pH 7.35 to 7.45. Blood glucose levels were also measured preischemically, and if the value exceeded 10 mmol/L, the experiment was discontinued. Core temperature was measured with a flexible probe, with the sensor tip placed into the rat's bowel at 10 cm from the anus. Skull temperature was measured with a needle probe placed subcutaneously above the skull bone. Both temperatures were kept at 37.5±0.5°C during ischemia. Core temperature spontaneously increased to 38.0°C at 30 minutes after ischemia.

Postischemic Hypothermia
In all rats treated with hypothermia, anesthesia was induced again with 3.5% halothane in N₂O/O₂ (70:30) at 2 hours into recirculation and maintained at 1.0% halothane. Core temperature was reduced to 33.0°C within 15 to 25 minutes by spraying alcohol onto the chest and abdominal skin, which was thereafter placed in contact with a metallic surface. The 33.0±0.5°C range was maintained by keeping part of the ventral skin in contact with a metallic surface and part on a heating pad set at 33.0°C. Anesthesia was then discontinued, and the rats were allowed to rewarm spontaneously, reaching a core temperature of 38.0°C within 90 to 120 minutes.

With a heating pad, core temperature was maintained in the range of 37.5° to 38.5°C in the normothermia group from 2 to 9 hours of recovery with rats under halothane anesthesia. Ventilatory assistance was maintained for an additional 30 minutes after either hypothermic or normothermic treatment.

Postischemic Temperature Measurements
After ischemia, the rats were maintained at a 12-hour light/dark cycle with lights off from 6 PM to 6 AM. Temperature was measured with a digital thermometer (Ellab) at 1 AM, 7AM, 1 PM, and 7 PM from the first to the last day of recovery. The probe was lubricated with tap water and the sensor tip introduced at 10 cm from the anus 20 to 30 seconds before core temperature measurement was made and just before the intraperitoneal injections during dipyrone or saline treatment.

Histopathology
At either 7 days or 2 months of survival, the rats were anesthetized and perfusion fixed with 0.9% NaCl followed by phosphate-buffered 4.0% formaldehyde. The brains were allowed to fix in situ for 24 hours and then were removed, dehydrated, and embedded in paraffin. Serial coronal sections at 8 mm were obtained and stained with celestine blue and acid fuchsin.¹⁰

Brain damage in the hippocampus was evaluated at a coronal level 3.8 to 4.0 mm caudal to bregma and quantified by visual counting of surviving intact (violet) cells at a magnification of x400. Each examined area of the CA1 region was 400 µm in length. The medial and middle CA1 region contained a mean value of 70 cells, and the medial part contained 75 cells. Each examined area of the CA1 region was 400 µm in length. The percentage of damaged neurons of the total neuronal population was calculated for lateral, medial, and middle CA1 hippocampal subsectors in each hemisphere, and a mean value was calculated. The mean damage of the three subsectors was also calculated. In rats perfused 2 months after ischemia, cortical thickness was measured from the external wall of the lateral ventricle to the cortical surface in the temporoparietal cortex above the entorhinal fissure at the same level as the evaluation of hippocampal damage. Similarly, the areas of the striatum and lateral ventricles were calculated at the level of 0.4 to 0.2 mm rostral from bregma by computerized image analysis with the Image program (Dr Wayne Rasband, National Institute of Mental Health, Bethesda, Md) and a Macintosh Quadra 950 computer. The mean of the two hemispheric values is provided.

Experimental Groups
In this study, 104 rats were distributed into 13 experimental groups, as shown in Table 1. Hypothermia (core temperature, 32.5° to 33.5°C) for 7 hours was initiated 2 hours after ischemia. The normothermia group was maintained at a core temperature of about 38.0°C, with a period of anesthesia similar to that of hypothermia-treated rats. Dipyrone-treated rats received 100 mg·kg^-1 dipyrone (0.6 mL of 5% solution), and the saline (vehicle)-treated rats received the same volume of 0.9% NaCl. Treatment was administered intraperitoneally every 3 hours from 14 to 72 hours after ischemia. The rats were perfused at either 7 days or 2 months of recovery.

View this table: [in this window] [in a new window]   Table 1. Experimental Rat Groups

If not otherwise stated, the Mann-Whitney U test was used for the evaluation of differences among experimental groups.

	Results

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Changes in Core Temperature Over 2 Months After Ischemia
The core temperature displayed characteristic circadian changes over 24 hours, with a peak value of about 39°C at 1 AM and a lowest value around 37°C at approximately 1 PM (data not shown). This pattern changed during the first 4 days after a transient ischemic episode. At 1 to 3 days of recovery after ischemia, a sustained elevation of temperature was seen, followed by a resumption of the temperature variations seen in control rats (Fig 1). After the transient hyperthermic reaction, the nocturnal core temperature levels were transiently depressed but progressively increased thereafter, resuming a steady state by 2 weeks after ischemia (Fig 2). When the specific temperature values at 1 AM and 1 PM are plotted separately, the significant (P<.01) difference between core temperature in normothermic saline-treated rats at night and during the day at 1 week of recovery and thereafter can be appreciated (Fig 2).

View larger version (34K): [in this window] [in a new window]   Figure 1. Mean core temperature at 1 AM, 7 AM, 1 PM, and 7 PM over 60 consecutive days after 10 minutes of transient ischemia (induced between 9 AM and 2 PM) in Wistar rats maintained on a 12-hour light/dark cycle (lights off 6 PM to 6 AM) and stable environmental temperature (24°C). Values are mean±SD (n=8).

View larger version (21K): [in this window] [in a new window]   Figure 2. Mean core temperature at 1 PM in rats subjected to 10 minutes of ischemia followed by saline treatment () and at 1 AM (). From 7 days of recovery, temperature differed significantly (P<.05) between 1 AM and 1 PM of the same day (Student's t test). Values are mean±SD (n=8).

Rats are active during the nighttime, so nocturnal core temperature could be affected by activity and food intake. Therefore, we compared the mean temperature at 1 PM in saline- and dipyrone-treated rats and in the combined hypothermia+saline- and hypothermia+dipyrone-treated rats (Fig 3). A sustained elevation in temperature was seen in saline-treated and hypothermia+saline-treated rats immediately after the induced hypothermia period, extending in duration to 3 to 4 days after ischemia. This hyperthermic episode was obliterated in both the dipyrone- and hypothermia+dipyrone-treated groups, in which temperature decreased to approximately 38°C. The temperature during the subsequent 60 days did not differ among the experimental groups.

View larger version (14K): [in this window] [in a new window]   Figure 3. Mean core temperature at 1 PM in rats subjected to 10 minutes of ischemia followed by 100 mg·kg-1 dipyrone () or saline () given intraperitoneally every 3 hours from 14 to 72 hours of recovery (b) or hypothermia (H) (33°C from 2 to 9 hours of recovery [a]) plus 100 mg·kg-1 dipyrone () or saline () given intraperitoneally every 3 hours from 14 to 72 hours of recovery. *Significant difference (P<.01, Student's t test) between saline-treated and dipyrone-treated groups; #significant difference (P<.01, Student's t test) between hypothermia+saline- and hypothermia+dipyrone-treated groups.

Neuronal Damage in the Hippocampus, Neocortex, and Striatum
The effects of the different treatments on neuronal damage in the lateral and medial CA1 subsectors of the hippocampus at 7 days and 2 months of survival are shown in Figs 4 and 5, respectively. At 7 days of recovery, dipyrone treatment alone decreased neuronal damage by 40% in the lateral CA1 region and by 26% in the medial CA1 region. This protection was abolished at 2 months of recovery (Fig 5), when dipyrone only slightly protected the lateral (damage decreased by 19%, P<.05) but not the medial CA1 subsector compared with saline treatment. Similarly, at 7 days of recovery, hypothermia provided a robust protective effect, with 53% protection in the lateral CA1 region and 34% protection in the medial CA1 region. In contrast, at 2 months of recovery, hypothermia alone provided moderate protection to the lateral (damage decreased by 23%, P<.01) and medial (damage decreased by 22%, P<.05) CA1 subsectors compared with normothermia. The combined hypothermia+dipyrone treatment, on the other hand, was markedly protective at both 7 days and 2 months. At 7 days, 37% and 51% neuronal necrosis was observed in the lateral and medial CA1 regions, respectively. At 2 months of recovery, neuronal damage was depressed to 23% and 56%, respectively.

View larger version (39K): [in this window] [in a new window]   Figure 5. Neuronal damage in the pyramidal layer of the lateral and medial CA1 regions of rat hippocampus evaluated at 2 months of survival. Experimental conditions and treatment paradigms are the same as in Fig 4.

Neuronal necrosis in the CA1 sector of the hippocampus can be assessed with reasonable accuracy at 2 months of recovery because the neurons display a homogeneous band of cell bodies in the stratum pyramidale. It is not possible to assess cell loss in the striatum and cortex by cell counting because degenerated cells are absorbed by glial cells, and subsequently, the tissue collapses and shrinks, causing an underestimation of cell damage. We therefore measured the thickness of the temporal cortex, an area of cortex where damage is mainly found following this duration of ischemia.¹¹ The cortex was significantly thinner in the saline-treated rats compared with both the hypothermia- and hypothermia+dipyrone-treated rats (Table 2). Similarly, in the dorsolateral striatum, damage was reflected as a shrinkage of the caudate nucleus and enlargement of the ventricles (Fig 6). Hypothermia and hypothermia+dipyrone significantly diminished ventricular enlargement, whereas hypothermia alone significantly prevented striatal shrinkage (Table 2).

View this table: [in this window] [in a new window]   Table 2. Changes in Striatum and Lateral Ventricle Sizes and the Thickness of the Temporal Cortex

View larger version (63K): [in this window] [in a new window]   Figure 6. Striatum of rat subjected to 10 minutes of ischemia and 2 months of recovery (a) and similar rat treated with hypothermia (33°C) at 2 to 9 hours of recovery plus 100 mg·kg-1 dipyrone IP every 3 hours starting at 14 to 72 hours of recovery (b). Note enlarged ventricles (v) and shrunken striatum (s) (celestine blue–acid fuchsin stain, magnification x20).

At 7 days of recovery, brains treated with saline, hypothermia, or dipyrone or the combined regimen displayed variable degrees of gliosis and multiple red condensed neurons surrounded by dark-staining microglial nuclei. At this time, the thicknesses of the strata oriens and radiatum were not markedly changed compared with normal brain. At 2 months of recovery, extensive gliosis, microdeposits of amorphous acidophilic material, and red-staining shrunken neurons were seen in brains not treated with the combination of hypothermia and dipyrone (Fig 7b and 7c). Two types of red-staining cells could be identified. One was morphologically similar to that usually observed at 1 week of recovery and described above and was diffusely seen throughout the CA1 pyramidal layer (Fig 7b and 7c). The other, stained dark red, showed aberrant, multiple shapes, occasionally suggestive of entangled filaments and sometimes surrounded by small, rounded fragments of the same color (Fig 7c). The reason for the presence of light red residues of cell bodies and the appearance of an amorphous substance in the CA1 region (Fig 7b and 7c) of untreated and partially treated rats at 2 months of survival is unknown. The patchy aggregation of polymorphous dark red neuronal residues observed in the medial CA1 subsector of the hippocampus of some rats subjected to hypothermic treatment followed by spontaneous hyperthermia may represent calcified cells similar to those reported earlier.¹²

View larger version (185K): [in this window] [in a new window]   Figure 7. Photomicrographs of the pyramidal layer of the middle CA1 region of the dorsal hippocampus of rats killed at 2 months of recovery: Sham-operated rat (a) and rats subjected to 10 minutes of ischemia and treated with saline (b), treated with hypothermia (33°C) 2 to 9 hours after ischemia (c), or treated with a similar hypothermic episode plus 100 mg·kg-1 dipyrone IP every 3 hours starting at 14 to 72 hours of recovery (d). Note the similar appearance of cells in a and d. Degenerated shrunken neurons (large arrow) and microglial cells (large arrowheads) are seen. Microdeposits of amorphous substance (small arrowhead) and cell bodies with tangles (small arrows) are indicated (celestine blue–acid fuchsin stain, magnification x100).

At 2 months of recovery, the degeneration of neurons had affected the dendritic processes, causing a collapse of the strata oriens and radiatum of the CA1 region (Fig 8b and 8c). In rats treated with hypothermia, a treatment in which moderate protection was attained (Fig 5), the shrinkage of the CA1 region was less conspicuous (Fig 8d).

View larger version (94K): [in this window] [in a new window]   Figure 8. Middle CA1 sector of rats exposed to 10 minutes of ischemia and 2 months of recovery: Sham-operated rat (a) and rats subjected to 10 minutes of ischemia and treated with saline (b), treated with 100 mg·kg-1 dipyrone IP every 3 hours from 14 to 72 hours of recovery (c), treated with hypothermia (33°C) at 2 to 9 hours after ischemia (d), and treated with a hypothermic episode similar to that in d plus 100 mg·kg-1 dipyrone IP every 3 hours starting at 14 to 72 hours of recovery (e). Arrows indicate border between the hippocampal fissure/stratum lacunosum moleculare (top) and alveus/stratum oriens (bottom). p indicates stratum pyramidale.

In contrast, in rats subjected to combined hypothermia+dipyrone treatment, the general appearance of the CA1 region (Figs 7d and 8e) could not be distinguished from that of sham-operated rats (Figs 7a and 8a), apart from a lower density of cells per unit area. A major finding is the lack of extensive microglial infiltration at 2 months of recovery, contrasting with the high density of nuclei of these cells normally observed 7 days after the same ischemic insult. Also, the thicknesses of the strata radiatum and oriens were not markedly different from those of sham-operated rats (Fig 8e).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
In this study, we have demonstrated that postischemic hypothermia or treatment with the antipyretic and anti-inflammatory drug dipyrone delays neuronal damage in the rat hippocampus, cortex, and striatum. The combined treatment renders a long-lasting neuroprotection evaluated at 2 months of recovery. Based on these data, we will discuss the need for prolonged intervention for effective therapeutic effects against ischemic brain damage and also discuss mechanisms affected by the two neuroprotective treatment regimens.

In our earlier studies, we demonstrated that 5 hours of hypothermia (33°C) initiated at 2, 6, and 12 hours of recovery provided a robust protection of up to 50% assessed at 7 days after ischemia. In contrast, 5 hours of hypothermia induced at 24 and 36 hours of recovery did not provide any neuroprotective effect.² Also, 12 hours of hypothermia initiated at 1 hour of recovery after 5 minutes of ischemia in the gerbil delayed neuronal damage.⁴ However, when hypothermia was prolonged to 24 hours, a sustained protection was achieved.⁵ Three hours of hypothermia also prevented the development of neuronal damage, as seen at 7 days of recovery,¹³ a protection more or less abolished at 2 months.³ Here we demonstrate a smaller but still significant neuroprotective effect by 7 hours of hypothermia at 2 months of recovery. Taken together, these data show that if postischemic hypothermia is instituted within hours after reperfusion and is of sufficient duration, a long-lasting neuroprotection will be attained. However, the severity of the insult, the delay in the induction of hypothermia, and the duration of the hypothermic treatment as well as superimposed secondary insults or some other differences between animal models may all contribute to differences in the effectiveness of hypothermic treatments.

We have earlier observed a period of hyperthermia in rats subjected to the two-vessel occlusion model of forebrain ischemia, from the end of the 1st day to the end of the 3rd day of recovery.⁸ This hyperthermic episode evidently enhances neuronal loss because the anti-inflammatory and antipyretic drug dipyrone or cooling the rats to normothermic levels diminishes neuronal loss observed at 1 week of recovery.⁸ However, the dipyrone treatment merely delays neuronal damage (Figs 4 and 5), suggesting that slowly evolving pathogenic processes are activated during recovery and lead to neuronal degeneration over an extended period of time. These processes can be inhibited by immediate prolonged hypothermia because the combined treatment of hypothermia and dipyrone provides a long-lasting protection. Hypothermia alone also delays neuronal damage rather then provides a permanent protection, apparently because a hyperthermic episode also develops after the 7 hours of hypothermic treatment (Fig 3). The superimposed period of hyperthermia is obviously capable of initiating or reviving pathophysiological mediators made quiescent by the hypothermic treatment. In other words, the powerful and long-lasting protection provided by the combined hypothermia/antipyresis treatment may be the result of the hypothermia alone provided that the complication of a secondary hyperthermic insult is abolished. The failure of previous studies to achieve a sustained and marked protective effect in the hippocampal CA1 region³ may therefore have been due to a borderline or suboptimal duration of postischemic hypothermia and/or an unrecognized delayed hyperthermic episode.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of 100 mg·kg-1 dipyrone or saline given intraperitoneally every 3 hours from 14 to 72 hours of recovery, hypothermia (33°C from 2 to 9 hours of recovery) or normothermia (38°C under halothane anesthesia), or consecutive hypothermia+dipyrone (DIPY) or saline (SAL) treatments on neuronal necrosis induced by 10 minutes of ischemia. Neuronal damage was assessed in the pyramidal layer of the lateral and medial CA1 regions of rat hippocampus and evaluated at 2 months of survival. Neuronal damage is expressed as percentage of damaged neurons in the area investigated. Circles represent mean damage of the two brain hemispheres of the same rat. Gray bars represent mean damage for each group. Statistically significant differences (*P<.05, **P<.01) between experimental (dipyrone, hypothermia plus halothane, hypothermia followed by dipyrone) and respective control (saline, normothermia plus halothane, hypothermia followed by saline) paradigms are indicated. Dotted line indicates mean value of neuronal damage in control ischemia group.

The morphological data presented here indicate ongoing neurodegenerative processes late during recovery in rats treated with hypothermia or dipyrone only. In the rats treated with hypothermia, the demise of pyramidal cells is seen at 2 months of recovery since microglial infiltration occurs (Fig 7c), suggesting that neurodegeneration is progressing. We have identified the microglia by immunocytochemical staining with OX-42 antibody.¹⁴ The fact that ongoing neurodegeneration is occurring at 2 months of recovery would lead one to expect even less neuronal survival at later recovery times. A similar "postresuscitation encephalopathy" characterized by neuronal degeneration associated with dark and shrunken neurons and astrocytic-microglial proliferation was described in the cortex and hippocampus at 6 months after resuscitation in a rat model of brain ischemia induced by cardiac arrest.^{12 15} Also, a slowly progressing cell death has been reported after transient ischemia in the rat.¹⁶ In contrast, with combined hypothermia/antipyresis treatment, neurodegeneration is not increased at 2 months of recovery compared with that occurring at 1 week of recovery. Also, and more important, in this group there are no signs of infiltration by microglial cells (Fig 7d) at the late recovery time, suggesting that the treatment may provide a persistent neuroprotection.

The pathophysiological mediators affected by temperature late into the recovery period are unknown. It is possible that temperature manipulations may affect not a single but multiple pathophysiological entities. Glutamate release has been shown to be proportional to intraischemic temperature.¹⁷ Therefore, at least theoretically, hypothermia and hyperthermia may depress and enhance glutamate and calcium toxicity, respectively, late into the postischemic period. Also, hypothermia could protect mitochondrial function and reduce the accumulation of oxygen species, thereby preventing free radical damage.¹⁸

In vitro studies have shown that brain macrophages are able to release glutamate¹⁹ and other neurotoxins, including reactive oxygen species.^{20 21 22 23} Therefore, the inflammatory process that follows neuronal damage^{24 25} may represent a source of neurotoxic substances, such as superoxide, nitric oxide, or cytokines,^{26 27 28} which could induce a rise in temperature quite late after the ischemic episode. Interleukin-1ß induces fever²⁹ and is expressed in the postischemic brain.³⁰ Fever induced by interleukin-1ß is potently depressed by dipyrone.^{31 32} The microglial proliferation seen at 2 months of recovery suggests that the initial damage occurring during the first days after ischemia may trigger an inflammatory response that becomes a vicious cycle enhancing a slowly progressive neurodegeneration. Since cooling depresses and hyperthermia enhances inflammation,^{33 34} hypothermia may interrupt this cycle, and conversely, hyperthermia would enhance or reactivate this process.

In general, postischemic hyperthermia may have a dual detrimental effect on the survival of those neuronal populations that have not normalized cellular homeostasis after ischemia: an immediate and a sustained one. The immediate effect would trigger a spurt of cell death by acutely enhancing glutamate and calcium toxicity and free radical mechanisms. The sustained one would involve microglia-derived mediators of a chronic inflammatory response, maintaining a secondary, prolonged release of neurotoxic substances. Combined hypothermia and dipyrone treatment would prevent both the initial detrimental processes as well as processes enhanced and made self-sustained by the transient rise in temperature during the first days after ischemia.

The slow maturation of neuronal degeneration seen in this study may be relevant to the findings in stroke patients, in whom progressive mental deterioration, not seen at 3 weeks after the insult, is evident years after the stroke.³⁵ Also, the detrimental effects of hyperthermia on neuronal survival after ischemia are consistent with the observations of Hindfelt⁷ on the consequences of hyperthermia during the first week after ischemic stroke, as assessed by the neurological evolution from the initial examination to that observed 2 months later. According to Hindfelt, the occurrence of subfebrility (37.5° to 38.0°C) is related to a poorer neurological recovery, and fever (temperature higher than 38.5°C) is particularly detrimental. Likewise, fever is associated with a larger brain lesion on computed tomographic examination of stroke patients.^{36 37 38}

In summary, this study provides tangible evidence of a temperature-sensitive encephalopathic process during reperfusion after an ischemic insult. It also shows that proper temperature control late into the postischemic period prevents the activation of chronic neurodegeneration. Moreover, postischemic hypothermia provides powerful and long-lasting protection, if cooling is maintained for an adequate period and not excessively delayed and if it is followed by treatment with anti-inflammatory/antipyretic drugs. The data suggest a prolonged (life-long?) administration of anti-inflammatory/antipyretic drugs as an additional therapy after brain ischemia.

	Acknowledgments

 
This work was supported by the Swedish Medical Research Council (grant No. 08644), the Medical Faculty at Lund University, the Crafoord Foundation, the Åke Wibergs Foundation, the Gunnar and Martha Bergendahl Foundation, the Carola Henckels Foundation, the Brazilian Council for the Development of Science and Technology (CNPq), and the Foundation for Research Support in the State of Sao Paulo (FAPESP).

Received February 6, 1996; revision received May 10, 1996; accepted June 7, 1996.

	References

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1. Coimbra CG, Wieloch T. Hypothermia ameliorates neuronal survival when induced 2 hours after ischemia in the rat. Acta Physiol Scand.. 1992;146:543-544.[Medline] [Order article via Infotrieve]
2. Coimbra CG, Wieloch T. Moderate hypothermia mitigates neuronal damage when initiated several hours following transient cerebral ischemia. Acta Neuropathol.. 1994;87:325-331.[Medline] [Order article via Infotrieve]
3. Dietrich WD, Busto R, Alonso O, Globus MY-T, Ginsberg MD. Intraischemic but not postischemic brain hypothermia protects chronically following global forebrain ischemia in rats. J Cereb Blood Flow Metab.. 1993;13:541-549.[Medline] [Order article via Infotrieve]
4. Colbourne F, Corbett D. Delayed and prolonged post-ischemic hypothermia is neuroprotective in the gerbil. Brain Res.. 1994;654:265-272.[Medline] [Order article via Infotrieve]
5. Colbourne F, Corbett D. Delayed postischemic hypothermia: a six month survival study using behavioral and histologic assessments of neuroprotection. J Neurosci.. 1995;15:7250-7260.[Abstract]
6. Minamisawa H, Smith ML, Sieajo BK. The effect of mild hyperthermia and hypothermia on brain damage following 5, 10 and 15 minutes of forebrain ischemia. Ann Neurol.. 1990;28:26-33.[Medline] [Order article via Infotrieve]
7. Hindfelt B. The prognostic significance of subfebrility and fever in ischemic cerebral infarction. Acta Neurol Scand.. 1976;53:72-79.[Medline] [Order article via Infotrieve]
8. Coimbra CG, Boris-Moller F, Drake M, Wieloch T. Hypo- and hyperthermia affect the neuronal survival several hours after brain ischemia in rats. Abstr Am Soc Neurosci.. 1993;23:1670. Abstract.
9. Smith M-L, Bendek G, Dahlgren N, Rosen I, Wieloch T, Siesjo BK. Models for studying long-term recovery following forebrain ischemia in the rat, 2: a two vessel occlusion model. Acta Neurol Scand.. 1984;69:385-401.[Medline] [Order article via Infotrieve]
10. Auer RN, Olsson Y, Siesjo BK. Hypoglycemic brain injury in the rat: correlation of density of brain damage with the EEG isoelectric time: a quantitative study. Diabetes.. 1984;33:1090-1098.[Abstract]
11. Nellga°rd B, Wieloch T. Postischemic blockage of AMPA but not NMDA receptors mitigates neuronal damage in the rat brain following transient severe cerebral ischemia. J Cereb Blood Flow Metab.. 1991;12:2-11.
12. Zelman IB, Mossakowski MJ. Remote pathological brain changes in rats following experimentally induced clinical death. Neuropat Pol.. 1988;26:151-162.
13. Green EJ, Dietrich WD, van Dijk F, Busto R, Markgraf CG, McCabe PM, Ginsberg MD, Schneiderman N. Protective effects of neural hypothermia on behaviour following global cerebral ischemia in rats. Brain Res.. 1992;580:197-204.[Medline] [Order article via Infotrieve]
14. Coimbra CG, Sinigaglia R, Mazzacoratti MGN, Cavalheiro EA. Persistent macrophages in the rat brain two months after ischemia. Abstr Am Soc Neurosci.. 1995;25:789. Abstract.
15. Walski M, Borowicz J. Brain phagocytes and the microglia of the cerebral cortex of rats following an incident of total ischaemia. J Hirnforsch.. 1995;36:55-66.[Medline] [Order article via Infotrieve]
16. Nakano S, Kogure K, Fujikura H. Ischemia-induced slowly progressive neuronal damage in the rat brain. Neuroscience.. 1990;38:115-124.[Medline] [Order article via Infotrieve]
17. Takagi K, Ginsberg MD, Globus MY, Martinez E, Busto R. Effect of hyperthermia on glutamate release in ischemic penumbra after middle artery occlusion in rats. Am J Physiol.. 1994;267:H1770-H1776.[Abstract/Free Full Text]
18. Kil HY, Zhang J, Piantadosi CA. Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats. J Cereb Blood Flow Metab.. 1996;16:100-106.[Medline] [Order article via Infotrieve]
19. Piani D, Frei K, Do KQ, Cuenod M, Fontana A. Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate. Neurosci Lett.. 1991;133:159-162.[Medline] [Order article via Infotrieve]
20. Colton CA, Gilbert DL. Production of superoxide anion by a CNS macrophage, the microglia. FEBS Lett.. 1987;223:284-288.[Medline] [Order article via Infotrieve]
21. Frei K, Siepl C, Groscurth P, Bodmer S, Schwerdel C, Fontana A. Antigen presentation and tumor cytotoxicity by interferon--treated microglial cells. Eur J Immunol.. 1987;17:1271-1278.[Medline] [Order article via Infotrieve]
22. Giulian D. Reactive glia as rivals in regulating neuronal survival. Glia.. 1993;7:102-110.[Medline] [Order article via Infotrieve]
23. Banati RB, Gehrmann J, Schubert P, Kreutzberg GW. Cytotoxicity of microglia. Glia.. 1993;7:111-118.[Medline] [Order article via Infotrieve]
24. Morioka T, Kalehua AN, Streit W. The microglial reaction in the dorsal hippocampus following transient forebrain ischemia. J Cereb Blood Flow Metab.. 1991;11:966-973.[Medline] [Order article via Infotrieve]
25. Gehrmann J, Bonnekoh P, Miyazawa T, Hossmann K-A, Kreutzberg GW. Immunocytochemical study of an early microglial activation in ischemia. J Cereb Blood Flow Metab. 1992a;12:257-269.
26. Boje KM, Arora PK. Microglial produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res.. 1992;587:250-256.[Medline] [Order article via Infotrieve]
27. Popovich PG, Reinhardt JF, Flanagan EM, Stokes BT. Elevation of the neurotoxin quinolinic acid occurs following spinal cord trauma. Brain Res.. 1993;633:348-352.
28. Sawada M, Kondo N, Suzumura A, Marunouchi T. Production of tumor necrosis factor-alpha by microglia and astrocytes in culture. Brain Res.. 1989;491:394-397.[Medline] [Order article via Infotrieve]
29. Endres S, Van der Meer JWM, Dinarello CA. Interleukin-1 in the pathogenesis of fever. Eur J Clin Invest.. 1987;17:469-474.[Medline] [Order article via Infotrieve]
30. Minami M, Kuraishi Y, Yabuuchi K, Yamazaki A, Satoh M. Induction of interleukin-1ß mRNA in rat brain after transient forebrain ischemia. J Neurochem.. 1992;58:390-392.[Medline] [Order article via Infotrieve]
31. Brune K, Alpermann H. Non-acidic pyrazoles: inhibition of prostaglandin production, carrageenan oedema and yeast fever. Agents Actions.. 1983;13:360-363.[Medline] [Order article via Infotrieve]
32. Shimada SG, Otterness IG, Stitt JT. A study of the mechanism of action of the mild analgesic dipyrone. Agents Actions.. 1994;41:188-192.[Medline] [Order article via Infotrieve]
33. Janssen CW, Waller E. Body temperature, antibody formation and inflammatory response. Acta Pathol Microbiol Scand.. 1967;69:557-566.
34. Fujishima H, Yagi Y, Toda L, Shimazaki J, Tsubota K. Increased comfort and decreased inflammation of the eye by cooling after cataract surgery. Am J Ophthalmol.. 1995;119:301-306.[Medline] [Order article via Infotrieve]
35. Tetemichi TK, Paik M, Bagiella E, Desmond DW, Stern Y, Sano M, Hauser WA, Mayeux R. Risk of dementia after stroke in a hospitalized cohort: results of a longitudinal study. Neurology.. 1994;44:1885-1891.[Abstract/Free Full Text]
36. Przelomski MM, Roth RM, Gleckman RA, Markus EM. Fever in the wake of a stroke. Neurology.. 1986;36:427-429.[Abstract/Free Full Text]
37. Reith J, Jørgensen HS, Møller Pedersen P, Nakayama H, Raaschou HO, Jeppesen LL, Skyhøj Olsen T. Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome. Lancet. 1996. In press.
38. Azzimondi G, Bassein L, Nonino F, Fiorani L, Vignatelli L, Re G, D'Alessandro R. Fever in acute stroke worsens prognosis: a prospective study. Stroke.. 1995;26:2040-2043.[Abstract/Free Full Text]

Editorial Comment

Evidence for Chronic Encephalopathic Processes Following Ischemia

Dale Corbett, PhD, Guest Editor

Division of Basic Medical SciencesFaculty of MedicineMemorial University of NewfoundlandSt John's, NF, Canada

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The authors have noted that 10 minutes of global ischemia in the rat triggers a period of mild hyperthermia lasting 17 to 72 hours into the recovery period. Mild hypothermia (33°C) initiated 2 hours after occlusion and maintained for 7 hours produced a significant reduction in hippocampal CA1 cell death, with survival times of 7 days and 2 months. However, the degree of protection observed at 2 months was less than that obtained at 7 days, thereby raising the possibility that this duration of postischemic hypothermia may have delayed rather than prevented cell death. Administration of the antipyretic dipyrone every 3 hours from 14 to 72 hours in the recovery period also diminished CA1 cell loss, although less effectively than postischemic hypothermia. The combination of hypothermia plus dipyrone resulted in a 50% preservation of CA1 neurons at both survival times, suggesting permanent protection.

These results are in agreement with recent studies in the gerbil^{1R 2R} that achieved lasting protection (6-month survival time) with even longer durations of postischemic hypothermia. Taken together, these findings illustrate the necessity of prolonged rather than acute treatment with hypothermia for permanent protection to be achieved. Similar prolonged (perhaps days or weeks?) treatment regimens are likely to be required in pharmacological studies. Also, based on the above work, it is imperative that long survival times (>1 month) be used in all preclinical studies because several drug treatments^{3R 4R} have recently been shown to delay rather than prevent cell death. Finally, since hyperthermia markedly worsens ischemic injury and fever is commonly associated with stroke and other types of brain injury, the clinician should not only aggressively treat any incidence of hyperthermia but take steps to prevent its development.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Colbourne F, Corbett D. Delayed and prolonged postischemic hypothermia is neuroprotective in the gerbil. Brain Res.. 1994;654:265-272.
2. Colbourne F, Corbett D. Functional and histological protection following prolonged postischemic hypothermia: a six month survival study. J Neurosci.. 1995;15:7250-7260.
3. Nurse SM, Corbett D. Neuroprotection following several days of mild, drug-induced hypothermia. J Cereb Blood Flow Metab.. 1996;16:474-480.[Medline] [Order article via Infotrieve]
4. Valtysson J, Hillered L, Andine P, Hagberg H, Persson L. Neuropathological endpoints in experimental stroke pharmacotherapy: the importance of both early and late evaluation. Acta Neurochir.. 1994;129:58-63.[Medline] [Order article via Infotrieve]

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