Preventing hyperthermia decreases brain damage following neonatal hypoxic-ischemic seizures

Brain Research 1011 (2004) 48 – 57 www.elsevier.com/locate/brainres

Research report

Preventing hyperthermia decreases brain damage following neonatal hypoxic-ischemic seizures Jerome Y. Yager a,b,*, Edward A. Armstrong a, Cleo Jaharus a, Deborah M. Saucier c, Elaine C. Wirrell d b

a Department of Pediatrics, University of Saskatchewan, 103 Hospital Drive, Saskatoon, Saskatchewan, Canada S7N 0W8 Department of Pediatrics, Stollery Children’s Hospital, University of Alberta, Room 2C3, Walter C. Mackenzie Health Centre, Edmonton, Canada T6G 2R7 c Department of Psychology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada d Department of Pediatrics, Alberta Children’s Hospital, University of Calgary, Calgary, Alberta, Canada

Accepted 28 February 2004 Available online 27 April 2004

Abstract Neonatal seizures are the most common manifestation of underlying cerebral dysfunction. Hypoxic-ischemic encephalopathy is the cause of seizures in 40 – 60% of newborns. Previous work from our laboratory demonstrates that seizures associated with a hypoxicischemic insult results in aggravation of neuronal cell death, specifically within the hippocampus. The latter occurs in the setting of spontaneously occurring hyperthermia of 1.5 jC. The purpose of this study was to determine whether preventing the onset of seizure induced hyperthermia would be neuroprotective. Three groups of 10-day old rat pups received unilateral hypoxic-ischemic insults for 30 min followed by KA-induced seizures. Hyperthermia was prevented by lowering the environmental temperature (‘‘relative hypothermia’’) to 29 jC such that the seizuring rat pups were normothermic. In one group, the prevention of hyperthermia occurred immediately following hypoxia-ischemia, whereas in the other group it occurred at the onset of seizures. The third group of rat pups (controls) remained at their nesting temperature and therefore became hyperthermic during seizures. Early (3 days) and late (20 days) neuropathology was assessed. Rat pups in whom hyperthermia was prevented during seizures displayed a significant reduction in brain damage compared to controls ( p < 0.05). Assessment of hippocampal brain damage also showed a significant improvement in neuronal necrosis at 20 days of recovery compared to 3 days of recovery ( p < 0.05). The results indicate that preventing spontaneous hyperthermia in this model of hypoxic-ischemic seizures in the newborn is neuroprotective. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Neonatal; Seizures; Hypoxia-ischemia; Damage; Brain; Hyperthermia

1. Introduction Controversy remains as to whether or not seizures per se cause damage to the neonatal brain[3,46]. It has become increasingly clear that the brain damaging effect of seizures is an age dependant phenomenon. While the immature brain is highly epileptogenic[13], neuronal injury appears to occur predominantly in animals beyond 15 days of age * Corresponding author. Tel.: +1-780-407-7329; fax: +1-780-407-8283. E-mail addresses: [email protected], [email protected] (J.Y. Yager). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.070

[13,14,28,38,47]. In the newborn, seizures are the most frequent of neurological signs and usually reflect a serious underlying derangement of the brain. Perinatal hypoxiaischemia (HI) continues to be the most common cause of neonatal seizures, accounting for at least 40% of all cases [33,35]. When combined with other forms of perinatal cerebral ischemic events such as focal bland and hemorrhagic infarction, cerebrovascular disease accounts for 50– 60% of all seizures in the term and pre-term infant [27,33]. Seizures usually occur within the first 48 h of life [33,34] and are often prolonged and frequent, and status epilepticus is not uncommon [33].

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Since the most common clinical event is a neonatal seizure occurring as a result of perinatal hypoxia-ischemia, our laboratory has developed an animal model meant to specifically reflect this clinical scenario. This model exposed 10-day old rats to a hypoxic-ischemic insult, following which they were subjected to prolonged, electrographically confirmed kainic-acid (KA) induced seizures. When occurring on a background of HI, prolonged seizures significantly exacerbated brain damage, specifically within the region of the hippocampus [48]. Though others [4,45] have found neonatal animals to be resistant to brain damage following seizures, even in the context of hypoxia-ischemia, recent in vitro [8] and human studies [26] have supported our findings. Miller et al. [26] investigated 90 human newborns with perinatal asphyxia of whom 33 had seizures. In their study, seizure severity was significantly associated with MRI abnormalities as measured by an increase in the ratio of lactate/choline in both the inter-vascular boundary zone and the basal nuclei, suggesting that indeed the seizures were associated with additional brain damage in these infants. Though not specifically measured in other studies, our original data suggested that in those rat pups experiencing post-ischemic seizures, core temperatures increased by a mean of 1.47 jC, as compared to those rat pups not experiencing seizures. In this regard, it has been well known in the adult literature, that intra or post-ischemic hyperthermia can profoundly exacerbate the brain damaging effects of a HI insult. Reglodi et al. [31] showed that post-ischemic spontaneous hyperthermia to 39 – 40.5 jC significantly increased infarct volume following middle cerebral artery occlusion (MCAO) in adult rats. In humans, Hajat et al. [12] undertook a meta-analysis of post-stroke pyrexia. Of nine studies examined, 3790 patients were identified, revealing a highly significant correlation between pyrexia after stroke and a profound increase in morbidity and mortality. Given that our previous study demonstrated that seizures exacerbate hypoxic-ischemic brain injury and the apparent association with seizure induced hyperthermia, the current study was undertaken to determine whether ensuring normothermia post-ischemically would ameliorate the brain damaging effect of seizures. It should be noted that the purpose of this study was not to investigate the effects of hypothermia (below normal temperatures) but rather to ascertain whether preventing the spontaneously occurring hyperthermia during seizures would prevent their brain damaging effect.

2. Materials and methods Female Wistar rats (Charles River Laboratories, Montreal) were bred in the animal facilities at the University of Saskatchewan. Rat pups were reared with their Dams in a temperature controlled environment at 21 F 0.5 jC, with a relative humidity of 30%, and a 12 h on/off lighting schedule. The date of experimentation was postnatal day 10 (date of birth-day 1). This study received ethical approval

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from the Animal Care Committee at the University of Saskatchewan. 2.1. Induction of cerebral hypoxia-ischemia (HI) Cerebral hypoxia-ischemia was induced in 10-day old rat pups using the modified Levine preparation described by Rice et al. [32], and previously utilized by our laboratory [48,49]. Rat pups were anesthetized with halothane (4% induction; 1% maintenance), placed in the supine position, and their right common carotid artery was permanently ligated through a midline neck incision measuring no more than 1.0 cm. The incision was then sutured and the pups were allowed a 2-h recovery period with their Dam. Hypoxiaischemia was subsequently induced by placing each animal in individual 500 ml glass jars through which a gas mixture of 8% oxygen/balance nitrogen was delivered via inlet and outlet portals. The pups were exposed to hypoxia for a period of 30 min. Based on our previous study [48], this duration of exposure induced a ‘‘minimal lesion’’ involving the hippocampus, and to a lesser extent the cortex of the hemisphere ipsilateral to the common carotid artery ligation. Following the hypoxic-ischemic insult, the pups were again allowed to recover, within the glass chambers, for an additional 30 min prior to the induction of seizures. During this phase of the experiment, the environmental temperature was maintained at 34 F 0.5 jC (nesting temperature) [6,9]. 2.2. Induction of seizures As in our previous study [48] seizures were induced utilizing the pro-convulsant kainic-acid (KA [Ocean Produce International, PEI Canada]). Briefly, in order to ensure uninterrupted electrographic (EEG) status epilepticus, a continuous infusion of KA was required. Prior to the injection of KA, a small bore polyethylene catheter (PE 10) was inserted subcutaneously between the scapula of each rat pup, and held in place with acrocyanate adhesive. Rat pups each received a single subcutaneous injection of 3 mg/kg KA followed by a continuous subcutaneous infusion of 2 mg/kg/ h for 3 h. Control animals received normal saline in the same fashion, in a volume equal to that of the KA (150 Al followed by an infusion of 100 Al/hr for 3 h). Injection of KA in this fashion results in clinical seizures characterized by scratching, loss of balance, hyperactivity, upper limb tremors and salivation. Electrographic seizures are manifest by continuous high voltage spikes which contrasted markedly with the low voltage activity seen on the baseline recording (Fig. 1a and b). In those animals receiving KA, continuous EEG seizure activity persisted for a mean duration of 4 h and 42 min (range 4 h 0 min to 5 h 27 min). 2.3. Preventing seizure induced spontaneous hyperthermia Our previous study [48] showed that those rat pups experiencing prolonged KA induced seizures displayed an

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Fig. 1. Electroencephalograms of 10-day old rat pups under normal conditions (a); continuous KA-induced seizures (b); and KA-induced seizures during ‘‘controlled normothermia’’(c). Note the decreased amplitude in the EEG of that group of rat pups in which hyperthermia was prevented—though clinical seizure manifestations were unchanged.

increase in core (rectal) temperature of approximately 1.47 jC (throughout the duration of seizure activity) above those rats not experiencing KA-induced seizures. In this latter study, all pups were maintained in a single walled servocontrolled incubator, each within a 500-ml glass jar. The environmental temperature within the incubator was maintained at 34 F 0.5 jC. To determine the core temperature of normothermic rat pups maintained at nesting temperature, a group (6) of animals underwent sham surgical procedure, and placement of the subcutaneous catheter. This group received normal saline via the catheter, but no hypoxia-

ischemia or KA. In this group of animals it was determined that normothermia was 35.73 F 0.71 jC (Table 1). In order to determine the environmental temperature required to prevent hyperthermia in those pups experiencing KA induced seizures following the HI insult, groups of rat pups underwent the above described surgical procedure for the induction of HI, after which seizures were induced. The temperature was sequentially lowered in the incubator until such time as a stable ‘‘normothermic’’ core temperature, was obtained in the seizuring pups, and was able to be maintained throughout the period of status epilepticus. In that regard, an

J.Y. Yager et al. / Brain Research 1011 (2004) 48–57 Table 1 Mean temperature of rat pups

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(b) for late pathology. Rat pups from each litter were sampled both 3 and 20 days of recovery.

Treatment

Temperature (jC)

Post-HI Controlled Normothermia Pre KA Injection Controlled Normothermia Hyperthermic HI and KA Induced Seizures Normal Saline Injectedb (Normothermic)

35.81 F 0.54a 36.09 F 0.65a 37.46 F 0.38 35.73 F 0.71a

a Significantly different than hyperthermic (Control) group ( p < 0.05). There were no differences in temperature between other groups. All animals except the normal saline group received 30 min of HI followed by KA induced seizures. b The normal saline group illustrates temperature of a select group of animals utilized to determine ‘‘normothermic’’ levels.

incubator temperature of 29 F 0.5 jC was needed to lower the KA seizure animals to ‘‘normothermia’’ (Table 1). In all rat pups (6 per group) in whom temperature was monitored, core (rectal) temperatures were assessed by placing Physitemp Instruments temperature probes (IT-18) approximately 0.5 cm into the rectum of the rat pup, and stabilizing it in position by simply wrapping with adhesive tape to the proximal length of the tail. Temperature probes were connected to a multi-channel monitor (Acadia Clinical Corporation) which conditioned the signals for the computer (with A/D conversion, Dycor) by converting measured fluctuations in voltage of the various parameters to changes in either degrees centigrade. Temperature was displayed continuously throughout the experimental protocol, and the signals subsequently stored for statistical and graphic analyses (Lab Notebook 4.3). The average temperature as determined across that period of time during which seizures occurred was used in the experimental design (Table 1).

2.5. EEG recordings IN separate groups (6 animals in each group) of rat pups, EEG recordings were obtained representing animals exposed to controlled normothermic status epilepticus and spontaneous hyperthermic status epilepticus, and normothermic controls without seizures, in order to determine the effect of reducing the naturally occurring hyperthermia during seizures, on the EEG and clinical seizures (Fig. 1). The placement of the electrodes was as per Wirrell et al. [48]. Briefly, while under anesthesia, a bifronto-occipital scalp flap was made and temporarily secured with sutures. The skull landmark bregma was identified, and using the stereotaxic coordinates from Snead and Stephens [37], burr holes were made in the calvarium with a 25guage needle. Electrodes were placed in the right hemispheric sensorimotor cortex, and the left hemispheric dorsal hippocampus, and held in place with glass ionomer (Fuji II LC, GC, Tokyo). Referential electrodes were placed along the nasal ridge. Electrodes were connected to a Grass polygraph (Electroencephalograph Model 8– 18D, Grass Instrument Quincy, MA) and recordings were made for 2 h during the stabilization period prior to the onset of seizures (baseline), continuing throughout the kainic acid infusion, and until the electrographic seizure activity became intermittent or abated. Paper speed was 30 mm/s and sensitivity was 10 AV/mm. Electrocardiograms were simultaneously recorded by placing an electrode subcutaneously within the abdominal wall of selected animals.

2.4. Experimental paradigm 2.6. Neuropathologic analysis Three experimental groups were identified, all of which were exposed to 30 minutes of HI, followed 1/2 h later by KA induced seizures: (1) Group I [Controls] (n = 33 survivors)-underwent the above experimental paradigm (no alteration in environmental temdperature which was maintained at 34 F 0.05 jC [nesting temperature]); (2) Group II (n = 42 survivors)-immediately following HI, controlled normothermia was induced by lowering the environmental temperature; and (3) Group III (n = 38 survivors)-controlled normothermia was induced at the onset of KA injection. Seizures were also monitored for clinical characteristics in separate pups during the maintenance of ‘controlled normothermia’, and in those pups who became spontaneously hyperthermic. At the end of the KA injection and seizures, all rat pups were then allowed to recover with their Dams until the date of neuropathologic analysis on either 3 days of recovery (early) or 20 days of recovery (late). Since separate animals from each of the above groups had neuropathology assessed in the early or late categories, groups were designated as either (a) for early pathology or

Surviving animals in all groups were examined neuropathologically at 3 (early) and 20 (late) days of recovery. All pups were recovered with their Dams. On the day of sacrifice, each animal was deeply anaesthetized with 4% halothane in a 20% O2/80% N2 environment. The animals were then decapitated and the brains rapidly removed and placed in formaldehyde-glacial acetic acid-methanol 1:1:8 (FAM) for a minimum of 48 h before being embedded in paraffin. Brains were then sectioned coronally from poste-

Table 2 Grading of brain damage Grade

Gray matter

0 1 2 3 4 5

Normal Few neurons damage (1 – 5%) Several neurons damaged (6 – 25%) Moderate number of neurons damaged (26 – 50%) Greater than one-half of neurons damaged (51 – 75%) Majority or all of neurons damaged including infarction (>75%)

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Fig. 2. Line graph depicting core temperature of rat pups during KAinduced seizures under the various environmental circumstances. Note: there is a significant difference between the temperature of rat pups allowed to become spontaneously hyperthermic and the other groups. Time 0 is the time at which seizures began to manifest. All pups were at the same temperature post-ischemically.

rior to anterior. Serial sections of 6 Am in thickness were taken every 0.5 mm and stained with hematoxylin and eosin (H and E) and glial fibrillary acidic protein (GFAP). Evaluation of brain injury was performed on each section

by two of the investigators (EA and JY) who were blinded to group assignment. Given that our previous work had indicated that enhanced damage occurred exclusively in the hippocampus, we assessed the degree of brain damage on a section of the brain through the posterior splenium of the corpus callosum, inclusive of all four regions of the hippocampus. Damage ranged from 0% to >75% including infarction, using a modification of the method described by Cataltepe et al. [4], and as described in our previous study [48]. The histologic evaluation was made under a 10  objective (100  magnification) and each subdivision of the sections was scored on a scale of 1– 5 (Table 2). The posterior sections were divided into cerebral cortex (subdivided as described above), thalamus, striatum, and the hippocampus (further subdivided into dentate gyrus, CA1, medial and lateral CA2, medial and lateral CA3 and CA4). The total sum of the scores given each of the segments were added together, in order to derive a score which represented the mean histologic damage score for all regions examined in the brain section. For more in depth determination of the extent to which cell damage occurred, a designated area (0.724 mm2) of the CA3 region of the hippocampus was outlined, using an image analysis system (NIH Image). Viable cells were marked, counted and eliminated. The non-viable cells were

Fig. 3. Photomicrographs of brain damage in control rat pups (A and C) and those during which post-ischemic hyperthermia was prevented (B and D). A and B are hematoxylin and eosin representations of coronal sections of the hippocampus at 3 days of recovery. In the group of photos, the pink staining cells are those that have died. Photographs C and D are GFAP representations of the hippocampus at 20 days of recovery. In this set of photographs, the darkly staining regions are abnormal astrocyte infiltration, depicting the area of damage. Note the very significant decrease in damage in those hippocampi of rats in which hyperthermia was prevented. Arrows point to representative cells that have died.

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then counted. The damage was assessed as a percentage of viable cells remaining compared to the total number of cells counted within the region (no. of non-viable cells/no. of viable cells + no. of non-viable cells X 100=% cells damaged). Evidence of irreversible neuronal injury included eosinophilia or clearing of the perikaryon, pyknosis and karyorrhexis of the nucleus at 3 and 20 days post-insult, and/or positive GFAP staining at 20 days post-insult. 2.7. Statistical analysis Data regarding neuropathologic outcome was analyzed using the non-parametric Kruskal – Wallis test for comparisons of ranking. Between group analysis and data regarding cell counting was compared utilizing Welch’s paired t-test for groups with unequal standard deviations. Temperatures were compared with two-tailed analysis of variance (ANOVA) followed by Tukey comparison. Differences in mortality rates among the age groups were

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analyzed using Fischer’s exact test. Significance was considered at p < 0.05.

3. Results In order to prevent spontaneous hyperthermia for those animals receiving KA, an environmental temperature of 29 F 0.5 jC was required, compared to the normal nesting temperature of 34 F 0.5 jC. Once accomplished, there was no significant difference in the core temperatures of normothermic animals, and those in the controlled normothermia group (see Table 1 and Fig. 2). Animals receiving 30 min HI plus KA, and not being controlled for normothermia, reached significantly higher temperatures (37.46 F 0.38 jC) throughout the duration of the seizures, compared to the other groups ( p < 0.05). Clinically, the characteristics of the seizure activity in those groups of rat pups (II and III) in who hyperthermia was

Fig. 4. Mean Brain Damage Score of rat pups as assessed at 3 (early) and 20 (late) days following the induction of hypoxia-ischemia and KA-induced seizures. Neuroprotection was afforded by preventing seizure induced hyperthermia either immediately post-ischemia or at the onset of seizures. Also note that the protection afforded was long-lasting. **p < 0.01 compared to homologous control (hyperthermic) brain region. *p < 0.05 compared to homologous control (hyperthermic) brain region.

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prevented, remained unchanged compared to controls (rat pups in whom spontaneous hyperthermia was allowed to occur). Electrographic monitoring of these rat pups (6 per group) again revealed no alteration in the duration nor severity of seizures. As in our previous study, and compared to controls, electrographic seizures were continuous, though there was a decrement in the amplitude of the discharges (Fig. 1c). Interestingly, mortality rates were significantly greater in those rat pups in whom hypothermia was induced immediately after the HI event [42.42%] (30 min prior to KA injection) compared to either the control animals who were hyperthermic [13.16%], or those in whom hypothermia was induced at the time of KA injection [14.28%]. There was no significant difference in mortality between the latter two groups of rat pups.

3.1. Neuropathology Photomicrographs of the hippocampal injury following seizures under normothermic and controlled normothermia conditions are shown in Fig. 3. Controlling the temperature of rat pups either immediately after the HI event, or prior to the onset of seizures significantly decreased ( p < 0.05) the amount of damage experienced by the immature brain exposed to HI and seizures (Fig. 4a and b). Neuroprotection was provided in a stepwise fashion, such that those animals in whom spontaneous hyperthermia was prevented immediately following the HI insult displayed the greatest decrease in mean rank score for brain damage (3.65 F 1.22 SEM vs. 14.25 F 1.48) at 3 and (2.38 F 1.06 vs. 12.44 F 3.34) at 20 days of recovery, compared to control values. In those rat pups receiving controlled normothermia prior to the onset of seizures, brain damage was also decreased, though to a lesser extent (Fig. 4). Comparison of the two controlled hypothermia groups did not reveal a significant difference in the extent or neuroprotection afforded, overall. There was however, a significant difference in hippocampal damage of those animals in which hyperthermia was prevented immediately following HI ( p < 0.05) (Fig. 5). Analysis of the data derived from cell counting within the CA3 region of the hippocampus mimicked the findings based on ranking the brains according to the extent of injury. No difference existed in the degree to which controlling for normothermia provided neuroprotection to the different regions of the hippocampus. Correlation between the two methods of pathologic assessment was strong with r2 values varying between 0.78 and 0.93.

4. Discussion

Fig. 5. Brain damage score of rat pups undergoing spontaneous hyperthermia during seizures (Group I), controlled normothermia immediately after HI (Group II), and controlled normothermia at the onset of seizures (Group III). In each graph letters (a) and (b) refer to early and late neuropathology. Note the significant decrease in hippocampal damage when the animals were assessed for late neuropathology as compared to early neuropathology, in those rats in whom controlled normothermia was induced immediately following hypoxia-ischemia.

These experiments provide important information with respect to the role that temperature plays in the newborn infant with HI encephalopathy. Our findings confirm those of our previous study [49], which showed that immature rat pups exposed to HI complicated by KA induced seizures become hyperthermic during the episode of seizures. Moreover, the findings of the current study indicate that (1) maintaining core temperature within normal values following HI and during post-ischemic seizures is neuroprotective, and (2) the induction of normothermia is time sensitive, such that a greater reduction in brain damage occurs if spontaneous hyperthermia is prevented immediately following HI, as opposed to at the onset of seizures. The latter finding requires clarification, in that the improved brain protection seen in those animals whose temperature was controlled immediately following hypoxia-ischemia, occurred in the setting of a higher mortality. It is possible that had the pups survived instead, that the extent of injury (or protection) would have been similar in the two groups. Currently, the authors cannot explain this dichoto-

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my, though the findings are clearly noteworthy and point out the important clinical implications that arise from timing of the induction of post-ischemic hypothermia. With respect to the improved neuroprotection seen in those animals in whom hyperthermia was prevented immediately following the ischemic event, some explanation may arise from previous work done in this animal model [51]. In this regard, using an identical animal model, recovery of ATP was found to be delayed in those rat pups that later demonstrated enhanced brain damage following hypoxiaischemia and seizures. These latter findings were further associated with a marked elevation of extracellular fluid concentrations of glutamate, as measured within the CA3 region of the hippocampus. If the prevention of hyperthermia early on in the course of recovery enhances ATP recovery following hypoxia-ischemia, or reduces the extent of hypermetabolism that occurs with seizures, the relative improvement in energy reserves within the hippocampus of these rat pups may alter the direction of cell death, and in some instances lead to complete recovery. Spontaneously occurring hyperthermia of 1 – 2 jC above normal has been well documented in the gerbil and rat models of adult stroke [17,52]. Several authors have shown a relationship between post-ischemic hyperthermia and the induction of immediate early genes (hsp70) [5,19]. In the immature rat model of hypoxia-ischemia, the animal becomes poikilothermic during the early recovery phase, and as such will reflect its surrounding environmental temperature [40,50]. Indeed, such is the case for the human newborn infant as well, and a prominent reason for the development of thermoregulatory control of the sick neonate [7,50]. However, controversy remains regarding whether or not post-ischemic hyperthermia exacerbates the damage that occurs as a result of ischemia [17,18,20,30]. In one of the few studies that have examined the effects of hyperthermia on epileptic brain damage, Meldrum and Brierly [25] noted that following bicucullineinduction, baboons who sustained seizures of between 1 and 5 h duration became hyperthermic. The authors of this study concluded that the hyperthermia aggravated the brain damage that was seen. In studies in which hyperthermia has been intentionally maintained during seizures, again in adult animals, brain damage has been markedly exacerbated [2,23]. Though there are no studies which have determined the effects of maintaining normothermia during post-ischemic seizures, several investigators have studied the neuropathologic effects of post-ischemic hypothermia on seizure induced brain damage. Gunn et al. [10,11] selectively cooled the brains of near-term fetal sheep to between 30 and 33 jC following 30 min of cerebral ischemia. In this experiment, hypothermia was induced prior to the onset of spontaneously occurring seizures. They found that while the hypothermia did not prevent the seizures, it did markedly reduce neuronal loss in the parasagittal and lateral cortex, compared to controls. In a subsequent study, the authors tested whether

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the induction of hypothermia after the onset of seizures in this experimental paradigm would be neuroprotective, and found that it was not. Importantly, these studies, as in the current investigation did not prevent the onset of seizures, indicating that the effect of hypothermia, or ‘‘relative hypothermia’’ acts to prevent or inhibit the secondary metabolic perturbations caused by the seizures, as opposed to simply acting as an anticonvulsant. Other investigators have similarly shown that hypothermia ameliorates, and hyperthermia exaggerates the brain damaging effect of seizures in adult animals [22,36], with variable effects on seizure propagation itself. Our finding that the prevention of hyperthermia (relative hypothermia) following HI and prior to the onset of seizures is neuroprotective is consistent with the work of Gunn et al. [10,11]. Since seizures alone do not result in neuronal necrosis in the immature brain [1,28,38,39,48], a pre-requisite requirement of injury appears necessary for seizures to cause further damage [12]. The protective effect afforded by the ‘‘relative post-ischemic hypothermia’’ may be related to its influence on cerebral energy metabolism and rate of recovery. Elegant work done by Laptook et al. [21] determined the rate of cerebral energy utilization in piglets which had been exposed to systemic temperatures ranging from 28 jC to 41 jC during 30 minutes of complete ischemia. This group of investigators found that energy utilization was reduced by 5.3% per 1 jC of hypothermia. During recovery, Thoresen et al. [42] found that post-ischemic systemic hypothermia in newborn piglets delayed the secondary energy failure that occurs within 48 h of the ischemic insult under normothermic conditions. Tasker et al. [41] found that the induction of post-ischemic hypothermia of 3 jC, in neonatal rat brain slices, improved the recovery of ATP by 20% compared to controls. With respect to the effect of hypothermia on KA induced seizures, Maeda et al. [24], showed a reduction in cerebral glucose utilization in the limbic structures of hypothermic compared to normothermic rats. Prolonged seizures following HI preferentially damaged the hippocampus in our animals [48]. It was intriguing to note that assessment of the brain damage in our groups revealed a reduction in damage to the hippocampus at 20 days of recovery compared to 3 days of recovery. These findings may be an indication of the degree to which the additive seizure induced injury may be recoverable in the immature brain. Certainly, others have witnessed a similar resolution of injury. Owens et al. [29] reported on the morphologic and electrophysiologic consequences of hypoxia-induced seizures. In their group of immature animals (P8 – P10), 3% oxygen causing [15] near complete energy depletion [16] and seizures, resulted in neuronal injury detected acutely in the dentate and hilar regions of the hippocampus, but not when the animals were examined at 60 – 80 days of recovery. Similarly, Toth et al. [43] found that in their model of febrile seizures in the immature rat, hyperthermic seizures resulted in the appearance of cell

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damage in the limbic system within 24 h, but by 4 weeks of recovery no significant neuronal dropout was evident. With respect to hypoxia-ischemia alone, data from the work by Towfighi et al. [44] also revealed an overall decrease in damage with time. In their study, 7-day rat pups were exposed to 2 h of hypoxia-ischemia and assessed histopathologically at recovery periods of from 0 to 3 weeks. Damage was seen almost immediately and peaked in the hippocampal region at 24 h of recovery. Subsequently, the overall extent of neuronal injury diminished. These results may be indicative of the reversible nature of seizure induced brain damage, and implies a role for successful intervention. In summary, our findings have important implications with respect to further understanding the mechanisms which underly HI related seizures in the newborn. Given that thermoregulation plays such a significant role in the treatment of the sick term and pre-term neonate, our findings further substantiate the need to prevent abnormal increases in brain temperature that may arise as a result from hypermetabolic pathologic states, such as neonatal seizures or sepsis. While most neonatal intensive care units thermoregulate the newborn to ensure that hypothermia does not occur, the same degree of vigilance does not apply to hyperthermic states. Indeed, there are no studies which have documented the role of pyrexia in the newborn sick infant. Our findings suggest that maintaining normothermia in the neonate following a hypoxic-ischemic event will diminish the damage that will occur as a result of subsequent seizures. Moreover, there finding further suggest a role for the apoptosis in seizure induced brain damage of the newborn, which because of its delayed occurrence, may provide therapeutic window during which intervention may take place. Further studies are clearly required to determine the mechanisms of brain damage involved in hypoxic-ischemic induced seizures of the newborn, and the interventions that may reduce their effect.

Acknowledgements The authors gratefully acknowledge the support for this research by grants from the Toronto Hospital for Sick Children Foundation and the Health Services Utilization and Research Commission of Saskatchewan. Dr. J.Y. Yager is the recipient of the Henry J.M. Barnett Scholarship from the Heart and Stroke Foundation of Canada.

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