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The progressive events after HI injury to the developing brain are complex and dependent upon the nature and conditions of the insult. Both pathophysiologic and histopathologic data suggest that further neuronal death may occur many hours and even days after the primary injury(1, 2). Studies of the time course of high energy phosphate metabolites and of loss of membrane function suggest that there are two phases of injury. The first phase of injury is characterized by energy failure and occurs during the hypoxia-ischemia and is followed 12 to 48 h later by the second phase of energy failure in neonates(3) and piglets(4). Similarly there are primary and secondary phases of loss of membrane function in the parasagittal cortex of fetal sheep with the secondary phase developing from about 7 h after severe HI injury(5). Molecular evidence of delayed death may also be inferred from the time course of neuronal DNA degradation in the infant rat brain after HI injury(6). After severe HI injury in the rat, the DNA is intact at 5 h but is degraded in the striatum, cortex and in the hippocampus by 24 h. After moderate injuries, these processes are slowed and occur over a period of about 24 to 72 h(6).

The importance of cerebral temperature during and immediately after the injury in determining outcome has been highlighted(7–11). Hypothermia during the period of the insult has been shown to prevent or retard HI brain injury(10, 12–14). Conversely, mild hyperthermia during or immediately after an insult can exacerbate an injury(15). Hypothermia is thought to protect the brain by reducing metabolic demands and suppressing cytotoxic processes, such as excitatory amino acid accumulation(16) and free radical activity(17). However, it is uncertain as to whether the protective effect of hypothermia is restricted to the insult period or whether it extends into the period of delayed cell death. If the latter was the case, then this may be of therapeutic advantage. Therefore, we designed an experiment to examine the effects of prolonged environmental temperature modifications after a moderate HI injury during the period when delayed damage may occur.

MATERIALS

HI injury preparation. The experimental approach is based on the Levine(18) preparation and involves inducing a transient unilateral HI injury in the brain of an infant rat. This approach has been previously described(6, 19) and offers the advantage of allowing chronic instrumentation for monitoring cerebral temperature(19). The experimental protocol followed guidelines approved by the Animal Ethics Committee of the University of Auckland. Weaned 21-d-old Wistar rats of both sexes weighing between 40 and 49 g were used for the study. The rats were maintained on a 12-h cycle of light and dark with free access to food and water throughout the study. Rats were anesthetized with halothane, and the right common carotid artery was exposed through a midventral neck incision. The carotid artery was double ligated using 4 “0” silk, and the neck incision was closed. A randomized complete block design was used, and studies were performed in batches of litter mates of 8 to 12 infant rats. After surgery the rats were held for 1 h in a prewarmed infant incubator maintained at 34°C with >80% relative humidity, then exposed to 8% O2 for 15 min. After hypoxia the rats were kept caged in groups of four to eight until they were killed.

Study 1. Effect of temperature on histopathologic outcome. The effect of environmental temperature on histologic outcome was investigated in 62 rats. After the hypoxia, the rats were divided into four temperature treatment groups.

  • Group 1. Group 1 was held in an environment of 34°C for 72 h after the hypoxia (n = 15).

  • Group 2. Group 2 was held at 34°C for 6 h and then at 22°C for a further 66 h (n = 14).

  • Group 3. Group 3 was held at 22°C for 6 h and then at 34°C for 66 h (n = 17).

  • Group 4. Group 4 was held at 22°C for 72 h after the hypoxia(n = 16).

Study 2. Effect of temperature regime on long term histopathologic outcome. At the end of the hypoxia, the rats were divided into two groups corresponding to group 1 (34°C for 72 h, n = 11) and group 4(22°C for 72 h, n = 10) of study 1. Subsequently both groups of rats were maintained at 22°C for the next 19 d.

Study 3. Effect of environment on cortical temperature. Cortical temperature was recorded in a separate group of rats (n = 13) using telemetric temperature probes (XM-FH-BP, Mini Mitter Co., Inc., Sunriver, OR). For the cortical temperature measurements the thermistors were positioned on the dura 2 mm anterior to the ear bars(20) and 3 mm lateral to the midline and fixed in place with dental cement. The thermistors were calibrated in the 22°C environment at 34 and 40°C in a precision water bath. The measurements made in the 34°C environment were corrected by -0.4°C to compensate for the systematic error due to the effect of the 34°C environmental temperature on the probe body. Temperature was continuously recorded during the experiments and averages were calculated and stored to disk at 1-min intervals(21). Histologic outcome was not determined in these rats. Cortical temperature measurements were made over 72 h after hypoxia in ligated rats for each of the temperature regimes (see Table 1 and Fig. 2, a-d). Cortical temperature measurements were also made in a group of sham controls(n = 3) that were not ligated but exposed to hypoxia then kept at 22°C (see Table 1 and Fig. 2e).

Table 1 Cortical temperature measurements for 0-6 h and 6-72 h after HI injury
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Figure 2
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The effect of environmental temperature upon cortical temperature. (a) Rats maintained at 34°C for 72h (n = 2). (b) Rats maintained at 34°C for 6 h then 22°C for 66 h(n = 3). (c) Rats maintained at 22°C for 6 h then 34°C for 66 h (n = 3). (d) Rats maintained at 22°C for 72 h (n = 2). (e) Sham-ligated rats maintained at 22°C for 72 h (n = 3).

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Histologic preparation. The brains of the rats that died after the hypoxia were processed for histology only if they were collected within 6 h of death. Otherwise all rats were euthanized with sodium pentobarbitone 72 h or 21 d after hypoxia for studies 1 and 2, respectively. The brains were fixedin situ by transcardial perfusion with a 0.9% saline solution followed by 10% neutral buffered formalin. The fixed brains were sliced into 2-mm thick coronal sections using a rat brain matrix (RBM-2000C, Activational Systems, Inc., Warren, MI), dehydrated through graded alcohols, and embedded in paraffin. Serial 4-μm sections were cut and stained with thionine-acid fuchsin as described previously(19).

Histologic analysis. The quantification of histologic damage was carried out by a researcher blinded to the treatment protocols. The live and dead neurons were discriminated by the respective absence or presence of acidophilia in the cytoplasm as previously described(22–25). Light microscopy on these sections was carried out at 40×, 100×, and 400×. Only the extent of infarction was analyzed after study 2.

Cortex. The cortical damage was evaluated by measurement of the area of pannecrosis using an image analyzer (Java, Jandel Scientific, San Rafael, CA). The coronal brain slices analyzed were taken from between 2.9 and 3.5 mm anterior to the ear bars. The indirect technique was used(26), and outcome was calculated as follows:Equation where RI = infarct area of right ligated hemisphere, LT = total cortex area of nonligated (left) hemisphere, and RN = total area of viable cortex of the ligated (right) hemisphere in the same slide. These infarct measurements were taken from three slices in each brain. The measurement with the largest area was used for subsequent statistical analysis(26).

Hippocampus. The damage was measured by counting the neurons surviving in the CA1-CA3 regions of the dorsal hippocampus of both hemispheres (Fig. 1). These counts were done in a single slide of a coronal section taken from between 2.9 and 3.5 mm anterior to the ear bars. The percentage of surviving hippocampal neurons was calculated by using the following formula:

Figure 1
figure 1

Schematic diagram of areas analyzed for histopathologic changes.

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Striatum. Damage within the striatum was determined in coronal sections taken from between 6.2 and 7 mm anterior to the ear bars. The severity of striatal neuronal loss was assessed using a four-point scale as reported elsewhere(27). Grade 0, no neuronal loss; grade 1, between 1 and 5% neuronal loss; grade 2, between 6 and 50% neuronal loss; and grade 3, between 51 and 100% neuronal loss.

Statistical analysis. All data are presented as mean ± SEM. The histologic data in study 1 were compared nonparametrically by applying ANOVA on the rank-transformed data followed by Student-Newman-Keulspost hoc tests. The incidence of cortical infarction was compared using the χ2 test with the Bonferroni correction for multiple comparisons. The area of cortical infarction in study 2 was compared by Mann Whitney U test. Cortical temperature measurements were compared byt test (Sigmastat, Jandel Scientific, San Rafael, CA).

RESULTS

Temperature. The results of the temperature recording data are described in Table 1 and Figure 2. During the period 0-6 h after the HI, all rats (n = 5) kept at 22°C had a lower cortical temperature of 35.5 ± 0.1°C compared with 37.9 ± 0.2°C for all those (n = 5) kept at 34°C(p < 0.01). A similar trend was recorded during the period from 6-72 h and those kept at 22 or 34°C had cortical temperatures of 35.4± 0.2°C and 37.9 ± 0.4°C, respectively (p < 0.01).

Study 1. Histologic outcome. Three group 3 rats were excluded from analysis as their brains could not be collected within 6 h of death. These rats died between 24 and 48 h after hypoxia.

Cortex. The rats exposed to the 34°C environment for either the first 6 h or subsequently (groups 1-3) had an increased incidence and area of cortical infarction compared with group 4 rats that were kept at 22°C for the period 0-72 h (p < 0.05) (Table 2, Fig. 3, a and b). The extent of cortical infarction was 6.5 ± 1.7 mm2 in group 1, 4.5 ± 1.8 mm2 in group 2, 5.1 ± 1.5 mm2 in group 3, and 0.3 ± 0.3 mm2 in group 4. In the group 1 rats, the coronal sections showed shrinkage of cortex within the ligated hemisphere (Fig. 4a). The neuronal damage was apparent as a loss of viable neurons and the presence of acidophilic and pyknotic neurons. Most rats had laminar necrosis with loss of cortical neurons in the more superficial layers (layers 1-3) of the cortex. This neuronal loss extended through all the layers of the cortex in the more severely affected rats (Fig. 4a). The extracellular matrix was vacuolated and uneven, and there were reactive glia and an abundance of dilated capillaries. These findings are consistent with pannecrosis or infarction (Fig. 4b). The histologic outcome in groups 2 and 3 rats were similar to that of group 1 rats but with a lesser degree of severity. In contrast the coronal sections from the ligated hemisphere of group 4 rats did not show these gross morphologic changes (Fig. 5a). The histopathologic changes in the cortex generally consisted of selective neuronal loss predominantly occurring in cortical layers 4 and 5 within the middle cerebral artery territory of the lateral cortex. There were no gross histologic changes to the extracellular matrix (Fig. 5b).

Table 2 Cortical infarction and mortality
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Figure 3
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The effect of environmental temperature on histologic outcome after the HI injury. (a) Incidence of cortical infarction(group 4 p < 0.05). (b) Area of cortical infarction(group 4 p < 0.05). (c) Number of surviving CA1-CA3 hippocampal neurons. (d) Striatal loss (group 4 p = 0.05).(e) Area of cortical infarction from rats kept 21 d (p< 0.01).

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Figure 4
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Photomicrographs of a representative brain from a rat kept at 34°C from 0 to 72 h after hypoxia (group 1). (a) Coronal section showing loss of definition between layer 1 (molecular layer) and layer 2 of the cortex (solid arrow) due to shrinkage resulting from infarction and presence of dilated capillaries (arrowheads). Bar = 1 mm. (b) High power photomicrograph of the cortex (boxed area) taken from (a). Examples of pyknotic neurons(arrows). The extracellular matrix is vacuolated and shows increased vascularization. An increased glial reaction (solid arrows) is evident around some of the pyknotic neurons. Thionine-acid fuchsin(400×). Bar = 100 μm.

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Figure 5
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Photomicrographs of a representative brain from a rat kept at 22°C from 0 to 72 h after the hypoxia (group 4). (a) Coronal section showing loss of hippocampal Nissl staining confined to the ligated hemisphere but no other gross changes to the cortical morphology. Bar= 1 mm. (b) High powered photomicrograph of the injured cortex(boxed area from a) showing several pyknotic neurons(arrows) scattered between a population of viable cortical neurons. The extracellular matrix appears normal. Thionine-acid fuchsin (400×). Bar = 100 μm.

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Hippocampus. Environmental temperature did not significantly alter neuronal loss within CA1-3 region of the hippocampus, although there was a trend to reduced loss in group 4 (Fig. 3c). At low magnifications, the hippocampal damage in whole brain sections appeared as a unilateral loss of neuronal Nissl staining. The damaged neurons within the hippocampus were acidophilic and pyknotic. Compared with the group 4 rats, group 1 rats had increased gliosis and vacuolation in areas adjacent to CA1 region.

Striatum. The temperature treatments modified the severity of damage within the striatum and the group 4 rats maintained at 22°C had less damage (p = 0.05) (Fig. 3d). Injured striatal neurons were pyknotic or acidophilic, and the severity of injury in groups 1, 2, and 3 appeared to be similar.

Study 2. Three group 1 rats were excluded from analysis because their brains were not collected within 6 h of death. The rats exposed to the 34°C environment for 72 h after the hypoxia had an increased area of cortical infarction (12.1 ± 3 mm2) compared with those kept at 22°C (3.4 ± 1.5 mm2) for the period 0-72 h (p < 0.01) when assessed 21 d after the injury (Fig. 3e). In the former group, the coronal sections showed shrinkage of cortex within the ligated hemisphere. Rats in group 1 were lighter at 1 but not 3 wk postinjury(p < 0.01) (Table 3).

Table 3 Study 2 body weights (g)
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DISCUSSION

The results of this study suggest that moderate reductions in cerebral temperature of about 2°C throughout the period 0-72 h after HI injury can markedly reduce the severity of damage. However, reducing cerebral temperature for either the first 6 h or from 6 to 72 h did not significantly improve outcome, although there was a trend for cooling during the early and late neuronal injury phases to reduce cortical neuronal loss. These data suggest that prolonged reductions in cerebral temperature initiated after the insult itself are necessary to improve outcome.

Moderate hypothermia of about 2-3°C during hypoxic-ischemic injury is clearly neuroprotective in the mature and developing brain(28, 29). A recent study in adult gerbils showed that the efficacy of postischemic hypothermia is dependent on the severity of the primary insult. Twelve hours of hypothermia increased the number of surviving hippocampal neurons with more neurons surviving after 3 compared with 5 min of ischemia. Furthermore, there was a lesser percentage reduction in neuronal loss after the longer period of ischemia. It was further observed that increasing the duration of the hypothermia from 12 to 24 h produced much greater and long-term protection of hippocampal neurons after the 5 min of ischemia(30). A similar trend toward reduced loss of hippocampal neurons was observed when the duration of hypothermia was raised from 30 min to 5 h, starting 2 h after 10 min of ischemia in adult rats(31). In contrast a shorter duration (3 h) of hypothermia after 10 min of ischemia in adult rats(10) was not protective. Similarly 3 h of hypothermia after a prolonged (3 h) HI injury in 7-d-old rats(29) did not provide long-term protection. In the present study, 3 d of moderate hypothermia markedly reduced the extent of cerebral damage after a brief injury. Our results are compatible with observations in the mature brain that suggest prolonged hypothermia after HI injury provides a greater neuroprotective effect.

The cortex and striatum, but not the hippocampus, showed less damage when the rats were maintained at the lower temperature (Fig. 3,b-e). A similar regional neuroprotective effect occurs when moderate hypothermia is applied during ischemia in piglets(28). Given the superficial location of the cortex, it will have cooled to a greater degree due to conduction of heat through the skull and scalp(32). In contrast, the temperature of deeper brain structures is primarily determined by core temperature, particularly in species larger than rats(32). Work by others(28, 29) indicates that core temperature falls to a lesser degree than the cortex; however, the magnitude of this difference is likely to be influenced by the size of the species(32). Presumably the deeper structures are influenced to a lesser degree by the environmental temperature changes. This difference may partially explain the lack of significant protection in the hippocampus compared with the cortex, although it still does not clearly account for the more significant response in the striatum. Possibly the hippocampus was more severely injured or that different mechanisms were involved in the hippocampal neuronal death. A greater reduction in core temperature of at least 3-4°C might be necessary to achieve significant protection in deeper structures such as the hippocampus(31).

Infarction apparent as laminar necrosis of the cortex was seen in rats maintained in the warm environment (Fig. 4). This pattern of damage is similar to that seen in late gestation fetal sheep after an ischemic injury in utero(33). These cortical infarcts are associated with the development of seizures(33, 34). Thus it is possible that the increased cerebral temperature that occurs in the fetal environment exacerbates encephalopathies resulting from HI injuries occurring in utero.

The mechanisms causing delayed damage have not been clearly identified. The cascade of processes triggered by the primary injury can lead to secondary energy failure(35) and further neuronal death(6) in the developing brain. Apoptosis(6), excitoxicity(33), seizure activity(33), free radicals(36), and microglial reactions(6) may be involved in this cascade. Hypothermia is likely to interfere with several of these cytotoxic processes and can, at least around the period of the primary injury, reduce accumulation of excitatory amino acids(16), phospholipid degradation(37), the actions of free radicals(17), and cerebral metabolism. Clearly a difference of about 2°C can strongly modulate the cytotoxic mechanisms leading to cortical infarction. This effect may have important implications for some pharmacologic neuronal rescue studies because it is possible that some therapeutic agents may alter outcome by inducing moderate hypothermia. Surprisingly moderate hypothermia during either the first 6 h or from 6 to 72 h, when much of the neuronal death occurs in this preparation(6), did not significantly alter outcome. Only prolonged hypothermia was effective, suggesting that there is a critical period preceding and during the period of delayed neuronal death when cytotoxic processes need to be suppressed. In contrast to our results, Coimbra and Wieloch(31) observed that a delayed period (5 h) of cooling initiated at 6 and 12 h after 10 min ischemia in adult rats reduced the loss of hippocampal but not striatal neurons. This difference may be due to the use of lower temperatures (33°C) in the study by Coimbra and Wieloch(31). Alternatively regional differences in the rate of cell death(6, 31) or differences in neurologic maturation and the nature of the injury could account for this disparity.

The observations in this study may have important ramifications. Clinical studies suggest that sick premature newborn infants maintained in a warmer environment display a lower mortality(38–40). Thus current practice aims at maintaining thermoneutrality in all babies. However, this study suggests that exposure to a thermoneutral environment in the hours after HI injury can markedly worsen neurologic outcome (Fig. 3). In contrast, prolonged and mild cerebral hypothermia of about 2°C was protective. In terms of neurologic development, the rat preparation used in these studies is more relevant to the term or more mature infant(19). These findings raise questions regarding the routine management of temperature in asphyxiated term newborn infants. Further investigations are needed to evaluate the relevance of these observations.