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(Stroke. 1995;26:1273-1278.)
© 1995 American Heart Association, Inc.

Articles

Deep Hypothermia Diminishes the Ischemic Induction of Heat-Shock Protein-72 mRNA in Piglet Brain

Ellen G. Shaver, MD; Frank A. Welsh, PhD; Leslie N. Sutton, MD; Gerardo Mora, MD; Laura M. Gennarelli, BS Carole R. Norwood, PhD

From the Division of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia (E.G.S., F.A.W.); and the Division of Neurosurgery (L.N.S., L.M.G.) and the Cardiac Surgical Research Laboratories (G.M., C.R.N.), The Children's Hospital of Philadelphia (Pa).

Correspondence to Dr Frank A. Welsh, 313 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104.

	Abstract

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Background and Purpose Expression of the 72-kD heat-shock protein (HSP72) has served as a useful indicator of ischemic stress after cerebral ischemia. Moderate hypothermia (30°C) has been reported to block the induction of HSP72 after a brief episode of forebrain ischemia. The objective of the present study was to examine the effects of deep hypothermia (15°C) on expression of HSP72 after a prolonged period of cerebral ischemia.

Methods Piglets, 19 to 23 days old, were placed on cardiopulmonary bypass, and brain temperature was lowered to 23°C (n=9) or 15°C (n=9) before circulatory arrest for 1 hour. In an additional group of animals (n=5), the temperature was lowered to 29°C before arrest for 45 minutes. All animals were reperfused at 37°C for 2 hours, and the regional expression of HSP72 mRNA was assessed using in situ hybridization.

Results After ischemia at 15°C, expression of HSP72 mRNA was limited to a few scattered regions of cerebral cortex; the percentage of cortex exhibiting HSP72 mRNA was 23±7% (mean±SEM). Ischemia at 23°C triggered expression of HSP72 mRNA in a significantly larger portion of the cortex (68±8%, P<.001). Ischemia at 29°C failed to induce substantial expression of HSP72 mRNA in the cerebral cortex.

Conclusions These results suggest that, relative to ischemia at 23°C, deep hypothermia (15°C) diminishes ischemic alterations leading to induction of HSP72 mRNA. The lack of cortical expression of HSP72 mRNA following ischemia at 29°C may be secondary to inadequate recovery of energy metabolism.

Key Words: cardiopulmonary bypass • cerebral ischemia • heat-shock proteins • hypothermia • pigs

	Introduction

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Cerebral ischemia induces the selective synthesis of a number of proteins, including the 72-kD heat-shock protein (HSP72).^{1 2 3} Although the cellular functions of HSP72 are not well understood, the induction of HSP72 has been associated with cellular resistance to ischemic and excitotoxic injury.^{4 5 6 7} Apart from its function, HSP72 has served as a useful indicator of cell stress in a number of experimental models.^{8 9 10 11 12} Thus, induction of HSP72 may be an indicator of potentially lethal cell stress, whether induced by heat, excitatory stimuli, or ischemia.

Recently, hypothermia (30°C) was reported to diminish the induction of HSP72 resulting from transient forebrain ischemia in rats.¹³ However, the mechanism by which hypothermia blocks the ischemic induction of HSP72 has not been elucidated. Hypothermia may simply reduce the intensity of ischemic stress below the threshold required to trigger expression of HSP72. If so, then extending the duration of ischemia might be expected to overcome the hypothermic reduction of expression. A model of prolonged ischemia in piglets subjected to cardiopulmonary bypass has been developed to investigate the effects of deep hypothermia on ischemic alterations.¹⁴ The objective of the present study was to examine the temperature dependence of HSP72 expression caused by an extended episode of circulatory arrest. Expression of HSP72 was examined regionally using in situ hybridization to detect the presence of the mRNA encoding HSP72. Preliminary results of this study have been presented previously.¹⁵

	Materials and Methods

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The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia. A full description of the animal preparation and cardiopulmonary bypass has been given previously.¹⁴ Briefly, 25 piglets, 19 to 23 days old and 4.5 to 6.7 kg in weight, were anesthetized with fentanyl and mechanically ventilated with room air enriched with oxygen. The femoral artery was cannulated for measurement of blood pressure and blood gases. Core temperature was monitored with a rectal or esophageal probe, and brain temperature was measured with a subdural thermistor. A median sternotomy was performed, the ribs were retracted, cannulas were placed into the right atrium and aorta, and cardiopulmonary bypass was initiated.

Two principal groups of animals were studied. In the first group (n=9), brain temperature was lowered to 15°C using the refrigeration system of the bypass apparatus. In the second group (n=9), brain temperature was lowered to 23°C before ischemia. In both groups, complete circulatory arrest was achieved by stopping the bypass pump. After a 1-hour period of complete ischemia, cardiopulmonary bypass was resumed using perfusate warmed to 37°C. Once the brain temperature had returned to 37°C, the piglets were removed from bypass for 2 hours of normothermic reperfusion. Additional animals were prepared to examine the effects of intraischemic temperatures warmer than 23°C on expression of HSP72 mRNA. Thus, in a group of 5 animals, brain temperature was lowered to 29°C before a 45-minute episode of circulatory arrest and 2-hour reperfusion. Longer periods of ischemia at 29°C or warmer were not attempted because preliminary studies indicated impaired recirculation after circulatory arrest. Finally, a group of 5 animals served as controls. Two of these underwent bypass for 1 hour at 15°C and 23°C without arrest, followed by restoration of brain temperature to 37°C for 2 hours. The remaining 3 animals served as unoperated controls.

In situ hybridization was used to detect the regional expression of HSP72 mRNA. After 2 hours of normothermic reperfusion, animals were killed by arresting the heart, and the brain was dissected free of the cranium and frozen on dry ice. The brain was sectioned in a cryostat (-11°C) at a thickness of 15 µm, and sections were mounted on glass slides, dried, fixed in 4% paraformaldehyde, and rinsed in 2x saline–sodium citrate (SSC). The mRNA for HSP72 was detected using a 30-mer oligodeoxynucleotide probe with the following sequence: 5'-C GAT CTC CTT CAT CTT GGT CAG CAC CAT GG-3'. This sequence has been reported to be specific for the inducible HSP72.^{16 17} The probe was labeled on the 3' end with ³⁵S-dATP using terminal transferase, and the labeled probe was hybridized to tissue sections overnight at 37°C in 4x SSC. After being rinsed with 1x SSC and 0.5x SSC at 37°C, the slides were dried and exposed to Kodak XAR film for 1 to 3 weeks.

The percentage of cerebral cortex exhibiting HSP72 mRNA was measured with image analysis (BRAIN, Drexel University Imaging and Computer Vision Center, Philadelphia, Pa). For each brain section, the autoradiographic image was digitized, and the total area of cerebral cortex was determined by visually outlining the cortical perimeter and integrating and subtracting the area of white matter. Regions of cortex in which the optical density was at least 50% higher than baseline cortex were then integrated and expressed as a percentage of the total cortex in the section. For each brain, at least eight autoradiographic images were analyzed, and the results were averaged.

An unpaired Student's t test was used to determine the statistical significance of the difference in cortical expression of HSP72 mRNA between the 15°C and 23°C groups. ANOVA was used to test for significant differences in arterial variables.

	Results

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All animals survived the period of circulatory arrest and reperfusion. There were no significant differences in arterial variables among the temperature groups, except for mild acidosis that developed during postischemic reperfusion in the 29°C group (Table 1). Arterial PO₂ was elevated in all animals because of the addition of oxygen to the ventilation mixture. After the piglets were placed on cardiopulmonary bypass, brain temperature fell to 23°C by 9 minutes and to 15°C by 17 minutes, remaining at these levels for the duration of the 1-hour period of arrest (Fig 1). During reperfusion, the temperature of the brain returned to 37°C by 30 minutes in both groups and was maintained at 37°C for the 2-hour recirculation period.

View this table: [in this window] [in a new window]   Table 1. Arterial Variables

View larger version (30K): [in this window] [in a new window]   Figure 1. Graph shows brain temperature during ischemia and reperfusion. Animals were placed on cardiopulmonary bypass, and brain temperature was lowered to 23°C (n=9) or 15°C (n=9) before ischemia. After circulatory arrest for 1 hour, the brain was reperfused and rewarmed to 37°C within 30 minutes for an additional 2 hours of normothermic recirculation. Each point is a mean value, with vertical brackets representing standard errors.

In control animals not undergoing surgery, expression of HSP72 mRNA was not detected in cerebral cortex or white matter (data not shown). Likewise, in control animals placed on cardiopulmonary bypass and rendered hypothermic without circulatory arrest, there was no evidence of HSP72 mRNA expression (Fig 2a and 2c). The intensity of hybridization was higher in the cerebellar folia than in cerebral cortex in both control and sham-operated animals.

View larger version (85K): [in this window] [in a new window]   Figure 2. Photomicrographs show regional expression of heat-shock protein 72 mRNA in piglet brain: a, 1 hour of cardiopulmonary bypass at 15°C (without arrest) followed by 2 hours of recovery at 37°C; b, 1 hour of ischemia at 15°C followed by 2 hours of reperfusion at 37°C (note isolated patches of cortical expression [arrow]); c, 1 hour of cardiopulmonary bypass at 23°C (without arrest) followed by 2 hours of recovery at 37°C; and d, 1 hour of ischemia at 23°C followed by 2 hours of reperfusion at 37°C.

In animals undergoing circulatory arrest at 15°C, expression of HSP72 mRNA occurred in a few scattered regions of cerebral cortex (arrow, Fig 2b). The cortical expression was typically patchy, often encompassing large portions of cortical gyri. However, there appeared to be no consistent anatomic localization to the cortical pattern of HSP72 mRNA expression. The percentage of cerebral cortex expressing HSP72 mRNA in this group was 23±7% (mean±SEM, n=9). In subcortical regions, expression of HSP72 mRNA was detected in the basal ganglia and hippocampus in several of the animals (Table 2). In the cerebral white matter, expression of HSP72 mRNA was not observed, with the exception of 1 animal.

View this table: [in this window] [in a new window]   Table 2. Regional Expression of Heat-Shock Protein 72 mRNA After Hypothermic Complete Ischemia

Circulatory arrest at 23°C induced widespread expression of HSP72 mRNA in the cerebral cortex (Fig 2d). The percentage of cortex expressing HSP72 mRNA after ischemia at 23°C (68±8%) was significantly greater than that after ischemia at 15°C (Fig 3). The regional pattern of cortical expression again was patchy, with no consistent anatomic localization. In regions showing HSP72 mRNA expression, the intensity of hybridization appeared to be similar in both the 23°C and 15°C groups. In subcortical regions, robust expression of HSP72 mRNA was also detected in the cellular layers of the hippocampus in all animals of the 23°C group (Table 2). In addition, a moderate level of expression was detected in the basal ganglia in 6 of 9 animals, whereas in the white matter, expression of HSP72 mRNA was noted in only 3 of 9 animals of this group.

View larger version (25K): [in this window] [in a new window]   Figure 3. Bar graph shows percentage of cerebral cortex expressing heat-shock protein 72 (Hsp72) mRNA after 1 hour of ischemia at 23°C or 15°C. For each animal, the area of cortex exhibiting increased hybridization was measured and expressed as a percentage of total area of cortex. The height of the bars represents the mean value for each temperature group (n=9 animals per group), and the vertical brackets denote standard errors. The asterisk indicates a significant difference from the 23°C group, P<.001, Student's t test.

In the 29°C group, expression of HSP72 mRNA was remarkably absent in the cerebral cortex in 3 of 5 animals (Fig 4 and Table 2). In the remaining 2 animals of this group, a moderate level of expression was detected in the deep portions of several sulci (data not shown). Diffuse expression of HSP72 mRNA occurred throughout the subcortical white matter expression, with fingerlike projections extending into cortical gyri. In the cerebellum, intense HSP72 mRNA expression was noted (Fig 4). Expression of HSP72 mRNA also occurred in the basal ganglia and hippocampus in the 29°C group of animals (Table 2).

View larger version (119K): [in this window] [in a new window]   Figure 4. Photomicrograph shows the effect of ischemia at 29°C on regional expression of heat-shock protein 72 (HSP72) mRNA. Brain temperature was lowered to 29°C before 45 minutes of ischemia followed by 2 hours of reperfusion at 37°C. Expression of HSP72 mRNA was absent in cerebral cortex but was detected in subcortical white matter, basal ganglia, and hippocampus.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present results demonstrate that the ischemic induction of HSP72 mRNA is strongly influenced by temperature of the brain during circulatory arrest. Thus, ischemia at 15°C induced the expression of HSP72 mRNA in a smaller percentage of the cerebral cortex compared with that induced by ischemia at 23°C. However, ischemia at 29°C failed to induce cortical expression of HSP72 mRNA, except in a few isolated regions. Thus, in the present model of prolonged ischemia, cortical expression of HSP72 mRNA was most extensive in the 23°C group of animals.

The biological significance of the ischemic induction of HSP72 mRNA requires some discussion. Because HSP72 is not normally expressed in brain, the presence of HSP72 and its mRNA has been interpreted as an indication of ischemic stress in several models of cerebral ischemia.^{8 9 10 11 18 19} Furthermore, induction of HSP72 in hippocampal neurons after afferent stimulation was reported to closely parallel selective neuronal injury in this model, suggesting that the presence of HSP72 indicates potentially lethal cell stress.¹² This interpretation is consistent with the general correlation between expression of HSPs and the intensity of other forms of cell stress, such as heat.²⁰ Thus, expression of HSP72 mRNA after cerebral ischemia is likely to be related to the intensity of ischemic stress. It should be cautioned, however, that the presence of HSP72 does not reliably predict whether a given cell will survive the ischemic insult.¹¹

The decreased extent of cortical expression of HSP72 mRNA after ischemia at 15°C versus 23°C is consistent with a previous report showing that reduction of temperature from 36°C to 30°C markedly reduced the subsequent appearance of immunoreactive HSP72 after transient forebrain ischemia in rats.¹³ Furthermore, a reduction in temperature has been reported to decrease HSP induction in vitro after exposure to metabolic inhibitors.²¹ Taken together, these results suggest that hypothermia reduces the expression of HSP72 by diminishing the ischemic alterations that activate expression of the HSP72 gene.

What then is the explanation for the paucity of HSP72 mRNA expression after ischemia at 29°C? The duration of ischemia at 29°C (45 minutes) was less than that used for 23°C (60 minutes). It is possible but not likely that the difference in duration of ischemia accounts for the difference in expression between 23°C and 29°C. Alternatively, ischemia at 29°C may have caused irreversible alterations in cellular metabolism that interfered with expression of HSP72 mRNA. Previous studies in this model have demonstrated marked impairment in the restoration of high-energy phosphates after 30 minutes of ischemia at brain temperatures between 33°C and 37°C.¹⁴ Although brain metabolites were not measured in the present study, it is possible that restoration of ATP after 45 minutes of ischemia at 29°C was not adequate to support transcriptional activity. Furthermore, the variability in cortical expression of HSP72 mRNA after ischemia at this temperature may be due to small differences in ATP restoration. Indeed, impaired reperfusion could easily limit the availability of metabolic energy needed for expression as well as account for the variability of expression in cortex. However, without additional information, the reasons for the limited expression of HSP72 mRNA in cerebral cortex after ischemia at 29°C must remain speculative. Finally, the prominent expression of HSP72 mRNA in white matter may be related to the enhanced restoration of ATP in white matter compared with that in cortex as demonstrated previously.¹⁴

After ischemia at both 15°C and 23°C, the regional pattern of expression of HSP72 mRNA in cerebral cortex was remarkably patchy. The factors responsible for the regionally heterogeneous expression are not immediately apparent. Because ischemia was complete, the possibility of residual perfusion in isolated regions of the brain is unlikely. Alternatively, temperature gradients within the brain during ischemia might have caused uneven expression of HSP72 mRNA. However, the apparently random location and size of the regions exhibiting expression were not suggestive of temperature gradients. Patchy reperfusion has been described in previous studies of prolonged periods of cerebral ischemia.^{22 23} Thus, it is possible that the cortical pattern of HSP72 mRNA expression observed in the present study is related in some way to the pattern of reperfusion.

In conclusion, the present results demonstrate that reduction of brain temperature from 23°C to 15°C before 1 hour of complete ischemia markedly diminished the extent of expression of HSP72 mRNA in cerebral cortex. This result suggests that deep hypothermia reduces the intensity of ischemic cell stress even after extended periods of ischemia. Finally, the paradoxical lack of HSP72 mRNA expression after prolonged ischemia at 29°C may be due to impaired energy metabolism.

	Acknowledgments

 
This work was supported by National Institutes of Health grant NS-29331. We thank Valerie A. Harris and Ralph Franks for their valuable technical assistance.

Received June 16, 1994; revision received February 23, 1995; accepted March 22, 1995.

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