Journal of Applied Physiology AJP: Cell Physiology
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J Appl Physiol 84: 1269-1277, 1998;
8750-7587/98 $5.00
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Vol. 84, Issue 4, 1269-1277, April 1998

HSP70 expression in the CNS in response to exercise and heat stress in rats

T. J. Walters1, K. L. Ryan2, M. R. Tehrany2, M. B. Jones2, L. A. Paulus3, and P. A. Mason1

1 Veridian, Inc. at Air Force Research Laboratory, Human Effectiveness Directorate, Directed Energy Bioeffects Division, Brooks Air Force Base; 2 Department of Biology, Trinity University, San Antonio 78212; and 3 Air Force Research Laboratory, Human Effectiveness Directorate, Directed Energy Bioeffects Division, Brooks Air Force Base, Texas 78235-5324

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We have previously documented the regional distribution of 70-kDa heat shock protein (HSP70) in brains of rats made hyperthermic by brief exposure to high-powered microwaves (HPM; 2.06 GHz). We now compare HSP70 expression induced by HPM exposure to that induced by exertional and/or environmental heat stress. Rats were chronically implanted with a temperature probe guide in the hypothalamic region of the brain (Tbr). After recovery, the following treatment groups were examined: HPM; sham exposed; treadmill exercise at room temperature (24°C; Ex-1); treadmill exercise in a warm environment (34°C; Ex-2); and sedentary groups (Sed-1 and Sed-2), in which ambient temperature was adjusted so that the Tbr mimicked the Tbr in the corresponding exercise groups. Significant HSP70 expression occurred only in the hyperthermic (Ex-2, Sed-2, and HPM) groups. The pattern of HSP70 expression was similar among Ex-2 and Sed-2 rats but differed from that in HPM rats. We conclude that 1) the pattern of HSP70 expression differs between HPM and nonmicrowave heating, and 2) exercise alone was not sufficient to induce central HSP70 expression.

heat shock protein 70; central nervous system; brain; electromagnetic fields; microwaves; thermoregulation; treadmill

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

TO GAIN A BETTER UNDERSTANDING of the health and safety concerns of microwave exposure, we have been involved in a series of investigations examining the impact of microwave exposure on the central nervous system (CNS). Preliminary data have been reported showing the regional distribution of 70-kDa heat shock protein (HSP70) in the brains of rats exposed to brief periods of hyperthermia induced by high-powered microwave (HPM; 2.06 GHz, 1.7-2.0 W/cm2) exposure (21, 22, 35). These investigations revealed a nonuniform HSP70 distribution after HPM exposure. These results could be due to either regional differences in the capacity of the brain to express HSP70 or to some other factor unique to HPM heating. To address these possibilities, it was necessary to compare HPM heating with nonmicrowave modes of inducing heat stress.

Few naturally occurring conditions result in the magnitude of temperature increase produced by HPM exposure, with the exception of exertional heat stress. This would seem to make exertional heat stress an ideal nonmicrowave modality with which to compare the HSP70 response after HPM-induced hyperthermia. However, it is unknown whether exercise, independent of an increase in temperature, results in the induction of HSP70. Human and animal exercise studies have reported the induction of expression of HSP70 mRNA, HSP70 protein, as well as heat-shock transcription factor in skeletal muscle, cardiac muscle, liver, spleen cells, lymphocytes, and leukocytes after acute exercise (8, 9, 13, 16, 17, 24, 27, 28, 30, 32). It is important to note, however, that these studies did not measure the local temperature in the tissue of interest. This temperature may have been elevated by exercise. HSP70 induction has been shown to take place in the CNS in response to a number of nonthermal stressors, such as ischemia (for review, see Refs. 7, 15, 25, and 34). If exercise alone is a sufficient stimulus for HSP70 expression in the brain, the pattern and magnitude of HSP70 expression may differ after acute exercise in the presence and absence of heat stress (i.e., heat-dependent and -independent expression).

To address these questions, the following null hypothesis was tested: similar levels of heat stress [i.e., similar brain temperatures (Tbr)] induced by 1) passive heat stress, 2) exertional heat stress, or 3) HPM-induced heat stress will result in qualitatively and quantitatively similar HSP70 induction in the rat brain. During these experiments, Tbr was measured continuously throughout the treatment. After animals were euthanized, immunohistochemical techniques were used to identify, with single-cell resolution, the pattern of HSP70 immunoreactive nuclei (HSP IN) expression in the rat brain.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal care. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee of Armstrong Laboratory, Brooks Air Force Base, Texas, and were conducted according to the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" [DHEW Publication No. (NIH) 86-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892].

Male Sprague-Dawley rats were obtained from the colonies of Charles River (Wilmington, MA). They were individually housed in standard plastic cages (26 × 23 × 20.5 cm) with water available ad libitum. Rats received Formulab 5008 (Purina Meals, St. Louis, MO) and were weighed twice a week. From 10 wk of age, all rats were maintained on a calorically restricted diet, receiving 60-63% of the diet consumed by rats fed ad libitum (29). Food restriction was required to maintain all rats within the same weight range. This was especially critical in the HPM group because the specific absorption rate of microwave energy is dependent on animal mass (6). Rats were 4-5 mo old and weighed 319-385 g at the time of the experiments. A 12:12-h light-dark cycle (light on at 0600) was used, and the room temperature was maintained at 22-24°C.

Stereotaxic surgery. All rats [except the home cage (HC) animals] were anesthetized with a combination of ketamine (70 mg/kg ip), xylazine (10 mg/kg ip), and atropine (0.01 mg/kg ip) and placed on a thermostatically controlled water-filled heating pad set to maintain a rectal temperature (Tre) of 37°C. Rats then had a Vialon guide (Becton Dickinson) stereotaxically implanted into the hypothalamus. With the mouthpiece of the stereotaxic instrument set at -3.3, the coordinates for the tip of the guide were 1.8 mm posterior to bregma, 1.5 mm lateral from midline, and 8.3 mm below dura (26). The bottom of the guide was sealed, and the top of the guide was equipped with a threaded head cap. The cap was mated with a Tygon tether that contained the thermal probe used to measure Tbr during experimentation. The guide was held in place with cranioplastic cement (Plastic One, Roanoke, VA) anchored to nylon screws.

Treadmill familiarization. Two weeks after surgery, rats were familiarized with treadmill exercise by running on a motorized treadmill (Dual Economy model, Columbus Instruments, Columbus, OH) for 10 min at a speed of 13 m/min (0% grade). Each rat received a minimum of four sessions, spaced no less than 2 days apart. However, to further guard against a possible confounding effect of treadmill training, rats were not assigned to the sedentary and exercise treatment groups until after treadmill familiarization was complete; thus all of the rats were exposed to treadmill running. HC rats and rats that were exposed or sham exposed to HPM did not undergo treadmill familiarization.

Treatment groups. After treadmill familiarization, rats were assigned to one of four treatment groups: treadmill exercise at room temperature (24°C; Ex-1, n = 6); treadmill exercise in a warm environment (34°C; Ex-2, n = 6); and two sedentary groups [Sed-1 (n = 6) or Sed-2 (n = 6)]. For the two sedentary groups, rats were placed on the environmentally controlled treadmill but were not run. Instead, environmental temperature (Tenv) was manipulated so that the Tbr in the Sed-1 and Sed-2 rats mimicked the rise in Tbr measured in the Ex-1 and Ex-2 rats, respectively. Because the Sed-1 and Sed-2 groups were intended to mimic the thermal profiles of their respective exercise groups, it was necessary to first complete the experiments with Ex-1 and Ex-2 groups to provide the profiles. Other groups of implanted rats were exposed to HPM (HPM; n = 5) or were sham exposed (Sham; n = 4). HC rats (n = 4) were also included to control for any possible effect of the technical manipulations (e.g., surgery, temperature measurements) on HSP IN expression. These rats were not exposed or sham exposed to any treatment but remained in their HC until euthanization.

Experimental procedure. All experiments were carried out between 0730 and 1130. On the day of the experiment, rats were brought into the laboratory (room temperature: 22-24°C) and placed on a rubber cart top. Each rat was then instrumented with a lubricated rectal probe that was inserted 5 cm beyond the rectum and attached to the tail with silk surgical tape. Tre was measured by using a nonmetallic temperature probe (±0.1°C, model 101, Vitek, Boulder, CO) during HPM exposure and a YSI temperature probe (model 700, Yellow Springs Instruments, Yellow Springs, OH) during all other procedures. The guide cannula fixed to the rat's skull was then connected to a tether containing a Vitek temperature probe that was advanced until it reached the end of the guide within the brain. Baseline Tre and Tbr were recorded for 10 min. Tympanic temperature (Tty) was recorded immediately before and as soon as possible after all experimental treatments. Tty measurements then continued at 2.5-min intervals for another 10 min.

Rats were placed on a treadmill (8% grade) where Tenv had been adjusted to the target temperature (see Environmental control). In the exercise groups, the initial speed of the treadmill belt was set at 13 m/min. After 2.5 min, the speed was increased to 18 m/min for another 2.5 min. After this warm-up period, the treadmill speed was further increased to 21 m/min. Many rats were not able to complete the entire treatment period (60 min) at this speed. When they became unable to keep pace with the treadmill, the treadmill speed was reduced to 18 m/min. If a rat was still unable to keep pace, the speed was further reduced to 13 m/min; however, the speed was returned to 21 m/min when possible. Sed-1 and Sed-2 rats sat on the treadmill for 60 min. Immediately on completion of the treatment, the rat was removed from the treadmill and replaced on the rubber cart top. In addition to the manual recording of the temperatures at 5-min intervals, the instrument displays and a profile view of each rat on the treadmill were recorded on videotape for data backup and more detailed analysis at a later time.

Environmental control. One lane of the treadmill was modified as an environmental chamber that was heated with a commercially available heating source. During experimentation, the chamber temperature was monitored continuously and was controlled manually to within ±1°C of the target environmental temperature by turning the heating source on or off while airflow was kept constant.

Microwave dosimetry. Incident power density was determined before and after the experiment by using a Loral-Narda Electromagnetic Radiation Monitor (model 8616) with an Isotropic Probe (model 8623D). Measurements were made in front of the restrainer's headgate (see Microwave exposure). To confirm that the probe was isotropic in this application, the average incident power density (6) was determined by making eight separate readings, rotating the probe 45° between each reading.

Microwave exposure. Rats were exposed for <30 s to 2.06-GHz radiation delivered in the far field at a power density of 1.7 W/cm2 in the k-polarization (i.e., face toward the transmitter) with a vertical E-field (6) by using an L-band Klyston source (model 2852, Colber Electronics, Stamford, CT) in an anechoic chamber (Emerson and Cuming, Canton, MA). By using the Tbr, the time-averaged specific absorption rate value was 983 mW/g (calculated by using Eq. 7.7 in Ref. 6). The temperature and humidity inside this chamber were 23-25°C and 60-80%, respectively. To maintain a consistent orientation with respect to the transmitter, rats were placed in a Styrofoam restrainer (9 cm internal width × 6 cm internal height × 27 cm length; each Styrofoam section was 4.3 cm thick) equipped with a polyvinyl chloride headgate and Plexiglas tailgate immediately before exposure. The headgate was positioned between the head and neck so that the head of the rat extended outside of the restrainer. Rats were removed from the restrainer immediately after the termination of exposure.

Immunohistochemistry. Rats were euthanized with pentobarbital sodium (80 mg/kg ip) 6 h after termination of exposure. The time of euthanasia was based on previous investigations that demonstrated intense HSP70 expression between 30 min and 24 h after hyperthermia produced by treadmill exercise, nonexertional heating, or injections of D-lysergic acid diethylamide (2, 4, 8, 13, 14, 20, 25, 32). Preliminary observations have demonstrated similar HSP70 expression at 6 and 24 h after HPM exposure (22). When rats were deeply anesthetized and unresponsive to tail pinch, they were perfused intracardially with heparinized saline, followed by a solution of 4% paraformaldehyde-0.1 M phosphate buffer-0.01% thimerosal. Brains were removed and stored in this solution for 12 h, followed by soaking them in 30% sucrose-0.1 M phosphate buffer until they sank. Brains were then frozen rapidly on dry ice and sliced into 30-µm sections on a refrigerated cryostat (Leica, Deerfield, IL). Coronal sections corresponding to pages in the rat brain atlas of Paxinos and Watson (26) were stored in 0.01 M PBS until the sections were immunocytochemically stained for HSP70 (72/73 kDa). The staining method used was compiled from that described by Dragunow and Robertson (5) and the guidelines produced by Oncogene Science (Manhassett, NY) and Vector Laboratories (Burlingame, CA). The latter was based on the immunoperoxidase procedure devised by Hsu et al. (12).

Staining consisted of incubating sections in a solution of 1.0% horse serum-0.01 M PBS-0.2% Triton X-100 for 20 min, followed by washing with 0.01 M PBS. Sections were then incubated in primary antibody [1:200 dilution of mouse monoclonal HSP72 (RPN 1197, Amersham, Arlington Heights, IL)] or 1.0 mg/ml mouse monoclonal HSP72/73 (Oncogene Science) for 24 h at 4°C. In negative control sections, incubation in the primary antibody was omitted. Sections were then rinsed in 0.01 M PBS and incubated in biotinylated secondary antibody (mouse IgG, PK-4002, Vector Laboratories) for 60 min at room temperature. After being rinsed with 0.01 M PBS, sections were incubated in the Vectastain avidin-biotin-peroxidase complex mixture from the above kit for 60 min at room temperature. After sections were washed with 0.01 M PBS and rinsed with a 0.5% Triton X-100-0.01 M PBS solution, a 0.03% 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO)-0.0015% hydrogen peroxide solution was placed on the sections for 1-3 min. To stop the reaction, sections were placed in distilled water and then mounted on gelatin-coated slides. After air drying, sections were dehydrated in 70 and 95% alcohol and 100% 2-propanol for 1 min each. Sections were soaked in AmeriClear (Baxter Scientific) and coverslipped by using Permount (Fisher Scientific). Sections were visually scanned and photographed by using a light microscope (Vanox, Olympus, Lake Success, NY). The density of HSP IN (no. of positively stained cells/10,000 µm2) in each selected brain region was determined in a blind manner, in that the microscopist did not know the experimental condition to which each rat had been assigned.

Brain region selection. Brain regions were selected on the basis of their location and purported roles in motor behavior and balance control (caudate putamen, medial habenular nucleus, cerebellum, dorsal paragigantocellular nucleus, and medial vestibular nucleus), somatosensory control (parietal cortex, area 1, cerebellum, raphe nuclei), and autonomic control (medial forebrain bundle, medial habenular nucleus) (see Fig. 1). The distances (in mm) of the 10 selected brain regions, posterior to bregma, were the following: caudate putamen (0.3); parietal cortex, area 1 (2.3); corpus callosum (2.3); medial forebrain bundle (2.3); medial habenular nucleus (2.3); dorsal raphe nucleus (7.3); median raphe nucleus (7.3); cerebellum (white matter; 10.8); dorsal paragigantocellular nucleus (10.8); and medial vestibular nucleus (10.8). Nomenclature and distances are from the work of Paxinos and Watson (26).


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  		Fig. 1.   Atlas pages from Paxinos and Watson (26) showing location of 10 brain regions in which 70-kDa heat shock protein (HSP70) densities were determined. Distances of these regions posterior to bregma were the following: 1) caudate putamen (0.3); 2) parietal cortex, area 1 (2.3); 3) corpus callosum (2.3); 4) medial habenula nucleus (2.3); 5) medial forebrain bundle (2.3); 6) dorsal raphe nucleus (7.3); 7) median raphe nucleus (7.3); 8) cerebellum (10.8); 9) medial vestibular nucleus (10.8); and 10) dorsal paragigantocellular nucleus (10.8). Scale bar above 1st brain section, 1 mm. Nomenclature and distances are from Paxinos and Watson (26).

Statistical analyses. With the use of Statistica (version 5.0, StatSoft, Tulsa, OK) software, one-way ANOVA and least-significant difference post hoc tests were conducted on the peak Tre for each experimental group. This was repeated for Tbr and Tty. For each 5-min period, the treadmill speeds of the two exercise groups were compared by using an independent t-test. One-way ANOVA and least-significant difference post hoc tests were also conducted on the density of HSP IN within a 100 × 100-µm area of each selected brain region as a function of experimental group. Pearson product-moment correlation analysis was conducted on density of HSP IN, as revealed by the Oncogene Science (HSP72/73) vs. Amersham (HSP72) antibodies. All values reported are means ± SE. Statistical significance was considered attained if P <=  0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The mean thermal profiles for the nonmicrowave treatments are shown in Fig. 2. All experimental conditions resulted in significant increases in peak temperatures (Figs. 2-4). Inspection of Fig. 2 shows that the thermal profiles displayed during exercise (Fig. 2, B and D) were mimicked in their respective sedentary groups (Fig,. 2, A and C) via manipulation of Tenv. The mean Tenv values were 42.8 ± 1.1 and 46.5 ± 0.7°C for Sed-1 and Sed-2 groups, respectively. Also shown in Fig. 2, B and D, are the mean treadmill speeds for Ex-1 and Ex-2 groups. Comparison of treadmill speeds between the Ex-1 and Ex-2 conditions revealed a significant difference only during the 50- to 55-min period. Neither group was able to maintain the target pace of 21 m/min for the entire 1-h period. There were no significant differences between the temperatures recorded for the Sed-1 and Ex-1 groups or Sed-2 and Ex-2 groups. However, the temperatures for the Sed-2 and Ex-2 groups were significantly higher than those for the Sed-1 and Ex-1 groups. The mean thermal profile for the HPM group is shown in Fig. 3. Both peak Tbr (Fig. 4) and Tty were significantly higher in the HPM group than in any other treatment group. In contrast, peak Tre was similar to those in the Sed-1 and Ex-1 groups and significantly less than those in the Sed-2 and Ex-2 groups. The peak increase in all measured temperatures for the Sham rats was <0.4°C.


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  		Fig. 2.   Thermal profiles for nonmicrowave treatments. A: passive heating (Sed-1) to brain temperatures (Tbr; black-square) that mimics those of exercise at room temperature [24°C; Ex-1 (B)]. C: passive heating (Sed-2) to Tbr that mimics those of exercise in a warm environment [34°C; Ex-2 (D)]. Gray areas, treatment period. Rectal temperature (Tre; bullet ) and Tbr were plotted at 5-min intervals. It was not technically possible to measure tympanic temperatures (Tty; black-triangle) during treatment period. Treadmill speeds (bars) are shown for exercise treatment conditions (B and D). Any adjustment to treadmill speed was made only at beginning of each 5-min period, in 3-m/min increments.


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  		Fig. 3.   Thermal profiles for rats exposed to high-powered microwaves (HPM). Symbols and grey area are defined as in Fig. 2. Tre (bullet ) and Tbr (black-square) temperatures were plotted at 5-min intervals. It was not technically possible to measure Tty (black-triangle) during treatment period.


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  		Fig. 4.   Mean peak Tbr values for each treatment group. Sham, sham exposed. 1Significantly >Sham; 2Significantly >Sed-1; 3Significantly >Ex-1; 4Significantly >Sed-2; 5Significantly >Ex-2. P <=  0.05.

With the use of the Oncogene Science antibody, the density of HSP IN within each of the 10 brain regions examined is shown in Fig. 5 as a function of experimental condition. There were significant increases in HSP70 expression in all brain regions examined, except the caudate putamen, after Ex-2 or Sed-2 conditions. It should be noted, however, that HSP70 expression in the majority of these regions (exceptions: dorsal raphe nucleus and cerebellum) did not differ between Ex-2 and Sed-2 groups. These increases were significantly greater than those observed after HPM exposure in some brain regions, usually those located in the anterior or dorsal regions. Significant increases in the density of HSP IN were observed in posterior brain regions (e.g., dorsal raphe nucleus, median raphe nucleus, cerebellum, dorsal paragigantocellular nucleus, and medial vestibular nucleus) after HPM exposure. There were no significant differences in the density of HSP IN between the Sed-1 and Ex-1 groups, and HSP70 expression in these groups was not significantly greater than those observed in the HC or Sham groups. Figure 6 shows photomicrographs of HSP70 expression as a function of brain region and experimental condition. Figure 7 shows photomicrographs of Purkinje cells in the cerebellum that were stained with the HSP72/73 antibody (Oncogene Science) after the Ex-2 or HC conditions.


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  		Fig. 5.   Number of positively stained grains/10,000 µm2 from 7 fore- and midbrain regions and 3 hindbrain regions as a function of experimental condition. HC, home cage. 1Significantly >HC; 2Significantly >Sham; 3Significantly >Sed-1; 4Significantly >Ex-1; 5Significantly >Sed-2; and 6Significantly >HPM. P <=  0.05.


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  		Fig. 6.   Photomicrographs of coronal brain sections of caudate putamen (CPu) and parietal cortex, area 1 (Par1; A), and dorsal raphe nucleus (DR) and dorsal paragigantocellular nucleus (DPGi; B) stained for Oncogene Science antibody (HSP72/73) after treadmill exercise by rats in Ex-2, HPM exposure, or HC conditions. Also shown are sections stained for HSP72 (Amersham antibody) from high-powered microwave (HPM-A) condition. Nomenclature was used according to Paxinos and Watson (26). External capsule (ec) and cortical layer 3 (3) are used as landmarks on CPu and Par1 sections, respectively. Magnification was ×105.6 at film plane, except for that of Par1, which was ×80.


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  		Fig. 7.   Photomicrographs of Purkinje cells (PN), located between molecular layer (ML) and granular layer (GL) of cerebellum just ventral to primary fissure, which were stained with HSP72/73 antibody (Oncogene Science) after Ex-2 or HC conditions. Magnification was ×128 at film plane.

To determine whether the HSP70 expression being observed with the Oncogene Science antibody was primarily HSP72 or HSP73, additional sections from each experimental condition were stained with the Amersham antibody (HSP72). The HSP densities observed with the Amersham antibody were then compared with those observed with the Oncogene Science antibody. In hyperthermic animals, there was a significant correlation (r = 0.77, P < 0.001) between the number of positively stained cells revealed by the Oncogene Science and Amersham antibodies, indicating that HSP72 was the primary protein being expressed.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present investigation is the first to compare the induction of HSP70 in the brain after microwave and nonmicrowave heating. It is also the first study to examine regional HSP70 expression in the brain in response to aerobic exercise.

HSP70 expression was not significantly elevated in response to exercise at room temperature (Fig. 5). In addition, passive and exertional heat stress resulted in similar increases in HSP70 expression (Fig. 5). The observation that exertional heat stress induces HSP70 expression is consistent with reports of HSP70 induction in spleen and lymphocytes when exercise was accompanied by hyperthermia (16). Our observation that HSP70 expression is not altered by exercise at room temperature contrasts, however, with previous reports that HSP70 expression in rat skeletal muscle, heart, and liver is induced by exercise alone (9, 16, 32). Possible explanations advanced for these presumably nonthermal, exercise-induced increases in HSP70 include increases in free radical production, glycogen depletion, calcium release, hypoxia, ATP depletion, and the production of metabolic inhibitors (17, 28, 32). Furthermore, a recent study of human skeletal muscle reports an increase in HSP70 mRNA after acute exercise without a concomitant increase in HSP70 protein (27).

Although it is possible that one or more of these factors may be responsible for stimulating HSP70 expression, the absence of an elevated Tre in these previous studies does not eliminate heat as a (co)factor because the temperature of the tissues under scrutiny was not actually measured (9, 32). This is particularly important in the case of skeletal muscle, in which the temperature of the active muscle is known to increase significantly above Tre during exercise (31). Although peripheral tissues were not studied herein, it is evident from this study that exercise alone does not produce significant HSP70 expression in the brain in the absence of pronounced hyperthermia.

An important feature of the present study is the measurement of Tbr and its subsequent correlation with HSP70 induction. As indicated above, examination of the question of whether exercise alone is sufficient to induce HSP70 expression is strengthened by measurement of the temperature of the tissue under study. Additionally, this assumes even greater importance when the effects of exercise on the brain are being dealt with, in that selective brain cooling has been demonstrated in rats during exercise (3). Therefore, the use of Tre as the only measurement of core temperature may result in an overestimation of Tbr and makes measurement of Tbr an essential element of a study such as this. Indeed, Fig. 2, B and D, show that selective brain cooling did occur in the present study during both Ex-1 and Ex-2 treatments.

The pattern of HSP70 expression was similar after Sed-2 and Ex-2 conditions, with dense concentrations of HSP70 throughout the brain. HSP70 expression in these rats was similar to that found by Blake et al. (1). They found that increased ambient temperatures induced HSP70 expression in brain regions coordinating neuroendocrine responses to stress. It is therefore evident that, under the conditions examined in this study, a certain degree of hyperthermia, whether produced passively by environmental heating alone or by the combination of environmental heating and exercise, is necessary to induce HSP70 expression in the brain. HSP70 expression after HPM exposure was similar to that in the Sed-2 and Ex-2 conditions only in the ventral mid- and hindbrain regions. This type of regional HSP70 expression aptly demonstrates the divergent patterns of brain heating resulting from HPM vs. nonmicrowave sources. It is important to note that if methods using whole brain homogenates had been used rather than immunohistochemical methods in brain sections, these interesting regional differences in HSP70 expression could not have been resolved.

Previously, we were not sure whether the pattern of HSP70 expression obtained in the brain after HPM exposure was due to factors unique to HPM (e.g., nonuniform brain heating) or to regional differences in the ability of the CNS to express HSP70. It is clear from the present study that the regions in question are capable of induced HSP70 expression and that heat appears to be the primary stimulus. This observation is important for two reasons: 1) it indirectly suggests that HPM exposure results in nonuniform heating in the brain; and 2) it indicates that a qualitative pattern of cellular stress caused by HPM exposure can be acquired by comparing, on a region-to-region basis, the pattern of HSP70 expression produced by nonmicrowave heating to that produced by HPM heating.

It is interesting to note that HPM resulted in similar or even smaller levels of HSP70 expression compared with those in the nonmicrowave hyperthermic groups (Fig. 5), despite significantly greater peak Tbr in the HPM group (Fig. 4). However, inspection of the timescale in Fig. 3 shows that, despite significantly higher Tbr resulting from HPM, the duration of the hyperthermic event was small compared with those in the Sed-2 and Ex-2 groups (Fig. 2, C and D). Using HSP70 density as a marker of cellular stress, the results of this investigation demonstrate that normally lethal Tbr levels can be tolerated with relatively little stress when the duration of severe hyperthermia is very brief.

Two types of HSP70 antibodies were used in the present experiment. The Amersham antibody recognizes only the inducible (HSP72) form. The Oncogene Science antibody (clone W27, E. Harlow, Cold Spring Harbor Laboratory, NY) should recognize both the inducible and constitutive (HSP73) forms. HSP73 might be involved in cellular repair and protection of stressed cells, and its function may be augmented by HSP72 (10), a protein not normally observed under control conditions and considered to be stress induced (23). The Oncogene Science antibody (HSP72/73) was used on all sections to keep the experimental design consistent with our previous and ongoing research. However, because HSP73 may be heat inducible (33, 36), the Amersham antibody (HSP72) was subsequently used on selected sections from each experimental condition to determine whether HSP72 or HSP73 was being observed primarily with the use of the Oncogene Science antibody. In hyperthermic animals, the Amersham and Oncogene Science antibodies revealed similar patterns of HSP IN expression, indicating that HSP72 was the primary protein being observed with the Oncogene Science antibody.

In a comparison of the treatment groups, light HSP70 expression was found with the Oncogene Science antibody in the brains of the HC and Sham rats. Staining was pronounced in the neurons (e.g., Purkinje cells) in the hindbrain. These results are consistent with those of Manzerra et al. (18-20) showing constitutive HSP70 expression in the cerebellum (Purkinje cells) of control animals.

It is important to note that exercise at room temperature increased Tre, Tbr, and Tty. The treadmill speed used in the present study was less than that used in many chronic exercise training studies, inasmuch as speeds of at least 28 m/min are typically reported in the literature (11). At these higher workloads, it is possible that many chronic exercise studies routinely, but unknowingly, induce hyperthermia during daily training. If this is the case, it would be expected that significant HSP70 expression may take place in the brain, as well as in other tissues, during chronic exercise training.

In summary, the results of this investigation demonstrate that HSP70 expression was not induced in the brain by exercise in the absence of hyperthermia. The general pattern of HSP70 expression was quantitatively and qualitatively similar after either passive or exertional heat stress, providing additional evidence that heat is the major factor responsible for HSP70 expression after exertional heat stress. Nonmicrowave-induced hyperthermia produced HSP70 expression throughout the brain; in contrast, HPM-induced HSP70 expression was confined primarily to the ventral regions of the mid- and hindbrain. Taken together, these results suggest that the nonmicrowave treatments and HPM treatment may induce different patterns of heating.

    ACKNOWLEDGEMENTS

Technical assistance from Joanne Doyle, Janet Roe, and Kevin Kosub and animal care by George Lantrip are greatly appreciated.

    FOOTNOTES

Address for reprint requests: T. J. Walters, USAF/AFRL/HEDR, Bldg. 1184, 8308 Hawks Rd., Brooks AFB, TX 78235-5324 (E-mail: thomas.walters{at}aloer.brooks.af.mil).

Received 12 September 1997; accepted in final form 10 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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