Heatstroke induces c-fos expression in the rat hypothalamus

Neuroscience Letters 262 (1999) 41–44

Heatstroke induces c-fos expression in the rat hypothalamus Huey-Jen Tsay a, Hui-Yun Li b, Chia-Hsuan Lin a, Yi-Ling Yang c, Jeng-Yi Yeh d, Mao-Tsun Lin c ,* a

Institute of Neuroscience, National Yang-Ming University, School of Life Sciences, Taipei, Taiwan 11221 b Department of Anatomy, Chang-Gung University, Tao-Yuan, Taiwan 333 c Institute of Physiology, National Yang-Ming University, School of Life Sciences, Taipei, Taiwan 11221 d Department of Physiology, National Cheng-Kung University, Tainan, Taiwan 70101 Received 30 November 1998; accepted 7 January 1999

Abstract We induced heat stress in urethane-anesthetized rats (the animals were exposed to an ambient temperature at 42°C), and monitored their colon temperature, mean arterial pressure and local cerebral blood flow. Rats 0, 20, 40 or 80 min after heat stress were sacrificed for determination of c-fos mRNA and protein expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and preoptic nucleus (PON). The heatstroke, which appears as profound decreases in both mean arterial pressure and local cerebral blood flow and increases in colon temperature, is produced 80 min after heat stress. We show the c-fos mRNA and protein is strongly induced in all these nuclei of rat hypothalamus after the onset of heatstroke. We conclude that c-fos expression in the hypothalamus during rat heatstroke is associated with hyperthermia, arterial hypotension and cerebral ischemia.  1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Heatstroke; c-Fos; Hyperthermia; Hypothalamus; Cerebral ischemia

The expression of proto-oncogene c-fos is rapidly induced in the hypothalamus or other brain regions by a variety of physiologic processes and experimental manipulations such as hyperthermia (due to heat stress or pyrogen administration) [4,5,14,16,17], cerebral ischemia [1] and baroreceptor activation [6]. Our previous results have shown that, after the onset of heatstroke, animals exhibit hyperthermia, cerebral ischemia, arterial hypotension, intracranial hypertension, neural damage, and augmented interleukin-1 level in plasma [2,3,7]. These observations prompted us to hypothesize that c-fos expression may be induced in the regions of hypothalamus during heatstroke. In the present study, we therefore conducted an in situ hybridization and immunocytochemical study to ascertain the timing and site of c-fos expression in the hypothalamus during rat heatstroke. Adult male Sprague–Dawley rats weighing 250–300 g were purchased from National Yang-Ming University Animal Center (Taipei, Taiwan), and housed in a temperatureand light-controlled animal room (23 ± 1.0°C; lights on * Corresponding author. Fax: +886-2-28264049.

0304-3940/99/$ - see front matter PII: S03 04-3940(99)000 30-0

from 06:00 to 20:00 h) with free access to rat chow and water. The right femoral artery and vein of these rats, under urethane (1400 mg/kg i.p.) anesthesia, were cannulated with polyethylene tubing (PE 50). Systemic arterial blood pressure was monitored continually using a pressure transducer and chart recorder (Gould model 481). The animals were then fixed to a stereotaxic frame. Local cerebral blood flow was monitored with a Laserflo BPM2 laser Doppler flowmeter (Vasamedics, St. Paul MN, USA). A 24 gauge stainless steel needle probe (diameter, 0.58 mm; length, 40 mm) was inserted into right paraventricular nuclei (PVN) of the hypothalamus using the coordinates of A, −1.8 mm; L, 0.6 mm; and H, 8.2 mm [12]. The Laserflo BPM2 records at a bandwidth of 30 Hz, and a low bandpass of 20 kHz. The display is digital only, collects eight data points per second, and was set to give a moving average of data every 0.3 s. Blood perfusion was recorded until the flux value leveled off at a stable reading, which usually occurred after about 1 min. That value was then recorded and used as the flux measurement for the period. The colon temperature was continuously monitored by thermocouples.

 1999 Elsevier Science Ireland Ltd. All rights reserved.

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H.-J. Tsay et al. / Neuroscience Letters 262 (1999) 41–44

Two groups of urethane-anesthetized rats were used. (A) Normothermic rats were exposed to an ambient temperature of 24°C for 40 and 80 min to serve as control animals for immunocytochemistry and in situ hybridization assay, respectively. Their colon temperatures were maintained at about 36°C using an electric thermal mat. (B) The experimental group of rats were exposed to an ambient temperature of 42°C to induce heatstroke. The moment at which the mean arterial blood pressure or local cerebral blood flow began to decrease from its peak level was taken as the onset of heatstroke [19]. We sacrificed the animals at 0, 20, 40 and 80 min after the onset of heat stress and studied the c-fos expression using standard in situ hybridization and immunohistochemical methods. At the end of experiments, rats were perfused transcardically with 0.9% NaCl via the left ventrical to wash out the blood vessels, and followed by ice-cold 4°C (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brain were removed, postfixed for 4 h, and cryoprotected with 20% glycerol in PBS (phosphate buffered saline) at 4°C for 24–48 h. Immunocytochemistry and in situ hybridization was performed on 30 mm sections cut by a dry-ice sliding micotome (HM400R, Microm, Germany). Sections were stored in antifreeze buffer containing 5.7 mM NaH2PO4, 19 mM Na2HPO4, 20% glycerol, 30% ethylene glycerol at −20°C. Immunocytochemistry was performed as described by Liu et al. [8]. Briefly, sections were rinsed with 0.2% Triton X-100 in PBS (PBS-TX) for 5 min. Endogenous peroxidase activity was quenched by pretreating sections with 10% methanol/3% H2O2 in PBS, three washes in PBS, followed by 2% normal goat serum in PBS-TX for 1 h. Sections were incubated in 1:5000 rabbit polyclonal anti-Fos antiserum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 1:1600 rabbit polyclonal anti-vasopressin antiserum (Chemicon International Inc.) at 4°C for 24–28 h. Sections were rinsed three times by PBS, then incubated in 1:500 biotinylated goat anti-rabbit IgG in PBS for 1 h, rinsed three times with PB. Immunoreactivity was visualized by the avidinbiotin complex method (Vectastain, Vector, Burlingame, CA, USA). After incubated avidin-biotin complex for 1 h, sections were developed in the mixture of 0.02% 3.3′ diaminobenzidine (DAB; Sigma, Saint Louis, MO, USA) and

0.08% nickel ammonium sulfate in PBS for 5 min. Hydrogen peroxide was added to a final concentration of 0.00018%. The color of precipitate formed by DAB is monitored microscopically. Sections were mounted onto gelatincoated slides, dehydrated a serial alcohol gradient and xylene, then coverslipped with Entellan (Merck, Germany). The preparation of 35S labeled c-fos antisense riboprobe and in situ hybridization were preceeded as described by Simmons et al. [15]. The full-length rat c-fos coding region was released by EcoRI and subcloned into PBS SK + plasmid (Stratagene, La Jolla, CA, USA). The plasmid was linearized by BglII and transcribed by T3 RNA polymerase to synthesize antisense riboprobe. 35S labeled antisense riboprobe was diluted to 107 pm/ml in hybridization buffer containing 50% formamide, 10% dextran sulfate, 1 × denhardt’solution, 0.3 M NaCl, 1 M EDTA, 10 M Tris–HCl (pH 7.5), 10 mM DTT, 10 mg/ml yeast tRNA. The sections were mounted onto gelatin-coated slides and air-dried. Slides were incubated in 4% paraformaldehyde for 2 h, then rinsed four times by PBS. Sections were digested by 10 mg/ml proteinase K for 30 min at 37°C, then acetylated with 0.1 M triethanolamine and 0.25% acetic anhydride for 10 min. Sections were dehydrated, air dried, then incubated with 100 ml hybridization mixture at 57°C for 12–16 h. After washed with 4 × SSC, sections were treated with 20 mg/ml RNase A in 2 × SSC containing 1 mM DTT. Sections were washed in solutions with increasing stringency (2 × SSC, 1 × SSC, 0.5 × SSC, and 0.1 × SSC) at room temperature for 5 min, followed by 0.1 × SSC at 57°C for 30 min. After dehydration through a graded alcohol (50, 70, 90, 100, 100%), slides were exposed to Kodax BioMax film for 24 h. After defat in xylene, slides were dipped in NTB2 emulsion (Kodax diluted 1:1 with distilled water), exposed for 3 weeks, then developed in Kodas D19 and fixed. Sections were counterstained by thionin, dehydrated, then coverslipped with Entellan. Cells contain c-fos immunoreactivity in the PVN area was counted through a microscopy. The relative levels of c-fos mRNA for each group was quantified by the microcomputer imaging device (Imaging Research, Canada). The difference of the c-fos mRNA levels and c-fos positive cells among control and heated animals were determined by oneway ANOVA followed by Student–Newman-Keul’s multi-

Table 1 Effects of heat stress (42°C) on colon temperature (TCO), mean arterial pressure (MAP), local cerebral blood flow (CBF), and c-fos expression in the paraventricular nucleus (PVN) of the hypothalamus in rats. Data are the mean ± SEM with the number of animals used in parentheses. Physiological parameters

TCO (°C) MAP (mmHg) CBF (% baseline) Fos mRNA (arbitrary unit) Fos protein (no. cells/section)

Time after heat stress (42°C) 0 min

20 min

40 min

80 min

36.5 ± 0.4 (6) 85 ± 5 (6) 100 ± 4 (6) 1 (3) 44.3 ± 1.9 (6)

37.6 ± 0.3 (6)* 90 ± 6 (6) 118 ± 6 (6) 27 ± 2 (3)* 127.8 ± 8.4 (6)*

39.5 ± 0.2 (6)* 105 ± 5 (6)* 151 ± 9 (6)* 57 ± 3 (3)* 184.5 ± 25.6 (6)*

41.8 ± 0.4 (8)* 35 ± 4 (8)* 25 ± 3 (8)* 42 ± 2(3)* 478.9 ± 48.6 (8)*

*P , 0.05, significantly different from the corresponding control values (0 min group), ANOVA.

H.-J. Tsay et al. / Neuroscience Letters 262 (1999) 41–44

Fig. 1. The expression of c-fos mRNA in the region of the paraventricular nucleus in a normothermic control rat (A), a rat 20 min after the start of heat exposure (B), a rat 40 min after the start of heat exposure (C), and a rat after the onset of heatstroke (or 80 min after the start of heat exposure) (D). Scale bar, 200 mm.

ple range test. A value of P , 0.05 was considered significant. Table 1 summaries the time course changes of various parameters induced by an acute heat stress. The latency for the onset of heatstroke was found to be about 80 min after

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heat stress (42°C). The parameters including TCO, MAP, CBF, c-fos mRNA and protein in the PVN increased at 20–40 min after heat stress. At the onset of heatstroke (or 80 min after heat stress), the animals displayed hyperthermia (41.8°C), arterial hypotension (35 mmHg), cerebral ischemia (25% of the baseline control) and strong c-fos expression (Figs. 1 and 2) in the PVN. The mRNA level of c-fos peaks at 40 min after heat stress, and its immunoreactivity reaches the maximum level at the onset of heatstroke. The similar regulation pattern of c-fos mRNA and protein after heat stress appeared in SON and PON (data not shown). In order to identify the cell type of c-fos-positive neurons in the PVN after heatstroke, we stained the adjacent sections with c-fos and vasopressin polyclonal antibodies. As shown in Fig. 3, c-fos-positive cells included a major population of magnocellular neurons (vasopressin-positive) and a small population of parvocellular neurons. Although the vasopressin immunoreactivity was observed in normothermia control rats, they are not altered by heatstroke. In fact, hyperthermia induced by exposing the animals to a high ambient temperature [4,13,14] or administration of lipopolysaccharide, prostaglandins or interleukin-1 [16–18] produced c-fos expression in the regions of PON, PVN and SON of the hypothalamus. In addition, it is well known that c-fos is induced rapidly and transient in brain cells after cerebral ischemia [1]. Twenty-four hours after dehydration,

Fig. 2. The immunoreactivity of c-fos protein in the paraventricular nucleus in a normothermic control rat (A), a rat 20 min after the start of heat exposure (B), a rat 40 min after the start of heat exposure (C), and a rat after the onset of heatstroke (or 80 min after the start of heat exposure) (D). Scale bar, 50 mm.

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[9] Fig. 3. The c-fos and vasopressin immunoreactivities and the cell morphology (by hematoxylin staining) in the PVN of the hypothalamus in normothermic control rats (A–C) and rats with heatstroke (D–F). Note that there was no difference in vasopressin immunoreactivity of the PVN between normothermic, control rats and rats with heatstroke. Scale bar, 300 mm.

c-fos protein was also expressed in the regions of PVN, SON, and PON of the hypothalamus [9–11]. Furthermore, baroreceptor activation induced by intravenous administration of phenylephrine was able to induce s-fos expression in either the PVN, SON or PON of the rat hypothalamus [6]. In the present results, animals with heatstroke exhibited hyperthermia, arterial hypotension, cerebral ischemia, and c-fos expression in the PVN, SON, and PON of rat hypothalamus. However, heatstroke did not enhance the vasopressin immunoreactivity in the PVN of the rat hypothalamus. Putting these observations together, it suggests that the c-fos expression in the hypothalamus is associated with hyperthermia, arterial hypotension, and/or cerebral ischemia during rat heatstroke. However, the role of c-fos protein plays either as protective of or detrimental to brain cells after its induction is not clear at present [1]. This work was supported by the National Science Council of the Republic of China (NSC 88-2314-B-010083). We thank Dr. F.C. Liu and Dr. F.D. Wu for help with immunocytochemistry. [1] Akins, P.T., Liu, P.K. and Hsu, Y.H., Immediate early gene expression in response to cerebral ischemia, Stroke, 27 (1996) 1682–1687. [2] Chiu, W.T., Kao, T.Y. and Lin, M.T., Interleukin-1b receptor antagonist attenuates the heatstroke-induced neuronal damage

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