Brain Research Bulletin 108 (2014) 94–99
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Involvement of endogenous hydrogen sulﬁde (H2 S) in the rostral ventrolateral medulla (RVLM) in hypoxia-induced hypothermia Alberto F. Donatti, Renato N. Soriano 1 , João P. Sabino, Luiz G.S. Branco ∗ Department of Morphology, Physiology and Basic Pathology, Dental School of Ribeirão Preto, University of São Paulo, 14040-904 Ribeirão Preto, SP, Brazil
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Article history: Received 29 April 2014 Received in revised form 1 August 2014 Accepted 28 August 2014 Available online 26 September 2014 Keywords: Aminooxyacetate Cystathionine beta-synthase Central nervous system Body temperature
a b s t r a c t Hypoxia evokes a regulated decrease in deep body temperature (Tb). Hydrogen sulﬁde (H2 S), a signaling molecule that belongs to the gasotransmitter family, has been demonstrated to participate in several brain-mediated responses. Rostral ventrolateral medulla (RVLM) is a brainstem region involved in thermoregulation. Recently, it has been shown that exogenous H2 S modulates RVLM activity. In the present study, we investigated whether endogenously produced H2 S in the RVLM plays a role in the control of hypoxia-induced hypothermia. Tb was measured before and after bilateral microinjection of aminooxyacetate (AOA, 0.2, 1 and 2 pmol/100 nl, a cystathionine ␤-synthase, CBS, inhibitor) or vehicle into the RVLM followed by a 60-min normoxia (21% inspired O2 ) or hypoxia (7% inspired O2 ) exposure. Microinjection of AOA or vehicle did not change Tb during normoxia. Exposure to hypoxia evoked a typical decrease in Tb. Microinjection of AOA (2 pmol) into the RVLM followed by hypoxia signiﬁcantly attenuated the decrease in Tb. Thus, endogenous H2 S in the RVLM seems to play no role in the maintenance of basal Tb, whereas during hypoxia this gas plays a cryogenic role. Moreover, RVLM homogenates of rats exposed to hypoxia exhibited a decreased rate of H2 S production. Our data are consistent with the notion that during hypoxia H2 S synthesis is diminished in the RVLM facilitating hypothermia. © 2014 Elsevier Inc. All rights reserved.
1. Introduction It is extremely spread among taxa the ﬁndings that organisms respond to hypoxia by reducing their temperature (Malvin and Wood, 1992; Wood, 1991). This regulated reduction in body temperature (Tb) has been reported to be beneﬁcial since it increases the afﬁnity of hemoglobin for oxygen and reduces the oxygen demand of tissues (Wood, 1991). Hypothermia induced by hypoxia is a brain-mediated response (Steiner and Branco, 2002), and involves a number of mediators, such as adenosine (Barros and Branco, 2000, 2002; Barros et al., 2006), dopamine (Barros et al., 2004), opioids (Scarpellini Cda et al., 2009), serotonin (Gargaglioni et al., 2005), and the gaseous neuromodulators nitric oxide (NO) (Steiner et al., 2000), carbon monoxide (CO) (Paro et al., 2001, 2002), and hydrogen sulﬁde (H2 S) (Kwiatkoski et al., 2012). The latter has been shown to be synthesized mainly by two pyridoxal-5 -phosphate-dependent enzymes, cystathionine ␤-synthase (CBS) or cystathionine ␥-lyase (CSE)
(Abe and Kimura, 1996), and capable of altering neuronal activity (Kimura et al., 2005). CBS predominates in the brain, whereas CSE is mainly expressed in the periphery (Yang et al., 2008). Rostral ventrolateral medulla (RVLM) is a brainstem region that has been reported to play an inhibitory role in the sympathetic outﬂow to brown adipose tissue (Cao et al., 2010), a thermogenic tissue whose activity is reduced during exposure to hypoxia (Cannon and Nedergaard, 2004; Gautier, 1996; Hinrichsen et al., 1998; Mortola and Naso, 1997; Mortola et al., 1999). Therefore, taking into consideration that the RVLM may participate in hypoxia-induced hypothermia and that H2 S modulates RVLM neurons activity (Guo et al., 2011), the present study was undertaken to test the hypothesis that H2 S endogenously produced in the RVLM plays a modulatory role in hypoxic hypothermia. Our results indicate that RVLM H2 S has a cryogenic effect during the hypothermic response to hypoxia and its synthesis is reduced in this medullary region. 2. Materials and methods
∗ Corresponding author. Tel.: +55 16 3602 4051; fax: +55 16 3633 0999. E-mail address: [email protected]
(L.G.S. Branco). 1 Present address: Federal University of Juiz de Fora, 35020-220 Governador Valadares, MG, Brazil. http://dx.doi.org/10.1016/j.brainresbull.2014.08.010 0361-9230/© 2014 Elsevier Inc. All rights reserved.
2.1. Animals Adult male Wistar rats were group-housed (four to ﬁve animals per cage) and acclimated (25 ◦ C; 12:12-h light–dark cycle) for 1
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Fig. 1. Representative photomicrography (inset) of a microinjection into the RVLM (intra-RVLM), and schematic drawings of medullary slices (adapted from Paxinos and Watson, 2005) showing the sites of microinjection intra-RVLM (solid circles) and peri-RVLM (gray circles). Landmarks: AmbC, ambiguous nucleus; 4 V, forth ventricle; py, pyramidal tract.
week before experimental use. The rats had free access to water and food, and were housed in a temperature-controlled chamber at 25 ◦ C (model: ALE 9902001; Alesco Ltda, Monte Mor, SP, Brazil). Experiments were performed on fully conscious, freely moving animals weighing 270–300 g. Animal care was carried out in compliance with the guidelines set by the Brazilian College of Animal Experimentation (COBEA), an afﬁliate of the international Council for Laboratory Animal Science (ICLAS), which included minimizing the number of animals used and their suffering, and had the approval of the Animal Care and use Committee of the University of São Paulo (n◦ 064/2012).
2.2. Drug Aminooxyacetate (AOA; 0.2, 1 and 2 pmol/100 nl, commonly used as a CBS inhibitor) (Kimura, 2010; Kwiatkoski et al., 2012, 2014) was purchased from Sigma (St. Louis, MO, USA), and dissolved in phosphate-buffered saline (PBS).
2.3. Surgical procedures Surgical procedures were performed under ketamine–xylazine anesthesia (100 and 10 mg/kg; respectively; 1 ml/kg, i.p.). Antibiotics (160,000 U/kg benzylpenicilin, 33.3 mg/kg streptomycin, and 33.3 mg/kg dihydrostreptomycin, i.m.; prophylactically) and analgesic medication (Flunexine; 2.5 mg/kg, s.c.) were provided immediately after the end of surgeries. The animals were ﬁxed (prostrate) on a stereotaxic frame to be implanted with stainless steel guide cannulas (15 mm long, 22 gauge outer diameter) toward the RVLM (for intra-RVLM microinjection), according to the following stereotaxic coordinates (Paxinos and Watson, 2005): 3.2 mm caudal to the lambda; 1.8 mm lateral to the midline; 6.5 mm ventral to the skull surface). The guide cannulas were attached to the bone with stainless steel screws and acrylic cement. Tightﬁtting stylets were kept inside the cannulas to prevent occlusion. Afterwards, a median laparotomy was performed so as to insert a temperature datalogger capsule (SubCue, Calgary, AB, Canada) into the peritoneal cavity. All animals were kept under deep anesthesia throughout the surgical procedures, receiving a supplementary dose of anesthetic whenever necessary. Before the experimental procedure, the animals were allowed to recover from surgical interventions for at least 5 days.
2.4. Tb recordings The animals had their Tb recorded at 5-min intervals with the temperature datalogger capsule (SubCue, Calgary, AB, Canada) inserted into the peritoneal cavity. 2.5. Microinjection To perform microinjection within the RVLM we used a microinjection device (model 310, Stoelting, Wood Dale, IL, USA) and a 10-l syringe (Hamilton, Reno, NV, USA) connected to a microinjection needle (30-gauge outer diameter) with a polyethylene tube (PE-10). Microinjection was performed at a ﬂow rate of 50 nl min−1 . The microinjection needle, 3.5 mm longer than the guide cannula, was inserted into the cannula solely at the moment of the microinjection. The animals in which the microinjection of AOA did not reach the RVLM were grouped during the data analysis process, and then were used to compose the group peri-RVLM to demonstrate that the signiﬁcant effect of the drug is statistically signiﬁcant if, and only if, the drug reaches RVLM cells. 2.6. Histology At the end of the experiments, the animals were deeply anesthetized with ketamine–xylazine (100 and 10 mg/kg; respectively; 1 ml/kg, i.p.), and then microinjected with Evan’s blue (0.1 l) to ease the visualization of microinjection sites (see Fig. 1). Afterwards, the rats were transcardially perfused with phosphatate-buffered saline followed by 10% buffered formalin solution. The brains were removed and stored in 10% formalin for at least 2 days. After ﬁxation, the brains were sectioned in a microtome (30 m), and stained using the Cresyl–Nissl method for light microscopy. The rats in which the microinjection reached regions close to, but not within, the RVLM were included in the group ‘periRVLM’. Data obtained from the group peri-RVLM indicate that the effects of the drugs are restricted to the RVLM. The stereotaxic atlas by Paxinos and Watson (2005) was used to determine whether the microinjection sites were or not within the RVLM. 2.7. Experimental protocols In all experimental protocols, the rats were habituated to experimental conditions for 40 min in plethysmographic chambers
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continuously ventilated with humidiﬁed room air. After the habituation, the animals were microinjected into the RVLM and exposed to normoxia (21% O2 , N2 balance) or hypoxia (7% O2 , N2 balance) for 60 min. Room temperature was maintained at 22 ◦ C throughout the experiment. Doses of AOA tested in pilot experiments were based on the literature (Kwiatkoski et al., 2012, 2013, 2014). All gas conditions were ﬂushed by a ﬂow meter gas-mixing pump (Cameron Instruments GF-3/MP; Guelph, ON, Canada). O2 gas analyzer (Raytech quadralyser 224A; Middletown, CT, USA) was used to monitor gas composition inside the chamber in all experimental protocols.
2.7.1. Effect of AOA within the RVLM on Tb during normoxia or hypoxia To investigate whether endogenous H2 S in the RVLM plays a role in hypoxia-induced hypothermia, unanesthetized, freely moving rats were microinjected into the RVLM with AOA at three different doses: 0.2, 1 and 2 pmol 100 nl−1 or vehicle (PBS, 100 nl) and kept under normoxia or exposed to hypoxia. Baseline of Tb was measured for 30 min, after the acclimatization (40 min), and subsequently the rats received intra-RVLM microinjection of AOA or PBS. Tb was measured at 5-min intervals for 60 min after the microinjection and exposure to normoxia or hypoxia.
2.7.2. H2 S levels in the RVLM of rats exposed to normoxia or hypoxia After performing the in vivo approaches, we measured H2 S production in the RVLM of animals exposed to normoxia or hypoxia. We assessed the levels of H2 S in the RVLM of rats that were acclimatized (40 min) and then exposed to normoxia or hypoxia for 60 min. Immediately after the exposure, the animals were decapitated and the brains processed. RVLM samples were excised in a cryostat by a punch needle (0.9 mm inner diameter) from a 500-m slice of the brainstem at the level of the RVLM. Bilateral punches of the RVLM were taken above the ventral boundary of the medullary surface. RVLM samples were homogenized in potassium phosphate buffer (100 mM; pH 7.4) as previously described (Francescato et al., 2011; Kwiatkoski et al., 2012, 2013, 2014; Singh et al., 2009).
2.8. Statistical analysis The results are expressed as mean ± S.E.M. Tb values (◦ C) plotted at 5-min intervals, are reported as changes () from basal (initial) values (Tbi). Tbi is Tb measured at 5-min intervals averaged over the last 30 min of the 40-min acclimatization period. Thermal indexes (◦ C min), calculated from area under curve of the 60-min experimental period, clarify the differences among the groups. Statistical differences in thermal indexes were analyzed by one-way ANOVA followed by Tukey’s post hoc test; differences in Tb, by oneway ANOVA for repeated measures followed by Tukey’s post hoc test; and RVLM H2 S levels, by Student’s t-test. Differences were considered statistically signiﬁcant when P value was