Luiz G. S. Branco

Neural Substrate of Cold-Seeking Behavior in Endotoxin Shock

Systemic inflammation is a leading cause of hospital death. Mild systemic inflammation is accompanied by warmth-seeking behavior (and fever), whereas severe inflammation is associated with cold-seeking behavior (and hypothermia). Both behaviors are adaptive. Which brain structures mediate which behavior is unknown. The involvement of hypothalamic structures, namely, the preoptic area (POA), paraventricular nucleus (PVH), or dorsomedial nucleus (DMH), in thermoregulatory behaviors associated with endotoxin (lipopolysaccharide [LPS])-induced systemic inflammation was studied in rats. The rats were allowed to select their thermal environment by freely moving in a thermogradient apparatus. A low intravenous dose of Escherichia coli LPS (10 µg/kg) caused warmth-seeking behavior, whereas a high, shock-inducing dose (5,000 µg/kg) caused cold-seeking behavior. Bilateral electrocoagulation of the PVH or DMH, but not of the POA, prevented this cold-seeking response. Lesioning the DMH with ibotenic acid, an excitotoxin that destroys neuronal bodies but spares fibers of passage, also prevented LPS-induced cold-seeking behavior; lesioning the PVH with ibotenate did not affect it. Lesion of no structure affected cold-seeking behavior induced by heat exposure or by pharmacological stimulation of the transient receptor potential (TRP) vanilloid-1 channel (“warmth receptor”). Nor did any lesion affect warmth-seeking behavior induced by a low dose of LPS, cold exposure, or pharmacological stimulation of the TRP melastatin-8 (“cold receptor”). We conclude that LPS-induced cold-seeking response is mediated by neuronal bodies located in the DMH and neural fibers passing through the PVH. These are the first two landmarks on the map of the circuitry of cold-seeking behavior associated with endotoxin shock.

Central chemoreceptor drive to breathing in unanesthetized toads, Bufo paracnemis

Central chemoreceptor drive to breathing was studied in unanesthetized toads, equipped with face masks to measure pulmonary ventilation and arterial catheters to analyze blood gases. Two series of experiments were performed. Expt. 1: The fourth cerebral ventricle was perfused with solutions of mock CSF, adjusted to stepwise decreasing pH values. Concomitant perfusion-induced increases of pulmonary ventilation, pHa and PaO2 were measured. Expt. 2: Inspiration of hypercapnic gas mixtures was applied to stimulate both central and peripheral chemoreceptors. Subsequently, only peripheral chemoreceptors were stimulated. This was accomplished by repeating the hypercapnic conditions while the fourth ventricle was perfused with mock CSF at pH 7.7. This procedure reduced the slope of the ventilatory response curve by about 80%. Taken together, the experiments suggest a highly dominant role of central chemoreceptors in the ventilatory acid-base regulation of the toad.

Thermoeffector neuronal pathways in fever: a study in rats showing a new role of the locus coeruleus

It is known that brain noradrenaline (norepinephrine) mediates fever, but the neuronal group involved is unknown. We studied the role of the major noradrenergic nucleus, the locus coeruleus (LC), in lipopolysaccharide (LPS)‐induced fever. Male Wistar rats had their LC completely ablated electrolytically or their catecholaminergic LC neurones selectively lesioned by microinjection of 6‐hydroxydopamine; the controls were sham‐operated. Both lesions resulted in a marked attenuation of LPS (1 or 10 μg kg−1, i.v.) fever at a subneutral (23°C) ambient temperature (Ta). Because electrolytic and chemical lesions produced similar effects, the role of the LC in fever was further investigated using electrolytic lesions only. The levels of prostaglandin (PG) E2, the terminal mediator of fever, were equally raised in the anteroventral third ventricular region of LC‐lesioned and sham‐operated rats during the course of LPS fever, indicating that LC neurones are not involved in febrigenic signalling to the brain. To investigate the potential involvement of the LC in an efferent thermoregulatory neuronal pathway, the thermoregulatory response to PGE2 (25 ng, i.c.v.) was studied at a subneutral (23°C, when fever is brought about by thermogenesis) or neutral (28°C, when fever is brought about by tail skin vasoconstriction) Ta. The PGE2‐induced increases in metabolic rate (an index of thermogenesis) and fever were attenuated in LC‐lesioned rats at 23°C, whereas PGE2‐induced skin vasoconstriction and fever normally developed in LC‐lesioned rats at 28°C. The LC‐lesioned rats had attenuated PGE2 thermogenesis despite the fact that they were fully capable of activating thermogenesis in response to noradrenaline and cold exposure. It is concluded that LC neurones are part of a neuronal network that is specifically activated by PGE2 to increase thermogenesis and produce fever.

Cardiovascular responses to chemoreflex activation with potassium cyanide or hypoxic hypoxia in awake rats.

Although intravenous (iv) injection of potassium cyanide (KCN) activates the arterial chemoreflex, it has been questioned whether cytotoxic hypoxia reproduces a physiological stimulus such as hypoxic hypoxia (low inspired O2 tension). Thus, the goal of the present study was to compare the cardiovascular responses elicited by intravenous injection of KCN to those caused by hypoxic hypoxia in awake rats before and after bilateral ligature of carotid body arteries. We tested the hypothesis that hypoxic hypoxia activates the cardiovascular chemoreflex just as KCN does, causing an increase in arterial pressure and bradycardia. Intact adult Wistar rats received an intravenous injection of KCN (160 microg/kg) and were exposed to hypoxic hypoxia (7-5% O2 breathing) for 10-15 s at random while mean arterial pressure (MAP) and heart rate (HR) were measured. After the experiments, the animals were submitted to bilateral ligature of carotid body arteries or sham operation and the protocol was repeated on the subsequent day. Before surgery, all rats showed an abrupt rise in arterial pressure accompanied by a marked bradycardia in response to KCN or hypoxic hypoxia, with a very similar pattern. After surgery, these responses persisted only in the sham-operated group and were totally abolished in the ligature group. In conclusion, our data show that KCN is an appropriate stimulus to activate arterial chemoreflex because its cardiovascular responses are comparable to those induced by hypoxic hypoxia. Thus, the use of KCN as a tool to evaluate different aspects of the complex pattern of cardiovascular, respiratory, and behavioural responses to chemoreflex activation seems to be physiologically acceptable.

Role of nitric oxide in systemic vasopressin-induced hypothermia

It has been reported that arginine vasopressin (AVP) plays a thermoregulatory action, but very little is known about the mechanisms involved. In the present study, we tested the hypothesis that nitric oxide (NO) plays a role in systemic AVP-induced hypothermia. Rectal temperature was measured before and after AVP, AVP blocker, or N G-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) injection. Control animals received saline injections of the same volume. The basal body temperature (Tb) measured in control animals was 36.53 ± 0.08°C. We observed a significant ( P < 0.05) reduction in Tb to 35.44 ± 0.19°C after intravenous injection of AVP (2 μg/kg) and to 35.74 ± 0.10°C after intravenous injection ofl-NAME (30 mg/kg). The systemic injection of the AVP blocker [β-mercapto-β,β-cyclopentamethylenepropionyl1, O-Et-Tyr2,Val4,Arg8]vasopressin (10 μg/kg) caused a significant increase in Tb to 37.33 ± 0.23°C, indicating that AVP plays a tonic role by reducing Tb. When the treatments with AVP and l-NAME were combined, systemically injected l-NAME blunted AVP-induced hypothermia. To assess the role of central thermoregulatory mechanisms, a smaller dose ofl-NAME (1 mg/kg) was injected into the third cerebral ventricle. Intracerebroventricular injection ofl-NAME caused an increase in Tb, but when intracerebroventricular l-NAME was combined with systemic AVP injection (2 μg/kg), no change in Tb was observed. The data indicate that central NO plays a major role mediating systemic AVP-induced hypothermia.It has been reported that arginine vasopressin (AVP) plays a thermoregulatory action, but very little is known about the mechanisms involved. In the present study, we tested the hypothesis that nitric oxide (NO) plays a role in systemic AVP-induced hypothermia. Rectal temperature was measured before and after AVP, AVP blocker, or NG-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor) injection. Control animals received saline injections of the same volume. The basal body temperature (Tb) measured in control animals was 36.53 +/- 0.08 degreesC. We observed a significant (P < 0.05) reduction in Tb to 35.44 +/- 0.19 degreesC after intravenous injection of AVP (2 micrograms/kg) and to 35.74 +/- 0. 10 degreesC after intravenous injection of L-NAME (30 mg/kg). The systemic injection of the AVP blocker [beta-mercapto-beta, beta-cyclopentamethylenepropionyl1,O-Et-Tyr2,Val4,Arg8]vasopressin (10 micrograms/kg) caused a significant increase in Tb to 37.33 +/- 0.23 degreesC, indicating that AVP plays a tonic role by reducing Tb. When the treatments with AVP and L-NAME were combined, systemically injected L-NAME blunted AVP-induced hypothermia. To assess the role of central thermoregulatory mechanisms, a smaller dose of L-NAME (1 mg/kg) was injected into the third cerebral ventricle. Intracerebroventricular injection of L-NAME caused an increase in Tb, but when intracerebroventricular L-NAME was combined with systemic AVP injection (2 micrograms/kg), no change in Tb was observed. The data indicate that central NO plays a major role mediating systemic AVP-induced hypothermia.

Temperature and central chemoreceptor drive to ventilation in toad (Bufo paracnemis)

The central chemoreceptor drive to ventilation was assessed in unanesthetized toads, Bufo paracnemis, exposed to three different temperatures: 15, 25 and 35 degrees C. The acid-base status of the fourth ventricle was manipulated by mock CSF perfusion. In additional experiments, arterial pH was varied by inspiration of hypercapnic gas mixtures. Ventilation was measured directly by pneumotachography and arterial blood samples were analyzed using electrodes for pH and PO2. Regardless of temperature, the ventilatory control of acid-base status was predominantly central. Moreover, an increase in temperature was accompanied by a proportional increase in the ventilatory response to chemoreceptor stimulation by either lowered mock CSF pH or hypercapnia. The alphastat hypothesis could not adequately account for the temperature effects on the ventilatory responses to hypercapnia or on air convection requirements in the toad.

Carbon monoxide as a novel mediator of the febrile response in the central nervous system

Heme oxygenase catalyzes the metabolism of heme to biliverdin, free iron, and carbon monoxide (CO), which has been shown to be an important neuromodulatory agent. Recently, it has been demonstrated that lipopolysaccharide (LPS) can induce the enzyme heme oxygenase in glial cells. Therefore, the present study was designed to test the hypothesis that central CO plays a role in LPS-induced fever. Colonic body temperature (Tb) was measured in awake, unrestrained rats (basal Tb= 36.8 ± 0.2°C). Intracerebroventricular injection of zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG; 75 nmol), a heme oxygenase inhibitor, caused no significant change in Tb, indicating that the central heme oxygenase pathway plays no tonic role in Tb under the experimental conditions used. Intraperitoneal injections of LPS (50-100 μg/kg) evoked dose-dependent increases in Tb. Intracerebroventricular injection of ZnDPBG in febrile rats attenuated LPS-induced fever (thermal index with ZnDPBG = 1.1 ± 0.2°C, thermal index with vehicle = 2.3 ± 0.4°C), suggesting that the central heme oxygenase pathway plays a role in fever generation. The antipyretic effect of ZnDPBG could be reversed by intracerebroventricular administration of heme-lysinate or CO-saturated saline. Collectively, our data indicate that CO arising from heme oxygenase may play an important role in fever generation by acting on the central nervous system.

Nitric oxide in the regulation of body temperature and fever

(1) In this article, the aspects regarding the role of nitric oxide (NO) in thermoregulation and fever is reviewed. (2) It is currently believed that fever results from de novo synthesis of cytokines and subsequent stimulation of the generation of prostaglandins in the central nervous system. However, the mechanisms underlying fever still remain only partly understood. (3) Recently, a new biologically active molecule has been described, i.e., the gaseous compound NO. This molecule started a revolution in the understanding of several physiological and pathophysiological processes, including thermoregulation and fever.

Role of central adenosine in the respiratory and thermoregulatory responses to hypoxia

No reports are available about the role of central adenosine in the respiratory and thermoregulatory responses to hypoxia in conscious rats. We therefore measured ventilation (VE) and body temperature (Tb) before and after intracerebroventricular injection of saline or aminophylline (adenosine antagonist), followed by a 30-min period of hypoxia exposure. Aminophylline did not change VE or Tb during normoxia; however, during hypoxia, it caused a significant increase in VE, and significantly attenuated hypoxic hypothermia. The present data indicate that central adenosine has an inhibitory effect on hypoxic hyperventilation and partially causes hypoxic hypothermia, suggesting that the ventilatory and metabolic interaction during hypoxia does not involve opposing mechanisms.

Evidence for thermoregulation by dopamine D1 and D2 receptors in the anteroventral preoptic region during normoxia and hypoxia

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been called anapyrexia. Stimulation of dopamine receptors in the central nervous system (CNS) reduces Tb in rats, and dopamine D1 and D2 receptors seem to be involved in this response. Thus, we predicted that injection of SCH 23390 and haloperidol, D1 and partly D2 receptor antagonists, respectively, into the anteroventral preoptic region (AVPO, the thermointegrative region of the CNS) would lessen the hypoxia-induced anapyrexia. We measured Tb of conscious Wistar rats before and after injection of SCH 23390 (50 and 100 ng/100 nl) or haloperidol (50 e 500 ng/100 nl) or their respective vehicles (saline and DMSO 5%) into the AVPO followed by 30 min of hypoxia (7% O2). Vehicles and the lower doses of SCH 23390 and haloperidol had no effect on Tb during normoxia or hypoxia. The higher doses of SCH 23390 and haloperidol attenuated (P<0.05) the drop in Tb elicited by hypoxia. However, this higher haloperidol dose also increased Tb during normoxia. The present data is consistent with the notion that dopamine is an important thermoregulatory neurotransmitter in a way that D2 receptors are mainly involved with maintenance of Tb in euthermia, while D1 receptors are activated to induce hypoxic anapyrexia in the AVPO.