Glial vascular endothelial growth factor overexpression in rat brainstem under tolerable hypoxia: evidence for a central chemosensitivity

The ventilatory response to hypoxia is mediated by peripheral inputs arising from the arterial chemoreceptors. In their absence, hypoxic adaptation can be achieved, possibly as a result of central cellular reorganization. To study this reorganization, we used chemodenervated rats to investigate the expression and localization of vascular endothelial growth factor (VEGF) in the brainstem. VEGF is a target gene of hypoxia-inducible factor (HIF) that is responsible for the morphofunctional remodeling induced by hypoxia. Intact and chemodenervated rats were subjected to normoxia or hypoxia for 6 hr (10% O(2) in N(2)). VEGF protein was quantified in micropunches of brainstem tissue. Only chemodenervated animals showed an increased VEGF expression in response to hypoxia, whereas, in normoxia, VEGF expression was not modified by chemodenervation. The same hypoxic condition was repeated for 8 days before immunocytochemical staining with anti-VEGF; antiglial fibrillary acidic protein (GFAP), a marker of astrocytes; and anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells. Confocal analysis showed a cellular colocalization of GFAP and VEGF, indicating that VEGF was overexpressed predominantly in astrocytes. Increased RECA-1 immunolabeling indicated an enhanced angiogenesis in chemodenervated rats subjected to hypoxia. These results indicate that glial cells and the vascular network contribute to the brainstem remodeling. The peripheral chemodenervation reveals a central O(2) chemosensitivity involving a cascade of gene expression triggered by hypoxia, which in intact animals may act synergically with peripheral chemosensory inputs.     

Regional specificity of the long-term regulation of tyrosine hydroxylase in some catecholaminergic rat brainstem areas. I. Influence of long-term hypoxia

The present study was carried out to investigate the influence of long-term hypoxia on tyrosine hydroxylase (TH) protein quantity in some catecholaminergic rat brainstem areas such as the dorsomedial medulla (DMM), the ventrolateral medulla (VLM) and the locus coeruleus (LC). TH protein quantity was also measured in a dopaminergic structure, the substantia nigra (SN). Male Sprague-Dawley rats were exposed to normobaric hypoxia (10% O₂/90% N₂) for 3, 7, 14 or 22 days. Controls were kept in normoxia for the same period. This study demonstrates that: (1) 3 days of hypoxia produced a 50% and a 26% increase in the quantity of TH protein in the rostral and caudal LC, respectively; (2) 14 days of hypoxia produced a 44% increase of TH protein content exclusively in the caudal part of DMM and a 31% increase in the VLM area; and (3) the stimulus failed to alter the TH protein quantity in the SN. After 14 and 22 days of hypoxia respectively, the TH protein content in the LC and DMM returned to the level of controls. To determine whether the increase in TH protein quantity could be related to a change in norepinephrine (NE) content, the rate constant of disappearance (k) of NE was measured in the catecholaminergic areas of intact or chemodenervated rats submitted to long-term hypoxia. Our results show that hypoxia causes an increase of TH protein quantity within the subpopulations of catecholaminergic areas additionally with an elevation in the NE content. These data suggest a selective response of the TH regulation to long-term hypoxia within the caudal DMM catecholaminergic area which receives chemosensory inputs.     

Functional recovery of the ventilatory chemoreflexes after partial chronic denervation of the nucleus tractus solitarius

In pentobarbitone-anesthetized cats breathing spontaneously, we studied whether excision of one petrosal ganglion would modify the reflex efficacy of the remaining carotid and aortic chemoafferences in ventilatory control. Resting ventilation was not affected shortly after the ganglionectomy, but decreased sensitivities and reactivities for changes in tidal volume and respiratory frequency were revealed by dose-response curves for ventilatory chemoreflexes evoked by NaCN i.v. After 2 weeks of ganglionectomy, basal tidal volume was increased, being slightly reduced by contralateral carotid neurotomy, but persisting above control after section of all buffer nerves. The ventilatory chemosensory drive--tested by breathing 100% O2--was unmodified with respect to the acute condition, but the tonic ventilatory influence exerted by the right carotid nerve was diminished. Dose-response curves for reflex changes in tidal volume exhibited increased sensitivity, while those for changes in respiratory frequency showed increased reactivity. Thus, partial chemosensory denervation of the nucleus tractus solitarius triggers a slowly developing increase in the reflex efficacy of the remaining chemosensory inputs. The recovery of sensitivity for reflex changes in tidal volume required the presence of contralateral carotid afferents, while the increased reactivity in respiratory frequency needed the integrity of aortic afferents. The results also suggest an enhanced contribution of central structures other than chemosensory inputs in respiratory control after partial deafferentation.     

颈动脉注射辣椒素激活脑干心血管相关核团的儿茶酚胺神经元

在外周压力感受器去神经支配的大鼠上，用Ｆos蛋白和酪氨酸羟化酶（ＴＨ）的双重免疫组化方法，研究辣椒素的效应是否通过激活脑干核团内儿茶酚胺能神经元而诱发。结果显示，颈动脉注射辣椒素诱发脑干中最后区（ＡＰ）、孤束核（ＮＴＳ）、巨细胞旁外侧核（ＰＧＬ）和蓝斑（ＬＣ）等多个部位出现大量ＦOS样免疫反应（ＦＬＩ）神经元和双标神经元，辣椒素受体阻断剂钌红（ＲＲ）或ＮＭＤＡ受体阻断剂ＭＫ－８０１可明显减弱此效应。以上结果表明，辣椒素的兴奋效应通过激活儿茶酚胺能神经元而诱发，辣椒素受体和／（或）谷氨酸介导这一效应。     

Hypoxia-induced cellular and vascular changes in the nucleus tractus solitarius and ventrolateral medulla

Major changes in arterial pressure, autonomic, and respiratory activity occur in response to hypoxia. We analyzed structural damage and increased vascular permeability in the ventrolateral medulla and nucleus tractus solitarius, which control autonomic, respiratory, and cardiovascular functions in adult Wistar rats subjected to 2 hours of hypoxia (7% oxygen + 93% nitrogen) for up to 14 days after hypoxicexposure. Brainstem tissue levels of vascular endothelial growth factor (VEGF), nitric oxide (NO), and glutamate were significantly increased over control levels after hypoxic injury. By electron microscopy, swollen neurons and dendrites, degenerating axons, disrupted myelin sheaths, and swollen astrocyte processes were observed in the nucleus tractus solitarius and ventrolateral medulla. Leakage of intravenously administered horseradish peroxidase was observed through vascular walls in hypoxic rats. These results suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, which, along with increased levels of glutamate, may have induced structural alterations of the neurons, dendrites, and axons. Administration of the antioxidant neurohormone melatonin (10mg/kg) before and after the hypoxia reduced VEGF, NO, and glutamate levels and improved ultrastructural abnormalities induced by hypoxia exposure, suggesting that it may have a therapeutic potential in reducing hypoxia-associated brainstem damage.     

Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus

We have recently used Fos expression in adult rats to map neuronal populations activated in the brainstem and hypothalamus during the acute ventilatory response to moderate hypoxia (O(2) 11%). Although present at birth, this response evolves postnatally. The present investigation aimed at a better understanding of these maturational processes by delineating structures that might functionally develop after birth. The developmental pattern Fos expression evoked by hypoxia was analysed in rats aged from 0 to 26 postnatal days. The numbers of Fos positive neurons markedly increased with the age in the medullary areas related to respiratory control during the 2 first postnatal weeks. Thereafter, the response plateaued in the nucleus tractus solitarius and attenuated in the ventral medulla. In the upper brainstem (parabrachial area, central grey) and the hypothalamus (posterior and dorsomedial nuclei, ventral zone), Fos response to hypoxia was absent or weak at birth and increased until late development. The significance of the development of evoked Fos expression in these rostral sites is discussed together with their possible contribution to the maturation of O(2)-sensitive chemoreflex pathways.     

Enhancement of respiratory response to carbon dioxide produced by lesioning caudal regions of the nucleus of the tractus solitarius

Bilateral lesions of the caudal region of the nucleus of the tractus solitarius produced a significant enhancement of the ventilatory response to carbon dioxide in awake rats. The result indicates release of an inhibitory influence normally operating at hindbrain level. Respiration in air or oxygen was not affected while hypoxic responses were depressed insignificantly.     

Nicotine activates NPY and catecholaminergic neurons in brainstem regions involved in ACTH secretion

Nicotine rapidly and potently stimulates ACTH secretion via a centrally mediated mechanism. The purpose of the current study was to identify the phenotype of nicotine-sensitive neurons in brainstem catecholaminergic regions previously shown to be responsive to nicotine. Immunocytochemical double-labeling was used to detect c-Fos expression in neurons positive for activin, galanin, or neuropeptide Y (NPY), in comparison to those containing tyrosine hydroxylase (TH, catecholaminergic biosynthetic enzyme). These neuropeptides were chosen because (1) each is located in nicotine-sensitive brainstem regions, (2) neurons containing each of these peptides project to the hypothalamic paraventricular nucleus, and (3) each has been shown to affect ACTH secretion. Freely moving, adult, male rats received an intravenous (i.v.) infusion of saline or nicotine (0.045 mg/kg over 30 s or 0.135 mg/kg over 90 s) and were cardiac perfused 60 min thereafter. Nicotine significantly increased c-Fos expression in a dose-dependent manner in the brainstem regions examined. In nucleus tractus solitarius (NTS)-A2 and NTS-C2, both NPY⁺ and TH⁺ neurons responded to the lower dose of nicotine, whereas the activin and galanin neurons in these regions were unresponsive to either dose of nicotine. In contrast, the higher dose of nicotine was required to activate NPY⁺ neurons in the A1 region and both NPY⁺ and galanin⁺ neurons in the locus coeruleus; the C1 region was unresponsive to nicotine. Since plasma ACTH is elevated by the low dose of nicotine and only NTS neurons are activated by this dose, NPY projections from the NTS are likely to contribute to nicotine-stimulated ACTH secretion, in addition to the previously described catecholaminergic neurons.     

Fos-determined distribution of neurons activated during the Bezold-Jarisch reflex in the medulla oblongata in conscious rabbits and rats

Experiments were performed in unanaesthetized rabbits and rats to investigate the distribution, within the medulla oblongata, of neurons activated during the Bezold-Jarisch reflex. Repeated intravenous injections of phenylbiguanide evoked depressor and bradycardic responses in both rabbits and rats. Fos-positive neurons were present in the nucleus tractus solitarius and in the caudal ventrolateral medulla oblongata. Double-label tyrosine hydroxylase (TH) immunohistochemical studies in the ventrolateral medulla showed that most Fos-positive neurons in the caudal ventrolateral medulla were TH-negative neurons scattered between A1 noradrenaline cells, in the rabbit and in the rat. Approximately 20% of neurons in the caudal ventrolateral medulla in rabbits, and 50% in rats, were immunoreactive for both Fos and TH. Some Fos-positive, TH-negative neurons in the caudal ventrolateral medullawere retrogradely labelled with cholera toxin B-Gold after injection of this tracer into the sympathoexcitatory region of the rostral ventrolateral medulla. Our data suggests that neurons in the nucleus tractus solitarius, and rostrally projecting TH-negative neurons in the caudal ventrolateral medulla, are part of the pathway by which stimulation of cardiopulmonary receptors inhibits sympathetic vasomotor tone to decrease blood pressure during the Bezold-Jarisch reflex.     

Developmental plasticity of the carotid chemoafferent pathway in rats that are hypoxic during the prenatal period

The chemoreflex pathway undergoes postnatal maturation, and the perinatal environment plays a critical role in shaping respiratory control system. We investigated the role of prenatal hypoxia on the maturation of the chemoreflex neural circuits regulating ventilation in rat. Effects of hypoxia (10% O2) from the 5th to the 20th day of gestation were studied on male offspring at birth and on postnatal days 3, 7, 21 and 68. Maturation of the respiratory control system was assessed by in vivo tyrosine hydroxylase (TH) activity measurement in peripheral chemoreceptors (carotid bodies, petrosal ganglia), and in brainstem catecholaminergic cell groups (A2C2c and A1C1 areas in the medulla, A5 and A6 areas in the pons). Resting ventilation and ventilatory response to hypoxia were evaluated as functional sequelae. In peripheral structures, prenatal hypoxia reduced TH activity within the first postnatal week and enhanced it later. In contrast, in central areas, prenatal hypoxia upregulated TH activity within the first postnatal week and downregulated it later. The in vivo TH activity impairment is therefore tissue specific, with an opposite effect on the peripheral and central neural circuits. A shift of the effect of prenatal hypoxia occurred between 1 and 3 weeks, indicating a postnatal temporal effect of prenatal hypoxia. An important period in the development of the chemoafferent pathway occurred between the first and the third postnatal week. Functionally, prenatal hypoxia impaired resting ventilation and ventilatory response to hypoxia. The alterations of the catecholaminergic components of the chemoafferent pathway resulting from prenatal hypoxia might contribute to impair postnatal respiratory behaviour.     

Neonatal maternal separation and enhancement of the inspiratory (phrenic) response to hypoxia in adult rats: disruption of GABAergic neurotransmission in the nucleus tractus solitarius

Neonatal maternal separation (NMS) alters respiratory control development. Adult male rats previously subjected to NMS show a hypoxic ventilatory response 25% greater than controls. During hypoxia, γ-aminobutyric acid (GABA) release within the nucleus tractus solitarius (NTS) modulates the magnitude of the ventilatory response. Because development of GABAergic receptors is sensitive to NMS, we tested the hypothesis that in adults, a change in responsiveness to GABA within the NTS contributes to NMS-related enhancement of the inspiratory (phrenic) response to hypoxia. Pups subjected to NMS were placed in an incubator for 3 h/day for 10 consecutive days [postnatal days 3 to 12]. Controls were undisturbed. Adult (8–10 weeks old) rats were anaesthetized (urethane; 1.6 g/kg), paralysed and artificially ventilated to record phrenic activity. Rats either received a 50-nL microinjection of GABA (5 µ m ) or phosphate-buffered saline (sham) within the caudal NTS, or no injection prior to being exposed to hypoxia (FiO₂ = 0.12; 5 min). NMS enhanced both the frequency and amplitude components of the phrenic response to hypoxia vs controls. GABA microinjection attenuated the phrenic responses in NMS rats only. This result is supported by ligand binding autoradiography results showing that the number of GABA_A receptors within the NTS was 69% greater in NMS vs controls. Despite this increase, the phrenic response to hypoxia of NMS rats is larger than controls, suggesting that the higher responsiveness to GABA microinjection within the NTS is part of a mechanism that aims to compensate for: (i) a deficient GABAergic modulation; (ii) enhancement of excitatory inputs converging onto this structure; or (iii) both.     

Identification of brainstem neurons responding to hypoxia in fetal and newborn sheep

Hypoxia causes a reversible decrease in the level of respiratory, oculomotor and postural muscle activity in fetal sheep, an effect not seen in newborn lambs. We have used Fos immunohistochemistry to identify neurons which are activated by hypoxia and which may mediate this motor inhibition in the fetus. Pregnant sheep of either 117 or 138 days gestation were made hypoxic by allowing them to breathe 8–9% O₂ for 2 h. Compared to age-matched control fetuses, hypoxia caused a significant increase in Fos-immunoreactivity in several medullary nuclei including the nucleus tractus solitarius, lateral reticular nucleus and the rostral ventrolateral medulla and also in the lateral parabrachial nucleus, locus coeruleus and subcoeruleus region in the pons. Hypoxia in newborn lambs, 7–18 days old, resulted in Fos staining in the same medullary and pontine nuclei with the exception of the subcoeruleus region which was devoid of Fos-immunoreactivity. In newborn lambs in which the carotid sinus nerves had been sectioned bilaterally, Fos-immunoreactivity was increased in the nucleus tractus solitarius in the medulla and in the locus coeruleus, lateral parabrachial and Kölliker–Fuse nuclei in the pons when compared to intact control newborn lambs. When carotid sinus nerve denervated-lambs were subjected to hypoxia the pattern of Fos-ir was similar to the pattern seen in the denervated control lambs but in addition staining was present in the subcoeruleus. These results suggest that a specific set of pontine neurons are activated by low oxygen levels in the fetus but not in the newborn lamb in the presence of an intact innervation from the carotid sinus. We hypothesise that: (a) in the fetus hypoxia activates neurons in the region of the subcoeruleus and this causes cessation of breathing movements and muscle atonia; and (b) that after birth stimulation of the carotid chemoreceptors by hypoxia normally inhibits activation of these subcoeruleus neurons.     

Expression of Fos immunoreactivity in some catecholaminergic brainstem neurons in rats following high-altitude exposure

This study examined the response of neurons of the cardiorespiratory centers, i.e., the nucleus tractus solitarius and the ventrolateral medulla as well as the area postrema in adult and postnatal rats subjected to high-altitude exposure at 4,000 m and 8,000 m. In adult control rats, sporadic Fos-positive neurons were detected in the above-mentioned areas. On exposure to 4,000 m altitude, the number of Fos-positive neurons was noticeably increased. At 8,000 m, the incidence of labeled cells was markedly increased, with many of them doubly labeled for tyrosine hydroxylase. In postnatal rats, Fos expression was not detected in these areas in either control rats or rats exposed to 4,000 m altitude. Fos-positive cells, however, were observed in the these areas in postnatal rats exposed to 8,000 m. In the latter, tyrosine hydroxylase labeling was observed in some Fos-positive cells in the nucleus tractus solitarius and ventrolateral medulla. In rats killed at 24 hr after exposure to high altitude, Fos expression in both the adult and the postnatal rats was comparable to that in their corresponding control rats. Present results suggest that Fos expression in various brainstem areas was induced by reduced oxygen tension in the ambient air at high altitude. Double labeling of some Fos-positive neurons with tyrosine hydroxylase indicates an increased sympathetic activation, which may be involved in the mediation of cardiorespiratory responses to hypoxia. This, however, was less evident in the postnatal animals. It is possible that the peripheral chemoreceptors or the regulation of autonomic functions is not fully developed in this age group.     

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

Fos expression within regions of the preoptic area, hypothalamus and brainstem during pregnancy and parturition

Vaginocervical stimulation, that occurs during mating or with the birth of pups, is believed to induce specific sexual and maternal behaviours in the rat as well as stimulating a number of neuroendocrine responses including the secretion of oxytocin, prolactin and luteinizing hormone. Since the medial preoptic area has been implicated in the induction of maternal behaviour, the expression of the immediate-early gene product Fos was compared between non-pregnant, late pregnant and parturient rats. Although no difference was detected in the number of Fos-positive neuronal profiles in the preoptic area of non-pregnant and late-pregnant rats, a large increase was observed in the medial preoptic nucleus and the anteroventral periventricular region, as well as in the hypothalamic supraoptic nucleus, of parturient rats. Double labelling for Fos and tyrosine hydroxylase immunoreactivity in the brainstem of parturient rats showed the activation of catecholaminergic neurons in both the nucleus of the tractus solitarius and in the ventrolateral medulla that may form part of the afferent pathway from the uterus and cervix to the preoptic area and hypothalamus.     

Effects of intermittent hypoxia on cat petrosal ganglion responses induced by acetylcholine, adenosine 5'-triphosphate and NaCN

Exposure to chronic intermittent hypoxia (CIH) for 4 days enhances the cat carotid body (CB) chemosensory responses to acute hypoxia. However, it is not known if CIH enhances the responses of the petrosal ganglion (PG) neurons that innervate the CB chemoreceptor cells. Accordingly, we studied the effects of the CB putative excitatory transmitter acetylcholine (ACh) and adenosine 5 -triphosphate (ATP), and the effects of citotoxic hypoxia (NaCN) applied to the isolated PG from cats exposed to CIH for 4 days. The dose-dependent curve parameters of the frequency of discharges evoked in the carotid sinus nerve by the application of ACh, ATP and NaCN to the isolated PG in control condition were not significantly modified in the CIH-treated cats. Present results suggest that CIH enhances the chemosensory responses to acute hypoxia acting primarily at the chemoreceptor cells, without major changes in the response of PG neurons evoked by the application of putative CB excitatory transmitters to their somata.     

Regulatory role of glucocorticoids and glucocorticoid receptor mRNA levels on tyrosine hydroxylase gene expression in the locus coeruleus during repeated immobilization stress

Sustained responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis during chronic or repeated stress is associated with continuous activation of ascending noradrenergic neurons from the brainstem to the hypothalamic paraventricular nucleus (PVN). The fact that glucocorticoid receptor (GR) exists in the brainstem noradrenergic neurons including locus coeruleus (LC) suggests that glucocorticoids play a modulatory role in maintaining the activity of these neurons during chronic stress. To determine whether alterations in the sensitivity of noradrenergic neuronal activity to endogenous CORT occur during chronic or repeated stress, tyrosine hydroxylase (TH) and GR mRNA expressions in the LC were examined in acute (2 h) and repeated (2 h daily, 14 days) immobilization stress, using sham-operated rats and adrenalectomized rats with a moderate dose of CORT replacement (ADX+CORT group). In acute stress, TH mRNA in the LC increased in the ADX+CORT rats, but not in sham operated rats. In repeated stress, however, elevated endogenous CORT failed to inhibit TH mRNA responses in sham rats; LC TH mRNA in sham rats responded to the same extent as in ADX+CORT rats. A reduction of GR mRNA in the LC was observed in the acutely stressed and repeatedly stressed sham group, but not in the ADX+CORT groups. The decrease in LC GR mRNA levels in sham rats tended to be greater after repeated than after acute stress. LC GR mRNA levels decreased in response to systemic CORT treatment (200 mg pellet sc, for 14 days) and increased in response to adrenalectomy; neither CORT treatment nor adrenalectomy influenced TH mRNA levels in the LC. These results suggest that glucocorticoid responses to acute immobilization prevent LC TH mRNA levels from rising significantly, while glucocorticoids appear to decrease their capacity to restrain LC TH mRNA during repeated immobilization. Although the results clearly show glucocorticoid-dependent alterations in LC GR mRNA expression, the association between increased TH mRNA and decreased GR mRNA in the LC remains to be elucidated.