Autoregulation of blood flow drives early hypotension in a rat model of systemic inflammation induced by bacterial lipopolysaccharide.

Uncontrolled vasodilation is known to account for hypotension in the advanced stages of sepsis and other systemic inflammatory conditions, but the mechanisms of hypotension in earlier stages of such conditions are not clear. By monitoring hemodynamics with the highest temporal resolution in unanesthetized rats, in combination with ex-vivo assessment of vascular function, we found that early development of hypotension following injection of bacterial lipopolysaccharide is brought about by a fall in vascular resistance when arterioles are still fully responsive to vasoactive agents. This approach further uncovered that the early development of hypotension stabilized blood flow. We thus hypothesized that prioritization of the local mechanisms of blood flow regulation (tissue autoregulation) over the brain-driven mechanisms of pressure regulation (baroreflex) underscored the early development of hypotension in this model. Consistent with this hypothesis, an assessment of squared coherence and partial-directed coherence revealed that, at the onset of hypotension, the flow-pressure relationship was strengthened at frequencies (<0.2 Hz) known to be associated with autoregulation. The autoregulatory escape to phenylephrine-induced vasoconstriction, another proxy of autoregulation, was also strengthened in this phase. The competitive demand that drives prioritization of flow over pressure regulation could be edema-associated hypovolemia, as this became detectable at the onset of hypotension. Accordingly, blood transfusion aimed at preventing hypovolemia brought the autoregulation proxies back to normal and prevented the fall in vascular resistance. This novel hypothesis opens a new avenue of investigation into the mechanisms that can drive hypotension in systemic inflammation.

SOCS3 Ablation in Leptin Receptor-Expressing Cells Causes Autonomic and Cardiac Dysfunctions in Middle-Aged Mice despite Improving Energy and Glucose Metabolism.

Leptin resistance is a hallmark of obesity. Treatments aiming to improve leptin sensitivity are considered a promising therapeutical approach against obesity. However, leptin receptor (LepR) signaling also modulates several neurovegetative aspects, such as the cardiovascular system and hepatic gluconeogenesis. Thus, we investigated the long-term consequences of increased leptin sensitivity, considering the potential beneficial and deleterious effects. To generate a mouse model with increased leptin sensitivity, the suppressor of cytokine signaling 3 (SOCS3) was ablated in LepR-expressing cells (LepR∆SOCS3 mice). LepR∆SOCS3 mice displayed reduced food intake, body adiposity and weight gain, as well as improved glucose tolerance and insulin sensitivity, and were protected against aging-induced leptin resistance. Surprisingly, a very high mortality rate was observed in aging LepR∆SOCS3 mice. LepR∆SOCS3 mice showed cardiomyocyte hypertrophy, increased myocardial fibrosis and reduced cardiovascular capacity. LepR∆SOCS3 mice exhibited impaired post-ischemic cardiac functional recovery and middle-aged LepR∆SOCS3 mice showed substantial arhythmic events during the post-ischemic reperfusion period. Finally, LepR∆SOCS3 mice exhibited fasting-induced hypoglycemia and impaired counterregulatory response to glucopenia associated with reduced gluconeogenesis. In conclusion, although increased sensitivity to leptin improved the energy and glucose homeostasis of aging LepR∆SOCS3 mice, major autonomic/neurovegetative dysfunctions compromised the health and longevity of these animals. Consequently, these potentially negative aspects need to be considered in the therapies that increase leptin sensitivity chronically.

Amygdala rapid kindling impairs breathing in response to chemoreflex activation.

Temporal lobe epilepsy is often accompanied by behavioral, electroencephalographic and autonomic abnormalities. Amygdala kindling has been used as an experimental model to study epileptogenesis. Although amygdala kindling has been extensively investigated in the context of its clinical relevance to the epilepsies, potential associated respiratory alterations are not well known. Here, our main objective was to better investigate the mechanisms involved in respiratory physiology impairment in the amygdala rapid kindling (ARK) model of epileptogenesis. Male Wistar rats with electrodes implanted into the amygdaloid complex were used. After recovery from surgery, the rats were subjected to electrical stimulation of basolateral amygdala for 2 consecutive days (10 stimuli/day). The ventilatory parameters were evaluated by whole body plethysmography. Thereafter, animals were also exposed to hypercapnia (7% CO2) for 3 h to evaluate fos protein expression in several nuclei involved in respiratory control. We observed a significant reduction in ventilation during the ictal phase elicited by ARK. We also found that 10 days after ARK, baseline ventilation as well as the hypercapnia ventilatory response (7% CO2) were reduced compared to control rats. The number of fos-immunoreactive neurons in the retrotrapezoid nucleus, raphe magnus and nucleus of the solitary tract were also reduced after ARK. Our results showed that ARK was able to impair breathing function, demonstrating a strong coupling between amygdala and the respiratory neurons in the brainstem, with potential impact in respiratory failures, frequently fatal, during or after epileptic seizures in chronic animal models and in patients.

Breathing responses produced by optogenetic stimulation of adrenergic C1 neurons are dependent on the connection with preBötzinger complex in rats.

Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBötzinger complex (preBötC) is a group of neurons located in the intermediate aspect of the ventrolateral medulla, critical for respiratory rhythmogenesis, and is modulated by glutamate and catecholamines. Our hypothesis is that selective activation of C1 neurons leads to breathing responses by excitatory connections with the preBötC neurons. Anatomical connection between C1 cells and preBötC was evaluated using retrograde (Cholera Toxin b; preBötC) and anterograde (LVV-PRSx8-ChR2-eYFP; C1 region) tracers. LVV-PRSx8-ChR2-eYFP (viral vector that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter (PRSx8) was also injected into the C1 region of male Wistar rats for the functional experiments. Anatomical results demonstrated that preBötC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBötC was evaluated by photostimulation of ChR2-transduced C1 neurons before and after unilateral injection of the ionotropic glutamate antagonist, kynurenic acid (kyn), or cocktail of adrenergic antagonists in the preBötC. Kyn injection into preBötC blocked the increase in DiaEMG frequency without changing the MAP increase elicited by photostimulation of C1 neurons, while the injection of adrenergic antagonists into the preBötC did not change DiaEMG frequency and MAP increase induced by photostimulation of C1 cells. Our results suggest that the increase in breathing produced by photostimulation of C1 neurons can be caused by a direct glutamatergic activation of preBötC neurons.

Impaired chemosensory control of breathing after depletion of bulbospinal catecholaminergic neurons in rats.

Bulbospinal catecholaminergic neurons located in the rostral aspect of the ventrolateral medulla (C1 neurons) or within the ventrolateral pons (A5 neurons) are involved in the regulation of blood pressure and sympathetic outflow. A stimulus that commonly activates the C1 or A5 neurons is hypoxia, which is also involved in breathing activation. Although pharmacological and optogenetic evidence suggests that catecholaminergic neurons also regulate breathing, a specific contribution of the bulbospinal neurons to respiratory control has not been demonstrated. Therefore, in the present study, we evaluated whether the loss of bulbospinal catecholaminergic C1 and A5 cells affects cardiorespiratory control during resting, hypoxic (8% O2), and hypercapnic (7% CO2) conditions in unanesthetized rats. Thoracic spinal cord (T4-T8) injections of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP-2.4 ng/100 nl) and the retrograde tracer Fluor-Gold or ventrolateral pontine injections of 6-OHDA were performed in adult male Wistar rats (250-280 g, N = 7-9/group). Anti-DßH-SAP or 6-OHDA eliminated most bulbospinal C1 and A5 neurons or A5 neurons, respectively. Serotonergic neurons and astrocytes were spared. Depletion of the bulbospinal catecholaminergic cells did not change cardiorespiratory variables under resting condition, but it did affect the response to hypoxia and hypercapnia. Specifically, the increase in the ventilation, the number of sighs, and the tachycardia were reduced, but the MAP increased during hypoxia in anti-DßH-SAP-treated rats. Our data reveal that the bulbospinal catecholaminergic neurons (A5 and C1) facilitate the ventilatory reflex to hypoxia and hypercapnia.

Impaired central respiratory chemoreflex in an experimental genetic model of epilepsy.

KEY POINTS: It is recognized that seizures commonly cause apnoea and oxygen desaturation, but there is still a lack in the literature about the respiratory impairments observed ictally and in the post-ictal period. Respiratory disorders may involve changes in serotonergic transmission at the level of the retrotrapezoid nucleus (RTN). In this study, we evaluated breathing activity and the role of serotonergic transmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We conclude that the respiratory impairment in the WAR could be correlated to an overall decrease in the number of neurons located in the respiratory column. ABSTRACT: Respiratory disorders may involve changes in serotonergic neurotransmission at the level of the chemosensitive neurons located in the retrotrapezoid nucleus (RTN). Here, we investigated the central respiratory chemoreflex and the role of serotonergic neurotransmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We found that naive or kindled WARs have reduced resting ventilation and ventilatory response to hypercapnia (7% CO2 ). The number of chemically coded (Phox2b+ /TH- ) RTN neurons, as well as the serotonergic innervation to the RTN, was reduced in WARs. We detected that the ventilatory response to serotonin (1 mm, 50 nl) within the RTN region was significantly reduced in WARs. Our results uniquely demonstrated a respiratory impairment in a genetic model of tonic-clonic seizures, the WAR strain. More importantly, we demonstrated an overall decrease in the number of neurons located in the ventral respiratory column (VRC), as well as a reduction in serotonergic neurons in the midline medulla. This is an important step forward to demonstrate marked changes in neuronal activity and breathing impairment in the WAR strain, a genetic model of epilepsy.

GABA mechanisms of the nucleus of the solitary tract regulates the cardiovascular and sympathetic effects of moxonidine.

The antihypertensive drugs moxonidine and clonidine are α2-adrenoceptor and imidazoline (I1) agonists. Previous results from our laboratory have shown that moxonidine can act in the commissural nucleus of the solitary tract (commNTS). In addition, some studies have shown that GABA or glutamate receptor blockade in the RVLM blunted the hypotension produced by these antihypertensive agents in spontaneously hypertensive rats. Therefore, in the present study we verify whether the cardiovascular and sympathetic effects produced by moxonidine in the commNTS are dependent on GABAergic or glutamatergic mechanisms. Mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (sSNA) were recorded in urethane-anesthetized, and artificially-ventilated male Wistar rats (250-350 g). Injection of the GABAA antagonist bicuculline (25 pmol/50 nL) into the commNTS reduced the hypotension as well as the sympathoinhibition elicited by moxonidine. Prior injection of the glutamate receptor antagonist kynurenic acid (2.5 nmol/50 nL) into the commNTS was not effective in reducing the hypotension and sympathoinhibition elicited by moxonidine. Therefore, we conclude that the hypotensive and sympathoinhibitory effects elicited by microinjection of moxonidine into the commNTS are dependent on GABA receptors, but not ionotropic glutamate receptors.