Guus H.M. Schoorlemmer

Consequences of subchronic and chronic exposure to intermittent hypoxia and sleep deprivation on cardiovascular risk factors in rats

Since studies suggest that both hypoxia and sleep fragmentation are related to cardiovascular alterations induced by obstructive sleep apnea, the present study was designed to evaluate the effects of hypoxia, sleep deprivation, and their combination on biochemical blood parameters in rats. In subchronic experiments (4 days), rats were exposed to intermittent hypoxia (IH) during the light period (2min room air-2min 10% O(2) for 12h/day) and/or paradoxical sleep deprivation (PSD, 24h/day). Consequences of chronic intermittent hypoxia (CIH) exposure were examined after 21 consecutive days of hypoxia protocol from 10:00 to 16:00 followed by a sleep restriction (SR) period of 18h (16:00-10:00). Rats were randomly assigned to seven treatment groups: (1) control (2) IH (3) PSD (4) IH-PSD (5) SR (6) CIH and (7) CIH-SR. PSD reduced triglycerides and very low-density lipoprotein (VLDL) cholesterol concentrations and increased total cholesterol and high-density lipoprotein (HDL) cholesterol. IH did not alter any of these parameters. The combination of IH-PSD did not modify the values of total cholesterol and HDL compared to control group. In the chronic experiment, the animals exposed to CIH displayed a reduction of Vitamin B(6) and an increase of triglycerides and VLDL. Our findings show a duration-dependent effect of hypoxia on triglycerides. Rats in the SR and CIH-SR groups showed a diminished concentration of triglycerides and VLDL. SR rats showed a reduction in the concentration of homocysteine but the animals in the CIH-SR treatment condition did not display any alterations in this parameter. In this latter group, an augmentation of cysteine concentration was observed. These results suggest that sleep deprivation and hypoxia modify biochemical blood parameters in distinct ways.

Ablation of NK1 receptor bearing neurons in the nucleus of the solitary tract blunts cardiovascular reflexes in awake rats.

The nucleus of the solitary tract (NTS) receives primary afferents involved in cardiovascular regulation. We investigated the role of NK(1)-receptor bearing neurons in the NTS on cardiovascular reflexes in awake rats fitted with chronic venous and arterial cannulae. These neurons were lesioned selectively with saporin conjugated with substance P (SP-SAP, 2 microM, bilateral injections of 20 nL in the subpostremal NTS, or 200 nL in both the subpostremal and the commissural NTS). Before, and 7 and 14 days after injection of SP-SAP, we measured changes in blood pressure and heart rate induced by i.v. injection of phenylephrine and nitroprusside (baroreceptor reflex), cyanide (arterial chemoreceptor reflex), and phenylbiguanide (Bezold-Jarisch reflex). The smaller injections with SP-SAP completely abolished NK1 receptor staining in the subpostremal NTS. The larger injections abolished NK1 receptor immunoreactivity in an area that extended from the commissural NTS to the rostral end of the subpostremal NTS. The lesions seemed to affect only a limited number of neurons, since neutral red stained sections did not show any obvious reduction in cell number. The smaller lesions reduced the gain of baroreflex bradycardia and the hypotension induced by phenylbiguanide. The larger lesions completely abolished the response to phenylbiguanide, blocked the baroreflex bradycardia induced by phenylephrine, severely blunted the baroreflex tachycardia, and blocked the bradycardia and reduced the hypertension induced by cyanide. Thus, these responses depend critically on NK(1)-receptor bearing neurons in the NTS.

Hindbrain mineralocorticoid mechanisms on sodium appetite

Aldosterone acting on the brain stimulates sodium appetite and sympathetic activity by mechanisms that are still not completely clear. In the present study, we investigated the effects of chronic infusion of aldosterone and acute injection of the mineralocorticoid receptor (MR) antagonist RU 28318 into the fourth ventricle (4th V) on sodium appetite. Male Wistar rats (280-350 g) with a stainless-steel cannula in either the 4th V or lateral ventricle (LV) were used. Daily intake of 0.3 M NaCl increased to 46 ± 15 and 130 ± 6 ml/24 h after 6 days of infusion of 10 and 100 ng/h of aldosterone into the 4th V (intake with vehicle infusion: 2 ± 1 ml/24 h). Water intake fell slightly and not consistently, and food intake was not affected by aldosterone. Sodium appetite induced by diuretic (furosemide) combined with 24 h of a low-sodium diet fell from 12 ± 1.7 ml/2 h to 5.6 ± 0.8 ml/2 h after injection of the MR antagonist RU 28318 (100 ng/2 μl) into the 4th V. RU 28318 also reduced the intake of 0.3 M NaCl induced by 9 days of a low-sodium diet from 9.5 ± 2.6 ml/2 h to 1.2 ± 0.6 ml/2 h. Infusion of 100 or 500 ng/h of aldosterone into the LV did not affect daily intake of 0.3 M NaCl. The results are functional evidence that aldosterone acting on MR in the hindbrain activates a powerful mechanism involved in the control of sodium appetite.

Increased renal sympathetic nerve activity leads to hypertension and renal dysfunction in offspring from diabetic mothers

The exposure of the fetus to a hyperglycemic environment promotes the development of hypertension and renal dysfunction in the offspring at adult age. We evaluated the role of renal nerves in the hypertension and renal changes seen in offspring of diabetic rats. Diabetes was induced in female Wistar rats (streptozotocin, 60 mg/kg ip) before mating. Male offspring from control and diabetic dams were studied at an age of 3 mo. Systolic blood pressure measured by tail cuff was increased in offspring of diabetic dams (146 ± 1.6 mmHg, n = 19, compared with 117 ± 1.4 mmHg, n = 18, in controls). Renal function, baseline renal sympathetic nerve activity (rSNA), and arterial baroreceptor control of rSNA were analyzed in anesthetized animals. Glomerular filtration rate, fractional sodium excretion, and urine flow were significantly reduced in offspring of diabetic dams. Two weeks after renal denervation, blood pressure and renal function in offspring from diabetic dams were similar to control, suggesting that renal nerves contribute to sodium retention in offspring from diabetic dams. Moreover, basal rSNA was increased in offspring from diabetic dams, and baroreceptor control of rSNA was impaired, with blunted responses to infusion of nitroprusside and phenylephrine. Thus, data from this study indicate that in offspring from diabetic mothers, renal nerves have a clear role in the etiology of hypertension; however, other factors may also contribute to this condition.

A new method to produce obstructive sleep apnoea in conscious unrestrained rats

We developed a new method to produce obstructive apnoea in conscious rats. An inflatable balloon contained in a rigid Teflon tube was implanted in the trachea to allow the induction of apnoea without inducing pain. We also developed a balloon‐tipped catheter that was advanced along the trachea into the mediastinum for the measurement of intrathoracic pressure. Rats recovered well from implantation of these balloons. The tracheal implant, while deflated, did not significantly impair normal breathing (thoracic pressure swing during rest was 4.5 ± 0.4 mmHg before implantation and 5.8 ± 0.5 mmHg 4 weeks after implantation; P > 0.2; n= 7). Apnoeas of up to 16 s could be made during rapid eye movement sleep without awakening the rat. During 15 s of balloon inflation, arterial O2 saturation fell from 98 ± 0 to 80 ± 2% and partial pressure of CO2 increased from 35 ± 1 to 44 ± 1 mmHg (n= 9; P < 0.001). Intrathoracic pressure changes during the respiratory cycle increased from 6.3 ± 0.2 to 38.5 ± 6.0 mmHg (P < 0.001; n= 4), indicating increased breathing effort. Heart rate fell from 373 ± 23 to 141 ± 18 beats min−1 (P < 0.001; n= 4), and the heart beat became irregular, with few beats during expiratory effort. These responses remained intact after 60 apnoea episodes. Responses developed slightly more slowly when apnoea started at the end than at the beginning of the respiratory cycle. As these balloons last for a long time, cause few complications, allow induction of apnoea during sleep, allow induction of apnoeas that start at a fixed point in the respiratory cycle and elicit cardiorespiratory responses similar to those observed in humans, these balloons may aid investigation of both acute apnoea and chronic intermittent sleep apnoea.

Repeated amygdala-kindled seizures induce ictal rebound tachycardia in rats

It is thought that cardiovascular changes may contribute to sudden death in patients with epilepsy. To examine cardiovascular alterations that occur during epileptogenesis, we measured the heart rate of rats submitted to the electrical amygdala kindling model. Heart rate was recorded before, during, and after the induced seizures. Resting heart rate was increased in stages 1, 3, and 5 as compared with the unstimulated control condition. In the initial one third of the seizures, we observed bradycardia, which increased in intensity with increasing stage and was blocked by injecting methyl atropine. During stage 5 seizures, a rebound tachycardia was observed that also increased in intensity with increasing number of seizures. This study demonstrated the influence of seizure frequency on cardiac autonomic modulation, providing a basis for discussion of potential mechanisms that cause patients with epilepsy to die suddenly.

Cardiovascular adjustments induced by hypertonic saline in hemorrhagic rats: Involvement of carotid body chemoreceptors.

The peripheral hyperosmolarity elicited by intravenous infusion of hypertonic saline brings potential benefits to the treatment of hemorrhage. The neural mechanisms involved in these beneficial effects remain unknown. The present study examines the role of carotid chemoreceptors in cardiovascular responses induced by hypertonic saline after hypovolemic hemorrhage in rats. Male Wistar rats (300-400 g) were anesthetized with thiopental, and instrumented for recording of mean arterial pressure. Arterial pressure was reduced to 60 mm Hg by withdrawal of arterial blood over 10 min, and maintained at this level for 60 min by withdrawal or infusion of blood. In control rats (n = 8) with intact chemoreceptors, the subsequent intravenous infusion of hypertonic saline (3M NaCl, 1.8 ml kg(-1) body weight, in 2 min) restored blood pressure (pressure increased from 61 ± 4 to 118 ± 5 mm Hg). In experimental rats (n = 8), the carotid body arteries were tied, 30 min after the beginning of the hypotensive phase, leaving the carotid chemoreceptors ischemic. In these rats, hypertonic saline failed to restore blood pressure (pressure increased from 55 ± 1 to 70 ± 6 mm Hg). These findings suggest that the restoration of blood pressure after hypovolemic hemorrhage induced by hypertonic saline depends on intact carotid chemoreceptors.

Changes in sodium appetite evoked by lesions of the commissural nucleus of the tractus solitarius

Ablation of the area postrema/caudal nucleus of the tractus solitarius (NTS) complex increases sodium intake, but the effect of selective lesions of the caudal NTS is not known. We measured depletion-induced sodium intake in rats with electrolytic lesions of the commissural NTS that spared the area postrema. One day after the lesion, rats were depleted of sodium with furosemide (10 mg/kg body weight, sc) and then had access to water and a sodium-deficient diet for 24 h when 1.8% NaCl was offered. Water and saline intakes were measured for 2 h. Saline intake was higher in lesioned than in sham-lesioned rats (mean +/- SEM: 20 +/- 2 vs 11 +/- 3 mL/2 h, P < 0.05, N = 6-7). Saline intake remained elevated in lesioned rats when the tests were repeated 6 and 14 days after the lesion, and water intake in these two tests was increased as well. Water intake seemed to be secondary to saline intake both in lesioned and in sham-lesioned rats. A second group of rats was offered 10% sucrose for 2 h/day before and 2, 7, and 15 days after lesion. Sucrose intake in lesioned rats was higher than in sham-lesioned rats only 7 days after lesioning. A possible explanation for the increased saline intake in rats with commissural NTS lesions could be a reduced gastrointestinal feedback inhibition. The commissural NTS is probably part of a pathway for inhibitory control of sodium intake that also involves the area postrema and the parabrachial nucleus.

BRAINSTEM AREAS ACTIVATED BY INTERMITTENT APNEA in AWAKE UNRESTRAINED RATS

We investigated the role of the autonomic nervous system to cardiovascular responses to obstructive apnea in awake, unrestrained rats, and measured expression of Fos induced by apnea in the brainstem. We implanted a tracheal balloon contained in a rigid tube to allow the induction of apnea without inducing pain in the trachea. During bouts of 15s of apnea, heart rate fell from 371±8 to 161±11bpm (mean±SEM, n=15, p<0.01) and arterial pressure increased from 115±2 to 131±4mmHg (p<0.01). Bradycardia was due to parasympathetic activity because it was blocked by the muscarinic antagonist, methylatropine. The pressor response was due to vasoconstriction caused by sympathetic activation because it was blocked by the α1 antagonist, prazosin. Apnea induced Fos expression in several brainstem areas involved in cardiorespiratory control such as the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM), and pons. Ligation of the carotid body artery reduced apnea-induced bradycardia, blocked heart rate responses to i.v. injection of cyanide, reduced Fos expression in the caudal NTS, and increased Fos expression in the rostral VLM. In conclusion, apnea activates neurons in regions that process signals from baroreceptors, chemoreceptors, pulmonary receptors, and regions responsible for autonomic and respiratory activity both in the presence and absence of carotid chemoreceptors.

The interstitial system of the trigeminal spinal tract projects to the red nucleus in mice

We studied projections from the interstitial system of the spinal trigeminal tract (InSy-S5T) to the red nucleus of the mouse with retrograde tracers (fluorogold and latex microbeads impregnated with rhodamine and fluorescein). Injections in the magnocellular part of the red nucleus caused labeling of cells in the rostral, intermediate, and caudal paratrigeminal nucleus (Pa5), dorsal paramarginal nucleus (PaMD), insular trigemeo-lateral cuneate nucleus (I5CuL), and the trigeminal extension of the parvocellular reticular formation (5RPC). All projections were bilateral, but contralateral projections were stronger. The number of retrogradely labeled cells in the InSy-S5T in 3-, 6-, and 12-month-old mice was similar. Injections restricted to the parvocellular red nucleus did not label the nuclei of the InSy-S5T. This projection from the InSy-S5T to the red nucleus may mediate modulation of the facial muscles by pain and other sensory information.