Vitamin D supplementation at different doses affects the vagal component of the baroreceptor reflex and the Bezold-Jarisch reflex in eutrophic rats.

Vitamin D has been used to prevent several diseases. The 1,25 (OH) 2D3, the active form of vitamin D (VitD), participates in calcium metabolism, and has direct action in various tissues as those of the cardiovascular system binding to the VitD receptor. We investigated whether the supplementation with different doses of VitD affect or not the resting mean arterial pressure (MAP) and heart rate (HR), heart rate variability (HRV), baroreceptor and Bezold-Jarisch reflexes in eutrophic rats. Adult male Wistar rats were randomly assigned in 4 groups (Control, VitD 15, 250, and 3,750 IU/day, n = 6/group). After 3 days of supplementation, MAP and HR recordings were performed in freely moving rats. Baseline (resting) MAP, HR, and HRV showed no difference in Control and VitD groups. Nevertheless, the index of the baroreceptor reflex showed that the bradycardic component of the baroreflex evoked by a pressor dose of phenylephrine (3 µg/kg of b.w.) in bolus injection had a significant increase in rats supplemented with VitD 15 IU/day for 3 days compared to Control animals. No difference was observed in the index of the baroreflex evaluated with phenylephrine in rats treated with VitD 250 and 3,750 IU/day for 3 days in comparison to the Control group. The index of the baroreceptor reflex evaluated with an intravenous bolus injection of a depressor dose of sodium nitroprusside (30 µg/kg of b.w.) showed that the tachycardic component of the baroreflex is not different comparing all groups supplemented with VitD and Control animals. Rats supplemented with VitD 15 IU/day presented exaggerated bradycardic responses to the intravenous injection of phenylbiguanide (PBG, 5 µg/kg of b.w.) compared to Control animals, despite the similar hypotension in both groups. Higher doses of supplementation of VitD (250 and 3,750 IU/day for 3 days) abolished the hypotension and bradycardia induced by PBG. The findings suggest that the supplementation with different doses of VitD (15, 250, and 3,750 IU/day) for 3 days did not affect the resting arterial pressure, heart rate and autonomic modulation on the heart in rats. Despite that, the supplementation with a low dose of VitD (15 IU/day for 3 days) improved the sensitivity of the bradycardic component of the baroreflex, whereas higher doses of supplementation with VitD (250 and 3,750 IU/day for 3 days) were unable to cause such effect. In addition, the Bezold-Jarisch reflex responses can be affected regardless the dose of VitD (15, 250 or 3,750 IU/day) supplementation for 3 days in rats.

Reliability and Agreement in the Cerebellar Tonsil Tip Localization: Two Methods Using the McRae Line Concept in MRI.

BACKGROUND: The cerebellar tonsil tip position (TP) is a common parameter used for the radiologic diagnosis of Chiari malformation type 1 (CM1). However, these paramedian structures are usually not properly visualized in the midsagittal section. Such mismatch may be a source of bias in TP measurements based on the McRae line (ML) traced between median craniometric points. This study aims to evaluate the intraoperator and interoperator reliability and agreement of 2 protocols to trace the ML in magnetic resonance imaging (MRI) for the cerebellar tonsil tip localization, adding a 3-dimensional multiplanar reconstruction (MPR) approach to the midsagittal plane. METHODS: Sixty-two T1-weighted head MRIs were obtained for 32 CM1 patients and 30 controls. Two operators independently applied 2 TP measurement protocols, one considering only the visualization of the sagittal plane and the other using MPR. The intraclass correlation coefficient was used to assess intraoperator and interoperator reliability, and the Bland-Altman graphical method was used to evaluate the agreement between the measurement protocols. RESULTS: The sagittal method significantly underestimated ML and tonsillar herniation when compared with the MPR method. The MPR method provided better reliability of the ML measurement when compared to the sagittal method, but this did not influence the reliability of the TP. Analysis of the Bland-Altman plot showed that the limits of agreement were close to acceptable for the ML, but not for measures of TP. CONCLUSIONS: The standardization of the LM tracing by the MPR method improves the acquisition of data regarding the position of the tonsils.

Respiratory sinus arrhythmia is a major component of heart rate variability in undisturbed, remotely monitored rattlesnakes, Crotalus durissus.

ECG recordings were obtained using an implanted telemetry device from the South American rattlesnake, Crotalus durissus, held under stable conditions without restraining cables or interaction with researchers. Mean heart rate (fH) recovered rapidly (<24 h) from anaesthesia and operative procedures. This preceded a more gradual development of heart rate variability (HRV), with instantaneous fH increasing during each lung ventilation cycle. Atropine injection increased mean fH and abolished HRV. Complete autonomic blockade revealed a cholinergic tonus on the heart of 55% and an adrenergic tonus of 37%. Power spectral analysis of HRV identified a peak at the same frequency as ventilation. This correlation was sustained after temperature changes and it was more evident, marked by a more prominent power spectrum peak, when ventilation is less episodic. This HRV component is homologous to that observed in mammals, termed respiratory sinus arrhythmia (RSA). Evidence for instantaneous control of fH indicated rapid conduction of activity in the cardiac efferent nervous supply, as supported by the description of myelinated fibres in the cardiac vagus. Establishment of HRV 10 days after surgical intervention seems a reliable indicator of the re-establishment of control of integrative functions by the autonomic nervous system. We suggest that this criterion could be applied to other animals exposed to natural or imposed trauma, thus improving protocols involving animal handling, including veterinarian procedures.

Baroreflex stimulation attenuates central but not peripheral inflammation in conscious endotoxemic rats.

We previously reported that activation of the baroreflex, a critical physiological mechanism controlling cardiovascular homeostasis, through electrical stimulation of the aortic depressor nerve attenuates joint inflammation in experimental arthritis. However, it is unknown whether baroreflex activation can control systemic inflammation. Here, we investigate whether baroreflex activation controls systemic inflammation in conscious endotoxemic rats. Animals underwent sham or electrical aortic depressor nerve stimulation initiated 10 min prior to a lipopolysaccharide (LPS) challenge, while inflammatory cytokine levels were measured in the blood, spleen, heart and hypothalamus 90 min after LPS treatment. Baroreflex activation did not affect LPS-induced levels of pro-inflammatory (tumor necrosis factor, interleukin 1ß and interleukin 6) or anti-inflammatory (interleukin 10) cytokines in the periphery (heart, spleen and blood). However, baroreflex stimulation attenuated LPS-induced levels of all these cytokines in the hypothalamus. Notably, these results indicate that the central anti-inflammatory mechanism induced by baroreflex stimulation is independent of cardiovascular alterations, since aortic depressor nerve stimulation that failed to induce hemodynamic changes was also efficient at inhibiting inflammatory cytokines in the hypothalamus. Thus, aortic depressor nerve stimulation might represent a novel therapeutic strategy for neuroprotection, modulating inflammation in the central nervous system.

Analysis of the respiratory component of heart rate variability in the Cururu toad Rhinella schneideri.

Beat-to-beat variation in heart rate (f H ) has been used as a tool for elucidating the balance between sympathetic and parasympathetic modulation of the heart. A portion of the temporal changes in f H is evidenced by a respiratory influence (cardiorespiratory interaction) on heart rate variability (HRV) with heartbeats increasing and decreasing within a respiratory cycle. Nevertheless, little is known about respiratory effects on HRV in lower vertebrates. By using frequency domain analysis, we provide the first evidence of a ventilatory component in HRV similar to mammalian respiratory sinus arrhythmia in an amphibian, the toad Rhinella schneideri. Increases in the heartbeats arose synchronously with each lung inflation cycle, an intermittent breathing pattern comprised of a series of successive lung inflations. A well-marked peak in the HRV signal matching lung inflation cycle was verified in toads whenever lung inflation cycles exhibit a regular rhythm. The cardiac beat-to-beat variation evoked at the moment of lung inflation accounts for both vagal and sympathetic influences. This cardiorespiratory interaction may arise from interactions between central and peripheral feedback mechanisms governing cardiorespiratory control and may underlie important cardiorespiratory adjustments for gas exchange improvement especially under extreme conditions like low oxygen availability.

The human salivary microbiome exhibits temporal stability in bacterial diversity.

The temporal variability of the human microbiome may be an important factor in determining its relationship with health and disease. In this study, the saliva of 40 participants was collected every 2 months over a one-year period to determine the temporal variability of the human salivary microbiome. Salivary pH and 16S rRNA gene copy number were measured for all participants, with the microbiome of 10 participants assessed through 16S rRNA amplicon sequencing. In February 2013, 16S rRNA gene copy number was significantly (P < 0.001) higher, with individual changes between time points significant (P = 0.003). Salivary pH levels were significantly (P < 0.001) higher in December 2012 than in October 2012 and February 2013, with significant (P < 0.001) individual variations seen throughout. Bacterial α-diversity showed significant differences between participants (P < 0.001), but not sampling periods (P = 0.801), and a significant positive correlation with salivary pH (R(2) = 7.8%; P = 0.019). At the phylum level, significant differences were evident between participants in the Actinobacteria (P < 0.001), Bacteroidetes (P < 0.001), Firmicutes (P = 0.008), Fusobacteria (P < 0.001), Proteobacteria (P < 0.001), Synergistetes (P < 0.001) and Spirochaetes (P = 0.003) phyla. This study charted the temporal variability of the salivary microbiome, suggesting that bacterial diversity is stable, but that 16S rRNA gene copy number may be subject to seasonal flux.

Role of Chemoreceptor Activation in Hemodynamic Responses to Electrical Stimulation of the Carotid Sinus in Conscious Rats.

Electric carotid baroreflex activation has been used to treat patients with resistant hypertension. It is hypothesized that, in conscious rats, combined activation of carotid baro- and chemoreceptors afferences attenuates the reflex hypotension. Rats were divided into 4 groups: (1) control group, with unilateral denervation of the right carotid chemoreceptors; (2) chemoreceptor denervation group, with bilateral ligation of the carotid body artery; (3) baroreceptor denervation group, with unilateral denervation of the left carotid baroreceptors and right carotid chemoreceptors; and (4) carotid bifurcation denervation group, with denervation of the left carotid baroreceptors and chemoreceptors, plus denervation of the right carotid chemoreceptors. Animals were subjected to 4 rounds of electric stimulation (5 V, 1 ms), with 15, 30, 60, and 90 Hz applied randomly for 20 s. Electric stimulation caused greater hypotensive responses in the chemoreceptor denervation group than in the control group, at 60 Hz (-37 versus -19 mm Hg) and 90 Hz (-33 versus -19 mm Hg). The baroreceptor denervation group showed hypertensive responses at all frequencies of stimulation. In contrast, the carotid sinus denervation group showed no hemodynamic responses. The control group presented no changes in heart rate, whereas the chemoreceptor denervation group and the baroreceptor denervation group showed bradycardic responses. These data demonstrate that carotid chemoreceptor activation attenuates the reflex hypotension caused by combined electric stimulation of the carotid sinus and the carotid sinus nerve in conscious rats. These findings may provide useful insight for clinical studies using baroreflex activation therapy in resistant hypertension and heart failure.

Aerobic physical training has little effect on cardiovascular autonomic control in aging rats subjected to early menopause.

We investigated and compared the effects of physiological menopause (PM) and early menopause (EM) and the adaptations promoted by physical training on the cardiovascular autonomic control of aged rats. Female Wistar rats (N=72) were assigned to 3 groups: control (22 weeks old rats, undergoing sham surgery in the 10th week of life), PM (82 weeks old rats, undergoing sham surgery in the 10th week of life) and EM (82 weeks old rats, undergoing ovariectomy in the 10th week of life). In each group, half of the rats were subjected to swimming training over a period of 10 weeks. Sedentary PM and EM groups had higher basal mean arterial pressure (MAP) and heart rate (HR) and lower intrinsic HR compared to the sedentary control group. Physical training reduced MAP in PM group. All trained groups had lower basal HR; however, only control and PM-trained groups showed decreased intrinsic HR. The assessment of cardiac autonomic balance showed that PM and EM sedentary groups exhibited sympathetic predominance compared to control group. After physical training, only EM group presented sympathetic predominance. HR variability (pulse interval) was similar among all sedentary groups. However, control and PM-trained groups showed lower power in low frequency band (LF; 0.2-0.75 Hz) and higher power in high frequency band (HF; 0.75-3.0 Hz). The analysis of systolic arterial pressure variability revealed that PM and EM sedentary groups had higher LF power. However, PM group showed lower LF power following physical training. Finally, PM and EM groups had a reduction in spontaneous baroreflex sensitivity, that was attenuated by physical training. The overall results suggest that PM or EM promotes similar negative effects on MAP, HR and cardiovascular autonomic control. However, unlike the PM group, physical training was not able to mitigate all negative effects of EM on cardiovascular autonomic control.

Diabetes mellitus in the rat: diminution in the number of cells in the thymus and of lymphocytes in the blood.

In diabetic rats there is a rapid fall in the weight of the thymus with a great loss in the numbers of thymocytes. In this paper we show that there is also a marked diminution in the number of lymphocytes in the peripheral blood, although the numbers of neutrophils and monocytes are not affected. Since the thymus is thought to be the site in which T-lymphocytes develop, it is suggested that the paucity of lymphocytes in the peripheral circulation reflects the loss of cells from the thymus. The diminution in the number of circulating lymphocytes in diabetes may account for the liability of diabetics to suffer severely from infections.

The influx of ascorbic acid into the rat's brain.

The influx of [14C]ascorbic acid into the rat's brain through the cerebral capillaries was measured by the steady-state initial-rate technique. Ascorbic acid was found to pass through the capillaries by a carrier-mediated process and by simple diffusion. The carrier system was found to have an apparent Vmax of 1.2 nmol . min-1 . g-1 of cerebral tissue and a Kt of 125 microM. The apparent constant of transfer, Kd, of the diffusional component was 0.98 microliter. min-1 . g-1. The contribution of the cerebral capillaries to the movement of ascorbic acid into the brain is discussed and was found to be the major route of entry into the brain for this vitamin.

Atrophy of the thymus in rats rendered diabetic by streptozotocin.

The thymus atrophied rapidly in rats rendered diabetic by a single injection of streptozotocin. The weight of the gland was reduced to one-fifth of its original weight within three weeks. Histologically there was a great loss of cortical thymocytes and a marked increase in mast cells lying in the thickened fibrous tissue. These findings may have a bearing on the state of immune deficiency observed in animals with diabetes induced by streptozotocin.

Comparison of the vascular permeability of the brain and the spinal cord to mannitol and inulin in rats.

The kinetics of the movement of [14C]mannitol and [14C]inulin from the blood into the CNS of the rat were measured. The spinal cord was found to have a higher permeability to these two substances than the brain. It is likely that the channels through which the tracers diffused are larger in the capillaries of the spinal cord than those in the brain.

The effects of fasting on core temperature, blood glucose and body and organ weights in rats.

Male Wistar rats of about 300 g body weight were fasted from 1 to 7 d. They were weighed and had their temperature taken daily. At the end of the experimental period, they were anaesthetized and blood was taken for measurement of the glucose content. The animals were killed and various organs were taken and weighed. Over the first 24 h there were striking reductions in body weight, core temperature and level of blood glucose. Throughout the succeeding days of fasting, there was a gradual decrease in body weight and core temperature, while the level of the blood glucose was maintained at about 3 mmol X l-1. The decrease in body weight and core temperature was matched by the weight loss of the liver and skeletal muscles. The heart and the kidney and spleen were not affected over the first day, but thereafter their weight declined steadily. The brain, the testes and the lungs retained their mass throughout.

Relation between the increase in the diffusional permeability of the blood-central nervous system barrier and other changes during the development of experimental allergic encephalomyelitis in the Lewis rat.

The reduction in the effectiveness of the blood-brain and blood-spinal cord barriers, previously seen in rats at the height of the acute episode of experimental allergic encephalomyelitis, has now been measured at various stages in the development of the disease up to 60 days after inoculation with guinea pig spinal cord in complete Freund's adjuvants. The marker of extracellular space, radioactively labelled mannitol, only crosses the blood-central nervous system barriers very slowly by passive diffusion in normal rats. An abnormal penetration of this marker into the central nervous system began to develop during the second week after inoculation, appearing first in the lower spinal cord, where it also reaches the highest level during the acute phase of the attack. The leak begins before either the clinical signs become evident or cuffing is seen around blood vessels in stained sections. As the clinical signs are disappearing, from about 15 days onwards, the permeability of the barrier returns steadily to its normal low value, starting in the spinal cord, especially the caudal part. The timing of the reduction in the effectiveness of the blood-central nervous system barrier in relation to other clinical and histological changes suggests that it may play a part in the development of the lesion. The relation between the timing of these changes in EAE and that in the development of a new lesion in (exacerbation of) multiple sclerosis is discussed.

The pathogenesis of transmissible spongiform encephalopathy: an ultrastructural study.

The brains of 9 spider monkeys, inoculated intracerebrally with brain suspension from kuru patients and of 3 normal control monkeys have been studied. All the animals were killed by perfusion with fixative, 8 during early incubation (ranging from 2 to 40 weeks) when healthy and free from neurological signs, one after 122 weeks when the disease was fully established. The most striking feature seen in every brain between the incubation times of 4 and 40 weeks was the formation of multilaminated membranes (ACPMs). These affected stretches of two apposed, mostly neuronal, plasma membranes over variable distances and created the impression of complex ribbon-or cord-like junctions. Their number varied with the length of incubation, reaching a peak at 13 weeks and declining thereafter. ACPMs were found throughout the grey matter, but they were most numerous in phylogenetically older regions of the brain, regions which also show the severest lesions in human kuru. It is suggested that ACPMs are initially due to an excessive synthesis of some membrane constituent by the perikaryon and various possibilities for their genesis are discussed. The hypothesis is advanced that they may be due to the reactivation of embryonic growth mechanisms and represent abortive junctions which, being formed in mature neurons, take a rather bizarre shape. Other changes such as the formation of somatic spines, an excessive number of dendritic spines including a high proportion with long tortuous necks, and the presence of binucleated neurons and numerous growth cones, point to the similarly immature pattern and would support this hypothesis. The material provided ample evidence that ACPMs, which can occupy as much as 26 per cent of a dendritic surface, give rise to intracytoplasmic vacuoles, which may therefore be regarded as secondary to a primary change in the permeability of the plasma membrane at the site of ACPMs. Individual single vacuoles could often build up into complex soap-bubble-like arrays, which were interpreted as the ultrastructural equivalent of histological status spongiosus. There is some evidence that the development of status spongiosus in other transmissible spongiform encephalopathies follows the same pattern.

Changes in the effectiveness of the blood-brain and blood-spinal cord barriers in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) was induced In Lewis rats by the intradermal inoculation of an homogenate of guinea pig spinal cord in complete Freund's adjuvant. In these animals the effectiveness with which the capillary barrier excluded mannitol (a substance which normally only crosses this barrier very slowly) from the brain and spinal cord involvement were at their height, 14 days after the inoculation, the effectiveness of the barrier was reduced since the rate of diffusion of the mannitol out of the blood into the brain-stem and spinal cord was approximately doubled. Even as early as 7 days after inoculation, before any clinical signs had appeared, the rate of diffusion was significantly increased in the region of the lumbo-sacral cord. These changes roughly paralleled the histological changes seen in many of the small vessels. We believe that the changes are due to an increase in the permeability of the vessels to mannitol. The bearing of these findings on multiple sclerosis is discussed.

The effect of insulin upon the influx of tryptophan into the brain of the rabbit.

1. The effect of hyperinsulinaemia upon the influx of tryptophan into the brain was determined. A raised level of insulin was maintained in the circulation of rabbits for periods of up to 120 min by means of a continuous, programmed intravenous injection of the hormone, given by an electronically controlled variable-drive syringe. A similar, appropriately programmed, intravenous injection of glucose, given simultaneously with the insulin, maintained the concentration of the blood glucose within normal limits throughout each experiment, so that the results were not vitiated by the development of hypoglycaemia. 2. Raised levels of insulin in the blood affect the supply of tryptophan to the brain in two opposing ways: (a) by increasing the binding of tryptophan to the albumin in the blood, thereby reducing the level of the free tryptophan in the circulation by about a half, which would decrease the influx of tryptophan into the brain; (b) by simultaneously reducing the levels in the blood of six or more of the amino acids which compete with tryptophan for transport carriers into the brain, which would increase the influx of tryptophan. The net result of these two opposing effects is that insulin causes only a slight increase in the influx of tryptophan into the brain. 3. To account in quantitative terms for the effect of insulin upon the influx of tryptophan into the brain it proved necessary to make one assumption. This assumption was that a predictable proportion of the tryptophan which is loosely bound to blood albumin is being stripped off this protein by the transport carrier located on the luminal surface membranes of the endothelial cells during the passage of the blood through the cerebral capillaries. If this assumption is accepted the work reported here explains adequately the effect of insulin on the influx of tryptophan into the brain.