Comparison between pial and intraparenchymal vascular responses to cervical sympathetic stimulation in cats. Part 1. Under normal resting conditions

To investigate the role of sympathetic regulation in both resistance and capacitance vessels in cerebral circulation, the response of pial and intraparenchymal vessels to sympathetic nerve stimulation were simultaneously examined in 14 cats by means of a newly developed video camera photoelectric system. The system consisted of a video camera system for measurement of pial vascular diameters and a photoelectric apparatus for estimating regional cerebral blood volume in the intraparenchymal vessels. The ipsilateral superior cervical ganglion was electrically stimulated for 5 min. Initially, both the pial and intraparenchymal vessels constricted. The large pial arteries (173 +/- 25 micron, mean +/- SEM) remained constricted throughout the stimulation, whereas the intraparenchymal vessels began to dilate after the initial constriction and exceeded the control level at 175 +/- 25 s despite continued stimulation. In conclusion, such sympathetic nerve stimulation is considered to exert a constrictive effect on the intraparenchymal as well as the pial vessels at the early stage. The compensatory dilation of the intraparenchymal vessels was delayed 3 min after initiation of the stimulation.     

Pial vascular behavior during bilateral and contralateral cervical sympathetic stimulation

The present study in cats investigates the effect of cervical sympathetic stimulation on changes of diameter of pial arteries and veins, CBF, and intracranial pressure (ICP) using the cranial window and hydrogen clearance techniques. During 20 min of bilateral stimulation, pial arteries maximally constricted by 12%, veins by 13-15%. While the constriction of the large arteries remained stable during the whole 20-min period of bilateral stimulation, small arteries escaped after some 2 min. A similar though weaker trend was noted for the veins. CBF was reduced at 2 min by 31%, and was not different from resting at 18 min. Contralateral stimulation for 20 min induced early constriction only in small arteries, while all other vessels remained more or less unreactive. This phenomenon is explained by interhemispheric arterial collaterals that bring sympathetic fibers mainly to small arteries contralaterally. ICP was lowered initially by 47 +/- 12% during bilateral and by 23 +/- 5% during contralateral stimulation. ICP escaped after 2 and 5 min during bilateral and contralateral stimulation, respectively, and even started to rise after some 10 min. From these data, it is concluded that the sympathoadrenergic system exerts a short-lasting protective effect upon cerebral vascular volume. Small arteries escape from constriction as a consequence of primarily myogenic counteraction of pial and intraparenchymal vessels, and probably additional metabolic dilatation of intraparenchymal vessels.     

Determinants of response of pial arteries to norepinephrine and sympathetic nerve stimulation.

Feline pial arteries larger than 100 mu in diameter constricted in response to cervical sympathetic nerve stimulation suggests or in response to topical application of norepinephrine. Smaller pial arteries were unresponsive to norepinephrine. This unresponsiveness persisted when norepinephrine was dissolved in CSF with high calcium ion concentration, or in CSF with both high calcium ion and zero magnesium ion concentration, or when it was dissolved in the acid fluid used by Wahl et al. and applied by constant infusion or by intermittent application. Comparison of the responses of the larger pial vessels to norepinephrine and to sympathetic nerve stimulation that maximal activation of sympathetic nerves achieves a concentration of released norepinephrine equal to 5.9 x 10(-6) M. The constriction of the larger pial vessels in response to sympathetic nerve stimulation could account for modest reductions in cerebral blood flow.     

Locus coeruleus stimulation exerts different influences on the dynamic changes of cerebral pial and intraparenchymal vessels.

The present experimental study was undertaken to investigate the effects of locus coeruleus stimulation on the dynamic changes of intraparenchymal vessels and pial vessels. Twelve cats were anaesthetized with alpha-chloralose and urethane. For stimulation of the locus coeruleus, a concentric stainless-steel needle electrode was inserted stereotaxically. During the stimulation, volumetric changes of the intraparenchymal vessels were monitored by a photoelectric method for estimating the cerebral blood volume (CBV) (6 cats), and the diameters of pial arteries were measured continuously using a video camera system (6 cats). The CBV followed a decreasing course during the stimulation of the locus coeruleus. The decrease in CBV from the control value (6.3 vol%) was 0.14 +/- 0.04 vol% at 80 s (p less than 0.05), 0.15 +/- 0.05 vol% at 100 s (p less than 0.05), and 0.15 +/- 0.03 vol% at 120 s (p less than 0.01). After cessation of the stimulation, CBV showed a gradual recovery. On the other hand, the diameters of the pial arteries did not change during or after the stimulation of the locus coeruleus. The above results suggest that the locus coeruleus has a vasoconstrictive effect on the intraparenchymal vessels, although it exerts no apparent influence on the pial arteries.     

Quantitative measurement of blood flow velocity in feline pial arteries during hemorrhagic hypotension and hypercapnia

Due to methodologic difficulties, few investigations have been made on the blood flow velocity in the cerebral microcirculation. Using a newly developed video camera method, we simultaneously measured the blood flow velocity and diameter of pial arteries during hemorrhagic hypotension, after blood pressure recovery, and during CO2 inhalation in cats. When the mean arterial blood pressure was lowered from 129.7 +/- 6.6 to 71.5 +/- 4.1 mm Hg, the blood flow velocity inevitably decreased from 36.6 +/- 5.3 to 27.0 +/- 3.9 mm/sec (p less than 0.001). The calculated blood flow rate [pi X (diameter/2)2 X flow velocity] was preserved in cases with concomitant vasodilation. Conversely, the blood flow velocity increased from 25.3 +/- 5.1 to 31.0 +/- 5.4 mm/sec (p less than 0.001) after mean arterial blood pressure recovery from 67.1 +/- 3.7 to 129.8 +/- 5.8 mm Hg. The blood flow rate was again preserved in vessels with a vasoconstrictive response. Each pial artery apparently dilated or constricted in proportion to the decrease or increase in flow velocity during blood pressure changes, maintaining a constant cerebral blood flow. This indicated the importance of the pial arteries in the mechanisms of cerebral blood flow autoregulation. During 5% CO2 inhalation, the blood flow velocity increased markedly from 25.4 +/- 4.6 to 37.2 +/- 10.0 mm/sec (p less than 0.05), while the pial artery diameter (85.0 +/- 13.7 microns) increased by 9.6 +/- 1.5% (p less than 0.01). The increased flow velocity might be attributable to preferential dilatation of small arterioles or intraparenchymal vessels during hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and release of serotonin in rat cerebrovascular nerves after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Serotonin released from platelets has been suggested as one substance causing the vasospasm following subarachnoid hemorrhage. We studied whether such serotonin is able to constrict pial vessels. METHODS: We studied the uptake of serotonin in pial perivascular nerves by immunohistochemistry. We measured the contractile response in rat basilar artery after in vitro incubation with serotonin and during electrical field stimulation of perivascular nerves following experimental subarachnoid hemorrhage. RESULTS: After incubation with serotonin, electrical field stimulation caused a tetrodotoxin- and ketanserin-blockable contractile response. We observed no such response in vessels from rats treated with 6-hydroxydopamine or after blockade of serotonin uptake. After subarachnoid hemorrhage, a pronounced network of serotonin-immunoreactive nerve fibers was demonstrated in the vessel wall. In vessels from control rats, no serotonin fibers were seen, and in vessels from 6-hydroxydopamine-treated animals with subarachnoid hemorrhage only a few such fibers were seen. Electrical field stimulation of the basilar artery from rats tested 2 or 16 hours (but not 10 minutes or 24 hours) after subarachnoid hemorrhage showed contractile responses that were prevented by tetrodotoxin, ketanserin, and prior 6-hydroxydopamine treatment. CONCLUSIONS: Our study demonstrates a capacity of the perivascular sympathetic nerves to take up serotonin both in vitro and during the early phase of subarachnoid hemorrhage. Such uptake may help to remove excess serotonin from the subarachnoid space. Only if serotonin is subsequently released upon nerve activation may minor smooth muscle contraction develop.     

The difference in vascular volume between cerebrum and cerebellum is in the pia mater

Previous studies using intravascular tracers have shown that the apparent vascular volume in the cerebellum is 10-60% higher than that in the cerebrum. We questioned whether the extravascular volume in the cerebellum could be accounted for by the vasculature of the pia mater that covers its highly infolded surface. Estimates of vascular volume were made using a previously reported point-counting method. Two counts were done: one in which only intraparenchymal vessels were included, and a second one in which both intraparenchymal vessels and pial vessels were included. We found no differences in intraparenchymal vascular volume between cerebellum and cerebrum. When the pial vessels are included, however, the cerebral vascular volume increases by less than 6%, whereas the cerebellar vascular volume increases by greater than 30%. We suggest that the higher cerebellar vascular volume measured using intravascular tracers is due to inclusion of the pial vasculature. Since pial vessels do not express blood-brain barrier characteristics as prominently as intraparenchymal vessels, we further suggest that estimates of barrier permeability in cerebellum should not be made using simple models developed for cerebral tissue.     

Autonomic nerve and cardiovascular responses to changing blood oxygen and carbon dioxide levels in the rat

Contribution of autonomic nervous system activity to the heart rate and blood pressure responses during chemoreceptor excitations by systemic hypoxia and hypercapnia and to hyperoxia and hypocapnia was analyzed in the urethane-anesthetized, artificially ventilated rats. Systemic hypoxia induced a co-activation of two antagonistic nerves: an increase in cardiac sympathetic and in cardiac vagal efferent nerve discharges. Increased heart rate was due to predominance of the cardiac sympathetic over the cardiac vagal activation. In spite of a marked reflex increase in the renal and cardiac sympathetic nerve activities, the local vasodilator effect of hypoxia prevented consistent changes in arterial blood pressure. Bilateral section of the carotid sinus nerves (CSN) mostly abolished autonomic nerve responses and produced a profound decreases in the blood pressure during hypoxia. Hyperoxia elicited a pressor response due to peripheral vasoconstriction with no significant change in the autonomic nerve activities except for a decrease in the cardiac sympathetic nerve discharges. Hypercapnia significantly increased blood pressure and renal nerve sympathetic activity. In contrast to hypoxia, hypercapnia excited cardiac sympathetic and inhibited cardiac vagal activity. This reciprocal effect did not elicit neurogenic cardioacceleration, because it was masked by the local inhibitory action of CO2 on the heart rate. The increase in sympathetic activities and in blood pressure during hypercapnia persisted after bilateral CSN section indicating that the responses were mediated by central rather than by peripheral chemoreceptors. Hypocapnia produced a significant increase in cardiac vagal discharges yet a cardioacceleratory response occurred due to the local effect upon heart rate. The present results indicate that in the rat, autonomic nervous responses differ depending on the type, i.e. hypoxic or hypercapnic, chemoreceptor stimuli. Reflex heart rate and blood pressure responses do not follow the autonomic nerve activities exactly. Circulatory responses are greatly modified by local peripheral effects of hypoxic, hyperoxic, hypocapnic or CO2 stimuli on the cardiovascular system. Species differences characterizing the autonomic nerve responsiveness to chemical stimuli in the rat are described.     

Kinetic studies on uptake of serotonin and noradrenaline into pial arteries of rats

A population of cerebrovascular nerve fibers have recently been found to store serotonin (5-hydroxytryptamine; 5-HT). There is reason to assume that these 5-HT-containing fibers have a sympathetic rather than an intracerebral origin. This was further elucidated in the present study in which the uptake mechanisms of 5-HT and noradrenaline (NA) were characterized and compared in rat pial arteries by measuring the accumulation of [3H]5-HT and [14C]NA under various experimental conditions in vitro. Sympathectomized vessels served as blanks. The uptake into the perivascular sympathetic nerves was dependent on time as well as concentration and was saturable. The Km values were similar, 0.17 microM for 5-HT and 0.15 microM for NA, but the Vmax value was 10 times higher for NA (2.38 and 25 pmol/mg/15 min, respectively). The two amines competed with each other in the sympathetic uptake, as studied by inhibition of the accumulation of one labeled amine by the other nonlabeled amine. Corticosterone, acting on the extraneuronal process, significantly inhibited the 5-HT uptake but had no substantial effect on NA. Reserpine, blocking the intraaxonal vesicular stores, markedly attenuated the accumulation of NA, but not of 5-HT. The selective uptake blocker paroxetine reduced the 5-HT uptake with much higher potency than the NA uptake, whereas desipramine predominantly inhibited NA uptake. The pial 5-HT uptake was not significantly affected by lesion of the raphe complex, whereas it was reduced to half following superior cervical ganglionectomy. The results suggest that the 5-HT present in nerves associated with pial vessels at the base of the brain is taken up through an efficient axonal mechanism, functionally related but not identical to the uptake process for NA.     

Effect of 2-chloroadenosine on cerebrovascular reactivity to hypercapnia in newborn pig.

The effect of local administration of vasodilative concentrations of the adenosine receptor agonist 2-chloroadenosine (2-CADO) on the hyperemic responses of the pial and parenchymal microcirculations to graded hypercapnia was determined. The cranial window and brain microdialysis-hydrogen clearance techniques were utilized in two groups of isoflurane-anesthetized newborn pigs to measure changes in pial diameters and local CBF, respectively, in response to graded hypercapnia in the absence and presence of 2-CADO. Progressive size-dependent dilations of pial arterioles [small = 41 +/- 7 microns (mean +/- SD), intermediate = 78 +/- 13 microns, and large = 176 +/- 57 microns in diameter] occurred in response to graded hypercapnia alone (PaCO2 = 58 and 98 mm Hg) and to superfusions of 2-CADO (10(-5) M) during normocapnia; the magnitude of the dilative response to each of these stimuli was inversely proportional to vessel size. When hypercapnia was induced concomitantly with 2-CADO superfusion, the dilative effects of each stimulus were directly additive. Similarly, local microdialysis infusion of 10(-5) M 2-CADO, which doubled CBF during normocapnia, did not affect the hyperemic response of the parenchymal circulation to graded hypercapnia (PaCO2 = 69 and 101 mm Hg). Our findings are consistent with the participation of adenosine in the mediation of cerebral hypercapnic hyperemia. If, however, adenosine is not involved in this dilative response, our results indicate that concomitant vascular and neuromodulatory actions induced by adenosine receptor stimulation do not affect the mechanism responsible for the hypercapnic hyperemic response.     

Immunocytochemical localization of the erythroid glucose transporter: abundance in tissues with barrier functions

We investigated the cellular localization and tissue distribution of the glucose transporter protein in the nervous system of the monkey and rat, and in other tissues of the rat, by immunocytochemical methods with monoclonal and polyclonal antibodies to the glucose transporter of human erythrocytes. We found intense immunostaining, indicating a high density of the glucose transporter, in all intraparenchymal blood vessels of the brain and spinal cord, in pial vessels, and in endoneurial capillaries of peripheral nerves, nerve roots, and dorsal root ganglia. Larger blood vessels at the base of the brain and in major fissures did not stain. The only intraparenchymal brain microvessels that did not immunostain were in circumventricular organs. There was no specific immunostaining of neurons or glia, except for tanycytes in the floor of the third ventricle, which immunostained intensely. Vessels of the choroid plexus did not stain, but the choroid epithelium, especially its basal membranes, stained. The only non-neural organ where immunostaining was evident in its microvessels was the testis. In addition to the endothelium of neural and testicular tissues, there was immunostaining in certain epithelial tissues, such as the perineurium of peripheral nerves and nerve roots, the epithelium of the ascending loop of Henle in the kidney, and the epidermis of the skin. Based on these findings, we hypothesize that a high density of the erythroid-type glucose transporter is inherent to many endothelial and epithelial cells that are joined by occluding junctions. However, other epithelial tissues with known occluding intercellular junctions that lack the erythroid-type of glucose transporter may have other types of glucose transporter proteins.     

Hypercapnia induces long-term changes in postganglionic renal nerve activity in the piglet

In developing swine, time and frequency domain analyses were used to compare changes in discharge features of efferent phrenic and postganglionic renal nerve activities evoked by prolonged (1 h) exposure to severe hypercapnia (10% CO2, balance O2), before and after combined carotid sinus and aortic depressor nerve (CSN-AOD) sectioning. With intact CSN-AOD innervation, respiration-related activity in renal nerve discharge was rare (3 of 11 animals) during baseline periods with intact innervation, but was observed in most cases (10 of 11 animals) during baseline following denervation. Renal nerve respiration-related activity was recruited by hypercapnic stimulation in animals with intact CSN-AOD innervation, and was augmented in denervated animals with ongoing respiratory activity. Phrenic nerve discharge was markedly augmented during hypercapnia, whether CSN-AOD innervation was intact or not, and it did not exhibit a post-hypercapnic depression. Autopower spectra of renal nerve activity revealed the presence of two coexisting rhythms, 2-6 and 7-13 Hz, which were present whether CSN-AOD innervation was intact or not. The hypercapnic-induced increases of activity in the 2-6 and 7-13 Hz bands were not comparable, with the latter region exhibiting a much more robust response to hypercapnia, especially following CSN-AOD denervation. Thus, prolonged exposure to hypercapnia evoked changes in renal nerve discharge that involved increased coupling to neuronal ensembles shaping central inspiratory activity and those generating central sympathetic outflows, especially to networks generating 7-13 Hz rhythm. Such changes may permit more efficient modulation of innervated structures during exposure to stressors.     

Alpha-adrenoreceptors and muscarine receptors in human pial arteries and microvessels: a receptor binding study

Human pial arteries and intraparenchymal microvessels were isolated for enzyme assays and radioligand binding studies of receptors. Special attention was paid to contamination with brain tissue, which was assessed by luxol staining and cerebroside assays for myelin and by scanning electron microscopy. The amount of contamination was approximately 1% for pial vessels and 14% for microvessel preparations. Significant levels of alpha 1-adrenoreceptors (binding sites for [3H]prazosin) and alpha 2-adrenoreceptors (sites labeled by [3H]azidoclonidine) were found in both types of vessels, suggesting that each receptor can modify contractility in these human vessels. Levels of muscarine receptors (sites labeled with [3H]quinuclidinyl benzilate) and choline acetyltransferase activity were considered significant only in pial vessels.     

Pial artery pressure after one hour of global ischemia

Pial artery pressure was measured in anesthetized control cats and in animals subjected to 1 h of global ischemia and 6 h of recirculation. Cerebral blood flow (CBF) was measured with the intraarterial 133Xe technique before and after ischemia, and lumped segmental resistances upstream and downstream to the pial artery were calculated. In the control brain, upstream resistance was 1.30 +/- 0.28 and downstream resistance 0.94 +/- 0.1 mm Hg ml-1 100 g min. During the postischemic hypoperfusion period, both resistances significantly increased, indicating that hypoperfusion constitutes a dysregulation of both large extracerebral and small intracerebral vessels. Hypercapnia induced an increase of CBF in the control brain and was accompanied by a fall in downstream resistance, demonstrating intracortical vasodilation. By contrast, hypercapnia did not provoke changes in either CBF or segmental resistances in the hypoperfusion period. In conclusion, during the postischemic hypoperfusion period, both extra- and intracortical resistances are increased and vascular reactivity to CO2 is abolished.     

Autonomic cardiovascular responses to hypercapnia in conscious rats: the roles of the chemo- and baroreceptors

The role of the autonomic nervous system, the central and peripheral chemoreceptors, and the arterial baroreceptors was examined in the cardiovascular response to hypercapnia in conscious rats chronically instrumented for the measurement of arterial blood pressure (ABP), heart rate (HR), and renal sympathetic nerve activity (RSNA). Rats were exposed to hypercapnia (6% CO2), and the cardiovascular and autonomic nervous responses in intact and carotid chemo- and/or aortic denervated rats were compared. In intact and carotid chemo-denervated rats, hypercapnia induced significant increases in mean ABP (MABP) and RSNA, and a significant decrease in HR. The HR decrease was reversed by atropine and eliminated by bilateral aortic denervation, which procedure, however, did not affect the MABP or RSNA response. Bilateral carotid chemo-denervation did not affect the baroreflex control of HR, although this control was attenuated by aortic denervation. Hypercapnia did not affect baroreflex sensitivity in intact rats. These results suggest that hypercapnia induces an increase in MABP due to an activation of sympathetic nervous system via central chemoreceptors and a decrease in HR due to a secondary reflex activation of the parasympathetic nervous system via arterial baroreceptors in response to the rise in ABP. In addition, carotid chemoreceptors do not play a major role in the overall cardiovascular response to hypercapnia in conscious rats. The mechanism responsible for the parasympatho-excitation may also involve CO2 induced aortic chemoreceptor simulation.     

Pharmacological FMRI: measuring opioid effects on the BOLD response to hypercapnia.

Opioid binding to the cerebral blood vessels may affect vascular responsiveness and hence confound interpretation of blood oxygen level-dependent (BOLD) responses, which are usually interpreted as neuronal in origin. Opioid binding varies in different brain regions. It is unclear whether opioids alter neurovascular coupling, or whether their effects are purely neuronal. This study used BOLD functional magnetic resonance imaging (FMRI) to investigate the effect of a mu-opioid agonist remifentanil, on cerebrovascular CO(2) reactivity (being one component of neurovascular coupling). Hypercapnic challenges were delivered to human volunteers, while controlling potential opioid-induced respiratory depression. The BOLD signal increase to hypercapnia was compared before and during remifentanil administration. Remifentanil was shown not to have a generalised effect on CO(2) responsiveness in the cerebral vasculature. However, it caused a significant reduction in the positive BOLD response to hypercapnia in the bilateral primary sensorimotor cortices, bilateral extrastriate visual areas, left insula, left caudate nucleus, and left inferior temporal gyrus. We conclude that remifentanil does not modulate cerebrovascular CO(2) reactivity, as we saw no difference in BOLD response to hypercapnia in areas with high opioid receptor densities. We did however see a focal reduction in areas related to motor control and putative task activation, which we conclude to be related to changes in neuronal activity related to the sedative effects of remifentanil. Our method of controlling CO(2) levels effectively mitigated the potential confound of respiratory depression and allowed comparison over a similar range of CO(2) levels. We suggest that similar methodology should be used when investigating other potentially vasoactive compounds with FMRI.     

Pial arterial reactions to hyper- and hypocapnia: a dynamic experimental study in cats.

In 29 cats, the extent and time-course of the pial arterial reactions to hypo- and hypercapnia were studied by means of the skull-window technique. The typical, well-known dilatations and constrictions during hyper- and hypocapnia were seen. The latent period for dilatation after the beginning of CO2-inhalation was ca. 20 sec. There was no stable relation observable between vessel diameter and arterial carbon dioxide tension (paCO2). Diameter changes lagged behind CO2-changes, indicating that CO2 acts via metabolic regulation, probably extracellular pH.     

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.