Antal G. Hudetz

Production of 20-HETE and Its Role in Autoregulation of Cerebral Blood Flow

In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

Blood Flow in the Cerebral Capillary Network: A Review Emphasizing Observations with Intravital Microscopy

Capillary perfusion in the brain is characterized by an essentially continuous flow of erythrocytes and plasma in almost all capillaries. Rapid fluctuations and spatial heterogeneity or red blood cell (RBC) velocity (0.5–1.8 mm/s) within the capillary network are present. In addition, low‐frequency (4–8 cpm) synchronous oscillations in RBC velocity in the capillary network emerge when perfusion to cerebral tissue is challenged. Despite the tortuous, three‐dimensional architecture of microvessels, functional intercapillary anastomoses are absent. At rest, red cells travel through the capillary network in 100–300 ms along 150‐ to 500‐μm‐long paths. Physiological challenges elicit sizable changes in RBC velocity with a minor role for capillary recruitment, change in capillary diameter, or flow shunting. During acute hypoxia, RBC velocity increases in all capillaries; the corresponding response to hypercapnia is more complex and involves redistribution of capillary flow toward more homogeneous perfusion. The response of capillary flow to decreased perfusion pressure reflects autoregulation of cerebral blood flow but also involves intranetwork redistribution of RBC flow between two populations of capillaries, postulated as thoroughfare channels and exchange capillaries. Flow reserve may be provided by the thoroughfare channels and may help maintain flow velocity and capillary exchange and protect the microcirculation from perfusion failure. Isovolemic hemodilution increases RBC velocity three‐ to fourfold and increases RBC flux to a moderate degree with a relatively small decrease in capillary hematocrit, under normal and compromised arterial blood supply. In cerebral ischemia, leukocyte adhesion is enhanced and appears reversible when the ischemia is moderate but may be progressive when the injury is severe. The observed flow behavior suggests the presence of a physiological regulatory mechanism of cerebral capillary flow that may involve communication among various microvascular and parenchymal cells and utilize locally acting endothelial and parenchymal mediators such as endothelium‐derived relaxing factor or nitric oxide.

Increased Expression of Ca2+-Sensitive K+ Channels in the Cerebral Microcirculation of Genetically Hypertensive Rats Evidence for Their Protection Against Cerebral Vasospasm

The Ca2+-sensitive K+ channel (K(Ca) channel) plays a key role in buffering pressure-induced constriction of small cerebral arteries. An amplified current through this channel has been reported in vascular smooth muscle cells obtained from hypertensive animals, implying that the expression or properties of K(Ca) channels may be regulated by in vivo blood pressure levels. In this study, we investigated this hypothesis and its functional relevance by comparing the properties, expression levels, and physiological role of K(Ca) channels in cerebral resistance arteries from normotensive and genetically hypertensive rats. Whole-cell patch-clamp experiments revealed a 4.7-fold higher density of iberiotoxin-sensitive K(Ca) channel current at physiological membrane potentials in spontaneously hypertensive rat (SHR) compared with Wistar-Kyoto (WKY) rat cerebrovascular smooth muscle cells (n = 18 and 21, respectively). However, additional single-channel analysis in detached patches showed similar levels of unitary conductance, voltage, and Ca2+ sensitivity in K(Ca) channels from WKY and from SHR membranes. In contrast, Western analysis using an antibody directed against the K(Ca) channel alpha-subunit revealed a 4.1-fold increase in the corresponding 125-kD immunoreactive signal in cerebrovascular membranes from SHR compared with WKY rats. The functional impact of this enhanced K(Ca) channel expression was assessed in SHR and WKY rat pial arterioles, which were monitored by intravital microscopy through in situ cranial windows. Progressive pharmacological block of K(Ca) channels by iberiotoxin (0.1 to 100 nmol/L) dose-dependently constricted pial arterioles from SHR and WKY rats (n = 6 to 8). The arterioles in SHR constricted 2- to 4-fold more intensely, and vasospasm occurred in some vessels. These data provide the first direct evidence that elevated levels of in situ blood pressure induce K(Ca) channel expression in cerebrovascular smooth muscle membranes. This homeostatic mechanism may critically regulate the resting tone of cerebral arterioles during chronic hypertension. Furthermore, the overexpression of distinct K+ channel types during specific cardiovascular pathologies may provide for the upregulation of novel disease-specific membrane targets for vasodilator therapies.

Spontaneous flow oscillations in the cerebral cortex during acute changes in mean arterial pressure.

The purpose of this study was to characterize spontaneous oscillations of blood flow in the cerebral cortex of anesthetized rats under control conditions and after mean arterial pressure was altered by various means. Blood flow was monitored using a laser–Doppler flowmeter through the closed cranium. Spontaneous flow oscillations with amplitudes of 14–30% of the mean flow and frequencies of 4–11 cycles/min were recorded when arterial pressures were less than 90 mm Hg. Stepwise hemorrhagic hypotension and unilateral carotid occlusion increased the amplitude of oscillations. The amplitude of oscillations was negatively correlated with the level of mean arterial pressure after manipulation with norepinephrine or sodium nitroprusside. The oscillations were reversibly abolished during dilation of the cerebral circulation by elevating the inspired carbon dioxide content to 5%. The frequency of flow oscillations was very stable during all of the above maneuvers except during the infusion of norepinephrine, which increased the oscillation frequency slightly. The results suggest that flow oscillations are determined primarily by cerebral arterial pressure.

Transient Focal Ischemia in Subhuman Primates: Neuronal Injury as a Function of Local Cerebral Blood Flow

Unilateral, transient (30, 60, and 120 minutes (min)) middle-cerebralartery (MCA) occlusion was induced via transorbital craniotomy in 11 waking subhuman primates. Local cerebral blood flow (LCBF) was calculated from hydrogen clearance curves obtained through the use of intracerebral platinum microelectrodes. Unilateral MCA occlusion decreased LCBF in the territory of the ipsilateral MCA. Within minutes of the arterial occlusion all monkeys developed contralateral neurologic deficits that began disappearing three hours (h) after reopening the MCA. Regional ischemia, followed by 24 h of reperfusion, produced varying degrees of tissue vacuolation which correlated (r=0.60, p<0.01, n=49) with the percent reduction in LCBF multiplied by the occlusion time. Neurons were classified according to the structural features of their perikaryon. A plot of neuron types versus percent vacuolation suggested that normal neurons become increasingly scalloped under increasingly severe ischemic conditions. The number of scalloped neurons decreased precipitously in areas of marked sponginess coincident with the appearance of irreversibly damaged neurons. Local tissue edema values exceeding 30% correlated with irreversible injury to all neurons in the same area. Regional cerebral ischemia of increasing severity was acompanied by increasing numbers of lethally injured neurons.

Contribution of 20-HETE to Vasodilator Actions of Nitric Oxide in the Cerebral Microcirculation

BACKGROUND AND PURPOSE The present study examined the contributions of a rise in cGMP versus a fall in 20-HETE levels to the vasodilator response to nitric oxide (NO) in the cerebral circulation of the rat. METHODS Intact rat middle cerebral and basilar arteries were bathed in physiological saline solution containing indomethacin (5 micromol/L) and baicalein (0.5 micromol/L) and pressurized at 90 mm Hg. Relaxations to sodium nitroprusside (SNP) were studied before and after addition of [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ, a guanylyl cyclase blocker), 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT5823, a protein kinase G blocker), and 20-hydroxyeicosatetraenoic acid (20-HETE). Cerebral blood flow was measured by using a laser Doppler flow probe over a thin cranial window in anesthetized rats, and the effects of intracerebroventricular infusion of 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl (MAHMA nonoate) and dibromododecenyl methylsulfimide (DDMS) were determined. RESULTS SNP-induced dilation of serotonin-preconstricted (0.2 micromol/L) middle cerebral arteries (10(-7) to 10(-3) mol/L) was attenuated in arteries treated with ODQ (10 micromol/L) or KT5823 (1 micromol/L) by 52% and 27%, respectively. Preventing the NO-induced fall in intracellular 20-HETE, by adding 20-HETE (100 nmol/L) to the bath, reduced the dilation to SNP by 62%. Simultaneous administration of ODQ and 20-HETE markedly attenuated the SNP-induced dilation by 90%. In basilar arteries, ODQ (10 micromol/L) alone completely blocked the response to SNP. Infusion of MAHMA nonoate (10 nmol/min ICV) in anesthetized rats increased cerebral blood flow by 52% before and 8% after blockade of the endogenous production of 20-HETE with DDMS (50 pmol/min). CONCLUSIONS These results suggest that NO dilates cerebral arteries through both cGMP-dependent and cGMP-independent pathways and that inhibition of 20-HETE formation contributes to the cerebral vasodilator response to NO both in vitro and in vivo.

Role of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in Endotoxin-Induced Cerebral Hyperemia

BACKGROUND AND PURPOSE Bacterial lipopolysaccharide (LPS), an endotoxin, has been reported to induce the expression of inducible isoforms of both nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in various cell types. LPS is also known to dilate systemic vasculature, including cerebral vessels. This study aimed to determine to what extent LPS induces iNOS and COX-2 expression in the brain and whether NO and/or cyclooxygenase metabolites derived from iNOS and/or COX-2 contribute to the LPS-induced cerebral hyperemia. METHODS Regional cerebral blood flow (rCBF) was measured by laser-Doppler flowmetry in halothane-anesthetized, artificially ventilated rats for 4 hours after intracerebroventricular administration of LPS. RESULTS LPS at doses of 0.01 mg/kg to 1 mg/kg caused dose-dependent, progressive increases in rCBF at 1 to 4 hours after administration. The increase in rCBF was attenuated by systemic administration of the selective iNOS inhibitor aminoguanidine (100 mg/kg IP) or the selective COX-2 inhibitor NS-398 (5 mg/kg IP), and it was abolished by preventing induction of these isoforms with dexamethasone (4 mg/kg IP). LPS significantly increased iNOS and COX-2 mRNA, iNOS protein, and iNOS and cyclooxygenase enzyme activity. The increases in iNOS and cyclooxygenase enzyme activity were eliminated by aminoguanidine and NS-398, respectively. Dexamethasone also prevented the increase in iNOS and cyclooxygenase activity. CONCLUSIONS These results indicate that induction of iNOS and COX-2 expression and the increased production of NO and vasodilator prostanoids in the brain contribute to the elevation in CBF after intracerebroventricular administration of LPS.

Incremental elastic modulus for orthotropic incompressible arteries

Abstract Mechanics of incremental deformations has been applied to introduce an elastic modulus (H) for characterizing incremental elastic properties of thick walled, straight, cylindrically orthotropic, incompressible blood vessels pressurized at fixed length. The modulus H characterizes the radial and tangential stiffness complexly. Volume distensibility, wave velocity and characteristic impedance are directly related to H. For isotropic incompressible vessels, modulus H is equal to 4 3 of the isotropic incremental Youngs modulus. Formulae of both moduli contain an additional term representing the influence of the initial stress. This implies that the classical formula of the isotropic Youngs modulus introduced by Bergel for arteries is not applicable to incremental deformations. The incremental modulus H is proposed as a suitable parameter describing arterial elasticity under in vivo circumstances.

Architectural alterations in rat cerebral microvessels after hypobaric hypoxia

We performed 3-dimensional studies of vascular casts of the microvasculature of the cerebral cortex of rats that were exposed to three weeks of hypobaric hypoxia and of control rats. Scanning electron microscopy of the casts gave the qualitative impression of increased vascularity of the cerebral cortex, particularly the deeper layers, in hypoxic rats. Quantitative analysis of capillary segment lengths revealed a significant shift in the frequency distribution to longer lengths (from 77 +/- 8 to 90 +/- 14 microns) in the deep, but not in the superficial, layers of the cerebral cortex of hypoxic rats. These findings agree with previous results reporting increased capillary density in the brain after exposure to prolonged hypobaric hypoxia and suggest that capillary segment elongation plays a role in the increased capillary density in the deeper layers of the cerebral cortex.

Nitric oxide from neuronal NOS plays critical role in cerebral capillary flow response to hypoxia

We investigated, using a direct, intravital microscopic technique, whether nitric oxide (NO) from neuronal nitric oxide synthase (nNOS) plays a role in the cerebral capillary flow response to acute hypoxia. Erythrocyte flow in subsurface capillaries of the frontoparietal cortex of adult Sprague-Dawley rats was visualized using epifluorescence videomicroscopy after fluorescent labeling of red blood cells (RBC) in tracer concentrations. The velocity of labeled RBC in individual capillaries was measured off-line using an image analysis system. Hypoxia was produced by lowering the inspired O2 concentration to 15% for 5 min in control animals and in those pretreated with the selective nNOS inhibitor 7-nitroindazole (7-NI; 20 mg/kg ip). In the control group, hypoxia increased RBC velocity by 34 +/- 8%. In the group treated with 7-NI, this response was reversed to a statistically significant 8 +/- 3% decrease. This paradoxical response to hypoxia after 7-NI was observed in nearly all capillaries. 7-NI itself decreased the baseline RBC velocity by 12 +/- 4%. The cerebral hyperemic response to hypoxia was also assessed with the laser Doppler flow (LDF) technique. In control animals, hypoxia produced a 33 +/- 6% increase in LDF, similar to the increase in RBC velocity. After 7-NI treatment, the response to hypoxia was moderately attenuated but still significant at a 19 +/- 2% increase in LDF. These results support the role of NO from nNOS in the cerebral hyperemic response to hypoxia. They imply that 7-NI interfered with a physiological mechanism that was fundamental to cerebral capillary flow regulation and provide direct evidence that cerebral capillary perfusion may be dissociated from a concurrent change in regional tissue perfusion as reflected by LDF. In conclusion, NO from nNOS contributes to the maintenance of RBC flow in cerebral capillaries and plays a critically important role in the selective regulation of cerebral capillary flow during hypoxia.We investigated, using a direct, intravital microscopic technique, whether nitric oxide (NO) from neuronal nitric oxide synthase (nNOS) plays a role in the cerebral capillary flow response to acute hypoxia. Erythrocyte flow in subsurface capillaries of the frontoparietal cortex of adult Sprague-Dawley rats was visualized using epifluorescence videomicroscopy after fluorescent labeling of red blood cells (RBC) in tracer concentrations. The velocity of labeled RBC in individual capillaries was measured off-line using an image analysis system. Hypoxia was produced by lowering the inspired O2 concentration to 15% for 5 min in control animals and in those pretreated with the selective nNOS inhibitor 7-nitroindazole (7-NI; 20 mg/kg ip). In the control group, hypoxia increased RBC velocity by 34 ± 8%. In the group treated with 7-NI, this response was reversed to a statistically significant 8 ± 3% decrease. This paradoxical response to hypoxia after 7-NI was observed in nearly all capillaries. 7-NI itself decreased the baseline RBC velocity by 12 ± 4%. The cerebral hyperemic response to hypoxia was also assessed with the laser Doppler flow (LDF) technique. In control animals, hypoxia produced a 33 ± 6% increase in LDF, similar to the increase in RBC velocity. After 7-NI treatment, the response to hypoxia was moderately attenuated but still significant at a 19 ± 2% increase in LDF. These results support the role of NO from nNOS in the cerebral hyperemic response to hypoxia. They imply that 7-NI interfered with a physiological mechanism that was fundamental to cerebral capillary flow regulation and provide direct evidence that cerebral capillary perfusion may be dissociated from a concurrent change in regional tissue perfusion as reflected by LDF. In conclusion, NO from nNOS contributes to the maintenance of RBC flow in cerebral capillaries and plays a critically important role in the selective regulation of cerebral capillary flow during hypoxia.