Richard Sercombe

Cortical blood flow increases induced by stimulation of the substantia innominata in the unanesthetized rat.

The possible implication of projections from the substantia innominata (SI) to the cerebral cortex in the control of local cortical blood flow (CoBF) was studied in adult Fischer rats. Local blood flow (by helium clearance) and tissue gas partial pressures (pO2, pCO2) as metabolic indices, were measured in the frontal and parietal cortices in unanesthetized animals via chronically implanted probes connected to a mass spectrometer. Stimulating electrodes, also implanted chronically, were placed in the region of the SI. Out of 37 correctly located sites, 28 gave rise to cerebrovascular responses without significant hypertension or agitation. Both frontal (+114%) and parietal CoBF (+28%) increased significantly during ipsilateral 50 microA stimulation, but did not further significantly increase at 100 microA. Contralateral stimulation induced only small, non-significant effects. SI stimulation simultaneously increased cortical pO2 and decreased cortical pCO2, significantly more so in the frontal compared to the parietal cortex, and ipsilaterally compared to contralaterally. Both the CoBF and the tissue gas changes induced by SI stimulation were strongly potentiated by infusion of 0.15 mg/kg/h of the cholinomimetic agent physostigmine. The electrocorticogram (ECoG) was not systematically activated during the SI stimulation. The evidence presented favors a role for the cholinergic projections of the SI in control of CoBF (particularly frontal cortex), especially since the flow changes observed showed no obvious dependence on changes in local pCO2 or on paCO2, and could not be attributed to hypertension or behavioral changes.

Both ischemic preconditioning and ghrelin administration protect hippocampus from ischemia/reperfusion and upregulate uncoupling protein-2.

BackgroundA major endogenous protective mechanism in many organs against ischemia/reperfusion (I/R) injury is ischemic preconditioning (IPC). By moderately uncoupling the mitochondrial respiratory chain and decreasing production of reactive oxygen species (ROS), IPC reduces apoptosis induced by I/R by reducing cytochrome c release from the mitochondria. One element believed to contribute to reduce ROS production is the uncoupling protein UCP2 (and UCP3 in the heart). Although its implication in IPC in the brain has been shown in vitro, no in vivo study of protein has shown its upregulation. Our first goal was to determine in rat hippocampus whether UCP2 protein upregulation was associated with IPC-induced protection and increased ROS production. The second goal was to determine whether the peptide ghrelin, which possesses anti-oxidant and protective properties, alters UCP2 mRNA levels in the same way as IPC during protection.ResultsAfter global forebrain ischemia (15 min) with 72 h reperfusion (I/R group), we found important neuronal lesion in the rat hippocampal CA1 region, which was reduced by a preceding 3-min preconditioning ischemia (IPC+I/R group), whereas the preconditioning stimulus alone (IPC group) had no effect. Compared to control, UCP2 protein labelling increased moderately in the I/R (+39%, NS) and IPC+I/R (+28%, NS) groups, and substantially in the IPC group (+339%, P < 0.05). Treatment with superoxide dismutase (10000 U/kg ip) at the time of a preconditioning ischemia greatly attenuated (-73%, P < 0.001) the increase in UCP2 staining at 72 h, implying a role of oxygen radicals in UCP2 induction.Hippocampal UCP2 mRNA showed a moderate increase in I/R (+33%, P < 0.05) and IPC+I/R (+40%, P < 0.05) groups versus control, and a large increase in the IPC group (+333%, P < 0.001). In ghrelin experiments, the I/R+ghrelin group (3 daily administrations) showed considerable protection of CA1 neurons versus I/R animals, and increased hippocampal UCP2 mRNA (+151%, P < 0.001).ConclusionWe confirm that IPC causes increased expression of UCP2 protein in vivo, at a moment appropriate for protection against I/R in the hippocampus. The two dissimilar protective strategies, IPC and ghrelin administration, were both associated with upregulated UCP2, suggesting that UCP2 may often represent a final common pathway in protection from I/R.

Is there an active mechanism limiting the influence of the sympathetic system on the cerebral vascular bed? Evidence for vasomotor escape from sympathetic stimulation in the rabbit.

The influence of the cervical sympathetic chain on cerebral circulation in the rabbit was studied by means of 3 complementary techniques. Two dynamic techniques involving chronically implanted probes were used: blood flow in the caudate nucleus (CN) was measured by thermal clearance; tissue PO2 and PCO2 in the same structure were measured by mass spectrometry. Other variables measured continuously and simultaneously included arterial blood pressure (BP), PaO2 and PaCO2. The third technique was a tissue sampling method based on the Fick principle and using 14C1 ethanol as tracer. Blood flow in 7 regions was measured at stable BP, PaO2 and PaCO2. Stimulation of the sympathetic chain at 15 Hz induced mean maximal decreases in CN blood flow of 23.9% (thermal clearance) and 24.4% (ethanol technique). Mean decrease of PO2 in the CN at 15 Hz was 16.6%. Significant falls in blood flow were observed with the ethanol technique in all 7 structures measured. During prolonged stimulation (greater than 1 min) CN blood flow and PO2 were found to escape towards the baseline level, which was sometimes even exceeded during the stimulation (blood flow). Stimulation frequency had only a very moderate influence on the rate of escape, and no evidence of a metabolic mechanism was found, although injection of barbiturate decreased the escape. These results are discussed with respect to the conflicting evidence on the effects of sympathetic stimulation in the brain, and to possible mechanisms for the escape phenomenon.

Randomized, double-blind, placebo-controlled, pilot trial of high-dose methylprednisolone in aneurysmal subarachnoid hemorrhage

OBJECT The object of this study was to determine the efficacy of methylprednisolone in reducing symptomatic vasospasm and poor outcomes after subarachnoid hemorrhage (SAH). METHODS Ninety-five patients with proven SAH were recruited into a double-blind, placebo-controlled, randomized trial. Starting within 6 hours after angiographic diagnosis of aneurysm rupture, placebo or methylprednisolone, 16 mg/kg, was administered intravenously every day for 3 days to 46 and 49 patients, respectively. Deterioration, defined as development of a focal sign or decrease of more than 1 point on the Glasgow Coma Scale for more than 6 hours, was investigated by using clinical criteria and transcranial Doppler ultrasonography, cerebral angiography, or CT when appropriate. The end points were incidence of symptomatic vasospasm (delayed ischemic neurological deficits associated with angiographic arterial narrowing or accelerated flow on Doppler ultrasonography, or both) and outcome 1 year after entry into the study according to a simplified Rankin scale (Functional Outcome Scale [FOS]) in living patients and the Glasgow Outcome Scale in all patients included. RESULTS All episodes of deterioration and all living patients with a 1-year outcome were assessed by a review committee. In patients treated with methylprednisolone, the incidence of symptomatic vasospasm was 26.5% compared with 26.0% in those given placebo. Poor outcomes according to FOS were significantly reduced in the Methylprednisolone Group at 1 year of follow-up; the risk difference was 19.3% (95% CI 0.5-37.9%). The outcome was poor in 15% (6/40) of patients in the Methylprednisolone Group versus 34% (13/38) in the Placebo Group. CONCLUSIONS A safe and simple treatment with methylprednisolone did not reduce the incidence of symptomatic vasospasm but improved ability and functional outcome at 1 year after SAH.

Monoamine Oxidase Activity in Brain Microvessels Determined Using Natural and Artificial Substrates: Relevance to the Blood—Brain Barrier

The possible contribution of cerebrovascular monoamine oxidase (MAO) to the blood–brain barrier to catecholamines was studied in isolated porcine and rat microvessels by determining its activity with various substrates. Michaelis-Menten kinetic constants, Km and Vmax, were determined using noradrenaline (NA) as substrate in a Tris medium. Km values were 0.25 ± 0.05 mM in control and 0.16 ± 0.09 mM in ultrasonically disintegrated (USD) preparations (difference not significant); Vmax in USD preparations (1.83 ± 0.20 n.atoms O2 min−1 mg protein−1) was slightly higher (p < 0.05) than in control preparations (1.35 ± 0.11 n.atoms O2 min−1 mg protein−1), suggesting a certain restriction by the plasma membrane of substrate access to the enzyme. This phenomenon was confirmed in a more physiological, ionic medium; the activity was then approximately doubled for 1 mM NA, whereas that for 1 mM β-phenylethylamine (β-PEA), a lipid-soluble substrate, tended to decrease with USD treatment. These results show that this highly active form of MAO is unlikely to be saturated by physiological concentrations of catecholamine. It can be estimated that, for a plasma concentration of NA of 1 μM, a facilitated diffusion accelerating the entry of the catecholamine into the cells by at least 15-fold would be necessary in order to exceed the catabolic capacity of MAO. It is concluded that circulating catecholamines are not likely to cross the endothelial barrier of cerebral microvessels intact, and that the small quantities of radioactivity detected in the parenchyma in measurements of the brain uptake index essentially represent metabolites due to MAO activity.

Histamine-Induced Constriction and Dilatation of Rabbit Middle Cerebral Arteries in vitro: Role of the Endothelium

We studied the effects of histamine on perfused rabbit middle cerebral arteries in vitro. Intact and endothelium-denuded preparations were compared. Histamine caused concentration-dependent constrictions in intact vessels which were competitively inhibited by an H1 receptor antagonist. This constriction was potentiated by either H2-receptor blockade or endothelium denudation. The greatest potentiation was observed with intraluminal as opposed to extraluminal administration. The H1 receptor agonist pyridylethylamine induced similar concentration-dependent constriction in intact and denuded preparations. After preconstriction, histamine, in the presence of an H1 receptor antagonist, dilated intact vessels to a maximum of 45.1%, and endothelium-denuded vessels to a maximum of 22% (p less than 0.02). We conclude that rabbit middle cerebral arteries possess H1 constrictory and H2 dilatory receptors, and that many of the H2 dilatory receptors are situated on the endothelial cells.

Pial Artery Responses to Norepinephrine Potentiated by Endothelium Removal

The effect of endothelium removal on pial artery constriction in response to norepinephrine (NE) was studied in vitro using a perfused vessel setup in which pressure increases indicate vasoconstriction. In deendothelialized rabbit arteries, the reaction to extraluminal NE was found to be characterized by a much higher Emax (2.0 times) and a slight (but significant) leftward shift of the concentration-response curve (lower EC50) compared with control vessels. In cat arteries subjected to either extra- or intraluminal NE, the Emax was also substantially higher in deendothelialized preparations (4.4 and 5.1 times, respectively), but there was no significant difference in the EC50 values. Anatomical verification and functional tests (acetylcholine-induced dilatation) confirmed the presence and the absence of the endothelium in control and lesioned arteries, respectively. This modulatory influence of the endothelium may be of importance in cerebrovascular pathology.

Effect of the Muscarinic Agonist Carbachol on Pial Arteries in vivo after Endothelial Damage by Air Embolism

Reactions of pial arteries to the muscarinic agonist carbachol were tested in vivo in chloralose anesthetized cats before and after endothelial damage. Moderate endothelial damage was induced by arterial air embolism and verified by electron microscopy for the vessels tested. The experiments had three phases: First, the normal reactivity of pial arteries to carbachol (10−7 to 10−5 M) was tested using the microapplication technique, then, after air embolism, the reactivity was reinvestigated at the same vessel. Finally, pial arteries were taken out for scanning electron microscopy. The results show carbachol (10−6 and 10−5 M) induced significant dilatations under control conditions, also after repetition at the same vessel. After air embolism, the reactions to carbachol were abolished. Morphologic data revealed that whereas control pial arteries showed intact endothelium, the embolized vessels revealed various degrees of endothelial alterations. All showed flattening of endothelial nuclei, to a greater or lesser degree, and in many cases, the endothelium had a wrinkled appearance; several arteries showed severe degradation of the intercellular junctions. It is concluded that (a) carbachol-induced muscarinic vasodilatation of pial arteries in vivo can be abolished after a morphologically verified endothelial lesion—thus confirming in vitro studies in larger arteries and (b) disturbed vascular function does not require rubbing of the endothelium, but occurs already with moderate endothelial damage.

Evidence for regional differences in the effect of beta-adrenergic stimulation on cerebral blood flow

Chronic preparations of non-anaesthetized rabbits were used to test the effects of alpha- and beta-adrenergic stimulation on cerebral blood flow (CBF). The following variables were recorded simultaneously and continuously during several experimental sessions over a period of 1 month. (1) CBF by a thermal technique in (a) the caudate nucleus (CN), and (b) the lateral geniculate body (LGB); their irrigation depends on two different vascular beds. (2) Brain temperature. (3) Pa O2 and Pa CO2 by mass spectrography. (4) Aortic blood pressure (PB). (5) ECoG (electrocorticogram). The effects of drug injections were compared with the action of 5% CO2 inhalation which provoked a faster increase in flow in the LGB than in the CN. Injections of isoproterenol ranging from 0.5 to 3 μg/kg were made. None of these doses provoked an increase in blood flow in the LGB. In contrast, and without exception, there was a marked increase in the CN which was dose-dependent. This drug (2 μg/kg) produced an increase in flow approximately equivalent to 2.5 times the effect of 5% CO2 inhalation; at the same time we observed a decrease in blood pressure (10–15%) and in Pa CO2 (20–25%) and an increase in Pa O2 (25–30%). Likewise, injections of the alpha and beta stimulant, adrenaline (2 μg/kg), acted only on the CN, causing a decrease in flow. The isoproterenol-induced vasodilatation was verified by injection of the beta-blocking agent propranolol in doses ranging from 0.5 to 1 mg/kg, which themselves did not significantly affect the variables measured. The effects of isoproterenol were reversibly abolished. Similarly, the beta stimulation due to injected adrenaline was diminished giving rise to a greater decrease in flow in the CN. It is concluded that the two structures differ in that the CN vessels seem to be highly innervated and the LGB vessels poorly innervated by the sympathetic nervous system.

Parasympathomimetic influence of carbachol on local cerebral blood flow in the rabbit by a direct vasodilator action and an inhibition of the sympathetic-mediated vasoconstriction.

1 Two sorts of effect of carbachol on local cerebral blood flow (caudate nucleus) have been studied: (a) a direct action on the arterial smooth muscle; (b) an interaction with the adrenergic (sympathetic) constrictor system which innervates the vascular system of this nucleus. 2 Continuous measurements of the following variables were performed in lightly anaesthetized rabbits: local blood flow (caudate nucleus), arterial blood pressure, PaO2, PaCO2. 3 After blockade of the nicotinic synapses in the superior cervical sympathetic ganglion by local hexamethonium injection, carbachol was infused into the common carotid artery, thus minimizing systemic effects of this drug. Infusions of 0.6, 1.3 and 2.5 μg kg−1 min−1 induced mean increases in caudate blood flow of about 8, 17 and 37% respectively, without notable modifications of other variables measured. 4 The dilator effect of 2.5 μg kg−1 min−1 carbachol was reduced to a mean of 12% after intravenous injection of 0.5 mg/kg atropine, and could be totally abolished by higher doses (1 to 1.5 mg/kg). 5 Administration of 2.5 μg kg−1 min−1 of carbachol diminished by more than 50% the reduction in caudate blood flow induced by postganglionic stimulation of the cervical sympathetic chain, but did not affect the reduction of flow obtained by intravenous infusion of noradrenaline (2.5 to 5.0 μg kg−1 min−1). This inhibition of the adrenergic (sympathetic) system by carbachol was not modified by high doses of atropine (1 mg/kg i.v.). 6 We conclude that: (a) the local cerebral blood flow of a deep structure can be significantly modified by activation of vascular muscarinic receptors; (b) activation of non‐muscarinic prejunctional cholinoceptors can cause inhibition of the sympathetic fibres innervating the vascular bed of the same structure.