Jacques Seylaz

Local Uncoupling of the Cerebrovascular and Metabolic Responses to Somatosensory Stimulation After Neuronal Nitric Oxide Synthase Inhibition

It has recently been shown, using either genetically engineered mutant mice (nitric oxide synthase [NOS] knockout) or specific pharmacological tools, that type I NOS (neuronal isoform of NOS, [nNOS]) participates in coupling cerebral blood flow to functional activation. However, it has not been clearly established whether the associated metabolic response was preserved under nNOS inhibition and whether this action was exerted homogeneously within the brain. To address these issues, we analyzed the combined circulatory and metabolic consequences of inhibiting the nNOS both at rest and during functional activation in the rat anesthetized with α-chloralose. Cerebral blood flow and cerebral glucose use (CGU) were measured autoradiographically using [14C]iodoantipyrine and [14C]2-deoxyglucose during trigeminal activation induced by unilateral whiskers stimulation in vehicle- and 7-nitroindazole-treated rats. Our data show that inhibition of nNOS globally decreased CBF without altering CGU, indicating that NO-releasing neurons play a significant role in maintaining a resting cerebrovascular tone in the whole brain. During whisker stimulation, nNOS inhibition totally abolished the cerebrovascular response only in the second order relay stations (thalamus and somatosensory cortex) of the trigeminal relay without altering the metabolic response. These findings provide evidence that the involvement of neurally-derived NO in coupling flow to somatosensory activation is region-dependent, and that under nNOS inhibition, CBF and CGU may vary independently during neuronal activation.

Neurogenic influence on local cerebral blood flow Effect of catecholamines or sympathetic stimulation as correlated with the sympathetic innervation

Local cerebral blood flow was measured continuously in conscious rabbits (thermoclearance technique), and PaO2 and PaCO2 were recorded by mass spectrometry. Though inhalation of CO2 increased flow in caudate nucleus and lateral geniculate body, catecholamines only had effect on caudate nucleus where isoproterenolenhanced and epinephrine and norepinephrine reduced flow. Reduction by electrical stimulation of the neck sympathetic trunk was particularly evident in the caudate. Blood flow increased markedly in both regions after preganglionic conduction blockade. The effects were correlated with a significantly lower degree of sympathetic arteriolar innervation (fluorescence histochemistry) in the lateral geniculate body compared with the caudate nucleus.

Long-term in vivo investigation of mouse cerebral microcirculation by fluorescence confocal microscopy in the area of focal ischemia

This study was designed to assess that mouse pial and cortical microcirculation can be monitored in the long term directly in the area of focal ischemia, using in vivo fluorescence microscopy. A closed cranial window was placed over the left parieto-occipital cortex of C57BL/6J mice. Local microcirculation was recorded in real time through the window using laser-scanning confocal fluorescence microscopy after intravenous injection of fluorescent erythrocytes and dextran. The basal velocity of erythrocytes through intraparenchymal capillaries was 0.53 ± 0.30 mm/sec (n = 121 capillaries in 10 mice). Two branches of the middle cerebral artery were topically cauterized through the window. Blood flow evaluated by laser-Doppler flowmetry in two distinct areas indicated the occurrence of an ischemic core (15.2% ± 5.9% of baseline for at least 2 h) and a penumbral zone. Magnetic resonance imaging and histology were used to characterize the ischemic area at 24 h after occlusion. The infarct volume was 7.3 ± 3.2 mm3 (n = 6). Microcirculation was repeatedly videorecorded using fluorescence confocal microscopy over the next month. After the decrease following arterial occlusion, capillary erythrocyte velocity was significantly higher than baseline 1 week later, and attained 0.74 ± 0.51 mm/sec (n = 76 capillaries in six mice, P<0.005) after 1 month, while venous and capillary network remodeling was assessed, with a marked decrease in tortuosity. Immunohistochemistry revealed a zone of necrotic tissue into the infarct epicenter, with activated astrocytes at its border. Such long-term investigations in ischemic cortex brings new insight into the microcirculatory changes induced by focal ischemia and show the feasibility of long-term fluorescence studies in the mouse cortex.

Is α-chloralose plus halothane induction a suitable anesthetic regimen for cerebrovascular research?

The aim of this study was to determine whether alpha-chloralose, when associated with an initial period of halothane, is a suitable anesthetic regimen for cerebrovascular studies. For this purpose, rats anesthetized with alpha-chloralose plus halothane induction were first subjected to noxious stimuli, and the behavior, EEG and systemic variables were recorded. During a second step, cortical blood flow was measured with laser-Doppler flowmetry and the time-course of the cerebrovascular reactivity to hypercapnia were measured in artificially ventilated rats anesthetized with either alpha-chloralose (40 mg.kg-1, s.c.) plus halothane induction (1.5% given during the first 45-60 min) or halothane alone (1.5%). Finally, an experimental paradigm was developed that allowed the comparison of the hypercapnic reactivity, both in awake and anesthetized conditions in the same animal. Our results show that the association of alpha-chloralose with halothane leads to stable cardiovascular parameters and immobility of ventilated rats, placed in ear bars without curare, for 3 h without any sign of discomfort. Based on EEG criteria, we found that halothane induction lengthens the duration of alpha-chloralose anesthesia (253 +/- 19 vs. 200 +/- 15 min, P < 0.01). Under alpha-chloralose alone or in association with halothane induction, the vascular reactivity to hypercapnia was considerably impaired (-85% compared to the awake state, P < 0.01), but this impairment was transient, since a control reactivity was restored 150-190 min after induction of anesthesia. Under halothane alone, the vascular reactivity remained reduced throughout the experiment. These results provide evidence that alpha-chloralose plus halothane induction is a suitable anesthetic regimen which displays a temporal window of normal cerebrovascular reactivity.

Digestibility and bulking effect of ispaghula husks in healthy humans.

The digestibility of ispaghula, a mucilage from Plantago ovata composed mainly of arabinoxylans, and its faecal bulking effect were studied in seven healthy volunteers who ingested a low fibre controlled diet plus either placebo or 18 g/day of ispaghula for two 15 day periods. Whole gut transit time and gas excretion in breath and flatus were not different during the periods of ispaghula and placebo ingestion. Faecal wet and dry weights rose significantly, however, during ispaghula ingestion. Faecal short chain fatty acid concentrations and the molar proportions of propionic and acetic acids also increased. Most of the ispaghula had reached the caecum four hours after ingestion in an intact highly polymerised form. During ispaghula ingestion, the increase in the faecal output of neutral sugars was accounted for by the faecal excretion of arabinose and xylose in an intact highly polymerised form; the apparent digestibilities of these sugars were 24 (11) and 53% (6) respectively (mean (SEM)). In conclusion, ispaghula is more resistant to fermentation than previously reported in humans, and its bulking effect largely results from intact material.

Effects of an A1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia

Untreated rats and rats given the A1 receptor adenosine agonist, R-phenylisopropyladenosine (R-PIA), were subjected to four vessel ischemia. The effect of R-PIA on hippocampal amino acid release, hippocampal neuronal damage, exploratory behavior, learning capacity and global neurological score were evaluated. R-PIA decreased by half the glutamate released during ischemia and improved the global neurological scores 3, 24, 48, 78 h and 7 days after ischemia. But R-PIA had no effect on taurine/GABA release (during ischemia), hippocampal neuronal damage (7 days post-ischemia), exploratory behavior (48 h post-ischemia) or learning capacity (7 days post-ischemia). Thus, a decrease in glutamate release by R-PIA is not systematically correlated with an improvement of histological damage or learning capacity. Reduced glutamate release is not therefore a sufficient criterion on which to evaluate the neuroprotective capacity of a drug.

Absence seizures induce a decrease in cerebral blood flow : human and animal data

Our previous studies on cerebral metabolic activity in genetic absence epilepsy rats from Strasbourg (GAERS) were in favor of decreased functional activity during absences and normal or increased interictal activity. To ascertain that hypothesis, in the present study we performed continuous measurements of CBF in both children with typical absence epilepsy and GAERS, using Doppler ultrasonography and laser-Doppler flowmetry, respectively. CBF fluctuations during absences were recorded in four children between 5 and 6 years of age and 16 adult GAERS. In both children and animals, CBF measured in the middle cerebral artery and cortical capillaries, respectively, significantly decreased by a median value of 20–24% under basal levels during spontaneous absences. In GAERS, CBF levels were continuously decreased during haloperidol-induced absence status epilepticus, while they were not affected by ethosuximide. Conversely, convulsive seizures induced in rats either by kainate or Picrotoxin led to a 175–664% increase in CBF levels. In conclusion, the present data show that during spontaneous absences, CBF decreases under basal levels in both cortical capillaries (GAERS) and the middle cerebral artery (children). Moreover, these fluctuations occur in vessels with normal vascular reactivity, are not mediated by changes in Po2, Pco2, or arterial blood pressure, and represent rather a response to reduced metabolic demand.

Spreading depression induces prolonged reduction of cortical blood flow reactivity in the rat.

The purpose of the present study was to examine the dynamic aspects of the cerebrovascular events occurring during and up to 2 h following cortical spreading depression (CSD) in the rat, using the mass spectrometry technique which enables continuous measurements of the cortical tissue PO2 and PCO2 and repeated blood flow measurements (CoBF) by helium clearance. We mostly sought to determine whether cortical perforation by a stimulation electrode induced long-lasting perturbation of the cortical vasoreactivity to hypercapnia and basal forebrain electrical stimulation. Cortical perforation in the animal under alpha-chloralose anesthesia, chronically implanted with mass spectrometry probes, was associated with biphasic changes in tissue gases. PO2 first briefly decreased (-7.8%) and then strongly increased (+79%) while PCO2 changed in the opposite direction (+7%, -13%) in the ipsilateral frontal cortex. Qualitatively similar changes occurred in the ipsilateral parietal cortex. The CoBF measurements showed a marked vasodilation (131 and 108% in the frontal and parietal cortex, respectively) in parallel with the PO2 increase, followed by a prolonged (60 min), moderate hypoperfusion (maximum -17% at 20 min after CSD). There was a pronounced reduction of vascular reactivity to both hypercapnia (20.3% of the control response) and substantia innominata stimulation (1/6 of the response obtained 80 min later) at 10 min after CSD. Both reactivities progressively recovered in approximately 2 h. Since the issue of CSD in human has become a popular hypothesis for migraine, the reduced cerebrovascular reactivity could constitute the basis of a test for the involvement of CSD in migraine.

Effects of kainate-induced seizures on cerebral metabolism: a combined1H and 31P NMR study in rat

The cerebral metabolic changes elicited by kainate-induced seizures in the rat were investigated by in vivo combined NMR spectroscopy of 31P and 1H. Systemic injection of kainate induced no significant changes in cerebral ATP or PCr levels during up to 90 min of continuous, generalised seizures, and the cerebral 31P spectra showed only a transient mild cerebral acidosis 30 min after kainate administration. In parallel with the changes in intracellular cerebral pH, the 1H spectra showed a significant increase in lactate, which remained elevated throughout the seizures. These findings indicate that oxidative metabolism does not completely match the increased glycolysis during seizures though the energy homeostasis is maintained. This suggests that oxidative metabolism has a limited capacity to satisfy the brains energy needs during the kainate-induced seizures, but that the different pathways of energy production in the brain cells can overcome this limitation. Thus the brain damage associated with this experimental model of epilepsy is not due to extended major failure of the energy supply.

Carbon monoxide regulates cerebral blood flow in epileptic seizures but not in hypercapnia.

CARBON monoxide (CO) is an endogenously produced gas sharing many properties with nitric oxide (NO), notably activating soluble guanylate cyclase and relaxing blood vessels. The brain can generate high quantities of CO from a constitutive enzyme, haem oxygenase (HO-2). To determine whether CO is involved in the regulatory mechanisms of cerebral blood flow (CBF), two conditions associated with a reproducible CBF increase were studied in rats: epileptic seizures induced by kainate, and hypercapnia. The HO inhibitor tin protoporphyrin (Sn-PP) did not modify the basal level of CBF, significantly reduced the increase in CBF during status epilepticus, and did not affect the cerebrovascular response to hypercapnia. It is concluded that CO participates in the regulation of CBF in specific conditions, notably those associated with glutamate release.