Elisabeth Pinard

Reduction of Infarct Volume by Magnesium after Middle Cerebral Artery Occlusion in Rats

The effects of magnesium, an endogenous inhibitor of calcium entry into neurons, upon ischemic brain damage were investigated using a well-characterized model of focal cerebral ischemia in rats. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride transcardiac perfusion 48 h after middle cerebral artery (MCA) occlusion. The area of ischemic damage was quantified by image analysis in coronal sections taken every 0.5 mm. MgCl2 (1 mmol/kg) was injected intraperitoneally just after MCA occlusion and again 1 h later. Posttreatment with MgCl2 (16 control and 16 treated rats) significantly reduced the cortical infarct volume. Compensation for the hyperglycemic effect of MgCl2 with insulin (17 rats) further reduced the infarct volume in the neocortex. No systemic effects of either treatment could account for the observed neuroprotection.

Dynamic in vivo measurement of erythrocyte velocity and flow in capillaries and of microvessel diameter in the rat brain by confocal laser microscopy

A new method for studying brain microcirculation is described, Both fluorescently labeled erythrocytes and plasma were visualized on-line through a closed cranial window in anesthetized rats, using laser-scanning two-dimension confocal microscopy, Video images of capillaries, arterioles, and venules were digitized off-line to measure microvessel diameter and labeled erythrocyte flow and velocity in parenchymal capillaries up to 200 μm beneath the brain surface, The method was used to analyze the rapid adaptation of microcirculation to a brief decrease in perfusion pressure, Twenty-second periods of forebrain ischemia were induced using the four-vessel occlusion model in eight rats, EEG, arterial blood pressure, and body temperature were continuously controlled, In all conditions, labeled erythrocyte flow and velocity were both very heterogeneous in capillaries, During ischemia, capillary perfusion was close to 0, but a low blood flow persisted in arterioles and venules, while EEG was flattening, The arteriole and venule diameter did not significantly change, At the unclamping of carotid arteries, there was an instantaneous increase (by about 150%) of arteriole diameter. Capillary erythrocyte flow and velocity increased within 5 seconds, up to, respectively, 346 ± 229% and 233 ± 156% of their basal value, No capillary recruitment of erythrocytes was detected, All variables returned to their basal levels within less than 100 seconds after declamping, The data are discussed in terms of a possible involvement of shear stress in the reperfusion period.

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.

Nitric Oxide of Neuronal Origin is Involved in Cerebral Blood Flow Increase During Seizures Induced by Kainate

In a previous study, we reported that the sustained increase in CBF concomitant with seizures induced by kainate is mainly due to the potent vasodilator nitric oxide (NO). However, the production site of NO acting at cerebral vessels was undetermined. In the present study, we investigated whether NO responsible for the cerebral vasodilation is of either neuronal or endothelial origin. We used a putative selective inhibitor of neuronal NO synthase, 7-nitro indazole (7-NI). CBF was measured continuously in parietal cortex by means of laser Doppler flowmetry in awake rats. Systemic variables and electroencephalograms were monitored. Kainate (10 mg/kg i.p.) was given to rats previously treated with saline (n = 8) or 7-NI (25 mg/kg i.p., n = 8) or L-arginine (300 mg/kg i.p., n = 8) followed 30 min later by 7-NI (25 mg/kg i.p.). Under basal conditions, 7-NI decreased CBF by 27% without modifying the mean arterial blood pressure. Under kainate, 7-NI prevented significant increases in CBF throughout the seizures despite sustained paroxysmal electrical activity. L-arginine, the substrate in the production of NO, prevented any decrease in CBF under 7-NI in basal conditions and partially, but nonsignificantly, reversed the cerebrovascular influence of 7-NI during seizures. In a separate group of rats (n = 6), inhibition of cortical NO synthase activity by 7-NI was assayed at 73%. The present results show that neurons are the source of NO responsible for the cerebrovascular response to seizure activity after kainate systemic injection.

The Selective Inhibitor of Neuronal Nitric Oxide Synthase, 7-Nitroindazole, Reduces the Delayed Neuronal Damage Due to Forebrain Ischemia in Rats

BACKGROUND AND PURPOSE The present study was designed to investigate whether neuronally derived nitric oxide (NO) plays a toxic role in the cascade of cellular events triggered by global cerebral ischemia in rats. METHODS 7-Nitroindazole (7-NI) was used as a selective inhibitor of neuronal NO synthase. Global ischemia was induced for 20 minutes in anesthetized rats following the four-vessel occlusion model. Electroencephalogram and brain and body temperatures were continuously monitored. All rats were thermoregulated for the entire duration of anesthesia. 7-NI (25 mg/kg) or its vehicle was given intraperitoneally just after the carotid clamping and again 1 hour later. Rats were randomly divided into four groups: (1) vehicle (n = 7); (2) 7-NI (n = 7); (3) L-arginine (300 mg/kg IP) +7-NI (n = 7); and (4) 7-NI associated with warming to 37 degrees C for 7 hours after disruption of anesthesia to compensate for the decrease in temperature induced by 7-NI (n = 9). Seven days after ischemia, hippocampal CA1 damage was evaluated by classic histology. The lesion was scored with the use of a point scale, and the surviving neurons were counted. RESULTS Lesion scores were significantly lower and neuron counts higher in the two (warmed and unwarmed) groups of rats in which 7-NI was given alone than in vehicle- and L-arginine +7-NI-treated rats. CONCLUSIONS The results indicate that 7-NI was neuroprotective in 20-minute global ischemia in rats and that the neuroprotective effect of 7-NI was mostly due to the blockade of NO synthesis, suggesting that NO released from neurons in ischemic conditions has a deleterious influence on hippocampal pyramidal neurons.

Inhibition of neuronal (type 1) nitric oxide synthase prevents hyperaemia and hippocampal lesions resulting from kainate-induced seizures

The possible roles for nitric oxide produced by neurons in epileptic conditions have been investigated from two different aspects: microcirculation and delayed damage. Our aim was to determine whether the selective inhibition of neuronal (type 1) nitric oxide synthase by 7-nitroindazole, during seizures induced by systemic kainate, modifies hippocampal blood flow and oxygen supply and influences the subsequent hippocampal damage. Experiments were performed in conscious Wistar rats whose electroencephalogram was recorded. 7-Nitroindazole (25 mg/kg, i.p.) or its vehicle was injected 30 min before kainate administration (10 mg/kg, i.p.) and then twice at 1-h intervals. Kainate triggered typical limbic seizures evolving into status epilepticus, identified by uninterrupted electroencephalographic spike activity. The seizures were stopped by diazepam (5 mg/kg, i.p.) after 1 h of status epilepticus. Three types of experiments were performed in vehicle- and 7-nitroindazole-treated rats. (1) Hippocampal nitric oxide synthase activity was measured under basal conditions, at 1 h after the onset of the status epilepticus and at 24 h after its termination (n = 4-6 per group). (2) Hippocampal blood flow and tissue partial pressure of oxygen were measured simultaneously by mass spectrometry for the whole duration of the experiment, while systemic variables and body temperature were monitored (n = 6 per group). (3) Hippocampal damage was revealed by Cresyl Violet staining and evaluated with a lesion score seven days after status epilepticus (n = 12 per group). Hippocampal nitric oxide synthase activity was not significantly modified during status epilepticus or the following day in vehicle-treated rats. In contrast, it was inhibited by 57% in 7-nitroindazole-treated rats, both in basal conditions and after 1 h of status epilepticus, but was not different from its basal level 24 h later. 7-Nitroindazole significantly decreased basal hippocampal blood flow and tissue partial pressure in oxygen by 30% and 35%, respectively without affecting any systemic or thermal variable. During status epilepticus, 7-nitroindazole significantly reduced the increase in hippocampal blood flow by 70% and prevented any increase in the tissue partial pressure of oxygen. Seven days later, the hippocampal damage in the CA1 and CA3 layers was significantly less in 7-nitroindazole-treated rats than in vehicle-treated rats. These results indicate that the inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects neurons from seizure-induced toxicity despite reducing blood flow and oxygen supply to the hippocampus.

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.

Effect of MK-801 on focal brain infarction in normotensive and hypertensive rats.

The effects of the noncompetitive N-methyl-D-aspartate antagonist MK-801 on infarct size and systemic variables after middle cerebral artery occlusion in spontaneously hypertensive and Fischer-344 rats were investigated. Two doses (0.5 and 5 mg/kg) administered before the induction of ischemia were studied. MK-801 significantly reduced the neocortical volume of infarction (by about 32% at both doses) in Fischer-344 rats and had no neuroprotective effects in the striatum. In contrast, MK-801 had no significant influence on either cortical or striatal infarcted volume in spontaneously hypertensive rats. The reduction or lack of MK-801-induced neuroprotection in spontaneously hypertensive rats, as compared with Fischer-344 rats, could be attributed to a reduced collateral supply in the marginal area due to difference in the morphology of the pial anastomoses and/or in the effects of ischemia and treatment on arterial pressure. The results may have major clinical implications since a great proportion of human strokes are associated with hypertension.

Blockade of nitric oxide synthesis inhibits hippocampal hyperemia in kainic acid-induced seizures

We investigated whether the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) affects the cerebrovascular changes occurring in seizures induced by kainic acid (KA) in awake, spontaneously breathing rats. Blood flow and tissue Po2 and Pco2 were continuously and simultaneously measured by mass spectrometry from a cannula chronically implanted into the dorsal hippocampus. l-NAME (20 mg/kg; n = 8) or saline (n = 9) was administered i.p. 30 min prior to i.p. KA (10 mg/kg) injection. l-NAME significantly decreased hippocampal blood flow and Po2 and increased mean arterial blood pressure (MABP). In l-NAME-treated rats, seizure activity occurred about 10 min sooner than in control rats, and status epilepticus was inevitably followed by a flat electroencephalogram and sudden death. In contrast, control rats survived KA-induced seizures. Hippocampal blood flow was significantly less elevated during the seizures in l-NAME-treated rats than in control rats (maximal levels, 170 and 450%, respectively, of baseline values), though MABP remained significantly higher. Hippocampal Po2 was significantly decreased at all times after KA injection in l-NAME-treated rats, whereas it remained at or above normoxic levels in control rats. The present results show that l-NAME markedly attenuates the hippocampal blood flow and tissue Po2 changes in response to enhanced metabolic activity due to limbic seizures and suggest that NO is of major importance in cerebral blood flow control during KA-induced seizures.

Dynamic Cerebral Microcirculatory Changes in Transient Forebrain Ischemia in Rats: Involvement of Type I Nitric Oxide Synthase:

The diameter of surface microvessels and the erythrocyte velocity and flux through intraparenchymal capillaries in the parietal cortex were measured during transient global cerebral ischemia and reperfusion using laser-scanning confocal fluorescence microscopy in anesthetized rats. The role of nitric oxide (NO) from neurons in the microcirculatory changes was also investigated using 7-nitro-indazole (7-NI, 25 mg/kg, IP). Wistar rats (4 per group) equipped with a closed cranial window were given fluorescein isothiocyanate (FITC)-Dextran and FITC-labeled erythrocytes intravenously to respectively visualize the microvessels and the erythrocytes in the capillaries. Experiments were videorecorded on-line. Forebrains were made ischemic for 15 minutes and then reperfused for 120 minutes under the microscope. Ischemia was associated with a flattened EEG, a low persistent blood flow, and a transient leakage of fluorescein across the arteriole wall. Unclamping the carotid arteries led to immediate high blood flow in the arterioles, but it was not until 5 minutes later that the arterioles dilated significantly (181% ± 27%) and erythrocyte velocity in the capillaries increased significantly (460% ± 263%). Neither nonperfused capillaries nor erythrocyte capillary recruitment occurred. 7-Nitro-indazole significantly reduced the arteriole dilatation and prevented the increase in erythrocyte velocity and flux through capillaries in early reperfusion. 7-Nitro-indazole had no influence on the fluorescein leakage. The current study suggests a partial role for NO released from neurons in the postischemic microcirculatory changes and provides new findings on the timing of arteriole dilatation and blood—brain barrier opening, and on erythrocyte capillary circulation in global ischemia.