Yolande Calando

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.

Effects of hyperinsulinemia on local cerebral insulin binding and glucose utilization in normoglycemic awake rats

The present study was carried out to characterize the effects of insulin, using the euglycemic hyperinsulinemic clamp, on insulin binding and glucose utilization in specific areas of rat brain, by autoradiographic methods. Binding of [125I]Insulin was significantly higher in the hippocampus CA1, the ventromedial and lateral hypothalamus nuclei of the hyperinsulinemic rats than in control rats. Glucose utilization was slightly but not significantly decreased in the hippocampus CA1, the ventromedial and lateral hypothalamus of hyperinsulinemic rats. These data suggest that insulin, via its specific receptors, may exert its central actions by affecting glucose utilization.

Subcortical cerebral blood flow and metabolic changes elicited by cortical spreading depression in rat

Changes in cerebral cortical perfusion (CBFLDF), local cerebral blood flow (ICBF) and local cerebral glucose utilization (ICGU) elicited by unilateral cortical spreading depression (SD) were monitored and measured in separate groups of rats anesthetized with a-chloralose. CBFLDF was recorded with laser Doppler flowmetry, while ICBF and ICGU were measured by the quantitative autoradiographic [14C]iodoantipyrine and [14C]-2-deoxyglucose methods, respectively. SD elicited a wave of hyperemia after a latency of 2 to 3 min followed by an oligemic phase. Ninety minutes following the onset of SD cortical (frontal, parietal and occipital) ICBF and ICGU were essentially the same as on the contralateral side and in sham-treated rats. However, alteration in the ICBF and ICGU in upper and lower brainstem persisted. The present results demonstrate, for the first time, that long-lasting cerebrovascular and metabolic alterations take place within the subcortical regions following SD. These regions provide an attractive site to integrate observations in man concerning spreading depression and the aura of migraine with the other features of the syndrome.

Limbic and/or generalized convulsive seizures elicited by specific sites in the thalamus

Convulsive seizures were elicited by a single unilateral microinjection of the cholinergic muscarinic agonist, carbachol, into the thalamus. Moreover, using systematic single microinjections of carbachol, we identified specific regions within the thalamus which were the origin of behavioural and electrocortical correlates associated with limbic and/or generalized convulsive seizures. Neither serotonin, noradrenaline nor glutamate had any convulsive effect when injected into the epileptogenic thalamic areas. The specific epileptogenic sites identified within the thalamus may provide a new experimental model which should prove useful for exploring the thalamic and thalamo-cortical mechanisms underlying limbic and generalized convulsive seizure disorders.

In vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse.

The aim of the study was to monitor in vivo and noninvasively the fate of single bone marrow cells (BMCs) transplanted into the ischemic brain of unirradiated mice. In vivo imaging was performed through a closed cranial window, throughout the 2 weeks following cell transplantation, using laser-scanning confocal fluorescence microscopy. The window was chronically implanted above the left parieto-occipital cortex in C57BL/6J adult mice. BMC (3 x 10(5) nucleated cells in 0.5 microL medium) from 5-week-old transgenic mice, ubiquitously expressing green fluorescent protein (GFP), was transplanted into the ipsilateral cortex 24 h after the induction of focal ischemia by coagulation of the left middle cerebral artery (n = 15). Three nonischemic mice served as controls. Repeated in vivo imaging, up to a depth of 200 microm, revealed that BMCs survived within the ischemic and peri-ischemic cortex, migrated significantly towards the lesion, proliferated and adopted a microglia-like morphology over 2 weeks. These results were confirmed using ex vivo imaging after appropriate immunocytochemical treatments. This study indicates that confocal fluorescence microscopy is a reliable and unique tool to repeatedly assess with cellular resolution the in vivo dynamic fate of fluorescent cells transplanted into a mouse brain. These results also provide the first in vivo findings on the fate of single BMCs transplanted into the ischemic brain of unirradiated mice.

Cerebrovascular changes elicited by electrical stimulation of the centromedian-parafascicular complex in rat

Electrical stimulation of the centromedian-parafascicular complex (CM-Pf) in anesthetized (chloralose) and paralyzed (tubocurarine) rats elicits a widespread cerebrovascular dilatation. Regional cerebral blood flow (rCBF) was measured in dissected tissue samples of 10 brain regions (medulla, pons, cerebellum, inferior colliculus, superior colliculus, frontal parietal and occipital cortices, caudate-putamen and corpus callosum) by [14C]iodoantipyrine method. In unstimulated and sham-operated rats rCBF ranged from 40 +/- 3 (ml/100 g/min) in corpus callosum to 86 +/- 6 (ml/100 g/min) in inferior colliculus. During CM-Pf stimulation, rCBF increased significantly (P less than 0.05, analysis of variance and Scheffes test) in all cerebral regions bilaterally ranging from +118% in parietal cortex to +38% in cerebellum. Although cerebral vasodilation elicited by CM-Pf stimulation persisted after unilateral transection of the cervical sympathetic trunk, the cortical CBF was significantly reduced (P less than 0.05) on the denervated side. Acute adrenalectomy significantly (P less than 0.05) decreased elevated rCBF during CM-Pf stimulation in all cortical regions (frontal-36%, parietal -34%, and occipital -27%) and in caudate nucleus (-37%). Thus, excitation of neurons originating in, or fibers passing through the CM-Pf can elicit a powerful cerebral vasodilation. The cerebral vasodilation is modulated by cervical sympathectomy and circulating adrenal hormones. We conclude that CM-Pf elicited vasodilation is at least partly mediated by intrinsic neural pathways.

Glucose utilization and insulin binding in discrete brain areas of obese rats

The present study was carried out to determine whether genetically obese Zucker rats present changes in brain glucose utilization and/or insulin binding when compared to their lean counterparts. Glucose utilization in the whole brain, determined by measurement of 2-deoxy(1-3H)glucose-6-phosphate, was significantly lower in obese than in lean Zucker rats. In order to precise the structure involved, we then used quantitative autoradiography methods after either (1-14C) 2-deoxyglucose injection or 125I-insulin incubation. In obese rats, local cerebral glucose utilization (LCGU) was significantly decreased in the external plexiform layer (-37%, p < 0.05), in the lateral hypothalamus (-23%, p < 0.05), and in the basolateral amygdaloid nucleus (-30%, p < 0.05). In contrast, no difference in specific insulin binding was found between the two genotypes in any of the areas studied. These results are consistent with some data showing a decrease of LCGU in hyperinsulinemic rats. All together, these data show perturbations of glucose utilization, particularly in structures linked to the regulation of body weight and food intake in obese Zucker rats.

Cerebrovascular and metabolic uncoupling in the caudate-putamen following unilateral lesion of the mesencephalic dopaminergic neurons in the rat

Changes in local cerebral blood flow (lCBF) and local cerebral glucose utilization (lCGU) were assessed in dopaminergic primary target areas in the rat 6 weeks after unilateral lesion of dopaminergic neurons within the substantia nigra pars compacta (SNc) and adjacent ventrotegmental area (VTA) using 6-hydroxydopamine (6-OHDA). lCBF and lCGU were determined using the autoradiographic [14C]iodoantipyrine and [14C]2-deoxyglucose method. Dopaminergic deafferentation provoked a marked unilateral lCBF decrease in the dorso-lateral portion of the rostral caudate-putamen. The decrease in lCBF was not associated with significant changes in glucose metabolism. Thus, lesions of dopaminergic afferents to the caudate-putamen appear to provoke a sustained decrease in basal blood flow with unchanged local metabolic activity.

FOS Induction in Brain Associated with Seizure and Sustained Cortical Vasodilation in Anesthetized Rat

Summary: Purpose: By estimating the anatomical distribution of neurons expressing c‐fos protein, we sought to establish whether the intrinsic neurol systems known to be implicated in the cerebrovascular regulation were activated during the increase in cortical blood flow associated with epileptic seizures.

A New, Segmental, Critical-Size Defect Model for Long Bones: A Sheep Study

In the present study, we (i) established a large animal model in sheep of a two-step surgical procedure for successful reconstruction of large segmental bone defects, based on a concept that proved successful in humans and (ii) explored the possibility of using skeletal stem cells loaded onto a coral scaffolds to repair such defects.