Pierre Lacombe

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: A confocal microscopy study

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three‐dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S‐100β] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S‐100β showed numerous well‐defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well‐defined rosette‐like structures lying on the vessel wall.

Serotonin in the regulation of brain microcirculation.

Manipulation of brainstem serotonin (5-HT) raphe neurons induces significant alterations in local cerebral metabolism and perfusion. The vascular consequences of intracerebrally released 5-HT point to a major vasoconstrictor role, resulting in cerebral blood flow (CBF) decreases in several brain regions such as the neocortex. However, vasodilatations, as well as changes in blood-brain barrier (BBB) permeability, which are blocked by 5-HT receptor antagonists also can be observed. A lack of relationship between the changes in flow and metabolism indicates uncoupling between the two variables and is suggestive of a direct neurogenic control by brain intrinsic 5-HT neurons on the microvascular bed. In line with these functional data are the close associations that exist between 5-HT neurons and the microarterioles, capillaries and perivascular astrocytes of various regions but more intimately and/or more frequently so in those where CBF is altered significantly following manipulation of 5-HT neurons. The ability of the microvascular bed to respond directly to intracerebrally released 5-HT is underscored by the expression of distinct 5-HT receptors in the various cellular compartments of the microvascular bed. Thus, it appears that while some 5-HT-mediated microvascular functions involve directly the blood vessel wall, others would be relayed through the perivascular astrocyte. The strategic localization of perivascular astrocytes and the different 5-HT receptors that they harbor strongly emphasize their putative pivotal role in transmitting information between 5-HT neurons and microvessels. It is concluded that the cerebral circulation has full capacity to adequately and locally adapt brain perfusion to changes in central 5-HT neurotransmission either directly or indirectly via the neuronal-astrocytic-vascular tripartite functional unit. Dysfunctions in these neurovascular interactions might result in perfusion deficits and might be involved in specific pathological conditions.

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.

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.

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.

High Sensitivity of Protoplasmic Cortical Astroglia to Focal Ischemia

The generally accepted concept that astrocytes are highly resistant to hypoxic/ischemic conditions has been challenged by an increasing amount of data. Considering the differences in functional implications of protoplasmic versus fibrous astrocytes, the authors have investigated the possibility that those discrepancies come from specific behaviors of the two cell types. The reactivity and fate of protoplasmic and fibrous astrocytes were observed after permanent occlusion of the medial cerebral artery in mice. A specific loss of glial fibrillary acidic protein (GFAP) immunolabeling in protoplasmic astrocytes occurred within minutes in the area with total depletion of regional CBF (rCBF) levels, whereas “classical” astrogliosis was observed in areas with remaining rCBF. Severe disturbance of cell function, as suggested by decreased GFAP content and increased permeability of the blood–brain barrier to macromolecules, was rapidly followed by necrotic cell death, as assessed by ultrastructure and by the lack of activation of the apoptotic protease caspase-3. In contrast to the response of protoplasmic astrocytes, fibrous astrocytes located at the brain surface and in deep cortical layers displayed a transient and limited hypertrophy, with no conspicuous cell death. These results point to a differential sensitivity of protoplasmic versus fibrous cortical astrocytes to blood deprivation, with a rapid demise of the former, adding to the suggestion that protoplasmic astrocytes play a crucial role in the pathogenesis of ischemic injury.

Local Injection of Antisense Oligonucleotides Targeted to the Glial Glutamate Transporter GLAST Decreases the Metabolic Response to Somatosensory Activation

The mechanisms responsible for the local increase in brain glucose utilization during functional activation remain unknown. Recent in vitro studies have identified a new signaling pathway involving an activation of glial glutamate transporters and enhancement of neuron–astrocyte metabolic interactions that suggest a putative coupling mechanism. The aim of the present study was to determine whether one of the glutamate transporters exclusively expressed in astrocytes, GLAST, is involved in the neurometabolic coupling in vivo. For this purpose, rats were microinjected into the posteromedial barrel subfield (PMBSF) of the somatosensory cortex with GLAST antisense or random phosphorothioate oligonucleotides. The physiologic activation was performed by stimulating the whisker-to-barrel pathway in anesthetized rats while measuring local cerebral glucose utilization by quantitative autoradiography in the PMBSF. Twenty-four hours after injection of two different antisense GLAST oligonucleotide sequences, and despite the presence of normal whisker-related neuronal activity in the PMBSF, the metabolic response to whisker stimulation was decreased by more than 50%. Injection of the corresponding random sequences still allowed a significant increase in glucose utilization in the activated area. The present study highlights the contribution of astrocytes to neurometabolic coupling in vivo. It provides evidence that glial glutamate transporters are key molecular components of this coupling and that neuronal glutamatergic activity is an important determinant of energy utilization. Results indicate that astrocytes should also be considered as possible sources of altered brain metabolism that could explain the distinct imaging signals observed in some pathologic situations

Impaired vascular mechanotransduction in a transgenic mouse model of CADASIL arteriopathy.

Background and Purpose— CADASIL is an inherited small-vessel disease responsible for lacunar strokes and cognitive impairment. The disease is caused by highly stereotyped mutations in Notch3, the expression of which is highly restricted to vascular smooth muscle cells (VSMCs). The underlying vasculopathy is characterized by degeneration of VSMCs and the accumulation of granular osmiophilic material (GOM) and Notch3 protein within the cell surface of these cells. In this study, we assessed early functional changes related to the expression of mutant Notch3 in resistance arteries. Methods— Vasomotor function was examined in vitro in arteries from transgenic mice that express a mutant Notch3 in VSMC. Tail artery segments from transgenic and normal wild-type male mice were mounted on small-vessel arteriographs, and reactivity to mechanical (flow and pressure) forces and pharmacological stimuli were determined. Mice were studied at 10 to 11 months of age when VSMC degeneration, GOM deposits, and Notch3 accumulation were not yet present. Results— Passive arterial diameter, contraction to phenylephrine, and endothelium-dependent relaxation to acetylcholine were unaffected in transgenic mice. By contrast, flow-induced dilation was significantly decreased and pressure-induced myogenic tone significantly increased in arteries from transgenic mice compared with wild-type mice. Conclusions— This is the first study to our knowledge providing evidence that mutant Notch3 impairs selectively the response of resistance arteries to flow and pressure. The data suggest an early role of vascular dysfunction in the pathogenic process of the disease.

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.

Alzheimer's disease-like cerebrovascular pathology in transforming growth factor-beta 1 transgenic mice and functional metabolic correlates

Abstract: Alzheimers disease (AD) is frequently associated with cerebrovascular changes, including perivascular astrocytosis, amyloid deposition, and microvascular degeneration, but it is not known whether these pathological changes contribute to functional deficits in AD. To characterize the temporal relationship between amyloid deposition, cerebrovascular abnormalities, and potential functional changes, we studied transgenic mice that express transforming growth factor‐β1 (TGF‐β1) at low levels in astrocytes. TGF‐β1 induced a prominent perivascular astrocytosis, followed by the accumulation of basement membrane proteins in microvessels, thickening of capillary basement membranes, and later, around 6 months of age, deposition of amyloid in cerebral blood vessels. At 9 months of age, various AD‐like degenerative alterations were observed in endothelial cells and pericytes. Associated with these morphological changes were changes in regional cerebral glucose utilization. Preliminary results showed that TGF‐β1 mice had significantly decreased glucose utilization in the mammillary bodies, structures involved in mnemonic and learning processes. Glucose utilization tended to be decreased in several other brain regions as well; however, in the inferior colliculus, it was markedly higher in TGF‐β1 mice than in controls. We conclude that chronic overproduction of TGF‐β1 triggers a pathogenic cascade leading to AD‐like cerebrovascular amyloidosis, microvascular degeneration, and local alterations in brain metabolic activity. Similar mechanisms may be involved in AD pathogenesis.