Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

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 mm(3) (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.

Penumbral microcirculatory changes associated with peri-infarct depolarizations in the rat.

BACKGROUND AND PURPOSE: This study was designed to investigate the influence of peri-infarct depolarization elicited by occlusion of the middle cerebral artery on the dynamics of the microcirculation. METHODS: The microcirculation in the frontoparietal cortex of 9 rats was visualized in real time through a closed cranial window with the use of laser-scanning confocal fluorescence microscopy combined with intravenous fluorescein isothiocyanate (FITC)-dextran and FITC-labeled erythrocytes. The direct current potential/electrocorticogram was continuously monitored. Intraluminal focal ischemia was induced for 2 hours in 6 rats anesthetized with halothane and mechanically ventilated. Reperfusion was monitored for 1 hour. Three rats underwent sham operation. Brains were removed 24 hours after occlusion and processed for histology. RESULTS: In control conditions, the velocity of fluorescent erythrocytes through capillaries was 0.51+/-0.19 mm/s (mean+/-SD), and the diameter of the arterioles studied was 33+/-12 microm. Under ischemia, erythrocyte velocity through capillaries was significantly decreased to 0.33+/-0.14 mm/s, while arteriole diameter did not change significantly. During spontaneous peri-infarct depolarizations, arteriole diameter was significantly increased (119+/-23% of baseline), while capillary erythrocyte velocity was further decreased by 14+/-34%. The direction of arteriolar blood flow episodically and transiently reversed during approximately half of the peri-infarct depolarizations. The decrease in capillary erythrocyte velocity was more pronounced (23+/-37%) in these cases. After reperfusion, the microcirculatory variables rapidly returned to baseline. All rats in the ischemic group had infarcts 24 hours after occlusion. CONCLUSIONS: Peri-infarct depolarization has an adverse influence on penumbral microcirculation, reducing capillary perfusion by erythrocytes, despite dilatation of arterioles. These findings suggest that a steal phenomenon contributes to the deleterious effect of these depolarizations.

Albumin therapy of transient focal cerebral ischemia: in vivo analysis of dynamic microvascular responses.

BACKGROUND AND PURPOSE: To study whether intravascular or hemodynamic factors contribute to the marked neuroprotective effect of albumin therapy in focal cerebral ischemia, 2 complementary methods were applied: laser-scanning confocal microscopy (LSCM) and laser-Doppler perfusion imaging (LDPI). METHODS: In the LSCM study, Sprague-Dawley rats were anesthetized with halothane/nitrous oxide, and a cranial window was placed over the dorsolateral frontoparietal cortex. Rats received 2-hour middle cerebral artery occlusion (MCAO) by an intraluminal suture and were treated with human albumin (1.25 g/kg; n=4) or saline (n=3) after 30 minutes of recirculation. Video images of cortical vessels were continually acquired and were digitized offline to measure diameters and fluorescent erythrocyte velocities. In the LDPI study, cortical perfusion was measured in anesthetized Sprague-Dawley rats that received 2-hour MCAO and were treated with albumin (2.5 g/kg; n=6) or saline (n=5) at 30 minutes after recirculation. RESULTS: In the LSCM study, MCAO was associated with arteriolar dilation and slowing of capillary and venular erythrocyte perfusion. During the first 15 to 30 minutes of postischemic recirculation, prominent foci of vascular stagnation developed within cortical venules, associated with thrombuslike foci and adherent corpuscular structures consistent in size with neutrophils. Saline administration failed to affect these phenomena, while albumin therapy was followed by significant increases in arteriolar diameter ( approximately 12%; P=0.007) and by a prompt improvement of venular and capillary erythrocyte perfusion and a partial disappearance of adherent thrombotic material. Albumin therapy increased erythrocyte flow velocity in both capillaries (288+/-73% versus 76+/-18% in the saline group; P=0.023) and venules (2.7-fold [P=0.001] versus 1.0-fold in the saline group [P=NS]). In the LDPI study, cortical perfusion declined during MCAO and rose initially with recirculation (to approximately 135% of baseline) in both groups. Mean cortical perfusion improved slightly (approximately 14%; P=NS) in albumin-treated animals. CONCLUSIONS: These results reveal a beneficial effect of albumin therapy in reversing stagnation, thrombosis, and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia and support its utility in the treatment of acute ischemic stroke.

Long-term, repeated measurements of mouse cortical microflow at the same region of interest with high spatial resolution.

A method for long-term, repeated, semi-quantitative measurements of cerebral microflow at the same region of interest (ROI) with high spatial resolution was developed and applied to mice subjected to focal arterial occlusion. A closed cranial window was chronically implanted over the left parieto-occipital cortex. The anesthetized mouse was placed several times, e.g., weekly, under a dynamic confocal microscope, and Rhodamine B-isothiocyanate-dextran was each time intravenously injected as a bolus, while microflow images were video recorded. Left and right tail veins were sequentially catheterized in a mouse three times at maximum over a 1.5 months' observation period. Smearing of the input function resulting from the use of intravenous injection was shown to be sufficiently small. The distal middle cerebral artery (MCA) was thermocoagulated through the cranial window in six mice, and five sham-operated mice were studied in parallel. Dye injection and video recording were conducted four times in this series, i.e., before and at 10 min, 7 and 30 days after sham operation or MCA occlusion. Pixelar microflow values (1/MTT) in a matrix of approximately 50×50 pixels were displayed on a two-dimensional (2-D) map, and the frequency distribution of the flow values was also calculated. No significant changes in microflow values over time were detected in sham-operated mice, while the time course of flow changes in the ischemic penumbral area in operated mice was similar to those reported in the literature. This method provides a powerful tool to investigate long-term changes in mouse cortical microflow under physiological and pathological conditions.

Magnetic targeting of nanometric magnetic fluid loaded liposomes to specific brain intravascular areas: a dynamic imaging study in mice.

PURPOSE: To prospectively determine, by using dynamic imaging, whether a magnet placed over a specific area of the mouse brain could target systemically administered rhodamine-labeled magnetic fluid-loaded liposomes (MFLs) to that brain region. MATERIALS AND METHODS: Experiments were performed with a French Ministry of Agriculture permit and regional ethics committee authorization. In seven anesthetized C57BL/6 mice, a closed cranial window was implanted above the left parieto-occipital cortex. A laser-scanning confocal fluorescence microscope (LSCFM) was used to track the intravenously injected rhodamine-labeled MFLs within this cortical area, through the cranial window. The MFLs were video monitored for 2 minutes every 15 minutes for 1 hour after injection. A magnet was placed on the cranial window implanted in four mice, while no magnet was placed in three (control) mice. After dynamic in vivo imaging, static in vivo imaging was performed with a different LSCFM. Ex vivo fluorescence histologic analysis was then performed. Paired Student t testing was used to compare the cerebral blood flow and two-dimensional flow values before and 1 hour after MFL injection. For image analysis, intergroup comparisons were performed by using an independent t test. RESULTS: In vivo video monitoring through the window revealed that the rhodamine-labeled MFLs accumulated in the mouse brain microvasculature exposed to the magnet-first within superficial brain venules and then within intracerebral venules-with no significant change in blood flow (P > .05). MFLs accumulated neither in the arterioles of the mice with a magnet nor in the arterioles of the control mice. Static in vivo imaging findings confirmed the microvascular localization of the rhodamine-labeled MFLs, and histologic findings specified their accumulation on the side of the magnet only. CONCLUSION: Real-time in vivo imaging of rhodamine-labeled MFLs in the mouse brain cortex revealed that these nanosystems can be magnetically targeted, through microvessels, to selected brain areas.

Vascular fate of adipose tissue-derived adult stromal cells in the ischemic murine brain: A combined imaging-histological study.

Increasing evidence indicates that fat tissue can provide a novel source of progenitor cells with therapeutic potential. Here, the fate of adipose tissue-derived stromal cells (ADSCs) transplanted into the mouse ischemic cortex was monitored in the long term using in vivo imaging, and subsequently characterized. The left middle cerebral artery (MCA) was occluded in C57BL/6J mice equipped with a closed cranial window chronically implanted over the left parietal cortex (n = 20). ADSCs expressing the green fluorescent protein (GFP) (approximately 18 x 10(3) cells in 0.5 microl) were transplanted into the ipsilateral cortex, 24 h after MCA occlusion. GFP+-ADSCs were monitored through the window using confocal fluorescence microscopy to assess their single fate in vivo. Co-localization of GFP with vascular, neuronal, glial or proliferation markers was investigated immunohistochemically. Repeated in vivo imaging revealed that GFP+-ADSCs migrated over 1 week toward the lesion, survived for at least 4 weeks, and exhibited a particular tropism for vessels. About 5% of the transplanted GFP+-ADSCs were scattered in the peri-ischemic area on histological sections. Immunohistochemistry evidenced that perivascular GFP+-ADSCs enfolded CD31-labeled endothelial cells, always outside their basal lamina, and occasionally expressed smooth muscle alpha-actin. Less than 1% GFP and BrdU co-labeling indicated a low proliferation rate of ADSCs. These results demonstrate that cerebral ischemia induces ADSCs survival, migration toward the lesion, especially toward microvessels, and occasional differentiation into smooth muscle cells.

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.