A method for microscopic studies of cerebral angioarchitecture and vascular- parenchymal relationships, based on the demonstration of ·paravascular’ fluid pathways in the mammalian central nervous system (original) (raw)
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Brain Research, 1974
A new method for tracing connections in the central nervous system is described. Horseradish peroxidase is injected into the area of interest where it is taken up by cell bodies and dendrites and transported to their axons and terminals. The enzyme is then demonstrated histochemically, revealing the efferent connections of the injected area. The method has several advantages with regard to existing techniques: it is reliable, simple to perform, and requires very little time. Furthermore, the technique can be used for both light and electron microscopic analyses. In some cases the terminals of labeled axons failed to stain indicating that in its present form the method may not be appropriate for use in all brain systems.
Tubular profiles do not form transendothelial channels through the blood-brain barrier
Journal of Neurocytology, 1987
The contribution of tubular profiles within the mammalian cerebral endothelium to the formation of transcellular channels was analysed following exposure of the endothelium to native horseradish peroxidase (HRP) dissolved in saline or dimethyl sulphoxide (DMSO) administered intravenously in mice. Within 5-15 min, but not at 30 rain to 2h postinjection, peroxidase-positive extravasations were evident within the parenchyma of the forebrain and brainstem of mice exposed and not exposed to DMSO. The extravasations may be associated with the rupture of interendothelial tight junctions at the level of arterioles as a consequence of the perfusion-fixation process. Ultrastructural inspection of endothelia within and away from areas of peroxidase extravasation revealed the following intraendothelial, peroxidase-positive organelles: presumptive end0cytic vesicles, endosomes (a prelysosomal compartment), multivesicular and dense bodies, and tubular profiles. Statistical analysis of the concentration of HRP-labelled presumptive endocytic vesicles, which may coalesce to form tubules, within endothelia from mice injected intravenously with HRP-DMSO compared to mice receiving HRP-saline revealed no significant difference. HRP-positive tubular profiles were blunt-ended, variable in length and width, and appeared free in the cytoplasm or in continuity with dense bodies. Labelled tubules free in the cytoplasm were positioned parallel to the luminal and abluminal plasma membranes and were less frequently oblique or perpendicular to these membranes. Tubular profiles analysed in serial thin sections or with a goniometer tilt stage did not establish membrane continuities with the luminal and abluminal plasma membranes. Peroxidase-positive tubular profiles were similar morphologically to those exhibiting acid hydrolase activity but did not share morphological and enzyme cytochemical similarities with the endoplasmic reticulum that stained for glucose-6-phosphatase (G6Pase) activity. G6Pase-positive profiles of endoplasmic reticulum were not observed to contribute to a transendothelial canalicular network. Our results suggest that: (i) peroxidase-labelled tubules, acid hydrolase-positive tubules, and G6Pase-positive endoplasmic reticulum do not form transcellular channels through the cerebral endothelium; (ii) tubular profiles labelled with blood-borne HRP in the cerebral endothelium are, associated with the etidosome apparatus and/or the lysosomar system of organelles; and (iii) DMSO does not appear to alter the permeability of the blood-brain barrier to blood-borne protein.
Visualization of macroscopic cerebral vessel anatomy—A new and reliable technique in mice
Journal of Neuroscience Methods, 2012
Visualizing rodent cerebral vasculature is an important tool in experimental stroke research. Intravascular perfusion with colored latex has been the method of choice until recently. However, latex perfusion has some technical limitations which compromise its reproducibility. We therefore describe a simple and reproducible method to visualize cerebral vessels in mice. A mixture of two commercially available carbon black inks is injected into the thoracic aorta resulting in efficient filling and high contrast visualization of cerebral vessels. Feasibility of this technique has been validated by identifying anastomotic points between anterior and middle cerebral arteries. Furthermore, perfusion with combined carbon inks allows visualization of significantly smaller vessel diameters at a higher vessel density in comparison to perfusion with diluted/undiluted latex. Thus, perfusion with combined carbon inks offers a simple, cost-effective and reproducible technique in order to visualize cerebral vasculature.
Vascular Supply of the Cerebral Cortex is Specialized for Cell Layers but Not Columns
Cerebral cortex (New York, N.Y. : 1991), 2014
The vascular supply to layers and columns was compared in macaque primary visual cortex (V1) by labeling red blood cells via their endogenous peroxidase activity. Alternate sections were processed for cytochrome oxidase to reveal "patches" or "blobs," which anchor the interdigitated column systems of striate cortex. More densely populated cell layers received the most profuse blood supply. In the superficial layers the blood supply was organized into microvascular lobules, consisting of a central venule surrounded by arterioles. Each vessel was identified as an arteriole or venule by matching it with the penetration site where it entered the cortex from a parent arteriole or venule in the pial circulation. Although microvascular lobules and cytochrome oxidase patches had a similar periodicity, they bore no mutual relationship. The size and density of penetrating arterioles and venules did not differ between patches and interpatches. The red blood cell labeling in...
Isolation of Rodent Brain Vessels
BIO-PROTOCOL
The prevalence of neurodegenerative diseases is increasing worldwide. Cerebrovascular disorders and/or conditions known to affect brain vasculature, such as diabetes, are well-known risk factors for neurodegenerative diseases. Thus, the evaluation of the brain vasculature is of great importance to better understand the mechanisms underlying brain damage. We established a protocol for the isolation of brain vessels from rodents. This is a simple, non-enzymatic isolation protocol that allows us to perform comparative studies in different animal models of disease, helping understand the impact of several pathological conditions on brain vasculature and how those alterations predispose to neurodegenerative conditions.
In situ analysis of microvascular pericytes in hypertensive rat brains
Tissue and Cell, 1988
We used immunofluore\cence nucroscopy and Iwactin-specific antlhodies to characterue the pattern and prevalence of pcricytes wthin the brain mxrocirculat~on. Blood pressures of normotensive. Wistar-Kyoto (WKY) and spontaneously hypertenwe (SHR) rat\ were measured prmr to sacrifice and pressure-perfusion fixation. WKY and SHR brains wrc whdivided into ten major regions prior to ultracryomlcrotomy. Sections O..?-0.5 ym wide M'CIC treated wth l&40 pg/ml affimty-purified antihodie\ to the muscle and non-muscle actin isoforms. These localizatmn studies show that there are four tlrnch the number of pcricytc-rich capillaries m the SHR motor cortex compared to WKY counterparts (59.9 \b. 15.35) In contrast. the sensory cortex of both rat strains is deficient m muscle actin staining burroundmg the capillaries. The most striking difference in pericyte presence and muscle actm antihod! staining between the SHR and WKY was ohservcd in the tcgmentum of the hrainstem. There I\ nearly a one-to-one coincidence ohservcd m perlcyte and capillary profiles present within thin. frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched WK\ microvessels of these hramatem regions were pericyte-poor. Tranumssion electron nwroscop~c analyses ot plastic embedded thin sections confirmed the prcwnce of perxytca and their filament-enriched processes encircling the capillaries of the hvpcrtcnswe hrams. These roultr suggest that pericytes may play Important role\ in hyperts&n and cerehrovascular dl\ca\c processe,
Ultrastructural studies on cerebrovascular permeability in acute hypertension
Acta Neuropathologica, 1975
Acute hypertension, experimentally induced by intravenous injection of metaraminol in adult rabbits, rapidly induced a damage of the blood-brain barrier in the cerebral cortex, as visualized by Evans-blue-conjugated albumin and horseradish peroxidase. Extravasation of these two exogenous tracers was demonstrated to occur in arterioles, in capillaries and, rarely, in venules. Peroxidase passed the endothelial cell into the nervous tissue in either of three different ways, i. e. through channels, often sigmoidshaped, in the cytoplasm, and transendothelial pinocytosis. The third pathway could, although rarely, be demonstrated between adjacent endothelial cells after cleavage of junctional complexes. The tracer peroxidase was further spread along the blood vessel within the basement membrane and in the extracellular space of the brain. Damaged endothelial cells with diffuse cytoplasmic peroxidase aetivity and large vesicles were occasionally observed within the areas with blood-brain barrier injury. There were also signs of increased pinocytotic activity in endothelial cells outside the barrier damaged cortical areas. Nerve cells and neuroglial cells could show either a diffuse cytoplasmic peroxidase activity or a vesicular location of the tracer, or sometimes both.
Annals of The New York Academy of Sciences, 1988
Blood vessels throughout the brain and spinal cord are surrounded by longitudinal fluid pathways that are in continuity with cerebrospinal fluid (CSF) in the subarachnoid space (SAS).'.' These pathways can be demonstrated light microscopically by infusion of the tracer protein, horseradish peroxidase (HRP), into the SAS and subsequent localization of the enzyme in brain sections using the highly sensitive chromogen, tetramethylbenzidine (TMB)? HRP enters the perivascular spaces (PVS) around penetrating arterioles and, within minutes, is distributed throughout the CNS along the basal laminae (BL) of capillaries. This rapid, unidirectional fluid/tracer movement along the vascular network appears to be facilitated by arteriolar pulsations? From these "paravascular pathways," HRP spreads into the tissue spaces and also selectively delineates localized groups of neurons.