Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia.

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

Subcellular localization of transporters along the rat blood-brain barrier and blood-cerebral-spinal fluid barrier by in vivo biotinylation.

Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Time course for expression of VEGF and its receptor and regulator levels of contraction and relaxation in increased vascular permeability of lung induced by phosgene.

Acute lung injury (ALI) induced by phosgene increases risk of serious edema and mortality. Increased permeability of the microvascular endothelium is implicated in the progression of ALI, but the processing interaction and time course activity of the vascular regulators in exudation are still not understood. The main aim of this study was to investigate the time course and potential role for vascular endothelial growth factor (VEGF), its receptors, and some vascular function regulators related to increased vascular permeability of lung induced by phosgene. Sprague Dawley rats were randomly divided into seven groups according to time post phosgene exposure (control, and 1, 3, 6, 12, 24, and 48 h groups). Lung tissue was removed to evaluate VEGF isoforms, fms-like tyrosine kinase receptor 1 (Flt-1), and kinase insert domain containing region (KDR/Flk-1) by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Blood samples were collected for measurement of plasma endothelin-1 (ET-1) and nitric oxide (NO) level. The results showed that the mRNA and protein expression profile of the VEGF system after phosgene exposure was time dependent. The VEGF system expression in lung tissue was related closely to the level of ET-1 and NO. In conclusion, increased permeability of the lung microvascular endothelium induced by phosgene was primarily a result of differential expression of VEGF and its receptors, and was related to the level of ET-1 and NO. The results suggest that the cooperation of VEGF system, ET-1, and NO plays a critical role, and all those parameters emerge as time dependent in the early phase of the permeability process induced by phosgene exposure.

Histochemical study of the definitive erythropoietic foci in the chicken yolk sac.

It is well known that avian yolk sac is involved in both primitive and definitive erythropoiesis during embryonic development. Definitive erythropoiesis is first detected at about 4-5 days incubation and its maximum activity is reached between day 10 and 15 of incubation, ending between days 18 and 20 of incubation. We confirmed the definitive erythropoietic foci in the chicken yolk sac throughout the 5th to 19th day of incubation by histochemical light and electron microscopy. The definitive erythropoietic foci were observed in the yolk sac endodermal layer from day 5 until day 19, just before hatching. Ultrastructurally, definitive erythropoietic foci were observed extravascularly in the yolk sac endodermal cell layer in direct contact with the vitellolysis zone. These findings provide a basis for clarifying definitive erythropoiesis in vertebrates.

Gap filling of 3-D microvascular networks by tensor voting.

We present a new algorithm which merges discontinuities in 3-D images of tubular structures presenting undesirable gaps. The application of the proposed method is mainly associated to large 3-D images of microvascular networks. In order to recover the real network topology, we need to fill the gaps between the closest discontinuous vessels. The algorithm presented in this paper aims at achieving this goal. This algorithm is based on the skeletonization of the segmented network followed by a tensor voting method. It permits to merge the most common kinds of discontinuities found in microvascular networks. It is robust, easy to use, and relatively fast. The microvascular network images were obtained using synchrotron tomography imaging at the European Synchrotron Radiation Facility. These images exhibit samples of intracortical networks. Representative results are illustrated.

NADPH-diaphorase activity and neurovascular coupling in the rat cerebral cortex.

The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.

Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium.

Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. Ultrasound bursts (1.5-MHz) in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJ-specific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of i.v. administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La(3+), m.w. approximately 139 Da). Leakage of these agents was observed at 1 and 2 h and, in a few vessels, at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain.

Microvascular preconditioning is not detectable by corrosion casting in the isolated perfused rat heart after 30 minutes of ischaemia.

OBJECTIVE: Ischaemic preconditioning protects the myocardium from ischaemic injury and may also protect the vascular endothelium from the deleterious effects of ischaemia and reperfusion. We examined the possibility that ischaemic preconditioning might preserve the integrity of the coronary microcirculation following ischaemia and reperfusion. METHODS: Isolated rat hearts were perfused in Langendorff mode for 30 minutes and then subjected to 30 minutes of global ischaemia with or without ischaemic preconditioning (threexthree minute cycles). Some hearts underwent an additional 60 minutes of reperfusion. At the end of each protocol, microvascular corrosion casts were made by methylmethacrylate injection. RESULTS: Median left ventricular capillary density [interquartile range] after ischaemia was slightly but not significantly better with preconditioning at 6.8 [4.0-14.7]x10(-2) mm3.mg(-1) vs. 5.2 [2.6-7.1]x10(-2) mm3.mg(-1) (p=0.13). After 60 min of reperfusion, capillary density in preconditioned left ventricles was 20.7 [10.7-22.8]x10(-2) mm3.mg(-1) vs. 16.0 [10.2-23.0]x10(-2) mm3.mg(-1) for untreated ventricles (p=0.47). Coronary blood flow and heart rate were unchanged from before ischaemia. CONCLUSIONS: Ischaemia for 30 minutes induced global left ventricular capillary loss which was unmodified by preconditioning. We did not demonstrate vascular preconditioning using this model.

Transient regulation of transport by pericytes in venular microvessels via trapped microdomains.

A phenomenon that has defied explanation for two decades is the time scale for transient reabsorption in the classic experiments of Michel and Phillips on individually perfused frog mesentery microvessels. One finds that transient reabsorption lasts <2 min before a new steady state of low filtration is established when the lumen pressure is abruptly dropped from a high to a low value. Our experiments in frog and rat venular microvessels under a variety of conditions revealed the same time trend for new steady states to be established as in Michel and Phillips' experiments. In contrast, one theoretically predicts herein that the time required for the tissue albumin concentration to increase to values for a new steady state to be achieved through reabsorption is in the order of several hours. In this paper we propose a new hypothesis and theoretical model for this rapid regulation, namely that pericytes covering the interendothelial cleft exits create small trapped microdomains outside the cleft exits which regulate this transient behavior. Our electron microscopy studies on rat mesenteric venular microvessels reveal an average pericyte coverage of approximately 85%. The theoretical model based on this ultrastructural study predicts an equilibration time on the order of 1 min when the lumen pressure is abruptly lowered. The basic concept of a trapped microdomain can also be extended to microdomains in the interstitial space surrounding skeletal muscle capillaries.

Bridging mucosal vessels associated with rhythmically oscillating blood flow in murine colitis.

Oscillatory blood flow in the microcirculation is generally considered to be the result of cardiopulmonary influences or active vasomotion. In this report, we describe rhythmically oscillating blood flow in the bridging vessels of the mouse colon that appeared to be independent of known biological control mechanisms. Corrosion casting and scanning electron microscopy of the mouse colon demonstrated highly branched bridging vessels that connected the submucosal vessels with the mucosal plexus. Because of similar morphometric characteristics (19 +/- 11 microm vs. 28 +/- 16 microm), bridging arterioles and venules were distinguished by tracking fluorescent nanoparticles through the microcirculation using intravital fluorescence videomicroscopy. In control mice, the blood flow through the bridging vessels was typically continuous and unidirectional. In contrast, two models of chemically induced inflammation (trinitrobenzenesulfonic acid and dextran sodium sulfate) were associated with a twofold reduction in flow velocity and the prominence of rhythmically oscillating blood flow. The blood oscillation was characterized by tracking the bidirectional displacement of fluorescent nanoparticles. Space-time plots and particle tracking of the oscillating segments demonstrated an oscillation frequency between 0.2 and 5.1 cycles per second. Discrete Fourier transforms demonstrated a power spectrum composed of several base frequencies. These observations suggest that inflammation-inducible changes in blood flow patterns in the murine colon resulted in both reduced blood flow velocity and rhythmic oscillations within the bridging vessels of the mouse colon.

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells.

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.

A further observation of the structural changes of microvessels in the extensor digitorum longus muscle of the aged rat.

We further examined the structural changes of microvessels in the extensor digitorum longus muscle of the aged (18 months) rat. Muscle bundles in this aged muscle constantly consisted of numerous large muscle fibers 50-60 mum in diameter and a few small muscle fibers <30 mum in diameter. Neuromuscular junctions (NMJs) in large muscle fibers often showed degenerative figures, thus degenerating muscle fibers. On the other hand, NMJs in small muscle fibers were mainly characterized by sparse and short junctional folds, being possibly in the course of regeneration. In some muscle bundles, the extracellular matrix was a little widened. Microvascular networks from arterioles to venules via capillaries seemed to vary in structural features between muscle bundles. In addition to the normal microvascular network consisting of microvessels with a round or oval vascular lumen during their course, two different types of microvascular networks were found. One type was characterized by the constriction of arterioles, capillaries and venules, probably representing a degenerative process of the microvascular network. In fact, uneven and compressed scaffolds of basal laminae of capillaries were often observed around these constricted microvessels. The other type consisted of arterioles and capillaries with an irregular slit-like vascular lumen and venules with a round or oval vascular lumen, and these capillaries had thick or two-layered basal laminae, being probably in the course of remodeling of the microvascular network. From these findings, it is suggested that the constriction and/or contraction of microvessels by smooth muscle cells and pericytes may be involved in the degeneration and remodeling of the microvascular network in the muscle bundles following degeneration and regeneration of the muscle fibers.

In vivo dynamic light scattering microscopy of tumour blood vessels.

We present a study investigating the use of dynamic light scattering microscopy based on the temporal laser speckle's contrast that is produced over time by red blood cells (RBCs) flowing inside tumour blood vessels. The proposed noninvasive methodology is capable of producing high-resolution images of tumour vasculature. The technique is effective at producing images from tissue at a significant depth, as well as potentially having the ability to monitor tumour perfusion. An advantage of this methodology is that it has improved depth penetration compared with conventional imaging techniques (such as reflected-light microscopy), and one can avoid the use of any fluorescent or artificial chemicals for labeling. This is advantageous since labeling materials can affect imaging and animal welfare with respect to experiments that require continuous and repetitive monitoring.

Structural changes of microvessels in the extensor digitorum longus muscle of the aged rat.

We examined the structural changes of microvessels (arterioles, capillaries, and venules) in the extensor digitorum longus muscle of the aged (27 months) rat. Muscle bundles in the aged muscle almost consisted of medium-sized muscle fibers which were peculiar in the aged EDL muscle. However, microvessels in and around these muscle bundles varied in shape. Degenerating capillaries and scaffolds of basal laminae remaining after necrosis of preexisting capillaries were frequently observed around these medium-sized muscle fibers. In addition, the vascular lumen was often very narrow or irregular slit-like. In terminal and precapillary arterioles, the endothelium and smooth muscle cells showed a constricted appearance and their vascular lumen was often irregular slit-like, probably playing an important role in intercepting the blood flow into the disrupted capillaries. Moreover, some venules had the slit-like vascular lumen, sawtooth-like endothelium and thick or multilayered basal laminae, and occasional erythrocytes were found outside the endothelium, probably indicating that these venules are in the course of regeneration. These findings suggest that in addition to the frequent destruction of capillaries, the structural changes of arterioles and venules may be involved in remodeling the microvasculature of the muscle bundles after maturation of regenerating muscle fibers.

A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix.

The fenestrated microvasculature of the area postrema shows a less restrictive blood-brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10-70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood-brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema.

Scanning electron microscopy of vascular corrosion casts--standard method for studying microvessels.

Scanning electron microscopy (SEM) of vascular corrosion casts (microvascular corrosion casting/SEM method) is a standard method, which allows three-dimensional visualization with good resolution of the normal and abnormal microvessels, including the capillaries of various organs and tissues. SEM of vascular corrosion casts can obtain qualitative as well as quantitative informations important to anatomists, pathologists and clinicians. Considering these, the history, the advantages and the main steps of this technique including general morphological characteristics of vascular casts observed in SEM are reviewed in this paper. Corrosion casts done by the author representing the microvascular organization of the rat liver and kidney observed in SEM are, also, presented.

Alterations in cerebral cortex microvessels and the microcirculation in a rat model of traumatic brain injury: a correlative EM and laser Doppler flowmetry study.

OBJECTIVES: We sought to establish the temporal association of fluctuations in cortical cerebral blood flow (CBF) with ultrastructural alterations of microvessels in rat sensorimotor cortex (smCx) following administration of a rodent acceleration impact model of traumatic brain injury (TBI). METHODS: Laser Doppler flowmetry (LDF) and electron microscopy (EM) were used in parallel experiments that lasted for up to 48 hours after induction of TBI. RESULTS: Compared to sham-operated control, there was a 37% reduction of cortical CBF between 12 and 24 hours, this reduction remaining unchanged for up to 48 hours post-TBI. Ultrastructural alterations in the lumen and wall of smCx microvessels, including endothelial cell distortion and luminal collapse, were seen at hour 1 and continued up to 48 hours after trauma. Compared to control, there was a 40% decrease in the average microvascular luminal area 4 hours and a trend to recover (21%) by 48 hours after trauma. Smooth muscle (SM) in the wall of reacting microvessels showed evidence of increase contractility that coincided temporally with the decreased perfusion of cortical CBF. DISCUSSION: Based on these observations, it is proposed that TBI causes alterations in the vascular tone of reacting microvessels which leads to prolonged vasoreactivity and restriction of the lumen in many but not all microvessels.

Spatial alterations in endothelin receptor expression are temporally associated with the altered microcirculation after brain trauma.

OBJECTIVES: To study the cellular distribution of endothelin receptors A and B (ETrA and ETrB) in the post-traumatic sensorimotor cortex and hippocampus. MATERIALS AND METHODS: We inflicted closed head trauma to male Sprague-Dawley rats and visualized ETrA and ETrB immunoreactivity with 3,3'-diaminobenzidine. RESULTS: ETrA immunolabeling was the most prominent in pyramidal neurons 24 and 48 hours post-trauma, while it reached its peak in the microvasculature at hour 4. ETrB immunolabeling was observed in endothelial cells, perivascular neurons, smooth muscle cells (SM) and pericytes, the expression being the most pronounced 24 hours post-trauma. DISCUSSION: The results suggest that the vasoconstrictor effect of endothelin-1 (ET-1) is mediated primarily by ETrA. The dual effects of ETrB are reflected in its vasoconstrictor role at the vascular bed and conversely, in the attenuation of ET-1 availability and synthesis. We conclude that both receptors play a role in the disturbed microvascular autoregulation and in the sustained reduction of blood flow following trauma to the brain.

Microvascular architecture of the near-term uterine caruncles in water buffaloes (Bubalus bubalis).

The present investigation was carried out on five near-term pregnant water buffaloes for studying the microvascular architecture of the uterine caruncles. The vascular casts were obtained by injection of 4:1 mixture of mercox and methylmethacrylate through the branches of the uterine arteries. After complete polymerization of the plastic, corrosion was conducted in 20% potassium hydroxide, then the vessel casts were immersed in distilled water, cut into small pieces, sputter coated with gold, and examined by using a scanning electron microscope. The buffalo uterine caruncle is highly vascularized through two slightly convoluted arteries and a single less tortuous vein. The arteries branch into several stem arteries at the base of the uterine caruncle, which follow nearly straight course in the primary septa towards the fetal side. During the courses of these stem arteries arterioles of variable diameters arise. The arterioles run in the secondary and tertiary septae and at this location arterioles and venules are connected through a voluminous capillary complex. The latter consists of capillaries of greatly variable diameters with vigorous coiling and sinusoidally dilated zones. From the capillary complexes the blood is driven through postcapillary venules back to the tertiary, secondary and primary septa, respectively, and then converge into stem veins which leave the caruncles through the branches of the uterine vein.