Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain barrier leakage and active demyelination

Blood-brain barrier (BBB) hyperpermeability in multiple sclerosis (MS) is associated with lesion pathogenesis and has been linked to pathology in microvascular tight junctions (TJs). This study quantifies the uneven distribution of TJ pathology and its association with BBB leakage. Frozen sections from plaque and normal-appearing white matter (NAWM) in 14 cases were studied together with white matter from six neurological and five normal controls. Using single and double immunofluorescence and confocal microscopy, the TJ-associated protein zonula occludens-1 (ZO-1) was examined across lesion types and tissue categories, and in relation to fibrinogen leakage. Confocal image data sets were analysed for 2198 MS and 1062 control vessels. Significant differences in the incidence of TJ abnormalities were detected between the different lesion types in MS and between MS and control white matter. These were frequent in oil-red O (ORO)+ active plaques, affecting 42% of vessel segments, but less frequent in ORO- inactive plaques (23%), NAWM (13%), and normal (3.7%) and neurological controls (8%). A similar pattern was found irrespective of the vessel size, supporting a causal role for diffusible inflammatory mediators. In both NAWM and inactive lesions, dual labelling showed that vessels with the most TJ abnormality also showed most fibrinogen leakage. This was even more pronounced in active lesions, where 41% of vessels in the highest grade for TJ alteration showed severe leakage. It is concluded that disruption of TJs in MS, affecting both paracellular and transcellular paths, contributes to BBB leakage. TJ abnormality and BBB leakage in inactive lesions suggests either failure of TJ repair or a continuing pathological process. In NAWM, it suggests either pre-lesional change or secondary damage. Clinically inapparent TJ pathology has prognostic implications and should be considered when planning disease-modifying therapy

Altered pericyte-endothelial relations in the rat retina during aging: Implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of a-smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte-endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels.

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle.

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37 degrees C using the perforated patch-clamp technique with Cs(+)- and K(+)-filled pipettes. Depolarizing steps evoked currents that consisted of L-type Ca(2+) [I(Ca(L))] current and a slowly developing current. The slow current reversed at 1 +/- 1.5 mV with symmetrical Cl(-) concentrations compared with 23.2 +/- 1.2 mV (n = 5) and -34.3 +/- 3.5 mV (n = 4) when external Cl(-) was substituted with either glutamate (86 mM) or I(-) (125 mM). Nifedipine (1 microM) blocked and BAY K 8644 enhanced I(Ca(L)), the slow-developing sustained current, and the tail current. The Cl(-) channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl(-) equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca(2+)-activated Cl(-) current plays an important role in the electrical activity underlying spontaneous activity in this tissue. PMID: 11029279 [PubMed - indexed for MEDLINE]

Hyperpolarisation-activated inward current in isolated sheep mesenteric lymphatic smooth muscle.

1. Freshly isolated sheep lymphatic smooth muscle cells were studied using the perforated patch-clamp technique. Hyperpolarisation with constant-current pulses caused a time-dependent rectification evident as a depolarising 'sag' followed by an anode-break overshoot at the end of the pulse. Both sag and overshoot were blocked with 1 mM Cs+. 2. Cells were voltage clamped at -30 mV and stepped to -120 mV in 10 mV steps of 2 s duration. Steps negative to -60 mV evoked a slowly activating, non-inactivating inward current which increased in size and rate of activation with increasing hyperpolarisation. 3. The slowly activating current was reduced in Na+-free bathing solution but enhanced when the extracellular K+ concentration was increased to 60 mM. The current was significantly reduced by 1 mM Cs+ and 1 microM ZD7288 but not by 1.8 mM Ba2+. 4. The steady-state activation curve of the underlying conductance showed a threshold at -50 mV and half-maximal activation at -81 mV. Neither threshold nor half-maximal activation was significantly affected by increasing the external K+ concentration to 60 mM. 5. The frequency of spontaneous contractions and fluid propulsion in isolated cannulated segments of sheep mesenteric lymphatics were decreased by 1 mM Cs+ and by 1 microM ZD7288. 6. We conclude that sheep lymphatics have a hyperpolarisation-activated inward current similar to the If seen in sinoatrial node cells of the heart. Blockade of this current slows spontaneous pumping in intact lymphatic vessels suggesting that it is important in normal pacemaking.

Tetrodotoxin-sensitive sodium current in sheep lymphatic smooth muscle.

1. Fast inward currents were elicited in freshly isolated sheep lymphatic smooth muscle cells by depolarization from a holding potential of -80 mV using the whole-cell patch-clamp technique. The currents activated at voltages positive to -40 mV and peaked at 0 mV. 2. When sodium chloride in the bathing solution was replaced isosmotically with choline chloride inward currents were abolished at all potentials. 3. These currents were very sensitive to tetrodotoxin (TTX). Peak current was almost abolished at 1 microM with half-maximal inhibition at 17 nM. 4. Examination of the voltage dependence of steady state inactivation showed that more than 90% of the current was available at the normal resting potential of these cells (-60 mV). 5. The time course of recovery from inactivation was studied using a double-pulse protocol and showed that recovery was complete within 100 ms with a time constant of recovery of 20 ms. 6. Under current clamp, action potentials were elicited by depolarizing current pulses. These had a rapid upstroke and a short duration and could be blocked with 1 microM TTX. 7. Spontaneous contractions of isolated rings of sheep mesenteric lymphatic vessels were abolished or significantly depressed by 1 microM TTX.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.

Fast Retinal Vessel Detection and Measurement Using Wavelets and Edge Location Refinement

The relationship between changes in retinal vessel morphology and the onset and progression of diseases such as diabetes, hypertension and retinopathy of prematurity (ROP) has been the subject of several large scale clinical studies. However, the difficulty of quantifying changes in retinal vessels in a sufficiently fast, accurate and repeatable manner has restricted the application of the insights gleaned from these studies to clinical practice. This paper presents a novel algorithm for the efficient detection and measurement of retinal vessels, which is general enough that it can be applied to both low and high resolution fundus photographs and fluorescein angiograms upon the adjustment of only a few intuitive parameters. Firstly, we describe the simple vessel segmentation strategy, formulated in the language of wavelets, that is used for fast vessel detection. When validated using a publicly available database of retinal images, this segmentation achieves a true positive rate of 70.27%, false positive rate of 2.83%, and accuracy score of 0.9371. Vessel edges are then more precisely localised using image profiles computed perpendicularly across a spline fit of each detected vessel centreline, so that both local and global changes in vessel diameter can be readily quantified. Using a second image database, we show that the diameters output by our algorithm display good agreement with the manual measurements made by three independent observers. We conclude that the improved speed and generality offered by our algorithm are achieved without sacrificing accuracy. The algorithm is implemented in MATLAB along with a graphical user interface, and we have made the source code freely available.