Spinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair

doi:10.1006/exnr.1996.6405

Experimental Neurology

Volume 144, Issue 2, April 1997, Pages 381-399

https://doi.org/10.1006/exnr.1996.6405 Get rights and content

Abstract

This study examines morphological changes of the blood–brain barrier (BBB) after spinal cord compression. The lowest thoracic segment (T13) of female guinea pigs was injured and the BBB was tested from 7 days to 5.5 months postinjury using intravenously injected horseradish peroxidase (HRP) as a tracer. Tracer leakage in the injured segment was verified with the light microscope and the fine structure of capillaries was examined. Diffuse tissue staining was observed at T13 up to 2 weeks following injury. A leaky BBB correlated with expected changes in the fine structure of endothelial cell junctions. These were predominantly nonoverlapping cell junctions which, in many instances, were separated by clefts between adjacent cells. At early survival times, numerous capillary profiles with juxtaposed astrocyte foot processes were noted in addition to altered cell associations. Complete sealing of the BBB against interstitial HRP leakage was not observed until 17 days postinjury. After the first week, some of the endothelial cells were contacted by macrophages, processes of perivascular microglia, and processes of swollen and degenerating astrocytes. Perivascular spaces varied in extent and contained amorphous deposits of extracellular materials in addition to supernumerary layers of basal lamina. The early changes were followed by profound tissue restructuring due to loss of both neurons and glia. At longer survival times the BBB to HRP repaired. Endothelial cells formed complex overlapping junctions with zonulae occludentes. Most of the capillaries in the injured segment were no longer in direct contact with astrocyte foot processes, although reactive astrocytes constituted the predominant cell type in the remaining gray matter. Substantial expansion of perivascular spaces was evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular spaces contained layers of assembled collagen arranged perpendicularly to each other in addition to amorphous matrix materials. The findings suggest that decoupling of astrocyte foot processes from endothelial cell surfaces does not prevent reformation of tight junctions. It remains to be examined what effects the larger perivascular spaces, extracellular matrix deposits, and changes of cell associations may have on transport systems and ionic buffering. The data are relevant for estimating an opportune time for application of barrier-impermeable drugs to the lesion area.

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The range of therapeutic treatment options for central nervous system (CNS) diseases is greatly limited by the blood-brain barrier (BBB). While a variety of strategies to circumvent the blood-brain barrier for drug delivery have been investigated, little clinical success has been achieved. Focused ultrasound (FUS) is a unique approach whereby the transcranial application of acoustic energy to targeted brain areas causes a noninvasive, safe, transient, and targeted opening of the BBB, providing an avenue for the delivery of therapeutic agents from the systemic circulation into the brain. There is a great need for viable treatment strategies for CNS diseases, and we believe that the preclinical success of this technique should encourage a rapid movement towards clinical testing. In this review, we address the versatile applications of FUS-mediated BBB opening, the safety profile of the technique, and the physical and biological mechanisms that drive this process.
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Over-expression of laminin correlates to recovery of vasogenic edema following status epilepticus
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In contrast to dystrophin expression, laminin expression is remarkably upregulated in the BMs of vessels after SE in parallel with the astroglial loss (Biagini et al., 2008; Gualtieri et al., 2012). This laminin over-expression is explained by an improved accessibility of laminin epitopes in damaged blood vessels (Szabó and Kálmán, 2004; Kálmán et al., 2011), since the astrocytic anchoring to BMs is exposed in consequence of brain damage and inflammation (Jaeger and Blight, 1997; Kálmán et al., 2011; Gualtieri et al., 2012). Therefore, up-regulation of laminin expression is one of the consequences from BBB modification and predicts astroglial damage following SE (Gualtieri et al., 2012).
In the present study, we addressed the question of whether the up-regulation of laminin expression represents the astroglio-vascular responses to status epilepticus (SE) in the rat brain to better understand the role of vasogenic edema in epileptogenic insult. In the hippocampus, vasogenic edema was observed in the hippocampus 12 h after SE when astroglial degeneration was undetected. Vasogenic edema in the hippocampus was more severe in the CA1 region where astroglial loss was absent than in the dentate gyrus showing astroglial degeneration. In the piriform cortex (PC), vasogenic edema was accompanied by appearance of astroglial degeneration 12 h after SE. Laminin expression in the hippocampus and the PC was increased 3 days and 4 days after SE, respectively. Laminin expression was up-regulated in the hippocampus and the PC with concomitant reduction of SMI-71 (the endothelial barrier antigen) expression. Four weeks after SE, laminin expression was reduced in vessels showing strong SMI-71 expression within vasogenic edema lesion. Inhibition of SE-induced vasogenic edema formation by BQ788 effectively prevented laminin over-expression. Therefore, our findings indicate that laminin over-expression may be one of consequences from vasogenic edema rather than astroglial loss, and that laminin over-expression may promote migration of astrocytes to damaged or newly generated vessels to repair brain–blood barrier (BBB) disruption accompanied by the reconstruction of endothelial barrier.
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The astrocytic end-feet are anchored to this capillary basal lamina by the so-called dystrophin–glycoprotein complex, which is linked to laminin and is crucial to BBB integrity (Abbott et al., 2006; del Zoppo et al., 2006; Wolburg et al., 2009). On the other hand, the astrocytic anchoring to capillary basal lamina is released in consequence of tissue damage and inflammation (Jaeger and Blight, 1997; Kálmán et al., 2011) or angiogenesis (Sixt et al., 2001). In view of these data, it appeared interesting to examine the relationship between the vascular changes that follow SE and glial cells, especially astrocytes.
Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet not systemically investigated astrocytic and vascular injuries. To investigate their possible association with neuronal damage, the changes in glial fibrillary acidic protein (GFAP), laminin and neuron-specific nuclear protein (NeuN) immunoreactivities were analyzed in rats treated with pilocarpine (380 mg/kg) or kainate (15 mg/kg), and receiving diazepam (20 mg/kg) after 10 min of SE. A different group of rats was injected with endothelin-1 (ET-1) in the caudate putamen to reproduce the changes in GFAP and laminin immunoreactivities associated with ischemia. Focal loss of GFAP immunostaining was accompanied by increased laminin immunoreactivity in blood vessels, in all the examined groups. Regression analysis revealed a significant (P < 0.01) relationship between astrocytic lesion and increased laminin immunoreactivity in the piriform cortex (Pir) of both pilocarpine (R² = 0.88) and kainate (R² = 0.94) groups of treatment. A significant relationship (P < 0.01; R² = 0.81) was also present in the cornu Ammonis 3 (CA3) hippocampal region of pilocarpine-treated rats. At variance, neuronal and glial lesions were significantly related (P < 0.05, R² = 0.74) only in the substantia nigra of pilocarpine-treated rats. The ratio between areas of GFAP and laminin changes of immunoreactivity in the ET-1 group was similar to those found in pilocarpine- and kainate-treated rats in specific brain regions, such as the hippocampal CA3 subfield, Pir and the anterior olfactory nucleus. The amygdala and submedius thalamic nucleus in the pilocarpine group, and the perirhinal and entorhinal cortices in the kainate group, also presented ischemic-like changes. These results indicate that laminin immunoreactivity is upregulated in the basal lamina of blood vessels after SE induced by pilocarpine or kainate. This phenomenon is significantly associated with lesions involving more glial than neuronal cells, in specific cerebral regions.
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Severance of posterior and anterior spinal veins and arteries that lie on the surface of the cord supplying the interior gray matter causes an ischaemic insult to the targeted segment as well as below the level of the lesion (Sandler and Tator, 1976). There is loss of the blood–brain barrier (BBB), internal hemorrhage (Jaeger and Blight, 1997; Maikos and Shreiber, 2007), altered blood flow (Senter and Venes, 1978), and endothelial cell death (Benton et al., 2008) at the site of injury. These events further prolong and exacerbate ischaemia and render the BBB permeable.
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^☆: Supported by the School of Veterinary Medicine, Purdue University and Public Health Service Grant RO1-NS 21122 to A.R.B.

^☆☆: A. J. SucklingM. G. RumsbyM. W. B. Bradbury, Eds.

²: To whom correspondence should be addressed at present address: Department of Basic Medical Sciences, Purdue University, School of Veterinary Medicine, Lynn Hall, West Lafayette, IN 47907-1246.

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Regular ArticleSpinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair☆,☆☆

Abstract

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Brain Res.

The role of the macrophage in micovascular regeneration following brain injury

J. Neuropathol. Exp. Neurol.

Transendothelial vesicular transport of protein following compression injury to the spinal cord

Lab. Invest.

Quinolinic acid accumulation and functional deficits following experimental spinal cord injury

Brain

Appraisal of the role of endothelial cells and glia in barrier breakdown

The Blood–Brain Barrier in Health and Disease

Implication of astroglia in the blood–brain barrier

Ann. N. Y. Acad. Sci.

Endothelial cell biology and the enigma of transcytosis through the blood–brain barrier

Adv. Exp. Med. Biol.

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Cell Tissue Res.

An Introduction to the Blood–Brain Barrier

Peptides and blood–brain barrier transport

Physiol. Rev.

Exudation of fibronectin and albumin after spinal cord injury in rats

Acta Neuropathol. (Berlin)

Receptor mediated transport of peptides and proteins across the blood–brain barrier

Regular Article
Spinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair☆,☆☆