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Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis

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Abstract

The endothelins (ETs) are a family of peptides that exert their biological effects via two distinct receptors, the endothelin A receptor (ETAR) and the endothelin B receptor (ETBR). To more clearly define the potential actions of ETs following spinal cord injury, we used immunohistochemistry and confocal microscopy to examine the protein expression of ETAR and ETBR in the normal and injured rat spinal cord. In the normal spinal cord, ETAR immunoreactivity (IR) is expressed by vascular smooth muscle cells and a subpopulation of primary afferent nerve fibers. ETBR-IR is expressed primarily by radial glia, a small population of gray and white matter astrocytes, ependymal cells, vascular endothelial cells, and to a lesser extent in smooth muscle cells. Fourteen days following compression injury to the spinal cord, there was a significant upregulation in both the immunoexpression and number of astrocytes expressing the ETBR in both gray and white matter and a near disappearance of ETBR-IR in ependymal cells and ETAR-IR in primary afferent fibers. Conversely, the vascular expression of ETAR and ETBR did not appear to change. As spinal cord injury has been shown to induce an immediate increase in plasma ET levels and a sustained increase in tissue ET levels, ETs would be expected to induce an initial marked vasoconstriction via activation of vascular ETAR/ETBR and then days later a glial hypertrophy via activation of the ETBR expressed by astrocytes. Strategies aimed at blocking vascular ETAR/ETBR and astrocyte ETBRs following spinal cord injury may reduce the resulting ischemia and astrogliosis and in doing so increase neuronal survival, regeneration, and function.

Introduction

The endothelins (ETs) are a family of 21-amino-acid peptides consisting of three isoforms: ET-1, ET-2, and ET-3. Within the normal mammalian CNS and spinal cord, ET-1 and ET-3 isoforms are expressed in vascular endothelial cells 4, 33, 65 and in a population of neurons (Giaid et al., 1989). Previous studies have shown that both CSF and plasma levels of ET-1 and ET-3 are low in the normal, uninjured CNS 60, 66, but significantly increase following traumatic brain injury 19, 58, spinal cord trauma 39, 52, 61, and stroke (Lampl et al., 1997). The cellular source of these elevated levels of ET is injury dependent and may be due initially to increased synthesis in neuronal and endothelial cells 39, 58, followed by delayed synthesis in reactive astrocytes 23, 34, 35, 58, 67, infiltrating leukocytes (Bertsch et al., 2001), or activated microglia and macrophages 13, 57, 58, 64.

In the mammalian CNS, ETs produce their biological effects via activation of two receptor subtypes, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). ET-1 and ET-2 have greater affinity for ETAR than does ET-3, whereas all three peptides have similar affinities for ETBR. Receptor binding studies in mammals, including humans 11, 24, 45, demonstrated that endothelin receptors are distributed throughout the normal spinal cord. In situ hybridization studies in rat forebrain describe distinct patterns of expression for the two receptor subtypes in the CNS, with ETAR mRNA abundantly expressed in vascular smooth muscle cells, whereas ETBR mRNA is predominantly expressed in glial cells and vascular endothelial cells 21, 42. Immunohistochemical studies have begun to address the cell type expression and protein distribution of these receptor subtypes within the forebrain 30, 58, but similar studies have not been conducted in the spinal cord.

Injury to the spinal cord triggers a myriad of secondary pathogenic events including ischemia, reactive gliosis, and blood–spinal cord barrier breakdown 2, 9. All of these events contribute to the cellular environment within the injured spinal cord and ultimately influence neuronal survival and regeneration. It has been suggested that endothelins are partially responsible for generating some of these events, with ETAR playing a prominent role in posttraumatic ischemia 5, 12, superoxide generation (Kasemsri and Armstead, 1997), and disruption of the blood–spinal cord barrier (McKenzie et al., 1995), whereas ETBR is involved in mediating reactive gliosis 22, 28. However, the exact cellular targets for ET in the normal and injured spinal cord remain to be defined. In the present study, we demonstrate that ETAR and ETBR are expressed on distinct populations of vascular, glial, and neuronal elements in the normal and injured spinal cord. After spinal cord injury there is a profound upregulation of ETBR expression by glial cells, whereas the vascular ETAR/ETBR expression does not appear to change. The present data suggest that ETAR/ETBR expressed by vascular cells and ETBR expressed by glial cells offer a therapeutic target for attenuating the ischemia and gliosis that follows spinal cord injury.

Section snippets

Spinal cord injury surgery

All procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee. Spinal cord injury surgeries were performed on eight adult female Sprague–Dawley rats (Charles River, Wilmington MA), weighing 225–250 g. The procedure was modified from a method established by Martin and Moonen (Martin et al., 1992). Animals were anesthetized using an intramuscular injection of a 2:1 mixture (100 mg/kg) of ketamine and xylazine. The animals were placed in a sterotaxic frame

Localization of endothelin A receptor in the normal and injured rat spinal cord

In the normal rat spinal cord, ETAR-IR was associated with the vasculature throughout the parenchyma of the cord (Fig. 1A). ETAR-IR was associated primarily with vascular smooth muscle cells as determined by double labeling with the marker α-actin (data not shown). The cell type expression of ETAR was also confirmed using morphological criteria. ETAR-IR cells possessed a banded appearance with cells forming ring-like structures around the endothelial cell layer of blood vessels typical of

The expression of ETAR and ETBR in the normal and injured spinal cord

In the present report we have focused on ETR expression in the spinal cord and the potential role these receptors may play in spinal cord injury. In general, the pattern of ETR expression in spinal cord vasculature is very similar to that reported in peripheral vascular beds where ETARs are expressed primarily by smooth muscle cells and ETBRs are expressed by vascular smooth muscle cells and endothelial cells 41, 46, 55. The present study also agrees with previous studies in rat forebrain

Acknowledgements

This work was supported by a VA Merit Review, National Institute of Neurological Disorders and Stroke Grant NS23970, and National Institute on Drug Abuse Grant DA11986. The authors thank Dr. Mary Ann Sabino for helpful discussion.

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