Intraspinal Application of Endothelin Results in Focal Ischemic Injury of Spinal Gray Matter and Restricts the Differentiation of Engrafted Neural Stem Cells

Abstract

Previous data have shown that pluripotent stem cells engrafted into the contused spinal cord differentiate only along an astrocytic lineage. The unknown restrictive cues appear to be quite rigid as even neuronal-restricted precursors fail to differentiate to the mature potential they exhibit in vitro after similar grafting into the contused spinal cord. It has been hypothesized that this potent lineage restriction is, in part, the result of the significant loss of both gray and white matter observed following spinal contusion, which elicits a massive acute inflammatory response and is manifested chronically by dramatic cystic cavitation. To evaluate the gray matter component, we developed a clinically relevant model of focal gray matter ischemic injury using the potent vasoconstrictor endothelin (ET-1) and characterized the differentiation of pluripotent stem cells transplanted into this atraumatic vascular SCI. Results demonstrate that low dose ET-1 microinjection into cervical spinal gray matter results in an inflammatory response that is temporally comparable to that observed following traumatic SCI, as well as chronic gray matter loss, but without significant cystic cavitation or white matter degeneration. However, despite the preservation of host spinal parenchyma, no elaboration of neuronal phenotypes was observed from engrafted stem or precursor cells. These results suggest that a common pathologic component responsible for this lineage restriction exists between contusive SCI and ET-1 mediated focal ischemic SCI.

Transcriptional Activation of Endothelial Cells by TGFβ Coincides with Acute Microvascular Plasticity following Focal Spinal Cord Ischaemia/Reperfusion Injury

“…Although this implicates direct activation of neurons, little data exist demonstrating receptor-mediated neurotoxic actions of ET-1. Indeed, previous results show that direct application of 1 μM ET-1 to mixed cultures of neonatal neurons and astrocytes results in neither cell death nor potentiation of excitotoxicity, whereas as little as 50 nM is sufficient for cellular activation, primarily via ET B (Benton et al, 2005). In the present study, acute grey matter pathology following ET-1 microinjection is limited to areas corresponding to patterns of intrinsic blood supply of targeted penetrating microvasculature (Mautes et al, 2000).…”

“…Focal ischaemic grey matter SCI was accomplished by slight modification of methods described previously (Benton et al, 2005). A total of 70 adult (approx.…”

“…Using a previously described model of focal spinal ischaemia (Benton et al, 2005), we sought to replicate the non-traumatic stress/ischaemia present in penumbral spinal tissue (i.e. immediately surrounding the traumatic injury site).…”

Vascular Pathology as a Potential Therapeutic Target in SCI

“…In this lesion, picomolar quantities of the potent vasoconstrictor endothelin-1 (ET-1) are applied to penetrating branches of the anterior spinal artery within the medial spinal sulcus. This results in a transient vasospasm and resultant ischemic episode leading to significant loss of gray matter perfused by the affected arterioles [12,42]. While this focal ischemia leads to acute loss of neurons and astroglial activation akin to what is observed acutely following traumatic SCI [7], quantitative assessment of microvascular density in affected spinal gray matter shows that this ischemia-reperfusion injury does not result in changes in the number of microvessels in/ around the lesion site acutely (RL Benton, unpublished observations).…”

“…In the normal spinal cord, ECs express low levels of ET-1 [81], while higher basal expression of the major ET-1 receptor isoforms (i.e., ET-R A and ET-R B ) is observed [108]. This relatively high ET-1 receptor expression profile has been exploited to develop a novel model of focal spinal ischemia whereby small amounts of ET-1 is applied to the spinal microvasculature, resulting in a transient focal ischemia and subsequent loss of neuropil [7,12,42]. This model is likely clinically relevant as ET-1 is significantly increased in cerebrospinal fluid (CSF), plasma, and tissue in response to traumatic SCI [94,133,153].…”