The Role of EphB3 in Cortical Vascular Integrity Following Traumatic Brain Injury

Traumatic brain injury (TBI) continues to be a major health concern worldwide. One of the earliest and most profound deficits comes from vascular damage and breakdown of the blood-brain barrier (BBB). Despite the consequences of blood vessel destruction, little is known of the cellular responses that occur in vessel integrity following TBI. B class ephrins and Eph receptors are membrane bound proteins that initiate bidirectional signals during cell-cell interactions, and are indispensable for developmental and pathological neovascularization. The role of EphB3 and its ligand ephrinB3 was investigated in vascular integrity in a murine controlled cortical impact (CCI) injury model of TBI. We identified a novel pro-apoptotic role for EphB3 in cerebral vascular endothelial cells (cvECs) that lead to increased BBB permeability and immune cell infiltration after CCI injury. In addition, EphB3 also regulated the degree of cvECastrocytic end-feet membrane association in the gliovascular unit, as well as the expression of the endothelial intercellular junction proteins ZO-1 and VE-cadherin. In short, our findings demonstrate that blocking EphB3 receptor signaling in the cerebral vasculature increases cvEC survival and reduces BBB permeability representing a potentially important therapeutic strategy for vessel repair and/or stability following TBI.</p

A flow cytometric approach to analyzing mature and progenitor endothelial cells following traumatic brain injury

Traumatic brain injury (TBI) continues to be a major source of death and disability worldwide, and one of the earliest and most profound deficits comes from vascular damage and breakdown of the blood-brain barrier (BBB). Cerebral vascular endothelial cells (cvECs) and endothelial progenitor cells (EPCs) have been shown to play essential roles in vessel repair and BBB stability, although their individual contributions remain poorly defined. We employ TruCount beads with flow cytometry to precisely quantify cvECs, EPCs, and peripheral leukocytes in the murine cortex after controlled cortical impact (CCI) injury. We found a significant reduction in the number of cvECs at 3 days post-injury (dpi), whereas the EPCs and invading peripheral leukocytes were significantly increased compared with sham controls. Proliferation studies demonstrate that both cvECs and EPCs are undergoing cell expansion in the first week post-injury. Furthermore, analysis of protein expression using mean fluorescence intensity found increases in PECAM-1, VEGFR-2, and VE-Cadherin expression per cell at 3 dpi, which is consistent with western blot analysis. Classic methods of cell analysis, such as histological cell counts, in the traumatic injured brain are labor intensive, time-consuming, and potentially biased; whereas flow cytometry provides an efficient, non-biased approach to simultaneously quantify multiple cell types. However, conventional flow cytometry that employs capped events can provide misleading results in CNS injured tissues. We demonstrate that TruCount quantification using flow cytometry is a powerful tool for quantifying mature and progenitor endothelial cell changes after TBI

Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

Abstract Background In the vertebrate spinal cord, motor neurons (MN) are generated in stereotypical numbers from a pool of dedicated progenitors (pMN) whose number depends on signals that control their specification but also their proliferation and differentiation rates. Although the initial steps of pMN specification have been extensively studied, how pMN numbers are regulated over time is less well characterized. Results Here, we show that ephrinB2 and ephrinB3 are differentially expressed in progenitor domains in the ventral spinal cord with several Eph receptors more broadly expressed. Genetic loss-of-function analyses show that ephrinB2 and ephrinB3 inversely control pMN numbers and that these changes in progenitor numbers correlate with changes in motor neuron numbers. Detailed phenotypic analyses by immunostaining and genetic interaction studies between ephrinB2 and Shh indicate that changes in pMN numbers in ephrin mutants are due to alteration in progenitor identity at late stages of development. Conclusions Altogether our data reveal that Eph:ephrin signaling is required to control progenitor identities in the ventral spinal cord

Additional file 2: Table S1. of Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

Figure-by-figure details on sample size (DOCX 13 kb

Additional file 1: of Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

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Additional file 3: Figure S1–3. of Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord

Expression of Shh is not changed in ephrin mutants. (PDF 330 kb