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

Abstract

Background 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. New Method 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. Results 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 fluorescent intensity found increases in PECAM-1, VEGFR-2, and VE-Cadherin expression per cell at 3 dpi, which is consistent with western blot analysis. Comparison with Exiting Methods 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. Conclusions We demonstrate that TruCount quantification using flow cytometry is a powerful tool for quantifying mature and progenitor endothelial cell changes after TBI.

EphB3 signaling propagates synaptic dysfunction in the traumatic injured brain

“…To examine the effects of traumatic brain injury (TBI) on synaptic stability and function, we took advantage of a well-described controlled cortical impact (CCI) injury model ( Edward Dixon et al, 1991 ; Osier et al, 2015 ; Smith et al, 1995 ; Theus et al, 2014 ). While other TBI models exist, CCI injury allows for accurate replication of biomechanics similar to human TBI ( Peña et al, 2005 ; Xiong et al, 2013 ; Zhang et al, 2014 ), and can be adapted to different injury severities depending on the experimental paradigm ( BRODY et al, 2007 ; Chen et al, 2014 ; Saatman et al, 2006 ; Assis-Nascimento et al, 2016 ). To examine synaptic dysfunction in the TBI environment, we established a moderate mouse controlled cortical impact (CCI) injury model where hippocampal synaptic damage is independent of neuronal loss.…”

Enhanced astrocytic d-serine underlies synaptic damage after traumatic brain injury

“…Our CCI injury model produces a moderate cortical injury with only mild damage to subcortical tissues, such as the hippocampus, where synaptic damage and loss within the hippocampus underlie learning and memory deficits (27). This enabled us to examine discrete and reversible synaptic responses while maintaining the overall environmental characteristics associated with TBI, including distal tissue losses (34), reactive gliosis (27), a compromised blood-brain barrier, and inflammatory responses (35). The presence of astrocytosis in the CCI-injured hippocampus allowed us to examine how gliotransmitters from reactive astrocytes may regulate synaptic function and/or dysfunction.…”

EphB3 signaling induces cortical endothelial cell death and disrupts the blood–brain barrier after traumatic brain injury

“…The gating strategy was based on using forward and side scatter to exclude cellular debris and select for homogeneity of size and granularity of individual cells isolated from a cortical hemisphere. The selection marker CD45 was used to exclude infiltrating leukocytes and residential microglia from the analysis, where cvECs were also identified as CD45 - /CD144 + cells 36 . We observed a 36% reduction in the number of cvECs in the WT CCI injured (1.65 × 10 5 ± 140 cell/μL; P < 0.001) cortex at 3 dpi as compared with WT sham (2.59 × 10 5 ± 230 cell/μL) cortex (Fig.…”

“…We next examined whether deficiencies in EphB3 or ephrinB3 affected BBB permeability to infiltrating peripheral cells 36 . Infiltrating macrophages express high levels of CD45 (i.e., CD45 high ) and CD11b markers, which were significantly increased in all groups at 3 dpi (Fig.…”

“…In the adult CNS, ECs are thought to be relatively quiescent; however, during adult angiogenesis latent proliferative and apoptotic processes can be re-initiated. In pathological conditions, such as stroke and traumatic injury, the proliferative angiogenic response is activated within hours to days after the initial insult 44 , 45 , where EC proliferation and EPC infiltration are thought to contribute to vessel repair and regeneration 36 , 45 – 50 . Our findings support a small yet significant proliferative response in cvECs within the first 3 days after CCI injury as well as enhanced EPC infiltration at the same time point.…”