Pre-Clinical Common Data Elements for Traumatic Brain Injury Research: Progress and Use Cases.

Traumatic brain injury (TBI) is an extremely complex condition due to heterogeneity in injury mechanism, underlying conditions, and secondary injury. Pre-clinical and clinical researchers face challenges with reproducibility that negatively impact translation and therapeutic development for improved TBI patient outcomes. To address this challenge, TBI Pre-clinical Working Groups expanded upon previous efforts and developed common data elements (CDEs) to describe the most frequently used experimental parameters. The working groups created 913 CDEs to describe study metadata, animal characteristics, animal history, injury models, and behavioral tests. Use cases applied a set of commonly used CDEs to address and evaluate the degree of missing data resulting from combining legacy data from different laboratories for two different outcome measures (Morris water maze [MWM]; RotorRod/Rotarod). Data were cleaned and harmonized to Form Structures containing the relevant CDEs and subjected to missing value analysis. For the MWM dataset (358 animals from five studies, 44 CDEs), 50% of the CDEs contained at least one missing value, while for the Rotarod dataset (97 animals from three studies, 48 CDEs), over 60% of CDEs contained at least one missing value. Overall, 35% of values were missing across the MWM dataset, and 33% of values were missing for the Rotarod dataset, demonstrating both the feasibility and the challenge of combining legacy datasets using CDEs. The CDEs and the associated forms created here are available to the broader pre-clinical research community to promote consistent and comprehensive data acquisition, as well as to facilitate data sharing and formation of data repositories. In addition to addressing the challenge of standardization in TBI pre-clinical studies, this effort is intended to bring attention to the discrepancies in assessment and outcome metrics among pre-clinical laboratories and ultimately accelerate translation to clinical research.

GGF2 (Nrg1-ß3) treatment enhances NG2+ cell response and improves functional recovery after spinal cord injury.

The adult spinal cord contains a pool of endogenous glial precursor cells, which spontaneously respond to spinal cord injury (SCI) with increased proliferation. These include oligodendrocyte precursor cells that express the NG2 proteoglycan and can differentiate into mature oligodendrocytes. Thus, a potential approach for SCI treatment is to enhance the proliferation and differentiation of these cells to yield more functional mature glia and improve remyelination of surviving axons. We previously reported that soluble glial growth factor 2 (GGF2)- and basic fibroblast growth factor 2 (FGF2)-stimulated growth of NG2(+) cells purified from injured spinal cord in primary culture. This study examines the effects of systemic administration of GGF2 and/or FGF2 after standardized contusive SCI in vivo in both rat and mouse models. In Sprague-Dawley rats, 1 week of GGF2 administration, beginning 24 h after injury, enhanced NG2(+) cell proliferation, oligodendrogenesis, chronic white matter at the injury epicenter, and recovery of hind limb function. In 2',3'-cyclic-nucleotide 3'-phosphodiesterase-enhanced green fluorescent protein mice, GGF2 treatment resulted in increased oligodendrogenesis and improved functional recovery, as well as elevated expression of the stem cell transcription factor Sox2 by oligodendrocyte lineage cells. Although oligodendrocyte number was increased chronically after SCI in GGF2-treated mice, no evidence of increased white matter was detected. However, GGF2 treatment significantly increased levels of P0 protein-containing peripheral myelin, produced by Schwann cells that infiltrate the injured spinal cord. Our results suggest that GGF2 may have therapeutic potential for SCI by enhancing endogenous recovery processes in a clinically relevant time frame.

Increased growth factor expression and cell proliferation after contusive spinal cord injury.

The damage caused by traumatic central nervous system (CNS) injury can be divided into two phases: primary and secondary. The initial injury destroys many of the local neurons and glia and triggers secondary mechanisms that result in further cell loss. Approximately 50% of the astrocytes and oligodendrocytes in the spared white matter of the epicenter die by 24 h after spinal cord injury (SCI), but their densities return to normal levels by 6 weeks. This repopulation is largely due to the proliferation of local progenitors that divide in response of CNS injury. Previous studies indicate that the secondary events that cause cell death after SCI also increase the local levels of several growth factors that stimulate the proliferation of these endogenous progenitors. We compared the spatial pattern of the post-injury up-regulation of the pro-mitotic growth factors with that of 5-bromodeoxyuridine (BrdU) incorporation to determine if each could play a role in proliferation. Three days after a standard contusive SCI or laminectomy, animals received intraperitoneal BrdU injections to label dividing cells and were perfused 2 h after the last injection. Immunohistochemistry for BrdU and basic fibroblast growth factor (FGF2) and in situ hybridization for ciliary neurotrophic factor (CNTF) and glial growth factor (GGF2) mRNA were used to compare the number of dividing cells with growth factor levels in sections 2 and 4 mm from the epicenter. All three growth factors are significantly up-regulated 3 days after SCI, when cell proliferation is maximal. The increase in GGF2 and FGF2 levels is highest in sections 2 mm rostral to the epicenter, mimicking BrdU incorporation. Addition of rhGGF2 to cultured cells isolated from the spinal cord 3 days after SCI increased the number of NG2+ glial progenitors. These data suggest that FGF2 and GGF2 may contribute to the spontaneous recovery observed after SCI by stimulating the proliferation of local progenitors that help repopulate the injured cord.

Cell proliferation and replacement following contusive spinal cord injury.

After spinal cord injury (SCI), about 50% of the oligodendrocytes and astrocytes in the residual white matter at the injury site are lost by 24 h. However, chronically after SCI, the density of oligodendrocytes is normal. Previous studies have shown that the adult rat spinal cord contains a pool of proliferating glial progenitors whose progeny could help restore cell density after injury. To study proliferation in response to injury, we performed SCI on adult female rats at the T8 level, using a standardized contusion model. Animals received bromodeoxyuridine (BrdU) injections during the first week after SCI, and were perfused within 2 h for acute studies, and at 6 weeks for chronic studies. The tissue was analyzed using immunohistochemical detection of BrdU and cell marker antigens. We demonstrate that cell proliferation in the residual white matter is increased at 1-7 days after SCI, peaking on day 3. Dividing cells include oligodendrocytes, astrocytes, microglia/macrophages, and a high proportion of NG2(+) glial precursors. By 6 weeks, some cells that had been labeled 2-4 days after SCI were still present. Double immunohistochemistry showed that while very few of these cells expressed NG2 or the microglia/macrophage marker OX42, about 50% expressed CC1 or glial fibrillary acidic protein (GFAP), markers of mature oligodendrocytes and astrocytes, respectively. The post-injury environment represented by residual white matter is thus permissive to the differentiation of glial precursors. Cells that are stimulated to divide during the first week after SCI develop chronically into mature phenotypes that replace macroglia lost after injury.

Chronic alterations in the cellular composition of spinal cord white matter following contusion injury.

Spinal cord injury (SCI) involves the loss of neurons and glia due to initial mechanical and secondary biochemical mechanisms. Treatment with the sodium channel blocker tetrodotoxin (TTX) reduces acute white matter pathology and increases both axon density and hindlimb function chronically at 6 weeks after injury. We investigated the cellular composition of residual white matter chronically to determine whether TTX also has a significant effect on the numbers and types of cells present. Rats received an incomplete thoracic contusion injury, in the presence or absence of TTX (0.15 nmole) injected focally, beginning at 15 min prior to injury. Six weeks later, cell density was significantly increased in the residual white matter of the dorsal, lateral, and ventral funiculi, both rostral and caudal to the injury site in both TTX-treated and injury control groups. Oligodendrocyte and astrocyte density was similar to normal but large numbers of cells expressing microglia/macrophage markers were present. Labeling with the progenitor markers nestin and NG2 showed that precursor cell density had also doubled or tripled as compared with uninjured controls. Some of these cells were also labeled for antigens that indicate their possible progression along an oligodendrocyte or astrocyte lineage. Our results support the hypothesis that the beneficial effect of TTX in SCI is related to its preservation of axons per se; no effect on chronic white matter cell composition was detected. They highlight the profound changes in cellular composition in preserved white matter chronically at 6 weeks after injury, including the accumulation of endogenous progenitor cells and the persistence of activated macrophages/microglia. The manipulation of these endogenous cells may be used in the future to enhance recovery after SCI.