The proposed study will investigate the differences in neuronal morphology and astrocyte proliferation between groups of rats that received either pre-injury EE or SE and either EE or SE post-injury. Samples will be optically cleared using SeeDB and IHC will be used in order to visualize morphological and structural differences between groups. To investigate differences in total dendritic length, total bifurcations, and dendritic spine density, anti- microtubule-associated protein (MAP-2) will be used. The antibody anti-synaptophysin will be used to quantify synapses in the sample. Neuronal nuclei will be labeled with NeuN and counted in order to quantify cell loss in the injured brain. To quantify the immune response to the injury astrocytes will be labeled with glial fibrillary-associated protein (GFAP) (Beonisch, Farmilo, & Stead, 1989). After IHC, samples will be imaged using confocal microscopy. The series of images produced will be stitched into single 3-dimensional images using Imaris. Imaris will then be used to volumetrically render portions of the tissue that are immunohistochemically-labeled. The software may then be used to calculate dimension, such as combined length of dendritic arbors; or to count cellular and morphological features such as neural cell counts and dendritic spines. Three regions will be analyzed: the CA1 region of the hippocampus, the medial dorsal thalamic nucleus (MDN), and the lesion site. CA1 is an important region of interest for two reasons. Firstly, CA1 is a site of migration of progenitor cells from the dentate gyrus. Secondly, the hippocampus plays a role in learning and memory and cellular differences in this region may facilitate observed recovery in these cognitive functions following TBI (Gaulke et al., 2005). The MDN is associated with executive functioning and MFC contusion results in substantial neural loss and astrocytosis in this region (Sato, Chang, Igarashi, & Noble, 2001; Bigler & Maxwell, 2011). It is hypothesized that rats reared in EE and that are returned to EE following MFC contusion will show increases in dendritic length, total bifurcations, dendritic spines, and synapses when compared to rats that experience EE only before or only after injury. Animals experiencing any EE will show an improvement in these morphological features compared to animals that receive only SE.
The SRCI committe awarded $9957.73 to support Garrick's project.
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