The effect of enriched environment on the outcome of traumatic brain injury; a behavioral, proteomics, and histological study.

De novo hippocampal neurogenesis contributes to functional recovery following traumatic brain injury (TBI). Enriched environment (EEN) can improve the outcome of TBI by positively affecting neurogenesis. Blast induced traumatic brain injury (bTBI) characterized by memory impairment and increased anxiety levels, is a leading cause of chronic disability among soldiers. Using a rodent model of bTBI we asked: (a) whether long-term exposure to EEN after injury can ameliorate behavioral abnormalities and (b) what the effects of EEN are at the molecular and cellular levels and on de novo neurogenesis. We found that housing injured animals in EEN resulted in significantly improved spatial memory while animals in normal housing (NH) showed persistent memory impairment. VEGF and Tau protein but not Interleukin-6 (IL-6) levels were normalized in the dorsal hippocampus (DHC) of EEN rats while all three markers remained elevated in NH rats. Interestingly, after peaking at 6 weeks post-injury, anxiety returned to normal levels at 2 months independent of housing conditions. Housing animals in EEN had no significant effect on VEGF and Tau protein levels in the ventral hippocampus (VHC) and the amygdala (AD). We also found that EEN reduced IL-6 and IFNγ levels in the VHC; these markers remained elevated following NH. We observed an increase in GFAP and DCX immunoreactivities in the VHC of NH animals at 2 months post-injury. Conversely, injured animals housed in EEN showed no increase in GFAP or DCX immunoreactivity in their VHC. In summary, long-term exposure of injured animals to EEN appears to play a positive role in the restoration of memory functions but not on anxiety, which returned to normal levels after a significant period of time. Cellular and molecular changes in response to EEN appear to be a part of neurogenesis-independent as well as dependent recovery processes triggered by bTBI.

Stress and traumatic brain injury: a behavioral, proteomics, and histological study.

Psychological stress and traumatic brain injury (TBI) can both result in lasting neurobehavioral abnormalities. Post-traumatic stress disorder and blast induced TBI (bTBI) have become the most significant health issues in current military conflicts. Importantly, military bTBI virtually never occurs without stress. In this experiment, we assessed anxiety and spatial memory of rats at different time points after repeated exposure to stress alone or in combination with a single mild blast. At 2 months after injury or sham we analyzed the serum, prefrontal cortex (PFC), and hippocampus (HC) of all animals by proteomics and immunohistochemistry. Stressed sham animals showed an early increase in anxiety but no memory impairment at any measured time point. They had elevated levels of serum corticosterone (CORT) and hippocampal IL-6 but no other cellular or protein changes. Stressed injured animals had increased anxiety that returned to normal at 2 months and significant spatial memory impairment that lasted up to 2 months. They had elevated serum levels of CORT, CK-BB, NF-H, NSE, GFAP, and VEGF. Moreover, all of the measured protein markers were elevated in the HC and the PFC; rats had an increased number of TUNEL-positive cells in the HC and elevated GFAP and Iba1 immunoreactivity in the HC and the PFC. Our findings suggest that exposure to repeated stress alone causes a transient increase in anxiety and no significant memory impairment or cellular and molecular changes. In contrast, repeated stress and blast results in lasting behavioral, molecular, and cellular abnormalities characterized by memory impairment, neuronal and glial cell loss, inflammation, and gliosis. These findings may have implications in the development of diagnostic and therapeutic measures for conditions caused by stress or a combination of stress and bTBI.

Reverse phase protein microarray technology in traumatic brain injury.

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

SATB2 interacts with chromatin-remodeling molecules in differentiating cortical neurons.

During our search for developmental regulators of neuronal differentiation, we identified special AT-rich sequence-binding protein (SATB)2 that is specifically expressed in the developing rat neocortex and binds to AT-rich DNA elements. Here we investigated whether the regulatory function of SATB2 involves chromatin remodeling at the AT-rich DNA site. In-vitro and in-vivo assays using a DNA affinity pre-incubation specificity test of recognition and chromatin immunoprecipitation showed that SATB2 specifically binds to histone deacetylase 1 and metastasis-associated protein 2, members of the nucleosome-remodeling and histone deacetylase complex. Double immunohistochemistry showed that, in the developing rat neocortex, SATB2 is coexpressed with both proteins. Using a cell culture model, we showed that trichostatin A treatment, which blocks the activities of histone deacetylases, reverses the AT-rich dsDNA-dependent repressor effect of SATB2. These findings suggested that the molecular regulatory function of SATB2 involves modification of the chromatin structure. Semi-quantitative chromatin immunoprecipitation analysis of cortices from SATB2 mutant and wild-type animals indicated that, in the knock-out brains, SATB2 is replaced in the chromatin-remodeling complex by AU-rich element RNA binding protein 1, another AT-rich DNA binding protein also expressed in differentiating cortical neurons. These results suggested that an altered chromatin structure, due to the presence of different AT-rich DNA binding proteins in the chromatin-remodeling complex, may contribute to the developmental abnormalities observed in the SATB2 mutant animals. These findings also raised the interesting possibility that SATB2, along with other AT-rich DNA binding proteins, is involved in mediating epigenetic influences during cortical development.