High‐density microarray analysis of hippocampal gene expression following experimental brain injury

Abstract

Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. Traumatic brain injury (TBI) often causes spatial memory deficits, which are thought to arise from the death as well as the dysfunction of hippocampal neurons. Cell death and dysfunction are commonly associated with and often caused by altered expression of specific genes. The identification of the genes involved in these processes, as well as those participating in postinjury cellular repair and plasticity, is important for the development of mechanism-based therapies. To monitor the expression levels of a large number of genes and to identify genes not previously implicated in TBI pathophysiology, a high-density oligonucleotide array containing 8,800 genes was interrogated. RNA samples were prepared from ipsilateral hippocampi 3 hr and 24 hr following lateral cortical impact injury and compared to samples from sham-operated controls. Cluster analysis was employed using statistical algorithms to arrange the genes according to similarity in patterns of expression. The study indicates that the genomic response to TBI is complex, affecting approximately 6% (at the time points examined) of the total number of genes examined. The identity of the genes revealed that TBI affects many aspects of cell physiology, including oxidative stress, metabolism, inflammation, structural changes, and cellular signaling. The analysis revealed genes whose expression levels have been reported to be altered in response to injury as well as several genes not previously implicated in TBI pathophysiology.

Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury

“…Oxidative stress contributes to synaptic dysfunction and disconnection, with impaired mitochondrial transport to the synapses contributing to both neuronal death and neuritic degenerative cascades [15,16]. Exitotoxicity contributes to energy failure resulting in further oxidative stress by enhanced metabolism of excitatory amino acids, leading to secondary cascades of injuries following TBI [46].…”

“…As a consequence of oxidative stress both the function and transport of mitochondrial to synaptic regions is impaired, decrease synaptic function [15,16], which results in neurodegeneration after brain injury [17,18]. A recent study reported that oxidative stress alters the function of PSD-95 by decreasing a voltage-gated potassium channel that is closely linked to the PSD [19], suggesting that synapse loss and replacement, known to occur following TBI [20], could be modulated by the levels of oxidative stress.…”

Methodological Considerations Regarding Single-Cell Gene Expression Profiling for Brain Injury

“…Several examples of studies that have used microarray analysis of brain homogenates during the past few years to examine gross regional gene expression profiles in rodent models of such complex brain disorders as TBI, stroke, and epilepsy are summarized in Table I, along with the species, conditions, and types of arrays that were used for each comparison. [25][26][27][28][29][30][31][32][33] All five of the TBI studies listed in Table I employed a controlled cortical impact (CCI) model of injury, but different strains and species were used in the different studies. The microarrays most frequently used for all of the rat studies were the Affymetrix U34 Rat Neurobiology array and U34 Rat Genome Set, whereas different arrays were used for each of the mouse studies.…”

“…Although Kobori et al 28 analyzed gene expression at five intervals between 2 h and 14 days, all of the other studies evaluated relatively acute time points after injury. 28 CCI (male c57/BL6 Cortex (2 h, 6 h, 24 h, 3 days, Incyte GEM 2 cDNA Ipsilateral injured frontal mice, 20 g-27 g) a and 14 days postinjury) microarray chips 6 mm of cortex vs. naive (n ϭ 10 pooled) Matzilevich et al 29 CCI (male Long-Evans Hippocampus (3 h and Affymetrix Rat Genome Ipsilateral injured rats, 250 g-275 g) a 24 h postinjury) U34A GeneChip, aRNA hippocampus vs. sham (T7) amplification (n ϭ 10 were pooled) Rao et al 30 MCAO (male spontaneously Frontoparietal cortex Affymetrix Rat Neurobiology Ipsilateral injured cortex hypertensive rats, 280 g-320 g) b (6 h and 24 h postinfarction) U34 GeneChip vs. contralat. cortex (n ϭ 3 per each of 3 chips per group were pooled) Schmidt-Kastner et al 31 MCAO (male Sprague-Dawly Peri-ischemic cortex Incyte Mouse UniGene 1 Ipsilateral injured cortex vs. rats, 263 g-370 g) b (3 h postinfarction) microarray sham (n ϭ 2 per each of 3 chips per group were pooled) Elliott et al 32 Drug-induced and terminated Dentate gyrus: 14 days after Affymetrix Rat Genome Bilateral dentate gyrus after SE status epilepticus (male status epilepticus (SE) and U34A GeneChip vs. P3 (n ϭ 2 to 3 from adult Sprague-Dawley rats, 180 g-200 g) 3 days postnatal or n ϭ 10 to 12 from P3 animals pooled) Lukasiuk et al 33 Amygdala stimulation with Temporal lobe and hippocampus Research Genetics GF300 Bilateral hippocampus and electrode (male Sprague-Dawley (1 day, 4 days, 1 week, 2 weeks filter cDNA microarrays temporal lobes after SE vs. rats, 290 g-360 g) with seizures and 2 weeks without controls (n ϭ 2 to 3 were seizures after SE) pooled per chip per group) a CCI-Controlled Cortical Impact brain injury.…”

“…21,[34][35][36] Instead, it combines all of the different cell types present in a sample tissue (neurons, glia, blood cells and vessels, and so forth) at potentially disparate stages of injury and disease processes. [25][26][27][28][29][30][31][32][33] As a result, the gene expression profiles of selectively vulnerable subpopulations of cells may be largely obscured, and the detection of potentially important, less abundantly expressed genes (such as transcription factors) may be overlooked. Identification of the source of an observed pattern of differential gene expression can also be more difficult when tissue homogenates are used for gene expression profiling.…”