Temporal course of changes in gene expression suggests a cytokine-related mechanism for long-term hippocampal alteration after controlled cortical impact

Abstract

Mild traumatic brain injury (mTBI) often has long-term effects on cognitive function and social behavior. Altered gene expression may be predictive of long-term psychological effects of mTBI, even when acute clinical effects are minimal or transient. Controlled cortical impact (CCI), which causes concussive, but nonpenetrant, trauma to underlying (non-cortical) brain, resulting in persistent changes in hippocampal synaptic function, was used as a model of mTBI. The hippocampal transcriptomes of sham-operated or injured male rats at 1, 7, and 30 days postinjury were examined using microarrays comprising a comprehensive set of expressed genes, subsequently confirmed by quantitative reverse-transcriptase polymerase chain reaction. Transcripts encoding the chemokines, chemokine (C-C motif) ligand (Ccl)2 and Ccl7, inflammatory mediators lipocalin-2 (Lcn2) and tissue inhibitor of metalloproteinase 1 (Timp1), immunocyte activators C-C chemokine receptor type 5 (Ccr5) and Fc fragment of IgG, low affinity IIb, receptor (CD32) (Fcgr2b), the major histocompatibility complex II immune response-related genes, Cd74 and RT1 class II, locus Da (RT1-Da), the complement component, C3, and the transcription factor, Kruppel-like factor 4 (Klf4), were identified as early (Ccl2, Ccl7, Lcn2, and Timp1), intermediate (Ccr5, Fcgr2b, Cd74, RT1-Da, and C3), and late (Klf4) markers for bilateral hippocampal response to CCI. Ccl2 and Ccl7 transcripts were up-regulated within 24 h after CCI, and their elevation subsided within 1 week of injury. Other transcriptional changes occurred later and were more stable, some persisting for at least 1 month, suggesting that short-term inflammatory responses trigger longer-term alteration in the expression of genes previously associated with injury, aging, and neuronal function in the brain. These transcriptional responses to mTBI may underlie long-term changes in excitatory and inhibitory neuronal imbalance in hippocampus, leading to long-term behavioral consequences of mTBI.

FIG. 1.

Characterization of injury severity caused by CCI. Mild CCI does not cause a significant loss of neurons in the CA1 region 24 hours or 7 days after injury. (A) Photomicrograph of Nissl-stained brain coronal slice. Indicated are the sites of impact and the ipsilateral (Ipsi) and contralateral (Contra) CA1. (B) Representative photomicrographs from Nissl-stained sections showing BLA cells from the ipsilateral (top) and contralateral (bottom) sides of sham, 1-day CCI, and 7-day CCI animals, respectively. Total magnification is 630×; scale bar, 50 μm. (C) Group data (mean±standard error; n=8 for each group) of stereological estimation of the total number of Nissl-stained neurons in the CA1. There were no significant differences in neuronal number between sham and 1-day or 7-day CCI, on either ipsilateral or contralateral sides, using Student's t-test. CCI, controlled cortical impact.

FIG. 2.

Regulatory networks for CCI-dependent transcripts in rat hippocampal tissue. Transcripts with an abundance of 1.5 or greater in cortically injured (CCI) male rats at 1, 7 and 30 days post-injury compared to sham-operated by microarray analysis (see Supplementary Table 2) (see online supplementary material at http://www.liebertpub.com) were used as the input for analysis of potential networks using the signal transduction knowledge environment of Ingenuity (http://www.ingenuity.com). A, B, and C are the highest score networks involving the CCI-up-regulated transcripts at day one, seven and 30 respectively. CCI-regulated transcripts are depicted in gray and linked components in white. Circular lines above symbols indicate auto-regulation; connecting lines without arrows indicate direct protein interaction; dashed and solid arrows indicate indirect (e.g. regulation of messenger RNA levels) and direct (e.g. enzymatic) activation. CCI, controlled cortical impact.

FIG. 3.

Chemokine transcript up-regulation in hippocampal tissue from the ipsilateral (i.d.) dorsal or contralateral (c.d.) dorsal side of sham-operated (Sham) or cortically injured (CCI) male rats at 1, 7 and 30 days post-injury. Ccl2 (A, i.d. and C, c.d.) and Ccl7 (B, i.d. and D, c.d.) mRNA levels, determined by qRT-PCR, are expressed as fold increase over corresponding control values and represent means±standard error of the mean of 3 samples (animals) for each condition. *p<0.05; **p<0.01; versus the corresponding control (Student's t-test). CCI, controlled cortical impact; mRNA, messenger RNA; Ccl2, chemokine (C-C motif) ligand.

FIG. 4.

Intermediary and longer-term transcriptome changes after CCI. Cd74 (A), C3 (B), RT1-Da (C), and Klf4 (D) mRNA levels, determined by quantitative reverse-transcription polymerase chain reaction, are expressed as fold increase over corresponding control values and represent means±standard error of the mean of 3 samples (animals) for each condition. *p<0.05; **p<0.01; versus the corresponding control (Student's t-test). CCI, controlled cortical impact; mRNA, messenger RNA; RT1-Da, RT1 class II, locus Da; Klf4, Kruppel-like factor 4.