Early and sustained increase in the expression of hippocampal IGF-1, but not EPO, in a developmental rodent model of traumatic brain injury

Abstract

Pediatric traumatic brain injury (pTBI) is the leading cause of traumatic death and disability in children in the United States. Impaired learning and memory in these young survivors imposes a heavy toll on society. In adult TBI (aTBI) models, cognitive outcome improved after administration of erythropoietin (EPO) or insulin-like growth factor-1 (IGF-1). Little is known about the production of these agents in the hippocampus, a brain region critical for learning and memory, after pTBI. Our objective was to describe hippocampal expression of EPO and IGF-1, together with their receptors (EPOR and IGF-1R, respectively), over time after pTBI in 17-day-old rats. We used the controlled cortical impact (CCI) model and measured hippocampal mRNA levels of EPO, IGF-1, EPOR, IGF-1R, and markers of caspase-dependent apoptosis (bcl2, bax, and p53) at post-injury days (PID) 1, 2, 3, 7, and 14. CCI rats performed poorly on Morris water maze testing of spatial working memory, a hippocampally-based cognitive function. Apoptotic markers were present early and persisted for the duration of the study. EPO in our pTBI model increased much later (PID7) than in aTBI models (12 h), while EPOR and IGF-1 increased at PID1 and PID2, respectively, similar to data from aTBI models. Our data indicate that EPO expression showed a delayed upregulation post-pTBI, while EPOR increased early. We speculate that administration of EPO in the first 1-2 days after pTBI would increase hippocampal neuronal survival and function.

FIG. 1.

Graph depicting IGF-1 mRNA in the CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA levels. The dashed line represents 100% of sham IGF-1 mRNA. CCII IGF-1 mRNA increased relative to both sham and CCIcon at PID 1, 2, 3, and 7, and relative to sham at PID 14. Results are presented as percentages of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham animals; &p < 0.05 relative to CCI con animals; IGF-1, insulin-like growth factor-1; CCI, controlled cortical impact).

FIG. 2.

Graph depicting IGF-1 receptor (IGF-1R) mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA level. The dashed line represents 100% of sham IGF-1R mRNA. IGF-1R decreased in CCII relative to sham animals at PID 1 and 2, and then increased at PID 3, after which expression was unchanged. CCII IGF-1R decreased and increased, relative to CCIcon, at PID 1 and 7, respectively. Results are presented as percentages of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham animals; &p < 0.05 relative to CCIcon; IGF-1, insulin-like growth factor-1; CCI, controlled cortical impact).

FIG. 3.

Graph depicting EPO mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA levels. The dashed line represents 100% of sham EPO mRNA. CCII EPO trended towards an increase at PID 3 (p = 0.05), and was increased at PID 7, relative to sham animals. CCII EPO increased relative to CCIcon at PID 3 and 7. Results are presented as percentages of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham animals; &p < 0.05 relative to CCIcon; EPO, erythropoietin; CCI, controlled cortical impact).

FIG. 4.

Graph depicting EPOR mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA levels. The dashed line represents 100% of sham EPOR mRNA. EPOR was increased relative to sham and to CCIcon as early as PID 1, peaked at PID 3, and remained elevated at least until PID 14. Results are presented as percentage of sham mRNA ± standard error of the mean; n = 6; **p < 0.05 relative to Sham; &p < 0.05 relative to CCIcon; EPOR, erythropoietin receptor; CCI, controlled cortical impact).

FIG. 5.

Graph depicting p53 mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of the corresponding sham mRNA level. The dashed line represents 100% of sham p53 mRNA. CCII p53 mRNA increased at PID 3 and PID 7 relative to both sham and CCIcon, and at PID 14 relative to sham animals. Results are presented as percentage of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham; &p < 0.05 relative to CCIcon; CCI, controlled cortical impact).

FIG. 6.

Graph depicting bax mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA levels. The dashed line represents 100% of sham bax mRNA. CCII bax mRNA was increased at PID 2 and at PID 14. Results are presented as percentage of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham animals; CCI, controlled cortical impact).

FIG. 7.

Graph depicting bcl2 mRNA in CCI hippocampus ipsilateral to injury (CCII) obtained at post-injury days (PID) 1, 2, 3, 7, and 14, presented as a percentage of corresponding sham mRNA levels. The dashed line represents 100% of sham bcl2 mRNA. CCII bcl2 mRNA was decreased relative to sham and to CCIcon at PID 1. Results are presented as percentages of sham mRNA ± standard error of the mean; n = 6; *p < 0.05 relative to sham; &p < 0.05 relative to CCIcon; CCI, controlled cortical impact).

FIG. 8.

Graph depicting EPO protein levels in CCI hippocampus ipsilateral to injury (CCII), expressed as densitometry units relative to GAPDH, at PID 2 and 7. CCI EPO protein levels increased relative to sham levels at PID 7. Representative immunoblot images are shown for CCI and sham animals. Results are presented as EPO:GAPDH ratio ± standard error of the mean; n = 6; *p < 0.05 relative to sham; CCI, controlled cortical impact; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PID, post-injury day).

FIG. 9.

Graph depicting the average latency to find the hidden platform as a function of day of testing (PID days 14–18) for each group of animals. CCI animals were significantly slower on PID 16 (relative to sham), and on PID 17 (relative to naïve). Latency is expressed in seconds ± standard error of the mean (n = 6–8; *p < 0.05 relative to sham; %p < 0.05 relative to naïve; CCI, controlled cortical impact; PID, post-injury day).