Central Infusion of Insulin-Like Growth Factor-1 Increases Hippocampal Neurogenesis and Improves Neurobehavioral Function after Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) produces neuronal dysfunction and cellular loss that can culminate in lasting impairments in cognitive and motor abilities. Therapeutic agents that promote repair and replenish neurons post-TBI hold promise in improving recovery of function. Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor capable of mediating neuroprotective and neuroplasticity mechanisms. Targeted overexpression of IGF-1 enhances the generation of hippocampal newborn neurons in brain-injured mice; however, the translational neurogenic potential of exogenously administered IGF-1 post-TBI remains unknown. In a mouse model of controlled cortical impact, continuous intracerebroventricular infusion of recombinant human IGF-1 (hIGF) for 7 days, beginning 15 min post-injury, resulted in a dose-dependent increase in the number of immature neurons in the hippocampus. Infusion of 10 μg/day of IGF-1 produced detectable levels of hIGF-1 in the cortex and hippocampus and a concomitant increase in protein kinase B activation in the hippocampus. Both motor function and cognition were improved over 7 days post-injury in IGF-1-treated cohorts. Vehicle-treated brain-injured mice showed reduced hippocampal immature neuron density relative to sham controls at 7 days post-injury. In contrast, the density of hippocampal immature neurons in brain-injured mice receiving acute onset IGF-1 infusion was significantly higher than in injured mice receiving vehicle and equivalent to that in sham-injured control mice. Importantly, the neurogenic effect of IGF-1 was maintained with as much as a 6-h delay in the initiation of infusion. These data suggest that central infusion of IGF-1 enhances the generation of immature neurons in the hippocampus, with a therapeutic window of at least 6 h post-injury, and promotes neurobehavioral recovery post-TBI.

Keywords: IGF-1; TBI; cognition; hippocampus; neurogenesis.

FIG. 1.

Dose-dependent effects of insulin-like growth factor (IGF-1) delivered by central infusion on hippocampal neurogenesis and memory retention after controlled cortical impact (CCI). (A) Representative images of doublecortin (DCX; green) immunoreactivity in the ipsilateral hippocampus of vehicle (Veh) and IGF-1–infused CCI-injured mice at 7 days post-injury. CCI + Veh mice exhibited a robust loss of DCX immunoreactivity in the dentate gyrus granular layer. DCX immunoreactivity appeared to increase in a dose-dependent manner in CCI + IGF-1 mice. Granular layer (GL) and hilus (H). Scale bar represents 50 μm. (B) In the hippocampus ipsilateral to impact, immature neuron density increased as a function of IGF-1 infusate concentration, peaking at 3 μg/day of IGF-1, which produced a significantly higher density of DCX⁺ cells compared to vehicle infusion (*p < 0.05). Immature neuron density in the hippocampus contralateral to impact was not significantly changed. (C) Recognition indices (% time exploring novel object) at 3 and 7 days post-CCI. Brain-injured mice receiving vehicle or 0.3 μg/day of IGF-1 performed at chance levels, whereas CCI mice receiving 1, 3, or 10 μg/day of IGF-1 performed above chance at either 3 days, 7 days, or both time points (⁺p < 0.05 compared to 50%). Data are presented as mean + SEM (n = 4 vehicle-treated CCI-injured and n = 5/dose IGF-1–treated CCI-injured mice). SEM, standard error of the mean. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Central infusion of 10 μg/day human insulin-like growth factor-1 (hIGF-1) over 7 days elevates brain levels of hIGF-1 and enhances Akt activation in the hippocampus after controlled cortical impact (CCI). (A) hIGF-1 was detected in the ipsilateral (ipsi) cortex (CTX), and ipsilateral and contralateral (contra) hippocampus (HP) in sham and CCI mice at 7 days post-injury (*p < 0.05, contra HP compared to ipsi CTX or HP). (B) Representative western blot images of phosphorylated Akt (pAkt) and actin, as a control protein, for vehicle (Veh) and IGF-1–treated sham and CCI mice at 7 days post-injury. (C) Infusion of IGF-1 in CCI-injured mice resulted in a significant increase in Akt phosphorylation within the hippocampus at 7 days post-injury, compared to vehicle infusion (*p < 0.005). Optical density (OD) for each pAkt band was normalized to its respective control protein actin band OD; the mean of the triplicate samples for each mouse was normalized to the mean OD value of the vehicle-treated sham group. The results are presented as mean + SEM (n = 5 sham-injured/treatment and n = 9 CCI-injured/treatment). Akt, protein kinase B; SEM, standard error of the mean.

FIG. 3.

Central infusion of 10 μg/day of insulin-like growth factor-1 (IGF-1) attenuates cognitive and motor impairment after controlled cortical impact (CCI). (A) At 3 days post-injury, novel object recognition (NOR) task performance varied across groups (ANOVA, p < 0.05), but memory retention of injured cohorts was not significantly less than sham cohorts in post-hoc testing. (B) At 7 days post-injury, CCI + Veh mice exhibited a significant reduction in the recognition index compared to either sham group (*p < 0.005). In contrast, memory function of CCI + IGF-1 mice was significantly improved when compared to CCI + Veh mice (^#p < 0.05) and was not statistically different from that of sham groups. (C) CCI + Veh mice exhibited significant impairment in motor function at 1, 2, 3, and 5 days post-injury as assessed by a modified neurological severity score (NSS) (*p < 0.001, compared to sham + Veh mice). CCI + IGF-1 mice also exhibited a significant motor impairment at 1, 2, and 3 days post-injury when compared to sham + IGF-1 (*p < 0.001). However, CCI + IGF-1 mice showed improved motor function at 3 and 5 days post-injury compared to CCI + Veh mice (^#p < 0.01). Data are presented as mean ± SEM (n = 9 sham-injured/treatment and n = 19 CCI-injured/treatment). ANOVA, analysis of variance; SEM, standard error of the mean; Veh, vehicle.

FIG. 4.

Central infusion of 10 μg/day of insulin-like growth factor-1 (IGF-1) increases immature neuron density in the injured hippocampus after controlled cortical impact (CCI). (A) Representative images of doublecortin (DCX; green) immunoreactivity in the ipsilateral hippocampus of vehicle (Veh) and IGF-1–infused sham- and CCI-injured mice at 7 days post-injury. Scale bar represents 50 μm. Granular layer (GL) and Hilus (H). (B) Brain injury resulted in a significant decrease in DCX⁺ cell density in vehicle-treated mice (*p < 0.005, compared to sham + Veh), but not in CCI mice infused with IGF-1. DCX⁺ cell density was significantly greater in IGF-1–treated brain-injured mice than in vehicle-treated counterparts at 7 days after CCI injury (^#p < 0.005, compared to CCI + Veh mice). Immature neuron counts obtained from the ipsilateral granular layer were normalized to the volume of the ipsilateral granular layer to calculate cellular density (1000/mm³). Data are expressed as mean + SEM (n = 4 sham-injured/treatment, n = 10 CCI + Veh, and n = 9 CCI + IGF-1). SEM, standard error of the mean. Color image is available online at www.liebertpub.com/neu

FIG. 5.

Gross histopathology and regional water content at 7 days after controlled cortical impact (CCI). (A–E) Representative images of cresyl violet staining of the cortex (CTX) and hippocampus (HP) ipsilateral (ipsi) to impact at 7 days post-injury from vehicle (Veh) and IGF-1–treated mice subjected to sham or CCI injury. Sham-injured mice infused with either (A) vehicle or (B) IGF-1 exhibited brain swelling at the craniotomy site, which was not present at the time of surgery. (C) Injured mice treated with vehicle exhibited cortical cavitation and hippocampal cell loss consistent with CCI. (D) The majority of IGF-1–infused brain-injured mice (7 of 10) exhibited characteristic histopathology with minimal or no brain swelling. (E) However, a subset of IGF-1–infused CCI-injured mice (3 of 10) showed distortion of the hippocampus at 7 days post-injury, raising concerns that central infusion of IGF-1 could exacerbate cerebral edema (n = 4 sham-injured/treatment and n = 10 CCI-injured/treatment). Scale bar represents 500 μm. (F) Water content was quantified in mice subjected to CCI injury without cannulation or infusion (CCI), CCI injury with central infusion of vehicle (Veh CCI), or CCI injury with central infusion of 10 μg/day of IGF-1 (IGF-1 CCI; n = 6/group). Comparison by one-way ANOVA revealed infusion of IGF-1 did not result in significant regional cerebral edema; however, a small subset of mice (2 of 6) had markedly increased water content in the ipsilateral (ipsi) hippocampus. Contralateral (contra). Individual data points are superposed with bars representing mean + SEM. ANOVA, analysis of variance; SEM, standard error of the mean.

FIG. 6.

Delayed infusion of 10 μg/day of insulin-like growth factor-1 (IGF-1) increased hippocampal immature neuron density 1 week after controlled cortical impact (CCI). (A) Compared to CCI + Veh mice, CCI + IGF-1 mice showed increased doublecortin (DCX; green) immunoreactivity. Scale bar represents 100 μm. (B) Infusion of IGF-1 post-CCI resulted in significantly increased immature neuron density at 7 days post-injury, compared to vehicle treatment (^#p < 0.05). Data are presented as mean + SEM (n = 3 CCI + Veh and n = 5 CCI + IGF-1). SEM, standard error of the mean. Color image is available online at www.liebertpub.com/neu