Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) survivors often suffer from long-lasting cognitive impairment that stems from hippocampal injury. Systemic administration of insulin-like growth factor-1 (IGF-1), a polypeptide growth factor known to play vital roles in neuronal survival, has been shown to attenuate posttraumatic cognitive and motor dysfunction. However, its neuroprotective effects in TBI have not been examined. To this end, moderate or severe contusion brain injury was induced in mice with conditional (postnatal) overexpression of IGF-1 using the controlled cortical impact (CCI) injury model. CCI brain injury produces robust reactive astrocytosis in regions of neuronal damage such as the hippocampus. We exploited this regional astrocytosis by linking expression of hIGF-1 to the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, effectively targeting IGF-1 delivery to vulnerable neurons. Following brain injury, IGF-1Tg mice exhibited a progressive increase in hippocampal IGF-1 levels which was coupled with enhanced hippocampal reactive astrocytosis and significantly greater GFAP levels relative to WT mice. IGF-1 overexpression stimulated Akt phosphorylation and reduced acute (1 and 3d) hippocampal neurodegeneration, culminating in greater neuron survival at 10d after CCI injury. Hippocampal neuroprotection achieved by IGF-1 overexpression was accompanied by improved motor and cognitive function in brain-injured mice. These data provide strong support for the therapeutic efficacy of increased brain levels of IGF-1 in the setting of TBI

Early Microglial Activation Following Closed-Head Concussive Injury Is Dominated by Pro-Inflammatory M-1 Type

Microglial activation is a pathological hallmark of traumatic brain injury (TBI). Following brain injury, activated microglia/macrophages adopt different phenotypes, generally categorized as M-1, or classically activated, and M-2, or alternatively activated. While the M-1, or pro-inflammatory phenotype is detrimental to recovery, M-2, or the anti-inflammatory phenotype, aids in brain repair. Recent findings also suggest the existence of mixed phenotype following brain injury, where activated microglia simultaneously express both M-1 and M-2 markers. The present study sought to determine microglial activation states at early time points (6–72 h) following single or repeated concussive injury in rats. Closed-head concussive injury was modeled in rats using projectile concussive impact injury, with either single or repeated impacts (4 impacts, 1 h apart). Brain samples were examined using immunohistochemical staining, inflammatory gene profiling and real-time polymerase chain reaction analyses to detect concussive injury induced changes in microglial activation and phenotype in cortex and hippocampal regions. Our findings demonstrate robust microglial activation following concussive brain injury. Moreover, we show that multiple concussions induced a unique rod-shaped microglial morphology that was also observed in other diffuse brain injury models. Histological studies revealed a predominance of MHC-II positive M-1 phenotype in the post-concussive microglial milieu following multiple impacts. Although there was simultaneous expression of M-1 and M-2 markers, gene expression results indicate a clear dominance in M-1 pro-inflammatory markers following both single and repeated concussions. While the increase in M-1 markers quickly resolved after a single concussion, they persisted following repeated concussions, indicating a pro-inflammatory environment induced by multiple concussions that may delay recovery and contribute to long-lasting consequences of concussion

Temporal and Regional Changes in IGF-1/IGF-1R Signaling in the Mouse Brain after Traumatic Brain Injury

Although neurotrophic factors such as nerve growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin 4/5 are elevated after traumatic brain injury (TBI), little is known about the endogenous response of insulin-like growth factor-1 (IGF-1). We evaluated IGF-1, IGF-1 receptor (IGF-1R), and total and phosphorylated Akt (p-Akt), a known downstream mediator of IGF-1 signaling, using ELISA, Western blotting, and immunohistochemistry at 1, 6, 24, 48, and 72 h following 0.5-mm controlled cortical impact brain injury in adult mice. IGF-1 was transiently upregulated in homogenates of injured cortex at 1 h, and cells with increased IGF-1 immunoreactivity were observed in and around the cortical contusion site up to 48 h. IGF-1R and total Akt levels in cortical homogenates were unchanged, although immunohistochemistry revealed regional changes. In contrast, serine p-Akt levels increased significantly in homogenates at 6 h post-injury. Interestingly, delayed increases in vascular IGF-1R, total Akt, and p-Akt immunostaining were observed in and around the cortical contusion. IGF-1 and its downstream mediators were also upregulated in the subcortical white matter. Our findings indicate that moderate TBI results in a brief induction of IGF-1 and its signaling components in the acute post-traumatic period. This may reflect an attempt at endogenous neuroprotection or repair

Trauma-induced astrocyte-specific IGF-1 overexpression.

<p><b>A)</b> Coronal sections from IGF-1Tg mice show a marked increase in IGF-1 immunolabeling in the hippocampal dentate gyrus (DG) and CA-3 regions in the ipsilateral (ipsi) hemisphere compared to the contralateral (contra) after lateral controlled cortical impact (CCI) brain injury. Immunohistochemical staining was performed using an antibody that detects both human and mouse IGF-1. Representative picture is taken from an IGF-1Tg mouse 72 h after CCI. Scale bar = 100 µm. <b>B)</b> Quantification of IGF-1 expression using a human-specific IGF-1 ELISA. IGF-1 concentrations increased progressively in the ipsilateral hippocampus of IGF-1Tg mice at 24 h and 72 h after severe CCI brain injury. Data plotted as mean+SEM. * p<0.05 comparing injured with sham, <b>!</b> p<0.05 comparing ipsilateral to contralateral, and # p<0.05 comparing 72 h to 24 h. <b>C)</b> Confocal images taken from ipsilateral hemisphere bordering the contused cortex demonstrate widespread colocalization of IGF-1 (green) with the astrocyte-specific marker GFAP (red) in brain-injured IGF-1Tg mice. No co-localization of IGF-1 (green) and neuron-specific NeuN (red) was observed. Scale bar = 50 µm for C.</p

IGF-1 overexpression promoted hippocampal neuronal survival at 72 h after severe brain injury.

<p>Representative images of the ipsilateral hippocampus (HP) from Nissl-stained brain sections illustrate pallor or thinning of CA-3 and CA-1 areas of pyramidal layer (arrows) in wildtype (WT) mice. Higher magnification images from DG, CA-3 and CA-1 areas demonstrate hilar and CA-3 neuronal loss and thinning of the CA-1 pyramidal layer. IGF-1 overexpressing (IGF-1Tg) mice showed marked neuroprotection in each hippocampal subregion. Arrowheads point to hilar neurons. Scale bars = 500 µm (top HP panel), 100 µm for DG panel, 50 µm for CA-3 and CA-1 panels.</p

IGF-1 expression reduced post-traumatic behavioral impairment after severe brain injury.

<p><b>A)</b> In the modified neurological severity score (NSS), a test series of beam walking tasks, IGF-1 overexpressing (IGF-1Tg) mice exhibited significantly better coordinated motor function compared to wildtype (WT) mice over the first 4d after severe (1.0 mm) CCI. <b>B)</b> In the novel object recognition (NOR) does task, the recognition index represent the percent exploration time spent on the novel object. IGF-1 overexpression prevented posttraumatic memory impairment at 7d post-injury. Data expressed as mean+SEM. * p<0.05 comparing WT CCI and WT sham, # p<0.05 comparing IGF-1 Tg CCI and IGF-1Tg sham, and ! p<0.05 comparing IGF-1Tg CCI to WT CCI.</p

Enhanced astrocytosis in IGF-1Tg mice after brain injury.

<p><b>A)</b> Immunoreactivity for GFAP was similar in the hippocampus of WT and IGF-1Tg sham mice. Controlled cortical impact (CCI) brain injury induced astrocytosis in the ipsilateral hippocampus which was more profound in IGF-1Tg mice than in WT mice (shown for 72 h, moderate severity). Boxed areas in the molecular layer of the DG and the apex of the dentate hilus (DH) are magnified in the two lower panels to illustrate hypertrophied astrocytes (arrows) and elongated astroglial processes (arrowheads). <b>B)</b> Representative confocal images from the DH of an IGF-1Tg mouse showing GFAP-stained processes arising from SOX-2 positive cells (arrows). DH: dentate hilus, GL: granule layer. Scale bars = 200 µm for low magnification images and 50 µm (A) or 100 µm (B) for high magnification images.</p

IGF-1 overexpression provided hippocampal neuroprotection 10d after severe brain injury.

<p><b>A)</b> Representative images of the ipsilateral hippocampus from NeuN-immunostained brain sections after severe (1.0 mm) brain injury showing CA-3 neuronal loss (arrow). Scale bar = 200 µm. <b>B)</b> Stereological estimate of total number of CA-3 pyramidal neurons within the dorsal hippocampus (−1.0 to −2.2 mm Bregma) and <b>C)</b> Dentate hilar neuronal counts (total number of cells in 4 sections/animal) from wildtype (WT, open bars) and IGF-1 transgenic (IGF-1Tg, closed bars) mice. IGF-1 overexpression significantly increased neuronal survival in both regions. Data are expressed as mean+SEM. * p<0.05 and # p<0.05 comparing CCI with sham, and <b>!</b> p<0.05 comparing IGF-1Tg to WT.</p

IGF-1 overexpression increased hippocampal GFAP levels following brain trauma.

<p><b>A)</b> Representative western blot images for ipsilateral hippocampal samples probed with anti-GFAP and anti-actin antibodies. Blots illustrate expression at 72 h after severe controlled cortical impact (CCI) brain injury or sham injury (Sh). Relative expression of hippocampal GFAP at 24 h (<b>B,C</b>) and 72 h (<b>D,E</b>) following either 0.5 mm (<b>B,D</b>) or 1.0 mm (<b>C,E</b>) depth CCI in wildtype (WT, open bars) and IGF-1 transgenic (IGF-1Tg, closed bars) mice. Optical density from each band was normalised to its respective actin band and then group means were normalised to the mean of the WT sham group. Data represented as mean+SEM. * p<0.05 and # p<0.05 comparing CCI with sham, and <b>!</b> p<0.05 comparing IGF-1Tg to WT.</p