Sonia Villapol

Temporal dynamics of cerebral blood flow, cortical damage, apoptosis, astrocyte-vasculature interaction and astrogliosis in the pericontusional region after traumatic brain injury.

Traumatic brain injury (TBI) results in a loss of brain tissue at the moment of impact in the cerebral cortex. Subsequent secondary injury involves the release of molecular signals with dramatic consequences for the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The mechanisms behind the progression of tissue loss remain under investigation. In this study, we analyzed the spatial–temporal profile of blood flow, apoptotic, and astrocytic–vascular events in the cortical regions around the impact site at time points ranging from 5 h to 2 months after TBI. We performed a mild–moderate controlled cortical impact injury in young adult mice and analyzed the glial and vascular response to injury. We observed a dramatic decrease in perilesional cerebral blood flow (CBF) immediately following the cortical impact that lasted until days later. CBF finally returned to baseline levels by 30 days post-injury (dpi). The initial impact also resulted in an immediate loss of tissue and cavity formation that gradually increased in size until 3 dpi. An increase in dying cells localized in the pericontusional region and a robust astrogliosis were also observed at 3 dpi. A strong vasculature interaction with astrocytes was established at 7 dpi. Glial scar formation began at 7 dpi and seemed to be compact by 60 dpi. Altogether, these results suggest that TBI results in a progression from acute neurodegeneration that precedes astrocytic activation, reformation of the neurovascular unit to glial scar formation. Understanding the multiple processes occurring after TBI is critical to the ability to develop neuroprotective therapeutics to ameliorate the short and long-term consequences of brain injury.

Commercially Available Angiotensin II At2 Receptor Antibodies Are Nonspecific

Commercially available angiotensin II AT2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available AT2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and AT2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or AT2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of AT2 receptor binding or gene expression. We conclude that none of these commercially available AT2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study AT2 receptor expression.

Candesartan, an Angiotensin II AT1-Receptor Blocker and PPAR-γ Agonist, Reduces Lesion Volume and Improves Motor and Memory Function After Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) results in complex pathological reactions, the initial lesion worsened by secondary inflammation and edema. Angiotensin II (Ang II) is produced in the brain and Ang II receptor type 1 (AT1R) overstimulation produces vasoconstriction and inflammation. Ang II receptor blockers (ARBs) are neuroprotective in models of stroke but little is known of their effect when administered in TBI models. We therefore performed controlled cortical impact (CCI) injury on mice to investigate whether the ARB candesartan would mitigate any effects of TBI. We administered candesartan or vehicle to mice 5 h before CCI injury. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%, decreased the number of dying neurons, lessened the number of activated microglial cells, protected cerebral blood flow (CBF), and reduced the expression of the cytokine TGFβ1 while increasing expression of TGFβ3. Candesartan-treated mice also showed better motor skills on the rotarod 3 days after injury, and improved performance in the Morris water maze 4 weeks after injury. These results indicate that candesartan is neuroprotective, reducing neuronal injury, decreasing lesion volume and microglial activation, protecting CBF and improving functional behavior in a mouse model of TBI. Co-treatment with a peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist significantly reduced some of the beneficial effects of candesartan after CCI, suggesting that PPARγ activation may contribute to part or to all of the neuroprotective effect of candesartan. Overall, our data suggest that ARBs with dual AT1R-blocking and PPARγ activation properties may have therapeutic value in treating TBI.

Neurorestoration after traumatic brain injury through angiotensin II receptor blockage

See Moon (doi:10.1093/awv239) for a scientific commentary on this article.Traumatic brain injury frequently leads to long-term cognitive problems and physical disability yet remains without effective therapeutics. Traumatic brain injury results in neuronal injury and death, acute and prolonged inflammation and decreased blood flow. Drugs that block angiotensin II type 1 receptors (AT1R, encoded by AGTR1) (ARBs or sartans) are strongly neuroprotective, neurorestorative and anti-inflammatory. To test whether these drugs may be effective in treating traumatic brain injury, we selected two sartans, candesartan and telmisartan, of proven therapeutic efficacy in animal models of brain inflammation, neurodegenerative disorders and stroke. Using a validated mouse model of controlled cortical impact injury, we determined effective doses for candesartan and telmisartan, their therapeutic window, mechanisms of action and effect on cognition and motor performance. Both candesartan and telmisartan ameliorated controlled cortical impact-induced injury with a therapeutic window up to 6 h at doses that did not affect blood pressure. Both drugs decreased lesion volume, neuronal injury and apoptosis, astrogliosis, microglial activation, pro-inflammatory signalling, and protected cerebral blood flow, when determined 1 to 3 days post-injury. Controlled cortical impact-induced cognitive impairment was ameliorated 30 days after injury only by candesartan. The neurorestorative effects of candesartan and telmisartan were reduced by concomitant administration of the peroxisome proliferator-activated receptor gamma (PPARγ, encoded by PPARG) antagonist T0070907, showing the importance of PPARγ activation for the neurorestorative effect of these sartans. AT1R knockout mice were less vulnerable to controlled cortical impact-induced injury suggesting that the sartans blockade of the AT1R also contributes to their efficacy. This study strongly suggests that sartans with dual AT1R blocking and PPARγ activating properties have therapeutic potential for traumatic brain injury.

Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice.

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2+ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1+ microglial cells were proliferating more. A smaller, but significant number of GFAP+ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX+ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP+ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.

TGF-β Superfamily Gene Expression and Induction of the Runx1 Transcription Factor in Adult Neurogenic Regions after Brain Injury

Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.

Sexual dimorphism in the inflammatory response to traumatic brain injury

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate‐to‐severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro‐ (IL‐1β and TNFα) and anti‐inflammatory (Arg1 and TGFβ) cytokines with a single‐phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro‐inflammatory response peaks at 4 h and 7 days, and a delayed anti‐inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post‐TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.

Smad3 deficiency increases cortical and hippocampal neuronal loss following traumatic brain injury

Transforming growth factor-β (TGF-β) signaling is involved in pathological processes following brain injury. TGF-β signaling through Smad3 contributes significantly to the immune response and glial scar formation after brain injury. However, TGF-β is also neuroprotective, suggesting that Smad3 signaling may also be involved in neuroprotection after injury. We found expression of the TGF-β type II receptor (TβRII) and Smad3 protein to be strongly and rapidly induced in neurons in the ipsilateral cortex and CA1 region of the hippocampus after stab wound injury. In contrast, astrocytic expression of TβRII and Smad3 was induced more slowly. Comparison of the response of wild-type and Smad3 null mice to cortical stab wound injury showed a more pronounced loss of neuronal viability in Smad3 null mice. Neuronal density was more strongly reduced in Smad3 null mice than in wild-type mice at 1 and 3days post lesion in both the ipsilateral cortex and hippocampal CA1 region. Fluoro-Jade B, TUNEL staining, and cleaved caspase-3 staining also demonstrated increased neuronal degeneration at early time points after injury in the ipsilateral hemisphere in Smad3 null mice. Taken together, our results suggest that TGF-β cytokine family signaling through Smad3 protects neurons in the damaged cortex and hippocampus at early time points after injury.

Protease induced plasticity: Matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism.

Hepatic Expression of Serum Amyloid A1 Is Induced by Traumatic Brain Injury and Modulated by Telmisartan

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.