P7C3-A20 treatment one year after TBI in mice repairs the blood-brain barrier, arrests chronic neurodegeneration, and restores cognition

Abstract

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Keywords: blood–brain barrier; inflammation; neurodegeneration; neuroprotection; traumatic brain injury.

Fig. 1.

P7C3-A20 restores cognition in chronic TBI. (A) Experimental schematic. (B) All groups showed equal learning at 15 mo. At 19 mo, TBI-Veh exhibited learning deficits on day 4 compared to Sham-Veh. (C) Platform crossings in the memory probe test show expected aging-related decline in Sham-Veh mice, and TBI-Veh showed significant impairment relative to Sham-Veh. Memory function was fully restored in TBI-P7C3-A20 at both time points. Values are mean ± SEM. Individual data points represent individual animals. Significance was determined by repeated-measure two-way ANOVA for learning, one-way ANOVA for memory, and Dunnett’s post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001, relative to TBI-Veh.

Fig. 2.

P7C3-A20 restores the BBB and arrests neurodegeneration in chronic TBI. (A) Neuronal cell loss measured by immunostaining for NeuN in cortex (CTX) and hippocampal dentate gyrus (DG) reveals reduced neurons in TBI-Veh and TBI-P7C3-A20 relative to Sham-Veh. (Scale bars: 200 μm.) (B) Ongoing neurodegeneration measured by silver staining of CTX, corpus callosum (CC), fimbria, internal capsule, and hippocampus (HPC) demonstrate significant increase in TBI-Veh relative to Sham-Veh, with TBI-P7C3-A20 restored to Sham-Veh levels. (Scale bars: 20 μm.) (C) Transmission electron microscopy of CTX and HPC shows BBB capillary endothelium breaks (red arrows) in TBI-Veh, but not in Sham-Veh or TBI-P7C3-A20. (Scale bars: 0.5 μm and 1 μm). (D) Peripheral IgG infiltration into the brain, and neuroinflammation via Iba1 microglial activation, are increased in CTX, CC, and HPC in TBI-Veh compared to Sham-Veh, and restored to Sham-Veh levels in TBI-P7C3-A20. (Scale bars: 20 μm.) (E) Quantification of NeuN. (F) Quantification of silver staining. (G) Quantification of BBB capillary endothelium breaks was determined by number of capillary endothelial cell (EC) breaks per an average of 113 capillaries. (H) Quantification of IgG infiltration and Iba1 microglial activation. All values are mean ± SEM. Individual data points represent individual animals. Significance was determined by one-way ANOVA and Dunnett’s post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001 relative to TBI-Veh.

Fig. 3.

P7C3-A20 restores BBB capillary endothelium length and tight junction protein expression in chronic TBI and also increases basal pericyte abundance. (A–F) CD31 staining was used to determine capillary endothelium length, and PDGFRβ staining was used to quantify pericytes. Representative pictures (A and B) and quantification (C–F) from CTX and HPC of Sham-Veh, TBI-Veh, and TBI-P7C3-A20 show reduced capillary endothelium length in TBI-Veh relative to Sham-Veh, which is restored to normal by P7C3-A20. TBI-P7C3-A20 shows an increase in the number of pericytes per capillary length in the CTX and the HPC relative to both Sham-Veh and TBI-Veh. (Scale bars: 25 μm.) (G and H) Western blot analysis of tight junction proteins reveals that TBI-P7C3-A20 animals have higher levels of claudin-5 in the CTX and HPC, and of ZO1 in the CTX. Values are mean ± SEM. Individual data points represent individual animals. Significance was determined by one-way ANOVA and Dunnett’s post hoc analysis, n = 3; *P < 0.05, ***P < 0.001 relative to TBI-Veh. a.u., arbitrary unit.

Fig. 4.

P7C3-A20 protects endothelial cells in vivo and in vitro. (A) LPS-damage to the BBB was measured by brain permeability of fluorescent-conjugated 3-kDa dextran, with greater dextran entry in LPS-Veh relative to Veh-Veh. LPS-P7C3-A20, however, showed significant reduction relative to LPS-VEH. Values are mean ± SEM. Individual data points represent individual animals. Significance was determined by one-way ANOVA and Tukey’s post hoc analysis. ***P < 0.001, ****P < 0.0001 relative to Veh-LPS. (B) Cultured human brain microvascular endothelial cells exposed to 0.1 mM H₂O₂ showed 50% reduction in cell viability, which was blocked by P7C3-A20 in a dose–response manner. Values are presented as mean ± SEM. Individual data points represent individual experiments of six replicates. Significance was determined by one-way ANOVA and Dunnett’s post hoc analysis, n = 5; **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to H₂O₂ cells treated only.

Fig. 5.

P7C3-A20 treatment acutely protects BBB integrity after TBI. (A) Acute damage to the BBB was determined by brain permeability of fluorescent-conjugated 3-kDa dextran at different time points after TBI, with significantly higher dextran permeability at 3 h after the injury compared against Sham group, and returning to normal thereafter. Values are mean ± SEM. Individual data points represent individual animals. Significance was determined by one-way ANOVA and Dunnett’s post hoc analysis, *P < 0.05 relative to Sham. (B) Experimental schematic. Efficacy of P7C3-A20 in protecting the BBB was determined at (C) 3 h and (D) 6 h after TBI. Values are mean ± SEM. Individual data points represent individual animals. Significance was determined by one-way ANOVA and Dunnett’s post hoc analysis, *P < 0.05 relative to TBI-Veh.