Fibrinogen-cellular prion protein complex formation on astrocytes.

Traumatic brain injury (TBI) is one of the most common neurological disorders causing memory reduction, particularly short-term memory (STM). We showed that, during TBI-induced inflammation, increased blood content of fibrinogen (Fg) enhanced vascular protein transcytosis and deposition of extravasated Fg in vasculo-astrocyte interfaces. In addition, we found that deposition of cellular prion protein (PrPC) was also increased in the vasculo-astrocyte endfeet interface. However, association of Fg and PrPC was not confirmed. Presently, we aimed to define whether Fg can associate with PrPC on astrocytes and cause their activation. Cultured mouse brain astrocytes were treated with medium alone (control), Fg (2 mg/mL or 4 mg/mL), 4 mg/mL of Fg in the presence of a function-blocking anti-PrPC peptide or anti-mouse IgG, function-blocking anti-PrPC peptide, or anti-mouse IgG alone. After treatment, either cell lysates were collected and analyzed via Western blot or coimmunoprecipitation was performed, or astrocytes were fixed and their activation was assessed with immunohistochemistry. Results showed that Fg dose-dependently activated astrocytes, increased expressions of PrPC and tyrosine (tropomyosin) receptor kinase B (TrkB), and PrP gene. Blocking the function of PrPC reduced these effects. Coimmunoprecipitation demonstrated Fg and PrPC association. Since it is known that prion protein has a greater effect on memory reduction than amyloid beta, and that activation of TrkB is involved in neurodegeneration, our findings confirming the possible formation of Fg-PrPC and Fg-induced overexpression of TrkB on astrocytes suggest a possible triggering mechanism for STM reduction that was seen previously during mild-to-moderate TBI.NEW & NOTEWORTHY For the first time we showed that fibrinogen (Fg) can associate with cellular prion protein (PrPC) on the surface of cultured mouse brain astrocytes. At high levels, Fg causes upregulation of astrocyte PrPC and astrocyte activation accompanied with overexpression of tyrosine receptor kinase B (TrkB), which results in nitric oxide (NO) production and generation of reactive oxygen species (ROS). Fg/PrPC interaction can be a triggering mechanism for TrkB-NO-ROS axis activation and the resultant astrocyte-mediated neurodegeneration.

Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg), which results in enhanced cerebrovascular permeability and leads to short-term memory (STM) reduction. Previously, we showed that extravasated Fg was deposited in the vasculo-astrocyte interface and was co-localized with cellular prion protein (PrPC) during mild-to-moderate TBI in mice. These effects were accompanied by neurodegeneration and STM reduction. However, there was no evidence presented that the described effects were the direct result of the HFg during TBI. We now present data indicating that inhibition of Fg synthesis can ameliorate TBI-induced cerebrovascular permeability and STM reduction. Cortical contusion injury (CCI) was induced in C57BL/6J mice. Then mice were treated with either Fg antisense oligonucleotide (Fg-ASO) or with control-ASO for two weeks. Cerebrovascular permeability to fluorescently labeled bovine serum albumin was assessed in cortical venules following evaluation of STM with memory assessement tests. Separately, brain samples were collected in order to define the expression of PrPC via Western blotting while deposition and co-localization of Fg and PrPC, as well as gene expression of inflammatory marker activating transcription factor 3 (ATF3), were characterized with real-time PCR. Results showed that inhibition of Fg synthesis with Fg-ASO reduced overexpression of AFT3, ameliorated enhanced cerebrovascular permeability, decreased expression of PrPC and Fg deposition, decreased formation of Fg-PrPC complexes in brain, and improved STM. These data provide direct evidence that a CCI-induced inflammation-mediated HFg could be a triggering mechanism involved in vascular cognitive impairment seen previously in our studies during mild-to-moderate TBI.

Fibrinogen and Neuroinflammation During Traumatic Brain Injury.

Many neurodegenerative diseases such as Alzheimer's disease (AD), multiple sclerosis, and traumatic brain injury (TBI) are associated with systemic inflammation. Inflammation itself results in increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg). Fg is not only considered an acute phase protein and a marker of inflammation, but has been shown that it can cause inflammatory responses. Fibrin deposits have been associated with memory reduction in neuroinflammatory diseases such as AD and TBI. Reduction in short-term memory has been seen during the most common form of TBI, mild-to-moderate TBI. Fibrin deposits have been found in brains of patients with mild-to-moderate TBI. The vast majority of the literature emphasizes the role of fibrin-activated microglia as the mediator in the neuroinflammation pathway. However, the recent discovery that astrocytes, which constitute approximately 30% of the cells in the mammalian central nervous system, manifest different reactive states warrants further investigations in the causative role of HFg in astrocyte-mediated neuroinflammation. Our previous study showed that Fg deposited in the vasculo-astrocyte interface-activated astrocytes. However, little is known of how Fg directly affects astrocytes and neurons. In this review, we summarize studies that show the effect of Fg on different types of cells in the vasculo-neuronal unit. We will also discuss the possible mechanism of HFg-induced neuroinflammation during TBI.

Positive Allosteric Modulation of Cholinergic Receptors Improves Spatial Learning after Cortical Contusion Injury in Mice.

We examined benzyl quinolone carboxylic acid (BQCA), a novel M1 muscarinic-positive allosteric modulator, for improving memory and motor dysfunction after cerebral cortical contusion injury (CCI). Adult mice received unilateral motorsensory cortical CCI or sham injury. Benzyl quinolone carboxylic acid (BQCA; 5, 10, and 20 mg/kg, intraperitoneally [i.p.] × 2/day × 3-4 weeks) or vehicle (Veh) were administered, and weekly evaluations were undertaken using a battery of motor tests, as well as the Morris water maze. Thereafter, cerebral metabolic activation was investigated in awake animals during walking with [14C]-2-deoxygIucose autoradiography, comparing CCI mice previously treated with BQCA (20 mg/kg) or vehicle. Relative changes in local cerebral glucose uptake (rCGU) were evaluated in three-dimensional-reconstructed brains using statistical parametric mapping. CCI resulted in mild hyperactivity in the open field, and modest significant motor deficits, as well as significantly decreased spatial learning at 3 weeks. BQCA in CCI mice resulted in significantly improved spatial recall during the third week, with minimal effects on motor outcomes. CCI significantly decreased rCGU in the ipsilesional basal ganglia-thalamocortical circuit and in somatosensory regions, with relative increases noted contralaterally, as well as in the cerebellum. Significant decreases in rCGU were noted in subregions of the ipsilesional hippocampal formation, with significant increases noted contralesionally. BQCA compared to vehicle-treated mice showed modest, though significantly increased, rCGU in motor regions, as well as a partial reversal of lesion-related rCGU findings in subregions of the hippocampal formation. rCGU in ipsilesional posterior CA1 demonstrated a significant inverse correlation with latency to find the submerged platform. BQCA at 20 mg/kg had no significant effect on general motor activity, body weight, or acute motor, secretory, or respiratory symptoms. Results suggest that BQCA is a candidate compound to improve learning and memory function after brain trauma and may not suffer the associated central nervous system side effects typically associated with even modest doses of other cholinergic enhancers.

Effects of DHA on Hippocampal Autophagy and Lysosome Function After Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers endoplasmic reticulum (ER) stress and impairs autophagic clearance of damaged organelles and toxic macromolecules. In this study, we investigated the effects of the post-TBI administration of docosahexaenoic acid (DHA) on improving hippocampal autophagy flux and cognitive functions of rats. TBI was induced by cortical contusion injury in Sprague-Dawley rats, which received DHA (16 mg/kg in DMSO, intraperitoneal administration) or vehicle DMSO (1 ml/kg) with an initial dose within 15 min after the injury, followed by a daily dose for 3 or 7 days. First, RT-qPCR reveals that TBI induced a significant elevation in expression of autophagy-related genes in the hippocampus, including SQSTM1/p62 (sequestosome 1), lysosomal-associated membrane proteins 1 and 2 (Lamp1 and Lamp2), and cathepsin D (Ctsd). Upregulation of the corresponding autophagy-related proteins was detected by immunoblotting and immunostaining. In contrast, the DHA-treated rats did not exhibit the TBI-induced autophagy biogenesis and showed restored CTSD protein expression and activity. T2-weighted images and diffusion tensor imaging (DTI) of ex vivo brains showed that DHA reduced both gray matter and white matter damages in cortical and hippocampal tissues. DHA-treated animals performed better than the vehicle control group on the Morris water maze test. Taken together, these findings suggest that TBI triggers sustained stimulation of autophagy biogenesis, autophagy flux, and lysosomal functions in the hippocampus. Swift post-injury DHA administration restores hippocampal lysosomal biogenesis and function, demonstrating its therapeutic potential.

A behavioral and histological comparison of fluid percussion injury and controlled cortical impact injury to the rat sensorimotor cortex.

Our primary goal was to evaluate the behavioral and histological outcome of fluid percussion injury (FPI) and cortical contusion injury (CCI) to the sensorimotor cortex (SMC). The SMC has been used to evaluate neuroplasticity following CCI, but has not been extensively examined with FPI. In both the CCI and FPI models, a mechanical force of 4mm in diameter was applied over the SMC, allowing for a direct comparison to measure the relative rates of histology and recovery of function in these models. Gross behavioral deficits were found on the sensory task (tactile adhesive removal task) and multiple motor assessments (forelimb asymmetry task, forelimb placing task, and rotorod). These sensorimotor deficits occurred in the absence of cognitive deficits in the water maze. The CCI model creates focal damage with a localized injury wheras the FPI model creates a more diffuse injury causing widespread damage. Both behavioral and histological deficits ensued following both models of injury to the SMC. The neuroplastic changes and ease at which damage to this area can be measured behaviorally make this an excellent location to assess traumatic brain injury (TBI) treatments. No injury model can completely mimic the full spectrum of human TBI and any potential treatments should be validated across both focal and diffuse injury models. Both of these injury models to the SMC produce severe and enduring behavioral deficits, which are ideal for evaluating treatment options.

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury.

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1(+) microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32(+) microglia or macrophages, but an increased CD206(+) phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1(+) microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1(+) microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.