The tissue-type plasminogen activator-plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans.

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma.

Sex-dependent effects of tranexamic acid on blood-brain barrier permeability and the immune response following traumatic brain injury in mice.

BACKGROUND: Tranexamic acid (TXA) is an anti-fibrinolytic agent used to reduce bleeding in various conditions including traumatic brain injury (TBI). As the fibrinolytic system also influences the central nervous system and the immune response, TXA may also modulate these parameters following TBI. OBJECTIVES: To determine the effect of TXA on blood-brain barrier (BBB) integrity and changes in immune and motor function in male and female mice subjected to TBI. METHODS: Wild-type and plasminogen deficient (plg-/-) mice were subjected to TBI then administered either TXA/vehicle. The degree of BBB breakdown, intracerebral hemorrhage (ICH), motor dysfunction, and changes in inflammatory subsets in blood and brain were determined. RESULTS AND CONCLUSIONS: Tranexamic acid significantly reduced BBB breakdown, and increased blood neutrophils in male mice 3 hours post-TBI. In contrast, TXA treatment of female mice increased BBB permeability and ICH but had no effect on blood neutrophils at the same time-point. TXA improved motor function in male mice but still increased BBB breakdown in female mice 24 hours post-TBI. Brain urokinase-type plasminogen activator (u-PA) antigen and activity levels were significantly higher in injured females compared to males. Because TXA can promote a pro-fibrinolytic effect via u-PA, these sex differences may be related to brain u-PA levels. TXA also increased monocyte subsets and dendritic cells in the injured brain of wild-type male mice 1 week post-TBI. Plg-/- mice of both sexes had reduced BBB damage and were protected from TBI irrespective of treatment indicating that TXA modulation of the BBB is plasmin-dependent. In conclusion, TXA is protective post-TBI but only in male mice.

Tranexamic Acid Influences the Immune Response, but not Bacterial Clearance in a Model of Post-Traumatic Brain Injury Pneumonia.

The antifibrinolytic agent, tranexamic acid (TXA), an inhibitor of plasmin formation, currently is evaluated to reduce bleeding in various conditions, including traumatic brain injury (TBI). Because plasmin is implicated in inflammation and immunity, we investigated the effects of plasmin inhibition on the immune response after TBI in the presence or absence of induced pneumonia. Wild-type mice treated with vehicle or TXA or mice deficient in plasminogen (plg-/-) underwent TBI using the controlled cortical impact model. Mice were then subjected to Staphylococcus aureus induced pneumonia and the degree of immune competence determined. Significant baseline changes in the innate immune cell profile were seen in plg-/- mice with increases in spleen weight and white blood cell counts, and elevation in plasma interleukin-6 levels. The plg-/- mice subjected to TBI displayed no additional changes in these parameters at the 72 h or one week time point post-TBI. The plg-/- mice subjected to TBI did not exhibit any further increase in susceptibility to endogenous infection. Pneumonia was induced by intratracheal instillation of S. aureus. The TBI did not worsen pneumonia symptoms or delay recovery in plg-/- mice. Similarly, in wild type mice, treatment with TXA did not impact on the ability of mice to counteract pneumonia after TBI. Administration of TXA after TBI and subsequent pneumonia, however, altered the number and surface marker expression of several myeloid and lymphoid cell populations, consistent with enhanced immune activation at the 72 h time point. This investigation confirms the immune-modulatory properties of TXA, thereby highlighting its effects unrelated to inhibition of fibrinolysis.

Activated CD8+ T Cells Cause Long-Term Neurological Impairment after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8+ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17+CD4+ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8+ T cells, but not depletion of CD4+ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.

Tranexamic acid modulates the cellular immune profile after traumatic brain injury in mice without hyperfibrinolysis.

BACKGROUND: Traumatic brain injury (TBI) is known to promote immunosuppression, making patients more susceptible to infection, yet potentially exerting protective effects by inhibiting central nervous system (CNS) reactivity. Plasmin, the effector protease of the fibrinolytic system, is now recognized for its involvement in modulating immune function. OBJECTIVE: To evaluate the effects of plasmin and tranexamic acid (TXA) on the immune response in wild-type and plasminogen-deficient (plg-/- ) mice subjected to TBI. METHODS: Leukocyte subsets in lymph nodes and the brain in mice post TBI were evaluated by flow cytometry and in blood with a hemocytometer. Immune responsiveness to CNS antigens was determined by Enzyme-linked Immunosorbent Spot (ELISpot) assay.  Fibrinolysis was determined by thromboelastography and measuring D-dimer and plasmin-antiplasmin complex levels. RESULTS: Plg-/-  mice, but not plg+/+  mice displayed increases in both the number and activation of various antigen-presenting cells and T cells in the cLN 1 week post TBI. Wild-type mice treated with TXA also displayed increased cellularity of the cLN 1 week post TBI together with increases in innate and adaptive immune cells. These changes occurred despite the absence of systemic hyperfibrinolysis or coagulopathy in this model of TBI. Importantly, neither plg deficiency nor TXA treatment enhanced the autoreactivity within the CNS. CONCLUSION: In the absence of systemic hyperfibrinolysis, plasmin deficiency or blockade with TXA increases migration and proliferation of conventional dendritic cells (cDCs) and various antigen-presenting cells and T cells in the draining cervical lymph node (cLN) post TBI. Tranexamic acid might also be clinically beneficial in modulating the inflammatory and immune response after TBI, but without promoting CNS autoreactivity.

The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis.

Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE.

Traumatic brain injury opens blood-brain barrier to stealth liposomes via an enhanced permeability and retention (EPR)-like effect.

The opening of the tight junctions in the blood-brain barrier (BBB) following traumatic brain injury (TBI) is hypothesized to be sufficient to enable accumulation of large drug carriers, such as stealth liposomes, in a similar manner to the extravasation seen in tumor tissue via the enhanced permeability and retention (EPR) effect. The controlled cortical impact model of TBI was used to evaluate liposome accumulation in mice. Dual-radiolabeled PEGylated liposomes were administered either immediately after induction of TBI or at increasing times post-TBI to mimic the likely clinical scenario. The accumulation of radiolabel in the brain tissue ipsilateral and contralateral to the site of trauma, as well as in other organs, was evaluated. Selective influx of liposomes occurred at 0-8 h after injury, while the barrier closed between 8 and 24 hr after injury, consistent with reports on albumin infiltration. Significantly enhanced accumulation of liposomes occurred in mice subjected to TBI compared to anaesthetized controls, and accumulation was greater in the injured versus the contralateral side of the brain. Thus, stealth liposomes show potential to enhance drug delivery to the site of brain injury with a wide range of encapsulated therapeutic candidates.

The neuroprotective actions of hypoxic preconditioning and postconditioning in a neonatal rat model of hypoxic-ischemic brain injury.

Perinatal hypoxic-ischemic (HI) brain injury remains a major contributing factor to newborn mortality and morbidity. Preconditioning with mild hypoxia has been shown to protect the brain against HI insults and it has recently been shown that mild hypoxia administered after a brain injury, termed 'postconditioning' can protect the adult mouse brain. Here, we have investigated the neuroprotective effects of hypoxic pre- and postconditioning in a neonatal rat model of HI brain injury. 7-Day-old Sprague-Dawley rat pups underwent unilateral common carotid artery ligation in combination with 3h at 5.5% oxygen. Hypoxic treatments consisted of either 3h of 8% oxygen performed 24h prior to injury (preconditioning); or 1h of 8% oxygen 24h post-injury, performed once a day for 5 days (postconditioning). Brains were removed 1 week post-injury for histological analysis. HI caused an increase in lesion volume compared to controls and both hypoxic pre- and postconditioning reduced the degree of brain damage following HI injury. To specifically examine neuronal loss, NeuN immunohistochemistry and regional brain area analysis were performed. HI injury caused a loss in NeuN staining in all brain regions examined. Preconditioning with hypoxia resulted in a significant reduction in cortical, hippocampal and striatal neuronal loss, compared with HI alone. Hypoxic postconditioning resulted in a reduction in cortical and striatal neuronal loss, compared to HI alone. Our results further support the clinical potential for mild hypoxia in the treatment of brain injuries, either as a pre- or post-injury treatment strategy.

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice.

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25-30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.