fibrin and Demyelinating-Diseases

Cortical demyelination is a common finding in patients with chronic multiple sclerosis (MS) and contributes to disease progression and overall disability. The exact pathomechanism that leads to cortical lesions is not clear. Research is limited by the fact that standard animal models of multiple sclerosis do not commonly affect the cortex, or if they do in some variants, the cortical demyelination is rather sparse and already remyelinated within a few days. In an attempt to overcome these limitations we implanted a tissue-compatible catheter into the cortex of Dark Agouti rats. After 14days the rats were immunized with 5μg myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's Adjuvant, which did not cause any clinical signs but animals developed a stable anti-MOG antibody titer. Then the animals received an injection of proinflammatory cytokines through the catheter. This led to a demyelination of cortical and subcortical areas starting from day 1 in a cone-like pattern spreading from the catheter area towards the subarachnoid space. On day 3 cortical demyelination already expanded to the contralateral hemisphere and reached its peak between days 9-15 after cytokine injection with a widespread demyelination of cortical and subcortical areas of both hemispheres. Clinically the animals showed only discrete signs of fatigue and recovered completely after day 15. Even on day 30 we still were able to detect demyelination in subpial and intracortical areas along with areas of partial and complete remyelination. Loss of cortical myelin was accompanied with marked microglia activation. A second injection of cytokines through the catheter on day 30 led to a second demyelination phase with the same symptoms, but again no detectable motor dysfunction. Suffering of the animals appeared minor compared to standard Experimental Autoimmune Encephalomyelitis and therefore, even long-term observation and repeated demyelination phases seem ethically acceptable.

Topics: Animals; Calcium-Binding Proteins; Caspase 3; Cerebral Cortex; Cytokines; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Fibrin; Freund's Adjuvant; Functional Laterality; Immunization; Lipids; Male; Microfilament Proteins; Microscopy, Confocal; Motor Activity; Myelin Proteolipid Protein; Myelin-Associated Glycoprotein; Nerve Tissue Proteins; Rats; Statistics, Nonparametric

Neuronal loss, a key substrate of irreversible disability in multiple sclerosis (MS), is a recognized feature of MS cortical pathology of which the cause remains unknown. Fibrin(ogen) deposition is neurotoxic in animal models of MS, but has not been evaluated in human progressive MS cortex. The aim of this study was to investigate the extent and distribution of fibrin(ogen) in progressive MS cortex and elucidate its relationship with neurodegeneration.. A postmortem cohort of pathologically confirmed MS (n = 47) and control (n = 10) cases was used. The extent and distribution of fibrin(ogen) was assessed and related to measures of demyelination, inflammation, and neuronal density. In a subset of cases (MS, n = 20; control, n = 10), expression of plasminogen activator inhibitor 1 (PAI-1), a key enzyme in the fibrinolytic cascade, was assessed and related to the extent of fibrin(ogen).. Motor cortical fibrin(ogen) deposition was significantly over-represented in MS compared to control cases in all compartments studied (ie, extracellular [p = 0.001], cell body [p = 0.003], and neuritic/glial-processes [p = 0.004]). MS cases with high levels of extracellular fibrin(ogen) had significantly upregulated PAI-1 expression in all cortical layers assessed (p < 0.05) and reduced neuronal density (p = 0.017), including in the functionally-relevant layer 5 (p = 0.001).. For the first time, we provide unequivocal evidence that fibrin(ogen) is extensively deposited in progressive MS motor cortex, where regulation of fibrinolysis appears perturbed. Progressive MS cases with severe fibrin(ogen) deposition have significantly reduced neuronal density. Future studies are needed to elucidate the provenance and putative neurotoxicity of fibrin(ogen), and its potential impact on clinical disability. Ann Neurol 2017;82:259-270.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Demyelinating Diseases; Female; Fibrin; Fibrinogen; Humans; Inflammation; Male; Middle Aged; Motor Cortex; Multiple Sclerosis, Chronic Progressive; Nerve Degeneration; Plasminogen Activator Inhibitor 1

Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood-brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b(+) antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity.

Topics: Adaptive Immunity; Animals; Antigen Presentation; Autoimmunity; Blood-Brain Barrier; Brain; CD11b Antigen; Cell Proliferation; Chemokine CCL2; Chemokine CXCL10; Chemokines; CX3C Chemokine Receptor 1; Demyelinating Diseases; DNA-Binding Proteins; Encephalomyelitis, Autoimmune, Experimental; Fibrin; Fibrinogen; Flow Cytometry; Gene Expression Profiling; Genes, MHC Class II; Homeodomain Proteins; Immunohistochemistry; Macrophages; Mice; Mice, Knockout; Microglia; Myelin-Oligodendrocyte Glycoprotein; Rats; Receptors, Antigen, T-Cell; Receptors, Chemokine; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Th1 Cells

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.

Topics: Animals; Axons; Blood Coagulation Factors; Connexin 30; Connexins; Demyelinating Diseases; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Fibrin; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Poly I-C; Thrombin

Early signs of inflammatory demyelination include entry of fibrin(ogen) into the central nervous system (CNS), which is normally excluded by the blood-brain barrier, and up-regulation of components of the plasminogen activator system. Using mice deficient in tissue-type plasminogen activator (tPA-/-) and urokinase plasminogen activator receptor (uPAR-/-), we investigated the involvement of the PA system on the clinical and pathological features of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. tPA-/- mice suffered an early and a more severe acute disease characterized by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of plasminogen activator inhibitor-1. This correlated with fibrin accumulation, which co-localized with nonphosphorylated neurofilament on thickened axons in experimental allergic encephalomyelitis tissue. In contrast, uPAR-/- mice had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells. These animals developed chronic disease as a result of steadily increased inflammation, increased levels of urokinase-type plasminogen activator (uPA), and greater degree of demyelination. Thus, the plasminogen activator system can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA and uPAR on fibrinolysis and cell adhesion/migration, manipulation of which may have therapeutic implications for multiple sclerosis.

Topics: Animals; Axons; Central Nervous System; Demyelinating Diseases; Encephalomyelitis, Autoimmune, Experimental; Female; Fibrin; Fibrinolysis; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Nerve Degeneration; Plasminogen Activator Inhibitor 1; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

In multiple sclerosis, in which brain tissue becomes permeable to blood proteins, extravascular fibrin deposition correlates with sites of inflammatory demyelination and axonal damage. To examine the role of fibrin in neuroinflammatory demyelination, we depleted fibrin in two tumor necrosis factor transgenic mouse models of multiple sclerosis, transgenic lines TgK21 and Tg6074. In a genetic analysis, we crossed TgK21 mice into a fibrin-deficient background. TgK21fib(-/-) mice had decreased inflammation and expression of major histocompatibility complex class I antigens, reduced demyelination, and a lengthened lifespan compared with TgK21 mice. In a pharmacologic analysis, fibrin depletion, by using the snake venom ancrod, in Tg6074 mice also delayed the onset of inflammatory demyelination. Overall, these results indicate that fibrin regulates the inflammatory response in neuroinflammatory diseases. Design of therapeutic strategies based on fibrin depletion could potentially benefit the clinical course of demyelinating diseases such as multiple sclerosis.

Topics: Animals; Cell Line; Demyelinating Diseases; Fibrin; Inflammation; Macrophage Activation; Mice; Mice, Transgenic; Multiple Sclerosis; Plasminogen Activators; Tumor Necrosis Factor-alpha; Up-Regulation

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.

Topics: Animals; Axons; Blood Coagulation; Demyelinating Diseases; Extracellular Matrix; Fibrin; Fibrinogen; Fibrinolysis; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Denervation; Muscle, Skeletal; Nerve Degeneration; Plasminogen; Schwann Cells; Sciatic Nerve; Tissue Plasminogen Activator

We examined the role of coagulation-fibrinolysis system in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). The degree of fibrin deposition around the vessels in the spinal cord was significantly higher in susceptible SJL/J mice on 30 days post intracerebral injection (i.c.) than resistant C57BL/6 mice on 30 days post i.c. or mock infected SJL/J mice. Treatment with batroxobin (30 BU/kg/day), which is a thrombin-like defibrinogenating enzyme, causing a profound degree of afibrinogenemia, suppressed clinical signs of TMEV-IDD. Plasma fibrinogen concentration was significantly decreased in batroxobin-treated mice. Histologically, though the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in batroxobin-treated mice compared to saline-treated control mice, fibrin deposition was markedly suppressed in batroxobin-treated mice. These findings suggest that batroxobin suppresses TMEV-IDD through its defibrination effect, and provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of the blood-brain barrier (BBB), are prerequisite events for clinical manifestations of TMEV-IDD.

Topics: Animals; Antibodies, Viral; Demyelinating Diseases; Disease Susceptibility; Female; Fibrin; Hypersensitivity, Delayed; Injections, Intraperitoneal; Injections, Intraventricular; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Poliomyelitis; Sodium Chloride; Spinal Cord; T-Lymphocytes; Theilovirus

Sural nerve biopsy was performed in twenty-four diabetic patients, with clinical and electrophysiological evidence of diabetic neuropathy. Material from an autopsy case was also examined. Vessels plugged with fibrin were seen within nerve in nine cases. In three cases fibrin was observed tracking into the vessel wall and in four, older thrombus was observed in vessels. Areas of necrosis in nerve bundles were seen in two of the latter. In two patients there had been a preceding episode of intravascular coagulation. Fibrin deposition within small vessels could well play a part in damaging the diabetic nerve and a disturbance of the balance between deposition and removal by fibrinolysis could explain phasic variation in the symptoms of neuropathy.

Topics: Adult; Aged; Blood Vessels; Demyelinating Diseases; Diabetic Neuropathies; Female; Fibrin; Fibrinolysis; Humans; Male; Middle Aged; Neural Conduction; Peripheral Nerves; Sural Nerve

Topics: Animals; Antigens; Bacterial Infections; Brain; Cross Reactions; Demyelinating Diseases; Disease Models, Animal; Edema; Encephalomyelitis, Autoimmune, Experimental; Exudates and Transudates; Fibrin; Guinea Pigs; Haplorhini; Hemorrhage; Heparin; Humans; Immunization, Passive; Inflammation; Leukocytes; Lymphocytes; Multiple Sclerosis; Myelin Sheath; Pertussis Vaccine; Proteins; Rats; Virus Diseases