The Veterinary Journal 298–299 (2023) 106018 Contents lists available at ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl Immunohistochemical evaluation of fibrin/fibrinogen, D-dimers, and intravascular thrombosis in brains of dogs with meningoencephalitis of unknown origin M. Raposo a, b, C. de la Fuente b, M. Pumarola c, J. Ríos d, S. Añor a, b, * a Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain b Fundació Hospital Clínic Veterinari, Facultad de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain c Unitat de Patologia Murina i Comparada (UPMiC) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain d Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Granulomatous meningoencephalitis (GME) and necrotizing encephalitides (NE) are the most common immune- Fibrinolysis mediated inflammatory diseases of the central nervous system in dogs. Activation of the fibrinolytic system in Granulomatous meningoencephalitis multiple sclerosis, a similar immune-mediated disease affecting the central nervous system in humans, seems to Immunohistochemistry be related to disease progression. The aim of this study was to identify fibrin/fibrinogen and D-dimer deposition, Necrotizing encephalitis as well as presence of intravascular thrombosis (IVT) in brains of dogs with a diagnosis of GME or NE. Immu­ Thrombi nohistochemical studies using antibodies against fibrin/fibrinogen and D-dimers were performed. Statistical analyses were performed to determine whether there were differences in the presence and location of fibrin/ fibrinogen, D-dimers deposits, and IVT between GME and NE. Samples from sixty-four dogs were included in the study: 32 with a diagnosis of GME and 32 with a diagnosis of NE. Fibrin/fibrinogen depositions were detected in all samples and D-dimers were detected in 43/64 samples. IVT was present in 29/64 samples, with a significantly higher score in samples from dogs with NE than in samples from dogs with GME (P = 0.001). These data support hemostatic system activation in both diseases, especially NE. This finding might be related to the origin of the necrotic lesions seen in NE, which could represent chronic ischemic lesions. Further studies are needed to investigate the association between vascular lesions and the histopathological differences between GME and NE and the hemostatic system as a potential therapeutic target. Introduction which are differentiated by the distribution and appearance of the microscopic lesions (Summers et al., 1995; Vandevelde et al., 2012). The Inflammatory diseases are among the most common causes of central asymmetric necrotic lesions affecting the cerebral white or gray matter nervous system (CNS) dysfunction in dogs (Fluehmann et al., 2006). in the NE are the main histopathological difference between these and Granulomatous meningoencephalitis (GME) and necrotizing encepha­ GME. litides (NE) are different subtypes of meningoencephalitis of unknown Inflammation and coagulation are two systems that cannot be origin (MUO) and histopathological analysis is essential for a definitive considered separately, since extensive cross-talk exists between them diagnosis (Tipold, 1995; Granger et al., 2010; Talarico and Schatzberg, (Cicala and Cirino, 1998; Esmon, 2008; Levi and Van Der Poll, 2005, 2010; Coates and Jeffery, 2014). GME is characterized by concentric 2010; O’Brien, 2012). The inflammatory response causes release of tis­ perivascular accumulations of mononuclear inflammatory cells, mainly sue factor and a downregulation of the physiological anticoagulant macrophages with smaller numbers of lymphocytes and plasma cells, pathways. At the same time, coagulation proteases and anticoagulant predominantly affecting the CNS white matter. NE include necrotizing proteins interact with cell receptors inducing the synthesis of meningoencephalitis (NME) and necrotizing leukoencephalitis (NLE), pro-inflammatory cytokines (Levi and Van Der Poll, 2005, 2010). * Corresponding author at: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. E-mail address: sonia.anor@uab.cat (S. Añor). https://doi.org/10.1016/j.tvjl.2023.106018 Accepted 30 July 2023 Available online 1 August 2023 1090-0233/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). M. Raposo et al. The Veterinary Journal 298–299 (2023) 106018 Thereby, inflammation influences coagulation while coagulation acti­ normal distribution of fibrin/fibrinogen and D-dimers in the CNS. vates the inflammatory response. Fibrin/fibrinogen deposition was graded using a semiquantitative Similarities have been noted between MUOs in dogs and multiple score for intensity of immunostaining and location of deposits. All sclerosis (MS) in people such as their idiopathic origin, the female pre­ samples were evaluated under 20x objective lens. Fibrin/fibrinogen dilection, young age of the patients, and the same MHC class II genetic deposition was graded as follows: score 0, no staining; score 1, mild association between NME in Pugs and human MS (Greer et al., 2010; (pale) staining; score 2, moderate (evident) staining; and score 3, severe Church et al., 2021). MS is one of the most common causes of neurologic (strong) staining. Location of deposits was classified as intracellular disability in young adults that results from an autoimmune disease of the (inside glial cells), extracellular (extracellular space), or mixed (both CNS (World Health Organization. and Multiple Sclerosis International intra- and extracellular in the same sample). Federation., 2008). An association between the dysregulation of the D-dimer immunoreactivity was classified as positive if D-dimer de­ hemostatic system and the development and progression of has been posits were observed in the sample, or negative if there were no D-dimer described in both humans with MS (Wakefield et al., 1994; Gverić et al., deposits. IVT was considered to be present when a vessel was occluded 2003; Vos et al., 2005; Aksungar et al., 2008; Göbel et al., 2016; Yates by a deposit of D-dimers. Twenty high power fields (HPF; 40x objective et al., 2017; Petersen et al., 2018) and in the most commonly used lens) within the main lesion were evaluated and were graded as follows: experimental model for MS, experimental autoimmune encephalomy­ score 0: no vessels occluded; score 1: at least 1 but < 33% of total vessels elitis (EAE) in rodents (Han et al., 2008; Davalos et al., 2014). occluded; score 2: 33–66% of total vessels occluded; and score 3: > 66% Blood-brain barrier (BBB) disruption in MS causes extravasation and of total vessels occluded. deposition of serum fibrin/fibrinogen in the brain parenchyma. In addition to their role in the coagulation cascade, fibrin/fibrinogen Statistical analysis induce activation of the microglia, axonal damage, and demyelination, and hinders axonal regeneration (Yates et al., 2017; Petersen et al., Variables were tabulated according to sample GME / NE classifica­ 2018). Deposition of fibrin/fibrinogen and D-dimers, and presence of tion. Statistical analyses were performed using a Fisher’s Exact test to intravascular thrombosis (IVT) in canine gliomas (de la Fuente et al., compare the location of fibrin/fibrinogen deposits and the presence of 2014) and meningiomas (Font et al., 2015) has been described, but in­ D-dimer deposits between GME and NE samples. For ordinal variables, a formation is lacking about their occurrence in canine MUOs. Mann-Whitney U test was used to compare the scores of fibrin/fibrin­ Coagulation factor dysregulation and impaired fibrinolysis is also ogen immunostaining and the grade of IVT between GME and NE sam­ important in the development of MS (Gverić et al., 2003; Aksungar et al., ples. All statistical analyses were performed using a statistical software 2008; Han et al., 2008; Davalos et al., 2014; Göbel et al., 2016). The package (SPSS version 26, IBM Corp). Statistical significance was set at upregulation of some factors in the coagulation cascade, such as factor X P < 0.05. or thrombin, is linked to neuroinflammation and progression in MS and EAE (Davalos et al., 2014; Göbel et al., 2016). D-dimers, a specific Results degradation product of the turnover of cross-linked fibrin, are a sensitive marker of activation of coagulation and fibrinolysis (Weitz et al., 2017). Case selection Serum D-dimer concentrations are increased in MS patients (Aksungar et al., 2008). While in dogs, 54% (n = 20/37) of dogs with inflamma­ Samples from 64 dogs were included in the study. Dogs were aged tory/infectious CNS disease had detectable D-dimer concentrations in between 6 months and 11 years (median 4 years). Breeds included their CSF, whereas D-dimers were not detectable in the CSF of control Yorkshire terrier (n = 13), Crossbreed (n = 9), Chihuahua (n = 6), dogs (de la Fuente et al., 2012). Maltese (n = 6), French bulldog (n = 5), Alaskan Husky (n = 2), Belgian The aim of this study was to evaluate the presence and pattern of shepherd (n = 2), Cocker spaniel (n = 2), Shih tzu (n = 2), Pug (n = 2), fibrin/fibrinogen and D-dimer deposition in brain samples of dogs with a West Highland white terrier (n = 2), Miniature poodle (n = 2), and one diagnosis of GME and NE using immunohistochemical techniques, to of each of German shepherd, Pomeranian, Greyhound, Labrador evaluate the presence of IVT in these samples, and to compare the retriever, English Springer spaniel, Rottweiler, Miniature Pinscher, presence and location of these features between the two conditions. Parson Russell terrier, Dachshund, Samoyed, and Old English sheepdog. Thirty-two dogs had a diagnosis of GME, and 32 dogs a diagnosis of NE. Materials and methods Within the NE group, 15 had a diagnosis of NME and 17 a diagnosis of NLE. Case selection Fibrin/fibrinogen immunostaining Our veterinary neuropathology database was searched to identify dogs with a histopathological diagnosis of GME and NE, excluding dogs Fibrin/fibrinogen deposits were detected in all samples, with a score with focal inflammation involving the optic nerve or spinal cord, made of 3 in 35/64 (55%) samples, a score of 2 in 22/64 (34%) samples, and a by an ECVP board-certified pathologist (MP), for which there was score of 1 in 7/64 (11%) samples (Table 1). No fibrin/fibrinogen de­ archival tissue suitable for immunohistochemical studies between 2001 posits were detected in the control tissue. There was no significant dif­ and 2019. ference in fibrin/fibrinogen scores between the GME and the NE groups (P = 0.801). Immunohistochemistry Two different patterns of fibrin/fibrinogen depositions were detec­ ted. In one, fibrin/fibrinogen deposits were primarily located inside glial Formalin-fixed, paraffin-embedded tissue sections (3 µm thick) were cells (reactive astrocytes/gemistocytes and microglia/macrophages) used. The immunohistochemical procedures, using antibodies against combined with a more diffuse staining usually associated with areas of fibrin/fibrinogen (A0080, Dako) and D-dimers (ABS 015–22–02, loss of brain parenchyma (Fig. 1A, B). The other was a reticular pattern Thermo Fisher Scientific) were the same as those described previously of fibrin/fibrinogen deposits in the extracellular space, mainly sur­ for canine gliomas and meningiomas (de la Fuente et al., 2014; Font rounding blood vessels and forming perivascular cuffs (Fig. 1C). When et al., 2015). Sections from a canine brain with intra-parenchymal both patterns were present, deposits were classified as mixed (Fig. 1D). hemorrhage were used as a positive control. For a negative control, an There was a significant difference in the location of fibrin/fibrinogen isotype-specific immunoglobulin was used as a substitute for the pri­ deposits between GME and NE groups (P < 0.001; Table 1), with mary antibody. Healthy canine brain tissue was used to determine extracellular deposits being present in 18/32 (56%) of GME samples as 2 M. Raposo et al. The Veterinary Journal 298–299 (2023) 106018 Table 1 intra- and extravascular D-dimer deposition. Extravascular D-dimer Comparison of fibrin/fibrinogen deposition, fibrin/fibrinogen pattern, presence immunostaining showed a reticular pattern in the extracellular space of extracellular D-dimers, and intravascular thrombosis (IVT) between granu­ and co-localized with the fibrin/fibrinogen deposition (Fig. 2A-C). lomatous meningoencephalitis (GME) and necrotizing encephalitides (NE). Intracellular D-dimer immunoreactivity was not observed in any sam­ GME (n = 32) NE (n = 32) P ple. D-dimers were not detected in control tissue. There was no signifi­ Fibrin/fibrinogen Score 1: 4 (13%) Score 1: 3 (9%) 0.801 cant difference in extravascular D-dimer presence between GME and NE depositiona Score 2: 11 (34%) Score 2: 11 (34%) groups (P = 0.782). Score 3: 17 (53%) Score 3: 18 (56%) IVT was detected in the microvasculature within the lesions of 29/64 Fibrin/fibrinogen pattern Extracellular: 18 Extracellular: 6 < (45%) samples, comprising 8 with GME and 21 with NE (Fig. 2D, F). (56%) (19%) 0.001 Intracellular: 5 Intracellular: 20 Only five (17%) of the positive samples had a score of 3, of which four (16%) (63%) had NE (Table 1). The IVT score was significantly higher in the NE group Mixed: 9 (28%) Mixed: 6 (19%) than the GME group (P = 0.001). Presence of extracellular Yes: 10 (31%) Yes: 8 (25%) 0.782 D-dimers No: 22 (69%) No: 24 (75%) Discussion IVTb Score 0: 24 (75%) Score 0: 11 (34%) 0.001 Score 1: 4 (13%) Score 1: 9 (28%) Score 2: 3 (9%) Score 2: 8 (25%) Fibrin/fibrinogen deposits were found in all samples of the study, as Score 3: 1 (3%) Score 3: 4 (13%) was found in previous studies on MS and EAE (Vos et al., 2005; Marik GME, Granulomatous meningoencephalitis; NE, Necrotizing encephalitides; et al., 2007; Yates et al., 2017). The presence of fibrin/fibrinogen in the IVT, Intravascular thrombosis extracellular compartment in GME, mostly perivascular, could be a Samples with an absence of staining received a score of 0; samples with mild related to the acute damage of blood vessels and the breakdown of the staining received a score of 1; samples with moderate staining received a score of BBB. This is similar to what happens in the early stages of MS, preceding 2; and samples with strong staining received a score of 3. the demyelinating stage (Marik et al., 2007). In NE, the fibrin/fibrinogen b Samples with no vessels occluded received a score of 0; samples with at least deposits were located mostly within glial cells, particularly reactive one but < 33% of total vessels occluded received a score of 1; samples with astrocytes and gitter cells. The presence of fibrin/fibrinogen deposits 33–66% of total vessels occluded received a score of 2; and samples with > 66% within astrocytes is suspected to be a result of phagocytosis (Hsiao et al., of total vessels occluded received a score of 3. 2013) and is consistent with the findings of a study on progressive MS, in which fibrin/fibrinogen were found in glial cell processes and within cell compared to 6/32 (19%) of NE samples and glial cell deposits in 20/32 bodies in the more chronic stages of the disease (Yates et al., 2017). The (63%) of NE samples as compared to 5/32 (28%) of GME samples. results of the study described herein support GME and NE as naturally-occurring disease models of MS and evidenced the more D-dimers immunostaining and IVT chronic nature of the NE lesions compared with the GME lesions. Some workers have reported an increase in serum D-dimer concen­ D-dimer deposition was detected in 43/64 (67%) samples (Table 1). trations in patients with MS, which reflects an alteration in the coagu­ Extravascular D-dimer deposition was observed in 14 samples, intra­ lation system (Aksungar et al., 2008). Patients with relapsing-remitting vascular deposition in 25 samples, and a further four samples with both Fig. 1. Images showing fibrin/fibrinogen immunohistochemistry. Brown color indicates immunopositivity. (A) Fibrin/fibrinogen immunolabelling inside activated astrocytes in a necrotic lesion of a dog with necrotizing leu­ koencephalitis (NLE) (score 3). (B) Fibrin/ fibrinogen deposition accumulated by gitter cells in a necrotic lesion of a dog with NLE (score 2). (C) Fibrin/fibrinogen immunolabel­ ling with reticular patterns filling the extracel­ lular space and surrounding the perivascular cuffs in a dog affected with granulomatous meningoencephalitis (score 3). (D) Fibrin/ fibrinogen deposits in a dog affected with a necrotizing meningoencephalitis showing a mixed pattern, comprising a focal extracellular reticular pattern surrounding blood vessels and deposits inside gitter cells associated with a necrotic lesion (score 3). 3 M. Raposo et al. The Veterinary Journal 298–299 (2023) 106018 Fig. 2. (A) Hematoxylin and eosin staining, (B) fibrin/fibrinogen immunostaining, and (C) D-dimer immunostaining of the same region in a dog affected with granulomatous meningoencephalitis. Brown color indicates immunopositivity. Note the presence of both (fibrin/fibrinogen and D-dimer) deposits with a reticular pattern, but with more focal D-dimer immunostaining. (D) D-dimer immunohistochemistry in a dog with necrotizing meningoencephalitis, demonstrating D-dimer deposits inside blood vessels producing vascular occlusion close to a necrotic lesion (score 3). (E) Higher magnification of the dog in (D) with positive staining for D- dimer structure compatible with a thrombus in a larger vessel. MS also have increased intrathecal CSF D-dimer concentrations, when disease, especially with NE, and this could explain the necrotic lesions compared with disease controls (Schaller-Paule et al., 2022), as was also being secondary to ischemic damage. noted in patients with other inflammatory diseases such as bacterial and Most infarcts occur because of obstruction of a blood vessel, although viral meningitis and central nervous system neoplasia. Serum and CSF some of them might be secondary to inflammatory lesions within the D-dimer concentrations have been measured in dogs with various vessel wall (Vandevelde et al., 2012). Capillary damage caused by per­ neurological diseases (De la Fuente et al., 2012), with MUOs included in ivascular cuffs can be associated with the presence of thrombi, which a diverse group of inflammatory CNS diseases that included bacterial can lead to ischemic insults and may explain the presence of necrotic meningomyelitis, distemper virus meningoencephalomyelitis, and acute lesions in NE. There has only been one case report of isolated angiitis in idiopathic polyradiculoneuritis. Twenty of the 37 dogs in this group had the CNS of a dog (Sasaki et al., 2003). In that case, the microscopic detectable D-dimer concentrations in the CSF, but this was not signifi­ appearance of the lesions shared greater similarity with GME than NE. cantly different from the control group. D-dimer immunostaining was Lympho-histiocytic meningoencephalitis of unknown etiology with found in two-thirds of the cases in our study, suggesting local activation vasculitis of the small and medium vessels of the brain parenchyma has of the coagulation and fibrinolytic system in GME and NE. Future studies recently been described in three dogs, and is considered a new subtype could assess CSF D-dimer concentrations in MUO patients and compare of MUO (Zdora et al., 2022). The higher frequency of IVT in NE could the results between GME and NE patients. indicate a greater impact on blood vessels than GME. However, endo­ The main histopathological difference between GME and NE is the thelial / vascular damage in GME and NE samples was not investigated presence of necrotic lesions in the latter. The main hypothesis to explain in this study, therefore no definitive explanation was elucidated for the this is the chronicity of the inflammation in the NE, which entails origin of increased IVT in NE. phagocytosis of the affected nervous tissue forming cavitary lesions Recognition of the relationship between the hemostatic system and (Higgins et al., 2008). There are no other inflammatory CNS diseases the development of MS lesions led to the investigation of different with large necrotic areas as those seen in NE, regardless of the chronicity therapeutic targets to slow down progression of disease and thus of the condition. Only chronic ischemic lesions show a microscopic improve prognosis. Fibrin depletion in transgenic mouse models of MS appearance resembling necrotic NE lesions (Kuwamura et al., 2002; led to decreased inflammation and reduced demyelination (Akassoglou Vandevelde et al., 2012). IVT was detected in half of the samples in this et al., 2004), and the use of recombinant activated protein C decreased study, and most of them were NE samples. This result indicates that the severity of lesions in EAE, due to its anticoagulant action (Han et al., abnormal thrombotic activity is present in dogs with inflammatory CNS 2008). In human medicine, people with MS are at increased risk of 4 M. Raposo et al. The Veterinary Journal 298–299 (2023) 106018 cerebrovascular accidents, especially ischemic ones (Belliston et al., Font, C., de la Fuente, C., Pumarola, M., Blasco, E., Fernández, F., Viu, J., Añor, S., 2015. Canine intracranial meningiomas: Immunohistochemical evaluation of tissue factor, 2018; Hong et al., 2019). Treatment with acetylsalicylic acid shows fibrin/fibrinogen and D-dimers. The Veterinary Journal 206, 426–428. positive effects, such as decreased possibility of ischemic stroke and de la Fuente, C., Monreal, L., Cerón, J., Pastor, J., Viu, J., Añor, S., 2012. Fibrinolytic thromboembolism or reduced inflammation, but also shows negative activity in cerebrospinal fluid of dogs with different neurological disorders. Journal effects, such as increased risk of hemorrhagic stroke (Tsau et al., 2015). of Veterinary Internal Medicine 26, 1365–1373. de la Fuente, C., Pumarola, M., Blasco, E., Fernández, F., Viu, J., Añor, S., 2014. Our results suggest that the coagulation and fibrinolytic systems are Immunohistochemical evaluation of tissue factor, fibrin/fibrinogen and D-dimers in activated in GME and NE, and this opens a field of research for future canine gliomas. The Veterinary Journal 200, 387–392. studies to elucidate potential therapeutic targets in the coagulation Göbel, K., Kraft, P., Pankratz, S., Gross, C.C., Korsukewitz, C., Kwiecien, R., Mesters, R., Kehrel, B.E., Wiendl, H., Kleinschnitz, C., et al., 2016. Prothrombin and factor X are cascade. elevated in multiple sclerosis patients. Annals of Neurology 80, 946–951. There are some limitations associated with this study. Due to its Granger, N., Smith, P.M., Jeffery, N.D., 2010. Clinical findings and treatment of non- retrospective nature, antemortem treatments were not documented and infectious meningoencephalomyelitis in dogs: a systematic review of 457 published cases from 1962 to 2008. The Veterinary Journal 184, 290–297. could have included glucocorticoids, cytosine arabinose, nonsteroidal Greer, K.A., Wong, A.K., Liu, H., Famula, T.R., Pedersen, N.C., Ruhe, A., Wallace, M., anti-inflammatory drugs, and others that could have affected the Neff, M.W., 2010. Necrotizing meningoencephalitis of pug dogs associates with dog microscopic findings and the immunostaining results. The subjective leukocyte antigen class II and resembles acute variant forms of multiple sclerosis. Tissue Antigens 76, 110–118. semi-quantitative nature of the assessment of the immunohistochemical Gverić, D., Herrera, B., Petzold, A., Lawrence, D.A., Cuzner, M.L., 2003. Impaired results may lead to small inaccuracies. Additionally, due to the long fibrinolysis in multiple sclerosis: a role for tissue plasminogen activator inhibitors. period chosen to obtain the greatest number of cases possible, the Brain 126, 1590–1598. Han, M.H., Hwang, S.-I., Roy, D.B., Lundgren, D.H., Price, J.V., Ousman, S.S., Fernald, G. preservation of the oldest samples may have influenced the immuno­ H., Gerlitz, B., Robinson, W.H., Baranzini, S.E., et al., 2008. Proteomic analysis of histochemical staining. active multiple sclerosis lesions reveals therapeutic targets. Nature 451, 1076–1081. Higgins, R.J., Dickinson, P.J., Kube, S.A., Moore, P.F., Couto, S.S., Vernau, K.M., Conclusions Sturges, B.K., Lecouteur, R.A., 2008. Necrotizing meningoencephalitis in five Chihuahua dogs. Veterinary Pathology 45, 336–346. Hong, Y., Tang, H.R., Ma, M., Chen, N., Xie, X., He, L., 2019. Multiple sclerosis and IVT is present in GME and NE, suggesting activation of the coagu­ stroke: a systematic review and meta-analysis. BMC Neurology 19, 1–11. lation and fibrinolytic system in these diseases. This finding is especially Hsiao, T.W., Swarup, V.P., Kuberan, B., Tresco, P.A., Hlady, V., 2013. Astrocytes specifically remove surface-adsorbed fibrinogen and locally express chondroitin evident in NE and could be due to ischemic damage causing the necrotic sulfate proteoglycans. Acta Biomaterialia 9, 7200–7208. lesions present in these diseases. Future studies should be performed to Kuwamura, M., Adachi, T., Yamate, J., Kotani, T., Ohashi, F., Summers, B.A., 2002. elucidate the presence of vascular lesions in NE antemortem, and the Necrotising encephalitis in the Yorkshire terrier: a case report and literature review. Journal of Small Animal Practice 43, 459–463. hemostatic system as a potential therapeutic target in GME and NE. Levi, M., Van Der Poll, T., 2010. Inflammation and coagulation. Critical Care Medicine 38, S26–S34. Declaration of Competing Interest Levi, M., Van Der Poll, T., 2005. Two-way interactions between inflammation and coagulation. Trends in Cardiovascular Medicine 15, 254–259. Marik, C., Felts, P.A., Bauer, J., Lassmann, H., Smith, K.J., 2007. Lesion genesis in a None of the authors of this paper has a financial or personal rela­ subset of patients with multiple sclerosis: A role for innate immunity? Brain 130, tionship with other people or organizations that could inappropriately 2800–2815. O’Brien, M., 2012. The reciprocal relationship between inflammation and coagulation. influence or bias the content of the paper. Topics in Companion Animal Medicine 27, 46–52. Petersen, M.A., Ryu, J.K., Akassoglou, K., 2018. Fibrinogen in neurological diseases: Acknowledgments mechanisms, imaging and therapeutics. Nature Reviews Neuroscience 19, 283–301. Sasaki, M., Pool, R., Summers, B.A., 2003. Vasculitis in a dog resembling isolated angiitis of the central nervous system in humans. Veterinary Pathology 40, 95–97. We gratefully acknowledge Ester Blasco and Tamara Rivero for Schaller-Paule, M.A., Yalachkov, Y., Steinmetz, H., Friedauer, L., Hattingen, E., technical assistance. Preliminary results were presented as an Oral Miesbach, W., Weber, F., Kirchmayr, K., Schaefer, J.H., Foerch, C., 2022. Analysis of Presentation at the 34th ESVN–ECVN Symposium, Palma de Mallorca, CSF D-dimer to identify intrathecal fibrin-driven autoimmunity in patients with multiple sclerosis. Neurology Neuroimmunology and Neuroinflammation 9, 1–10. 23–24 September 2022. Summers, B.A., Cummings, J.F., De Lahunta, A., 1995. Veterinary Neuropathology. Mosby, St. Louis, Missouri, USA. References Talarico, L.R., Schatzberg, S.J., 2010. Idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: A review and future perspectives. Journal of Small Animal Practice 51, 138–149. Akassoglou, K., Adams, R.A., Bauer, J., Mercado, P., Tseveleki, V., Lassmann, H., Tipold, A., 1995. Diagnosis of inflammatory and infectious diseases of the central Probert, L., Strickland, S., 2004. Fibrin depletion decreases inflammation and delays nervous system in dogs: a retrospective study. Journal of Veterinary Internal the onset of demyelination in a tumor necrosis factor transgenic mouse model for Medicine 9, 304–314. multiple sclerosis. Proceedings of the National Academy of Sciences of the United Tsau, S., Emerson, M.R., Lynch, S.G., LeVine, S.M., 2015. Aspirin and multiple sclerosis. States of America 101, 6698–6703. BMC Medicine 13, 1–16. Aksungar, F.B., Topkaya, A.E., Yildiz, Z., Sahin, S., Turk, U., 2008. Coagulation status Vandevelde, M., Higgins, R., Oevermann, A., 2012. Inflammatory diseases. Veterinary and biochemical and inflammatory markers in multiple sclerosis. Journal of Clinical Neuropathology: Essentials of Theory and Practice. John Wiley and Sons, Ames, Neuroscience 15, 393–397. Iowa, USA, pp. 48–80. Belliston, S.M., Dubinsky, R.M., Lynch, S.G., 2018. Cerebrovascular comorbidities in Vos, C.M.P., Geurts, J.J.G., Montagne, L., van Haastert, E.S., Bö, L., van Der Valk, P., multiple sclerosis: analysis of the nationwide inpatient sample. Neurological Barkhof, F., de Vries, H.E., 2005. Blood-brain barrier alterations in both focal and Disorders and Stroke International 1, 1012. diffuse abnormalities on postmortem MRI in multiple sclerosis. Neurobiology of Church, M.E., Ceja, G., McGeehan, M., Miller, M.C., Farias, P., Sánchez, M.D., Swain, G. Disease 20, 953–960. P., Assenmacher, C.-A., Stopa, E.G., Vite, C.H., et al., 2021. Meningeal B cell clusters Wakefield, A.J., More, L.J., Difford, J., McLaughlin, J.E., 1994. Immunohistochemical correlate with submeningeal pathology in a natural model of multiple sclerosis. The study of vascular injury in acute multiple sclerosis. Journal of Clinical Pathology 47, Journal of Immunology 207, 44–54. 129–133. Cicala, C., Cirino, G., 1998. Linkage between inflammation and coagulation: an update Weitz, J.I., Fredenburgh, J.C., Eikelboom, J.W., 2017. A test in context: D-dimer. Journal on the molecular basis of the crosstalk. Life Sciences 62, 1817–1824. of the American College of Cardiology 70, 2411–2420. Coates, J.R., Jeffery, N.D., 2014. Perspectives on meningoencephalomyelitis of unknown World Health Organization, Multiple Sclerosis International Federation, 2008. Atlas origin. Veterinary Clinics of North America - Small Animal Practice 44, 1157–1185. multiple sclerosis resources in the world 2008. WHO Press, Geneva, Switzerland. Davalos, D., Baeten, K.M., Whitney, M.A., Mullins, E.S., Friedman, B., Olson, E.S., Ryu, J. Yates, R.L., Esiri, M.M., Palace, J., Jacobs, B., Perera, R., DeLuca, G.C., 2017. Fibrin K., Smirnoff, D.S., Petersen, M.A., Bedard, C., et al., 2014. Early detection of (ogen) and neurodegeneration in the progressive multiple sclerosis cortex. Annals of thrombin activity in neuroinflammatory disease. Annals of Neurology 75, 303–308. Neurology 82, 259–270. Esmon, C.T., 2008. Crosstalk between inflammation and thrombosis. Maturitas 61, Zdora, I., Raue, J., Söbbeler, F., Tipold, A., Baumgärtner, W., Nessler, J.N., 2022. Case 122–131. report: Lympho-histiocytic meningoencephalitis with central nervous system Fluehmann, G., Doherr, M.G., Jaggy, A., 2006. Canine neurological diseases in a referral vasculitis of unknown origin in three dogs. Frontiers in Veterinary Science 9, hospital population between 1989 and 2000 in Switzerland. Journal of Small Animal 944867. Practice 47, 582–587. 5