Astroglial Dysfunction, Demyelination and Nodular inflammation in Necrotizing Meningoencephalitis

Astroglial Dysfunction, Demyelination and Nodular inflammation in Necrotizing Meningoencephalitis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Astroglial Dysfunction, Demyelination and Nodular inflammation in Necrotizing Meningoencephalitis Sara I Hernandez , Charles A Assenmacher , Molly E Church , Jorge I Alvarez doi: https://doi.org/10.1101/2024.11.11.623107 Sara I Hernandez 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles A Assenmacher 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Molly E Church 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: churchm{at}vet.upenn.edu alvaj{at}vet.upenn.edu Jorge I Alvarez 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: churchm{at}vet.upenn.edu alvaj{at}vet.upenn.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Necrotizing Meningoencephalitis (NME), a form of Meningoencephalitis of Unknown Origin (MUO), is a progressive neuroinflammatory disease that primarily affects young, small-breed dogs. Due to limited understanding of its pathophysiology, early detection and the development of targeted therapies remain challenging. Definitive ante-mortem diagnosis is often unfeasible, and dogs with NME are frequently grouped under the broader MUO category. Our long-term objective is to identify distinct disease mechanisms within each MUO subtype to improve diagnostic accuracy, therapeutic approaches, and prognostic outcomes. To establish unique inflammatory patterns as they relate to neuropathologic changes in NME, we studied we studied the degree of immune cell infiltration, astrogliosis, demyelination, and microglial activation, comparing these factors with granulomatous meningoencephalomyelitis (GME), a closely related MUO subtype. We found that in the leptomeninges, NME is characterized by mild immune cell infiltration, in contrast to the prominent, B cell-rich aggregates seen in GME. In the neuroparenchyma, both diseases exhibit a comparable degree of lymphocyte infiltration; however, demyelination is more pronounced in NME, particularly within the subcortical white matter. Notably, areas of the brain affected by NME display a reduction in astrogliosis, which is associated with a marked decrease in the expression of the water channel protein aquaporin-4 (AQP4), a reduction not observed in GME. Additionally, we found that AQP4 expression levels correlate with the extent of microglial and macrophage activation. These findings suggest that astrocyte dysfunction in regions of microglial inflammation is a driver of NME and with adaptive immune responses likely playing a supportive role. Introduction Meningoencephalitis of Unknown Origin (MUO) refers to a group idiopathic neuroinflammatory diseases in canines. They include Granulomatous Meningoencephalomyelitis (GME), Necrotizing Meningoencephalitis (NME), and Necrotizing Leukoencephalitis (NLE). Clinical diagnosis of MUO is made by ruling out infectious, neoplastic, or other neurologic diseases affecting the Central Nervous System (CNS) 1 . The treatment for MUOs typically involves immunosuppressive drugs, such as corticosteroids, often combined with chemotherapy, along with supportive care to manage symptoms 1 . While, signalment, MRI findings, and clinical signs are associated with specific MUO subtypes, definitive diagnosis of each MUO subtype is only made post-mortem by histopathological confirmation of distinct patterns of change in the brain 2 . In GME, inflammation has classically been described as occurring in the white matter of the cerebrum, optic chiasm, midbrain, brainstem, and spinal cord, with a predilection toward caudal portions of the neuroaxis. Leptomeningeal and neuroparenchymal inflammation is characterized by the presence of aggregates of lymphocytes, plasma cells, and epithelioid macrophages extending from around blood vessels 1 & 3 . Perivascular infiltration in GME is typically more pronounced compared to other MUOs 1 , 3 & 4 . Our group recently demonstrated that in GME the extent of B cell aggregation in leptomeningeal infiltrates correlates with the severity of sub-meningeal demyelination 5 . NME mainly affects young, small breed dogs such as Pugs, Shih Tzus, Yorkshire Terriers, and Chihuahuas 1 & 6 . Genetic predispositions have been described in Pugs, Chihuahuas, Maltese, and other small breed dogs 7 , 8 & 9 . Lesions are found more rostrally in the neuroaxis, mainly in the grey matter of the cerebral cortex and sometimes extend to the subcortical white matter 1 , 4 & 6 . Leptomeningeal, neuroparenchymal, and perivascular inflammation is characterized by lymphocytes and macrophages, with some plasma cells documented. The characteristic histologic changes are typified by regions of parenchymal necrosis and rarefaction with associated activated macrophages. Moderate to large numbers of astrocytes are often observed at the periphery of these lesions 3 & 10 . In NLE, Yorkshire Terriers and French Bulldogs are overrepresented though it occurs in other small breed dogs 1 . Necrotizing lesions of NLE are localized more rostrally in the neuroaxis, like NME, but are found mainly in the subcortical white matter with associated activated macrophages. In contrast to GME and NME, there is minimal leptomeningeal and neuroparenchymal lymphoplasmacytic inflammation 1 & 6 . Dogs diagnosed with MUO have a guarded prognosis. Moreover, those with NME typically have a rapid decline after even with treatment 11 . Our understanding of NME immunopathogenesis is limited, though studies have identified a mutation in the canine major histocompatibility complex, also known as dog leukocyte antigen (DLA), in Pug, Chihuahua, and Maltese dogs that predisposes to NME 9 , 12 & 13 . The DLA class I and class II antigen-presenting molecules play a key role in presenting non-self antigens to mount immune responses as well as regulate reactivity to self, a process essential for maintaining tolerance 9 , 12 & 13 . Interestingly, there is evidence supporting an autoimmune component in NME, as autoantibodies against Glial Fibrillary Acidic Protein (GFAP), an intermediate filament protein expressed by astrocytes and upregulated during inflammation or injury, have been detected in the CNS of affected dogs 14 & 15 . Given the limited understanding of the pathophysiology of MUO, particularly NME, this study aimed at elucidating the role of immune-mediated components in driving the neuropathology of NME. Methods NME and GME Cases The archives of the Penn Vet Diagnostic Labs at the Ryan Veterinary Hospital of the School of Veterinary Medicine were searched for NME and GME cases diagnosed post mortem by ACVP board certified pathologists based on signalment, clinical signs, and histologic findings 5 . Formalin-fixed and paraffin embedded (FFPE) blocks from NME ( n = 11) and GME ( n = 11) cases were utilized for this study ( Table 1 ). FFPE blocks sampled from the cerebrum from five neurologically normal dogs were utilized as controls ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Clinical features of cases studied Tissue Processing and Immunostainings Tissue samples were fixed for at least 24 h and FFPE blocks were sectioned 5-m thick, mounted on glass slides, and stained with H&E. Tissue sections were processed for immunohistochemistry (IHC) as previously described 5 . In brief, cortical areas of seven NME, eleven GME, and five control dogs were evaluated with antibodies (Abs) against CD3 (T cells) (M7254, Ms mAb; Agilent Dako), CD79b (B cells) (96024, Rb mAb; Cell Signaling Technology), Iba1 (macrophages/microglia) (019-19741, Rb pAb; WAKO), myelin-oligodendrocyte glycoprotein (MOG) (myelin) (96457, Rb mAb; Cell Signaling Technology), glial fibrillary acidic protein (GFAP) (GA52461-2, Rb pAb; Agilent Dako), and aquaporin-4 (AQP4) (AB3594, Rb pAb; Sigma Millipore). All the stains were performed using a LEICA Bond RXm automated IHC stain according to the manufacturer’s recommendations. Isotype control Abs were used as negative controls. Positive controls were normal canine brain tissue and lymph node. All NME, GME, and control IHC slides stained immunohistochemically were scanned at 20x using a Leica Aperio AT2 slide scanner (Leica Biosystems, Buffalo Grove, IL) and image acquisition was performed with ImageScope (Leica Biosystems, Buffalo Grove, IL). For the quantification of the MOG, GFAP, and AQP4 staining to assess demyelination, astrocyte activation, and AQP4 expression respectively. Image analysis was performed using a positive pixel count algorithm using ImageScope software. A single algorithm was developed using appropriate thresholds for the staining intensity and was applied to specific annotated regions on each slide. Neuropathological Analysis Fulminant NME lesions are characterized by large areas of necrosis and neuroparenchymal loss mainly within the cerebral hemispheres. In contrast, GME is composed of single or multiple foci of inflammation mainly in caudal regions of the brain, including the spinal cord, though cerebral lesions can also be seen. To address the pathological differences between both diseases, our analysis was focused to areas of immune cell infiltration lacking necrosis and neuroparenchymal loss to reflect the potential early events leading to fulminant pathology in NME. Equivalent areas were also studied in GME and control cases. Leptomeningeal inflammatory infiltrates were scored using a four-tiered scale (absent= no inflammatory cells present; mild =fewer than 20 diffusely distributed inflammatory cells or leptomeningeal perivascular cuffing up to 3 layers; moderate = 20-50 diffusely distributed inflammatory cells or leptomeningeal perivascular cuffing three to seven layers; marked >50 diffusely distributed inflammatory cells or leptomeningeal perivascular cuffing greater than seven layers). Parenchymal cortical inflammation was scored using a similar scale: 0 =absent, 1 = mild (vessels with one cuff), 2 =moderate (many vessels with two cuffs), 3= marked (scattered or many vessels with >3cuffs). Evaluation of brain slides stained for for B cells (CD79b) and T cells (CD3) were based on previous methods 5 . Glial nodules were scored using a four-tired scale (absent= 0 nodules; mild= 1 to 2 nodules; moderate= 3 to 4 nodules; severe= >5). To account for the variation in nodule size a three-tier scaled was added to the aforementioned score (0= 4 to 5 glial cells within the nodule; 1= 6 to 9 glial cells withing the nodules; 2= >10 glial cells withing the nodules. Statistical Analysis Data were analyzed using GraphPad Prism v.10 software. Welch’ T test was used for comparisons between number of cell populations within leptomeningeal infiltrates. Brown-Forsythe and Welch ANOVA was used to determine the differential expression of MOG, GFAP, and AQP4 amongst control, GME, and NME dogs. Two tailed (95%) Pearson correlation analysis was used to determine associations between inflammation, lymphocyte infiltration, glial nodules, and algorithm outputs for the stains mentioned above. Results Mild leptomeningeal inflammation in NME To investigate the underlying pathologic mechanisms of NME, we analyzed postmortem NME and GME cases with diagnoses confirmed post-mortem by a board-certified veterinary pathologist (MEC). As previously described, GME cases contained robust perivascular cuffing by lymphocytes 5 & 16 . Histologically, perivascular cuffing was accompanied by adjacent aggregates as well as diffuse distribution of inflammatory cells within the leptomeninges ( Figure 1A ). In contrast, NME presented minimal perivascular immune cell aggregates within the leptomeninges and was characterized by a milder, diffuse infiltration of lymphocytes, which was most often associated with necrotic regions within the underlying brain neuroparenchyma ( Figure 1B ). Thus, NME and GME differ in the extent of leptomeningeal immune cell infiltration ( Figure 1C ). Download figure Open in new tab Figure 1. Leptomeningeal inflammation in NME and GME. (A & B) H&E stained slides of diffuse and perivascular inflammatory infiltrates at the leptomeninges. (C) Quantification of total leptomeningeal inflammation on H&E between GME and NME. (D & E) Leptomeningeal B infiltrates stained with anti-CD79b. (F) Quantification of total leptomeningeal B cells between GME and NME. (G & H) Leptomeningeal T cell infiltrates stained with anti-CD3. (I) Quantification of total leptomeningeal T cells between GME and NME. Tissues were counterstained with hematoxylin (blue) for nuclear staining. Error bars, mean ± SEM P ≤ 0.05* P≤0.01 ** by student t-test. Leptomeningeal B cells characterize GME from NME Given that overall inflammation in the leptomeninges was less severe in NME than GME, we next determined whether the immune cell composition varied between both MUOs. As expected, the extent of B cell infiltration was significantly higher in GME ( Figure 1D ) while in NME limited number of B cells were distributed throughout the leptomeninges, mainly in a diffuse manner ( Figures 1E-1F ). We found similar levels of leptomeningeal T cell accumulation across both groups, though mildly higher numbers of T cells in GME compared to NME ( Figures 1G-1I ). These findings confirm a unique pathophysiological role for leptomeningeal B cells in GME while suggesting T cell infiltration may be similar in both pathologies 5 . Comparable neuroparenchymal inflammation between NME and GME The leptomeningeal inflammation in NME is mild and is without a predominant lymphocyte type, when compared to GME, where there tend to be more B cells present. When examining the inflammation within the neuroparenchyma in regions away from rarefaction and loss that are common in NME, there was no significant difference in the extent of cortical inflammation between the two groups ( Figures 2A-2C ). The neuroparenchymal lymphocyte composition of both NME and GME was largely similar ( Figures 2D-2I ). Therefore, while the composition of the inflammatory infiltrates observed in the neuroparenchyma do not differ between GME and NME, the leptomeningeal infiltration presents an enrichment of B cells in GME and limited lymphocyte aggregation in NME. Download figure Open in new tab Figure 2. Neuroparenchymal inflammation in NME and GME. (A & B) H&E stained slides of diffuse and perivascular inflammatory infiltrates at the neuroparenchyma. (C) Quantification of total neuroparenchymal inflammation between GME and NME. (D & E) Neuroparenchymal B cells stained with anti-CD79b. (F) Quantification of neuroparenchymal B cells between GME and NME. (G & H) Neuroparenchymal T cell infiltrates stained with anti-CD3. (I) Quantification of total neuroparenchymal T cells between GME and NME. Error bars, mean ± SEM P ≤ 0.05* P≤0.01 ** by student t-test. Extensive subcortical white matter demyelination in NME The presence of leptomeningeal B cells correlates to the extent of subpial demyelination in GME 5 , and so we aimed to establish whether demyelination is also a component of NME. When compared to control groups, there was loss of MOG staining in the subpial, intracortical, leukocortical, and subcortical white matter layers of NME dogs ( Figures 3A-3B ). Specifically, demyelination was of the subpial grey matter and subcortical white matter in NME cases was greater than that present in GME cases ( Figures 3C-3F ). Sub-meningeal demyelination has been described as part of the pathophysiology of GME, however, distinct from GME, is the subcortical white matter demyelination in the cases of NME. Given the sparse lymphocyte infiltration observed in NME in the face of concurrent marked cortical and subcortical demyelination, we interrogated additional cellular components that may contribute to NME pathology. Download figure Open in new tab Figure 3. Demyelination in NME. (A & B) Representative images of control and NME dog brains stained with an anti-MOG antibody. The leptomeninges, subpial, intracortical, leukocortical, and subcortical white matter layers are indicated while the pia layer is labeled as a dotted lane. (C & F) Quantification of MOG expression between control ( n =5), GME ( n =11), and NME ( n =10) across different brain layers. Error bars,mean ± SEM. *P≤0.05 ** P≤0.01 by one way ANOVA. Astroglial component in NME Demyelinating and neuroinflammatory processes depend on astroglial activation to either propagate or subdue inflammation 17 . We anticipated a higher extent of astrogliosis in NME as it has been suggested that antibodies against GFAP are present in CSF and serum of NME-affected dogs. These antibodies might potentially be targeting and subsequently activating astrocytes 14 & 15 . Based on the considerable demyelination and the presence of inflammation, we suspected GFAP expression would be increased in both NME and GME at the cortical and subcortical level. We found that GFAP expression was significantly lower throughout the cortex and subcortical white matter in cases of NME when compared to control ( Figures 4A-4F ). In contrast, GFAP expression in GME was only significantly reduced at the intracortical and leukocortical layers ( Figures 4D-4E ). Throughout all layers GFAP levels trended downward in NME relative to GME ( Figures 4C-4F ). These findings indicate astrocytes might be a target of the inflammatory response seen in NME dogs. Download figure Open in new tab Figure 4. GFAP expression in NME. (A & B) Representative images of control and NME dog brains stained with an anti-GFAP antibody. Extent of GFAP expression at the subpial (C), intracortical (D), leukocortical (E), and subcortical white latter (F) in control ( n =5), GME ( n =11), and NME ( n =10) brains. Error bars, mean ± SEM. *P≤0.05, ** P≤0.01 by one way ANOVA. Loss of AQP4 Distinguishes NME As NME presents a reduction in GFAP expression, this led us to further examine the phenotype of astrocytes. We studied Aquaporin-4 (AQP4), a water channel protein expressed at astrocytic end-feet, involved in water homeostatic mechanisms, neuroexcitation, and astrocyte migration 18 . The expression of AQP4 was reduced in cortex and subcortical white matter when compared to GME with significant decreases found in the subpial and leukocortical areas ( Figures 5A-5C and 5E ). The reduction of AQP4 expression was specific to NME ( Figure 5C-5F ). Therefore, these findings suggest that astrocytes are particularly affected and that AQP4 depletion is a distinct feature of NME pathology. Download figure Open in new tab Figure 5. AQP4 expression in NME. (A & B) Representative images of control and NME dog brains stained with an anti-AQP4 antibody. Extent of AQP4 expression at the subpial (C), intracortical (D), leukocortical (E), and subcortical white latter (F) in control ( n =5), GME ( n =11), and NME ( n =10) brains. Error bars, mean ± SEM. *P≤0.05 ** P≤0.01 by one way ANOVA. Loss of AQP4 correlates with decrease GFAP and increase demyelination in NME The specific loss of AQP4 expression prompted us to determine if the demyelination observed in NME is associated with the extent of astrocyte damage across the cortex and within the subcortical white matter. In the cortex, we found no association between loss of MOG staining and the loss of GFAP staining ( Figure 6A ). Similarly, there was no correlation between the expression levels of GFAP and AQP4 ( Figure 6B ) nor between expression of MOG and AQP4 in cortical layers ( Figure 6C ). In contrast, within the subcortical white matter loss of GFAP expression was significantly correlated with a decrease in MOG expression ( Figure 6D ). Additionally, similar associations between AQP4-GFAP and AQP4-MOG expression ( Figures 6E-6F ) suggests that damage to both astrocytes and oligodendrocytes specifically within the subcortical white matter, may be related to a decrease in AQP4 expression as well. Download figure Open in new tab Figure 6. AQP4 loss in the white matter correlates with demyelination. We performed correlation analysis to determine the interdependence between astrocyte activation, demyelination, and AQP4 loss in the cortex (A-C) and subcortical white matter (D-F) of NME dogs ( n =10). *P≤0.05 Absence of AQP4 correlates to glial nodule and immune cell presence We also examined if microglial activation correlated with loss of AQP4 observed in NME. We used an antibody against Iba1 to detect microglia, the resident innate immune cells of the CNS, and macrophages which typically migrate into the CNS from the periphery in response to inflammation. We hypothesized that the severity of microglia/macrophage activation would be associated with loss of both MOG and AQP4 within the neuroparenchyma. Glial nodules were observed in both the cortical and subcortical white matter, varying in the size according to the number of cells they contained. These nodules ranged from small clusters of four cells to larger structures with more than ten cells per nodule. We found no significant association between the presence of glial nodules and loss of MOG staining in the cortical layers ( Figure 7A ). However, within the subcortical white matter, expression of MOG trended towards being decreased with increased number of glial nodules. Expression of AQP4 was negatively correlated with the presence of glial nodules in the cortical layers and trended towards a correlation in the subcortical white matter layer ( Figures 7C-7D ). This suggests that a change in function of astrocytes in regions with nodular inflammation, away from homeostasis of the BBB and neuronal function and shifted towards inflammation. Download figure Open in new tab Figure 7. Microglial activation associates with the loss of AQP4 expression. We performed correlation analysis to determine the link between demyelination, and AQP4 with the extent of microglial activation (glial nodules formation) in the cortex (A-B) and subcortical white matter (C-D) of NME dogs ( n =7). *P≤0.05, ** P≤0.01 This trend was also seen with severity of both B and T cell inflammation, where increased inflammation was correlated with decreased AQP4 expression in the cortex ( Figures 8A-8B ) but not in the subcortical white matter ( Figures 8C-8D ). Therefore, the presence of glial nodules was associated with loss of AQP4 in both layers, whereas the presence of lymphocytes correlated with AQP4 losses only at cortical layers. Download figure Open in new tab Figure 8. AQP4 loss correlates with the extent of lymphocyte infiltration in the cortex. We performed correlation analysis to determine the link between AQP4 and the extent of lymphocyte (B and T cells) infiltration in the cortex (A-B) and subcortical white matter (C-D) of NME dogs ( n =7). *P≤0.05 Discussion While the histologic features of MUO subtypes have been well defined over the years, the underlying etiology of MUOs is largely unknown 1 , 3 , 4 , 5 , 6 & 16 . Various hypotheses have been postulated regarding the mechanisms driving disease. Infectious etiologies have been suspected as causative, but none have been successfully detected in tissues 19 . The most abundant body of evidence suggests NME to be either a primary autoimmune disease or a multifactorial immune-mediated disease. Multiple studies have found genetic mutations at the DLA Class II gene which potentially predisposes dogs to develop NME 8 , 9 , 13 ,& 20 . Moreover, there is also evidence of an autoantibody against GFAP identified in CSF and serum samples 14 , 15 , & 21 , but it is still unknown whether these autoantibodies are directly causing the lesions or are secondarily produced from necrotic destruction exposing self-antigen, a mechanism known as epitope spreading 22 .In this study, we provide evidence demonstrating variation in the immune-mediated mechanisms between NME and GME. Additionally, we identified unique features to the NME disease process such as demyelination and astroglial dysfunction. Immune cell infiltrates comprising NME and GME lesions have been described in the past, though no studies have quantified the extent of immune cell infiltration in both the leptomeninges and neuroparenchyma. Similar to our findings here, T cells have been identified at the leptomeninges and neuroparenchyma of both NME and GME, with GME reported to have higher numbers of T cells, but with no reported statistical significance and in some cases with minimal presence in NME lesions 4 & 6 . Notably, one group found no differences in B cell numbers in the neuroparenchyma between MUO subtypes, which is in accordance with our data 6 . In the GME cases examined, B cells were significantly more abundant than T cells in the leptomeninges and show a trend toward higher numbers in the neuroparenchyma. While in the NME cases, there were similar amounts of T cells in both compartments. The detection of both T and B cells in the leptomeninges and neuroparenchyma in NME suggests an ongoing inflammatory (including infectious, autoimmune, or immune mediated) disease process. Importantly, the presence of B cells in both NME and GME is indicative not only of chronicity, but signals the production of antibodies, which could then exacerbate a cyclical immune mediated process. Indeed, CSF from dogs with MUO has been demonstrated to contain CSF-specific oligoclonal bands (a metric of intrathecal immunoglobulin (IgG) 10 . As previously described by our group, leptomeningeal B cell aggregates are associated with submeningeal demyelination in GME cases 5 . Although NME presented lesser leptomeningeal B cell infiltration, we interrogated whether demyelination was also present in association with immune cell aggregates. It is not widely agreed upon whether demyelination in NME represents a primary cause of the disease or a secondary response to loss of neuroparenchyma (and, thus, myelin) in cavitating lesions 9 & 11 . Therefore, in this study we examined areas adjacent to necrotic lesions to determine the degree of demyelination. We did find extensive demyelination especially in subpial grey matter and subcortical white matter. Typical NME lesions are characterized by marked astrogliosis in and around necrotic regions 1 , 3 & 6 . Astrocytes are the most abundant type of glial cells in the CNS, and they play a pivotal role in the maintenance of the neurovasculature as well as in nurturing neuronal function 23 & 24 . During inflammation or injury, astrocytes become activated and increase in numbers as well as the expression of GFAP, a structural component mainly found at astrocytic processes 25 . Our study contrasts the upregulation reported for GFAP during neuroinflammation and in combination with the focal loss of AQP4 suggest a potential immune targeting of astroglia unique to NME. If astrocytes are the direct or unintended target of antibodies, the resulting immune response could lead to astrocytic loss, which in turn would cause a reduction in the expression of GFAP and AQP4 26 . In this regard, the loss of AQP4 expression in NME correlated with the presence of microglial nodules, which form because of microglial activation needed to phagocytose neuroaxonal debris. Interestingly, the presence of microglial nodules does not correlate with demyelination in either cortical grey matter or subcortical white matter. In GME cases, demyelination was not associated with the presence of glial nodules but correlated with meningeal B cell infiltration 5 . Therefore, microglial nodules might play distinct roles in each disease process. Microglia are involved in homeostatic functions (like synapse formation and neuronal survival) and immune mediated mechanism of the CNS such as phagocytosis of aged or degenerated myelin as well as other apoptotic cellular debris. Moreover, microglia are capable of promoting or downregulating neuroinflammatory responses 27 & 28 .It is currently unclear whether microglial nodules are neuroprotective or neurodegenerative thus, further studies are required to elucidate the role of microglia in NME pathogenesis. It is possible that the demyelination detected in NME is secondary to neuroinflammation. Reduced AQP4 expression also correlated with increases in B and T cell immune infiltration in the cortex. These findings mirror neuropathological findings seen in human neuroinflammatory disorders where the activation of B and T cells correlates with the presence of microglial nodules and ongoing CNS damage 29 & 30 .Thus, primary astrocytic damage might be leading to a pro-inflammatory state, activation of microglia and perpetuation of inflammation. Such mechanism has also been postulated in humans with Autoimmune GFAP Astrocytopathy (AGA), a steroid responsive neuroinflammatory disease 31 . Similar to NME patients, humans with AGA experience neck discomfort, seizures, personality/behavioral changes 31 . Neuroparenchymal infiltrates in AGA consists of B and T cell with prominent microgliosis as well as loss of both GFAP and AQP4, and demyelination on histopathologic examination 32 . Diagnosis of AGA is predicated on the presence of GFAP-specific IgG in CSF samples 33 . Pre-mortem studies examining the specificity of IgG in CSF and serum of MUO dogs has the potential to identify a diagnostic biomarker and could aid in early diagnosis and targeted therapies for dogs suffering from NME. Acknowlegments We want to thank the anatomic pathology residents and histology technicians of the School of Veterinary Medicine at the University of Pennsylvania for their help in procuring and processing tissues used in this study. We also want to thank the clinical veterinary neurologists for their contribution on the clinical diagnosis of these canine patients. This study has been supported by the The National Institute of Health (NIH) of the United States by the grant NCATS 5UL1TR001878-04 to the Institute of Translational Medicine and Therapeutics (ITMAT) of the University of Pennsylvania and JIA. Bibliography 1. ↵ Coates , J. R. & Jeffery , N. D. Perspectives on meningoencephalomyelitis of unknown origin . Vet. Clin. North Am. Small Anim. Pract . 44 , 1157 – 1185 ( 2014 ). OpenUrl CrossRef PubMed 2. ↵ Cherubini , G. B. et al. Characteristics of magnetic resonance images of granulomatous meningoencephalomyelitis in 11 dogs . Vet. Rec . 159 , 110 – 115 ( 2006 ). OpenUrl Abstract / FREE Full Text 3. ↵ Uchida , K. , Park , E. , Tsuboi , M. , Chambers , J. K. & Nakayama , H. Pathological and immunological features of canine necrotising meningoencephalitis and granulomatous meningoencephalitis . Vet. J . 213 , 72 – 77 ( 2016 ). OpenUrl CrossRef PubMed 4. ↵ Suzuki , M. et al. A comparative pathological study on canine necrotizing meningoencephalitis and granulomatous meningoencephalomyelitis . J. Vet. Med. Sci . 65 , 1233 – 1239 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 5. ↵ Church , M. E. et al. Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis . J. Immunol. Baltim. Md 1950 207 , 44 – 54 ( 2021 ). OpenUrl 6. ↵ Park , E. S. , Uchida , K. & Nakayama , H. Comprehensive Immunohistochemical Studies on Canine Necrotizing Meningoencephalitis (NME), Necrotizing Leukoencephalitis (NLE), and Granulomatous Meningoencephalomyelitis (GME) . Vet. Pathol . 49 , 682 – 692 ( 2012 ). OpenUrl CrossRef PubMed 7. ↵ Greer , K. A. et al. Heritability and transmission analysis of necrotizing meningoencephalitis in the Pug . Res. Vet. Sci . 86 , 438 – 442 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ Greer , K. A. et al. Necrotizing meningoencephalitis of Pug dogs associates with dog leukocyte antigen class II and resembles acute variant forms of multiple sclerosis . Tissue Antigens 76 , 110 – 118 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 9. ↵ Schrauwen , I. et al. Identification of Novel Genetic Risk Loci in Maltese Dogs with Necrotizing Meningoencephalitis and Evidence of a Shared Genetic Risk across Toy Dog Breeds . PLoS ONE 9 , e112755 ( 2014 ). OpenUrl CrossRef PubMed 10. ↵ Prümmer , J. K. et al. Assessment of oligoclonal bands in cerebrospinal fluid and serum of dogs with meningoencephalitis of unknown origin . PLOS ONE 18 , e0280864 ( 2023 ). OpenUrl CrossRef PubMed 11. ↵ Windsor , R. , Stewart , S. , Huentelman , M. , Keller , S. & Khanna , C. An early clinical phenotype of necrotizing meningoencephalitis in the Pug reveals similarities to multiple sclerosis in humans . Am. J. Vet. Res. 1–9 ( 2023 ) doi: 10.2460/ajvr.23.07.0164 . OpenUrl CrossRef 12. ↵ Oshima , A. et al. Dog leukocyte antigen class II alleles and haplotypes associated with meningoencephalomyelitis of unknown origin in Chihuahuas . J. Vet. Med. Sci . ( 2022 ) doi: 10.1292/jvms.22-0116 . OpenUrl CrossRef 13. ↵ Barber , R. M. et al. Identification of Risk Loci for Necrotizing Meningoencephalitis in Pug Dogs . J. Hered . 102 , S40 – S46 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ Uchida , K. , Hasegawa , T. , Ikeda , M. , Yamaguchi , R. & Tateyama , S. Detection of an Autoantibody from Pug Dogs with Necrotizing Encephalitis (Pug Dog Encephalitis) . Vet. Pathol . 36 , 301 – 307 ( 1999 ). OpenUrl CrossRef PubMed 15. ↵ Miyake , H. , Inoue , A. , Tanaka , M. & Matsuki , N. Serum Glial Fibrillary Acidic Protein as a Specific Marker for Necrotizing Meningoencephalitis in Pug Dogs . J. Vet. Med. Sci . 75 , 1543 – 1545 ( 2013 ). OpenUrl CrossRef PubMed 16. ↵ Cordy , D. R. & Holliday , T. A. A necrotizing meningoencephalitis of pug dogs . Vet. Pathol . 26 , 191 – 194 ( 1989 ). OpenUrl CrossRef PubMed Web of Science 17. ↵ Wei , D. C. & Morrison , E. H. Histology, Astrocytes. in StatPearls ( StatPearls Publishing , Treasure Island (FL ), 2024 ). 18. ↵ Li , L. , Zhang , H. , Varrin-Doyer , M. , Zamvil , S. S. & Verkman , A. S. Proinflammatory role of aquaporin-4 in autoimmune neuroinflammation . FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol . 25 , 1556 – 1566 ( 2011 ). OpenUrl 19. ↵ Barber , R. M. et al. Broadly Reactive Polymerase Chain Reaction for Pathogen Detection in Canine Granulomatous Meningoencephalomyelitis and Necrotizing Meningoencephalitis . J. Vet. Intern. Med . 26 , 962 – 968 ( 2012 ). OpenUrl CrossRef PubMed 20. ↵ Van Renen , J. et al. Allele frequency of a genetic risk variant for necrotizing meningoencephalitis in pug dogs from Europe and association with the clinical phenotype . Front. Vet. Sci . 11 , 1407288 ( 2024 ). OpenUrl CrossRef PubMed 21. ↵ Shibuya , M. et al. Autoantibodies against Glial Fibrillary Acidic Protein (GFAP) in Cerebrospinal Fluids from Pug Dogs with Necrotizing Meningoencephalitis . J. Vet. Med. Sci . 69 , 241 – 245 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ McMahon , E. J. , Bailey , S. L. , Castenada , C. V. , Waldner , H. & Miller , S. D. Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis . Nat. Med . 11 , 335 – 339 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ Alvarez , J. I. et al. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence . Science 334 , 1727 – 1731 ( 2011 ). OpenUrl Abstract / FREE Full Text 24. ↵ Cheslow , L. & Alvarez , J. I. Glial-endothelial crosstalk regulates blood-brain barrier function . Curr. Opin. Pharmacol . 26 , 39 – 46 ( 2016 ). OpenUrl CrossRef PubMed 25. ↵ Alvarez , J. I. , Katayama , T. & Prat , A. Glial influence on the blood brain barrier . Glia 61 , 1939 – 1958 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 26. ↵ Hinson , S. R. et al. Molecular outcomes of neuromyelitis optica (NMO)-IgG binding to aquaporin-4 in astrocytes . Proc. Natl. Acad. Sci. U. S. A . 109 , 1245 – 1250 ( 2012 ). OpenUrl Abstract / FREE Full Text 27. ↵ Gao , C. , Jiang , J. , Tan , Y. & Chen , S. Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets . Signal Transduct. Target. Ther . 8 , 359 ( 2023 ). OpenUrl CrossRef PubMed 28. ↵ Prinz , M. , Jung , S. & Priller , J. Microglia Biology: One Century of Evolving Concepts . Cell 179 , 292 – 311 ( 2019 ). OpenUrl CrossRef PubMed 29. ↵ Tröscher , A. R. et al. Microglial nodules provide the environment for pathogenic T cells in human encephalitis . Acta Neuropathol. (Berl .) 137 , 619 – 635 ( 2019 ). OpenUrl CrossRef PubMed 30. ↵ Alvarez , J. I. et al. Focal disturbances in the blood-brain barrier are associated with formation of neuroinflammatory lesions . Neurobiol. Dis . 74 , 14 – 24 ( 2015 ). OpenUrl CrossRef PubMed 31. ↵ Kunchok , A. , Zekeridou , A. & McKeon , A. Autoimmune glial fibrillary acidic protein astrocytopathy . Curr. Opin. Neurol . 32 , 452 – 458 ( 2019 ). OpenUrl CrossRef PubMed 32. ↵ Long , Y. et al. Autoimmune glial fibrillary acidic protein astrocytopathy in Chinese patients: a retrospective study . Eur. J. Neurol . 25 , 477 – 483 ( 2018 ). OpenUrl CrossRef PubMed 33. ↵ Flanagan , E. P. et al. Glial fibrillary acidic protein immunoglobulin G as biomarker of autoimmune astrocytopathy: Analysis of 102 patients . Ann. Neurol . 81 , 298 – 309 ( 2017 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 14, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Astroglial Dysfunction, Demyelination and Nodular inflammation in Necrotizing Meningoencephalitis Sara I Hernandez , Charles A Assenmacher , Molly E Church , Jorge I Alvarez bioRxiv 2024.11.11.623107; doi: https://doi.org/10.1101/2024.11.11.623107 Share This Article: Copy Citation Tools Astroglial Dysfunction, Demyelination and Nodular inflammation in Necrotizing Meningoencephalitis Sara I Hernandez , Charles A Assenmacher , Molly E Church , Jorge I Alvarez bioRxiv 2024.11.11.623107; doi: https://doi.org/10.1101/2024.11.11.623107 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7651) Biochemistry (17746) Bioengineering (13928) Bioinformatics (42064) Biophysics (21499) Cancer Biology (18650) Cell Biology (25579) Clinical Trials (138) Developmental Biology (13409) Ecology (19947) Epidemiology (2067) Evolutionary Biology (24373) Genetics (15633) Genomics (22557) Immunology (17774) Microbiology (40504) Molecular Biology (17217) Neuroscience (88793) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4836) Physiology (7664) Plant Biology (15178) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9839) Zoology (2272)