Introduction
Meningoencephalomyelitis of unknown origin is an umbrella term for 3 histologically distinct, noninfectious inflammatory diseases of the CNS (NME, NLE, and GME).1,2,3 Although the etiology of MUO has yet to be elucidated, it is believed to be multifactorial and includes a combination of genetic predispositions, environmental factors, and various other triggers.1,2,3,4 The prognosis for dogs with MUO is poor if the condition remains untreated. The median survival time for dogs with MUO can range from weeks to months after the diagnosis, and the disease is ultimately fatal without appropriate treatment.5,6,7 Treatment with immunosuppressant drugs, such as corticosteroids, can alleviate clinical signs and delay disease progression. Investigators of a 2018 study8 involving dogs with idiopathic meningoencephalomyelitis used metagenomic sequencing of CSF and brain tissue to assess whether a less biased approach could detect infectious agents. A common infectious agent has yet to be identified in patients with MUO, which in combination with alleviation of clinical signs with administration of corticosteroids suggests that the condition is in fact an autoimmune disease.
For most affected dogs, the diagnosis of MUO is presumptive and made on the basis of a combination of patient signalment, clinical suspicion, characteristic results on MRI sequences and CSF analysis, and negative results on diagnostic tests for infectious diseases.2,3,7 Unfortunately, those findings do not definitively distinguish MUO from infectious or neo-plastic conditions of the CNS. Histologic examination of brain biopsy specimens is necessary for definitive diagnosis of MUO and to distinguish among the 3 disease processes (GME, NLE, and NME).1,9
The human medical literature contains descriptions of multiple antibody-mediated encephalitides, many of which are associated with unique syndromes. The most common antibody-mediated encephalitis occurs in patients with antibodies against N-methyl-d-aspartate receptors.10 The presence of autoantibodies against N-methyl-d-aspartate receptor–1 has been confirmed in dogs with neurologic disease.11
A form of antibody-mediated meningoencephalitis, termed autoimmune GFAP astrocytopathy, has recently been described in humans and is characterized by the expression of an astrocytic autoantibody called GFAP autoantibody.12,13,14 More specifically, IgG binding to the GFAP-α is more accurate for diagnosis of GFAP astrocytopathy than is IgG binding to other GFAP isoforms.13 Results of multiple veterinary studies15,16,17,18,19 indicate that GFAP and GFAP autoantibodies are detectable in the CSF, and GFAP is detectable in the serum of dogs.
An ELISA that targets bovine GFAP has been used to measure GFAP autoantibodies in canine CSF in multiple studies15,16,17; however, the diagnostic specificity of that ELISA is less than that of cell-based and tissue-based assays developed for humans. In human medical research, detection of autoantibody binding to individual GFAP isoforms appears to be a more sensitive diagnostic modality than the whole bovine GFAP ELISA that is commonly used in veterinary studies.12,13,14
Necrotizing meningoencephalitis in dogs appears to be similar to autoimmune GFAP astrocytopathy in humans in regard to the development of high GFAP autoantibody titers. Dogs with NME have a significantly higher GFAP autoantibody titer (as determined by an ELISA) in the CSF than do healthy dogs and dogs with other inflammatory diseases.17 Although other intracranial diseases of dogs can lead to elevated GFAP autoantibody titers, those titers are generally not as high as those in dogs with NME or GME, and it is unknown whether an extremely high GFAP autoantibody titer is characteristic of all types of MUO. If it can be determined that MUO or MUO subtypes are a naturally occurring form of autoimmune GFAP astrocytopathy in dogs, targeted treatments can be explored, and dogs could be used as a translational model for human patients with this condition. Furthermore, preferential binding of autoantibodies to the GFAP-α in dogs with MUO may be indicative of a more pathogenic autoantibody pheno-type, and that characteristic might be useful for diagnosis of the condition.
Identification of a minimally invasive technique for diagnosis of intracranial disorders is paramount for the development and prompt initiation of an appropriate treatment plan and for informing prognosis. If GFAP autoantibody titers or epitope binding sites vary significantly among patients with different intracranial disorders, brain biopsies might be avoidable, which would help alleviate both the emotional and financial burdens associated with that invasive procedure for clients. Furthermore, if it can be determined that a large number of dogs with MUO have autoantibodies that bind to the GFAP-α, that would help confirm the theory that MUO or some subclassification in dogs is similar to autoimmune GFAP astrocytopathy in humans.
The objectives of the study reported here were to assess whether cell-based and tissue-based IFAs run in parallel could be used to detect GFAP autoantibodies in the CSF of dogs with CNS disease and to determine whether the GFAP autoantibody titer in the CSF as measured by the cell-based IFA could be used as a biomarker for MUO or other CNS diseases of dogs.
Materials and Methods
CSF samples
Cisternal CSF samples (n = 15) obtained from 5 dogs with a presumptive diagnosis of MUO, 5 dogs with a CNS disease (infectious, metastatic, and primary neoplastic) other than MUO, and 5 dogs with idiopathic epilepsy (controls) were analyzed. All CSF samples were aseptically collected from 2014 to 2018 and stored frozen at −80°C until they were analyzed in 2019.
The 5 dogs with presumed MUO included 1 sexually intact male, 2 castrated males, and 2 spayed females, and had a median age of 44 months (range, 18 to 96 months). Breeds represented included 2 Yorkshire Terriers, 1 Bloodhound, 1 Border Collie, and 1 German Shorthair Pointer.
The 5 dogs with CNS disease other than MUO included a 7-year-old castrated male mixed-breed dog with high-grade oligodendroglioma, 5-month-old sexually intact female Labrador Retriever with suppurative meningoencephalitis, 2-year-old Staffordshire Terrier with neuronal degeneration and necrosis, 5-year-old castrated male Pembroke Welsh Corgi with histiocytic sarcoma, and 7-year-old castrated male Golden Retriever with disseminated mast cell tumors.
The 5 control dogs with idiopathic epilepsy were all castrated males with a median age of 36 months (range, 12 to 72 months) and included 2 mixed-breed dogs, 1 German Shepherd Dog, 1 Golden Retriever, and 1 Vizsla.
For all 15 dogs, a CBC, serum biochemical profile, MRI sequencing of the brain, and CSF cytologic analysis were performed. The diagnosis of idiopathic epilepsy was made with a tier II confidence level.20 The diagnosis of MUO was made on the basis of clinical signs, a CSF nucleated cell count > 5 cells/μL, and MRI changes consistent with GME, NLE, or NME as described.21 Diseases of the CNS other than MUO were diagnosed on the basis of necropsy findings. All MRI examinations were performed with a 1.5T scanner,a and T2-weighted, T1-weighted before and after contrast (gadopentetate dimeglumine, 1 mL/4.5 kg) administration, fast imaging employing steady-state acquisition, and gradient-recalled echo sequences were obtained in the sagittal, transverse, and dorsal planes. Additional MRI sequences were performed for some dogs at the discretion of the attending clinician.
Cell-based IFA
The CSF samples were shipped frozen to the Mayo Clinic in Rochester, Minn, for parallel analysis with a cell-based IFA that targeted the α isoform of human GFAP and a tissue-based IFA that involved mouse tissue cryosections. All assay steps were performed at room temperature (approx 22°C).
For the cell-based IFA, strain HEK 293 cells stably expressing green fluorescent protein–tagged human GFAP-α protein were grown on poly-l-lysine–coated chamber slides.b Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized in 0.2% Triton X-100 for 10 minutes, and blocked in 10% normal goat serum for 30 minutes. A 40-µL aliquot of each CSF sample was diluted 1:4 with normal goat serum. One hundred fifty microliters of the dilute CSF sample was then applied to the cells on a chamber slide, and the slide was incubated for 1 hour. Then, the cells were washed with PBS solution and incubated with 150 µL of rhodamine-conjugated goat anti-canine IgGc at a 1:100 dilution for 45 minutes. The cells were again washed with PBS solution and mounted in a liquid antifade reagent.d
Tissue-based IFA
For the tissue-based IFA, CSF samples were screened for reactivity to endogenous protein on 4-µm-thick cryosectionse of adult mouse cerebellum, midbrain, cerebral cortex, hippocampus, kidney, and stomach. The cryosections were permeabilized with 1% 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonatee and then fixed in 10% formalin for 4 minutes. The sections were incubated in 10% normal goat serum for 1 hour to block nonspecific staining. A 50-µL aliquot of each CSF sample was diluted 1:1 in 10% normal goat serum and 80 µL was incubated on each tissue section for 40 minutes. The tissue sections were washed with PBS solution then incubated with 100 µL of rhodamine-conjugated goat anti-canine IgGc for 35 minutes. The specimens were washed with PBS solution and mounted in a liquid antifade reagent.d
Data analysis
Descriptive data were generated. No statistical analyses were performed.
Results
Only 1 CSF sample yielded mildly positive results on the cell-based IFA; that sample was from 1 of the dogs with presumed MUO. The remaining 14 CSF samples tested negative on the cell-based IFA. All 15 CSF samples yielded negative results on the tissue-based IFA.
Discussion
In the present study, only 1 of the 5 CSF samples obtained from dogs with presumed MUO yielded mildly positive results on the cell-based IFA for detection of autoantibodies to the GFAP-α. The remaining CSF samples evaluated tested negative on the cell-based IFA, and all 15 CSF samples tested negative on the tissue-based IFA. Because IgG autoantibodies may bind to nonspecific proteins, a positive test result on both the cell-based and tissue-based IFAs is necessary to confirm a diagnosis of MUO. The findings of the present study contrasted with results of other studies15,16,17,19 in which GFAP autoantibodies were detected by ELISA or IFA in the serum or CSF of almost all dogs with NME or GME. Anti-astrocyte autoanti-bodies have also been identified in the CSF of dogs with certain brain tumors, canine distemper virus encephalitis, and cerebral infarction, but the anti-astrocyte autoantibody titers of those dogs were generally lower than those of dogs with NME or GME.16,17
In other studies,15,16,17 dogs, particularly Pugs, with NME made up a greater proportion of the subjects with inflammatory CNS disease than did dogs with GME. Furthermore, among the healthy control dogs of 1 study,17 Pugs were more likely to have detectable GFAP autoantibody titers than were dogs of other breeds. That finding raised the suspicion that GFAP autoantibodies might be a precursor to NME in Pugs and other breeds that are predisposed to the condition. Of the 15 CSF samples analyzed in the present study, none were obtained from Pugs or dogs with a histologically confirmed diagnosis of NME, which may have contributed to the discrepancy between the findings of this study and others.15,16,17
In a study13 involving human subjects, GFAP-α IgG was present in almost all patients with autoimmune GFAP astrocytopathy, but < 2% of control subjects without the condition. Results of that study13 also suggest that detection of autoantibodies that specifically bind to the GFAP-α is a more useful measure than detection of autoantibodies that bind to other isoforms of GFAP for identification of patients with autoimmune GFAP astrocytopathy. However, the specificity of GFAP-α as it relates to other intracranial diseases is unclear. Regardless, the specificity and sensitivity of the cell-based assay for autoimmune GFAP astrocytopathy should be much greater than that of an ELISA. Additionally, assays developed for humans are more likely to become commercially available than are the laboratory-specific assays used in veterinary research.14 In fact, a commercially available IFA designed to detect 6 antigenic biomarkers for autoimmune encephalitis in humans has been successfully used to detect those biomarkers in the CSF of dogs with signs of neurologic disease.11
In a study19 of Pugs with NME, the reactivity of CSF samples with bovine GFAP digested by α-chymotrypsin was variable, which suggested that the autoantibodies in those CSF samples recognized different epitopes of GFAP. The negative assay results observed in the present study might indicate that autoantibodies in the canine CSF samples did not react with the human and mouse GFAP-α present in the cell-based and tissue-based IFAs, respectively. Canine GFAP-α is 93% homologous with that of human GFAP-α and 85% homologous with that of mouse GFAP-α, whereas human GFAP-α is 88% homologous with mouse GFAP-α (on the basis of a BLASTp comparison of Homo sapiens GFAP isoform 1-GI:4503979, Canis lupus familiaris GFAP transcript variant α-GI:946724693, and Mus musculus GFAP isoform 1-GI:196115327).21 Thus, it is possible that the canine GFAP autoantibody has a different epitope than the human equivalent and does not bind to mouse GFAP. Additionally, it is important that the epitopes of GFAP autoantibodies be further elucidated to determine the extent to which they differ among species or vary among disease processes. Development of a canine GFAP autoantibody for use as a positive control is essential to discovering the binding site and developing and validating new detection techniques. Additionally, analysis of paired CSF and serum samples will help determine whether a noninvasive test for detection of GFAP autoantibodies is an accurate diagnostic tool, especially given that GFAP autoantibody titers in the systemic circulation are presumably lower than those in the CSF.
The cell-based and tissue-based IFAs used in the present study may have higher sensitivity and specificity than do other assays for GFAP and GFAP autoantibodies. Thus, it is possible that the CSF samples analyzed in this study truly did not contain GFAP autoantibodies. A future comparative study of dogs for which CSF or serum samples are analyzed by a GFAP ELISA and cell-based IFA in parallel with the tissue-based IFA would help assess the reliability of both assays. Additionally, further investigation of the binding of autoantibodies to specific GFAP isoforms would help elucidate the role of GFAP autoantibodies in CNS diseases of dogs.
Elevated serum concentrations of GFAP have been documented in human patients with traumatic brain injury and is a predictor of outcome.22 Human patients with severe traumatic brain injury develop elevated concentrations of GFAP and GFAP-BDP autoantibodies 4 to 10 days after the traumatic event, and the magnitude of those elevations can be used for prognostic purposes.23 Given the 4 to 10 days required for GFAP autoantibodies to develop in humans following a traumatic brain injury,23 we retrospectively reviewed the medical records for each patient of the present study to assess the duration between onset of clinical signs of CNS disease and collection of the CSF sample. Among the 5 dogs with presumed MUO, the CSF sample was collected 3, 6, and > 10 days after the onset of clinical signs for 1, 2, and 2 dogs, respectively. The CSF samples for 2 of the dogs with CNS neoplasia were collected 2 and 4 days after the onset of clinical signs. However, it was presumed that the CNS neoplasia or potential MUO in those dogs was present prior to the owners noticing clinical signs of CNS disease. It is possible that not enough time had elapsed for the development of autoantibodies before collection of CSF in some patients with presumed MUO. That was less probable for CSF samples collected from dogs with neoplasia because it is likely the disease was present well before clinical signs developed. However, if it is assumed that GFAP autoantibodies are the primary cause of disease, the time to CSF collection would be irrelevant because those antibodies would be expected to be present from disease onset.
In human medicine, disease processes other than autoimmune GFAP astrocytopathy have been associated with the development of GFAP autoantibodies. In a study13 of 102 human patients with autoimmune GFAP astrocytopathy, 35 (34%) had neoplasia diagnosed concurrently or within 2 years after diagnosis of autoimmune GFAP astrocytopathy. The types of tumors identified in the patients of that study13 included ovarian teratoma, adrenal carcinoma, glioma, squamous cell carcinoma, and multiple myeloma. Thyroid carcinoma, meningioma, and some benign tumors have also been identified concurrently with autoimmune GFAP astrocytopathy in human patients.12 It has been suggested that ectopic expression of GFAP in peripheral nervous tissue may contribute to the development of autoantibodies owing to neoplasia-induced cell lysis and immune exposure in those tissues.24
The pathogenesis of autoimmune GFAP astrocytopathy is poorly understood.23 Some research suggests that GFAP autoantibody is not the sole initiator of disease in affected patients. In a human study,21 15 of 96 (15.6%) healthy control subjects had detectable GFAP and GFAP-BDP autoantibody titers, and GFAP and GFAP-BDP autoantibodies in the serum of patients with traumatic brain injury reacted with GFAP in rat brain tissue sections. In a study17 involving dogs, 2 of 5 healthy Pugs had GFAP autoantibody titers that were similar to those of dogs with NME. Thus, it is unclear whether the presence GFAP autoantibody titers is a risk factor for autoimmune GFAP astrocytopathy or NME. It is also curious that some individuals with detectable GFAP autoantibody titers do not develop clinical signs of CNS disease. Given that human patients with autoimmune GFAP astrocytopathy produce many different autoantibodies24 and most of those autoantibodies have yet to be identified,25 it is possible that some of those other auto-antibodies may have a role in the pathogenesis of the disease and thus should be investigated in dogs with MUO.
To our knowledge, the present study was the first to describe the use of a tissue-based IFA involving mouse tissue cryosections for detection of GFAP autoantibodies in canine CSF samples. Previous descriptions18,19 of the measurement of GFAP autoantibodies in dogs by means of a tissue-based IFA involved the use of brain tissue cryosections from healthy dogs. We were surprised that the 1 CSF sample that yielded a positive test result on the cell-based IFA yielded a negative test result on the tissue-based IFA. The lack of congruent results between the 2 assays suggested that the cell-based IFA yielded a false-positive result for that CSF sample. Given the fact that tissue-based IFA for canine GFAP autoantibody has never been performed in mouse cryosections, it is possible that the canine GFAP autoantibodies in that sample failed to bind to mouse GFAP.
The present study had several limitations. The study population was small, and histologic findings were not available for any of the dogs with presumed MUO. Definitive diagnosis of GME, NME, and NLE is made on the basis of characteristic histologic findings. Therefore, it is possible, albeit unlikely, that none of the dogs with presumed MUO had NME or even meningoencephalomyelitis. One dog with presumed MUO was lost to follow-up after diagnosis. The clinical conditions for the remaining 4 dogs with presumed MUO improved after administration of long-term immunosuppressive therapy, which made infectious etiologies as the cause of disease in those dogs unlikely and supported the diagnosis of MUO. We would have ideally obtained CSF samples from dogs with a histologic diagnosis of MUO and subclassification of GME, NME, and NLE.
Results of the present study suggested that concurrent use of a cell-based IFA designed to target the human GFAP-α and a tissue-based IFA that involved mouse brain tissue cryosections was inadequate for detection of GFAP autoantibodies in canine CSF samples. On the basis of information provided in the current veterinary literature, it would be unlikely that GFAP autoantibodies were not present in any of the canine CSF samples analyzed. Our goal was to identify an established assay for detection of GFAP autoantibodies that could be easily adapted for use with canine CSF samples. Unfortunately, the findings of this study indicated that further research is necessary. That research should involve larger populations of dogs with confirmed GME, NME, or NLE and healthy control dogs than were evaluated in the present study. Additionally, the use of other cell-based and tissue IFAs to detect GFAP autoantibodies, evaluation of immunoreactivity to other isoforms of GFAP, use of the western blot technique for qualitative analysis or as a control, and assessment of other potentially pathogenic autoantibodies will be useful in future investigations into noninvasive identification and subclassification of dogs with MUO.
Acknowledgments
Supported by the Young Investigator Grant Program, Center for Companion Animal Studies, Colorado State University.
The authors declare that there were no conflicts of interest.
Abbreviations
| BDP | Breakdown product |
| GFAP | Glial fibrillary acidic protein |
| GFAP-α | α Isoform of glial fibrillary acidic protein |
| GME | Granulomatous meningoencephalomyelitis |
| IFA | Immunofluorescent assay |
| MUO | Meningoencephalomyelitis of unknown origin |
| NLE | Necrotizing leukoencephalitis |
| NME | Necrotizing meningoencephalitis |
Footnotes
Signa LX 1.5T HiSpeed Plus Shortbore (CXK4) MR Scanner (HDxt upgrade in 2015), GE Healthcare, Waukesha, Wis.
Corning Inc, Corning, NY.
Novus Biologicals LLC, Centennial, Colo.
ProLong Gold, Thermo Fisher Scientific, Waltham, Mass.
Scimedx Corp, Dover, NJ.
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