Successful diagnosis and treatment of canine polymyositis: utilizing MRI and immunohistochemistry for accurate detection

Abstract

Background

Inflammatory myopathy is generally categorized into generalized inflammatory myopathies (gIM), which affect muscles throughout the body, and focal inflammatory myopathies (fIM), which are localized to specific muscles or muscle groups. This report details a case of immune-mediated polymyositis in a dog, successfully diagnosed using MRI and IHC and managed with immunosuppressive therapy.

Case presentation

A 5-year-old castrated male Poodle was admitted to our hospital presenting with lethargy and exercise intolerance. Biochemical analysis revealed significantly elevated serum levels of aspartate aminotransferase (AST) and creatine kinase (CK). Physical examination showed muscle atrophy in the hind legs, but further orthopedic and neurological examinations identified no additional abnormalities. MRI demonstrated hyperintense and heterogeneous signal changes across the muscles, including contrast enhancement, suggesting inflammatory myopathy. This diagnosis was confirmed through histopathological examination, which revealed inflammatory lesions with fibrous tissue proliferation within the muscle tissue. To investigate the presence and type of inflammatory cells and vascular changes, aiding in the differential diagnosis of inflammatory myopathies, immunohistochemistry (IHC) was performed, revealing positive findings for CD8⁺, CD4⁺, and VEGF in the evaluated tissue, leading to a diagnosis of polymyositis.

Conclusions

The dog was diagnosed with immune-mediated polymyositis and treatment was initiated with prednisolone at 1 mg/kg twice daily and azathioprine at 2 mg/kg once daily. Following the administration of these immunosuppressive agents, CK levels returned to normal, and the dog’s exercise intolerance and lethargy resolved. The thickness of the hind legs also increased progressively. The dog has maintained an improved condition under continued immunosuppressive therapy for four months. This case highlights the critical role of MRI and immunohistochemistry in diagnosing immune-mediated polymyositis, demonstrating their alternative capability in cases where conventional electromyography (EMG) is not feasible in this context.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-024-04356-6.

Background

Inflammatory myopathy is a disease characterized by non-purulent infiltration of inflammatory cells in skeletal muscle. Similar to humans, myositis in veterinary medicine is classified based on its distribution. It is generally categorized into generalized inflammatory myopathies (gIM), which affect muscles throughout the body, including conditions such as polymyositis (PM), and focal inflammatory myopathies (fIM), which are localized to specific muscles or muscle groups [1]. Conditions such as extraocular myositis (EOM) and masticatory myositis (MMM) represent focal inflammatory myopathies in dogs. Dermatomyositis (DM), unclassified, and overlap syndromes will be individually categorized and characterized [1]. Aside from this classification, it can also be categorized into primary (immune-mediated) and secondary (bacterial agents, parasites, and pre/paraneoplastic syndromes). In human, gIM, including polymyositis is generally considered to be immune-mediated or para/preneoplastic syndromes [2]. However, in veterinary medicine, it exhibits similarities to human cases, yet al.so differs in certain potential causes including bacterial agents, parasites such as protozoa, rickettsia, and spirochetes [1]. Although specific data on the prevalence of polymyositis in dogs is lacking, it affects about 0.01% of the human population, indicating its rarity in both species. However, based on muscle biopsy data, the prevalence in dogs may be slightly higher but remains rare, with many cases potentially underreported due to the subtle onset of symptoms [3].

In veterinary medicine, clear diagnostic criteria for polymyositis have not been established, leading to the frequent application of human medicine. To diagnose polymyositis in human medicine, several criteria must be met: (1) clinical signs such as muscle pain or weakness, (2) elevated serum muscle enzymes, including creatine kinase (CK) and aspartate aminotransferase (AST), (3) electromyographic (EMG) abnormalities, and (4) histopathological evidence of muscle necrosis and inflammation [4, 5]. Additionally, magnetic resonance imaging (MRI) and immunohistochemistry (IHC) are valuable diagnostic tools for polymyositis [6–9].

This report details a case of immune-mediated polymyositis in a dog, successfully diagnosed using MRI and IHC and managed with immunosuppressive therapy.

Case presentation

A 5-year-old castrated male Poodle presented at the veterinary hospital exhibiting lethargy and exercise intolerance. The physical examination revealed muscle atrophy in the hind legs, with the circumference of the left hind leg measuring 11.7 cm and the right hind leg measuring 12.1 cm, as well as enlargement of the left popliteal lymph node, measuring approximately 10.6 × 5.2 mm. There were no notable abnormalities in the complete blood count (CBC). Biochemical analysis showed elevated serum levels of aspartate aminotransferase (AST) at 509 U/L (reference range 0–50 U/L) and CK levels that exceeded the measurable limit (reference range 10–200 U/L). Neurological assessment elicited a pain response at the spinal T12-L1 level, and spinal radiographs indicated slight narrowing of this area. Further orthopedic and neurological evaluations did not reveal any specific abnormalities. Given these findings, intervertebral disc disease (IVDD) was initially suspected. MRI was performed to confirm this diagnosis. However, MRI did not support IVDD but instead showed distinct hyperintensity on T2-weighted and T2-STIR images, and iso-to-hyperintensity on T1-weighted images across general muscle areas. There were signal changes throughout the skeletal muscles, including the bilateral masticatory, paravertebral, gluteal, and proximal tibial muscles. Post-contrast imaging revealed uneven but distinct enhancement following contrast administration (Fig. 1A and B), suggesting widespread muscle inflammation, potentially indicative of polymyositis. To verify this diagnosis, a muscle biopsy was taken from the most visibly affected areas, the bilateral biceps femoris muscles. Histopathological analysis of these sites showed moderate infiltration by mononuclear cells such as lymphocytes, plasma cells, and macrophages, alongside partial degeneration of the muscle tissue including mild necrosis (Fig. 2A). No neoplastic changes or pathogens were detected on hematoxylin and eosin (H&E) staining. To ensure clarity, tests for all known infectious agents were conducted, all of which returned negative results. The test is conducted by IDEXX Laboratories (Westbrook, Maine) and includes the following: Bartonella spp., Borrelia burgdorferi, Blastomyces dermatitidis, CDV, Coccidioides spp., Cryptococcus spp., Histoplasma capsulatum, Neospora spp., Toxoplasma gondii and West Nile Virus. All samples for the PCR panel were performed using blood and CSF samples. Immunohistochemistry was conducted on formalin-fixed paraffin-embedded tissue, using anti-CD8 antibody (ab17147, Abcam, Cambridge, UK), anti-CD4 antibody (10B5, Genetex, CA, USA), and anti-Vascular Endothelial Growth Factor antibody (M7273, Dako, Glostrup, Denmark) to further explore the nature of the inflammatory infiltrate. The immunohistochemistry results for CD4, CD8, and VEGF were positive, indicating a T cell-mediated immune response and angiogenesis in the inflamed lesions (Fig. 3A, B and C). Although abnormal findings were present in various regions on MRI, the biopsy was performed only on hind limb. Therefore, to rule out overlap syndrome with MMM, type 2 M antibody test (IDEXX Laboratories, Westbrook, Maine) was conducted. The antibody titer was less than 1:100, which indicates negative for MMM. To exclude myasthenia gravis, the acetylcholine receptor antibody was measured and the level was below the detection threshold. Based on the clinical presentation and diagnostic findings, the dog was diagnosed with immune-mediated polymyositis.

Fig. 1.

Photographs of MRI showing distinct hyperintensity on T2-weighted images, indicating muscle inflammation (red arrows). Includes the bilateral masticatory muscles (A) and gluteal muscles (B)

Fig. 2.

Histological analysis of the biceps femoris muscle using hematoxylin-eosin staining. The tissue was sectioned in the transverse plane. At low magnification (×100), the muscle tissue exhibits partial degenerative changes and infiltration of inflammatory cells (A). At high magnification (×250), there is infiltration of mononuclear cells (black arrows), including lymphocytes, plasma cells, and macrophages within the lesion. Additionally, it shows polyphasic necrosis and variability in the diameter of muscle fibers (B)

Fig. 3.

Histological and immunohistochemical analysis of the left biceps femoris muscle using hematoxylin-eosin staining and specific antibodies, respectively, both at high magnification (×250). The tissue was sectioned in the transverse plane. An H&E-stained image of a section adjacent to the one used for IHC, showing infiltration of various inflammatory cells (black arrows), provided to assist in the interpretation of the IHC findings by offering a detailed view of the tissue morphology and cellular context (A). CD8-positive staining indicates the presence of cytotoxic T cells (red arrows) (B), CD4-positive staining indicates helper T cells (red arrows) (C), and VEGF-positive staining suggests an angiogenic reaction in muscle tissue (black arrows) including capillary (arrow head) (D)

For the management of the condition, immunosuppressants were administered, including prednisolone (1 mg/kg PO bid; Solondo^® tablet, Yuhan, Seoul, Korea) and azathioprine (2 mg/kg PO sid; Immuthera^® tablet, Celltrion Pharmaceuticals, Incheon, Korea). One week following the administration of these agents, a significant reduction in serum CK levels was observed, decreasing from 2,815 to 108 (reference range: 10–200). Concurrently, the patient’s tolerance for walking during strolls extended from approximately 5 min to 20 min. Additionally, the circumference of the previously atrophied hind leg muscles increased from 11.7 cm to 17.1 cm on the left hind leg and from 12.1 cm to 19.2 cm on the right hind leg. The previously enlarged left popliteal lymph node has also reduced to normal size.

Azathioprine-induced myelosuppression led to a decrease in platelet count from 358 K/µL to 168 K/µL (reference range: 148 K/µL–484 K/µL), which lies at the lower limit of the normal range. Consequently, azathioprine was replaced with cyclosporine (5 mg/kg PO, BID; Cipol-N, Chong Kun Dang Pharm, Cheonan, Chungnam, Korea), resulting in a normalization of the platelet count to 454 K/µL. After about 13 months of management, there was a notable alleviation in exercise intolerance, and serum CK levels have stabilized within the normal range. The circumference of the bilateral hind legs has continued to increase consistently, remaining within normal limits with no abnormalities observed in gait (Fig. 4A, B and C).

Fig. 4.

The patient’s serum creatine kinase (CK) levels during the therapeutic monitoring period. After diagnosis of polymyositis, immunosuppressive drugs were administered (A). The patient’s bilateral hind limb circumference consistently increased during the therapeutic monitoring period (B)

Discussion and conclusions

This case report describes the successful diagnosis and clinical management of immune-mediated polymyositis in a Poodle. MRI played a crucial role in identifying muscle inflammation. While electromyography (EMG) is generally more practical, it is not commonly used in routine clinical practice [7]. In situations where EMG is not feasible, MRI is recommended for confirming suspected cases of myositis [9].

Typical MRI findings in polymyositis include hypointensity on T1-weighted images and hyperintensity on T2-weighted images. While comparing T1-weighted and T2-weighted images can help distinguish between fat and edema, differentiating edema superimposed on fat can be challenging. Therefore, it is advisable to also reference images obtained with STIR (Short Tau Inversion Recovery) sequences to emphasize the visualization of inflammation [9].

Immunohistochemical evaluation was also crucial in diagnosing this case. IHC involved the use of CD4 to identify helper T cells, CD8 for cytotoxic T cells, and vascular endothelial growth factor (VEGF) as a marker for potent endothelial angiogenic growth factors [1, 10, 11]. It is essential to recognize the presence of immune cell infiltration in immune-mediated polymyositis [12]. The goal of this evaluation is to detect the expression of major histocompatibility complex class I and II (MHC-1 and MHC-2) and the presence of T cells and macrophages in canine polymyositis [1, 12]. While the expression of MHC-1 is highly sensitive, it may also show positive results in other muscle disorders. In contrast, MHC-2 expression is more specific for the diagnosis of immune-mediated myositis [13, 14]. Additionally, B cell markers such as CD20 or CD21, and macrophage markers such as IBA-1, CD11, or CD68 are routinely used in the diagnosis and study of myositis in dogs [6, 7, 15]. However, a limitation of this study is that IHC was performed only for T cell lineages due to the lack of staining reagents applicable for IHC in dogs. To be sure, Previous research has shown that inflammatory cells infiltrating the muscles in cases of immune-mediated polymyositis are predominantly T lymphocytes, mainly CD8⁺ and to a lesser extent CD4⁺ [1, 10, 16].

The IHC results indicated that CD8⁺ T cells did not exhibit a significantly larger proportion of positive reactions compared to CD4⁺ T cells due to over-staining extending to the cytoplasm in CD4 staining. However, assessed only in the areas of inflammatory cell infiltration, CD8 and CD4 show similar levels of expression. In polymyositis, there is evidence that CD8⁺ T cells with cytotoxic properties target and attack muscle fibers expressing MHC-1 antigens [17]. Following activation, auto-aggressive CD8⁺ T cells release perforin granules, which cause muscle fiber necrosis [18]. CD4⁺ T cells are also implicated in autoimmune reactions, differentiating into cells capable of cytotoxic effects through the expression of perforin and granzymes [16]. In our case, the predominance of CD4 + T cells over CD8⁺ T cells likely indicates that the PM is in its early stage [19, 20]. CD4⁺ T cells are primarily involved in initiating and coordinating the immune response, recruiting other immune cells like CD8 + T cells. This early dominance of CD4⁺ cells suggests an initial immune activation phase before the cytotoxic response of CD8⁺ T cells becomes more pronounced as the disease progresses [19, 20].

VEGF plays a crucial role in angiogenesis, with particular importance in wound healing and tissue regeneration [21]. To date, VEGF has not been utilized for diagnosing polymyositis in dogs. In humans, however, research indicates that in the early phase without inflammatory infiltrates, the number of VEGF-expressing muscle fibers was found to be increased, in contrast to the control group and the chronic phase. This result supports hypothesis of hypoxic injury of muscle tissues without inflammatory infiltration. Based on these findings, extent of VEGF expression can differentiate between the acute and chronic stages of the disease [11, 22]. Drawing on these findings, and given the pathological similarities between canine and human polymyositis, in this case, the increased expression of VEGF in the presence of inflammatory infiltrates can be interpreted as a natural response by the body to repair damaged muscle fibers and capillaries, playing a crucial role in the early stages of recovery. However, it is important to consider that this VEGF expression, when combined with the inflammatory response, has the potential to progress to chronic inflammation. Therefore, treatment strategies should be developed based on this observation [11].

In human polymyositis, analysis of myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) plays a significant role in diagnosis [16]. However, studies on MSA and MAA in dogs are currently insufficient, necessitating further research [10]. Currently, the only blood test available for detecting antibodies related to inflammatory myopathy in dogs is the measurement of Type 2 M antibody, which are not detected in other muscle diseased except in cases of MMM [1]. In this case, while the MRI findings raise a suspicion of MMM, the absence of clinical symptoms specific to MMM and the negative type 2 M titer results do not confirm the diagnosis. It is important to note that type 2 M antibody testing alone may not be sufficient to rule out MMM entirely, as true MMM could involve the masticatory muscles while sparing the limb muscles [1]. The current results may be due to the early stage of the disease, where sufficient autoantibodies have not yet been produced, and thus the MRI may have detected these early changes before significant antibody formation. Previous reports suggest that a regimen combining immunosuppressive agents is preferable to minimize long-term side effects associated with corticosteroids [23]. Prednisolone and azathioprine are the most commonly used combination, constituting 86% of cases [23]. In human medicine, azathioprine is often the first choice as a steroid-sparing immunosuppressive agent [24, 25]. Similarly, the efficacy of cyclosporine in treating polymyositis has been well-documented in human medicine, with veterinary reports also confirming its effectiveness in canine polymyositis (11, 14, 26–27). Clinical improvement was noted after the administration of cyclosporine and steroids, with no specific side effects reported.

Electronic Supplementary Material

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Abbreviations

gIM

Generalized inflammatory myopathies

fIM

Focal inflammatory myopathies

EOM

Extraocular myositis

MMM

Masticatory myositis

DM

Dermatomyositis

CK

Creatine kinase

AST

Aspartate aminotransferase

EMG

Electromyographic

MRI

Magnetic resonance imaging

IHC

Immunohistochemistry

VEGF

Vascular endothelial growth factor

Author contributions

JW. H was the main contributor to the writing of the manuscript. KH. J, SB. C, SY. K, SJ. O and HJ. K performed the study. JW. H, SJ. O, and HJ. K analyzed the data and conducted clinical management of the case. HJ-K supervised the case management, edited the manuscript. All the authors have read and approved the final version of the manuscript.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2023R1A2C1005348) and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture and Food Convergence Technologies Program for Research Manpower development funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number: RS-2024-00398561).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Written informed consent was obtained from the dog’s owner.

Competing interests

The authors declare no competing interests.