Case Report

Antemortem Diagnosis of Polyneurit is Equi

M. Aleman, S.A. Katzman, B. Vaughan, J. Hodges, T.A. Crabbs, M.M. Christopher, G.D. Shelton,and R.J. Higgins

An 8-year-old Kentucky Mountain Saddle geldingwas examined for dysuria. The horse was clinically

normal when purchased 7 months before examination,and at this time was dewormed and vaccinated forinfluenza, rhinopneumonitis, eastern and western encep-halomyelitis, West Nile encephalomyelitis, tetanus, andrabies. Tail rubbing and dysuria were noted 1 month be-fore presentation. Upon palpation of the epaxial andgluteal muscles, the horse trembled repeatedly andmoved away from the examiner. Three weeks later thehorse developed marked hyperesthesia of the tail,dropped the penis often, and dribbled urine without pos-turing. The horse was treated for cystitis withtrimethoprim and sulfadiazine PO for 3 days with nobeneficial response. Because of concerns of urine voiding,the urinary bladder was catheterized and emptied beforereferral.Physical examination did not reveal abnormalities.

Neurologic abnormalities included subtly decreasedtongue tone, markedly decreased to absent tail, anal, andperineal tone, hypoesthesia to anesthesia of the tail, analsphincter and perineal region, penile paresis, urinary in-continence, anesthesia and hypotonicity over the leftgluteal and semimembranosus muscles, and hyperesthesiaover the right semimembranosus muscle to tuber ischium.The horse also had apparent left-sided atrophy of thegluteal, semimembranosus, and semitendinosus muscles.There was a moderately distended hypotonic bladderand hypotonic rectum with fecal retention. Grade 1 prop-rioceptive deficits were identified in all 4 limbs accordingto a standard classification system.1 Decreased urethraltone was noted during a cystoscopy. The urinary bladderwas red and contained thick yellow material. Treatmentwas initiated with trimethoprim sulfamethoxazole(30mg/kg PO q12h), 15% ponazuril (5mg/kg PO q24h),10% fenbendazole (10mg/kg PO q24h), flunixin meglu-mine (1mg/kg PO q12h), 10% dimethylsulfoxide (1 g/kgIV q24h), and vitamin E (26U/kg PO q24h). Urinary

catheterization and manual evacuation of the rectumwere also performed.

Clinical laboratory abnormalities included mildleukocytosis (WBC 12,170/uL; reference interval 5,000–11,600/uL), neutrophilia (9,675/uL; reference interval2,600–6,800/uL), hyperproteinemia (TP 8.3 g/dL; refer-ence interval 5.8–7.7 g/dL), hyperglobulinemia (5.6 g/dL;reference interval 1.6–5.0 g/dL), and hyperglycemia(125mg/dL; reference interval 50–107mg/dL). Alpha-1globulin concentration was increased, as assessed by pro-tein electrophoresis, suggesting an acute phase response.Serum IgG concentration was increased (3.5 g/L, refer-ence interval 1.3–3.0 g/dL) whereas IgM and IgAconcentrations were within reference limits. Urinalysisdid not reveal abnormalities. Results from serumimmunofluorescent antibody tests for Sarcocystis neuro-na and Neospora hughesii, and M-capture ELISA forantibodies to West Nile virus were negative. Polymerasechain reaction (PCR) assays for respiratory pathogensinfluenza A, herpes virus 1 and 4, equine arteritis virus,and Streptococcus equi equi were negative on a nasalswab. Additionally, a PCR on a buffy coat was negativefor equine herpes virus 1.

Results of lumbosacral cerebrospinal fluid (CSF) re-vealed moderate mixed (primarily lymphocytic)pleocytosis of 20 total nucleated cells/uL (reference in-terval 1–5/uL) with 19% nondegenerate neutrophils,72% lymphocytes, and 9% monocyte/macrophages.There was marked lymphoid reactivity with a few gran-ular lymphocytes admixed with many small lymphocytes,reactive lymphocytes, and plasma cells. Macrophageswere mildly vacuolated and rarely erythrophagia wasnoted. A few mitotic figures were seen. The CSF proteinconcentration (56mg/dL) was within reference values(o80mg/dL). From the CSF immunofluorescent anti-body tests for S. neurona and N. hughesii, and PCRassays for equine herpes virus 1 and West Nile virus werenegative. There was no growth on culture media for bac-teria and fungi.

A transrectal ultrasound examination with an8.5MHz curvilinear microconvex transducer revealedenlargement and diffusely mottled, hypoechoic appear-ance of the extradural sacral nerve roots as they exitedthe ventral sacral foramina and also iliac lymphadenopa-thy (Fig 1).

An antemortem biopsy of the left sacrocaudalis dorsal-is lateralis muscle was performed under sedation withxylazine hydrochloride (0.3mg/kg IV) and butorphanoltartrate (0.01mg/kg IV). A muscle biopsy at the base ofthe tail was selected because: (1) hyperesthesia followedby hypoesthesia and anesthesia of the tail are the pre-dominant signs in cases of polyneuritis equi (PNE); (2)innervation of the tail arises from the cauda equina; and(3) there is a high concentration of terminal intramuscu-

From the Department of Medicine and Epidemiology (Aleman),The William R. Pritchard Veterinary Medical Teaching Hospital(Katzman), Department of Surgical and Radiological Sciences(Vaughan), Department of Pathology, Microbiology and Immunol-ogy, the School of Veterinary Medicine; University of California,Davis, Davis, CA (Hodges, Crabbs, Christopher, Higgins), Depart-ment of Pathology, University of California, San Diego, CA(Shelton).

Corresponding author: Monica Aleman, MVZ, PhD, Dipl. AC-VIM; Department of Medicine and Epidemiology, Tupper Hall 2108,One Shields Avenue, University of California, Davis, CA 95616;e-mail: mraleman@ucdavis.edu.

Submitted October 9, 2008; Revised December 15, 2008;Accepted January 16, 2009.

Copyright r 2009 by the American College of Veterinary InternalMedicine

10.1111/j.1939-1676.2009.0285.x

J Vet Intern Med 2009;23:665–668

lar nerve branches within this muscle. Specifically, the sac-rocaudalis dorsalis lateralis muscle was selected based onease of collection with minimal adverse residual effects.After collection, biopsy tissue was flash-frozen in isopen-tane precooled in liquid-nitrogen, and stored at �80 1C orimmersion fixed in 10% formalin until further processingfor histological, histochemical, and immunohistochemicalevaluation. In frozen muscle biopsy sections, terminal in-tramuscular nerve branches contained intense lymphocyticand histiocytic cell infiltration that almost obliterated thenerve architecture with sparing of myofibers (Fig 2A). Ex-cessive epi- and perineurial fibrosis was confirmed with themodifiedGomori trichrome stain (Fig 2B). A predominantinfiltration of macrophages was evident with acid phos-phatase stain and confirmed with immunophenotypicidentification of CD11a1 and c1 ([1 : 10 dilution], Giftfrom Dr Peter F. Moore, Department of Pathology, Mi-crobiology and Immunology, School of VeterinaryMedicine, Davis, CA) immunoreactive macrophage pop-

ulations (not shown). Immunophenotyping of cellularinfiltrates in frozen sections identified immunoreactiveCD81 ([1 : 10 dilution], Gift from Dr Jeffrey Stott, De-partment of Pathology, Microbiology and Immunology,School of Veterinary Medicine, Davis, CA) cytotoxicT lymphocytes (CTL, Fig 2C) and rare CD41 helperT-lymphocytes ([1 : 10 dilution], Gift from Dr Peter F.Moore). There were also admixed some populations ofimmunoreactive CD201a plasma cells and fewer CD31 Tlymphocytes ([1 : 10 dilution], Gift from Dr Peter F.Moore). Supportive of nerve fiber damage, a pattern ofneurogenic atrophy was identified in the frozen musclebiopsy sections as shown by angular atrophy involv-ing both muscle fiber types, and some hypertrophic orsplit fibers (Fig 2D). Thus the provisional diagnosisof polyneuritis equi was made based on clinical, ultra-sound, CSF analysis, and histological demonstration ofextensive cellular infiltrations in intramuscular nervebranches.

The horse was euthanized and necropsied 7 days afteradmission because of financial constraints, progressionof neurologic deficits, and guarded to poor prognosis. Onmacroscopic examination, most intra- and extraduralspinal nerves of the cauda equina that exited the inter-vertebral foramen caudal to S1 were thickened, firm, anddiffusely white (Fig 3). Sacral lymph nodes were bilater-ally enlarged. The bladder wall was markedly distendedand red containing abundant thick, pasty and grittybrown yellow material consistent with sabulous cystitisand likely secondary to bladder atony. There were noobvious macroscopic abnormalities in the remaining ner-vous system. Bacterial and fungal cultures, acid faststain, and immunohistochemical reactivity to West Nilevirus, equine herpes virus 1, Neospora spp., and S.falcatula and S. neurona antigens on the affected tissuewere all negative.

Microscopic lesions consisted of severe necrotizingpyogranulomatous inflammatory cell infiltrate that wascentered in, and frequently effaced, individual nerve fas-cicles of both intradural and extradural spinal nerves ofthe cauda equina (Fig 4A). There was profound epi- andperineurial fibrosis. A continuum of lesions occurredwith early lesions in fascicles characterized by a predom-inantly lymphocytic infiltrate that was eventuallyreplaced with necrosis and abundant neutrophils andmacrophages as the lesion progressed (Fig 4B). Multinu-cleated syncytial cells were common. There was also anintense dorsal root ganglioneuritis. In spinal nerves distalto their gross involvement, moderate nerve fiber loss andregenerating axonal clusters indicated both degenerationand attempts at regeneration (not shown). Immunohis-tochemical findings in frozen cauda equina includedprofound infiltration with macrophages, moderate tomarked infiltration with CTL, mild infiltration of B-lym-phocytes, and rare helper T-lymphocytes in perineurialand endoneurial regions (not shown). There was no mi-croscopic evidence of any cranial or other spinal nerveinvolvement.

PNE is an uncommon neurologic disease of undeter-mined cause characterized by progressive, insidiousgranulomatous inflammation of peripheral nerves.2–14

Fig 1. Transrectal sonographic images of extradural sacral nerve

roots. (A) Transverse image of an abnormal sacral nerve root at left

S2 (arrows) from this horse demonstrating enlargement with a

diffusely mottled, hypoechoic appearance. (B) Image of an extra-

dural sacral nerve root at left S2 (arrows) from a normal horse.

666 Aleman et al

Results of CSF cytological analysis in PNE are variableand nonspecific ranging from mild mixed mononuclearto primarily neutrophilic pleocytosis, with normal to in-creased protein concentrations.5,15 The predominance oflymphocytes and marked lymphoid reactivity in the pres-ent case with reactive lymphocytes and plasma cells wassupportive of a humoral response to inflammation suchas that observed with viral encephalitis, chronic proto-zoal and rickettsial infections, and immune-mediateddiseases.16 Because relevant infectious etiologies wereruled out in this horse, immune-mediated disease wasconsidered the most likely explanation for the CSF find-

ings. These cytologic findings correlated closely with theinflammatory infiltrates seen in intramuscular nervebranches in the initial muscle biopsy specimen and inthe cauda equina at necropsy. In addition, ultrasono-graphic findings in affected extradural sacral nerve rootswere supportive of a diagnosis of PNE. Although serumanti-P2 myelin antibodies have been found in some casesof PNE9,17; these antibodies lack specificity for PNE9,15

and were not investigated in this horse.This report describes biopsy of a skeletal muscle inner-

vated by nerves arising from the cauda equina withimmunohistochemical abnormalities supportive of anantemortem diagnosis of PNE. This study furthercharacterized T-lymphocytes subpopulations with pre-dominant CD81 CTL and rare CD41 helper Tlymphocytes, which has not been described previously.Immunophenotyping of both frozen and formalin-fixedtissue demonstrated that various macrophage subpopu-lations and CTL were the predominant cell types in thiscase of PNE. Immunoreactive CD31 T lymphocyteswere individually scattered and fewer than the focal ag-gregates of CD201 immunoreactive B-lymphocytes andplasma cells in formalin-fixed tissue. The presence ofB-lymphocytes and plasma cells suggest a local produc-tion of antibodies. Early attempts at nerve regenerationwere detected, suggesting that enhanced regenerationcould be possible if the inflammatory response is con-trolled early in the course of the disease.

Fig 2. Sacrocaudalis dorsalis lateralis muscle biopsy. (A) Intramuscular nerve branches showed severe mixed inflammatory cell infiltration

and fibrosis on hematoxylin and eosin. (B) Intramuscular nerve branch stained with modified Gomori trichrome showing fibrosis and absence

of pink myelin staining. (C) Notice abundant red stain (chromogen: amino ethyl carbazole) uptake indicating positive immunhistochemical

staining results for T-cytotoxic lymphocytes (CD81) infiltrating intramuscular nerve branch. (D) A neurogenic pattern of myofiber atrophy

was evident with the myofibrillar ATPase reaction at pH 9.8 (note angular atrophy of both fiber types with type 1 fibers light pink and type 2

dark brown [no stain used]).

Fig 3. Cauda equina collected postmortem from the horse with

polyneuritis equi. Note thickened intra- and extradural spinal nerve

roots. The spinal cord is not shown. CR, cranial, CD, caudal, scale

bar 5 1 cm.

667Polyneuritis Equi

To date a confirmed diagnosis of PNE has only beenpossible through postmortem evaluation of the caudaequina and other peripheral nerves. In this report, wedemonstrate the value of the muscle biopsy and otherancillary testing to reach an antemortem diagnosis. Themuscle biopsy allowed the identification of massive cel-lular infiltrations into intramuscular nerve branches, afinding that has not been previously reported in PNE.Such cellular infiltrations into terminal intramuscularnerve branches have been described in experimentalmodels of autoimmune neuritis in mice.18 The presenceof CD81 T-cells in excess of CD41 T-cells is consistentwith underlying immune-mediated disease.18 The combi-nation of a muscle biopsy with the neurologicalexamination, CSF evaluation, ultrasonography should

enable a definitive clinical diagnosis of PNE to bereached premortem and allow early intervention andtreatment of the disease.

Acknowledgments

The authors thank Dr Bruce Daniels for the referral ofthis case, Ms Colette Williams and Mr John Doval fortechnical assistance, and Ms Diane Naydan for herimmunocytochemical expertise.

Footnote

a Labvision (1 : 10 dilution), Freemont, CA

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Fig 4. Immunohistological examination of spinal nerves from the

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CD11c1 immunostaining in formalin fixed tissue.

668 Aleman et al