S. Añor

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.

Magnetic resonance imaging findings in 40 dogs with histologically confirmed intracranial tumours.

Magnetic resonance (MR) images of 40 dogs with histologically confirmed primary and secondary intracranial tumours were reviewed. Forty-one tumours were diagnosed by means of MR imaging (MRI). MRI findings allowed diagnosis of a neoplastic lesion in 37/41 cases. Based on MRI features, differentiation between neoplastic and non-neoplastic lesions was possible in 24/27 (89%) primary brain tumours and in 13/14 (92%) secondary brain tumours. Diagnosis of tumour type based on MRI features was correct in 19/27 (70%) primary tumours and in 13/14 secondary tumours. The results of this study show that MRI is a good diagnostic imaging modality to detect neoplastic lesions and to diagnose tumour type in dogs. However, as some neoplasms show equivocal MRI features the technique has limitations in the detection of some intracranial tumours and in predicting tumour type.

Canine cutaneous spindle cell tumours with features of peripheral nerve sheath tumours: a histopathological and immunohistochemical study.

In veterinary medicine, the term peripheral nerve sheath tumour is usually restricted to neoplasms that are closely associated with an identified nerve. Thirty-three cases of canine cutaneous tumours previously classified as spindle cell tumours with features resembling peripheral nerve sheath tumours were examined. Two histological patterns were identified: dense areas of spindle shaped cells resembling the Antoni A pattern and less cellular areas with more pleomorphic cells resembling the Antoni B pattern. Immunohistochemically, all tumours uniformly expressed vimentin and 15/33 (45.4%) had scattered and patchy expression of S-100. Laminin expression was found in 25/33 (75.7%) tumours and collagen IV labelling occurred in 14/33 (42.4%). Expression of protein gene product 9.5 was detected in 31/33 (93.9%) of tumours and neuron specific enolase labelling was present in 27/33 (81.8%). Glial fibrillary acidic protein was only expressed within the cytoplasm of some large multinucleated cells in one tumour. These findings suggest that any cutaneous tumour with one of the two histopathological patterns described above should be described as a cutaneous peripheral nerve sheath tumour and that expression of S-100, laminin and collagen IV may be used to define a schwannoma.

Biochemical, Histological and Functional Correction of Mucopolysaccharidosis Type IIIB by Intra-cerebrospinal Fluid Gene Therapy

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the diseases main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.

Inert patch with bioadhesive for gentle foetal surgery of myelomeningocele in a sheep model

OBJECTIVE Current techniques used in foetal myelomeningocele repair can require considerable manipulation of fragile foetal tissues to obtain tension-free closure. The aim of this study was to assess the feasibility of a simple foetal coverage method without foetal tissue manipulation to provide closure of the neural tube defect in myelomeningocele. STUDY DESIGN This is an experimental study performed in 15 foetal sheep with lumbar myelomeningocele, surgically created on day 75 of gestation. Five foetuses remained untreated. Ten underwent coverage with inert sheeting (5 Silastic; 5 Silastic+Marlex) secured by surgical tissue adhesive without suturing on day 95; none of them underwent foetal muscle or skin manipulation. Clinical and subsequent histological examinations were performed at 48h after birth. The Chi-square, Fisher exact, and Mann-Whitney U tests, when appropriate, were used for the comparisons. RESULTS The mean operating time for foetal coverage was 7.1 (SD=1.6)min. All untreated animals were unable to walk, had sphincter incontinence, showed an open defect, histological spinal cord damage, and a large Chiari malformation. All covered animals were able to walk, had sphincter continence, showed almost complete closure of the defect with regeneration of several soft tissue layers, and minimum Chiari malformation. CONCLUSION In a surgical myelomeningocele model in sheep, a simple, fast and gentle coverage method using a sealed patch avoids foetal tissue manipulation and enables adequate closure of the neural tube defect, providing regeneration of several tissue layers that protect the spinal cord, and significantly reducing Chiari II malformation.

Presumed brain infarctions in two dogs with systemic leishmaniasis

Clinical signs and magnetic resonance imaging findings of multiple brain infarcts in two dogs infected with Leishmania spp. are reported. Clinical signs of intracranial dysfunction were peracute and there was no further deterioration. Magnetic resonance images of the brain were consistent with multifocal, non-haemorrhagic, ischaemic lesions. Routine serum biochemistry revealed hyperproteinaemia and hyperglobulinaemia. Serum antibody titres were highly positive for Leishmania infantum and Leishmania amastigotes were seen within bone marrow macrophages in both cases. Canine leishmaniasis can cause cerebrovascular alterations, such as vasculitis, that might predispose dogs to brain infarcts.

Acute postretinal blindness: ophthalmologic, neurologic, and magnetic resonance imaging findings in dogs and cats (seven cases)

OBJECTIVE To describe the ophthalmologic, neurologic, and magnetic resonance imaging (MRI) findings of seven animals with acute postretinal blindness as sole neurologic deficit. METHODS Medical records were reviewed to identify dogs and cats with postretinal blindness of acute presentation, that had a cranial MRI performed as part of the diagnostic workup. Only animals lacking other neurologic signs at presentation were included. Complete physical, ophthalmic, and neurologic examinations, routine laboratory evaluations, thoracic radiographs, abdominal ultrasound, electroretinography, and brain MRI were performed in all animals. Cerebrospinal fluid analysis and postmortem histopathologic results were recorded when available. RESULTS Four dogs and three cats met the inclusion criteria. Lesions affecting the visual pathways were observed on magnetic resonance (MR) images in six cases. Location, extension, and MRI features were described. Neuroanatomic localization included: olfactory region with involvement of the optic chiasm (n = 4), pituitary fossa with involvement of the optic chiasm and optic tracts (n = 1), and optic nerves (n = 1). Of all lesions detected, five were consistent with intracranial tumors (two meningiomas, one pituitary tumor, two nasal tumors with intracranial extension), and one with bilateral optic neuritis that was confirmed by cerebrospinal fluid analysis. Histologic diagnosis was obtained in four cases and included one meningioma, one pituitary carcinoma, one nasal osteosarcoma, and one nasal carcinoma. CONCLUSIONS Central nervous system (CNS) disease should be considered in dogs and cats with acute blindness, even when other neurologic deficits are absent. This study emphasizes the relevance of MRI as a diagnostic tool for detection and characterization of CNS lesions affecting the visual pathways.

NEOSPORA CANINUM IDENTIFICATION IN AN ABORTED BOVINE FETUS IN SPAIN

Neospora caninum tachyzoites were identified in areas of the cerebrum with lesions of non-suppurative encephalitis in an aborted bovine fetus in Spain.

Malignant Peripheral Nerve Sheath Tumor in a Water Moccasin (Agkistrodon Piscivorus)

Peripheral nerve sheath tumors (PNSTs) in domestic and wild animals are uncommon tumors that originate from cells that comprise the sheaths of peripheral nerves. One type of PNST originates from Schwann cells and is termed schwannoma. Two other types of cells, perineural cells and fibroblasts, are components of peripheral nerve sheaths and may give rise to tumors. These tumors, particularly the malignant variety, may have histologic patterns that closely mimic those of other fusiform tumors. Electron microscopic and/or immunohistochemical evaluation is often necessary to establish a diagnosis of PNST.11,13 PNSTs affecting cranial or spinal nerve roots and the brachial plexus have been described in several animal species (cattle, dog, cat, rat).11 Malignant cranial and spinal PNSTs are rare in animals and metastasize infrequently.3,11 In this report, a malignant PNST originating from a spinal nerve with hepatic and splenic metastasis is described in a snake. A 12-year-old captive-bred male water moccasin (Agkistrodon piscivorus) developed a firm nonulcerated and progressive swelling of the subcutaneous tissue on the right midbody. Flaccid paralysis and lack of propriocepcion was present distal to the lesion. The head and neck were displaced laterally to the left. The snake was anesthetized using a face mask with isofluoranea in oxygen for induction followed by tracheal intubation. A biopsy of the costal mass was obtained for evaluation. The histopathologic diagnosis was invasive undifferentiated spindle cell sarcoma. Neoplastic cells were observed in all the sample edges. The animal was again anesthetized to completely resect the cutaneous mass. The previous diagnosis was histologically confirmed, and neoplastic cells were observed in the deeper resection border associated with the ribs. Three months later, a new and larger subcutaneous swelling was observed in the same location. The snake’s general condition had markedly worsened: it was anorectic and the head displacement, flaccid paralysis, and lack of propriocepcion were more pronounced. The animal was again anesthetized and positioned in sternal recumbency to evaluate the extension of the new lesion and to detect possible metastasis by radiography and magnetic resonance imaging (MRI). Plain radiographs revealed a large, poorly defined soft tissue mass on the right costal wall that invaded the celomic cavity. On MRI, transverse sections revealed a hyperintense mass on the right side of the body. The mass was located in the paravertebral muscles and invaded the celomic cavity through the intercostal spaces. It also invaded the vertebral canal through intervertebral foramina and compressed the spinal cord (Fig. 1). Coronal sections demonstrated neoplas-

Antemortem diagnosis of a distal axonopathy causing severe stringhalt in a horse.

A 16-year-old Westfalian gelding was presented to the Equine Teaching Hospital of Barcelona for a 6week history of a sudden, progressive bilateral abnormal gait in the pelvic limbs. Previous treatment with phenylbutazone and paralumbar corticosteroid (triamcinolone) infiltration had not resulted in improvement. The horse had lived for 2 years in the same pasture with other unaffected horses. None of the other horses developed clinical signs consistent with stringhalt. No history of recent trauma was reported. The horse did not have any previous medical problems, apart from degenerative joint disease of the left radiocarpal and carpometacarpal joints, both metacarpophalangeal joints and both metatarsophalangeal joints that was diagnosed and treated 1 year before presentation. On admission, the horse was excited and reluctant to move, and sedation with xylazine (0.4mg/kg IV) was needed to move the horse out of the van. When eventually moved, the animal showed a bunny-hopping gait with severe and exaggerated flexion movements, and knuckled on both pelvic limbs (supporting information Video S1). Clinical signs were bilateral, but the right pelvic limb seemed more severely affected than the left. The horse kept the right pelvic limb hyperflexed while standing for several minutes until it relaxed. In addition, severe skin abrasions were found on the dorsal aspect of both carpal areas, and on the cannon bone and fetlock areas of both pelvic limbs caused by the abnormal gait. Complete physical and neurological examinations did not identify any other clinically relevant findings. The clinical diagnosis was bilateral stringhalt grade V/V, according to the gradation scale of Huntington et al. Examination of the horse’s pasture revealed very few dry plants, and no evidence of any toxic plant. The horse was fed festuca grass hay similar to other horses in the stable, and the food was in good condition. Results of CBC and serum biochemistry did not disclose any abnormalities. Lumbosacral cerebrospinal fluid collection was attempted, but a sample could not be obtained. Electromyographic (EMG) examination of the pelvic limb muscles including the long digital extensor was attempted with the horse awake to eliminate a peripheral neuropathy or primary myopathy. Unfortunately, the horse was extremely sensitive to any manipulation of the pelvic limbs and conclusive results could not be obtained. To reach a definitive diagnosis, biopsies from the long digital extensor muscle and the superficial peroneal nerve were obtained 4 days after admission. The horse was premedicated with romifidine (0.03mg/kg IV) and butorphanol (0.03mg/kg IV). General anesthesia was induced with diazepam (0.05mg/kg IV) and ketamine (2.2mg/kg IV) and maintained with isofluorane in 100% oxygen with intermittent positive pressure ventilation. The horse was positioned in left lateral recumbency to obtain biopsies from the right pelvic limb. The lateral surface proximal to the tarsus was clipped and surgically prepared. An 8-cm longitudinal incision was made at the level of the lateral and long digital extensor muscle bellies. Superficial tissues were bluntly dissected until the superficial peroneal nerve was identified. The nerve was carefully separated from surrounding connective tissue using sharp dissection and avoiding the use of gauze or blunt dissection to prevent artifact. A 2.5-cm-long fascicular nerve biopsy then was obtained. The sampled nerve then was divided longitudinally into 2 pieces. Dissection was continued cranially until the long digital extensor muscle could be visualized. Two muscle samples were obtained, 1 of standard dimensions (0.5 0.5 1.0 cm) and a smaller sample. The skin was sutured in a discontinuous single pattern using 0USP polypropylene. At the same time, a biopsy from the right middle gluteal muscle was obtained by a percutaneous punch biopsy procedure. One muscle and 1 nerve specimen were placed on a tongue depressor and immersed in 10% formalin. The 2nd specimens were wrapped in saline-moistened gauze sponges and placed into a dry, water-tight container. All samples were shipped overnight under refrigeration to the Comparative Neuromuscular Laboratory, University of California San Diego, La Jolla, California. Immediately upon receipt, unfixed, and chilled muscle and nerve specimens were flash frozen in isopentane precooled in liquid nitrogen, then stored at 801C until further proFrom the Servei de Medicina Interna Equina (Armengou, Monreal); Servei de Neurologia i Neurocirurgia (Añor); Servei de Cirurgia Equina (Climent), Hospital Clı́nic Veterinari, Universitat Autònoma de Barcelona, Barcelona, Spain; and Comparative Neuromuscular Laboratory, Department of Pathology, University of California, San Diego, La Jolla, CA (Shelton). This report was presented at the 2nd Congress of the European College of Equine Internal Medicine (ECEIM), February 2007, Naas, Ireland. The abstract was published in J Vet Intern Med 2007;21:882. Corresponding author: L. Armengou, DVM, Dipl ECEIM, Servei de Medicina Interna Equina, Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain; e-mail: lara.armengou@ uab.cat Submitted July 24, 2009; Revised September 25, 2009; Accepted October 14, 2009. Copyright r 2009 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2009.0437.x Abbreviation: