P.M. Smith

Histological and ultrastructural analysis of white matter damage after naturally-occurring spinal cord injury.

Detailed analysis of the structural changes that follow human clinical spinal cord injury is limited by difficulties in achieving adequate tissue fixation. This study bypasses this obstacle by examining the spinal cord from paraplegic domestic animals, enabling us to document the ultrastructural changes at different times following injury. In all but one case, injury resulted from a combination of contusion and compression. There was infarction and hemorrhage, followed by gray matter destruction and the rapid development of a variety of white matter changes including axon swelling and myelin degeneration. Axons greater than 5u2003µm in diameter were more susceptible to degenerative changes, whereas smaller axons, particularly those in the subpial region, were relatively well preserved. Demyelinated axons were seen within 2 weeks after injury and, at later time points, both Schwann cell and oligodendrocyte remyelination was common. More subtle white matter abnormalities were identified by examining sagittal sections, including focal accumulation of organelles in the axoplasm and partial and paranodal myelin abnormalities. These observations serve to validate observations from experimental models of spinal contusion but also highlight the complexity of naturally occurring (ie, clinical) spinal injury. They also raise the possibility that focal abnormalities such as paranodal demyelination may contribute to early axonal dysfunction and possibly to progressive tissue damage.

Cryopreserved cells isolated from the adult canine olfactory bulb are capable of extensive remyelination following transplantation into the adult rat CNS

Naturally occurring spinal cord injury in dogs provides a potentially powerful intermediate model for testing the efficacy of therapeutic strategies developed in experimental rodent models before phase 1 trials in human patients. A particularly promising strategy involves transplantation of olfactory ensheathing cells (OECs) that both promote axon regeneration and generate new myelin sheaths. As a first step in developing OEC transplantation in the canine intermediate model we describe the isolation, purification, and characterization of OECs from adult dog olfactory bulb. We also show that the canine OEC behaves in a manner similar to its rodent counterpart following transplantation into demyelinating lesions in rat spinal cord and that these properties are retained following cryopreservation.

SCIP/Oct‐6, Krox‐20, and desert hedgehog mRNA expression during CNS remyelination by transplanted olfactory ensheathing cells

Olfactory ensheathing cells (OECs), although having a separate developmental origin to Schwann cells, are able to generate myelin sheaths following transplantation into areas of CNS demyelination that are remarkably similar to those made by Schwann cells. The transcriptional control of Schwann cell myelination has been well documented, in particular the role of SCIP/Oct‐6 and Krox‐20. It is not known, however, whether these transcription factors are also expressed when OECs assume a myelinating phenotype. In this study, we addressed this question by using a transplantation approach to generate myelinating OECs and then examined the expression of SCIP/Oct‐6 and Krox‐20 mRNA by in situ hybridization using oligonucleotide probes. We also examined the expression of desert hedgehog (Dhh), a Schwann cell–derived signaling molecule that is responsible for regulating the development of the connective tissue elements in peripheral nerve, which bear similarities to the morphologies adopted by nonmyelinating transplanted cells. Our results indicate that both Krox‐20 and Dhh mRNA are strongly expressed by transplanted OECs, with SCIP mRNA present at much lower levels. The expression of Krox‐20 relative to the expression of P0 mRNA by the transplanted OECs is consistent with its playing a similar role in OEC myelination to that in Schwann cell myelination, while the expression of Dhh suggests a possible mechanism for the diverse morphologies that cells adopt following OEC transplantation into the damaged CNS. Taken together, our results provide further evidence for the close similarity of OECs and Schwann cells and suggest that, despite their separate origins, the manner in which they generate a peripheral‐type myelin sheath involves similar regulatory mechanisms. GLIA 36:342–353, 2001.

Acquired idiopathic laryngeal paralysis as a prominent feature of generalised neuromuscular disease in 39 dogs

A retrospective evaluation of 17 dogs treated surgically for idiopathic acquired laryngeal paralysis demonstrated a marked variability in outcome, with many dogs continuing to exhibit weakness and exercise tolerance. In a subsequent prospective study, 22 consecutive affected dogs were tested for neurological deficits by neurological examination, electrodiagnostic evaluation, and blood analysis to measure thyroxine and thyroid-stimulating hormone and to detect any evidence of myasthenia gravis. Clinical neurological deficits and/or electrodiagnostic abnormalities were found in each case. There was limited evidence that specific neurological deficits were associated with a poor prognosis for full recovery of exercise tolerance. None of the dogs was positive for anti-acetylcholine receptor antibodies, and only three showed evidence of thyroid dysfunction.

SpL201: A conditionally immortalized Schwann cell precursor line that generates myelin

Dramatic progress has been made over recent years toward the elucidation of the mechanisms regulating lineage determination and cell survival in the developing peripheral nervous system. However, our understanding of Schwann cell development is limited. This is partly due to the difficulties in culturing primary Schwann cell precursor cells, the earliest developmental stage of the Schwann cell lineage defined to date. Both the inability to maintain cultured Schwann cell precursor cells in an undifferentiated state and the technical difficulties involved in their isolation have hampered progress. We have conditionally immortalized rat Schwann cell precursor cells using a retrovirally encoded EGFR/neu fusion protein to circumvent these problems and to generate a source of homogeneous cells. The resulting SpL201 cell line expresses p75 and nestin, two proteins expressed by neural crest‐derived cells, as well as peripheral myelin protein 22, protein zero, and Oct‐6 as markers of the Schwann cell lineage. When cultured in EGF‐containing medium, the SpL201 cells proliferate and maintain an undifferentiated, Schwann cell precursor cell‐like state. The cell line is dependent on EGF for survival but can differentiate into early Schwann cell‐like cells in response to exogenous factors. Like primary rat Schwann cells, SpL201 cells upregulate Oct‐6 and myelin gene expression in response to forskolin treatment. Furthermore, the SpL201 cell line can form myelin in the presence of axons in vitro and is capable of extensively remyelinating a CNS white matter lesion in vivo. Thus, this cell line provides a valuable and unique tool to study the Schwann cell lineage, including differentiation from the Schwann cell precursor cell stage through to myelination. GLIA 36:31–47, 2001.

The effect of immunosuppressive protocols on spontaneous CNS remyelination following toxin-induced demyelination

Glial cell transplantation is a potential therapy for human demyelinating disease, though obtaining large numbers of human myelinating cells for transplantation remains a major stumbling block. Autologous transplantation is currently not possible, since the adult human CNS is not a good source of oligodendrocyte precursors, and long-term immunosuppression of engrafted allogeneic or xenogeneic cells is therefore likely to be necessary. Immunosuppressive drugs may need to be used in situations where more recent, active areas of demyelination are undergoing endogenous remyelination. It is therefore pertinent to establish the extent to which immunosuppressive protocols will suppress spontaneous remyelination. In order to investigate this issue, we created demyelinating lesions in the spinal cord of adult rats and compared the extent of remyelination in animals receiving different immunosuppressive treatments. In animals given only cyclosporin A, there was no difference in the extent of either Schwann cell or oligodendrocyte remyelination of ethidium bromide-induced demyelinating lesions. However, in animals given cyclophosphamide, either alone or in combination with cyclosporin, there was a significant reduction in the extent of oligodendrocyte-mediated remyelination. These results demonstrate that cyclophosphamide is deleterious to oligodendrocyte remyelination and for this reason should be used with caution in patients with demyelinating disease.

Comparison of Two Regimens for the Treatment of Meningoencephalomyelitis of Unknown Etiology

BACKGROUNDnThe optimal treatment for meningoencephalomyelitis of unknown etiology (MUE) remains unknown, despite the widespread use of a variety of immunosuppressive drugs.nnnOBJECTIVE/HYPOTHESISnTo compare the efficacy of prednisolone combined with either vincristine and cyclophosphamide (COP group; n= 10) or with cytosine arabinoside (AraC group; n= 9).nnnANIMALSnNineteen dogs with neurological deficits, neuroimaging, and cerebrospinal fluid abnormalities consistent with a diagnosis of MUE.nnnMETHODSnProspective, blinded, and randomized clinical trial. Dogs fulfilling the inclusion criteria were randomly allocated to receive 1 drug regimen.nnnRESULTSnFour of 10 dogs in the COP group and 5/9 in the AraC group survived > 12 months but neither the survival time nor the time-to-treatment failure differed between the 2 groups. Treatment with COP resulted in an unacceptable incidence of adverse effects.nnnCONCLUSIONSnThe adverse effects of COP make it an unsuitable treatment for MUE. Although survival of animals treated with AraC was broadly similar to that reported in recently published studies describing this treatment, it remains unclear whether it confers any benefit over using prednisolone alone.

Facial myokymia in a puppy

MYOKYMIA is a muscular disorder that causes a vermicular, writhing motion visible through the skin overlying the affected muscles, which is diagnosed by its characteristic clinical features and distinctive appearance on electromyography (Shapiro and others 2002). Generalised and focal forms are recognised in human beings; focal myokymia most commonly affects the face and is a rare sign associated with a variety of conditions, including intra-axial pontine lesions and demyelinating conditions such as multiple sclerosis and Guillain-Barre syndrome (Gutmann and Gutmann 2004). To the authors’ knowledge facial myokymia has not previously been described in dogs. This short communication describes a case of a puppy with facial myokymia. A six-month-old male cocker spaniel puppy was referred to the Queen’s Veterinary School Hospital, Cambridge University, with a two-month history of facial muscle twitching. This movement was continuous and persisted when the dog was sleeping; it had initially been restricted to the left side of the muzzle but by the time of presentation had progressed to involve the periorbital muscles and right side of the face. The owners did not report any other problems, and the puppy was able to eat normally. A possible diagnosis of otitis media had been investigated by the referring veterinary surgeon, but the condition had failed to respond to a course of antibiotics. During the clinical examination, there was a persistent, worm-like, writhing motion over the dog’s upper lip and eyelids, which was more pronounced on the left side. A similar pattern of muscle activity could also occasionally be seen over the left shoulder region. A neurological examination detected cranial nerve deficits: there was a bilaterally absent menace response, weak palpebral reflexes, abnormal vestibular eye movements and apparently poor hearing. The puppy had a tendency to tilt its head to the left and seemed generally slightly incoordinated; for example, it could not easily catch objects in its mouth. There were no deficits in postural reactions or segmental spinal reflexes. Electromyography (EMG) recorded characteristic neuromyokymic discharges (with an intraburst frequency of approximately 300 Hz) from the levator nasolabialis, orbicularis oris and orbicularis oculi muscles on the left and right sides (Fig 1a) (Anon 2006). A brainstem auditory evoked response could not be recorded at 100 dB from either ear. The electrodiagnostic tests were repeated two months later when clinical reassessment found that the dog’s condition was unchanged. The EMG recording (Fig 1b) demonstrated a distinct change in the pattern of activity: in addition to the previously observed myokymic activity there were positive sharp waves and fibrillation potentials, suggesting axonal disease. Routine haematology and serum biochemistry (including measurement of the activity of creatine kinase) were unremarkable. Serological tests were negative for immunoglobulin G and M antibodies to Toxoplasma species, but showed an elevated titre of antibodies to canine distemper virus (the puppy had been routinely vaccinated). Cerebrospinal fluid (CSF) contained an increased amount of protein (total protein 0·38 g/l, reference range 0·00 to 0·25 g/l) and increased numbers of nucleated cells (10/μl, reference range 0 to 8/μl); lymphocytes and monocytes were the predominant cell types. Magnetic resonance imaging (MRI) detected mild herniation of the caudal cerebellar tonsils through the foramen magnum and scattered equivocal areas of hypointensity were observed throughout the brain on T1-weighted and fluid-attenuated inversion recovery scans. Because the CSF analysis suggested inflammation, a trial treatment with corticosteroids was initiated, but did not eliminate the clinical signs; the owners declined further attempts at treatment. The clinical appearance and EMG findings in the puppy were diagnostic for focal myokymia. This focal condition has not previously been reported in dogs, but there have been previous reports of episodic involvement of the entire musculature, predominantly in adult Jack Russell terriers (Reading and McKerrell 1993, Van Ham and others 2004). In the series reported by Van Ham and others (2004), the dogs presented with generalised signs including ataxia and episodic collapse, during which there was continuous muscle activity. This generalised condition appears to be much more severe than the present case of focal myokymia, since four of the six cases described either died or were euthanased because of their ataxia or episodes of collapsing. There is considerable evidence to implicate abnormalities of the axonal voltage-gated potassium channels as the underlying cause of myokymia. These abnormalities can arise as a result of, for example, genetic abnormalities (Browne and others 1994), toxins (Lewis and Gutmann 2004) or autoimmune disease (Hart and others 2002). Continuous myokymia restricted to the face is an uncommon presentation in human medicine, and has been associated with structural lesions of the pons, demyelinating conditions, toxins, and genetic and autoimmune channelopathies; a benign, postfatigue form, affecting the periorbital musculature, is also recognised (Gutmann and Gutmann 2004). The duration of clinical signs is very variable, depending on the aetiology: in polyradiculoneuropathy it may last for several weeks or months, but in pontine glioma it may persist for the lifespan of the patient (Gutmann and Gutmann 2004). The diagnosis of hearing deficits and evidence of vestibular disturbances in combination with myokymia in the present case might suggest that a structural lesion of the caudal brainstem could underlie the clinical signs. However, although MRI did reveal a Chiari type I malformation, there were no other well defined anomalies. Nevertheless, many defects in neural development, such as neuronal migration disorders (Sarnat and Flores-Sarnat 2002), are not visible on MRI scans, and so a microscopic structural disorder cannot be ruled out. Indeed, defects in the formation of cranial nerve nuclei have been assoShort Communications