Shaking pups: A disorder of central myelination in the spaniel dog

Shaking pups: A disorder of central myelination in the spaniel dog

Journal of the Neurological Sciences, 1981.50:423-433 423 Elsevier/North-Holland Biomedical Press S H A K I N G PUPS: A D I S O R D E R OF C E N T ...

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Journal of the Neurological Sciences, 1981.50:423-433

423

Elsevier/North-Holland Biomedical Press

S H A K I N G PUPS: A D I S O R D E R OF C E N T R A L M Y E L I N A T I O N I N T H E SPANIEL DOG Part 1. Clinical, Genetic and Light-microscopical Observations

I. R. GRIFFITHS, I.D. DUNCAN*, M. McCULLOCH and M. J.A. HARVEY Department o[' Surgery, University o[' Glasgow VeterinaO, School, Bearsden Road, Glasgow G61 1QH (Great Britain)

(Received 20 October, 1980) (Accepted 20 November, 1980)

SUMMARY A new disorder of central myelination has been recognised in male Springer Spaniel pups which is probably inherited in a sex-linked recessive mode. The affected animals were much reduced in weight and size and showed gross generalised tremor, particularly when aroused, at about 10-12 days of age. Affected pups were studied between 1 and 3 months of age. There was severe hypomyelination throughout the CNS which was more marked in the cerebrum and optic nerves than in the spinal cord. The amount of myelin at each location increased with age. Axonal calibre also increased and there was no difference between the axonal diameters of affected and age-matched normal pups. Axons were either naked or surrounded by a disproportionately thin layer of myelin. Myelinated internodes tended to be short and heminodes were frequent. Vacuoles were present adjacent to axons or within glia but there was no evidence of demyelination. Total glial numbers were not reduced and numerous oligodendroglial and astrocytic nuclei identified. Peripheral, cranial and autonomic nerves were myelinated normally. It is suggested that there is an abnormality of oligodendroglial metabolism such that they cannot form and maintain normal myelin. Consequently the radial and longitudinal extensions of their plasma membranes are reduced. The vacuoles may represent a breakdown of defective myelin lipids as suggested in certain murine

This study was supported by the WellcomeTrust. *Department of Neurology, The Montreal General Hospital and McGill University, Montreal, Canada. Correspondence to: I.R. Griffiths, Department of Surgery, University of Glasgow Veterinary School, Bearsden Road. Glasgow G61 1 QH, Great Britain. 0022-510X/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press

424 mutants. This defect of myelination provides a further model in which normal and disordered myelinogenesis can be studied.

INTRODUCTION

Myelinogenesis of the central and peripheral nervous systems is a complex process which to date remains incompletely understood. Study of the developmental processes has benefited greatly from the investigation of animals with inherited disorders of myelination. Different mutations have separate defects in myelination leading to variations in distribution and severity of the abnormality. Mutations have usually been reported to affect either the central nervous system (e.g. the Jimpy mouse, Sidman et al. 1964) or the peripheral nervous system (e.g. the trembler mouse, Ayres and Anderson 1973) alone, although involvement of both in the same mutant has also been recorded (e.g. the quaking mouse, Sidman et al. 1964). While each mutant has been found to have different morphological and biochemical abnormalities, the precise abnormality has still to be defined. This present report concerns the clinical, genetic and light-microscopical observations on a family of Springer Spaniel dogs with a new, inherited myelin disorder of the central nervous system, found in association with congenital tremor. Ultrastructural observations on these dogs will be reported in a later communication. CASE REPORTS

The affected animals were all pedigree male Springer Spaniel pups born to the same mother. The first litter contained 7 live and 1 dead pup. Three females and 1 male were normal while 2 males developed clinical signs. These animals were not examined by us and were destroyed at 5 weeks of age. A second litter, delivered by Caesarean section, contained 6 live and 2 dead pups. Three females were normal and the 3 males developed clinical signs. A third litter was produced containing 5 males and 1 female. One male was stillborn and another died shortly after birthl Two of the remaining male pups were normal but the 3rd developed clinical signs. The sire of litter 1 was different from the sire used in litters 2 and 3. The clinical signs were identical in all dogs. At 10-12 days of age a gross generalised tremor developed involving body, limbs, head and eyes which made detailed neurological testing difficult. When the pups were lying quietly or sleeping the tremor decreased or ceased completely but intensified markedly when they were roused. The dogs could not stand or perform co-ordinated useful movements but if the head was held the animals could feed. When attempting to move around the animals usually progressed backwards or sideways without ever gaining their feet. Righting, pedal, and patellar reflexes were present. The photomotor reflexes were brisk. The affected dogs were approximately half the size and weight of normal littermates. As a result of progressive morbidity all affected pups were killed at varying time intervals (see below).

425 MATERIALSAND METHODS Four affected and one normal Spaniel pup together with 2 normal pups of other breeds were available for histological examination. In 2 affected animals (1 month and 3 months old) the CNS was immersion fixed in 10~ formal-saline and processed for paraffin-embedded sections. The stains used were haematoxylin and eosin, luxol fast blue-cresyl violet, cresyl violet and Holme's silver impregnation. The 2 remaining affected animals (1 month and 2 months old), the normal male Spaniel littermate (2 months old) and the further 2 normal pups (1 month and 2 months old) were fixed after heparinisation, by intracardiac perfusion of a glutaraldehyde/formaldehyde mixture in a cacodylate buffer (Karnovsky 1965). Blocks from selected areas of the spinal cord, brain and optic nerves were routinely processed for electron microscopy (EM) and embedded in Araldite. A number of peripheral, cranial and autonomic nerves from all animals were fixed under slight longitudinal tension in 2.5~ phosphate-buffered glutaraldehyde and processed for EM or single fibre teasing. Araldite-embedded material was cut at 1/~m and stained with toluidine blue and thin sections were cut with a diamond knife, double-stained and examined on a Philips 301 electron microscope. Samples of various thoracic and abdominal viscera were also immersion-fixed in 10}/o formal-saline and processed for paraffin sections. Frozen samples of fresh brain from an affected 1-month-old pup were prepared by cryostat section and examined for canine distemper virus antigen by indirect immunofluorescence. In addition portions of brain were inoculated into a continuous canine kidney cell line to detect any cytopathic effect. RESULTS Gross examination of the spinal cord and brain of affected pups prior to fixation demonstrated marked abnormalities of the white matter which was grey and gelatinous and contrasted sharply with the white colour of the nerve roots and peripheral and cranial nerves. Light microscopy demonstrated a severe deficiency of myelin throughout the entire CNS at all ages (Fig. 1). The degree of myelination present was always greater in the spinal cord than in the cerebral white matter and optic nerves. Within all areas of the CNS, especially the cerebrum and optic nerves numerous naked axons were seen (Figs. 2 and 3) while other axons were surrounded by a thin myelin sheath which at neither of the time intervals examined was comparable to the myelin sheath of normal animals. In the spinal cord, cerebrum and optic nerves the number of myelinated fibres and the thickness of myelin sheath increased with advancing age but was always markedly reduced compared to both the normal littermate and the other normal pups of the same age (Fig. 2). There was no obvious relationship between axonal diameter and the absence or presence and thickness of a myelin sheath. Longitudinal sections demonstrated that many myelinated internodes were much shorter than those of comparable

426

Fig. 1. Coronal section of brain at the level of the head of the caudate of a 2-month-old pup. No~c the severe lack of myelin throughout the white matter. Luxol fast blue-cresyl violet. × 5.

axons in normal pups and that the degree of myelination of consecutive internodes could vary (Fig. 4). Heminodes were found in far greater frequency than nodes of Ranvier. The majority of axons were normal but very occasionally swollen axons containing accumulations of organeltes were seen. The axonal diameter of fibres in both the spinal cord and optic nerve increased between 1 and 2 months of age and there was no obvious difference between the axonal calibre of the 2-month-old affected and normal pups (Figs. 2 and 3). Both transverse sections and teased fibre preparations confirmed the gross observation that myelination of the PNS was normal. The contrast between CNS and PNS myelin was well demonstrated at the spinal nerve root entry zones (Figs. 5 and 6). At the dorsal roots there was the outward dome of amyelinated/ hypomyelinated CNS tissue while the ventral roots showed the converse effect. Axons which were thickly myelinated by Schwann cells were either hypomyelinated or lacked myelin as they entered or left the CNS (Figs. 5 and 6). Visual inspection and preliminary counts of glial nuclei in transverse sections of cord and optic nerve did not suggest any paucity in total glial numbers. Both astrocytes and oligodendrocytes were identified on tight-microscopical and EM observations. Neurones appeared normal. There was no overt evidence of myelin

A

B

C Fig. 2. Areas of the cervical ventral columns from l-month-old (A) and 2-month-old (B) affected pups and a 2-month-old (C) normal littermate. There is severe hypomyelination in both (A) and (B) but the quantity of myelin has increased between I and 2 m o n t h s of age. The axonal diameters have also increased and appear similar between the 2-month-old affected and normal pups. Toluidine blue, x 500.

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Fig. 3. Optic nerves from affected (A) and normal (B) 2-month-old littermates showing hypomyelination. The degree of myelination in the affected optic nerve is less than in the spinal cord (Fig. 2B). Toluidine blue, × 300.

breakdown and macrophages were absent. Small vacuoles which sometimes coalesced were present adjacent to fibres and occasionally in glial (probably oligodendroglial) cytoplasm (Fig. 4). Their frequency was greater in the 1-month than in the 2-monthold pup and they were not seen in normal animals. No abnormality was seen in the thoracic or a~dominal viscera with the exception of a moderate thymic atrophy. No canine distemper virus was identified in the brain by immunofluorescence and a cytopathic effect was not seen in tissue culture.

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Fig. 4. Longitudinal sections o f spinal white matter. A : N u m e r o u s short hypomyelinated internodes terminating with heminodes are present, some of which are indicated (arrows). Toluidine blue, × 350. B: A single axon shows consecutive internodes with different degrees of myelination. Toluidine blue, × 400. C: N u m e r o u s vacuoles are present adjacent to axons and also with a glial cell, probably an oligodendrocyte (arrow). Toluidine blue, × 400.

DISCUSSION

This report describes a family of Springer Spaniel dogs with a primary disorder of myelination of the CNS. Myelination of the canine CNS has not been studied in detail but Fox et al. (1967) using celloidin sections reported "relative maturity of myelin" in the spinal cord at 6-10 weeks of age. Our preliminary EM studies of normal 2-month-old pups demonstrate some immaturity as indicated by occasional uncompacted lamellae and large lateral loops at paranodes. However, the well developed myelin of the normal pups contrasts markedly with the hypomyelinated white matter of affected dogs. Disorders of myelination, predominantly of an infectious or inherited origin

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Fig. 5. A: Dorsal nerve root entry zone showing the dome of amyclinated/hyponlyelinated area of oligodendroglial territory which extends out into the dorsal root. Toluidine blue, × 1t0. B: Single fibre showing the transition from Schwann cell myelin (left) to non-myelination as it enters the cord. Toluidine blue, x 350.

are common in animals (for review see Hogan 1977). Affected animals invariably show some form of involuntary movement, usually a tremor, and it appears likely that this results from the myelin abnormality. Breeding studies show that the present disorder is also inherited but with the limited numbers of animals it is impossible to be categorical about the mode of inheritance. The male bias fits well with a sex-linked recessive gene carried by the dam but at present the relationship between affected pups and their sex is not statistically significant. It does appear, however, that the inheritance is unlikely to be a single autosomal recessive gene as the distri-

431

Fig. 6. A : Ventral nerve root entry zone showing the normal inward extension of Schwann cell myelination and the hypomyelinationof the intramedullary portions of motor axons. Toluidine blue, x 110. B : Normal Schwann cell myelination of a motor axon (left) is contrasted with the hypomyelinatedintramedullary portion. Toluidine blue, × 1200. bution of affected pups is significantly different from that which would be expected. Many similar disorders in other species are known to be sex-linked, for example, Jimpy mice (Sidman et al. 1964), myelin deficiency of Wistar rats (Csiza and De Lahunta 1979) and congenital tremor type A I I I in pigs (Harding et al. 1973). Despite the frequency of these disorders in other species, they are uncommon in dogs. A single case report (Van den Akker 1958) described a male Spaniel pup with clinical signs similar to the present dogs although the animal had survived to 5 months at the time of destruction. Patchy deficiency of myelin was noted in the CNS of this dog but the more detailed morphology was not recorded. Generalised dysmyelination of the CNS in Chow dogs has also been described (Vandevelde et al. 1978), although certain tracts such as the fasciculi proprii were relatively better

432 myelinated. The neurological signs in these animals (including tremor) improved with age and eventually disappeared. It was postulated that a defect in gliat differentiation might underlie this condition. The CNS myelin defect in our cases is not as severe as that in Jimpy mice and is more like that of quaking mice which has an autosomal recessive inheritance. However, the PNS was normal in these dogs unlike quaking mice in which a myelin defect (less severe than in the CNS) is present (Samorajski et al. 1970; Suzuki and Zagoren 1977). The morphological appearance is also similar to the CNS hypomyelinating disorder of pigs, congenital tremor type AIII (Blakemore et al. 1974). These observations allow speculation on where the deficit lies in this new mutant. The normal myelination of the peripheral motor and sensory neurones by Schwann cells, compared to the marked hypomyelination of their intramedullary or intracerebral portions, indicates that the axonal "signal" which instructs the sheath cell to produce myelin (Aguayo et al. 1976) is normal. The axonal diameter within the CNS does not appear responsible for the defect as there is increase in axonal calibre with age and ensheathment and myelination is not confined to a particular axonal size. Preliminary counts of glial cells suggest that there is no gross deficit in oligodendroglial numbers. However, the variation in consecutive internodal lengths and thickness, as seen on longitudinal section, suggests that the oligodendrocytes are capable of initiating myelination but incapable of normal radial and longitudinal extension of their plasma membrane. Further evidence of possible oligodendroglial dysfunction is indicated by vacuoles adjacent to the myelin sheaths and within oligodendroglia which are similar to those reported in quaking mice (Wisniewski and Morell 1971; Watanabe and Bingle 1972) and as in the mice the frequency of vacuoles decreased with age. The absence of both macrophages and marked myelin debris indicates that this is not primarily a demyelinating disorder and that the vacuoles probably represent a breakdown of defective myelin lipids as suggested in quaking mice (Watanabe and Bingle 1972). The overall evidence from the light-microscopical studies suggests a defect in oligodendroglial metabolism with an inability to form and maintain normal myelin sheaths. This new canine model provides a further opportunity to study myelinogenesis and errors which may occur during myelin formation. The large size of the animal involved should enable useful correlative biochemical and morphological studies of the central nervous system to be carried out. ACKNOWLEDGEMENTS The authors are grateful to Miss M. Waterson for assistance with the histological preparations, to Dr. H. Thompson for performing the virological examinations and to Mr. A. Finnie for the photography.

433 REFERENCES Aguayo, A.J., J. Epps, L. Charron and G.M. Bray (1976) Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves - - Quantitative microscopy and radioautography, Brain Res., 104: 1-20. Ayres, M. M. and R. McD. Anderson (1973) Onion bulb neuropathy in the Trembler mouse - - A model of hypertrophic neuropathy (D6jerine-Sottas) in man, Acta neuropath. (Berl.), 25: 54-70. Blakemore, W.F., J. D.J. Harding and J.T. Done (1974) Ultrastructural observations on the spinal cord of a Landrace Pig with congenital tremor type AIII, Res. vet. Sci., 17:174-178. Csiza, C. K. and A. De Lahunta (1979) Myelin deficiency (md) - - A neurological mutant in the Wistar rat, Amer. J. Path., 95: 215-223. Fox, M.W., O. R. lnman and W.A. Himwich (1967) The postnatal development of the spinal cord of the dog, J. comp. Neurol., 130: 233-240. Harding, J. D. J., J.T. Done, J.F. Harbourne and F. R. Gilbert (1973) Congenital tremor type AIII in pigs - - An hereditary sex-linked cerebrospinal hypomyelinogenesis, Vet. Rec., 92 : 527-529. Hogan, H. L. (1977) Animal models of genetic disorders of myelin. In: P. Morell (Ed.), Myelin, Plenum Press, New York, NY, pp. 489-520. Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy, J. Cell Biol., 37: 137A. Samorajski, T., R. L. Friede and P. R. Reimer (1970) Hypomyelination in the quaking mouse A model for the analysis of disturbed myelin formation, J. Neuropath. exp. Neurol., 29 : 507-523. Sidman, R. L., M. M. Dickie and S.H. Appel (1964) Mutant mice (Quaking and Jimpy) with deficient myelination in the central nervous system, Science, 144:309-311. Suzuki, K. and J. C. Zagoren (1977) Quaking mouse An ultrastructural study of the peripheral nerves, J. Neurocytol., 6: 71-84. Van den Akker, S. (1958) A case of leukodystrophia in a dog, Folia psychiat, neurol, neurochir, neerl., 5 : 536-539. Vandevelde, M., K.G. Braund, T. L. Walker and J. N. Kornegay (1978) Dysmyelination of the central nervous system in the chow-chow dog, Acta neuropath. (Berl.), 42:211 215. Watanabe, I. and G.J. Bingle (1972) Dysmyelination in "quaking" mouse Electron microscopic study, J. Neuropath. exp. Neurol., 31 : 352-369. Wisniewski, H. and P. Morell (1971) Quaking mouse - - Ultrastructural evidence for arrest of myelinogenesis, Brain Res., 29 : 63 73.