Regional and Central Veterinary Laboratories, Department of Agriculture, Glenfield, New South Wales 2167, Australia. Summary. Ultrastructural findings in the ...
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Acta Neuropathol (Berl) (1986) 71:316-320
9 Springer-Verlag1986
Ultrastructural findings in maple syrup urine disease in Poll Hereford calves* P. A. W. Harper, P. J. Healy, and J. A. Dennis Regional and Central Veterinary Laboratories, Department of Agriculture, Glenfield, New South Wales 2167, Australia
Summary. Ultrastructural findings in the nervous systems of two Poll Hereford calves affected with maple syrup urine disease or branched chain ketoacid decarboxylase deficiency are described. The calves were affected within 2 days of birth with a severe generalised central nervous system (CNS) disorder characterised by dullness and weakness, progressing to recumbency and opisthotonus. The urine had an odour of burnt sugar. Analysis of plasma and cerebrospinal fluid demonstrated significantly elevated levels of the branched chain amino acids leucine, isoleucine and valine. Status spongiosus affecting mainly the white matter was recorded at microscopic examination of the CNS, with ultrastructural examination confirming the presence of intramyelinic vacuole formation, suggesting myelin oedema.
Key words: Maple syrup urine disease - Cattle Branched chain ketoacid decarboxylase deficiency Status spongiosus - Myelin oedema
Maple syrup urine disease ( M S U D ) in m a n is caused by an autosomal recessive error of catabolism of the branched chain amino acids (BCAA) leucine, isoleucine and valine. Cases c o m m o n l y present in the neonatal period with an overwhelming illness (Naughten et al. 1982; Tanaka and Rosenberg 1985). In the classical f o r m of the disease, persistent and dramatic increases in plasma BCAA's and branched chain ketoacids are observed. Most patients will die if untreated and survivors are frequently mentally retarded and fail to thrive. Spongy change in the central * Supported in part by a Wool Research Trust Fund postgraduate scholarship to one of us (P.A.W.H.) Offprint requests to." P. A. W. Harper (address see above)
nervous system (CNS) attributed to oedema and failure of myelination, have been recorded at autopsy (Silberman et al. 1961; Diezel and Martin 1964). We have recently recorded M S U D in calves associating a neonatal neurological disease with lesions o f CNS spongy change and elevated ratios of the BCAA's (Harper et al. 1986). This paper presents clinical, pathological, ultrastructural and biochemical findings in two confirmed cases of bovine M S U D .
Case Histories Calf 1 was a female calf presented at 2 days of age in right lateral recumbency with opisthotonus. Its hindlimbs were extended with reduced muscle tone. The calf was dull and responded poorly to tactile and auditory stimuli. When lifted to a standing position the calf was weak, could not support its weight unaided, and opisthotonus developed within 5 seconds of lifting. When returned to recumbency, the calf frequently developed disorganised paddling of all limbs. Calf 2 was a male Poll Hereford calf born overnight and was considered to be normal on day 1. The calf was from herd 2 and the result of mating of the son of Bull C with a granddaughter of Bull C (Harper et al. /986). On day 2 the calf was recumbent with its neck arched in opisthotonus. It was dull and weak, requiring assistance to achieve sternal recumbency or to stand. When assisted to its feet the calf could maintain an unsteady wide-based posture, with its head lowered and swaying for up to several minutes. Its condition deteriorated and by day 3 the calf was unable to support its weight unaided, collapsing to recumbency, frequently followed by disorganised limb paddling and opisthotonus. On day 4 the calf was moribund, in a decerebrate posture with neck arched in opisthotonus and the limbs extended and hypertonic with periodic muscle tremors. A continuous slow rotational nystagmus was present and all spinal reflexes including pain perception were severely diminished. The urine of both calves had a variable usually mild, bitter sweet odour of burnt sugar and was tested positive for ketoacids by the 2,4-dinitrophenylhydrazine test (Dancis and Levitz 1978). Cerebrospinal fluid collected by lumbosacral puncture from calf 1, and plasma from both calves were analysed by HPLC methods (Harper et al. 1986). Significant elevations of lencine, isolencine and valine were demonstrated in both cases when compared to 6 normal calves (Table 1).
P. A. W. Harper et al.: Bovine maple syrup urine disease
317
Table 1. Branched chain amino acid concentrations (~tM) in cerebrospinal fluid and plasma of maple syrup urine disease affected calves compared with six normal calves Amino acid
Cerebrospinal fluid
Plasma
Calf1
Calf/
Nor- (n real =6) calves SD mean
Calf2 Nor- (n mal =6) calves SD mean
Valine 457.79 3.12 1.82 1 6 0 8 5608 Isoleucine 154.:13 0.20 0.14 480 3071 Leucine 577.50 1.64 1.58 :1836 9383
214 56 89
32 13 18
Materials and methods Calf 1 was estimated to be 40 h old and calf 2 was considered to be 84 h old when killed. The calves were killed by exsanguination following intravenous barbituate anaesthesia and necropsied with the head sectioned along the midline, the brain blotted dry and weighed. The claw pads were worn and the abomasum contained milk clots in both cases, indicating that the calves had walked and sucked. From both calves, sections of the brain, spinal cord, eye, sciatic nerve, skeletal muscle, heart, lung, liver, kidney, spleen, thymus, adrenals, thyroid, mesenteric lymph nodes, small and large intestine, rumen, reticnlum, omasum and abomasum was fixed in 100% neutral buffered formalin and embedded in paraffin for light microscopy. Histological sections of neural tissues were routinely stained with haematoxylin and eosin (HaE) and luxol fast blue periodic acid Schiff (LFB-PAS), and selected sections by LFBHolmes Silver (LFB-Ag), Kluver-Barrera (KB) and phosphotungstic acid haematoxylin (PTAH) methods. Frozen sections of cerebral cortex, cerebellum and spinal cord were stained by Oil Red O. Portions of the cerebral cortex, ~aaidbrain, cerebellum, medulla oblongata, lumbar spinal cord, and sciatic nerve were collected immediately following exsanguination of both calves and fixed in chilled 3.0% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The tissue was postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer pH 7.1, then treated with uranyl acetate, dehydrated in an acetone series and embedded in epon. One micrometre sections were cut on an LKB-IV ultramicrotome, stained by toluidine blue and examined by light microscopy for screening. Ultrathin sections of selected areas were stained with uranyl acetate and lead citrate and examined in a Philips EM 300 electron microscope.
Results The brain of both calves appeared swollen, of increased wetness, and there was dilation and flattening of the cortical gyri, and narrowing of the sulci. The wet brain weight was 228 grams for calf1 and 226 grams for calf 2. A focal 1.5-cm-diameter area of ulceration o f the a b o m a s a l mucosa adjacent to the pylorus was noted in calf 1, and there was moderate atrophy of the fat reserves in calf 2. In both calves there was a severe spongy appearance or "status spongiosus" throughout the
Fig. 1. Cerebellum; diffuse severe status spongiosus of the white matter. Calf 2; PTAH, x 70
CNS, m o s t prominent in m a j o r white matter (WM) tracts of the cerebellum (Fig. 1) and cerebrum. In the cerebellum vacuolation was confined to the W M , but extended into the deeper grey matter ( G M ) layers of the cerebrum, and appeared to be associated with the presence of myelin. Severe vacuolation was present in the G M and adjacent W M of the spinal cord, midbrain and brain stem. The sponginess was attributed to variable but mainly large vacuoles, occasionally up to 200 gm in diameter, that compressed the adjacent neuropile. The vacuoles did not stain with PAS or Oil Red O methods. The web-like appearance of the vacuolated W M produced a rarefied appearance to the myelin and toluidine blue-stained sections o f e p o n embedded material demonstrated that the vacuoles were bounded by thin myelin lamellae, and were frequently crossed by myelin lamellar remnants. Increased n u m b e r or alterations in the m o r p h o l o g y of glial cells was not observed. In calf 2, an occasional swollen eosinophilic oval axon cylinder resembling an axonal "spheroid" was noted in the cerebellar W M tracts. Myelin loss was not observed. Ultrastructurally, multiple i n t r a m y e l i n vacuoles were present (Fig. 2), forming large interlamellar spaces. The vacuoles were due to splitting of the myelinic intraperiod line between the m a j o r dense lines. The majority of vacuoles were formed by separation of the outer lamellae (Fig. 3), however, midlamellar and occasional myelin-axon vacuoles were
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P. A. W. Harper et al.: Bovinemaple syrup urine disease
Fig. 2. Mid-brain; multiplevacuolationdue to enlargedmyelin interlamellar spaces. Calf 1; x 5770
Fig. 4. Brainstem; interlamellar myelin vacuole formation containingmyelindebris. Calf 1; x 12400
occasional myelinated axon was particularly prominent due to the common occurrence of outer lamellar vacuole formation, with sparing of the unmyelinated axons. Vacuole formation, clumping of organelles and mitochondrial swelling were observed in the cytoplasm of oligodendrocytes. No abnormalities of blood vessels were noted, and no myelin macrophages or swollen vacuolated astrocytes were observed.
Discussion
Fig. 3. Brainstem; separation of the outer lamella of the myelin sheath. Calf 1; x 42900
observed. Vacuoles were frequently traversed by single or multiple lamellae or contained myelin whorls or disintegrated myelin debris (Fig. 4). Some vacuoles appeared to have disrupted the myelin sheath completely. In areas dominated by unmyelinated axons, such as the deep cerebral cortical GM, the
The pathological findings indicate that the severe CNS status spongiosus observed in cases of bovine MSUD is due to splitting of the myelin sheath at the intraperiod line. "Status spongiosus" is a term applied to the seive-like appearance of neural tissue. It occurs in various neurological disorders (Adornato and Lampert 1971) and is not considered to represent a specific CNS lesion (Jellinger and Seitelberger 1970). The lesion has been reported in naturally occurring idiopathic diseases in cats (Kelly and Gaskell 1976), dogs (Zachary and O'Brien 1985; O'Brien and Zachary 1985), man (Adachi et al. 1973) and calves (Cordy et al. 1969), although it has been suggested that at least some of the bovine cases may have been MSUD (Harper et al. 1986). Status spongiosus characterised by the formation of intra-myelin vacuoles due to splitting of the myelin
P. A. W. Harper et al.: Bovine maple syrup urine disease lamellae at the intraperiod line is considered to indicate myelin oedema. This spongy myelinopathy has been described in a n u m b e r of experimental intoxications in animals including: a c t i n o m y c i n D (Rizzuto and G a m b e t t i 1976), cycloleucine (Greco et al. 1980), cuprizone (Suzuki and K i k k a n a 1969), hexachlorophene (Lampert et al. 1973), triethyl-tin (Suzuki 1971), isonicotinic acid hydrazide (Lampert and Schochet 1968), ethidium bromide (Yajima and Suzuki 1978), a m m o n i a (Cho and Leipold 1977; H o o p e r et al. 1974), and the plant Stypandra imbricata (Huxtable et al. 1980). Status spongiosus has been recorded in M S U D (Silberman et al. 1961; Diezel and Martin 1964), homocystinuria (Chou and Wais:man 1965), phenylketonuria and other aminoacidurias of m a n (Martin and Schlote 1972), although the fine structural studies have yet to be done (Greco et al. 1980). These aminoacidopathies are characterised by severe neurological s y m p t o m s and spongy myelinopathy, and it has been suggested that some o f the neurological abnormalities are due to the obvious structural damage of myelin sheaths (Agamanolis et al. 1982). The pathogenesis of myelin splitting is unresolved. F r o m experimental studies it would appear that of the possible mechanisms that could be involved in the development of myelin oedema, an interference in the enzymes necessary for electrolyte transport across cell m e m b r a n e s is m o s t likely to account for the destabilisation of myelin and formation of fluid-filled c o m p a r t m e n t s without changes in vascular permeability (Greco et al. 1980; L a m p e r t et al. 1973; Johnson 1976). A toxic effect directly on tile myelin sheath or mediated via the oligodendrocyte m a y account for the apparent failure in the integrity o f the central myelin sheath. The "toxin" involved in M S U D is unresolved (Dancis and Levitz 1978), however it is considered to be a metabolite resulting f r o m the oxidative decarboxylation of alpha-ketoacids (Stumpf et al. 1976).
Acknowledgements. The advice of Drs. Gary Brown, John Pollard, Clive Harper, Bill Hartley and Ross Annisson are gratefully acknowledged. The technical assistance of Ms Doreen Debono, Mr. Steve Wilson and staff of the department of Agriculture at CVL Glenfield, BCRI Rydalmere and the JMAI Camden park, is most appreciated.
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