CENTRAL PONTINE MYELINOLYSIS FOLLOWING SALINE TREATMENT OF A DIABETIC RAT FOR DEHYDRATION. J.R. WRIGHT, Jr., A.J. YATES, H.M. SHARMA and P.

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1 J. COMP. PATH VOL CENTRAL PONTINE MYELINOLYSIS FOLLOWING SALINE TREATMENT OF A DIABETIC RAT FOR DEHYDRATION J.R. WRIGHT, Jr., A.J. YATES, H.M. SHARMA and P. THIBERT BY The Ohio State University College of Medicine, Department of Pathology, Divisions of Neuropathology and Anatomical Pathology, Columbus, Ohio 43210, U.S.A. and Animal Resources Division, Health Protection Branch, Health and We&Tare, Canada, Ottawa, Ontario, Canada KIA OL2 INTRODUCTION The BB Wistar (BBW) rat, a promising new model for human juvenile-onset diabetes mellitus, was derived from an outbred line of Wistar rats at the Bio- Breeding laboratories in Ottawa, Canada (Nakhooda, Like, Chappel, Murray and Marliss, 1977). The diabetic syndrome in BBW rats is characterized by hyperglycaemia, glycosuria, ketoacidosis, insulinopenia, glucagonaemia, and hyperlipaemia (Nakhooda et al., 1977; Nakhooda, Like, Chappel, Wei and Marliss, 1978). Spontaneous diabetes occurs in 30 to 55 per cent of the offspring of diabetic matings. The onset is abrupt with rapid weight loss due to dehydration as a result of polyuria. If insulin therapy is not initiated immediately, death occurs within 1 to 2 weeks. Usually, the rats are easily maintained on insulin for 6 months to 1 year after onset of diabetes. However, at this point maintenance often becomes difficult; the rats lose weight rapidly (up to 25 to 40 per cent over a few weeks), become dehydrated and emaciated, and glycosuria disappears (unpublished observation). Ten of these rats were treated with subcutaneous injections of normal saline and their insulin treatment was discontinued. Despite therapy the animals died. One such casualty is the basis of this brief report. MATERIALS AND METHODS A male BBW insulin-dependent diabetic rat, 333 days old at the time of death, had been progressively losing weight over several weeks, and became severely dehydrated. The animal received a total of 15 ml of normal saline (0.9 per cent NaCl) as 2 to 3 ml subcutaneous injections per day during the week before its death in an attempt to rehydrate it. The rat died despite therapy (238 days after the onset of diabetes). At necropsy, the rat was thin, emaciated, and weighed only 255 g (normal is approximately 450 g) at death. A drop of blood placed on a Dextrostix (Ames Co., Elkhart, Indiana) reagent strip was negative for sugar. A complete gross post-mortem examination revealed only testicular atrophy, a common finding in older rats of this strain (Wright, Yates, Sharma, Shim, Tigner and Thibert, 1982). The brain appeared grossly normal and was of normal weight (l-67 g) _ T issue specimens from all organ systems were fixed in neutral phosphate buffered formalin, and processed for light microscopy. Sections of each organ were stained with haematoxylin and eosin (HE) ; /83/ $03.00/O Academic Press Inc. (London) Limited

2 510 J. R. WRIGHT &?t d. sections of brain were also stained with 1~x01 fast blue and Bodian s stain for axons. All organs were histologically normal except the testes (which were atrophic) and the brain. A lesion in the pons involved most of the basal portion and extended into the tegmentum and the base of the middle cerebellar peduncles (Fig. 1). The myelin in Fig. 1. Transverse section of pons and cerebellum show-ing marked pallor of my&n in basal region and tegmentum. Note the margin of normal myelin (arrow). Luxol fast blue. x 10. this area was almost completely destroyed and macrophages were common (Fig. 2). Necrotic cells with nuclei demonstrating pyknosis and karyorrhexis were scattered throughout the lesion but no oligodendrocytes could be identified with certainty. There were a few cells with large nuclei and a moderate amount of granular eosinophilic cytoplasm which may have represented degenerating neurons; however, neurons were numerous and a few axons traversed even the most damaged area (Fig. 3). There were no reactive astrocytes or evidence of either acute or chronic inflammation and the vessels were normal. One small focus with similar histological features was seen in the corpus callosum. Nine other rats were treated for dehydration with subcutaneous normal saline, but none of these received more than 9 ml. After a single dose of saline (2 to 8 ml) 4 died within 1 day and 1 within 2 days; 3 lived for 3 to 6 weeks after such treatment. One other received 9 ml normal saline over the last 4 days of life. Brains were examined histologically in all of these rats, but none had evidence of central pontine myelinolysis. DISCUSSION Central pontine myelinolysis (CPM), a rare disease of unknown aetiology, was first reported by Adams, Victor and Mancall ( 1959). Since then, over 150

3 PONTINE MYELINOLYSIS 511 Fig. 2. Central pans with macrophages and several unidentifiable necrotic cells showing karyorrhectic and pyknotic nuclei. Luxol fast blue. x 630. Fig. 3. Central pans with axons traversing the area. Bodian s silver stain. x 630. human cases have been reported (Wright, Lauren0 and Victor, 1979). The typical lesion is a singie symmetrical focus of grey softening in the centre of the upper and middle pons. Histologically, there is a sharply defined area of myelin breakdown with relative sparing of nerve cells and axons, although some axonal degeneration and nerve cell loss may be present in the very centre

4 512 J. R. WRIGHT& al. of the lesion. Macrophages containing sudanophilic lipids and reactive astrocytes may be numerous within the lesion, but oligodendrocytes are absent. Neither vascular disease nor inflammation is seen in CPM and therefore cannot be involved in the pathogenesis (Oppenheimer, 1976). The histological findings in this case are similar to those seen in human cases of CPM. In man, CPM is frequently associated with alcoholism and malnutrition. Recently, electrolyte abnormalities have been implicated in the pathogenesis of CPM (Wright et al., 1979). In fact, many investigators now believe that it is an iatrogenic disorder due to the rapid correction of hyponatraemia (Finlayson, Snider, Olivia and Gault, 1973 ; Tomlinson, Pierides and Bradley, 1976 ; Leslie, Robertson and Norenberg, 1980; Pogacar, 1980). Some evidence suggests that slower correction of hyponatraemia does not lead to CPM (Leslie et al., 1980). This pattern is consistent with the clinical course (i.e. rapid correction of dehydration) seen in this BBW rat prior to its death. A recent study by Kleinschmidt-DeMasters and Norenberg (1982) has shown that CNS demyelination can be induced by fluctuations in the serum electrolyte concentrations in rats. In that study, rats were made hyponatraemic by the administration of vasopressin and water for 3 days and then treated with hypertonic saline. Rats were killed 5 days later. They reported demyelination with sparing of neurons and axons in regions of the brain with a rich admixture of grey and white matter. Sites of lesions included corpus striatum, claustrum, external capsule, cerebral neocortex, anterior commissure, hippocampus and its fimbria, thalamus, periaqueductal grey, mid-brain reticular formation, red nucleus, and brainstem. In severely affected rats, neuronal loss and necrosis were present in the centre of the lesions. Although the lesions produced in that study are histologically similar to CPM, they are diffuse rather than localized to the pons as in our case. CPM has been reported in man several months after the onset of clinical diabetes and concurrent with fluid therapy for dehydration (Behar, Bental and Aviram, 1964). We can only speculate as to why CPM might occur in this BBW diabetic rat and yet has not been produced experimentally or observed incidentally in other animals. First, CPM is more common in chronic nutritionally deficient individuals (Oppenheimer, 1976)) and poorly controlled diabetes mellitus is such a condition. The emaciation and dehydration that frequently precede the death of BBW diabetic rats may set the stage for the development of CPM. Secondly, the diabetic state causes additional osmotic stress (Aloia and Nilakantan, 1973). We postulate that the combination of these conditions followed by rapid treatment with relatively large doses of saline caused CPM in this case. Nine other diabetic rats which did not develop CPM were treated with normal saline injections but none received as much saline as this animal, and their durations of therapy were all shorter. This suggests that both the dosage schedule of saline and the survival time following initiation of therapy may be critical. It is probable that many factors were involved in this incidental finding in a diabetic rat, but the significance of this report is that CPM has not, so far, been experimentally induced in animals (Oppenheimer, 1976). This study suggests that this can be done.

5 PONTINE MYELINOLYSIS 513 SUMMARY Central pontine myelinolysis (CPM) was observed at necropsy in an emaciated 333~day-old BB Wistar insulin-dependent diabetic rat that had been treated for dehydration by subcutaneous injection of normal saline. There was a large area of nearly complete demyelination involving most of the basal portion of the pons, part of the tegmentum, and the base of the middle cerebellar peduncles. There was a relative sparing of neurons and axons, but some unidentifiable large cells with degenerative changes in the cytoplasm were present. No oligodendrocytes or reactive astrocytes were seen and there was no evidence of inflammation or infarction. Although diffuse demyelination can be induced in rats by rapid fluctuation of serum electrolytes, no studies have experimentally induced central pontine myelinolysis nor has it been observed incidentally in animals previously. ACKNOWLEDGMENTS This study was supported in part by the Department of Pathology, the College of Medicine, and the Graduate School of the Ohio State University, by the Upjohn Company, and by NIH Grant NS J. R. Wright was supported by the Samuel J. Roessler Research Scholarship. REFERENCES Adams, R., Victor, M., and Mancoll, E. L. (1959). Central pontine myelinolysis. A hitherto undescribed disease occurring in alcoholic and malnourished patients. Archives of Neurology (Chicago), 81, 154-l 72. Aloia, J. E., and Nilakantan, S. (1973). The cerebrospinal fluid in uncontrolled diabetes mellitus. American Journal of the Medical Sciences, 266, Behar, A., Bentla, E., and Aviram, A. (1964). Central pontine myelinolysis (A case report). Acta fleuropathologica, 3, Finlayson, M., Snider, S., Oliva, L. A. and Gault, M. (1973). Cerebral and pontine myelinolysis. Two cases with fluid and electrolyte imbalance and hypotension. Journal of the Neurological Sciences, 18, Kleinschmidt-DeMasters, B. K., and Norenberg, M. D. (1982). Neuropathologic observations in electrolyte-induced myelinolysis in the rat. Journal of NeuropatholoD and Experimental fleurology, 41, Leslie, K. O., Robertson, A. S., and Norenberg, M. D. (1980). Central pontine myelinolysis: an osmotic gradient pathogenesis. Journal of Neurojathology and Experimental Neurology, 39, 370. Nakhooda, A. F., Like, A. A., Chappel, C. I., Murray, F. T., and Marliss, E. B. (1977). The spontaneously diabetic Wistar rat. Metabolic and Morphologic studies. Diabetes, 26, 100-I 12. Nakhooda, A. F., Like, A. A., Chappel, C. I., Wei, C. N., and Marliss, E. B. (1978). The spontaneously diabetic Wistar rat (the BB rat). Studies prior to and during development of the overt syndrome. Diabetologia, 14, Oppenheimer, D. R. (1976). Demyelinating diseases. In Green.zeld s Neuropathology. W. Blackwood and J. Corsellis, Eds, Edward Arnold, London, pp Pogacar, S. (1980). Iatrogenic precipitation of central pontine myelinolysis with formation of Rosenthal fibers. Journal of Neuropathology and Experimental NeuroloD, 39, 383. Tomlinson, B., Pierides, A., and Bradley, W. (1976). Central pontine myelinolysis.

6 514 J. R. WRIGHT et cd. Two cases with associated electrolyte disturbance. Quarterly Journal of Medicine (New Series), 40, Wright, D. G., Laureno, R., and Victor, M. (1979). Pontine and extrapontine myelinolysis. Brain, 102, Wright, J. R., Yates, A. J., Sharma, H., Sh im, C., Tigner, R. L., and Thibert, P. (1982). Testicular atrophy in spontaneously diabetic BB Wistar rats. American Journal of Pathology, 108, [Received for publication, July 16th, 19821