Case report

Vacuolation and chromatolysis of lower motoneurons in tetanus

A case report and review of the literature

In recent years, the pathophysiological action of tetanus toxin has been extensively studied by many investigators.1,2 The role of the toxin in decreasing Samuel M. Chou, M.D., Ph.D. inhibitory input to alpha motoneurons is well known. Morphological alterations in neurons in Department of Pathology both clinical and experimental tetanus have not been consistently described. Indeed, several authors William N. Payne, M.D.* simply state that no changes are found. There are, however, reports of nuclear capping, and either chromophobia or chromophilia3 as well as central or perinuclear chromatolysis in motoneurons4,5 in tetanus. The finding of striking concurrence of cen- tral chromatolysis and vacuolation confined to mo- toneurons in a case of human tetanus prompts this report and a review of the literature.

Case report A 58-year-old black man was admitted because of inability to swallow. The night before admission, he choked while trying to drink water; he also noted pain and stiffness in the neck and back. He was diabetic and took 40 units of insulin each morning. He had had an ulcer on his left heel for the preceding 10 months, but there was no history of recent wounds. He had not had a tetanus immunization for "many years." Chronic ulcers on the right leg had necessitated an above-the-knee am- putation l'/2 years before. The patient also had been * Section of Neuropathology, West Virginia taking phenytoin (Dilantin) and phenobarbital for a University Medical Center, Morgantown, West Virginia 26506. Supported in part by a grant from the Muscular Dystrophy Association.

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Downloaded from www.ccjm.org on September 24, 2021. For personal use only. All other uses require permission. 256 Cleveland Clinic Quarterly Vol. 49, No. 4 seizure disorder, which apparently followed dition of the patient after withdrawal of a stroke for which he had had a carotid curare. Curare was stopped and atropine bypass. and neostigmine (Prostigmin) were given. The patient initially went to the emer- The patient became bradycardic, developed gency room of a local hospital and was found atrioventricular block as well as asystole and to have risus sardonicus, opisthotonus, and died. dysphagia; the clinical impression was early tetanus. He was transferred to West Virginia Autopsy findings Medical Center. Autopsy was performed three hours Physical examination revealed marked after death. The right lower leg of this opisthotonus and masseter muscle spasm to obese, well-developed black man had the degree that the mouth could not be opened. The patient was moderately irrita- been surgically amputated, and the ble and exhibited muscle spasms when stump was well healed and clean. An touched. There was rigidity and increased ulcerative wound measuring 3X4 cm tone of the paraspinal muscles. Examination was present over the lateral left heel. of the heart, lungs, and abdomen was unre- The heart weighed 400 g; examination markable. A 2 X 1-cm ulcer was found on revealed severe arteriosclerotic changes the lateral aspect of the left heel; there was of the coronary arteries, focal athero- increased muscle tone in the left leg. The matous segments narrowing the lumen. right leg demonstrated good stump healing A proximal segment of the left circum- with no ulceration. were in- flex coronary artery was completely oc- tact with pupils equal and reactive; there cluded by two fresh brown thrombi. The was no papilledema. Sensory examination revealed decreased light touch and vibration posterior wall of the left ventricle dis- sense in the lower extremities; position sense played patchy areas of recent hemor- was normal. Deep tendon reflexes were nor- rhagic infarction. The aorta, however, mal and symmetrical; there was no Babinski had only mild atherosclerotic changes. sign on the left. The left lung showed marked consoli- Laboratory studies on admission revealed dation with microscopic findings of an elevated creatine phosphokinase (CPK) acute and subacute bronchopneumonia. of 700 U/L (normal, <125 U/L) that rose to Marked hepatosplenomegaly was pres- 1629 U/L five days after admission. The ent (liver, 23,500 g; spleen 650 g); mi- white blood cell count (WBC) was 11,200/ croscopically moderate chronic conges- mm3 with a left shift; hemoglobin and he- tion was noted. The gastrointestinal sys- matocrit were 13.8 g/dl and 43%, respec- tem was unremarkable. The kidneys tively. Serum electrolytes were normal; glu- showed the typical features of nodular cose was 70 mg/dl. Lumbar puncture re- vealed 880 erythrocytes, 3 leukocytes, and a intercapillary glomerular sclerosis char- glucose of 65 with a peripheral glucose of acteristic of Kimmelstiel-Wilson disease. 100. This was interpreted as a normal trau- The pancreas was both grossly and mi- matic tap. Culture of the ulcer on the left croscopically unremarkable, as were the heel grew Clostridium tetani. The patient was rest of the viscera. initially treated with 5000 units of tetanus Neuropathological findings. The brain immune globulin (Hyper-Tet) and intrave- weighed 1075 g, reflecting mild gener- nous penicillin (one million units every four alized cortical and focal cystic hours). The wound on the left heel was debrided. Following one generalized seizure encephalomalacia (2x3 cm) in the right he was transferred to the Intensive Care Unit temporo-occipital region. The basal cer- where he was treated with curare, and main- ebral arteries showed mild athero- tained on a respirator. Nine days after ad- sclerotic changes. The brain stem and mission, it was decided to evaluate the con- the spinal cord were macroscopically

Downloaded from www.ccjm.org on September 24, 2021. For personal use only. All other uses require permission. Winter 1982 Vacuolation and chromatolysis 257 normal. Microscopically, the cortical spared (Figs. IA and 2A). No astrocytic neurons, including those of Ammon's reaction was noted in the vicinity of horn, showed no ischemic or senile affected neurons; neuronal loss was not changes. The right temporo-occipital apparent. No foci of perivascular de- cortex showed focal cystic astro- myelinative lesions or gliosis were noted. gliosis. The Betz cells in the motor strips Finely granular intravacuolar mate- appeared intact. Ascending tract degen- rials were visualized on silver impreg- eration was found throughout the spinal nation (Fig. 2A and B). Transmission cord, especially the gracilis; and the pos- electron microscopy (TEM) revealed terior spinal roots showed diffuse inter- that the vacuoles contained finely gran- stitial fibrosis. The anterior spinal roots ular or amorphous material and mem- were spared and showed no sign of mye- brane-bound vesicles, 0.5 to 1.0 ¡x in lin or axonal damage. Most striking was diameter, containing parallel helical the vacuolation concurrent with central fibrillar material (Fig. 3A and B) and chromatolysis, which was confined to flocculant densities. These vesicles were the motoneurons of both the brain stem considered to be altered mitochondria and spinal cord. The anterior horn mo- in the vicinity of vacuoles (Fig. 3C) toneurons of the spinal cord, especially where little autolytic change was seen in in the lumbar cord, were most severely other subcellular . The vacu- involved; and almost all the anterior oles themselves were seldom lined by a horn motoneurons showed honeycomb- membrane. This impression was like multiple vacuolations (Fig. 1A and strengthened on scanning electron mi- B). The vacuoles measured 4-7 /x in croscopy (SEM) of the specimens pre- diameter and often contained fine gran- pared according to the method de- ules, which were readily seen in the scribed by Hamphery et al.6 Cryofrac- Bodian stain (Fig. 2A and B). They oc- tured control motoneurons (Fig. 4A) re- curred symmetrically throughout the vealed a finely granular fractured cyto- spinal cord; the severity of involvement plasmic surface without vacuoles; the increased caudally. The symmetric dis- nucleus with its chromatin and nucleo- tribution of lesions precluded the possi- lus was clearly identifiable. The vacu- bility of a secondary alteration from the oles in the honeycombed neurons (Fig. right above-the-knee amputation. The 4B) were lined by a finely granular yet neurons in Clark's columns, intermedi- relatively smooth surface without a olateral nuclei, and posterior horns were membrane. They infrequently dis- well preserved and free of vacuoles. The played fine linear or granular contents neurons in the brain stem motor nuclei over their surfaces (Fig. 4B, arrows), were also involved to a lesser extent; which corresponded in dimension to the motoneurons of XII, VII, and V dis- vesicles seen on TEM. played similar vacuolation. The neurons of sensory V, substantia nigra, reticular Discussion formation, locus ceruleus, and pontine gray were all intact. The vacuolation In his review of central nervous sys- was often superimposed on centrally tem (CNS) pathology in tetanus, Baker4 chromatolyzed large motoneurons. cited several investigators around the Chromatolysis (Fig. 1A and B), seldom turn of the century who had noted vac- without vacuoles, was also seen in the uolation in nerve cells. Miiller and anterior horns on both sides. Small neu- Jeschke7 concluded that for induction of rons in the anterior horns were generally the vacuolation in the anterior horn cells

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Fig. 1A. Large spinal motoneurons with multiple vacuoles in a honeycomb pattern; note relative sparing of small neurons; hematoxylin and eosin stain, X 360. B. Motoneurons demonstrating both vacuolation and central chromatolysis; Kluver-Barrera stain, X 360.

(which was seen in 14 of 22 tetanus cases toneurons. Tetanus toxin may not be and in 2 of 50 cases without tetanus) essential since transient, rhythmic, and two factors, hypoxia and tetanus toxin, synchronous myoclonic contraction of should act simultaneously upon the mo- muscles secondary to an increased excit-

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Fig. 2A. Honeycomb vacuolation accentuated by Bodian's silver impregnation; X 360. B. Higher magnification of vacuoles showing fine granules within them; Bodian stain, X 1408.

ability of motoneurons is sufficient to tion and has been reported in both hu- induce vacuolation and chromatolysis.8 man and animal tetanus. Baker4 de- The finding of chromatolysis in tetanus scribed it in his 12 clinical cases and is more consistent than that of vacuola- cited other investigators who have dem-

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Fig. 3A. Round or spindle-shaped, membrane-bound bodies containing granules, cristae, and parallel helical fibrils within the vacuole, which is not membrane bound; X 35,000. B. Detail of the membrane-bound round vesicular structure demonstrating parallel helical fibrillary material inside; X 52,500. C, Lower magnification showing the suspected origin of ihe vesicle (V) from a with flocculant densities (arrow) protruding into the vacuole. Note relatively well-preserved subcellular organ- elles around the vacuole; X 18,000.

onstrated it. Tarlov9 noted chromatoly- changes indicative of ischemic necrosis. sis in local tetanus experimentally and The authors noted that the vacuolation hypothesized the causal relationship of occurred in cells in which Nissl granules this change to the continuous muscle were still present and thus probably rigidity clinically observed. either preceded or was independent of The vacuolation of nerve cells seen in chromatolysis. Clinically, the patients the present case strikingly resembles demonstrated hypotonia and exhibited that seen in 2 cases of infantile moto- no rigidity or spasms, and both patients neuron disease.1"'11 This disease was died of severe bronchopneumonia. rapidly progressive with remarkable lo- Our patient had periods of hypoxia- calization of vacuolation and central secondary to both broncho- chromatolysis in the spinal and brain pneumonia and myocardial infarction stem motoneurons, although present in shortly before death. However, the pref- Clark's column as well. As in the present erential involvement of large motoneu- case, vacuoles were often multiple rons in both the brain stem and spinal within neurons and not associated with cord without morphologic changes of

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Fig. 4. Scanning electron micrograph of cryofractured spinal motoneuron. A. Nucleus and (N) without large vacuolation in the cytoplasm of a control motoneuron. B. Multiple vacuoles with granular or linear structures appear on the inner surfaces (arrows) of a honeycombed neuron.

Downloaded from www.ccjm.org on September 24, 2021. For personal use only. All other uses require permission. 262 Cleveland Clinic Quarterly Vol. 49, No. 4 ischemic necrosis is inexplicable on the The morphological changes in infan- basis of hypoxia-ischemia alone. Indeed, tile motoneuron disease13'14 correspond the selective involvement of large mo- closely to those in the tetanus cases de- toneurons argues against acute hypoxia- scribed previously,7 as well as to the ischemia12, 13 in which smaller neurons present case, and raise the possibility are more susceptible and large motoneu- that the mechanism induced by tetanus rons are resistant. Furthermore, other toxin could be related to motoneuron hypoxia-ischemia changes such as ho- disease. Chromatolysis, basophilic mogenization, shrinkage, and incrusta- clumping, and vacuolation have been tion do not coexist with vacuolation in observed in amyotrophic lateral sclero- the anterior horn or brain stem neurons sis.19'20 The hypothesis that certain eti- of this patient; in fact, they appear free ologic agents transported to the CNS of ischemic changes. Besides, micro- via retrograde axonal transport, as is the vacuolation characteristic of earliest case in tetanus poisoning,1'2 has been neuronal change in acute hypoxia-ische- considered in motoneuron disease. The mia13 differs markedly from the vacuoles vacuoles formed in tetanus motoneurons in tetanus. The microvacuoles (diame- fail to reveal any limiting membranes ter, 0.16-2.5 n) occur in small cortical on both TEM and SEM, thus differing neurons,14 whereas the vacuoles in teta- distinctively from those described in nus are exclusively in the lower moto- wobbler mouse,21 Creutzfeldt-Jakob dis- neurons and were never seen in Am- ease,22 or distended mitochondria in an- nion's horn, Purkinje cells, inferior oli- oxic-ischemia,14 when they are mem- vary, and dentate neurons. brane or multiple-membrane bound. Reported incidence of vacuolization The intravacuolar membrane-bound is greater in more recently published vesicles appear to derive from degener- tetanus cases. This may be because tet- ating mitochondria with flocculant anus patients today live longer because densities {Fig. 3C). The diameters of of better supportive care,7 as our patient 0.5-1.0 ¡1 are compatible with those of who lived about ten days. Therefore, mitochondria, and parallel helical fibrils time would permit longer repetitive dis- have been described23'24 in mitochon- charges from lower motoneurons. For- dria associated with nutritional defi- tunately, the phenomenon of vacuoliza- ciencies. The flocculant densities in the tion antedated tetanus toxoid so the vesicles resemble those within the mito- toxoid would appear not to be a factor. chondria (Fig. 3C) and those described The electrophysiological evidence15'16 by Zacks and Sheff25 as tetanus-specific that tetanus toxin decreases inhibitory "intramitochondrial dense granules." In input to alpha motoneurons to cause fact, similar intramitochondrial gran- increased firing and tetany is convinc- ules of high calcium content are typical ing. It appears as if the vacuolation and of cells under hypoxia-ischemia of early chromatolysis indicate severe damage autolysis. These mitochondrial changes by the toxin to motoneurons. However, may reflect a metabolic stress secondary these neurons must retain sufficient to continuous hyperexcitability result- function to have the repetitive neuronal ing from disinhibition of large motoneu- discharge or hyperexcitation necessary rons induced by tetanus toxin. The for tetanus. Indeed, base line physiolog- mechanism for cytoplasmic clearing and ical properties of motoneurons17 or in- vacuole formation around the altered terneurons18 in cats with local tetanus mitochondria remains obscure. The to- have been reported as normal. pistic restriction of the vacuolar changes

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superimposing on chromatolysis in the 6. Hamphery WJ, Spurlock BO, Johnson JS. lower motoneurons is puzzling and war- Critical point drying of ethanol-infiltrated cryofractured biological specimens for scan- rants further study in tetanus-intoxi- ning electron microscopy. In: Proc 7th An- cated animal models. nual SEM Symposium, Part 1. Chicago: IIT Summary Research Inst 1974: 275-282. 7. Mtiller HA, Jeschke R. Cytoplasmatische Vacuolation and central chromato- Vacuolen in den Vorderhornganglienzellen lysis overlapped and were found selec- des Menschen beim Tetanus. Virchows Arch tively in large motoneurons of the spinal 1971; 353:87-98. 8. Shavapour E, Teasdall RD. Spinal myoclonus cord and brain stem in a patient with with vacuolar degeneration of anterior horn clinically proved tetanus. From the his- cells. Arch Neurol 1980; 37: 451-453. topathologic findings described in pre- 9. Tarlov IM. Rigidity and primary motoneuron viously described tetanus patients, the damage in tetanus. Exp Neurol 1974; 44: inference was made that these concur- 246-254. 10. Kohn R. Clinical and pathological findings in rent conditions require the prolonged an unusual infantile disease. J repetitive uninhibited discharges of the Neurol Neurosurg Psychiat 1971; 34: lower motoneurons that are induced by 427-431. the effects of tetanus toxin. Ultrastruc- 11. Reif-Kohn R, Mundel G. Second case of an ture of the vacuoles in tetanus motoneu- infantile motor neuron disease. Confin Neurol 1974; 36: 23-32. rons was compared with those of other 12. Lindenberg R. Morphotrophic and morpho- conditions; they were not membrane- static necrobiosis; investigations on nerve cells bound and contained vesicles enclosing of the brain. Am J Pathol 1956; 32: granules and parallel helical fibrils, 1147-1168. probably products of degenerated mi- 13. Brierley J. Cerebral hypoxia. In: Blackwood W, Corsellis A, eds. Greenfield's Neuropa- tochondria. Hence the vacuoles were thology. London: Edward Arnold, 1976: distinctively different from those micro- 43-85. vacuoles formed by membrane-bound 14. Brown AW, Brierley JB. Anoxic-ischaemic distended mitochondria in acute hy- cell change in rat brain; light microscopic poxia. and fine-structural observations. J Neurol Sci 1972; 16: 59-84. Acknowledgment 15. Semba T, Kano M. Glycine in the spinal cord of cats with local tetanus rigidity. Science This research was carried out during Dr. 1969; 164: 571-572. Payne's postdoctoral fellowship from the 16. Curtis DR. The pharmacology of spinal post- Muscular Dystrophy Association. synaptic inhibition. Progr Brain Res 1969; 31: 171-189. References 17. Wiegand H, Wellhoner HH. Electrical excit- 1. Bizzini B. Tetanus toxin. Microbiol Rev 1979; ability of motoneurons in early local tetanus. 43: 224-240. Naunyn Schmiedebergs Arch Pharmacol 2. Habermann E. Tetanus. In: Vinken PJ, 1979; 308: 71-76. Bruyn GW, eds. Handbook of Clinical Neu- 18. Benecke R, Takano K, Schmidt J, Henatsch rology. Amsterdam: North Holland 1978: HD. Tetanus toxin induced actions on spinal 491-547. Renshaw cells and la-inhibitory interneurons 3. Foster JA, Matzke HA. The neurocytology of during development of local tetanus in the experimental ascending tetanus. World Neu- cat. Exp Brain Res 1977; 27: 271-286. rol 1961; 2: 22-35. 19. Chou SM. Pathognomy of intraneuronal in- 4. Baker AB. Medullary involvement in tetanus. clusions in ALS. In: Tsubaki T, Toyokura Y, Am J Pathol 1943; 19: 709-717. eds. Amyotrophic Lateral Sclerosis. Tokyo: 5. Tarlov IM, Ling H, Yamada H. Neuronal Univ Tokyo Press, 1979: 135-176. pathology in experimental local tetanus; clin- 20. Hirano A, Iwata M. Pathology of motor neu- ical implications. Neurology 1973; 23: 580- rons with special reference to amyotrophic 591. lateral sclerosis and related disease. In: Tsu-

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baki T, Toyokura Y, eds. Amyotrophic Lat- Hepatic changes associated with chronic al- eral Sclerosis. Tokyo: Univ Tokyo Press, 1979: coholism in rats. Lab Invest 1965; 14: 107-132. 1437-1455. 21. Andrews JM. The fine structure of the cervi- 24. Svoboda D, Higginson J. Ultrastructural cal spinal cord, ventral root and brachial changes produced by protein and related de- nerves in the wobbler (wr) mouse. J Neuro- ficiencies in the rat liver. Am J Pathol 1964; path Exp Neurol 1975; 34: 12-27. 45: 353-379. 22. Chou SM, Payne WN, Gibbs CJ Jr, Gajdusek 25. Zacks SF, Sheff MI. Studies on tetanus toxin. DC. Transmission and scanning electron mi- 1. Formation of intramitochondrial dense croscopy on spongiform change in Creutz- granules in mice acutely poisoned with teta- feldt-Jakob disease. Brain 1980; 103: 885-904. nus toxin. J Neuropath Exp Neurol 1964; 23: 23. Porta EA, Ilartroft WS, de la Iglesia FA. 306-323.

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