VIRUS-SIMULATING STRUCTURES IN T H E O P T I C NERVE HEAD IN CREUTZFELDT-JAKOB DISEASE A L A N M. R O T H , M.D., W I L L I A M G. E L L I S , M.D.,
J O H N L. K E L T N E R ,
M.D.,
AND M A N U E L A M A R T I N S - G R E E N , Davis,
Creutzfeldt-Jakob disease is a progres sive, fatal, degenerative disease of the central nervous system in man which, because of its typical pathologic lesions and its transmissible nature, has been included in the classification of subacute spongiform virus encephalopathies 1 or slow virus infections together with Kuru in man, scrapie in sheep, and transmissible encephalopathy in mink. Creutzfeldt-Jakob disease was first trans mitted to laboratory animals in 1968 2 by intracerebral injection of affected human brain and since has been transmitted with serial passages to a variety of species. Of particular interest to ophthalmologists was the report by Duffy and associates 3 of the possible transmission of CreutzfeldtJakob disease from person to person by corneal transplant and its experimental transmission in guinea pigs by intrao cular injection of corneal material from infected animals. 4 Virus-like particles have been found in brains in both natural and experimental Creutzfeldt-Jakob disease, 5 " 1 1 but there has been no attempt to identify such agents in the eye. We report herein the results of ultrastructural examination of ocular tissues from a patient with Creutzfeldt-Jakob disease with emphasis From the Departments of Ophthalmology (Drs. Roth and Keltner, and Mrs. Green), Pathology (Drs. Roth and Ellis), Neurology (Drs. Keltner and Ellis), and Neurological Surgery (Dr. Keltner), School of Medicine, University of California, Davis, and the University of California Davis, Medical Center, Sacramento, California. Reprint requests to Alan M. Roth, M.D., Eye Pathology Laboratory, 251 Professional Bldg.,4301 X St., Sacramento, CA 95817.
M.S.
California
on identification of possible virus-like particles. CASE REPORT A 68-year-old man was well until September 1976, at which time his daughter noted that he seemed to be having fewer temper tantrums. The patient had been a heavy wine drinker, up to a half gallon per day, but in December 1975 had changed to occa sional beers. He ate poorly while drinking. In Octo ber 1976, he had onset of vertigo, persisting for three weeks. The patient became ataxic and dysarthric, complaining of tinnitus, and his gait deteriorated with veering to the left. On Nov. 1, 1976, myoclonic jerks were observed during sleep, the patient was disoriented in his home, he was unable to work the water faucet, and had difficulty remembering recent events. Visual hallucinations were also observed b y the patient. On Nov. 6, 1976, the patient was admit ted here. Results of the physical examination were unre markable except for the neurologic examination. The patient was oriented to his name, knew the city, his birthdate, his age and address, but was unable to be more specific about recent events. Cranial nerve examination recorded vision as grossly normal; bi lateral sixth nerve weakness was noted; up-gaze weakness and bilateral nystagmus on horizontal gaze was noted. Ophthalmoscopic examination was unremarkable. The rest of the cranial nerves were intact. Strength and tone were normal in the extrem ities. The sensory examination revealed loss of vi bratory sense in both feet. Cerebellar testing showed marked dysmetria bilaterally with finger to nose testing. His gait was wide based, and the jaw-jerk was brisk. Plantar reflexes were normal. Results of tests were unremarkable except for an eosinophilia of 7%, increased alkaline phosphatase of 225 units, and lactic acid dehydrogenase of 400 units. Cerebrospinal fluid on Nov. 16, 1976, re vealed a protein of 50 mg/100 ml, glucose of 84%, and no cells. Skull x-rays were normal, and comput ed tomography scan was normal. Numerous electro encephalograms were performed, beginning on Nov. 8, 1976, and eventually showed deteriorating stereotyped periodic biphasic or triphasic discharg es. The patient's condition progressively deteriorat ed. On Nov. 16 1976, myoclonic jerks were detected. On Nov. 23, 1976 the patient was mute and his arms were rigid. He had intermittent conjugate deviation of the eyes to the left and right. Bilateral extensive
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plantar reflexes developed. Right-sided focal sei zures were observed on Dec. 4, 1976, and cortical blindness was recorded on Dec. 23, 1976. The patient's condition continued to deteriorate and he died on Feb. 25, 1977. M A T E R I A L AND M E T H O D S
We removed the eyes at autopsy imme diately after death and immersed the right globe in cold Karnovsky's fixative in 0.05M phosphate buffer. After hardening for 15 minutes, the eye was gross-sec tioned as previously described 1 2 ; gross examination was performed with the ca lottes immersed in fixative. Blocks of cor nea, retina, and optic nerve head were cut with a razor blade and fixed overnight in cold phosphate-buffered Karnovsky's so lution. The tissues were washed for sever al hours in a 0.05M phosphate buffer solution and postfixed for one hour in a 1% osmium tetroxide solution in the same buffer. They were washed in the buffer for IV2 hours, dehydrated in an ascending series of acetone solutions, and embed ded in Spurr resin. One micrometer sections were cut with an ultramicrotome and stained with toluidine blue. Ultrathin sections were col lected on uncoated copper grids, stained with an alcoholic solution of uranyl acetate and lead citrate, and examined and photographed with an electron mi croscope. Tissue from the left eye was frozen in liquid nitrogen for transmissability studies.
Fig. 1 (Roth and associates). Cortical atrophy present in frontal and occipital lobes (x0.6).
nuclei were markedly atrophic, and sub stantia nigra was depigmented. Microscopic findings included genera lized neuronal loss in the cerebral cortex, striatum and substantia nigra, and was most severe in the frontal cortex. In many areas the cortical neurons had been en tirely replaced by large gemistocytic astrocytes (Fig. 2). Status spongiosus was prominent in the deeper cortical laminae, particularly in areas of mild to moderate neuronal loss (Fig. 3). No inclusion bod ies or inflammatory cells were found. The right eye measured 24.8 mm in
RESULTS
General pathologic findings revealed marked cachexia and bronchopneumonia. On neuropathologic examination the brain weighed 1,025 g and externally showed only marked bifrontal cerebral cortical atrophy and occipital atrophy of moderate degree (Fig. 1). Coronal sections con firmed the frontal and occipital cortical atrophy. Cortex was tan and shrunken to less than 2 mm in width in many areas. Lateral ventricles were dilated, caudate
Fig. 2 (Roth and associates). Junction of cortex (left side of micrograph) and white matter (right side of micrograph) occipital lobe shows spongiform degeneration of neuropil most prominent in deep cortex. There is diffuse neuronal loss and marked astocytosis (hematoxylin and eosin, x80).
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anteroposterior diameter. The cornea was translucent. The anterior segment and vi treous cavity were otherwise unremark able. No retinal or macular lesions were seen grossly. The optic nerve head had a small conical cup. No gross optic nerve atrophy was apparent. On light microscopy the cornea showed marked changes in its epithelium (Fig. 4). This was diffusely thinned with dis organization of its cells. Superficial cells were flattened and stained densely with toluidine blue. Epithelial basal lamina was intact and Bowman's layer, stroma,
Descemet's membrane, and endothelium were unremarkable. The remainder of the ocular structures showed no significant pathologic changes. The macular area of the neurosensory retina and the optic nerve head were unaffected; the macular area showed no inner layer atrophy and the optic nerve head showed no evi dence of nerve fiber atrophy, gliosis, or spongiosis (Fig. 5). On electron microscopy, marked disor ganization and degenerative changes in corneal epithelium were found; these in cluded extreme osmiophilia, absence of cytoplasmic organelles, flattening of cell bodies and loss of nuclei in superficial layers and vacuolization of cytoplasm in basilar layers. The remainder of corneal structures were intact. There was no evi dence of virus-like particles in corneal sections. In the optic nerve head, however, we saw multiple particles within cell bodies and processes of many astroglia. These consisted of electron-dense cores sur rounded by lucent zones and concentric membranous rings having an overall di ameter of 125 to 200 u.M (Fig. 6 and 7). Occasional pockets of these structures contained budding particles (Fig. 8). No particles were evident inside of nuclei.
Fig. 4 (Roth and associates). Corneal epithelium is thinned with cellular disorganization. Its basal lam ina, Bowman's layer, and superficial stroma are intact (toluidine blue, x200).
Fig. 5 (Roth and associates). No nerve fiber atro phy or gliosis in temporal aspect of optic nerve head (toluidine blue, x80).
Fig. 3 (Roth and associates). Spongiform degener ation and gliosis in gray matter of occipital lobe (hematoxylin and eosin, x320).
Fig. 6 (Roth and associates). Virus-like particles (arrow) in astroglial process of optic nerve head. C designates the collagen of central connective tissue meniscus (x4,800).
Fig. 7 (Roth and associates). Particles (arrows) in glial cell body have electron-dense core and concentric membranous rings. Tip of cell nucleus (N) contains pores that may simulate virions (x9,600).
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Fig. 8 (Roth and associates). Pocket of virus-like particles (arrow) appears to contain budding virions (x 9,600).
The glial cells containing particles, as well as other glia and nerve fibers, showed no other ultrastructural changes. DISCUSSION
The apparent transmission of Creutzfeldt-Jakob disease between humans by corneal transplantation 3 implicates the presence of the infective agent in the eyes of affected patients. Herzberg and associates 13 suggested that the high inci dence of the disease in Israelis of Libyan background was related to their practice of eating sheep eyeballs. The presence of an infectious agent in the eye is sup ported by experimental infection of guinea pigs by inoculation of corneal tissue from affected animals into the anterior
chambers of healthy specimens. 4 It is also of interest that transmissible mink encephalopathy has been transmitted in hamsters by intracerebral injection of cor neal epithelium. 1 4 In the same study, nei ther cultured corneal epithelium nor aque ous humor was similarly infective. The difference between original and cultured epithelial infectivity was explained either by failure of infected epithelial cells to replicate in tissue culture or, more proba bly, that infectivity is associated with the free nerve fibers in corneal epithelium. We examined the ocular tissues from our patient specifically for the presence of an infective agent. The corneal changes we saw were nonspecific and probably related to the marked terminal morbidity
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of the patient. We found no morphologic evidence of virus-like particles in corneal tissue. However, we were able to identify only a few nerve endings in the altered epithelium and, if Marsh and Hanson's 1 4 hypothesis that infectivity is associated with the nerves is correct, this might account for the negative finding. We did identify numerous virus-like particles within glial cells of the optic nerve head. These structures were morphologically and dimensionally similar to those report ed in the numerous reports describing virus-like particles in brain tissue of pa tients with Creutzfeld-Jakob disease. 5 - 1 1 However, the significance of these parti cles in the pathogenesis of CreutzfeldtJakob disease is questionable. 1 5 The re ports may have shown virions that were incidentally present in the patient but had no relationship to the disease in question. Rogers and associates 16 described in chimpanzees with Kuru multiple latent viruses unassociated with the disease. Many regularly occurring cell compo nents and other unidentifiable structures may mimic viruses when viewed by the electron microscope. 17 We believe the particles we have described are such virion-simulating structures and it is probable that many of the particles previ ously reported are similar. If the structures reported as virus-like are not the causative agents of Creutzfeldt-Jakob disease, why have these not yet been found? They are atypical viruses which, although filterable, do not elicit host immune mechanisms, do not evoke viral-like inflammatory response in brain tissue, and possess physical properties unlike other viruses. 18 They show marked resistance to inactivation by many con ventional methods including heat, ultra violet radiation, and formaldehyde; this has led to recommendations of extreme caution in evaluation of patients having the disease. 1 9 " 2 3 Studies suggest that the virus might be small naked nucleic acids
JUNE, 1979
bound to membranes, 1 8 perhaps similar to the viroids causing plant diseases, 24 and could activate latent viruses in the host. 25 This would explain many of its atypical properties and the failure to identify it by the electron microscope. The blindness in patients with Creutzfeldt-Jakob disease has been classically described as cortical in nature. No clini cal ocular changes have been reported in such patients. Lesser and associates 26 found histologic atrophy of the optic nerve and the inner retinal layers at au topsy in a patient who showed no optic atrophy clinically, although visual loss was present during life. The authors were unable to explain this disparity. Barnett and Palmer 27 and Buyukmihci and asso ciates 28 described histologic degenerative changes in the outer retinal layers of animals infected with scrapie. Buyuk mihci and associates 28 additionally found vacuoles in the optic nerves. We found no evidence of retinal or optic atrophy either clinically or pathologically in our patient, and the severe occipital degeneration ac counted for his visual loss. The findings in our case indicate that there are no specific pathologic changes in the eyes of patients with CreutzfeldtJakob disease. The axons and glial ele ments in the nerve head, in the anterior optic nerve, and in the retina were struc turally intact. No evidence for an infec tious agent was found in any portion of the eye. SUMMARY
A 68-year-old man was treated for and died of Creutzfeldt-Jakob disease. At au topsy we found multiple virus-like parti cles in the optic nerve head, but saw no similar structures in the cornea. Although these particles were morphologically sim ilar to those previously reported in brain, we believe that they are not virions but unrelated cellular structures. We specu late that the causative agents may be
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naked membrane bound nucleic acids rather than true viruses. We found no optic atrophy or other specific pathologic changes in the eyes; severe occipital corti cal degeneration was responsible for the patient's visual loss.
REFERENCES 1. Gajdusek, D. C , and Gibbs, C. J.: Transmis sion of two subacute spongiform encephalopathies of man (Kuru and Creutzfeldt-Jakob disease) to New World monkeys. Nature 230:588, 1971. 2. Gibbs, C. J., Gajdusek, D. C , Asher, D. M., Alper, M. P., Beck, E., Daniel, P. M., and Matthews, W. B.: Creutzfeldt-Jakob disease (spongiform encephalopathy). Transmission to the chimpanzee. Science 161:388, 1968. 3. Duffy, P., Wolf, J., Collins, G., DeVoe, A. G., Streeten, B., and Cowen, D.: Possible person-toperson transmission of Creutzfeldt-Jakob disease. N. Engl. J. Med. 290:692, 1974. 4. Manuelidis, E. E., Angelo, J. N., Gorgacz, E. J., Kim, J. H., and Manuelidis, L.: Experimental Creutzfeldt-Jakob disease transmitted via the eye with infected cornea. N. Engl. J. Med. 296:1334, 1977. 5. Vernon, M. L., Horta-Barbosa, L., Fucillo, D. A., Sever, J. L., Baringer, J. R., and Birnbaum, G.: Virus-like particles and nucleoprotein-type fila ments in brain tissue from two patients with Creutzfeldt-Jakob disease. Lancet 1:964, 1970. 6. Allen, I. V., Dermott, E., Connolly, J. H., and Hurwitz, L. J.: A Study of a patient with the amyotrophic form of Creutzfeldt-Jakob disease. Brain 94:715, 1971. 7. Lambert, P. W., Gajdusek, D. C , and Gibbs, C. J.: Experimental spongiform encephalopathy (Creutzfeldt-Jakob disease) in chimpanzees. Elec tron microscope studies. J. Neuropathol. Exp. Neurol. 30:20, 1971. 8. Watanabe, I., Kolar, O., Horta-Barbosa, L. L., Fucillo, D., Sever, J. L., Thompson, J., and Leman, W.: Recent observations in Cruetzfeldt-Jakob dis ease. J. Neuropathol. Exp. Neurol. 30:123, 1971. 9. Bots, G. Th. A. M., de Man, J. C. H., and Verjaal, A.: Virus-like particles in brain tissue from two patients with Creutzfeldt-Jakob disease. Acta Neuropathol. 18:267, 1971. 10. Roy, C , Gupta, P. C., and Sethi, U.: Creutzfeldt-Jakob disease. An electron microscopic study with demonstration of virus-like particles. Neurol. India 20:226, 1972. 11. Bubis, J. J., Goldhammer, Y., and Braham, J.: Subacute spongiform encephalopathy. Electron mi croscope studies. J. Neurol. Neurosurg. Psychiatry 35:881, 1972. 12. Roth, A. M., and Foos, R. Y.: A system for the
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macroexamination of eyes in the laboratory. Am. J. Clin. Pathol. 59:674, 1973. 13. Herzberg, L., Herzberg, B. N., Gibbs, C. J., Sullivan, W., Amyx, H., and Gajdusek, D. C : Creutzfeldt-Jakob disease. Hypothesis for high inci dence in Libyan Jews in Israel. Science 186:848, 1974. 14. Marsh, R. F., and Hanson, R. P.: Transmissi ble mink encephalopathy. Infectivity of corneal epithelium. Science 187:656, 1975. 15. Matthews, W. B.: Creutzfeldt-Jakob disease as a transmissible encephalopathy. In Waterson, A. P., (ed.): Recent Advances in Clinical Virology, Edinburgh, Churchill Livingstone, 1977, p p . 51-60. 16. Rogers, N. G., Basnight, M., Gibbs, C. J., and Gajdusek, D. C : Latent viruses in chimpanzees with experimental Kuru. Nature 216:446, 1967. 17. Haguenau, F.: "Viruslike" particles as ob served with t-he electron microscope. In Dalton, A. J., and Haguenau, F. (eds): Ultrastructure of Animal Viruses and Bacterophages. An Atlas, New York, Academic Press, 1973, p p . 391-397. 18. Gajdusek, D. C : Kuru and Creutzfeldt-Jakob disease. Experimental models of noninflammatory degenerative slow virus disease of the central nerv ous system. Ann. Clin. Res. 5:254, 1973. 19. Traub, R. D., Gajdusek, D. C , and Gibbs, C. J.: Precautions in conducting biopsies and autop sies on patients with presenile dementia. J. Neuro surg. 41:394, 1974. 20. : Precautions in autopsies on Creutzfeldt-Jakob disease. Am. J. Clin. Pathol. 64: 287, 1975. 21. Gajdusek, D. C., and Gibbs, C. J.: Survival of Creutzfeldt-Jakob - disease virus in formol-fixed brain tissue. N. Engl. J. Med. 294:553, 1976. 22. Gajdusek, D. C , Gibbs, C. J., Asher, D. M., Brown, P., Diwan, A., Hoffman, P., Nemo, G., Rohwer, R., and White, L.: Precautions in medical care of, and in handling of material from, patients with transmissible virus dementia (CreutzfeldtJakob disease). N. Engl. J. Med. 297:1253, 1977. 23. Bernoulli, C , Siegfried, J., Baumgartner, G. Regli, F., Rabinowicz, T., Gajdusek, D. C , and Gibbs, C. J.: Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by sur gery. Lancet 2:478, 1977. 24. Diener, T. O.: Is the scrapie agent a viroid? Nature New Biol. 235:218, 1972. 25. Gajdusek, D. C : Slow virus infections and activation of latent virus infections in aging. Adv. Gerontol. Res. 4:201, 1972. 26. Lesser, R. L., Albert, D. M., Bobowick, A. R., and O'Brien, F. H.: Creutzfeldt-Jakob disease and optic atrophy. Am. J. Ophthalmol. 87:317, 1979. 27. Barnett, K. C , and Palmer, A. C : Retinopathy in sheep affected with natural scrapie. Res. Vet. Sci. 12:383, 1971. 28. Buyukniihci, N., Marsh, R. F., Albert, D. M., and Zelinski, K.: Ocular effects of scrapie agent in hamsters. Preliminary observations. Invest. Ophthal mol. Visual Sci. 16:319, 1977.
J O H N L. K E L T N E R ,
M.D.,
AND M A N U E L A M A R T I N S - G R E E N , Davis,
Creutzfeldt-Jakob disease is a progres sive, fatal, degenerative disease of the central nervous system in man which, because of its typical pathologic lesions and its transmissible nature, has been included in the classification of subacute spongiform virus encephalopathies 1 or slow virus infections together with Kuru in man, scrapie in sheep, and transmissible encephalopathy in mink. Creutzfeldt-Jakob disease was first trans mitted to laboratory animals in 1968 2 by intracerebral injection of affected human brain and since has been transmitted with serial passages to a variety of species. Of particular interest to ophthalmologists was the report by Duffy and associates 3 of the possible transmission of CreutzfeldtJakob disease from person to person by corneal transplant and its experimental transmission in guinea pigs by intrao cular injection of corneal material from infected animals. 4 Virus-like particles have been found in brains in both natural and experimental Creutzfeldt-Jakob disease, 5 " 1 1 but there has been no attempt to identify such agents in the eye. We report herein the results of ultrastructural examination of ocular tissues from a patient with Creutzfeldt-Jakob disease with emphasis From the Departments of Ophthalmology (Drs. Roth and Keltner, and Mrs. Green), Pathology (Drs. Roth and Ellis), Neurology (Drs. Keltner and Ellis), and Neurological Surgery (Dr. Keltner), School of Medicine, University of California, Davis, and the University of California Davis, Medical Center, Sacramento, California. Reprint requests to Alan M. Roth, M.D., Eye Pathology Laboratory, 251 Professional Bldg.,4301 X St., Sacramento, CA 95817.
M.S.
California
on identification of possible virus-like particles. CASE REPORT A 68-year-old man was well until September 1976, at which time his daughter noted that he seemed to be having fewer temper tantrums. The patient had been a heavy wine drinker, up to a half gallon per day, but in December 1975 had changed to occa sional beers. He ate poorly while drinking. In Octo ber 1976, he had onset of vertigo, persisting for three weeks. The patient became ataxic and dysarthric, complaining of tinnitus, and his gait deteriorated with veering to the left. On Nov. 1, 1976, myoclonic jerks were observed during sleep, the patient was disoriented in his home, he was unable to work the water faucet, and had difficulty remembering recent events. Visual hallucinations were also observed b y the patient. On Nov. 6, 1976, the patient was admit ted here. Results of the physical examination were unre markable except for the neurologic examination. The patient was oriented to his name, knew the city, his birthdate, his age and address, but was unable to be more specific about recent events. Cranial nerve examination recorded vision as grossly normal; bi lateral sixth nerve weakness was noted; up-gaze weakness and bilateral nystagmus on horizontal gaze was noted. Ophthalmoscopic examination was unremarkable. The rest of the cranial nerves were intact. Strength and tone were normal in the extrem ities. The sensory examination revealed loss of vi bratory sense in both feet. Cerebellar testing showed marked dysmetria bilaterally with finger to nose testing. His gait was wide based, and the jaw-jerk was brisk. Plantar reflexes were normal. Results of tests were unremarkable except for an eosinophilia of 7%, increased alkaline phosphatase of 225 units, and lactic acid dehydrogenase of 400 units. Cerebrospinal fluid on Nov. 16, 1976, re vealed a protein of 50 mg/100 ml, glucose of 84%, and no cells. Skull x-rays were normal, and comput ed tomography scan was normal. Numerous electro encephalograms were performed, beginning on Nov. 8, 1976, and eventually showed deteriorating stereotyped periodic biphasic or triphasic discharg es. The patient's condition progressively deteriorat ed. On Nov. 16 1976, myoclonic jerks were detected. On Nov. 23, 1976 the patient was mute and his arms were rigid. He had intermittent conjugate deviation of the eyes to the left and right. Bilateral extensive
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plantar reflexes developed. Right-sided focal sei zures were observed on Dec. 4, 1976, and cortical blindness was recorded on Dec. 23, 1976. The patient's condition continued to deteriorate and he died on Feb. 25, 1977. M A T E R I A L AND M E T H O D S
We removed the eyes at autopsy imme diately after death and immersed the right globe in cold Karnovsky's fixative in 0.05M phosphate buffer. After hardening for 15 minutes, the eye was gross-sec tioned as previously described 1 2 ; gross examination was performed with the ca lottes immersed in fixative. Blocks of cor nea, retina, and optic nerve head were cut with a razor blade and fixed overnight in cold phosphate-buffered Karnovsky's so lution. The tissues were washed for sever al hours in a 0.05M phosphate buffer solution and postfixed for one hour in a 1% osmium tetroxide solution in the same buffer. They were washed in the buffer for IV2 hours, dehydrated in an ascending series of acetone solutions, and embed ded in Spurr resin. One micrometer sections were cut with an ultramicrotome and stained with toluidine blue. Ultrathin sections were col lected on uncoated copper grids, stained with an alcoholic solution of uranyl acetate and lead citrate, and examined and photographed with an electron mi croscope. Tissue from the left eye was frozen in liquid nitrogen for transmissability studies.
Fig. 1 (Roth and associates). Cortical atrophy present in frontal and occipital lobes (x0.6).
nuclei were markedly atrophic, and sub stantia nigra was depigmented. Microscopic findings included genera lized neuronal loss in the cerebral cortex, striatum and substantia nigra, and was most severe in the frontal cortex. In many areas the cortical neurons had been en tirely replaced by large gemistocytic astrocytes (Fig. 2). Status spongiosus was prominent in the deeper cortical laminae, particularly in areas of mild to moderate neuronal loss (Fig. 3). No inclusion bod ies or inflammatory cells were found. The right eye measured 24.8 mm in
RESULTS
General pathologic findings revealed marked cachexia and bronchopneumonia. On neuropathologic examination the brain weighed 1,025 g and externally showed only marked bifrontal cerebral cortical atrophy and occipital atrophy of moderate degree (Fig. 1). Coronal sections con firmed the frontal and occipital cortical atrophy. Cortex was tan and shrunken to less than 2 mm in width in many areas. Lateral ventricles were dilated, caudate
Fig. 2 (Roth and associates). Junction of cortex (left side of micrograph) and white matter (right side of micrograph) occipital lobe shows spongiform degeneration of neuropil most prominent in deep cortex. There is diffuse neuronal loss and marked astocytosis (hematoxylin and eosin, x80).
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anteroposterior diameter. The cornea was translucent. The anterior segment and vi treous cavity were otherwise unremark able. No retinal or macular lesions were seen grossly. The optic nerve head had a small conical cup. No gross optic nerve atrophy was apparent. On light microscopy the cornea showed marked changes in its epithelium (Fig. 4). This was diffusely thinned with dis organization of its cells. Superficial cells were flattened and stained densely with toluidine blue. Epithelial basal lamina was intact and Bowman's layer, stroma,
Descemet's membrane, and endothelium were unremarkable. The remainder of the ocular structures showed no significant pathologic changes. The macular area of the neurosensory retina and the optic nerve head were unaffected; the macular area showed no inner layer atrophy and the optic nerve head showed no evi dence of nerve fiber atrophy, gliosis, or spongiosis (Fig. 5). On electron microscopy, marked disor ganization and degenerative changes in corneal epithelium were found; these in cluded extreme osmiophilia, absence of cytoplasmic organelles, flattening of cell bodies and loss of nuclei in superficial layers and vacuolization of cytoplasm in basilar layers. The remainder of corneal structures were intact. There was no evi dence of virus-like particles in corneal sections. In the optic nerve head, however, we saw multiple particles within cell bodies and processes of many astroglia. These consisted of electron-dense cores sur rounded by lucent zones and concentric membranous rings having an overall di ameter of 125 to 200 u.M (Fig. 6 and 7). Occasional pockets of these structures contained budding particles (Fig. 8). No particles were evident inside of nuclei.
Fig. 4 (Roth and associates). Corneal epithelium is thinned with cellular disorganization. Its basal lam ina, Bowman's layer, and superficial stroma are intact (toluidine blue, x200).
Fig. 5 (Roth and associates). No nerve fiber atro phy or gliosis in temporal aspect of optic nerve head (toluidine blue, x80).
Fig. 3 (Roth and associates). Spongiform degener ation and gliosis in gray matter of occipital lobe (hematoxylin and eosin, x320).
Fig. 6 (Roth and associates). Virus-like particles (arrow) in astroglial process of optic nerve head. C designates the collagen of central connective tissue meniscus (x4,800).
Fig. 7 (Roth and associates). Particles (arrows) in glial cell body have electron-dense core and concentric membranous rings. Tip of cell nucleus (N) contains pores that may simulate virions (x9,600).
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Fig. 8 (Roth and associates). Pocket of virus-like particles (arrow) appears to contain budding virions (x 9,600).
The glial cells containing particles, as well as other glia and nerve fibers, showed no other ultrastructural changes. DISCUSSION
The apparent transmission of Creutzfeldt-Jakob disease between humans by corneal transplantation 3 implicates the presence of the infective agent in the eyes of affected patients. Herzberg and associates 13 suggested that the high inci dence of the disease in Israelis of Libyan background was related to their practice of eating sheep eyeballs. The presence of an infectious agent in the eye is sup ported by experimental infection of guinea pigs by inoculation of corneal tissue from affected animals into the anterior
chambers of healthy specimens. 4 It is also of interest that transmissible mink encephalopathy has been transmitted in hamsters by intracerebral injection of cor neal epithelium. 1 4 In the same study, nei ther cultured corneal epithelium nor aque ous humor was similarly infective. The difference between original and cultured epithelial infectivity was explained either by failure of infected epithelial cells to replicate in tissue culture or, more proba bly, that infectivity is associated with the free nerve fibers in corneal epithelium. We examined the ocular tissues from our patient specifically for the presence of an infective agent. The corneal changes we saw were nonspecific and probably related to the marked terminal morbidity
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of the patient. We found no morphologic evidence of virus-like particles in corneal tissue. However, we were able to identify only a few nerve endings in the altered epithelium and, if Marsh and Hanson's 1 4 hypothesis that infectivity is associated with the nerves is correct, this might account for the negative finding. We did identify numerous virus-like particles within glial cells of the optic nerve head. These structures were morphologically and dimensionally similar to those report ed in the numerous reports describing virus-like particles in brain tissue of pa tients with Creutzfeld-Jakob disease. 5 - 1 1 However, the significance of these parti cles in the pathogenesis of CreutzfeldtJakob disease is questionable. 1 5 The re ports may have shown virions that were incidentally present in the patient but had no relationship to the disease in question. Rogers and associates 16 described in chimpanzees with Kuru multiple latent viruses unassociated with the disease. Many regularly occurring cell compo nents and other unidentifiable structures may mimic viruses when viewed by the electron microscope. 17 We believe the particles we have described are such virion-simulating structures and it is probable that many of the particles previ ously reported are similar. If the structures reported as virus-like are not the causative agents of Creutzfeldt-Jakob disease, why have these not yet been found? They are atypical viruses which, although filterable, do not elicit host immune mechanisms, do not evoke viral-like inflammatory response in brain tissue, and possess physical properties unlike other viruses. 18 They show marked resistance to inactivation by many con ventional methods including heat, ultra violet radiation, and formaldehyde; this has led to recommendations of extreme caution in evaluation of patients having the disease. 1 9 " 2 3 Studies suggest that the virus might be small naked nucleic acids
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bound to membranes, 1 8 perhaps similar to the viroids causing plant diseases, 24 and could activate latent viruses in the host. 25 This would explain many of its atypical properties and the failure to identify it by the electron microscope. The blindness in patients with Creutzfeldt-Jakob disease has been classically described as cortical in nature. No clini cal ocular changes have been reported in such patients. Lesser and associates 26 found histologic atrophy of the optic nerve and the inner retinal layers at au topsy in a patient who showed no optic atrophy clinically, although visual loss was present during life. The authors were unable to explain this disparity. Barnett and Palmer 27 and Buyukmihci and asso ciates 28 described histologic degenerative changes in the outer retinal layers of animals infected with scrapie. Buyuk mihci and associates 28 additionally found vacuoles in the optic nerves. We found no evidence of retinal or optic atrophy either clinically or pathologically in our patient, and the severe occipital degeneration ac counted for his visual loss. The findings in our case indicate that there are no specific pathologic changes in the eyes of patients with CreutzfeldtJakob disease. The axons and glial ele ments in the nerve head, in the anterior optic nerve, and in the retina were struc turally intact. No evidence for an infec tious agent was found in any portion of the eye. SUMMARY
A 68-year-old man was treated for and died of Creutzfeldt-Jakob disease. At au topsy we found multiple virus-like parti cles in the optic nerve head, but saw no similar structures in the cornea. Although these particles were morphologically sim ilar to those previously reported in brain, we believe that they are not virions but unrelated cellular structures. We specu late that the causative agents may be
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naked membrane bound nucleic acids rather than true viruses. We found no optic atrophy or other specific pathologic changes in the eyes; severe occipital corti cal degeneration was responsible for the patient's visual loss.
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macroexamination of eyes in the laboratory. Am. J. Clin. Pathol. 59:674, 1973. 13. Herzberg, L., Herzberg, B. N., Gibbs, C. J., Sullivan, W., Amyx, H., and Gajdusek, D. C : Creutzfeldt-Jakob disease. Hypothesis for high inci dence in Libyan Jews in Israel. Science 186:848, 1974. 14. Marsh, R. F., and Hanson, R. P.: Transmissi ble mink encephalopathy. Infectivity of corneal epithelium. Science 187:656, 1975. 15. Matthews, W. B.: Creutzfeldt-Jakob disease as a transmissible encephalopathy. In Waterson, A. P., (ed.): Recent Advances in Clinical Virology, Edinburgh, Churchill Livingstone, 1977, p p . 51-60. 16. Rogers, N. G., Basnight, M., Gibbs, C. J., and Gajdusek, D. C : Latent viruses in chimpanzees with experimental Kuru. Nature 216:446, 1967. 17. Haguenau, F.: "Viruslike" particles as ob served with t-he electron microscope. In Dalton, A. J., and Haguenau, F. (eds): Ultrastructure of Animal Viruses and Bacterophages. An Atlas, New York, Academic Press, 1973, p p . 391-397. 18. Gajdusek, D. C : Kuru and Creutzfeldt-Jakob disease. Experimental models of noninflammatory degenerative slow virus disease of the central nerv ous system. Ann. Clin. Res. 5:254, 1973. 19. Traub, R. D., Gajdusek, D. C , and Gibbs, C. J.: Precautions in conducting biopsies and autop sies on patients with presenile dementia. J. Neuro surg. 41:394, 1974. 20. : Precautions in autopsies on Creutzfeldt-Jakob disease. Am. J. Clin. Pathol. 64: 287, 1975. 21. Gajdusek, D. C., and Gibbs, C. J.: Survival of Creutzfeldt-Jakob - disease virus in formol-fixed brain tissue. N. Engl. J. Med. 294:553, 1976. 22. Gajdusek, D. C , Gibbs, C. J., Asher, D. M., Brown, P., Diwan, A., Hoffman, P., Nemo, G., Rohwer, R., and White, L.: Precautions in medical care of, and in handling of material from, patients with transmissible virus dementia (CreutzfeldtJakob disease). N. Engl. J. Med. 297:1253, 1977. 23. Bernoulli, C , Siegfried, J., Baumgartner, G. Regli, F., Rabinowicz, T., Gajdusek, D. C , and Gibbs, C. J.: Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by sur gery. Lancet 2:478, 1977. 24. Diener, T. O.: Is the scrapie agent a viroid? Nature New Biol. 235:218, 1972. 25. Gajdusek, D. C : Slow virus infections and activation of latent virus infections in aging. Adv. Gerontol. Res. 4:201, 1972. 26. Lesser, R. L., Albert, D. M., Bobowick, A. R., and O'Brien, F. H.: Creutzfeldt-Jakob disease and optic atrophy. Am. J. Ophthalmol. 87:317, 1979. 27. Barnett, K. C , and Palmer, A. C : Retinopathy in sheep affected with natural scrapie. Res. Vet. Sci. 12:383, 1971. 28. Buyukniihci, N., Marsh, R. F., Albert, D. M., and Zelinski, K.: Ocular effects of scrapie agent in hamsters. Preliminary observations. Invest. Ophthal mol. Visual Sci. 16:319, 1977.
