August 1981. Fig. 2. Left eye of monkey 1 on day 35. Fundus photograph (A) and fluorescein angiogram (B) dur- ing the late phase showing ODE (reflexes are.
Experimental allergic encephalomyelitis I. Optic nerve and central nervous system manifestations Sohan Singh Hayreh,* R. Michael Massanari,** Thoru Yamada,*** and Sant Manmohan Singh Hayreh*** Experimental allergic encephalomyelitis (EAE) was produced in six adult rhesus monkeys. The animals were evaluated serially by ocular, ophthalmoscopic,fluoresceinfundus angiographic, pupillary, visual evoked potential, neurologic, cerebrospinal fluid (CSF), and hematologic examinations and by postmortem detailed histopathologic examination. All the animals developed acute EAE. Four of the monkeys, surviving longer than 1 month, developed chronic relapsing EAE and were sacrificed 3 to 14 months after sensitization. All 12 eyes developed acute optic neuritis (with variable degrees of optic disc edema and visual loss). Later on, all the eyes of animals with chronic EAE developed optic atrophy with total or almost total blindness. Histopathologic examination of the optic nerve and central nervous system revealed inflammatory infiltrates, extensive demyelination, and axonal degeneration, without inflammation in the retina or optic nerve head (i.e., nonmyelinated neural tissue). Relapsing EAE was reflected in episodic increases of CSF proteins and pleocytosis. The various findings are correlated. Key words: optic nerve, brain, demyelination, optic neuritis, multiple sclerosis, encephalomyelitis
Lxperimental allergic encephalomyelitis (EAE) is an acute, immunologically mediated, inflammatory disease of the central nervous system.' The disease has been reproduced in a variety of mammalian and nonmammalian species.2 The acute, monophasic disease has served as a laboratory model of acute demyelinating diseases such as acute dissemiFrom the Departments of Ophthalmology*, Internal Medicine**, and Neurology***, University of Iowa College of Medicine, Iowa City. This study was supported by Public Health Service grant No. EY-01151 and by grants RG1087-A and RG 1220-A from the National Multiple Sclerosis Society. Submitted for publication Oct. 3, 1980. Reprint requests: Dr. S. S. Hayreh, Department of Ophthalmology, University Hospitals and Clinics, Iowa City, Iowa 52242.
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nated encephalomyelitis and postinfectious encephalomyelitis. Chronic relapsing forms of EAE have been described in monkeys,3' 4 rats, 5 guinea pigs, 6 " 8 and hamsters. 9 Histopathologic changes as well as the chronic relapsing clinical course resemble multiple sclerosis, a chronic demyelinating disease of humans. 10 Investigative efforts to date have focused on the pathogenesis of the central nervous system disease, with only passing references to lesions in optic nerves 3 ' n ~ 16 except in one paper dealing with optic neuritis in guinea pigs.17 The primary object of this study was to investigate in rhesus monkeys the ophthalmologic manifestations of EAE by modern diagnostic and investigative methods and to correlate the findings with neurologic ab-
0146-0404/81/080256+14$01.40/0 © 1981 Assoc. for Res. in Vis. and Ophthal., Inc.
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Fig. 1. Fundus photographs showing three of the four grades of ODE. A, Left eye of monkey 4 on day 49 with 1+ ODE. B, Right eye of monkey 3 on day 25 with 3+ ODE. C, Left eye of monkey 2 on day 65 with 4+ ODE. normalities. This is the first study in which animals were evaluated in detail over a long period with a variety of clinical and laboratory parameters. We report observations in animals with acute optic nerve and neurological lesions, as well as in animals with chronic relapsing EAE. Materials and methods EAE was produced in six adult rhesus monkeys weighing 3.3 to 4.4 kg. Sensitization regimen. Homologous spinal cord tissue was prepared as a 33% suspension (w/v) in sterile physiologic buffered saline and was homogenized through a double-hubbed syringe with
complete Freund's adjuvant (BCG, 4 mg/ml). Five animals were sensitized by a single in trad ermal injection of 0.1 ml of the sensitizing inoculum over the dorsal thorax, between the scapulae. The first animal (No. 5) to be sensitized received five 0.1 ml injections at the same site simultaneously. Because the subsequent neurologic disease was acute and rapidly fatal, the sensitizing dose was reduced to a single injection. Evaluation. All monkeys were examined serially, including prior to sensitization and immediately before sacrifice. The following investigations were conducted: 1. Complete ocular examination, including slitlamp examination, and testing for the pupillary light reaction and visual impairment.
Invest. Ophthalmol. Vis. Sci. August 1981
258 Hayreh et al.
temperature (37° C). The monkey was then cooled by cooling blanket and air conditioner until rectal temperature reached 35° C, and the VEP was recorded. The monkey was then heated to 40° C rectal temperature by heating blanket and the VEP was recorded. Postmortem studies. Five monkeys were sacrificed 31 to 433 days after sensitization and were perfused with neutral buffered formaldehyde. One animal died during the acute phase of EAE> 1 month after sensitization. Complete autopsies were performed on all the monkeys. Brain, spinal cord, eyeballs with entire length of the optic nerve, and portions of peripheral nerves and viscera were removed and fixed in buffered neutral formalin solution. Histopathologic sections were stained with hematoxylin-eosin. The optic nerve and eyeball were serially sectioned by cutting 8 fim thick longitudinal sections and were stained with hematoxylin-eosin, Luxol fast blue (for myelin), and Bodian's (for nerve fibers) stains. Multiple sections from various sites in the brain and spinal cord were examined by routine light microscopy. Results Fig. 2. Left eye of monkey 1 on day 35. Fundus photograph (A) and fluorescein angiogram (B) during the late phase showing ODE (reflexes are photographic artifacts). 2. Detailed fundus examination by direct and indirect ophthalmoscopy. 3. Color fundus photography, mostly stereoscopic. 4. Fluorescein fundus angiography. 5. Clinical neurologic evaluation for signs of neurologic disorder. 6. Cerebrospinal fluid was obtained by lumbar or cisternal puncture. It was analyzed by the standard clinical laboratory techniques. 7. Complete blood counts. 8. Recording of the visual evoked potential (VEP). VEP was recorded from a midoccipital electrode referred to the vertex region. Electrodes were silver-silver chloride discs attached with collodion over the scalp. Electrodes were soaked with electrocardiography paste to maintain the impedance at less than 5 Kfl. Flash stimulation was delivered at a rate of 1.5/sec by a Grass S8 photic stimulator. Stroboscope was placed 30 cm away from the eyes. Summation and averaging were accomplished by CAT-400. Sweep time was 250 msec. A total of 200 responses were averaged for each test. The first test was done with normal
Onset. All the animals developed clinical and histopathologic evidence of EAE. Clinically, the optic nerve involvement was seen within 2'/2 to 3Vfc weeks after the sensitization, except in one eye where it was seen on day 31. Simultaneous bilateral optic nerve involvement was seen in four animals. The central nervous system (CNS) lesions developed 2lk to 3Vi weeks after sensitization in animals 3 to 6 and after 5 weeks in animals 1 and 2, Clinically, the optic nerve was involved 2 to 12 days before the CNS involvement in animals 1 to 3; in animals 4 and 5 the CNS was involved 1 to 7 days before the optic nerve; in animal 6 the two developed simultaneously. Optic disc changes Initial changes. These consisted essentially of optic disc edema (ODE) (Figs. 1 and 2) and associated changes. The findings are summarized in Table I. Later changes. The ODE in eyes of animals that survived long enough (all except monkeys 5 and 6) subsided and was replaced by optic atrophy (Table I). The eyes fell into two groups.
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Table I. Optic disc changes Optic disc edema Reached maximum
Optic atrophy
Appeared (day)
Reached maximum
Animal
Eye
Appeared (day)
Day
Grade
Disappeared (day)
1
R L
