Reports Effects of chronic demyelination on axonal nerves showed primary focal demyelination with transport in experimental allergic optic perivasculitis and gliosis. Ultrastructural analysis neuritis. NARSING A. RAO, JOHN GUY, AND of the demyelinated axons revealed swollen axo- plasm with disarray of neurotubules and neu- PAMELAS. SHEFFIELD. rofilaments. In view of these morphologic altera- Axonal transport studies were undertaken to determine tions, the present study of axonal transport was the effect of chronic demyelination on axonal function in undertaken to detect any functional alterations in experimental allergic optic neuritis in the guinea pig, an chronic demyelinating (allergic) optic neuritis. animal model for multiple sclerosis. Fast and slow com- ponents of axonal transport over the prelaminar, lami- Materials and methods. Chronic demyelinating nar, and retrolaminar portions of the optic nerve head optic neuritis was produced in 12 strain 13 guinea 10 and at the foci of demyelination in the retrobulbar optic pigs as previously reported. One eye of each of nerve were evaluated by the autoradiographic grain- these experimental animals received an in- counting technique. At 6 hr there was a significant in- travitreal injection of 50 /xCi of tritiated leucine crease in grain counts over the demyelinated foci and in (L-leucine-5-4; 3H(N), sp. act. 58.5 Ci/mmol; New the regions proximal to the demyelination, including the England Nuclear Corp., Boston, Mass.) in 50 /u,l swollen disc. At day 1 there was no significant difference of sterile normal saline solution. Topical pro- in the grain counts at the site of demyelination when paracaine (E. R. Squibb & Co., Princeton, NJ.) compared to the myelinated portion of the nerve. How- and 0.5 ml of intraperitoneal pentobarbital were ever, at days 3 and 7 there was a decrease in the number used for anesthesia during the injection. Animals of grains over the demyelinated areas. These results indi- were sacrificed by intracardiac injection of 1 ml of cate impairment of axonal function in chronic demyeli- pentobarbital at intervals of 6 hr, 1 day, 3 days, nation. Moreover, in this pathologic process, most of the and 7 days after the intravitreal injection. The eyes synthesized materials appear to move in the fast trans- port phase, unlike in the normal optic nerve where the were enucleated with a segment of optic nerve bulk of materials move by slow transport. (INVEST measuring 6 mm or longer and fixed in 4% formal- OPHTHALMOL VIS SCI 21:606-611, 1981.) dehyde, 1% glutaraldehyde in 200 mOS phos- phate buffer at 4° C. The brain with the cranial Axoplanal transport of macromolecules and cel- portion of the optic nerve was removed from the lular organelles from the neuronal perikaryon to calvarium and fixed in 4% formaldehyde solution. the axon terminal includes a rapid phase moving at The sections with the optic disc and nerve were several hundred millimeters per day and a slow rinsed with 0.2M s-collidine buffer, postfixed in phase moving at a few millimeters per day. With 1% osmium tetroxide in 0.067M s-collidine, dehy- the tritiated amino acid leucine used as a substrate drated through a graded series of ethyl alcohol, for incorporation into synthesis of newly synthe- cleared in acetone, and embedded in Epon. Thin sized proteins, it has been found that the bulk of sections (1 /am) were cut on an ultramicrotome, transported materials normally moves in the slow placed on glass slides, and coated with Kodak phase.' NTB-2 emulsion (Eastman Organic Chemicals, Rochester, N.Y.). After a 3-week exposure, they Many factors have been shown to impair axo- were developed in Kodak D19 for 3 min, fixed in plasmic transport. Fast transport is interrupted by Regular Fix, and stained with toluidine blue. anoxia, by chemical depletion of energy supplied by ATP, mechanical compression,2 and by chemi- Autoradiographic analysis was performed by cal agents that interrupt intracellular micro- grain counting under 400X magnification using a tubules.3- 4 However, temperature increases in 0.5 by 0.5 mm grid (with 25 squares, 0.1 by 0.1 cold-blooded vertebrates as well as biogenic mm) over the prelaminar retina (point A), the monoamines in rabbits5 have been shown to en- lamina choroidalis (point B), the lamina scleralis hance fast axoplasmic transport. In addition, (point C), the postlaminar optic nerve head (point axonal transport is impeded at the scleral lamina in D), and the foci of demyelination (points E to I), experimental models of acute6 and chronic ocular located in the retrobulbar optic nerve. For the hypertension7 as well as in intracranial hyperten- internal controls, grains were counted at compa- sion8 and ocular hypotony.9 rable nondemyelinated areas of the optic nerve. We have previously reported an experimental The second control consisted in counting the model of optic neuritis resembling human mul- grains at the same topographical areas of the optic tiple sclerosis.10 Histopathologically, these optic nerve from normal guinea pigs (external controls) 606 Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 21 Number 4 Reports 607 Fig. 1. Autoradiographs of the guinea pig optic nerve showing increased numbers of grains at the site of demyelination at 6 hr (A) when contrasted against a topographically similar myelin- ated area at the same time interval (B). (Toluidine blue; X600.) at each of the four time intervals mentioned nificant increase (p < 0.01) in grain counts within above. Although grain counts were unbiased and the foci of demyelination when compared to simi- masked as to time interval, we could not mask the lar topographical areas along the nerve showing demyelinated areas within the same section. normal myelin staining (Fig. 1), as well as between Therefore we have built into our protocol an in- the demyelinated foci and normal control (p < ternal as well as an external control. 0.05). Analysis of variance with random block Results. At 6 hr there was a statistically sig- design was the statistical test used on points D to Downloaded from iovs.arvojournals.org on 10/02/2021 Invest. Ophthalmol. Vis. Sci. 608 Reports October 1981 Fig. 2. At day 3 there were a decreased number of grains at the focus of demyelination (A) when compared to a similarly matched myelinated area (B). (Toluidine blue; x600.) H, which represent the start of the myelinated the same at 1 day, and decreased at 3 and 7 days, optic nerve. Grain counts were also increased in no obvious blockade of axonal transport was ap- front of the demyelinated area (prelaminar retina, parent despite obvious disc edema, which was lamina choroidalis, and lamina scleralis) when com- noted in all experimental animals (Fig. 3). pared to areas in front of myelinated optic nerve Discussion. The demyelinating disease of the (Table I). optic nerve seen in chronic experimental optic At day 1 there was no statistically significant neuritis exhibits not only a loss of the myelin coat- difference in grain counts between demyelinated ing from the axon but also morphologic alterations areas and topographically similar myelinated areas within the axoplasm. Ultrastructurally, the de- within the experimental group or between the ex- myelinated axons were found to have swollen perimental group and the control group. axoplasm, with disarray of neurotubules and neu- At day 3 (Fig. 2) there was a statistically sig- rofilaments.l0 nificant decrease in grain counts in front of and Axonal transport is dependent on at least two within the demyelinated area when contrasted factors: (1) incorporation and synthesis within the against topographically similar myelinated areas. neuronal cell body and (2) transport of these syn- This was found both within the experimental thesized proteins from the cell body to the axonal group (p < 0.01) and between experimental and terminal. Any factor(s) that interferes with these control animals (p < 0.01). Statistical significance may affect axonal transport. was achieved from points A to H (Fig. 3.) In the experimental model of demyelinating At day 7 grain counts were slightly decreased optic neuritis, an increased rate of fast transport, within the demyelinated area (p < 0.10). Al- as noted at 6 hr, would give rise to heavier labeling though grain counts were increased at 6 hr, were in the demyelinated areas. However, if only the Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 21 Number 4 Reports 609 I C D £ f OllUnci A 6 C 0 t f G H Fig. 3. Mean grain counts of demyelinated areas compared to controls, at the lamina retinalis (A), lamina choroidalis (B), lamina scleralis (C), retrolaminar area (D), and the foci of demyeli- nation and comparable myelinated areas (E to H), fast phase were affected, slow transport might pro- slow transport phase. This hypothesis would ac- ceed normally, resulting in the same grain counts count for the heavier labeling seen at 6 hr. Assum- as in the comparable control group. This was not ing normal protein synthesis in ganglion cells, the case, since the number of grain counts de- with less labeled material left in the perikaryon to creased at days 3 and 7, which indicates that de- be carried by slow transport, one would expect a myelination causes an increase in the ratio of syn- decreased count at the later intervals because thesized materials moving in fast transport to most of the labeled material had already departed those moving in the slow phase, whereas normally with the fast phase. This explanation would ac- the bulk of synthesized materials moves in the count for the decrease in grain counts seen at 3 Downloaded from iovs.arvojournals.org on 10/02/2021 Invest. Ophthalmol. Vis. Sci. 610 Reports October 1981 Table I. Summary of grain counting data at various intervals at the sites of demyelination and myelinated parts of optic nerve Grains counted 6 hr after 3H* I clay after 3H* Exp.