In vivo test for myelinotoxicity of cerebrospinal fluid

Brain Research, 120 (1977) 103-112

103

© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

IN VIVO TEST FOR M Y E L I N O T O X I C I T Y OF C E R E B R O S P I N A L F L U I D

TAKESHI TABIRA, HENRY DeF. WEBSTER and SHIRLEY H. WRAY Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Md. and the Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. (U.S.A.)

(Accepted May 5th, 1976)

SUMMARY A quantitative double blind procedure is described to test the myelinotoxicity of cerebrospinal fluid (CSF) by using optic nerves of Xenopus tadpoles as an in vivo model of a myelinated CNS tract. Only 0.5 ml of unconcentrated CSF is needed for a test and the result is known in 5 days. Groups of 8-10 Xenopus tadpoles received a subcutaneous injection of 12-13 #1 of a coded CSF sample or of a saline control solution around the right optic nerve. After 48 h, whole mounts of the right optic nerves were prepared and the slides were randomized before using a differential-interference contrast microscope to count the myelin lesions. The myelinotoxicity of a CSF sample was considered positive (@) when it produced significantly higher (P < 0.01) counts than the saline control. When P was less than 0.05, the counts were recorded as borderline ( 2 ) and it was negative (--) when P was > 0.05.

INTRODUCTION Recently, we described a method for using tadpole optic nerves as an in vivo model for studying CNS demyelination quantitatively 24. Substances that are injected perineurally enter the nerve rapidly and surround its myelinated fibers is. If myelin lesions develop, they can be identified and counted by examining a whole mount of the nerve with a differential-interference contrast microscope 24. This method has been modified and used to show that unconcentrated cerebrospinal fluid (CSF) from a majority of patients hospitalized for an acute attack of chronic multiple sclerosis (MS) has myelinotoxic activity 22,2z. The purpose of this brief report is to describe the double blind procedure for testing CSF, to define the distribution of the myelin lesions in nerves of control and CSF injected tadpoles and to show that the lesions produced by perineural CSF injection are restricted mainly to CNS myelin sheaths.

104 MATERIALS AND METHODS

Xenopus tadpoles at stage 52-53 of development 16 were shipped from the A m p h i b i a n Facility (Univ. of Michigan, A n n Arbor, Mich.) by air express. After a 24 h c o n d i t i o n i n g period, shipments were divided into groups o f 100 tadpoles which were m a i n t a i n e d in 20 liter plastic bins c o n t a i n i n g 10-12 liters of dechlorinated, filtered aerated tap water that was changed twice weekly. The tadpoles were fed pea soup

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Fig. 1. Dorsal views of Xenopus tadpoles at stage 54 of development (E: eye, N: nostril). For the injections around the right optic nerve, the epidermis was pierced lateral to the right nostril (X shown in 1A). In 1B, the tip of the No. 33 needle (double arrow) is correctly positioned for an injectionand its relationship to the optic nerve (single arrow) is illustrated. Adjacent peripheral cranial nerves (IllVII) are also shown. Scale bars, 1 ram. Fig. 2. Right optic nerve whole mounts differential-interference contrast illumination, 48 h after a 12.5/~1 injection of a control solution (2A) and of a myelinotoxic CSF from a patient with multiple sclerosis (2B). In 2A, no abnormalities are apparent in the myelin sheaths which appear as if they were longitudinallysectioned. In 2B, there are two typical myelin lesions: the row of 3 large ovoids (left arrow) and the group of many small ovoids (right arrow). Scale bars, 10 #m.

105 powder daily and were used for tests 5-12 days after arrival as they reached stage 54 of development. In each test, groups of 8-10 tadpoles were randomly selected from the same shipment and injected with unconcentrated CSF or a saline control solution. The injection technique and the method used to prepare optic nerve whole mounts were similar to those already reported 24 except for the injection site. After light anesthesia with 0.02 ~ tricaine methanesulfonate, the tadpoles were placed under a dissecting microscope. The epidermis was pricked with a sharp bevelled number 30 needle just lateral to the right nostril (Fig. 1A). Then a blunt number 33 needle attached to a 25 #l syringe was inserted until the tip was next to the nerve at the point where it emerged from the optic foramen in the cranial cartilage (Fig. 1B). At this site, 12-13/~l of a saline control (Holtfreter's solution 19) or of a coded unconcentrated CSF sample were injected slowly. Careful needle positioning and a slow injection rate minimized leakage, optic nerve distortion and damage to adjacent tissue. After 2 days, the tadpoles were perfused with a fixative containing 1 . 5 ~ glutaraldehyde and 0.5 ~ formaldehyde in a 0.08 M phosphate buffer (pH 7.4). They were immersed in the same fixative overnight. Wedges of tissue containing the optic nerves were removed and soaked in a 70 ~ solution of glycerol in the same buffer. While immersed, a dissecting microscope was used to gently separate the optic nerves from surrounding tissue. After an hour of immersion, each nerve was placed in 100 glycerol for 1 h before it was mounted on a slide in a drop of glycerol. To prevent flattening of the whole mount, the coverslip was sealed with nail polish. On many slides in most tests, another cranial nerve (III, V or VII) was mounted beside the optic nerve to compare the incidence of lesions in central and peripheral myelin. In a few tests, spinal cord whole mounts were also prepared and examined. Before examining the slides, the entire set of slides was relabelled and randomized. To count myelin lesions in whole mounts of optic nerves, we used a Zeiss microscope, differential-interference contrast illumination 1, an image magnification of 1000 (40 × objective, 2 × optovar, 12.5 × oculars) and an ocular micrometer with a I0 mm scale. The scale was aligned parallel to the nerve; the central segment used for counts was 760/~m (6 lengths of the scale) from the cranial end of the nerve. There, the length of nerve between the ends of the micrometer scale was examined at each level of focus. We counted all of the myelin lesions (groups of small ovoids and other focal sheath abnormalities) that were 2.5 # m or larger. The lesions in two adjacent regions were counted in the same manner. The total was the number of myelin lesions in a 380/~m centrally located segment of optic nerve. After all of the slides had been examined, they were decoded and the counts were tabulated. In both control and experimental groups the lesion counts fitted the Poisson distribution; therefore, we calculated the square root of the sum of each count and 0.5 (i.e., ~/x q- 1/2). When transformed in this manner, the counts for each group were normally distributed. Student's t-test was then used to look for differences between control and experimental groups. The myelinotoxic activity of a CSF sample was considered positive (q-) when it produced significantly higher ( P < 0.01) lesion counts than the saline control. When P was less than 0.05, the myelinotoxic activity was recorded as borderline (4-) and it was negative (--) when P was > 0.05.

106 One or two tadpoles from each group were prepared for light and electron microscopic study using previously described methods 24. RESULTS AND DISCUSSION The optic nerves of Xenopus tadpoles at stage 54 of development are myelinating rapidly and the nerve in regions selected for counting lesions contained 100-300 myelinated fibers. The appearance of these fibers in whole mounts has been described 17, 24 and is illustrated in Fig. 2A. The myelin sheaths usually have a smooth contour and occasionally nodes of Ranvier can be identified. The appearance of myelin lesions in whole mounts of optic nerves from tadpoles injected with myelinotoxic CSF is shown in Fig. 2B. The most common abnormality was the presence of small ovoids which were randomly distributed along myelin internodes. The optic nerves of normal tadpoles also contained some myelin ovoids. When they were compared with the lesions found in nerves of tadpoles injected with a myelinotoxic CSF or a control solution, no distinctive morphological features were identified that were useful in our double blind tests. When present, the differences between myelin lesions in control and CSF injected groups were quantitative. The above double blind procedure was developed during our third series of CSF experiments and it has been used to test more than 30 CSF samples during a 6 month period. The results for 27 (MS-15; control patients-12) showed that 6 0 ~ of the MS CSFs were positive or borderline and 90 ~o of the CSFs from patients with other neurological diseases were negative. The counts obtained in one of these tests are shown in the top graph (A) of Fig. 3. Similar results were obtained when some of these CSF aliquots were recoded and tested again. This method was used also to retest CSFs that had been tested earlier with tadpoles from another source and with differences in sizes of tadpole groups, volumes injected, and regions of optic nerves used for lesion counts. In spite of these variables, which are described more fully below, CSFs had essentially the same myelinotoxic activity in each series of tests. The above test procedure was selected for routine use because it provided reproducible results in double blind tests and was simpler than earlier procedures. During the development of this method, several parameters of the test procedure were explored. Immediately after shipment, lesion counts in nerves of normal tadpoles were higher than they were 3-7 days later. No seasonal variation was observed and there was no significant difference in the number of lesions found in stage 52-53 and stage 55-56 tadpoles. Tests on groups of 8-10 normal tadpoles also showed a poor correlation between lesion counts in the right and left nerves of the same tadpole. In our first series of tests for CSF myelinotoxicity, there were 6-16 tadpoles (Rare Tropicals, Princeton, New Jersey) in each group; 5/~l of coded CSF or saline were injected around the right optic nerve, and the mye!in lesions were counted in 380/~m segments at the cranial ends of both optic nerves (Fig. 3B). This experiment showed that there was a poor correlation between lesion counts in right and left nerves of tadpoles injected with saline or CSF that did not produce significantly higher lesion counts. A good correlation of lesion counts in the right and left nerves was observed

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Fig. 3. Counts of myelin lesions in 380/~m segments of optic nerves from tadpoles that were normal or were injected with saline or CSF. G r a p h A illustrates raw and transformed counts obtained with the procedure described for the in vivo test (see text). The CSF of N.R., a patient with MS, is myelinotoxic (P < 0.01); CSF from W. M. is negative (P > 0.05). Graph B shows raw counts of myelin lesions in cranial segments of optic nerves on the uninjected (O) and injected (I) sides of 6--11 tadpoles that were normal or were sacrificed 48 h after an injection of 5 / d of saline or CSF. Graph C illustrates raw and transformed counts of myelin lesions in cranial segments of right optic nerves from 38-39 tadpoles, 48 h after an injection of CSF. J. T., myelinotoxic (P < 0.01); I. P., borderline (P < 0.05); G. F., negative (P > 0.05); saline control, not shown.

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Fig. 4. Average counts of myelin lesions in 380/~m segments of right optic nerves from 8 normal tadpoles and 8 injected with 12.5 ~1 of saline or a myelinotoxic CSF from a MS patient. In centrally located segments, the counts are highest and also, there is the greatest difference between counts produced by control and myelinotoxic CSF injections. in groups of tadpoles injected with CSFs that had positive myelinotoxic activity. In all groups of injected tadpoles, the means of transformed counts of lesions in right optic nerves were usually higher than those obtained from left nerves. Nevertheless, the differences were not significant. When lesion counts in nerves of saline and CSF injected tadpoles were compared, the greatest differences were found in the right nerves. Therefore, right optic nerve lesion counts were used to determine if a CSF sample had myelinotoxic activity. Of the 15 CSFs tested in the first series of experiments, (MS-7; control patients-8) 6 0 ~ of the CSF from MS patients were positive or borderline and 80 ~ of those from patients with other neurological diseases were negative. Since the ranges of counts for CSFs with positive and borderline activity were high in the first series, 7 of these CSFs were recoded and tested in the second series of experiments. The groups of tadpoles were larger (20-40), the volume injected was the same and only the right optic nerves were used for lesion counts (Fig. 3C). All 4 CSF samples from MS patients were positive and 2 of the 3 samples from patients with other neurological diseases were negative. The number of counts in each group was large enough to show clearly how the raw and transformed counts were distributed (Fig. 3C). Even though highly significant differences were observed, the procedure was too lengthy to be practical. For the third series of experiments, tadpoles had to be obtained from a different supplier (The Amphibian Facility). The optic nerves were more translucent and it was possible to study the distribution of myelin lesions along the nerves' entire length. Average lesion counts for one experiment are illustrated in Fig. 4 and are representative of our results. The number of lesions was highest in the middle segments of optic nerves from normal tadpoles and from those that received either saline or CSF injections. In repeat tests, we compared lesion counts in cranial and central segments of nerves from tadpoles injected with saline or a positive CSF. There was a much more significant difference in lesion counts in central segments of nerves from the two groups than there was in the cranial ends of the same nerves. CSFs that had been negative were retested in the same manner and lesion counts in the central regions of these nerves did not differ significantly from those in saline controls. Therefore, in this series of experiments and in all subsequent CSF tests, a centrally located 380/zm segment of

Fig. 5. Electron micrographs of longitudinally sectioned right optic nerves removed from tadpoles 48 h after an injection of a myelinotoxic CSF. In 5A, there are three small lamellar ovoids (arrows) and a vacuole (V) that form part of an internodal myelin lesion that was studied in serial sections. No abnormalities were found in the axon (A). In 5B, there is a paranodal myelin lesion characterized by loose lamellar whorls (upper arrow) and swollen lateral loops of a paranodal oligodendrocyte containing smaller ovoids and vacuoles (left arrows). The axon (A), paranodal loops (right arrows) and myelin sheath to the right of the node appear normal. Scale bars, 1 ,nm.

each right optic nerve has been used to count myelin lesions. The mechanisms responsible for the uneven distribution of lesions along optic nerves are not knownas. Perhaps some of the ovoids found in the central segments of normal and control nerves are associated with remodelling or turnover of myelin components in rapidly growing,

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Fig. 6. Whole mounts of nervous tissue removed from tadpoles 48 h after an injection of a myelinotoxic CSF. The right oculomotor nerve is shown in 6A and its peripheral myelinated fibers, including nodes of Ranvier (R), appear normal. In 6B, the neurons, their satellite cells, and the myelinated fibers of the right trigeminal ganglion appear normal. Part of the cervical spinal cord and its root fibers are shown in 6C. The neurons, the small CNS myelinated fibers and the much larger peripheral root fibers at the top of the figure appear normal. Scale bars, 10/~m. c o m p a c t sheaths. These immature sheaths may also be more easily damaged by myelinotoxic C S F than those closer to the chiasm. To determine if axonal and glial cell alterations were associated with the myelin lesions that were observed in whole mounts, we examined sections o f optic nerves that were prepared for phase and electron microscopic study. In electron micrographs, lamellar ovoids were found in internodal and paranodal areas o f focal myelin breakdown (Fig. 5). With rare exceptions, axons in these areas were normaJ and no lymphocytic infiltrates were found in the nerve parenchyma. In m a n y CSF tests, another cranial nerve (lII, V, VII) was mounted beside the optic nerve to compare the incidence of lesions in central and peripheral myelin o f the same tadpole. On these slides, including those with significantly elevated lesion counts in optic nerves, there were almost no abnormalities observed in peripheral myelinated fibers (Fig. 6), or ganglion cells (Fig. 6B) o f adjacent cranial nerves. Since experiments with tracers have demonstrated that perineurally injected substances diffuse rapidly 18, we prepared whole mounts o f spinal cords from a few tadpoles injected with a positive C S F (Fig. 6C). N o abnormalities were found in these C N S myelin sheaths. The concentration o f myelinotoxic CSF reaching these sheaths may have been too low to produce lesions. A n o t h e r possibility is that the observed distribution of C N S myelin lesions is similar to that described in hexachlorophene intoxication 25 and is additional evidence that optic nerve myelin sheaths are more readily damaged by myelinotoxic agents than those elsewhere in the CNS.

111 Studies using CNS tissue cultures have added significantly to our understanding of the pathophysiology of human and experimental demyelinating disease. Bornstein and his collaborators have shown that sera obtained from patients with MS and animals with experimental allergic encephalomyelitis produce demyelination in vitroS,6,L Since then, similar results have been obtained by other investigators2,11,12, 15,20,~6,27 and in addition, there have been reports describing myelination inhibition s, 21, electrical blocking activity 7,1°, and toxic effects on glia a,4. However, in tests of CSF from MS patients, demyelination has only been observed in cultures exposed to concentrated CSF la-15. At present, there is not enough evidence to relate these in vitro effects to the results obtained with our in vivo model 2a. In conclusion, this report describes a new in vivo test for CSF myelinotoxicity that is being used to explore the cellular mechanisms of myelin breakdown in MS. Only 0.5 ml of unconcentrated CSF is required and the result is known in 5 days. ACKNOWLEDGEMENTS The authors wish to thank Dr. Christina M. Richards from the Amphibian Facility at the University of Michigan for her help and advice in the care of the Xenopus tadpoles. Kathryn Winchell and Susan Larrick provided excellent technical assistance; Doris Sadowsky was helpful with the statistical analysis of the data. This study was supported in part by USPHS Grants Nos. NS 11551 and NS 03356 awarded by the National Institute of Neurological and Communicative Disorders and Stroke and also in part by a grant from Fight for Sight, Inc., New York, N.Y.

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