Electron Microscopic Studies of Cerebrospinal Fluid Sediment in Demyelinating Disease Robert M. Herndon, MD, and John Kasckow
Cerebrospinal fluid specimens from 31 patients with multiple sclerosis (MS) and 2 with progressive multifocal leukoencephalopathy (PML) were subjected to ultracentrifugation, and the resulting pellets were examined in an electron microscope. Cell types seen in the pellets included lymphocytes, occasional plasma cells, polymorphonuclear leukocytes, monocytes, eosinophils, lipid-laden macrophages, and fibroblasts. The most interesting noncellular elements were extracellular myelin fragments, recognizable by their characteristic alternation of major dense lines and intraperiod lines. Myelin fragments were seen in the CSF from 7 of 9 patients with MS in exacerbation involving areas other than the optic nerve. These fragments were not observed in 4 specimens from patients with acute attacks manifested by optic neuritis. Myelin fragments were present in 1 of the 2 patients with PML. These observations indicate that a portion of the myelin destruction seen in MS and PML occurs extracellularly, with release of myelin fragments and degradation products into the CSF. Herndon RM, Kasckow J: Electron microscopic studies of cerebrospinal fluid sediment in demyelinating disease. Ann Neurol 4:515-523, 1978
Although multiple sclerosis (MS)may be suspected at the time of the first attack, definite clinical diagnosis in remitting cases usually requires the occurrence of two or more episodes involving distinct areas of the central nervous system and separated in time by a month or more. In progressive cases, diagnosis requires at least six months of slow or stepwise progression [16]. These clinical criteria may be supplemented by the discovery of abnormalities in the cerebrospinal fluid, including pleocytosis, increased IgG, an increased IgG/albumin ratio, oligoclonal bands of IgG o n electrophoresis [ 101. or a combination of these findings. In practice, a firm diagnosis often proves extraordinarily difficult to make, and misdiagnosis is frequent and can be serious Ill. Thus, patients may be subjected to repeated studies, including angiograms, myelograms, and computerized axial tomography-with their attendant risks and expense-in an attempt to establish the cause of the neurological deficit. The cause of MS remains unknown. The evidence for and against viral and immunological theories has been extensively reviewed but remains inconclusive, and the search for a cause continues [ 5 , 11, 181. W e undertook an electron microscopic study of CSF sediment in order to see: (1) if there was any evidence for an infectious agent in the CSF and (2) if
there were any diagnostic features which might lead to new diagnostic approaches in MS. Details of the preparative method and preliminary results have been previously reported [BI.
From the Center for Brain Research and the Department of N e w rology, University of Rochester School of Medicine, Rochester, NY.
Accepted for publication M a y 16, 1978. Address requests Dr Hemdon, Box 601 Center, Rochester, N Y 14642.
Materials and Methods Specimens of CSF were obtained from 38 patients with MS. Diagnosis in the cases of MS in exacerbation was based on the classic remitting course, involving at least two separate areas in separate attacks, with the onset of new neurological symptoms within fourteen days preceding the lumbar puncture. Specimens from clinically inactive cases were also studied. These patients had shown a classic remitting course but had had no new symptoms in the preceding fourteen days. In a few of these cases the diagnosis of definite MS was not made until further attacks had occurred, after the lumbar puncture had been done. The patients with chronic progressive disease had shown at least six months of progression at the time of lumbar puncture. We also studied 2 patients with progressive multifocal leukoencephalopathy (PML), proved by biopsy, in whom the viruses were identified by culture and electron microscopic agglutination procedures. Biopsy results are the subject of a separate report [13]. More than 200 CSF specimens from patients with a variety of other neurological disorders, including cerebral thrombosis, epilepsy, brain tumors, drug withdrawal reaction, aseptic meningitis, and encephalitis, were examined and served as a comparison group. Although many of the specimens were normal
0 ~ ~ - ~ ~ ~ 4 1 ~ 8 1 ~ ~ 0 4 - 0 6@ 0 61978 $ 0 1by. 2Robert 5 M. Herndon
515
Findings on Electron Microscopic Examination of Cerebrospinal Fluid Specimens from 33 Patients with Multiple Sclerosis
Diagnosis
MS in exacerbation MS with acute optic neuritis MS inactive or in remission Chronic progressive MS Suspected MS
Transitional Lymphocytes
Plasma Cells
Ependymal Cells
8
4
2
1
0
4
2
1
1
9
3
10
4
2
0
3
4
1
3
1
0
0
1
2
0
2
0
0
0
Myelin Fragments
Myelin Figures
Neutrophils
EosinophiI.5
9
7
8
6
3
4
0
2
4
13
1
1
5
2
2
1
No.of Patients
Macrophages
in terms of cell count, sugar, and protein values, truly normal controls were not examined. CSF was obtained at the time of lumbar puncture and processed as previously described [8], with two exceptions. In the early part of the study the centrifuge tubes were not sterilized; since both bacteria and mycoplasma were found in some of the specimens, sterilization procedures were instituted. In the latter part of the study, Spurr lowviscosity embedding medium was substituted for Epon because its cutting characteristics are better for pellets. Semithin sections were stained with methylene blue-azure 11. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope. For purposes of measurement, the microscope was calibrated using a carbon grating replica. Enlargements were made, the final magnification was calculated, and measurements were done across three to eight laminae using a magnifying loupe and a scale calibrated at 0.1 mm. Initially, ultracentrifugation at 100,000 g was used rather than one of the gentler methods because we were primarily interested in precipitating any viral element that might be present. This method was then continued because of its success in precipitating the myelin fragments.
Results The results of our examination of 33 CSF specimens from patients with known or suspected MS are s u m marized in the Table; 5 of the 38 specimens submitted either were lost in processing or proved unsatisfactory for examination because of the presence of excessive numbers of erythrocytes or starch granules (glove powder). The pellets consisted of a layer of cells overlaid by a layer of the proteinaceous material, which occasionally contained cell fragments and other debris (Fig 1).
516 Annals of Neurology
Fig I . Section of a CSF pelkt fmm a patient w i t h multipk sckrosis in exacerbation. The bottom layer (right) is composed of cellr, mainly lymphocytes, with a f m macrophages andpolymorphonuckar kukocytes. Adjacent to that layer i s one composed largely of myelin fragments, which are unusually abundant in this pellet, and that layer is overlaid by one composed mainly of proteinaceous debris intermingled w i t h myelin fragments. (Methylme blue-azure I1 stain; X850.)
Vol 4 N o 6 December 1978
Pig 2. Electron micrograph ofthe myelin-containing region of the pelkt shown in Figum 1 . M o s t of the myelin akbri~has brohen down to a point whm it would be impossibk to recognize the periodicity of myelin, though we were able t o j n d a fcu, cleurly identtjable fragments in this specimen. Extensive arrnmulations offragments such as this are distinrtIy unusual. (X9,500.) NoncelIular Constituents
The filamentous portion of the pellet that overlies the cellular portion contained a variety of cell fragments, including occasional free mitochondria or fragments of pinched-off cytoplasm. The most important finding was the frequent presence of clearly recognizable myelin fragments (Figs 2, 3). These could occasionally be visualized on light microscopy (see Fig 1) but were identified by the alternation of major dense and intraperiod lines with a periodicity of 13 to 14 nm, as seen in the electron microscope (see Fig 3). In areas where the fragments were breaking up, the periodicity was sometimes increased to 15 to 17 nm. This appearance establishes the identity of the material as myelin. Myelin fragments were present in the CSF from 7 of 9 patients with exacerbations involving areas other than the optic nerve but were not seen in any of the 4 specimens from patients with exacerbations manifested as optic neuritis. Myelin fragments were present in 1 of 2 specimens from the patients with PML. Other laminated lipids, which may have been derived from
myelin but could have originated from other sources, were also frequently seen both intracellularly in macrophages and extracellularly. They are referred to as “myelin figures” (Fig 4). They were occasionally seen in specimens from other, nondemyelinating neurological diseases and, unlike myelin fragments, d o not appear to be specific for demyelination. Another frequent finding was the presence of clusters of small globules of varying size surrounded by and enmeshed in filamentous material. This picture was seen regularly in patients who had undergone myelography and probably represented residual contrast medium. Radioimmunoassay for myelin basic protein 13, 41 was carried out on stored supernatant fluid from 3 patients. (All the specimens in this series were collected before the radioimmunoassay procedure became available.) Of these 3 . 2 contained myelin fragments and were positive when tested for myelin basic protein; the third was negative on both tests. Cellular Constituents
Small lymphocytes that closely resembled those seen elsewhere in the body were the most common element in all pellets (Figs 1, 5 , 6). They had a diameter of 4 to 7 p in the electron micrographs. Their nuclei were round or indented, and the nuclear chromatin was in clumps. Their cytoplasm contained a few mitochondria, scattered free ribosomes, occasional channels of endoplasmic reticulum, and azurophil granules. Infrequently, small lymphocytes with scat-
Herndon and Kasckow: Electron Microscopy of CSP in MS 517
Pig 3. Tbe characteristic myelin periodicity is unusualIy well preserued in this myelinfragment, foundin a specimen from a pafient with progressive multipfe sclerosis. ( x 155,000.)
Pig 4.Myelinfigures of the type seen in this micrographare a nonspec$cfinding occurring in a variety of neurologicalconditions. They art ofren seen at cellsurfaces but may also lie free in tbe more superfinal Iayers of the pelfet. Tbe characmirtir alternation of major dcnse and intraperiod lines seen in myelin fragments is absent. (~200,000.)
518 Annals of Neurology Vol 4
No
tered microspikes (Fig 5 ) were seen and, occasionally, cells with a uropod and microspikes typical of migrating cells. Rarely, the nuclei of small lymphocytes displayed the characteristic fuzzy tubules which have been called "paramyxovirus-like particles." These probably are dependent upon the condition of the cell at the time of fixation and clearly are not of viral origin [7]. Medium-sized lymphocytes, 8 to 11 I.L in diameter, were also seen commonly, especially in specimens taken during an acute exacerbation (Fig 6). Many of these resembled antibody-producing lymphocytes [2, 171, and some were clearly transitional to plasma cells (see Fig 6). Typical mature plasma cells with an abundant endoplasmic reticulum and a small nucleus with densely clumped chromatin (see Fig 6) were occasionally seen. Neutrophilic leukocytes were present in many of the pellets (Fig 7) and often appeared to be phagocytic. They had a lobulated nucleus with densely aggregated chromatin, an abundant cytoplasm containing variable numbers of specific granules, a modnumber Of and a few scattered cistern= of &Vanulm endodasmic reticulum- Ph4W cytic vacuoles containing partially digested cell debris were common (see Fig 7) and were most frequent in
G December 1978
Fig 5 . Micrograph of a CSF pellet from a patient with multiple sclerosis in exacerbation illustrates the appearance of small lymphocytlr (L),macrophages (M), andan eosinophil (E).The macrophages art?large cells with a convoluted nuckus and a variablh amount ofphagocytizeddrbris. The eosinophilis idrntifid by the pmsence of the characteristic kzrge cosinophilic granuks. (X7,500.)
Herndon and Kasckow: Electron Microscopy of
CSF in MS 519
Fig 6. iCiicr-ographshowing a CSF pellet from u putienc with acute progressice multiple sclerosis. A lymphocyte undergoing mitosis can be seen at the center.Jnst above it is a rather typical p l m a cell (P)with numerous prrrallel channels of grurrulr endoplasmic reticulum. Several traasitional lymphocytes (T) are also visible (x3,OOO.)
F i g 7. Thispolymorphonuclear neutrophilic leukocyte contains numerous mitochondria and a number of specific granules along with sonre phagorytized material (arrows).(L = lymphocyte.) ( x I1,OOO.)
520
Annals of Neurology
Vol 4
cells that were largely degranulated. Eosinophilic leukocytes (see Fig 5 ) were present in 8 cases. These were slightly larger than the small lymphocytes, measuring 7 to 9 in diameter. They had a round or oval nucleus almost filled with dense chromatin aggregates, and their cytoplasm contained large eosinophil granules. They were never numerous, and only rarely was more than one seen in a given pellet. Nonphagocytic monocytes were rare. When present, they were usually oval cells, a bit larger than the lymphocytes, with multiple deep indentations in the nucleus which contained extensive dense aggregates of chromatin, much of it marginated along the nuclear membrane. The cytoplasm contained numerous free ribosomes, sparse granular endoplasmic reticulum, and a small Golgi apparatus. Actively phagocytic macrophages were more common. These were large cells, 11 to 15 p in diameter, with a large, irregular nucleus and marginated chromatin (Fig 8). They had a relatively small juxtanuclear Golgi apparatus, sparse granular endoplasmic reticulum, and scattered mitochondria. A variable number of phagocytic vacuoles containing cel1 debris in various
No 6 December 1978
Fig 8. Lurge macrophage containing a number of hpid bodies and vacuoles. The vacuoles probably contained lipid which waJ dissohed out i n processing. The numerous arrays of cytoplasmic membranes are characteristic of activated macrophages, as is the away of irregular uesicular structures in the cytoplasm. ( x 17,000.)
Herndon and Kasckow: Electron Microscopy of CSF in MS 521
stages of digestion were present. Aggregates of beaded filaments 90 to 120 nm in diameter were frequently found in these mononuclear cells regardless of whether or not they contained phagocytosed material. Pseudopodia were common. Specialized surface projections consisting of a thin film of cytoplasm about 250 nm thick, with the surface membrane o n either side, were also frequently seen. These often appeared to make a complete loop, forming a vacuole enclosing some cell debris, although at other times they appeared to open on the cell surface. This specialization is probably related to the process of phagocytosis.
Discussion Of the 9 specimens examined from patients with MS in exacerbation, 7 contained clearly recognizable myelin fragments, identified by the presence of characteristically alternating major dense and intraperiod lines. This alternation was the only criterion by which true myelin fragments could be reliably distinguished from the nonspecific laminated lipid structures commonly referred to as myelin figures. The latter were seen in 8 of the 9 specimens from patients with acute exacerbations but were considered to be nonspecific since they also occur in a variety of other conditions. Unlike myelin fragments, which were always extracellular, the myelin figures were frequently intracellular. Thus far, in over 200 specimens examined, we have not seen myelin fragments in any nondemyelinating neurological disease. This is not to imply that they will never be found in nondemyelinating diseases, but their occurrence in such other disorders clearly is rare. The 4 patients who had acute exacerbations manifested as optic neuritis were considered separately since the direction of CSF flow would be likely to carry any myelin fragments up over the convexity and away from the lumbar sac. Myelin fragments were not observed in any of these 4 specimens. We first reported the occurrence of myelin fragments in CSF sediment in 1970 [8]. Electron microscopy, though useful as a research tool, proved too cumbersome and expensive to use as a routine diagnostic test. For that reason we tried a variety of approaches, including several stains for light microscopic examination of sediment and the use of immunofluorescent staining with myelin antibody. None of these proved satisfactory, even though myelin fragments could occasionally be recognized in semithin sections of CSF pellets. We therefore reexplored the possibility of using a radioimmunoassay for myelin basic protein for this purpose, even though previous attempts by other groups to detect myelin breakdown products by this method had met with limited success [12, 141. After Cohen, McKhann, and Guarneri [4] developed an assay that 522
Annals of Neurology Vol 4
is sensitive into the nanogram range, we applied it to the detection of myelin in CSF with excellent results [3]. Whitaker [19] also developed a radioimmunoassay for the P1 fragment of myelin basic protein and has obtained similar results. Unless the pellets are serially sectioned- tedious and time-consuming procedure--electron microscopic examination will miss at least some of the cases in which breakdown products of myelin are present. Radioimmunoassay, in addition to being more efficient, appears distinctly more sensitive. The procedure extracts bound basic protein and thus detects any basic protein in the myelin fragments. It also detects basic protein in solution: in our study it picked up basic protein in the supernatant fluid of 2 specimens after centrifugation and removal of the pellet. How d o myelin fragments pass from tissue into CSF? First, we suspect that they are able to pass through breaks in the ependyma, because in 1 case, both myelin fragments and free ependymal cells were found in a specimen of CSF taken two to three days after the onset of an acute internuclear ophthalmoplegia. The finding suggested that a break had occurred in the ependyma overlying the medial longitudinal fasciculus, with passage of myelin fragments into the fourth ventricle. Second, the largest quantities of myelin fragments have been encountered in patients with acute spinal cord lesions, suggesting that the fragments can pass through the pia into the subarachnoid space. We d o not believe they are carried into the CSF by macrophages which then break down, as we have never found recognizable fragments within macrophages. We consistently find more degraded laminated lipids intracellularly. The regular occurrence of free myelin fragments in the CSF in MS has some implications for the disease process. In experimental allergic encephalomyelitis (EAE) the myelin is stripped from the axons by macrophages, a process which might not result in the release of free myelin fragments. This suggestion is supported by the absence of free myelin basic protein in the CSF of sheep with active EAE even though myelin basic protein bound to immunoglobulin has been found [6].This would not necessarily apply to all cases in which stripping occurs if other mechanisms of myelin breakdown are also active, as for example in infection of mice with the JHM strain of mouse hepatitis virus, in which stripping occurs but the primary process is one of oligodendrocyte destruction with secondary demyelination. On the other hand, it is probable that either myelin fragments, soluble basic protein, or fragments of the basic protein molecule released as part of the disease process get out of the CSF into the bloodstream, where they could induce an immunological response. Theoretically, such an autoimmune
N o 6 December 1978
response could account for some of the chronic progressive spinal cases of MS which bear a close resemblance to chronic EAE in the guinea pig [15]. Thus far, no one has been able to establish the consistent presence of an immune reaction to myelin basic protein in MS patients that is not also present in controls. In the initial stages of this investigation we had hoped to detect morphological evidence of a virus in the CSF; however, no such evidence was found. A few structures closely resembling mycoplasma were seen in the earlier specimens. The possibility that MS might be due to a mycoplasma was one that we found intriguing in view of the difficulty in detecting mycoplasma by routine techniques and the neurotoxicity of some of these organisms in animals 191. However, when we sterilized our ultracentrifuge tubes, the organisms no longer appeared in the pellets. O n the basis of our findings we conclude: 1. Fragments of myelin regularly occur free in the CSF in MS and appear to be specific for demyelinating diseases. 2. These fragments may contribute a portion of the myelin basic protein found by the radioimmunoassay procedure, but much of the basic protein remains in solution after the fragments are removed. 3. A number of hematogenous cells occur in the CSF in MS,including lymphocytes, polymorphonuclear leukocytes, lipid-laden macrophages, eosinophils, and, rarely, plasma cells. 4 . There is no good morphological evidence for the presence of a virus or other infectious agent in the CSF in MS. Supported by Grant 11080 from the NINCDS, NIH, and by a grant from the Multiple Sclerosis Society.
References 1. Bauer HJ. Orthner H. Poser S: Neurological aspects of false diagnosis and failure 10 diagnose multiple sclerosis. Adv Neurosurg 2:145-151, 1975
2. Cline MJ: The normal lymphocyte and plasma cell, in The White Cell. Cambridge. MA,Hacvard University Press, 1975. PP 225-303 3. &hen S, Herndon RM,McKhann GM: Radioimmunoassay of myelin basic protein in spinal fluid. An index of active demyelination. N Engl J Med 295:1455-1457, 1976 4. Cohen SR, McKhann GM. G u m e n M: A radioimmunoassay for myelin basic protein and its use for quantitative measurements. J Neurochem 25:371-376, 1975 5. Fraser KB: Multiple sclerosis: a virus disease? Br Med Bull 33~34-39,1977 6. Gutstein HS, Cohen SR: Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis. Science 199:301-303, 1977 7 . Hayano M, Sung JH, Mastri A R “Paramyxovirus-like” intranuclear inclusions occurring in the nervous system in diverse unrelated conditions. J N e w p a t h o 1 Exp Neurol 35:287294, 1976 8. Herndon RM,Johnson M: A method for the electron &KOscopic study of cerebrospinal fluid sediment. J Neuropathol Exp Neurol29:320-330, 1970 9. Hodges GR, Fass RJ. Saslaw S: Cenrral nervous system disease associated with Myropbma pncnmoniac infections. Arch Intern Med 130:277-282, 1972 10. Johnson KP, Nelson BJ: Multiple sclerosis: diagnostic usefulness of cerebrospinal fluid. Ann Neurol 2:425-431, 1977 11. Johnson RT: The possible viral etiology of multiple sclerosis. Adv Neurol 13:l-46, 1975 12. Lennon V, MacKay IR: Binding of lnSImyelin basic protein by serum and cerebrospinal fluid. Clin Exp Immunol 11:595602, 1972 13. Mazlo M, Herndon RM: Progressive mdtifocal leukoencephalopathy: ultrastructural findings in two brain biopsies. Neumpathol Appl Neurobiol 3:323-339, 1977 14. McPherson TA, Gelpin A, &land TP: Radioimmunoassay of CSF for encephalitogenic basic protein: a diagnostic test for MS? Can Med As= J 107:856-859, 1972 15. Raine CS, Snyder D H , Valsamis MP,er al: Chronic experimental allergic encephalomyelitis in inbred guinea pigs: an ultrastructural study. Lab Invest 31:369-380, 1974 16. Schumacher GA, Beebe G, Kibler RF, er al: Problems of experimental trials of multiple sclerosis: report by the Panel on the Evaluation of Experimental Trials of Therapy in Multiple Sclerosis. Ann N Y Acad Sci 122:552-568. 1965 17. Tanaka Y Electron Microscopy of H u m a n Blood Cells. New York, Harper & Row, 1972 18. Weiner LP, Johnson RT, Herndon RM: Viral infections and demyelinating disease. N Engl J Med 288:1102-1110, 1973 19. Whitaker JN: Myelinacing encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology (Minneap) 27:911-920, 1977
Herndon and Kasckow: Electron Microscopy of CSF in MS 523
Cerebrospinal fluid specimens from 31 patients with multiple sclerosis (MS) and 2 with progressive multifocal leukoencephalopathy (PML) were subjected to ultracentrifugation, and the resulting pellets were examined in an electron microscope. Cell types seen in the pellets included lymphocytes, occasional plasma cells, polymorphonuclear leukocytes, monocytes, eosinophils, lipid-laden macrophages, and fibroblasts. The most interesting noncellular elements were extracellular myelin fragments, recognizable by their characteristic alternation of major dense lines and intraperiod lines. Myelin fragments were seen in the CSF from 7 of 9 patients with MS in exacerbation involving areas other than the optic nerve. These fragments were not observed in 4 specimens from patients with acute attacks manifested by optic neuritis. Myelin fragments were present in 1 of the 2 patients with PML. These observations indicate that a portion of the myelin destruction seen in MS and PML occurs extracellularly, with release of myelin fragments and degradation products into the CSF. Herndon RM, Kasckow J: Electron microscopic studies of cerebrospinal fluid sediment in demyelinating disease. Ann Neurol 4:515-523, 1978
Although multiple sclerosis (MS)may be suspected at the time of the first attack, definite clinical diagnosis in remitting cases usually requires the occurrence of two or more episodes involving distinct areas of the central nervous system and separated in time by a month or more. In progressive cases, diagnosis requires at least six months of slow or stepwise progression [16]. These clinical criteria may be supplemented by the discovery of abnormalities in the cerebrospinal fluid, including pleocytosis, increased IgG, an increased IgG/albumin ratio, oligoclonal bands of IgG o n electrophoresis [ 101. or a combination of these findings. In practice, a firm diagnosis often proves extraordinarily difficult to make, and misdiagnosis is frequent and can be serious Ill. Thus, patients may be subjected to repeated studies, including angiograms, myelograms, and computerized axial tomography-with their attendant risks and expense-in an attempt to establish the cause of the neurological deficit. The cause of MS remains unknown. The evidence for and against viral and immunological theories has been extensively reviewed but remains inconclusive, and the search for a cause continues [ 5 , 11, 181. W e undertook an electron microscopic study of CSF sediment in order to see: (1) if there was any evidence for an infectious agent in the CSF and (2) if
there were any diagnostic features which might lead to new diagnostic approaches in MS. Details of the preparative method and preliminary results have been previously reported [BI.
From the Center for Brain Research and the Department of N e w rology, University of Rochester School of Medicine, Rochester, NY.
Accepted for publication M a y 16, 1978. Address requests Dr Hemdon, Box 601 Center, Rochester, N Y 14642.
Materials and Methods Specimens of CSF were obtained from 38 patients with MS. Diagnosis in the cases of MS in exacerbation was based on the classic remitting course, involving at least two separate areas in separate attacks, with the onset of new neurological symptoms within fourteen days preceding the lumbar puncture. Specimens from clinically inactive cases were also studied. These patients had shown a classic remitting course but had had no new symptoms in the preceding fourteen days. In a few of these cases the diagnosis of definite MS was not made until further attacks had occurred, after the lumbar puncture had been done. The patients with chronic progressive disease had shown at least six months of progression at the time of lumbar puncture. We also studied 2 patients with progressive multifocal leukoencephalopathy (PML), proved by biopsy, in whom the viruses were identified by culture and electron microscopic agglutination procedures. Biopsy results are the subject of a separate report [13]. More than 200 CSF specimens from patients with a variety of other neurological disorders, including cerebral thrombosis, epilepsy, brain tumors, drug withdrawal reaction, aseptic meningitis, and encephalitis, were examined and served as a comparison group. Although many of the specimens were normal
0 ~ ~ - ~ ~ ~ 4 1 ~ 8 1 ~ ~ 0 4 - 0 6@ 0 61978 $ 0 1by. 2Robert 5 M. Herndon
515
Findings on Electron Microscopic Examination of Cerebrospinal Fluid Specimens from 33 Patients with Multiple Sclerosis
Diagnosis
MS in exacerbation MS with acute optic neuritis MS inactive or in remission Chronic progressive MS Suspected MS
Transitional Lymphocytes
Plasma Cells
Ependymal Cells
8
4
2
1
0
4
2
1
1
9
3
10
4
2
0
3
4
1
3
1
0
0
1
2
0
2
0
0
0
Myelin Fragments
Myelin Figures
Neutrophils
EosinophiI.5
9
7
8
6
3
4
0
2
4
13
1
1
5
2
2
1
No.of Patients
Macrophages
in terms of cell count, sugar, and protein values, truly normal controls were not examined. CSF was obtained at the time of lumbar puncture and processed as previously described [8], with two exceptions. In the early part of the study the centrifuge tubes were not sterilized; since both bacteria and mycoplasma were found in some of the specimens, sterilization procedures were instituted. In the latter part of the study, Spurr lowviscosity embedding medium was substituted for Epon because its cutting characteristics are better for pellets. Semithin sections were stained with methylene blue-azure 11. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope. For purposes of measurement, the microscope was calibrated using a carbon grating replica. Enlargements were made, the final magnification was calculated, and measurements were done across three to eight laminae using a magnifying loupe and a scale calibrated at 0.1 mm. Initially, ultracentrifugation at 100,000 g was used rather than one of the gentler methods because we were primarily interested in precipitating any viral element that might be present. This method was then continued because of its success in precipitating the myelin fragments.
Results The results of our examination of 33 CSF specimens from patients with known or suspected MS are s u m marized in the Table; 5 of the 38 specimens submitted either were lost in processing or proved unsatisfactory for examination because of the presence of excessive numbers of erythrocytes or starch granules (glove powder). The pellets consisted of a layer of cells overlaid by a layer of the proteinaceous material, which occasionally contained cell fragments and other debris (Fig 1).
516 Annals of Neurology
Fig I . Section of a CSF pelkt fmm a patient w i t h multipk sckrosis in exacerbation. The bottom layer (right) is composed of cellr, mainly lymphocytes, with a f m macrophages andpolymorphonuckar kukocytes. Adjacent to that layer i s one composed largely of myelin fragments, which are unusually abundant in this pellet, and that layer is overlaid by one composed mainly of proteinaceous debris intermingled w i t h myelin fragments. (Methylme blue-azure I1 stain; X850.)
Vol 4 N o 6 December 1978
Pig 2. Electron micrograph ofthe myelin-containing region of the pelkt shown in Figum 1 . M o s t of the myelin akbri~has brohen down to a point whm it would be impossibk to recognize the periodicity of myelin, though we were able t o j n d a fcu, cleurly identtjable fragments in this specimen. Extensive arrnmulations offragments such as this are distinrtIy unusual. (X9,500.) NoncelIular Constituents
The filamentous portion of the pellet that overlies the cellular portion contained a variety of cell fragments, including occasional free mitochondria or fragments of pinched-off cytoplasm. The most important finding was the frequent presence of clearly recognizable myelin fragments (Figs 2, 3). These could occasionally be visualized on light microscopy (see Fig 1) but were identified by the alternation of major dense and intraperiod lines with a periodicity of 13 to 14 nm, as seen in the electron microscope (see Fig 3). In areas where the fragments were breaking up, the periodicity was sometimes increased to 15 to 17 nm. This appearance establishes the identity of the material as myelin. Myelin fragments were present in the CSF from 7 of 9 patients with exacerbations involving areas other than the optic nerve but were not seen in any of the 4 specimens from patients with exacerbations manifested as optic neuritis. Myelin fragments were present in 1 of 2 specimens from the patients with PML. Other laminated lipids, which may have been derived from
myelin but could have originated from other sources, were also frequently seen both intracellularly in macrophages and extracellularly. They are referred to as “myelin figures” (Fig 4). They were occasionally seen in specimens from other, nondemyelinating neurological diseases and, unlike myelin fragments, d o not appear to be specific for demyelination. Another frequent finding was the presence of clusters of small globules of varying size surrounded by and enmeshed in filamentous material. This picture was seen regularly in patients who had undergone myelography and probably represented residual contrast medium. Radioimmunoassay for myelin basic protein 13, 41 was carried out on stored supernatant fluid from 3 patients. (All the specimens in this series were collected before the radioimmunoassay procedure became available.) Of these 3 . 2 contained myelin fragments and were positive when tested for myelin basic protein; the third was negative on both tests. Cellular Constituents
Small lymphocytes that closely resembled those seen elsewhere in the body were the most common element in all pellets (Figs 1, 5 , 6). They had a diameter of 4 to 7 p in the electron micrographs. Their nuclei were round or indented, and the nuclear chromatin was in clumps. Their cytoplasm contained a few mitochondria, scattered free ribosomes, occasional channels of endoplasmic reticulum, and azurophil granules. Infrequently, small lymphocytes with scat-
Herndon and Kasckow: Electron Microscopy of CSP in MS 517
Pig 3. Tbe characteristic myelin periodicity is unusualIy well preserued in this myelinfragment, foundin a specimen from a pafient with progressive multipfe sclerosis. ( x 155,000.)
Pig 4.Myelinfigures of the type seen in this micrographare a nonspec$cfinding occurring in a variety of neurologicalconditions. They art ofren seen at cellsurfaces but may also lie free in tbe more superfinal Iayers of the pelfet. Tbe characmirtir alternation of major dcnse and intraperiod lines seen in myelin fragments is absent. (~200,000.)
518 Annals of Neurology Vol 4
No
tered microspikes (Fig 5 ) were seen and, occasionally, cells with a uropod and microspikes typical of migrating cells. Rarely, the nuclei of small lymphocytes displayed the characteristic fuzzy tubules which have been called "paramyxovirus-like particles." These probably are dependent upon the condition of the cell at the time of fixation and clearly are not of viral origin [7]. Medium-sized lymphocytes, 8 to 11 I.L in diameter, were also seen commonly, especially in specimens taken during an acute exacerbation (Fig 6). Many of these resembled antibody-producing lymphocytes [2, 171, and some were clearly transitional to plasma cells (see Fig 6). Typical mature plasma cells with an abundant endoplasmic reticulum and a small nucleus with densely clumped chromatin (see Fig 6) were occasionally seen. Neutrophilic leukocytes were present in many of the pellets (Fig 7) and often appeared to be phagocytic. They had a lobulated nucleus with densely aggregated chromatin, an abundant cytoplasm containing variable numbers of specific granules, a modnumber Of and a few scattered cistern= of &Vanulm endodasmic reticulum- Ph4W cytic vacuoles containing partially digested cell debris were common (see Fig 7) and were most frequent in
G December 1978
Fig 5 . Micrograph of a CSF pellet from a patient with multiple sclerosis in exacerbation illustrates the appearance of small lymphocytlr (L),macrophages (M), andan eosinophil (E).The macrophages art?large cells with a convoluted nuckus and a variablh amount ofphagocytizeddrbris. The eosinophilis idrntifid by the pmsence of the characteristic kzrge cosinophilic granuks. (X7,500.)
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Fig 6. iCiicr-ographshowing a CSF pellet from u putienc with acute progressice multiple sclerosis. A lymphocyte undergoing mitosis can be seen at the center.Jnst above it is a rather typical p l m a cell (P)with numerous prrrallel channels of grurrulr endoplasmic reticulum. Several traasitional lymphocytes (T) are also visible (x3,OOO.)
F i g 7. Thispolymorphonuclear neutrophilic leukocyte contains numerous mitochondria and a number of specific granules along with sonre phagorytized material (arrows).(L = lymphocyte.) ( x I1,OOO.)
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cells that were largely degranulated. Eosinophilic leukocytes (see Fig 5 ) were present in 8 cases. These were slightly larger than the small lymphocytes, measuring 7 to 9 in diameter. They had a round or oval nucleus almost filled with dense chromatin aggregates, and their cytoplasm contained large eosinophil granules. They were never numerous, and only rarely was more than one seen in a given pellet. Nonphagocytic monocytes were rare. When present, they were usually oval cells, a bit larger than the lymphocytes, with multiple deep indentations in the nucleus which contained extensive dense aggregates of chromatin, much of it marginated along the nuclear membrane. The cytoplasm contained numerous free ribosomes, sparse granular endoplasmic reticulum, and a small Golgi apparatus. Actively phagocytic macrophages were more common. These were large cells, 11 to 15 p in diameter, with a large, irregular nucleus and marginated chromatin (Fig 8). They had a relatively small juxtanuclear Golgi apparatus, sparse granular endoplasmic reticulum, and scattered mitochondria. A variable number of phagocytic vacuoles containing cel1 debris in various
No 6 December 1978
Fig 8. Lurge macrophage containing a number of hpid bodies and vacuoles. The vacuoles probably contained lipid which waJ dissohed out i n processing. The numerous arrays of cytoplasmic membranes are characteristic of activated macrophages, as is the away of irregular uesicular structures in the cytoplasm. ( x 17,000.)
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stages of digestion were present. Aggregates of beaded filaments 90 to 120 nm in diameter were frequently found in these mononuclear cells regardless of whether or not they contained phagocytosed material. Pseudopodia were common. Specialized surface projections consisting of a thin film of cytoplasm about 250 nm thick, with the surface membrane o n either side, were also frequently seen. These often appeared to make a complete loop, forming a vacuole enclosing some cell debris, although at other times they appeared to open on the cell surface. This specialization is probably related to the process of phagocytosis.
Discussion Of the 9 specimens examined from patients with MS in exacerbation, 7 contained clearly recognizable myelin fragments, identified by the presence of characteristically alternating major dense and intraperiod lines. This alternation was the only criterion by which true myelin fragments could be reliably distinguished from the nonspecific laminated lipid structures commonly referred to as myelin figures. The latter were seen in 8 of the 9 specimens from patients with acute exacerbations but were considered to be nonspecific since they also occur in a variety of other conditions. Unlike myelin fragments, which were always extracellular, the myelin figures were frequently intracellular. Thus far, in over 200 specimens examined, we have not seen myelin fragments in any nondemyelinating neurological disease. This is not to imply that they will never be found in nondemyelinating diseases, but their occurrence in such other disorders clearly is rare. The 4 patients who had acute exacerbations manifested as optic neuritis were considered separately since the direction of CSF flow would be likely to carry any myelin fragments up over the convexity and away from the lumbar sac. Myelin fragments were not observed in any of these 4 specimens. We first reported the occurrence of myelin fragments in CSF sediment in 1970 [8]. Electron microscopy, though useful as a research tool, proved too cumbersome and expensive to use as a routine diagnostic test. For that reason we tried a variety of approaches, including several stains for light microscopic examination of sediment and the use of immunofluorescent staining with myelin antibody. None of these proved satisfactory, even though myelin fragments could occasionally be recognized in semithin sections of CSF pellets. We therefore reexplored the possibility of using a radioimmunoassay for myelin basic protein for this purpose, even though previous attempts by other groups to detect myelin breakdown products by this method had met with limited success [12, 141. After Cohen, McKhann, and Guarneri [4] developed an assay that 522
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is sensitive into the nanogram range, we applied it to the detection of myelin in CSF with excellent results [3]. Whitaker [19] also developed a radioimmunoassay for the P1 fragment of myelin basic protein and has obtained similar results. Unless the pellets are serially sectioned- tedious and time-consuming procedure--electron microscopic examination will miss at least some of the cases in which breakdown products of myelin are present. Radioimmunoassay, in addition to being more efficient, appears distinctly more sensitive. The procedure extracts bound basic protein and thus detects any basic protein in the myelin fragments. It also detects basic protein in solution: in our study it picked up basic protein in the supernatant fluid of 2 specimens after centrifugation and removal of the pellet. How d o myelin fragments pass from tissue into CSF? First, we suspect that they are able to pass through breaks in the ependyma, because in 1 case, both myelin fragments and free ependymal cells were found in a specimen of CSF taken two to three days after the onset of an acute internuclear ophthalmoplegia. The finding suggested that a break had occurred in the ependyma overlying the medial longitudinal fasciculus, with passage of myelin fragments into the fourth ventricle. Second, the largest quantities of myelin fragments have been encountered in patients with acute spinal cord lesions, suggesting that the fragments can pass through the pia into the subarachnoid space. We d o not believe they are carried into the CSF by macrophages which then break down, as we have never found recognizable fragments within macrophages. We consistently find more degraded laminated lipids intracellularly. The regular occurrence of free myelin fragments in the CSF in MS has some implications for the disease process. In experimental allergic encephalomyelitis (EAE) the myelin is stripped from the axons by macrophages, a process which might not result in the release of free myelin fragments. This suggestion is supported by the absence of free myelin basic protein in the CSF of sheep with active EAE even though myelin basic protein bound to immunoglobulin has been found [6].This would not necessarily apply to all cases in which stripping occurs if other mechanisms of myelin breakdown are also active, as for example in infection of mice with the JHM strain of mouse hepatitis virus, in which stripping occurs but the primary process is one of oligodendrocyte destruction with secondary demyelination. On the other hand, it is probable that either myelin fragments, soluble basic protein, or fragments of the basic protein molecule released as part of the disease process get out of the CSF into the bloodstream, where they could induce an immunological response. Theoretically, such an autoimmune
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response could account for some of the chronic progressive spinal cases of MS which bear a close resemblance to chronic EAE in the guinea pig [15]. Thus far, no one has been able to establish the consistent presence of an immune reaction to myelin basic protein in MS patients that is not also present in controls. In the initial stages of this investigation we had hoped to detect morphological evidence of a virus in the CSF; however, no such evidence was found. A few structures closely resembling mycoplasma were seen in the earlier specimens. The possibility that MS might be due to a mycoplasma was one that we found intriguing in view of the difficulty in detecting mycoplasma by routine techniques and the neurotoxicity of some of these organisms in animals 191. However, when we sterilized our ultracentrifuge tubes, the organisms no longer appeared in the pellets. O n the basis of our findings we conclude: 1. Fragments of myelin regularly occur free in the CSF in MS and appear to be specific for demyelinating diseases. 2. These fragments may contribute a portion of the myelin basic protein found by the radioimmunoassay procedure, but much of the basic protein remains in solution after the fragments are removed. 3. A number of hematogenous cells occur in the CSF in MS,including lymphocytes, polymorphonuclear leukocytes, lipid-laden macrophages, eosinophils, and, rarely, plasma cells. 4 . There is no good morphological evidence for the presence of a virus or other infectious agent in the CSF in MS. Supported by Grant 11080 from the NINCDS, NIH, and by a grant from the Multiple Sclerosis Society.
References 1. Bauer HJ. Orthner H. Poser S: Neurological aspects of false diagnosis and failure 10 diagnose multiple sclerosis. Adv Neurosurg 2:145-151, 1975
2. Cline MJ: The normal lymphocyte and plasma cell, in The White Cell. Cambridge. MA,Hacvard University Press, 1975. PP 225-303 3. &hen S, Herndon RM,McKhann GM: Radioimmunoassay of myelin basic protein in spinal fluid. An index of active demyelination. N Engl J Med 295:1455-1457, 1976 4. Cohen SR, McKhann GM. G u m e n M: A radioimmunoassay for myelin basic protein and its use for quantitative measurements. J Neurochem 25:371-376, 1975 5. Fraser KB: Multiple sclerosis: a virus disease? Br Med Bull 33~34-39,1977 6. Gutstein HS, Cohen SR: Spinal fluid differences in experimental allergic encephalomyelitis and multiple sclerosis. Science 199:301-303, 1977 7 . Hayano M, Sung JH, Mastri A R “Paramyxovirus-like” intranuclear inclusions occurring in the nervous system in diverse unrelated conditions. J N e w p a t h o 1 Exp Neurol 35:287294, 1976 8. Herndon RM,Johnson M: A method for the electron &KOscopic study of cerebrospinal fluid sediment. J Neuropathol Exp Neurol29:320-330, 1970 9. Hodges GR, Fass RJ. Saslaw S: Cenrral nervous system disease associated with Myropbma pncnmoniac infections. Arch Intern Med 130:277-282, 1972 10. Johnson KP, Nelson BJ: Multiple sclerosis: diagnostic usefulness of cerebrospinal fluid. Ann Neurol 2:425-431, 1977 11. Johnson RT: The possible viral etiology of multiple sclerosis. Adv Neurol 13:l-46, 1975 12. Lennon V, MacKay IR: Binding of lnSImyelin basic protein by serum and cerebrospinal fluid. Clin Exp Immunol 11:595602, 1972 13. Mazlo M, Herndon RM: Progressive mdtifocal leukoencephalopathy: ultrastructural findings in two brain biopsies. Neumpathol Appl Neurobiol 3:323-339, 1977 14. McPherson TA, Gelpin A, &land TP: Radioimmunoassay of CSF for encephalitogenic basic protein: a diagnostic test for MS? Can Med As= J 107:856-859, 1972 15. Raine CS, Snyder D H , Valsamis MP,er al: Chronic experimental allergic encephalomyelitis in inbred guinea pigs: an ultrastructural study. Lab Invest 31:369-380, 1974 16. Schumacher GA, Beebe G, Kibler RF, er al: Problems of experimental trials of multiple sclerosis: report by the Panel on the Evaluation of Experimental Trials of Therapy in Multiple Sclerosis. Ann N Y Acad Sci 122:552-568. 1965 17. Tanaka Y Electron Microscopy of H u m a n Blood Cells. New York, Harper & Row, 1972 18. Weiner LP, Johnson RT, Herndon RM: Viral infections and demyelinating disease. N Engl J Med 288:1102-1110, 1973 19. Whitaker JN: Myelinacing encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology (Minneap) 27:911-920, 1977
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