Report on the second symposium concerning cerebrospinal fluid

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Society Proceedings R E P O R T O N THE SECOND SYMPOSIUM C O N C E R N I N G CEREBROSPINAL F L U I D (Miinster, Westphalia, July 4-6, 1963) The First Symposium devoted exclusively to Cerebrospinal Fluid was held in Hamburg (Germany) in 1960 (BAUER 1961). On the occasion of the second, about 40 participants from Belgium, the Netherlands, Sweden, Switzerland and Germany met in the Psychiatric and

Neurologic Clinic of the University of Miinster. Westphalia (Germany) 4th - 6th July 1963. They were welcomed by Professor Mauz, Director of the Clinic. The symposium was subdivided into 3 topics: the proteins, the enzymes and the cytology of the cerebrospinal fluid.

Proteins Considerable differences in the total protein values (Cu-Folin method) and colloid curves were found in different age groups by Wiithrich and Rieder (Basel). Investigating approximately 1200 samples of lumbar CSF from neurological patients, 51 ~ of the patients over 50 years of age and only 34 ~ of the younger patients showed pathological values of total protein. This difference was evident even in the diagnostically uniform groups, such as lumbar disc herniation. On the other hand, no age differences were found in 63 lumbar and 20 suboccipital samples of normal individuals. In general, males showed a greater tendency toward abnormal protein values, but statistically significant sex differences were found only in multiple sclerosis (1963). Sex differences with a higher mean value of total protein in males were found by Dencker (1962, 1963b) in 1600 psychiatric patients. But the Swedish males over 50 years of age showed a decrease of higher values of total proteins. To explain these differences geographical factors together with different disease entities were considered and discussed. In addition Dencker investigated the CSF proteins by paper electrophoresis in 29 males and 33 females without neurological symptoms. He found no differences in the mean values, only the standard deviations were a little greater in males. According to Berg and G~Stz (Erlangen) there are only quantitative differences in the electrophoretic protein pattern of serum and CSF with the exception of the prealbumins and the fitfraction. These differences concern especially the macroglobulins (1961). Special antisera allow the localisation of the lipid protein fractions. The results of staining methods seem to indicate that o~l-lipid proteins contain compo-

nents different from the other lipid proteins. Bammer (Wiirzburg) pointed out that the fllc-globulin is a physiological component in CSF. But it is important not to delay examination of the CSF (1963). In examinations made within 24 hours of spinal puncture this worker found fllc-globulin in 84 ~o of pathological CSF (from various neurological diseases) and in 760~;; of normal CSF, whereas in investigations after 24 hours identification was possible only in 19 and in 1 4 ~ of the samples respectively in spite of refrigerator storage at 4°C. In view of the different protein fractions in CSF, the y-globulins were discussed mostly. Frick (Munich) reported on fluorescence microscopical results using the method of Coons. Slides of guinea-pigs' brain and spinal cord were made and covered with a y-antiserum labelled with fluorescein-iso-thiocyanate. Normally a fine granulated fluorescing material was found only around the capillaries and little veins, probably indicating the presence of serum y-globulins. In animals affected with vaccinemeningitis or allergic encephalomyelitis, however, the cytoplasma of cells infiltrating the meninges and brain showed a great quantity of y-globulin. These results indicate that an intrathecal formation of y-globulins occurs in inflammatory diseases of the central nervous system. Quantitative immuno-studies on the CSFspecific yl-globulins were made by Dencker in agar gel (1963a). Using equal quantities of total protein from about 50 samples of lumbar CSF he noted considerable differences in various diseases. Precipitation in high degrees of dilution of the anti-CSF antigen (1:128, t :64, 1:52) had already been noted in psychiatric diseases. epilepsy and polyneuritis. Samples from patients with multiple sclerosis and amyotrophic lateral sclerosis showed precipation in dilutions from 1:32 to 1:8. In disc herniation, neurosypbilis, purulent meningitis and tumours a precipitation was found only in low degrees of dilution (1:8. 1:4, 1:2) These great quantitative differences are probably of clinical importance. In discussion of these results Frick demonstrated slides showing a characteristic y-fraction in CSF by the reaction of a CSF-protein antiserum. But absorption by y-globulins (from blood in very high concentration) effected a disappearance of this ;,-fraction. Therefore this ~,-fraction is

J. neurol. Sci. (1964) 1 : 590-598

SOCIETY PROCEEDINGS present in the blood in low concentrations too, and not a specific component of CSF, Dencker and Svenilson (Lund) then reported on the increase of the anodal part of 7S ?-globulin. In lumbar CSF from 24 patients with tumours of the central nervous system and from 47 cases of multiple sclerosis this fraction was increased in about 60 ~ of the investigated samples. But in tumours most of the precipitates of 7S ?-globulin were to be found in low values of total ?-globulin, whereas in multiple sclerosis precipitates were to be seen in high values of total ?-globulin. In addition two more ?-globulin precipitates, named ?E (extra) and ?c (cathode), were identified. These differences may be a sign of different origins of ?-globulin, in these two diseases. Perhaps in multiple sclerosis there is an intrathecal formation of ?-globulin, in turnouts however a damage in the blood-CSF barrier might be considered. L6wenthal, Karcher and van Sande (Berchem-Anvers) reviewed large series of electrophoretic runs on proteins of the CSF and serum from patients affected with infectious diseases and tumours of the nervous system. They have been able to demonstrate two different types of augmentation of the ?-globulins in the CSF: one in the form of a diffusely enlarged zone (tumour, Gulllain-Barr6 syndrome and meningeal diseases) and another type in the form of fragmentation of the ?-globulins (disseminated sclerosis, neurosyphilis and subacute sclerosing leucoencephalitis). The same fragmentation of the ?-globulins was found in the serum of patients affected with subacute sclerosing leucoencephalitis (1962). In the CSF of patients affected with this disease a spot staining with amidoblack was also demonstrated at the site of the fast fl-globulins and there was a relative increase of the r-globulins as compared with the ill-globulins. In patients affected with tumours of the nervous system on the other hand, there is a relative decrease of the r-globulins compared with the ill-globulins. In 4 cases of subacute sclerosing leucoencephalitis Booij (Amsterdam) had also found a considerable increase of the ?-globulins in the CSF. The ?-globulins increased only partially and in small degree in the serum. By agar electrophoresis this author distinguished 3-4 ?-subfractions in the CSF, and in the serum only 2 ?-subfractions could be separated by paper electrophoresis. Ursing (Lund) reported on immuno-electrophoretic studies in the CSF of 19 patients affected with bacterial meningitis. The increase of proteins in the CSF in this type of damage of the blood-CSF barrier was shown to be due not only to an increase of normally occurring proteins but also to the occurrence of highmolecular proteins as fibrinogen, ~x2-macro,

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fll-lipo- and fl2-macroglobulins. These findings of barrier damage persisted for up to 6 months in a case of tuberculous meningitis and at least for 2 months in pneumococcal and meningococcal meningitis. The persisting increase of fl2-macroand ?-globulins in the CSF in prolonged meningitis accompanied by a normal serum argues in favour of an intrathecal immunoglobulin production. Habeck, Lenz and Paal (Miinster) investigated the CSF of 83 neurological and psychiatric patients after poliomyelitis inoculation by Sabin's method (1963). Non-specific findings of the type seen in poliomyelitis (pleocytosis, increase of total protein, colloidal reactions) were demonstrated only occasionally and not in any marked degree. A certain accumulation, however, of increased albumin and decreased fl-globulin in paper electrophoresis was seen as long as 4-5 weeks after the inoculation. With regard to the specific findings after the inoculation by type 1 the number of samples showing neutralizing antibodies towards type 3 was a little greater than towards type 1 (in four cases there was even an increasing titer towards type 3). Sporadically antibodies were to be found in the blood only or in the CSF only. An intrathecal production of antibodies therefore could be considered. In the discussion Guadbeck (Homburg-Saar) remarked that there may be great differences of permeability even for similar substances in the blood-brain barrier. Berg doubted any intrathecal production of antibodies Berg and Braunhofer (Erlangen) demanded that the findings in examinations of the CSF in pathological case must be critically evaluated against the norm for healthy individuals. In this way it is possible to demonstrate an increase of fl-globulins in the CSF of epileptic patients. Habeck supported and interpreted these frequent findings in epilepsy. In addition Machetanz and Habeck (Miinster) investigated the CSF of 19 children affected with congenital heart failure. An increase of fl-globulins was found only in those children who showed a considerable cyanosis. Brain hypoxia therefore may be a possible cause of an increase of fl-globulins in the CSF. Using pneumoencephalograms, Habeck (1962) noted in 515 patients that in cases with displacement of ventricles, enlargement of the third ventricle or hydrocephalus externus the most abnormal CSF findings by paper electrophoresis were present. Progressive diseases of the brain in these cases frequently should be suspected. Most of the normal electrophoretical states were found in cases of congenital deformities or asymmetricaJ findings in the brain (displacements excepted). In these cases nonprogressive lesions of the

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brain have to be suspected. In 5 patients showing typical symptoms of an involutional disease of the brain a considerable increase of fl-globulins was found in spite of normal air encephalograms. Habeck and Suwelack (Miinster) noted in 484 cases that the most abnormal CSF findings corresponded with EEG findings of exceedingly abnormal slow activity and of ~- or ~%foci. Patients with epileptic seizure discharges frequently showed an increase of the albumin fraction as related to the time of the occurrence of the epileptic seizure. This "peri-ictal" increase of albumin may be interpreted as a temporary disturbance in the blood-CSF barrier. As the blood-CSF barrier had been discussed during the first symposium this subject was now touched on only by Hemmer, Marget and Hiendl (Freiburg-Breisgau). They investigated the effect of vaso-active drugs on the CSF level of chloramphenicol in normal persons. The additional injection of Eupaverin forte ® increased the chloramphenicol level up to nearly 100 %. The effect of Ronicol compositum ® and Priscol ® was smaller.

Enzymes The CSF and sera of about 200 neurological patients were investigated by Delank (Bochum) in relation to values of lactate dehydrogenase (LDH), malate dehydrogenase, creatine phosphokinase and some metabolites, such as pyruvic acid, o~-keto glutaric acid, and adenosin triphosphate(test combination Boehringer, Mannheim). Creatine phosphokinase was not found in the CSF. The enzyme activity in the CSF did not seem related to the age of the patients or to the content of cells in the CSF, but there was relation to the total protein values. The author supposed that there are also relations to the enzyme activity in the serum, at least when the serum activities were highly increased. Three constellations in the CSF became apparent: (1) in epileptic seizures only an increase of the enzyme activities; (2) in inflammatory diseases an increase of the enzyme activities and the metabolites in relation to the increase of the total protein; (3) in disseminated encephalomyelitis an increase of metabolites and a decrease of enzyme activities without relation to the total protein; in 7 of 25 cases no L D H activity was found at all (1963). Analyzing LDH-isoenzymes in CSF, sera, leucocytes and brain extracts, van der Helm (Santpoort near Haarlem) summarized the following conclusions. No relation exists between the LDH-activities in the serum and the CSF, because changes of LDH-isoenzymes in the serum were not accompanied by changes in the CSF. In CSF pleoeytosis, however, the LDH-

increase in the CSF can be explained by a release of the enzyme from the cells. We cannot expect any marked isoenzyme changes in the CSF if the brain tissue should lose LDH into the CSF or if plasma LDH should pass into CSF. The LDHisoenzyme analysis may have a diagnostical significance in carcinoma of the brain (19631. Studies of LDH-isoenzymes, according to the method of van der Helm were also reported by LOwenthal, Karcher and van Sande (Berchem-Anvers). Investigating the nervous tissue they found variations in the quantitative distribution. In the spinal cord the fastest fractions, especially the first fraction, were predominant. In the peripheral nerves on the other hand. the slowest fraction was by far the most pronounced. The results obtained from the spinal cord are comparable to those obtained by electrophoresis of the spinal cord hydrosoluble proteins, where a fast ~x-globulin fraction with the same mobility as the first fraction of the LDH-isoenzymc clearly predominated. The authors compared these results to the observations made in vascular syndromes of the brain. The CSF examined early in the developmenl of the disease showed a fast co-globulin fraction with the same mobility as the fast s-globulin of the spinal cord and ihe first fraction of the LDH-isoenzyme. These observations might originate new research of clinical use. Bischoff (Ziirich) stressed that the results and interpretations of enzyme findings still differ. Incompatible results may be caused by differences in method and in the choice of patients, by airencephalographic effects or by microhaemorrhages into the CSF. Investigations of the LDH activity (test combination Boehringer, Mannheim) in 170 samples of CSF by Kistler and Bischoff (1962) showed, that increase of age was not accompanied by increase of LDH activity. There was neither a relation of the LDH activity to the content of cells nor a statistically significant correlation to the total protein and globulin values. A statistically significant change of LDH activity characteristic of any disease was still not to be found. An occasional increase of LDH activity was seen in some diseases, e.g.. in vascular diseases of brain up to 25 times the values of the norm. Frick confirmed these results. In contradistinction to Delank, his own investigations of inflammatory diseases, especially multiple sclerosis, had not shown any decrease of LDH activity.

Cytology A simple, quick and careful way of demonstrating all the cells of a sample is the so-called Zellenfangverfahren (cell-catch-method), developed by Simon and Schr/Ser "(Wiirzburg). In this method the physiological coagulation is

.f. neurol. Sci. (1964) 1:590-598

SOCIETY PROCEEDINGS imitated by the addition of fibrinogen and thrombin to 1 to 3 ml of the sample CSF. The report on this method (1963) with an effective demonstration of slides was followed by a discussion on the question whether this new method is also practicable for cell counting in the CSF. Kalm (Dortmund) demonstrated lantern slides obtained by phase contrast microscopy. In this way living cells of CSF can be distinguished very quickly in negative-like representation, e.g., lymphocytes and granulocytes in meningitis or tumour cells. But the author once more stressed the precautions necessary in the diagnosis of tumour cells. Hrazdira (Brno) sent a paper on the phagocytic action of CSF cells. He found macrophagic giant cells (100-400 in width) with thick membrane-like peripheral structures and filled with many phagocytized cells. These macrophages are not identical with the macrophages phagocytizing red cells (described by Sayk and Wieczorek). They were to be found mostly in normal samples of CSF accompanied by a small number of autolyzed and non-phagocytized cells. The author therefore thinks that the macrophages phagocytize the cells just at the beginning of their disintegration. Thus a noxious effect of released enzymes might be prevented and therefore these macrophages may be termed 'regulatory macrophages'. Bischoff (Ziirich) reported on the LDH activity in CSF cells and their importance for the free L D H activity in the CSF. The LDH level in the CSF has to be maintained by parts of the central nervous system in a further sense because according to Fleischer et al. (1956) and Lieberman et al. (1957) the intact blood-brain barrier is impermeable to enzymes of the serum. In spite of a continuous splitting of LDH in the CSF the constant L D H level suggests that a great deal of LDH has its origin in cells of a short duration of life or in cells reversibly damaged. But Kistler and Bischoff (1962) could not verify any statistically significant correlation between the cell content and LDH activity up to 20 cells/mm 3. They therefore investigated the intracellular LDH activity in 136 samples according to the histochemical method of Pearse by the formation of formazane. Reticular cells only showed a striking activity of LDH. The histochemical reaction seems always to be localized near or in the mitochondria. A statistically significant correlation between the number of LDH-active reticular-cells and the activity of free LDH in the CSF did not seem to exist. Tumor cells showed a considerable and sometimes a very massive LDH activity. Most of the reports were followed by lively discussions throughout the Symposium, which

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indicated that several questions remain unanswered and that some communicated findings are still contested. The participants agreed to meet again in 1966 to continue their discussions. Summary

The reports and discussions of this Symposium on the CSF are summarized as follows. (1) There is still today no general agreement about the values of CSF total proteins. It is probable that males show values a little higher than females and that there is a certain increase of these values in advanced age. (2) The less refined method of paper electrophoresis and the finer method of agar electrophoresis of CSF proteins offer diagnostic clues apparently as yet not fully utilized in the clinic. Progressive disease of the central nervous system (non-inflammatory diseases included) often shows pathological changes in the CSF, whereas definite lesions mostly do not. There is a diffuse increase of ~,-globulins in tumours, meningitis, and in the Guillain-Barr6 syndrome, whereas some different 7-subfractions are increased in disseminated sclerosis, neurosyphilis, and subacute sclerosing leucoencephalitis. (3) Perhaps it will be possible to find more specific results in certain diseases by means of immuno-electrophoresis. Up to now many protein fractions of the serum could also be found by this method in the normal CSF. Some fractions of the serum were seen in the CSF but only in pathological conditions and this finding may indicate a damage of the blood-brain barrier. Some fractions seem to occur only in the CSF which might suggest their intrathecal formation. (4) In additon to a qualitative immunoelectrophoretic identification, the quantitative determination of CSF protein fractions may gain diagnostic importance, as has been demonstrated in 71- and 77S-globulins. (5) With regard to the origin of CSF proteins, the use of a 7-globulin conjugated with a fluorescing dye showed an intrathecal production of 7-globulins in vaccinal meningitis and allergic encephalomyelitis of guinea pigs. (6) The results and the diagnostic values of CSF enzyme studies are still contested. There are controversies about the correlations of the CSF enzyme activity with the cell content, the total protein value and the serum enzyme level. Clinically the occurrence of a decrease of L D H activity in multiple sclerosis is also contested. The investigation of iso-enzymes seems to have gained more importance. (7) The preparation of satisfactory slides of CSF cells no longer presents difficulties. This fact should aid greatly in diagnosis and in the investigation of special problems, e.g. for the differenJ. neurol. Sci. t1964) 1 : 590-598

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tiation of macrophages or the identification of enzymes. D. HABECK AND H. E. KEHRER

Re]erences BAMMER, H. (1963) Nachweis des tim-Globulins als normaler Bestandteil des Liquors durch lmmunoelektrophorese, Klin. Wschr., 41 : 1084. BAUER, H. (1961) Cerebrospinal fluid(report on a symposium), Wld Neurol., 2:254. BERG, G., H. G6TZ AND F. SCHEIFFARTH (1961) Das Proteinmuster des normalen und pathologischen Liquor cerebrospinalis, Med. Welt, 1961:369. DELANK, H. W. (1963) Enzyme des Kohlenhydratstoffwechsels im Liquor cerebrospinalis bei neurologischen Erkrankungen, Dtsch. Z. Nervenheilk., 184:632. DENCKER, S. J. (1962) Variation of total cerebrospinal fluid proteins and cells with sex and age, Wld Neurol., 3:778. DENCKER, S. J. (1963a) Studies on specific cerebrospinal y-globulin components, Acta neurol, scand., 39 (Suppl. 4):317. DENCKER, S. J. (1963b) Studies on variation of CSF total proteins and white ceils with sex and age, Kgl. Fysiograf. Siillskap. Lurid Forh., 33:1. FLEISHER, G. A., K. G. WAK1N, M. L. HANSON, M. J. AXELSON AND R. ARNS (1956) Transaminase in canine serum and cerebrospinal fluid after carbon tetrachloride poisoning and injection of transaminase concentrates, Proc. Mayo Clin., 31:640. HABECK, D. (1962) LiquoreiweiBbild und Pneumencephalogramm, Psychiat. Neurol. med. Psychol. (Lpz.), 14:185. HABECK, D., G. PAAL AND E. LENZ (1963)

Liquoruntersuchungen nach oraler Pol,,~myelitisschutzimpfung (Sabin) bei Erwach~ senen, Arch. Psychiat. Nervenkr., 204:389 HELM, H. J, VAN DER, H. A. ZONDAG AND lz'. KLEIN (1963) On the source of lactic dehydrogenase in cerebrospinal fluid. C/in. chim. Acta, 8:193. KISTLER, G. AND A. B1SCHOFF (1962) Zur exfoliativen Zytologie kleiner Flfissigkeitsmengen, Schweiz. reed. Wschr., 92:863. LIEBERMANN, J., O. DAIBER, S. J. DULKIN, 0 . [~. LOBSTEIN AND M. R. KAPLAN (1957) Glutamic

oxalacetic transaminase in serum and cerebrospinal fluid of patients with cerebrovascular accidents. Demonstration of a bloodcerebrospinal-fluid barrier, New End/. J. Med., 257:1201. L()WENTHAL, A. (1962) Acquisitions nouvclles par l'6tude electrophor6tique du liquide c6phalorachidien dans les affections d6myelinisantes de I'homme et de l'animal, Acta neurol, belg., 62:875. MACHETANZ, E. AND D. HABECK (1961) 1st die Hypoxie des Zentralnervensysten~s ein ursgchlicher Faktor der fl-Globulinvermehrung im Liquor? Arch. Psychiat. Nervenkr., 201:418. RIEDER, H. P. ANO R. W/JTHRICH 0962) Die Cu-Folin-Method zur Bestimmung des GcsamteinweiBgehaltes im Liquor, Kiln. Wschr_ 40:1070. SIMON, G. AND H. SCHR()ER (1963) Ein neues Verfahren zur vollst/indigen Erfassung der im Liquor cerebrospinalis vorhandenen Zellen (Zellenfangverfahren), Arch. Psyehiat. Nervenkr., 204:74. WOTHRICH, R., H. P. RIEDER AND J. B. MEYER (1963) Llber Geschlechtsdifferenzen in den Liquorbefunden bei Multiple Sklerose-Kranken, Nervenarzt, 34:32.

SOCII~TI~ FRAN(~AISE DE N E U R O L O G I E (S6ance du 6 f6vrier 1964)

Paralysies oculo-motrices et atteinte du trijumeau par volumineux andvrysme earotidien intraeaverneux, par M. ROUZAUD, A. GOUAZL P. VIGNERON ETC. J[Z~GABEL.

6t6 inftuenc6e favorablement par la ligature de la carotide primitive faite apr6s 6preuve de compression et 6tude des r6actions cliniques et 61ectro-enc6phalographiques.

Les auteurs rapportent un cas d'an6vrysme carotido-caverneux dont l'aspect clinique est assez courant, mais dont les caract~ristiques radiologiques sont tr~s particuli6res. La radiographie du erfine montre en effet une usure de la grande aile du sph6noide stir laquelle il a d6j~t 6t6 insist6 ant6rieurement, et surtout, l'art6riographie carotidienne met en 6vidence une image circulaire tr6s singuli6re. La n6vralgie t r i g 6 m i r ~ qui avait real r6agi aux traitements habituels a

Maladie de Von Hippel-Lindau avec h~mangioblastome kystique de la moelle dorsale, opdr& par M. LANGLOtS, P. DARLI~GUY, J. L. ARROUVIGNOD, M. TOGA ET R. Vmouaoox. Les auteurs out observ6 un syndrome de compression m6duilaire avec dissociation albumino-cytologique et blocage lipiodol6 qui a conduit ~ une intervention chirurgicale, Celle-ci a permis de d6couvrir un h6mangiobMstome kystique am61ior6 par l'intervention. A la suite J. neurol. Sci. (1964) I : 590 598