J. Neurol. Neurosurg. Psychiat., 1970, 33, 426-430

Experimental cerebral schistosomiasisjaponica in primates

JOHN A. JANE, KENNETH S. WARREN, AND STANLEY VAN DEN NOORT

From the Department of Neurosurgery, University of Virginia, School of Medicine, Charlottesville, Virginia22901, and the Department of Preventive Medicine and Medicine, Division of Neurology, Case Western

Reserve University, School of Medicine and University Hospitals, Cleveland, Ohio 44106, U.S.A.

SUMMARY Aspects of the pathogenesis of cerebral schistosomiasis have been elucidated. Arterialembolization alone, by either worms or eggs, does not appear to be likely as a major cause of thedisease. When large numbers of eggs enter the brain, sensitization may play a role in the occurrenceof neurological signs. These studies support but do not prove the theory that worms in the cerebralveins lay eggs that then cause the disease.

Schistosomiasis japonica is a parasitic worm diseaseof great potential severity which involves more than30,000,000 people in the Far East. While the mostcommon signs and symptoms are related to anacute febrile illness known as Katayama fever andchronic involvement of the intestine and liver, ithas been estimated that between 2 and 4% of allpatients with this disease may manifest symptoms ofcerebral involvement (Kane and Most, 1948;Ariizumi, 1963; Reyes, Yogore, and Pardo, 1964).The specific cause of the neurological complicationsof schistosomiasis remains unknown, although it isgenerally believed that the eggs of the parasite arethe major aetiological factor. The route by whichthe eggs enter the brain is also unknown. It has beenspeculated that either eggs produced by worm pairsin their usual sites embolize to the brain or thatworms present in cerebral vessels are producingeggs in situ. In view of the clinical importance ofcerebral schistosomiasis and the lack of knowledgeconcerning its aetiology, it is interesting to observethat no studies of cerebral schistosomiasis have everbeen performed in experimental animals (Warrenand Newill, 1967). There has been only an incidentalobservation of Fujinami as reported by Schmidzu(1935) of worms in the brain of hyperinfected experi-mental animals. In the present investigation, severalpossible aetiologies of cerebral schistosomiasisjaponica have been examined in the African greenmonkey, Cercopithecus aethiops.

MATERIALS AND METHODS

Twenty monkeys (Cercopithecus aethiops) of both sexes

weighing 2K1 to 4-6 kg were used in the present study.They were divided into the following five groups:

GROUP 1 Cranial exposure to cercariae Two monkeyswere anaesthetized with Semylan (10 mg/kg), their headsand faces shaved, and the skin moistened with saline.Using a bacteriologist's loop, 150 cercariae of a Japanesestrain of Schistosoma japonicum were placed in smallgroups on either side of the bridge of the nose in theupper inner quadrant of both orbits, along the sagittalsinus and the occiput ofeach animal.

GROUP 2 Intracarotid injection of worm pairs Twomonkeys were anaesthetized (Sernylan) and a smallpolyethylene catheter was threaded into the left commoncarotid artery. Seven actively motile worm pairs wereobtained by the perfusion method of Duvall and DeWitt(1967) from female Swiss albino mice infected for eightweeks with S. japonicum. The worms were suspended insterile saline and injected into the catheter within 20minutes of the perfusion procedure. During this proced-ure, and all those below involving injection of materialinto the carotid artery, electroencephalograms wererecorded from six surface electrodes with a standardclinical Grass electroencephalograph. The catheter wasremoved and the incision was closed with fine arterialsilk maintaining the continuity of the vessel. Thesemonkeys and all of those in the following experimentswere treated with 125 mg ampicillin (two doses intra-muscularly) on the day of the operative procedure.

GROUP 3 Jugular vein and sagittal sinus placement ofworm pairs Six monkeys premedicated with Semylanwere placed under general endotracheal anaesthesiawith halothane. The left jugular vein was ligated andfrom three to 12 worm pairs, obtained as described above,were injected into the vein above the ligation. Thirty-five days later two of the six monkeys had three worm

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pairs placed into a small segment of the anterior sagittalsinus isolated by ligatures at open craniotomy.

GROUP 4 Intracarotid injection of schistosome eggs S.japonicum eggs were isolated by the method of Browneand Thomas (1963) from the livers and intestines of miceinfected for eight weeks. These were injected into theleft carotid artery of eight monkeys twice, two weeksapart (Table). The eggs were ellipsoidal in shape and theirmean width, as measured by a Cooke Image SplittingEyepiece, was 600.

GROUP 5 Intracarotid injection of plastic beads In thesame manner as in group 2 and 4, divinylbenzene copoly-mer beads (Bio Rad) were injected into the carotid arteryof two monkeys on two occasions separated by twoweeks. On the first occasion one monkey received 84,000beads and the other 60,000 beads with a mean diameterof 45 A measured as described above. For the secondinjection both monkeys received 50,000 beads with a meandiameter of 57 j.

Approximately 12 weeks after the procedure describedabove, all the animals remaining alive had electroence-phalograms and then were killed by the rapid injectionof 10 ml Diabutol (50 mg/ml). In the case of the animalswhich had living worm pairs placed in the venous cir-culation, cranial venous drainage was occluded by acervical tourniquet within five seconds after loss ofconsciousness in order to maintain intracranial wormpairs in situ. The brains in all cases were removed andplaced in formalin. They were sectioned grossly andappropriate samples of tissue for microscope sectionswere obtained. At the time of death, portions of the liverand intestine of each animal were pressed between twoglass plates, except for the monkeys which had beenexposed to cercariae, and no sign of prior infection withschistosomes was observed.

RESULTS

The two monkeys exposed cranially to S. japonicumcercariae showed no cerebral symptomatology over a

period of 86 days after exposure. At the terminationof the experiment their electroencephalograms werenormal. Post-mortem examinationrevealed that bothmonkeys were infected with S. japonicum and thatthey had the usual intestinal and liver involvement.

Neither of the two monkeys which received sevenworm pairs into the carotid artery showed anyimmediate neurological manifestations, or EEGchanges, and both recovered completely from anaes-thesia. They both appeared completely well twodays after the procedure, but at four days one ofthem was anorexic. On the next day the animalwas found in a comatose state and shortly thereafterdied. At post-mortem examination left uncalherniation was observed. In addition, a largehaemorrhage extended from the level of the lateralgeniculate in the posterior temporal lobe to theexternal capsule and involved the putamen. Noparasitic elements were observed on microscopicexamination of sections taken from the haemorrhagicarea. The other monkey haa no neurological signsor symptoms and when killed 34 days after worminjection the brain was grossly normal.Of the six animals in which worm pairs were

implanted into the jugular vein or the sagittal sinus,none had any neurological signs, symptoms, orelectroencephalographic changes over a period of90 days nor were any striking changes noted in thebrains at necropsy.Of the eight monkeys injected for the first time

with eggs (4:50,000; 2:10,000; 1:1,000; 1:100) sixshowed no neurological signs or symptoms whatso-ever but two animals failed to regain consciousness;one of these received 50,000 eggs, the other 10,000eggs (Table). On post-mortem examination theformer animal had marked congestion and haemor-rhage over the left hemisphere and ecchymoses inthe Sylvian fissure (Fig. 1). In spite of these changes,

TABLEEFFECTS ON Cercopithecus aethiops OF PRIMARY AND SECONDARY INTRACAROTID INJECTIONS OF

Schistosoma japonicum EGGS

Animal First Result Second Result Grossinjection injection pathology

I 1 50,000 Death Gross subarachnoid bleed overleft hemisphere. Figs. I and 2

12 50,000 No signs 50,000 Death in 24 hr. Decerebrate Gross subarachnoid bleed overrigidity left frontal lobe

13 50,000 No signs 50,000 Mild right hemiparesis. Killed Normal6 days

14 50,000 No signs 50,000 Right hemiparesis, right Subarachnoid haemorrhage in lefthemianopsia. Killed 6 days hemiisphere

15 10,000 No signs 10,000 Right hemiparesis, right Normalhemianopsia. Killed 6 days

16 10,000 Death Slight subarachnoid bleed in lefthemisphere

17 1,000 No signs 1,000 Death 2 days, unrelated Normal18 100 Nosigns 100 Normal

3

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FIG. 1. Dorsal surface of brain with dura reflectedshowing subarachnoid haemorrhage.

remarkably little was observed on cross-section(Fig. 2). The brain of the latter monkey appearednormal. In neither animal were any parasitic ele-ments seen on microscopic examination.On the second injection of eggs, five of the six

animals either had severe neurological signs or died.Striking neurological signs, including decerebraterigidity and marked right hemiparesis accompaniedby hemianopsia, appeared immediately after injec-tion in the four animals injected with 50,000 or

10,000 eggs (Table). The monkeys which received

FIG. 2. Cut surface of brain showing poor grey whitedifferentiation and subarachnoid haemorrhage but very

little oedema.

FIG. 3. Granulomatous reaction arounda schistome egg.

1,000 and 100 eggs showed no immediate changesand by 24 hours both had recovered and were inexcellent condition. The former animal, however,died suddenly at seven days (see Table).The monkey with decerebrate rigidity (no. 12)

died at 24 hours and haemorrhage was observedover the left frontal lobe and at the base. Micro-scopic examination of tissue taken from the brainof this monkey revealed an egg in the cerebral sub-stance with a marked granulomatous reactionaround it (Fig. 3) and a group of eggs in a bloodvessel in the midst of an organizing thrombus (Fig.4). In one of the animals which developed hemi-paresis on second injection (no. 13) an egg wasfound with no reaction around it (Fig. 5). The threemonkeys with right hemiparesis recovered over thenext 24 hours, but were killed five days later.

FIG. 4. Organizing thrombus with schistosome eggs butvery little perivascular reaction.

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FIG. 5. Schistosome egg in a small vessel with no reaction.

Only one of these monkeys (no. 14) showed anygross cerebral changes-haemorrhage in the piaover the left hemisphere.None of the animals receiving the two injections

of glass beads showed any abnormal signs or symp-toms.

DISCUSSION

Cerebral schistosomiasis japonica has been estimatedto occur among 2 to 4% of the 30,000,000 Asiansinfected with Schistosoma japonicum (Kane andMost, 1948; Ariizumi, 1963; Reyes et al., 1964).The way in which the central nervous system isinvolved by this parasite of the gastrointestinaltract is unknown, but there are several aspects to thedisease picture which are of aetiological significance.Although overt cerebral involvement may occur atany time during the course of this prolonged chronicinfection, it may appear as early as four weeks afterexposure to the infective forms of the parasite (Kaneand Most, 1948). The clinical disease may be mani-fest by diffuse or focal signs of deficit or increasedexcitability, but commonly there is a focal deficit(Kane and Most, 1948; Ariizumi, 1963; Reyes et al.,1964). Most of the cerebral lesions found at surgeryin patients with localizing signs have been circum-scribed masses of eggs which are usually located inthe venules and not the arterioles (Kane and Most,1948; Ariizumi, 1963, Reyes et al., 1964). Finally,it should be pointed out that cerebral schistosomiasisdoes not merely denote the presence of eggs scat-tered about the brain, since this has been demon-strated at necropsy in many patients infected with S.japonicum who do not have signs or symptoms.Moreover, cerebral signs are extremely rare inpatients with schistosomiasis mansoni and haema-

tobia yet eggs have been found in the brains of morethan 50% of such patients by digestion procedures(Gelfand, 1950). Histological examination of brainsections also revealed S. mansoni eggs in patientswith no neurological signs and symptoms (Aleman,1966).When approached in the above manner, the most

likely explanation for most cases of cerebral schisto-somiasis is the presence of egg-producing wormpairs in the venous circulation of the brain (Green-field and Pritchard, 1937). Nevertheless, Kane andMost (1948) noted that most investigations sup-ported the theory of indirect (or embolic) depositionof eggs, either through arteriovenous anastomosesor from worms in the pulmonary venous circulation.In these conditions, it is also possible that the wormsthemselves may act as emboli. If adult worms are inthe cerebral veins, they may get there at threedifferent stages of their development: as cercariaewhen they penetrate the skin, as schistosomulaewhen they migrate from the lungs, or as adultworms migrating from the mesenteric veins.

All of the above aetiological possibilities wereconsidered in the experimental plan of the presentinvestigation. Thus cercariae were applied to thecranium, living worm pairs were injected into thecarotid artery or placed in the venous circulationof the brain, and schistosome eggs or plastic beadsof similar size were injected into the carotid artery.Consultation with several investigators in the fieldof schistosomiasis led to the suggestion that ex-posure of the cranial area to cercariae might lead topassage of schistosomulae through the emissaryveins and into the cerebral vessels where theywould develop into worms. Although no cerebralsigns or symptoms were observed in our animalstreated in this manner, only two monkeys werestudied. Nevertheless it was not unpredictable thatat necropsy the worms, which usually proceed fromthe site of infection to the lungs and then to themesenteric veins. were found in their usual finallocation. Adult worms (about 0-6 by 2 5 cm)injected into the cerebral arteries did not producethe expected result of neurological signs. One of thetwo animals treated in this drastic manner had nochanges whatsoever, while the effect on the otheranimal was delayed for approximately one weekand was perhaps related to the delayed onset of aninflammatory reaction around the dead worms(Warren, Domingo, and Cowen, 1967); it is unlikelythat conditions similar to those in these experimentsoccur in the natural course of infection. The responseto the second injection of eggs did not appear to berelated to granuloma formation as in some casesthere were profound inflammatory reactions aroundthe eggs (Fig. 3), but in others there were none

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(Fig. 5). Finally, the haemorrhagic lesions observedin the primates at necropsy bear no relation to thesurgical or post-mortem findings in man.

REFERENCES

Aleman, G. C. (1966). Localizacion ectopica aparentementeasintomatica de huevos de Schistosoma mansoni en elencefalo. Reporte de cuatro casos. Arch. Hosp. Vargas,8,71-84.

Ariizumi, M. (1963). Cerebral schistosomiasis japonica:Report of one operated case and fifty clinical cases.Amer. J. trop. Med. Hyg., 12,40-45.

Browne, H. G., and Thomas, J. E. (1963). A method forisolating pure, viable schistosome eggs from host tissues.J. Parasitol., 49, 371-374.

Duvall, R. H., and DeWitt, W. B. (1967). An improvedperfusion technique for recovering adult schistosomesfrom laboratory animals. Amer. J. trop. Med. Hyg., 16,483-486.

Gelfand, M. (1950). Schistosomiasis in South Central Africa.Juta: Cape Town.

Greenfield, J. G., and Pritchard, B. (1937). Cerebral infectionwith Schistosoma Japonicum. Brain, 60, 361-372.

Kane, C. A., and Most, H. (1948). Schistosomiasis of thecentral nervous system; experiences in World War 2and review of literature. Arch. Neurol. Psychiat., 59,141-183.

Reyes, V. A., Yogore, M. G., and Pardo, L. P. (1964).Studies on cerebral schistosomiasis. J. Philipp. med. Ass.,40,87-100.

Schmidzu, K. (1935). Ein Operationsfall von Schistosomiosiscerebri. Langenbecks Arch. klin. Chir., 182, 401-407.

Smithers, S. R., Terry, R. J., and Hockley, D. J. (1969).Host antigens in schistosomiasis. Proc. roy. Soc. B.,171,483-494.

Warren, K. S. (1970). Intestinal obstruction in murineschistosomiasis japonica. Gastroenterol. (In press.)

Warren, K. S., Domingo, E. O., and Cowan, R. B. T. (1967).Granuloma formation around schistosome eggs as amanifestation of delayed hypersensitivity. Amer. J.Path., 51, 735-756.

Warren, K. S. and Newill, V. A. (1967) Schistosomiasis: A

Bibliography of the World's Literature from 1852 to1962. The Press of the Western Reserve University:Cleveland, Ohio.

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