THE DISTRIBUTION OF MATERIAL FOLLOWING INTRACEREBRALINOCULATION INTO MACACUS RHESUS MONKEYS AND ITS

POSSIBLE INFLUENCE UPON THE RESULTS OFNEUTRALIZATION TESTS IN EXPERI-

MENTAL POLIOMYELITIS *

MoaRs SC<YFER, PHID., AND RALPH S. Mucxzmruss, MD.(From the Bureau of Laboratories, Department of Health, New York, N. Y.)

Intracerebral inoculations of monkeys with poliomyelitis virushave been employed extensively since Flexner and Lewis 1 in io9demonstrated that this route of inoculation is an effective methodof transmitting the disease to these animals. Because monkeyshave been most consistently infected with virus in this manner, theintracerebral inoculation has been considered the most reliablemeans of determining infectivity of the virus, especially after ithas been subjected to treatment with an inhibitory reagent such asimmune serum. However, despite the widespread use of injec-tions into the monkey brain, there is little knowledge concergthe fate of the inoculum subsequent to its deposition into the brainsubstance.Many investigators 2` have studied the diffusion of material

throughout the central nervous system by the injection of dyes or ofIndia ink; but as far as we could ascertain, no experiments havebeen conducted with a view toward determining to what degreeand extent material introduced into an area of the brain is eventu-ally distributed, and what bearing the resultant distribution mayhave upon the ultimate infectivity of infectious material thus de-posited.

Hurst,6 in a study on the pathogenesis of experimental polio-myelitis, as a preliminary step injected India ink into the cisternamagna of a monkey in order to follow the course of diffusion. Twodays later the ink had penetrated into the meninges along the wholelength of the cord, over large areas of the brain stem, cerebellum,base of the cerebrum, along certain of the fissures, about half wayup the lateral surfaces of the hemispheres, the choroid plexusesand the lateral ventricles. The nervous substance itself was not

* This work was aided by a grant from the President's Birthday Ball Commis-sion for Infantile Paralysis Research.

Received for publication December 2, 1937.

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SCHAEFFER AND MUCKENFUSS

discolored, except for slight staining in the floor of the fourthventride.When virus was inoculated intrathecally the earliest lesions were

usually situated in the floor of the fourth ventride. This suggestedto Hurst that the penetration of the virus through the nervoustissue may occur at the ependyma of the fourth ventride where,under experimental conditions, the virus is regurgitated at opera-tion; but the cerebrospinal fluid does not necessarily participatein its spread through the nervous system.

ExiimTALThe work to be presented here is an outgrowth of a series of

experiments conducted on the neutraizing action of immune serumupon the virus of poliomyelitis.* During the course of this studynumerous discrepancies in the results were observed, and an effortwas made to determine what possible factors ght be responsiblefor such variations. Among other things, the inoculation procedureand its effect upon the results were investigated.

Experimet I

To follow the course of distribution of substances from the siteof inoculation, material containing India ink was introduced intothe brains of Mcacus rhesus monkeys in the manner usually em-ployed in our previous experiments.

Technique: The customary serum-virus mixture used in ourneutralization tests was prepared. This consisted of I.5 cc. of aBerkefeld N filtered 5 per cent suspension of poliomyelitic monkeyspinal cords, mixed with I.5 cc. of human convalescent serum. Tothis mixture, i cc. of India ink was added. After thorough mixing,the material was injected into the right frontal lobes of 4 monkeys,each receiving 0.25 cc., 0.5 cc., i cc., and 2 CC., respectively, with atuberculin syringe carrying a 4 inch, 26 gauge needle, which wasinserted through a trephined opening made in the frontal boneapproximately i cm. to the lateral right of the midline and i cm.anterior to the coronal suture. After 2 hours the animals werechloroformed and their brains and cords emined at autopsy. Theresults of this experiment are summarized in Table I.

* The details of these experiments and a review of this subject, wich entailsome length, will be published esewhere.

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Excperiment II

A second series of 4 monkeys was inoculated in a manner similarto those in Experiment I. In this group 2 monkeys received i cc.and 0.25 CC., respectively, of the serum-virus-ink mixture with a4 inch, 26 gauge needle, and 2 others were injected with i cc. and

TABIx I

The Diffusion of Material Inoculated Intracerebraily (Right Frontal Lobe)Throughout the Central Nervous System, as Evidenced by the

Distribution of India Ink Contained in the Inoculum

Monkey Amount of mixture Appearance of the central nervous systemNo. injected 2 hours after injection

cc.I 0.25 There was evidence of seepage of the material from

the brain substance into the subarachnoid spaceabove the site of inoculation. Slight hemorrhage atthe site of inoculation was also noted. No India inkwas observed on the spinal cord (Flgs. I & 2)

2 o.5 When the monkey was chloroformed, leakage was ob-served to be still taking place externally at the siteof injection. On flapping back the scalp India inkwas found to be deposited around the trephinedopening and the surrounding area (Fig. 3). Thematerial had diffused over the surfaces of the brain,cerebellum and spinal cord (Figs. 4 & 5). Sec-tions at the site of inoculation showed that theinjection had probably been made directly into thelateral ventricle (Figs. 5 & 6)

3 I.0 This monkey had been used in a neutralization test2 months previously and therefore, as is often ob-served, had a sterile abscess in the right frontallobe at the previous site of inoculation. On ex-amination it was noted that although some Indiaink had seeped into the subarachnoid space, thebulk of the inoculum was found to be confined tothe necrotic cavity on the right side (Fig. 7)

4 2.0 The entire surface of the brain and cord of this ani-mal was covered with India ink, indicating theextensive seepage of the inoculum from the site ofinoculation into the cerebrospinal fluid (Figs. 8& 9)

0.25 CC., respectively, using a Y2 inch, 26 gauge needle. After 2hours the animals were sacrificed and their brains and cords ex-anined at autopsy. The findings are summarized in Table II.From the results of these experiments it was evident that the

distribution of material following intracerebral deposition varied

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SCHAEFFER AND MUCKENFUSS

to some extent. In most cases, however, little of the materialremained at the site of inoculation but rapidly entered the cere-

brospinal fluid either by seeping backward through the path ofinoculation into the subarachnoid space or via the ventricles intothe spinal canal. Except for areas reached by the needle no carbon

TABi.E II

Comparison of the Diffusion of Material Through the Central Nervous System afterIxtracerebral Ixoculatiox with Needles of Two Sizes

Monkey Amount of mix- Size of Appearance of the central nervous systemNo. ture injected needle 2 hours after injection

cc.I1.0 2 inch The entire surface of the right frontal lobe

(the side inoculated) up to the fissurecentralis was covered with India ink(Fig. Io). No ink was observed on theopposite hemisphere or on the spinalcord. Sections exm ed at the site ofinoculation indicated that the inoculumhad been deposited in the brain sub-stance at the site of inoculation belowthe cortex (Fig. iI), but some seepedbackward and entered the subarachnoidspace

2 0.2 ¼2 inch The general appearance of the brain andcord of this animal was similar to theone above, except that there was lessdistention of the brain tissue at the siteof inoculation (Flg. 12)

3 1.0 Y4 inch The entire cerebral hemisphere opposite tothe side inoculated was completely cov-ered with India ink (Flg. 13). Ink wasalso present in all of the ventrides, thebase of the brain and the spinal cord(Flg. 14). The photograph clearly showsthe path of the needle

4 0.25 ^4 inch No ink was observed on the surface of thebrain, but it was abundant on the sur-face of the spinal cord (Flg. I5). A sec-tion of the brain revealed a considerablequantity of ink in the lateral ventricle(Fig. i6)

particles were found deposited in the nervous substance itself, butthe India ink adhered to the surfaces of the brain or cord. Therewas some suggestion that with a shorter needle and a smalleramount of material the inoculum did not as readily diffuse throughthe cerebrospinal fluid and reach the spinal cord. On that basis,

230

EXPERIMENTAL POLIOMYELITIS

therefore, neutralization tests were performed to compare the re-sults of inoculation of a large and small volume of material, usingneedles of / inch and 4 inch length. The Y inch needle wasemployed in order that the inoculum might reach the lateral ven-tricle, and the Y4 inch needle in order that the material might bedeposited into the cerebral cortex. The volumes selected were i cc.,the usual amount inoculated in our neutralization tests, and 0.25cc., the injection of which in previous experiments had indicated atendency toward greater regularity.

Experiment IIITechnique: A set of io duplicate test tubes, each containing 1.5

cc. of a 5 per cent virus filtrate and I.5 cc. of pooled human con-valescent serum, was incubated for 2 hours at 370 C. and kept inthe refrigerator overnight. From each of 5 of these tubes 2 mon-keys were inoculated intracerebrally with i cc. and 0.25 cc., re-spectively, using a 7 inch needle. From each of the remin ng 5tubes 2 monkeys respectively received i cc. and 0.25 cc. with aY4 inch needle. Immediately before injection 0.25 cc. of sterileIndia ink was added to each tube in order that the dispersion ofthe inoculum could be followed in those animals that developedpoliomyelitis. The results are summarized in Table III.The variable manner in which material inoculated intracere-

brally diffuses is again illustrated by this experiment. The extentof the distribution is apparently not entirely governed by theamount inoculated or the length of the needle employed. It is in-teresting to note, however, that none of the monkeys receivingmaterial with the Y4 inch needle developed poliomyelitis, while 4of the io monkeys inoculated with similar mixtures, but with 36inch needles, became infected.

It has been observed 7-10 that upon dilution of a neutral mixtureof virus and immune serum a subsequent disruption of the virus-serum union, the so-called dilution phenomenon, takes place andthe mixture again becomes infective. The results of the aboveexperiments suggested the possibility that if some quantity of theinoculum escapes from the area of inoculation into the cerebro-spinal fluid, the dilution phenomenon may occur within the animalbody and thus account for the occasional infectivity of an other-wise apparently inactivated mixture.

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Experiment IVTo determine whether or not direct admixture with the cerebro-

spinal fluid would prove this point, a group of 5 monkeys wasinjected with i cc. of mixtures of virus and serum, prepared asdescribed above, but without India ink. The inoculations weremade below the dura and into the subarachnoid space above theright cerebral hemisphere. This was accomplished by surgicaltrephinig of the frontal bone at the usual site of inoculation. Thearea exposed was made large enough so that sufficient assurancecould be had that the brain substance was not touched uponsubdural insertion of the needle. All of these animals remainedwell during an observation period of 2 months, whereas a groupof 4 controls inoculated similarly, but receiving a mixture of thevirus and normal monkey serum, all developed poliomyelitis withinthe usual incubation period.Of another group of 4 monkeys, each inoculated with i cc. into

the cisterna magna, none showed evidence of infection while thefour controls became paralyzed.

Experiment VSince during the process of an intracerebral inoculation the brain

is traumatized, we decided to investigate the combined effect ofdirect inoculation into the spinal fluid and simultaneous braintrauma. Accordingly, a group of 12 monkeys was inoculated withi cc. of a neutral serum-virus mixture (4 cc. of undiluted pooledhuman convalescent serum and 4 cc. of 5 per cent virus suspen-sion) intracisternally. In 6 of these monkeys, immediately follow-ing inoculation, a sterile 36 inch needle was pushed into the brainat the usual site of inoculation and then withdrawn. The other 6monkeys were treated in a similar manner with a 4 inch needle.None of the 12 animals developed poliomyelitis, while the controlin each group became paralyzed.

Experiment VIHaving no indication from the above experiments that the dilu-

tion phenomenon took place in vivo, we attempted to determinewhether it would occur in vitro. Therefore, monkeys were injectedfrom 3 tubes containing mixtures which consisted of equalquantities (4 cc.) of 5 per cent virus filtrate and hutman con-

233

SCHAEFFER AND MUCKENFUSS

valescent serum, o.s cc. of which was diluted, after the usual incu-bation time, with 2 cc. of normal monkey spinal fluid, giving adilution ratio of I: 5. Twelve monkeys, 4 injected from each tube,received intracerebrally i cc. each of these mixtures prior to dilu-tion with the spinal fluid, and 6 monkeys, 2 injected from eachtube, received i cc. each of the mixtures following dilution. Noneof these animals developed the disease. Two controls receivingthe virus and normal monkey serum and 2 others injected withvirus, normal monkey serum and spinal fluid, all became infected.

DISCUSSION

Our experments indicate that the manner in which material,following intracerebral inoculation, is distributed, resembles inmany respects that noted after intrathecal inoculation, as describedby Hurst.6 While certain variations in the course and extent of thediffusion were observed, some admixture of the material with thecerebrospinal fluid occurred in almost every instance. Generally,little of the material was found in the brain substance at the site ofinoculation, except in monkeys that had received intracerebralinoculations in other experiments, in which case most of the inkwas usually confined in the necrotic area of the previous site ofinoculation. When larger amounts or longer needles were used, itappeared that the material more readily found its way into thecerebrospinal fluid. It may be stated, however, that the resultsare uncertain in so far as the final deposition of the material isconcerned, but, in any event, some seepage into the cerebrospinalfluid is to be expected.What, if any, correlation ets between the diffusion of material

and its ultimate effect upon the infectivity of the neutral virus-serum mixtures cannot be answered from the data at hand. Fromthe results of Experiment IH, it appears that intracerebral inocu-lations with a A inch needle tend towards greater regularity, sincemonkeys receiving the same mixtures were infected when the 34inch needle was used. At first, we were incined to attribute theconsistent infectivity of the neutral mixtures inoculated with ashort needle to the fact that less seepage might have taken place.However, admixture with the cerebrospinal fluid seems to have nobearing upon the infectivity of these mixtures since, as observed inExperiments IV and V, direct subarachnoid or cisternal introduc-

234

EXPERIMENTAL POLIOMYELITIS

tion or intracisternal inoculation with simultaneous brain traumacaused no reactivation of the inactive mixtures.We sought an explanation for irregularities on the ground that

the dilution phenomenon may occur. Although this has been re-ported to take place with a mixture of immune serum and polio-myelitis virus by Schultz and his collaborators,'1 we were unableto demonstrate, in our experiments, that the dilution phenomenonensued either sn vitro or within the animal body.

It is, therefore, difficult to state what factors may account forunexpected infections when a presumably neutral mixture of serumand virus is injected into a group of monkeys. So far as this workhas been carried, there is some indication that the use of a 4 inchneedle or direct intracisternal inoculation may be an improvementover the usual intracerebral method for performing neutralizationtests, but further studies on a larger scale are necessary to verifythis.

SUMM-ARY

i. The distribution of material throughout the central nervoussystem of Macacus rhesus monkeys, subsequent to intracerebralinoculation, was studied by the injection of India ink. The experi-ments indicated that certain variations in the degree and extent ofdiffusion occurred, but in most instances the inoculum rapidlyentered the cerebrospinal fluid either via the subarachnoid spaceor ventricles. Except for the site of inoculation, no carbon particleswere found in the brain substance itself. The India ink was de-posited on the surfaces of the brain or cord.

2. It appeared that when material was inoculated in largeramounts or with longer needles, it more readily entered the cere-brospinal fluid.

3. In an experiment where apparently neutral mixtures ofpoliomyelitis virus and immune serum were inoculated intracere-brally into monkeys with 4 inch needles and Y8 inch needles, noneof the io monkeys injected with the shorter needles developedpoliomyelitis, while 4 of io monkeys receiving the mixtures withY8 inch needles succumbed to the disease.

4. Direct inoculation of serum-virus mixtures into the sub-arachnoid space or cisterna magna did not render these neutralmixtures active, nor were these mixtures infective when inoculated

235

236 SCHAEFFER AND MUCKENFUSS

intracisternally accompanied by brain trauma, although controlssimilarly inoculated were- uniformly infected.

5. Experiments devised to demonstrate the occurrence of thedilution phenomenon in vivo or in vitro were negative.The authors gratefully acknowledge the technical assistance given by

Mr. Angelo Campagna.

REFERENCES

i. Flexner, Simon, and Lewis, Paul A. The transmission of acute poliomye-litis to monkeys. J.AM.A., I909, 53, I639.

2. Bula, I. Recherches sur la circulation du liquide c6phalo-rachidien aumoyen des injections de bleu de Prusse (methode du Professeur Gerotapour les lymphatiques) dans l'espace sous-arachnolidien. Compt. rend.Soc. de biol., I9I6, 79, 873-874.

3. Dandy, Walter E. Experimental hydrocephalus. Ann. Surg., I9I9, 70,I 29-I42.

4. Weed, Lewis H. The cerebrospinal fluid. Physiol. Rev., 1922, 2, I7I-203.

5. Sachs, Ernest, Wilkins, Harry, and Sams, Crawford F. Studies on cere-brospinal circulation by a new method. Tr. Am. Neurol. A., 1929, 55,252-273.

6. Hurst, E. Weston. Further observations on the pathogenesis of experi-mental poliomyelitis: intrathecal inoculation of the virus. J. Path. &Bact., 1930, 35, 41-52-

7. Bedson, S. P. Observations on the mode of action of a viricidal serum.Bnt. J. Exper. Path., I928, 9, 235-240.

8. Todd, Charles. On a dilution phenomenon observed in the titration of theserum of fowls immunized against the virus of fowl plague. Brit. J.Exper. Path., I928, 9, 244-252.

9. Andrewes, C. H. The action of immune serum on vaccinia and virus IIIin vitro. J. Path. & Bact., I928, 31, 67i-698.

io. Long, Perrin H., and Olitsky, Peter K. The recovery of vaccine virusafter neutralization with immune serum. J. Exper. Med., 1930, 5S,209-217.

ii. Schultz, E. W., Gebhardt, L. P., and Bullock, L. T. Studies on the anti-genic properties of the ultraviruses. VII. Nature of the viricidalantibody in antipoliomyelitis serum. J. Immunol., I93I, 2I, 17I-I98.

DESCRIPTION OF PLATES

PLATE 45

FIG. i. Dorsal view of the brain of Monkey No. 21. India ink is deposited onthe surface of the frontal lobe.

FIG. 2. Ventral view of the brain of Monkey No. 2I.

FIG. 3. Monkey No. 300 with skull exposed showing external leakage follow-ing injection. India ink is deposited in the area surrounding the trephinedopemng.

FIG. 4. Ventral view of brain and spinal cord of Monkey No. 300. India inkis deposited over the surfaces of the brain, cerebellum and spinal cord.

FIG. 5. Dorsal view of brain and spinal cord of Monkey No. 300 with sectionthrough the site of inoculation showing India ink deposited in the lateralventricle and on the surfaces of the brain and spinal cord.

FIG. 6. Brain of Monkey No. 300 sectioned through another plane at the siteof inoculation. Note the path of the needle and ink in the lateral ventricle.

FIG. 7. Brain of Monkey No. i8o sectioned through the site of inoculation.The India ink is confined to the necrotic cavity present as a result of aninoculation given 2 months previously.

FIG. 8. Dorsal view of brain and spinal cord of Monkey No. 88. The surfacesare heavily coated with India ink.

FIG. 9. Ventral view of the brain and spinal cord of Monkey No. 88 showingconsiderable deposit of India ink.

Aianc JORNAL OF PATHOLOGY. VOL. XI

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PLATE 45

Schaeffer and Muckenfuss Dx-perimental Poliomyehtis

PLATE 46

FIG. io. Dorsal view of brain of Monkey No. 295. India ink is deposited onthe entire surface of the right frontal lobe up to the fissure centralis.

FIG. ii. Sagittal section through the right hemisphere at the site of inocula-tion of the brain of Monkey No. 295. The area into which the inoculumhas been deposited is distended.

FIG. I2. Brain of Monkey No. 137 sectioned through the site of inoculation.Note the India ink on the surface and in the area inoculated. Distentionis also present here but to a lesser degree than that noted in MonkeyNo. 295.

FIG. I3. Dorsal view of the brain of Monkey No. 225 in situ with dura re-moved showing deposit of India ink over the entire surface of the cerebralhemisphere opposite the side inoculated.

FIG. 14. Brain of Monkey No. 225 sectioned through the site of inoculation.India ink is present in the ventricles and on the spinal cord. The path ofthe needle is made clearly visible by the deposit of India ink.

FIG. I . Brain and spinal cord of Monkey No. 207. No India ink is evidenton the surface of the brain but it is abundant on the spinal cord.

FIG. i6. Brain of Monkey No. 207 sectioned through the site of inoculation.A large amount of India ink is seen in the lateral ventricle and on thespinal cord.

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