original site of implantation was demonstrated by

THE FINE STRUCTURE OFCEREBRAL FLUID ACCUMULATION

IV. ON THE NATURE AND ORIGIN OF EXTRACELLULAR FLUIDSFOLLOWING CRYPTOCOCCAL POLYSACCHARIDE IMPLANTATION

ASAO HIRANO, M.D.*; H. M. ZIMMERMAN, M.D., AND SEYMOUR LEVINE, M.D.

From the Henry and Lucy Moses Research Laboratories of theLaboratory Division, Monteflore Hospital, New York, N.Y.,

and the St. Francis Hospital, Jersey City, N.J.

Implantation of cryptococcal or pneumococcal capsular polysaccha-rides mixed with graphite caused severe accumulation of fluid in the ratbrain.",2 The fluid was present at the site of the implant and spread intothe white matter of the callosal radiation. The presence of the implantedpolysaccharide in the fluid even at a considerable distance from theoriginal site of implantation was demonstrated by immunofluorescence.3Electron microscopic observations of perfusion-fixed, Vestopal-em-bedded brain proved that the fluid occupied an extracellular space dur-ing the acute phase.2 In addition to demonstrating the location of thefluid, the electron micrographs revealed that it was not homogeneous butconsisted of at least two types, one of which contained a reticulatedelectron-dense substance.2 In the present communication, we presentevidence that the reticulated substance is actually the implanted poly-saccharide, and that the two types of fluid are derived from the implantand from the blood vessels, respectively.

MATERIAL AND METHODSPellets of cryptococcal polysaccharide alone, graphite alone, or a mixture of equal

parts of polysaccharide and graphite were implanted in the anterior end of the hemi-spheric white matter in a total of i8 adult female rats. Pairs of rats were sacrificed at6 hours and then at I 2-hour intervals up to 48 hours, and their brains were fixed byperfusion. The opposite untreated cerebral hemispheres served as controls. VestopalW-embedded blocks from the implants, from the brain tissue adjacent to the implantand from the sites of fluid spread in distant white matter were sectioned and examinedwith an RCA EMU 3F electron microscope. In order to study the morphologicfeatures of the implanted chemicals under similar conditions, but without interferencefrom tissue reaction, the 3 types of pellets were implanted into the brains of 3 addi-tional freshly killed rats that had been pretreated intraperitoneally with i,ooo units ofheparin. The dead rats were cooled on ice, and the region of the implant was removed

This investigation was supported by United States Public Health Service ResearchGrant No. B-3533 from the National Institute of Neurological Diseases and Blindness, Na-tional Institutes of Health, and Grant No. 3 I7-I of the National Multiple Sclerosis Society.

Accepted for publication, March i6, I964.* Visiting Scientist, Epidemiology Branch, National Institute of Neurological Diseases

and Blindness of the National Institutes of Health, U. S. Public Health Service.

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HIRANO, ZIMMERMAN AND LEVINE

for electron microscopic study within 4 hours. Further details concerning implanta-tion, and other technical matters have been presented previously.2

OBSERVATIONS

Cryptococcal polysaccharide implanted after death consisted of homo-geneously distributed delicate substance, of low electron density in un-stained sections. The reticular structure was so faint as to be hardlydiscernible, even in relatively thick sections (approximately I,OOO A)(Fig. iA). However, the electron density increased dramatically afterstaining with uranyl acetate; lead hydroxide staining was slightly lesseffective. The stained polysaccharide appeared as a compactly distrib-uted, distinctly reticular substance (Fig. iB). Compactness and densityvaried from one area to another. The reticular substance did not have ascharacteristic a structure as ferritin, nor any specific structural arrange-ment like myelin lamellas (Figs. iC and D).The postmortem implant of graphite was extremely difficult to section

because of "chatter." The graphite appeared as a uniformly electron-dense or electron-opaque amorphous material in unstained sections. Itselectron density was uninfluenced by staining with uranyl acetate or leadhydroxide. It was observed commonly as large diffusely dense areassimilar to the heavily carbon-coated grid, less often as spots or bands ofvarious sizes and shapes.The implant of the polysaccharide-graphite mixture in the dead rat

exhibited a combination of the features noted with separate implants.The reticulated substance was partially obliterated by the increaseddensity of the diffusely distributed graphite.The electron microscopic appearances of cryptococcal polysaccharide,

of graphite, and of their mixture were identical in implants made in liv-ing and in dead rats. The staining reactions were also identical (Fig. 2B).However, the implants made during life contained, in addition to theimplanted chemicals, a few red cells, minute electron-dense particlesand other substances that occur in blood serum (Fig. 2A), and occa-sional leukocytes. A few areas in implants of polysaccharide-graphitemixture, examined at 6 and I2 hours, contained discrete zones that dif-fered in electron density. In unstained sections (Fig. 3A) the diffusetexture of the background (A) was interrupted by many small foci withhigher electron density (B). But the relative difference of electrondensity was completely reversed when the serial sections were stainedwith uranyl acetate or lead hydroxide (Fig. 3B). This reversal was dueto an absence of graphite staining and mild staining of other (hema-togenous) elements in the implant, in contrast to the strong affinity ofcryptococcal polysaccharide for the stains.

Similar observations were made in the cerebral tissue adjacent to the

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implant. Here, also, the cryptococcal polysaccharide could be identifiedby the presence in edema fluid of a uniformly distributed reticular sub-stance that stained strongly with uranyl acetate (Figs. 4A, B, C and D).The edema fluid was not uniform in appearance, however, in rats killedshortly after implantation. There were areas that were denser than thepolysaccharide-containing fluid in unstained sections, but less dense instained sections (Figs. 5A and B). This second type of fluid varied intexture from area to area, but had considerable resemblance to bloodplasma. A direct comparison of this fluid with plasma was impossiblebecause the vascular lumens had been emptied by perfusion.The two types of fluid differed in location as well as in appearance.

When they were observed in contiguity, the polysaccharide-containingfluid was always in the center, and the "plasma-like" fluid was locatedbetween the former and the tissue cells.2 The plasma-like fluid fre-quently appeared in the vicinity of blood vessels near the site of im-plantation. In this region, separation of perivascular astrocyte footprocesses occurred. The plasma-like fluid communicated with the vas-cular basement membrane through these gaps between foot processes(Figs. 6 to 8). Disruption of the astrocytic basement membrane was seenoccasionally, and there was continuity between the plasma-like fluid andthe material within the basement membrane (Fig. 9). These changeswere observed only in brain tissue close to the implant and only duringthe early stage of the reaction. The differentiation of two types of fluidwas very clear at 6 and I2 hours after implantation, less clear at 24 or36 hours, and absent after 48 hours.

Implants of cryptococcal polysaccharide alone and of polysaccharide-graphite mixtures gave the same appearances in the brain tissue. Im-plants of graphite alone produced some plasma-like fluid in extracellularspaces; needless to say, no polysaccharide-containing fluid was present.

DISCUSSION

Although we are dealing with excessive accumulation of fluid in thebrain, our data differ from other electron microscopic reports on cerebraledema or swelling. Reports of electron microscopy in cerebral edema orswelling vary considerably from one experimental situation to another.4The site of accumulated fluid has been reported to be in the glial cells ofthe cortex exclusively,58 almost exclusively in large clefts within themyelin sheath,9 or in great part in the extracellular space in the whitematter.4 In spite of such differences in respect to the site of involvementat the ultrastructural level, a common feature can be pointed out: theappearance of pale, clear and swollen areas in certain specific structures.Such enlarged spaces which displace pre-existing tissue structures areregarded as sites of fluid accumulation; accumulation of fluid is inter-

Aug., I964 I197


preted indirectly by way of structural alteration of the adjacent cerebraltissue components. Therefore, accumulated fluid presumably had a clear,structureless texture electron microscopically in these experiments.

This was not the case in our investigations. The accumulated fluidcould be identified in electron micrographs by its content of cryptococcalpolysaccharide. The polysaccharide was characterized by a reticularappearance and by strong staining with uranyl acetate or lead hydroxide,a feature not present in any normal central nervous system constituent inthe adult or infant rat. Chemical analysis of cryptococcal polysaccharidedemonstrated the presence of glucuronic acid, mannose, xylose and 3.72per cent ash, but no protein or lipid.10 Of particular importance is thefact that this water-soluble polysaccharide can combine and precipitatewith lead and uranyl salts.10 The affinity for metals may explain its strongstainability in our experiments.Our studies yielded another unique observation. As far as we know,

there has been no previous evidence that two different types of fluid canco-exist in the extracellular spaces of brain, morphologically distinct, yetnot separated by any membrane or other structure. How can this comeabout? We believe that the polysaccharide-containing fluid originatedfrom the implant and spread in the white matter by seepage throughthe extracellular spaces. This interpretation is supported by an earlierdemonstration that immunologically-specific fluorescence (representingpolysaccharide) was co-extensive with fluid distribution.3 The plasma-like fluid probably originated from blood vessels that had been damagedby contiguity with the implant or by the trauma of the implantation pro-cedure. The hematogenous origin was supported by the intimate relationof the plasma-like fluid to the vessels and by the separation or ruptureof astrocyte foot processes. Slight disruption of the blood-brain barrierin the immediate vicinity of the implant was not unexpected. However,the blood-brain barrier was essentially intact in other, more distant areasof fluid spread,' and neither plasma-like fluid nor separated foot proc-esses were detected in such areas. The two types of fluid remained dis-crete, then, only near the implant and early in the course of events.They mixed and blended as the fluid passed into more distant areas andas time progressed. The transitory co-existence of two different typesof fluid may be due, in part, to the viscous nature of polysaccharidesolutions which would impede mixing.

SUMMARYImplants of cryptococcal polysaccharide in rat brain produced an

accumulation of fluid distributed selectively in white matter. Electronmicroscopic identification of cryptococcal polysaccharide, and of poly-

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Aug., I964 CEREBRAL EDEMA 199

saccharide-containing fluid, was made possible by the strong affinity ofthis material for metals. The fluid originated from the implant andoccupied the extracellular space. In the early phase of fluid spread, andin areas close to the implant, the extracellular space contained a secondtype of fluid. The latter was thought to be of hematogenous origin be-cause of its structural features, staining reaction, proximity to vesselsand relation to gaps in the perivascular ring of astrocytic foot processes.The two types of fluid were able to co-exist for a brief time, contiguousbut unmixed, despite the absence of any physical barrier between them.

REFERENCESi. LEVINE, S.; ZIMMERMAAN, H. M.; WENK, E. J., and GONATAS, N. K. Experi-

mental leukoencephalopathies due to implantation of foreign substances. Am.J. Path., I963, 42, 97-II7.

2. HIRANO, A.; ZIMMERMAN, H. M., and LEVINE, S. The fine structure of cerebralfluid accumulation. III. Extracellular spread of cryptococcal polysaccharide inthe acute state. Am. J. Path., I964, 45, I-I9.

3. LEVINE, S. Cerebral white matter: selective spread of pneumococcal poly-saccharides. Science, I963, I39, 605-606.

4. GONATAS, N. K.; ZIMMERMAN, H. M., and LEVINE, S. Ultrastructure of in-flammation with edema in the rat brain. Am. J. Path., I963, 42, 455-469.

5. TORACK, R. M.; TERRY, R. D., and ZIMMERMAN, H. M. The fine structure ofcerebral fluid accumulation. I. Swelling secondary to cold injury. Am. J. Path.,I959, 35, II35-II47.

6. ToRACK, R. M.; TERRY, R. D., and ZIMMERMAN, H. M. The fine structure ofcerebral fluid accumulation. II. Swelling produced by triethyl tin poisoningand its comparison with that in the human brain. Am. J. Path., I960, 36, 273-287.

7. LUSE, S. A., and HARRIs, B. Electron microscopy of the brain in experimentaledema. J. Neurosurg., I960, I7, 439-446.

8. LuSE, S. A., and HAISis, B. Brain ultrastructure in hydration and dehydration.Arch. Neurol., I96I, 4, I39-I52.

9. ALEU, F. P.; KATZMAN, R., and TERRY, R. D. Fine structure and electrolyteanalyses of cerebral edema induced by alkyl tin intoxication. J. Neuropath. &Exper. Neurol., I963, 22, 403-4I3.

IO. EVANS, E. E., and THERIAULT, R. J. The antigenic composition of Crypto-coccus neoformans. IV. The use of paper chromatography for following puri-fication of the capsular polysaccharide. J. Bact., I953, 65, 57I-577.

[ Illustrations follow ]


LEGENDS FOR FIGURESFIG. i. An intracerebral implant composed of cryptococcal polysaccharide (in a

dead rat). The homogeneously distributed reticular substance is difficult toidentify in unstained sections (Fig. IA); it is distinctly stained with uranylacetate (Figs. iB, C and D). Figs. iA and C, X 50,000. Fig. iB, X 32,000. Fig.ID, X 75,000.

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1B

IC

ID

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202 HIRANOI ZIMMERMAN AND LEVINE Vol. 45, No. 2

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FIG. 2. Site of cryptococcal polysaccharide implantation. Rat sacrificed 6 hours afterimplantation. Part of a red cell (right) and minute electron-dense particles areseen in unstained sections, but cryptococcal polysaccharide is barely detectable(Fig. 2A). It becomes distinctly visible after staining with lead hydroxide (Fig.2B). Fig 2A X 40000 Fig. 2B, X 32,000.

FIG. 3. Six hours after implantation of cryptococcal polysaccharide mixed withgraphite. Implant site. Background with diffusely electron-dense texture (Fig.3A) is marked by many small areas showing higher electron density in unstainedsection. This relative difference in electron density is completely reversed afterstaining with uranyl acetate (Fig. 3B). Fig. 3A, X 3,700. Fig. 3B, X 30,000.

.4ug., I964 203


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FIG. 4. Accumulation of fluid containing cryptococcal polysaccharide in cerebral tis-sue near implant; 12 hours after implantation of cryptococcal polysaccharideand graphite. The fluid with homogeneous texture and even electron density sur-rounds a small vessel. Compare the density of fluid in two serial sections before(Figs. 4A and B) and after (Figs. 4C and D) staining with uranyl acetate. Figs.4A and C, X 7,000. Figs. 4B and D, X 24,500.

FIG. 5. Two types of fluid with different structural appearances and staining re-actions in cerebral tissue adjacent to an implant, I2 hours after introduction ofcryptococcal polysaccharide and graphite. The same reversal of electron densityshown in Figure 3 is evident in an unstained section (Fig. 5A) and a sectionstained with uranyl acetate (Fig. 5B). Many of the glycogen-like granules withinthe plasma membrane are also stained more clearly. X 2 2.900.

Aug., I964 20'

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FIG. 6, 7 and 8. Pools of extracellular fluid adjacent to small vessels, near the im-plant of cryptococcal polysaccharide and graphite at I 2 hours (Fig. 6), 24 hours(Fig. 7), and 36 hours (Fig. 8). As arrows indicate, there are gaps in the peri-vascular ring of astrocytic vascular feet. Extracellular fluid communicates withmaterial within the basement membrane through these separations. Stratificationof the two types of fluid is evident in Figure 6. Lead hydroxide stain. Fig. 6,X 2I,700 (insert, X 30,400). Fig. 7, X I7,600 (insert, X 35,200). Fig. 8,X 22,000.

FIG. 9. Intracerebral fluid near the margin of an implant. Electron-dense fluid (A)is surrounded by less dense fluid (B). The latter appears to be connected withmaterial within the basement membrane (arrow). X 30,000.

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original site of implantation was demonstrated by