Proc. Nat. Acad. Sci. USAVol. 70, No. 3, pp. 706-709, March 1973

Occlusion of Brain Capillaries by Endothelial Swelling inMycoplasma Infections

(turkeys/rats/encephalopathy/ischemic necrosis/electron microscopy)

ELIAS E. MANUELIDIS AND LEWIS THOMAS

Department of Pathology and Neurology, Yale University, School of Medicine, New Haven, Connecticut 06510

Contributed by Lewis Thomas, January 2, 1973

ABSTRACT Occlusion of cerebral capillaries due toacute swelling of the endothelial cells has been demon-strated in the brains of turkeys with encephalopathycaused by Mycoplasma gallisepticum (S-6 strain) infec-tion. Similar capillary lesions were seen in rats with rollingdisease, caused by M. neurolyticum toxin. Such lesionsmay be the basis of ischemic necrosis of the brain.

We report here a capillary lesion seen by electron microscopythat is associated with the neurotoxic action of two myco-plasma species: The S-6 strain of Aiycoplasma gallisepticum inturkey poults, and M. neurolyticum in rats. In turkeys, M.gallisepticum infection results in a fatal encephalopathy inwhich polyarteritis of the cerebral arteries is the most con-spicuous lesion seen by light microscopy (1, 2). In rats andmice, M. neurolyticum produces "rolling disease", an acuteneurological syndrome caused by the exotoxin elaborated bythis microorganism (3, 4, 5). In both syndromes, there are ex-tensive necrotizing lesions of the brain parenchyma, consistingof vacuolization, demyelination, and neuronal destruction.Previous ultrastructural studies of the M. neurolyticum diseasein rats and mice revealed extensive swelling of astrocytes andtheir processes, associated with necrosis of parenchymal struc-tures throughout the involved areas of the brain (6).

In turkeys, fibrinoid necrosis, which involved virtually allthe arteries in the brain, was assumed to be the basis forparenchymal necrosis (2). This destruction of brain tissue,often assuming the appearance of massive infarction, may infact be due to the capillary abnormality described below.

MATERIALS AND METHODS

Rats, weighing 150-200 g, were injected intravenously witha crude preparation of M. neurolyticum exotoxin, prepared byMillipore filtration of 18-hr broth cultures (4). The animalsbecame ill 1-2 hr after the injection, with convulsive move-ments of all four extremities, weakness or paralysis of the hindlimbs, and occasional episodes of repetitive rolling on the longaxis of the body.Turkey poults, 6 weeks of age, weighing about 1 kg, were

injected by vein with 1 ml of an overnight broth culture ofthe S-6 strain of M. gallisepticum, containing approximately5 X 108 organisms. With this dose, neurological symptomsoccurred 3 or 4 days later, that consisted of ataxia, weakness,torticollis, and, a few hours later, floundering convulsiveseizures and coma.Rats and turkeys with neurological manifestations and

healthy controls were killed by perfusion of the brain withKarnovsky's cacodylate-buffered formaldehyde-glutaralde-

hyde (7) through the left ventricle of the heart after ligation ofthe abdominal aorta and opening the right ventricle. The per-fusion was initiated with roughly 0.25 strength of the abovefixative followed by full-strength fixative, as suggested byBrightman (personal communication). Blocks of tissue fromvarious regions of the brain were post-fixed in osmium tetroxideand embedded in epon; ultrathin sections were stained withuranyl acetate-lead citrate and examined on an RCA 3Gelectron microscope.

In addition, sick turkeys were intravenously injected withhorseradish peroxidase, 200 mg/ml (type II Sigma) in 1 ml ofsaline (8). Five minutes later the birds were decapitated andthe brain was immediately removed, fixed in glutaraldehyde,and processed for tracer studies.

RESULTS

The endothelial cells of the affected capillaries in inoculatedrats were swollen to such an extent that the lumen was ob-literated partially or completely. Such an occlusive capillarylesion is illustrated in Fig. 1. There was marked swelling anddestruction of the pericapillary astrocytic processes, althoughtheir mitochondria were relatively spared. Neither the oc-clusive endothelial lesion nor the astrocytic swelling wasseen in any control rats, and their capillaries appeared similarto the normal vessel illustrated in Fig. 2.The capillaries in a control normal turkey and in two

inoculated birds are illustrated in Figs. 2-4. In healthy turkeys,the nuclei of endothelial cells were slender; the cytoplasmshowed a poorly developed Golgi apparatus, some thickprofiles of rough endoplasmic reticulum containing densegranular material, and mitochondria that were elongate andregular in appearance. Coated vesicles were seen and fibrilscould be detected in the cytoplasm. The normal endothelialcells were surrounded by a continuous basilar lamina, rvhichsplit to contain pericytes or their processes. The basal laminameasured approximately 20-35 nm (200-350 A). The innersurfaces of the endothelial cells were generally smooth, exceptfor a few cytoplasmic projections. The lumen, as illustratedin Fig. 2, was always patent regardless of the size of thecapillary. Tight junctions were readily visualized betweenmost endothelial cells.Remarkable changes were observed in the capillary endo-

thelial cells of the turkeys showing neurological symptoms.The cytoplasm and especially the nuclei were greatly en-

larged (Fig. 3). The pericytes were also swollen, and the basallamina was roughly 3 times as wide as in the normal controls.

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Endothelial Swelling in Mycoplasma Infections 707

.k~~~~~~~~''FIG. 1. Rat injected with myocoplasma toxin. An enlarged endothelial cell with a prominent nucleus (N) narrows the capillary

lumen (L). There is swelling of astrocytic processes around the basal lamina, in which relatively well-preserved mitochondria (m) floatX 12,000.

FGjeawattnn*StotblInato include cytplasm of a ericyte is oted (arrow). X 12,000

ki.~~~~~~~~~~~~~~~~

~~ ;¼~~~~(

FIG. 2. Control normal turkey. A capillary is seen with widely patent lumen. Cytoplasm of a typical endothelial cell (E) is seen, aswell as tight junctions (*). Splitting of the basal lamina to include cytoplasm of a pericyte is noted (arrows). X 12,000.

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708 Medical Sciences: Manuelidis and Thomas P

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FIG. 3. Turkey injected with M. gallisepticum killed on third day after injection. Distention of endothelial cells (E) with narrowingof the lumen (L) is noted. Both the pericyte (P) and the surrounding basal lamina (arrows) are enlarged. Mitochondria are more round,and the surface of the endothelial cells is irregular. Swelling of the astrocytic cytoplasm with the relative preservation of mitochondria isnoted. X12,000.fl@'ACE The chromatin of the endothelial cells was concentrated in

the peripheral part of the nucleus and the center was pale,suggesting distention of the chromatin matrix. Most of themitochondria were round in shape, but with intact cristae.The profiles of rough endoplasmic reticulum showed some de-gree of dilation. The tight junctions of the endothelial cellsappeared normal. The internal surfaces of the endothelialcells were irregular, and coated vesicles were more frequentlyseen than in normal vessels. Swollen endothelial cells pro-truded into the lumen of the capillary and caused marked

aft'¶''lF--Ss.f.^,4narrowing of the lumen. This event is illustrated in Fig. 4,taken from a turkey that received horseradish peroxidase in

4,t!1?s X .@J ' i Ad ;-'> ' order to visualize the vascular compartment. The tight junc-RW{9';are "gs A d tions appeared to be functionally intact, since no peroxidase, ;' ZZti; was extruded into the basal lamina. The lumen was occluded

almost completely by the swollen endothelial cells, and onlya small, irregular, slit-like lumen containing peroxidase was

-,tat. , Is * # # seen. In Figs. 3 and 4, swelling of the astrocytic processes.S~-VA ^-- D o around the basal lamina was also observed; the mitochondria

of these cells showed good preservation.

FIG. 4. Turkey infected with M. gallisepticum, injected withperoxidase before death. A markedly enlarged endothelial cell

Auset-t GUSHi u atalmost entirely occludes the capillary lumen. Its nucleus (N)is markedly distended. Peroxidase is seen in the lumen, which isreduced to a slit (arrows). Peroxidase does not appear in the basallamina or perivascular space. X 13,000.

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Endothelial Swelling in Mycoplasma Infections 709

DISCUSSIONAlthough endothelial proliferation has been described in thevessels of cerebral neoplasms, and in the encephalitidesassociated with rickettsial infections (9), the extraordinarydegree of endothelial swelling, not accompanied by any evi-dence of proliferation of endothelium, seems to be a uniquefinding in the rats and turkeys affected by mycoplasmas ortheir toxins. The remarkable speed with which this change canoccur is also noteworthy; in rats, fully developed lesions wereseen within 2 hr after the injection of M. neurolyticum toxin.The question of reversibility of the vascular lesions ob-

served in turkeys was investigated in tetracycline-treatedbirds. After the development of the neurological disease, at atime when the birds appear moribund, treatment with tetra-cycline reverses the disease process, and the cerebral arteriesappear to revert to normal (L. Thomas and W. Clyde, topublished). The rapidity with which the neurological signsdisappear in the treated birds suggests that a toxic property ofthe mycoplasma may be involved. Two turkeys that weretreated with tetracycline on the third day after infection,when neurological symptoms had appeared, were examined24 hr after the start of treatment, and the capillaries in thesebirds appeared entirely normal by electron microscopy.Therefore, it is likely that the capillary endothelial abnormal-ity is rapidly reversible.

Since the capillary endothelial swelling causes partial ortotal occlusion of the lumen, these endothelial changes mightin themselves be sufficient basis for ischemic changes in thebrain parenchyma. The rapid onset and reversibility of capil-lary occlusion provide a new and advantageous experimentalmodel for the study of anoxic injury in the nervous system.The existence of such a mechanism offers an approach to cer-tain spontaneous diseases of the brain in which ischemicnecrosis occurs without discernible thrombosis.

Sponsored by USPHS Grants A1-09721-04 and NB-05292-13and the Esther A. and Joseph Klingenstein Fund, Inc.

1. Cordy, D. R. & Adler, H. E. (1957) Avian Dis. 1, 235-245.2. Thomas, L., Davidson, M. & McCluskey, R. T. (1966) J.

Exp. Med. 123, 897-912.3. Tully, J. (1964) J. Bacteriol. 88, 381-388.4. Thomas, L., Aleu, F., Bitensky, M., Davidson, M. & Gesner,

B. (1966) J. Exp Med. 124, 1067-1082.5. Thomas, L. & Bitensky, M. (1966) J. Exp. Med. 124, 1089-

1098.6. Aleu, F. & Thomas, L. (1966) J. Exp. Med. 124, 1083-1088.7. Karnovsky, M. (1965) J. Cell. Biol. 27, 137 Abstr.8. Karnovsky, M. (1965) J. Cell. Biol. 27, 49 Abstr.9. Manuelidis, E. E. & Krigman, M. (1972) "Rickettsial

encephalitides," in Pathology of the Nervous System, ed. Minck-ler, J. (McGraw-Hill, New York), Vol. 3, pp. 2344-2362.

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