Mycoplasma pneumoniae-Induced Hydrocephalus …post-inoculation. M.pneumoniaeM-129-B14,obtainedfrom...
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INFECTION AND IMMUNITY, Nov. 1984, P. 619-624 0019-9567/84/110619-06$02.00/0 Copyright © 1984, American Society for Microbiology Vol. 46, No. 2 Mycoplasma pneumoniae-Induced Hydrocephalus in Hamsters DENNIS F. KOHN,* NARUMOL CHINOOKOSWONG, AND JO WANG Department of Comparative Medicine, Medical School, University of Texas Health Science Center, Houston, Texas 77030 Received 13 August 1984/Accepted 17 August 1984 Hydrocephalus was induced in neonatal hamsters after intracerebral inoculation of Mycoplasma pneumo- niae. Examination of the ependyma from affected animals by electron microscopy did not reveal mycoplasma. However, in an ependymal organ culture system, M. pneumoniae cytadsorbed to ependymal cells. The pathogenesis of congenital hydrocephalus in humans is often obscuire. In utero infection has been suggested as a cause in some instances (1, 11). Animals models have demonstrated that several viruses and Mycoplasma pul- monis can induce the disease in neonatal rodents (6, 7, 10, 12). The pathogenesis in several of the viral models is associat- ed with gliosis and subsequent obliteration of the aqueduct of Sylvius; however, M. pulmonis does not induce an inflammatory response within the central nervous system (CNS). Although the pathogenesis of the M. pulmonis- induced hydrocephalus is not defined, electron microscopy has revealed that M. pulmonis cytadsorbs to the ependymal cells in hydrocephalic rats and in organ culture (8, 9). It has been postulated that this localization is associated with the pathogenesis of the disease in neonatal rats. In this study, we used neonatal hamsters and hamster ependymal organ culture to determine whether the human pathogen, Mycoplasma pneumoniae, was capable of induc- ing hydrocephalus. The hamster was chosen since it has been used by others to study host-M. pneumoniae interac- tions in the trachea and lung (2, 5, 14). Pregnant hamsters, Eng:ELA (SYR), were purchased from Engle Laboratory Animals, Inc., Farmersburg, Ind. Their 2- to 4-day-old litters were inoculated intracerebrally with either 0.02 ml of an inoculum containing M. pneumo- niae or sterile saline. Neonates were killed at 7 to 14 days post-inoculation. M. pneumoniae M-129-B14, obtained from J. B. Baseman, University of Texas Health Science Center at San Anto'nio, was used for animal and organ culture studies. Mycoplasma were grown in Hayflick medium con- taining PPLO broth (Difco Laboratories), 20% gamma globu- lin-free horse serum (GIBCO Laboratories), 10% fresh yeast extract, 0.002% phenol red, 1% glucose, and 1,000 U of penicillin G per ml. Broth cultures were centrifuged at 10,000 x g for 30 min and suspended in phosphate-buffered saline to yield 106 to 108 CFU/ml. Ependymal explants were prepared from the cerebella of 2- to 3-day-old hamsters and placed onto cover slips as previously described (8). Explants were maintained in tubes containing Ham F12 medium supplemented with 20% fetal calf serum (GIBCO) and 1% glutamine. Cultures exhibiting areas of vigorous ciliary activity were selected for use. Cultures were inoculated with 0.1 ml of an M. pneumoniae suspension, and specimens were examined by electron mi- croscopy at 2 to 4 days. For light microscopy, brains intact within skulls were fixed in 10% buffered Formalin, decalcified, embedded in * Corresponding author. paraffin, and stained with hematoxylin-eosin. Serial sections of brains were cut every 100 ,um in four hamsters. Specimens for scanning electron microscopy (SEM) were fixed in 3% Millonig buffered glutaraldehyde (pH 7.4), dehydrated in ethanol series, critical point dried with liquid carbon dioxide, and coated with palladium gold. Epe'ndymal organ explants for transmission electron microscopy (TEM) were grown on plastic cover slips (Thermanox Flow Labora- tories, Inc.), fixed in 3% buffered glutaraldehyde (pH 7.4) for 1 h, and postfixed in 1% buffered 'osmium tetraoxide for 1 h. They were then dehydrated in graded ethanols and propyl- ene oxide and embedded in Araldite. Sections were cut on an MT2-B ultramicrotome and stained with uranyl acetate and Reynolds lead citrate. Of 117 hamsters inoculated intracerebrally with M. pneu- moniae, 46 developed hydrocephalus. Doming of the skulls occurred in those hamsters most severely affected. Hydro- cephalus was limited to dilatation of the lateral ventricles in all cases (Fig. la). The dilatation was mild in 24 hamsters, moderate in 16, and severe in 6. Histological examination of brains intact within skulls revealed no inflammatory re- sponse within the meninges, ventricles, or brain parenchy- ma. The aqueducts and foraminae of Monro were patent. A pyogenic inflammatory response occurred in the middle ear and Eustachian tube in one of four hamsters that were examined (Fig. lb). Hydrocephalus occurred in both male and female neo- nates. The occurrence according to sex was examined in 41 animals. Within this sample, 9 of 18 males and 7 of 23 females became hydrocephalic. Although M. pneumoniae-like bodies were occasionally observed on the ependyma of the lateral ventricles by SEM, their presence could not be confirmed by TEM. SEM of infected organ culture specimens demonstrated large num- bers of M. pneumoniae cells cytadsorbed to ependymal cells. Whereas uninfected cultures had a luxuriant covering of cilia and microvilli (Fig. 2a), infected cultures had various degrees of denuded surfaces (Fig. 2b). TEM revealed M. pneumoniae cytadsorbed to ependymal cells in the organ culture system (Fig. 3). M. pneumoniae was observed by TEM to be cytadsorbed to both ciliated and nonciliated epithelial cells of the middle ear (Fig. 4). To our knowledge, this is the first demonstration of M. pneumoniae-induced disease within the CNS of an animal model. The hydrocephalus produced in these hamsters was comparable to that induced by M. pulmonis in rats. Light microscopy indicated that there was not an inflammatory response within the CNS or meninges and that stenosis of the aqueduct and foraminae was not present. Ependymal organ culture demonstrated that M. pneumoniae cytadsorbs to ependymal cells; however, this tropism could not be confirmed by TEM in ependymal specimens from hamsters. 619 on October 23, 2020 by guest http://iai.asm.org/ Downloaded from
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INFECTION AND IMMUNITY, Nov. 1984, P. 619-6240019-9567/84/110619-06$02.00/0Copyright © 1984, American Society for Microbiology
Vol. 46, No. 2
Mycoplasma pneumoniae-Induced Hydrocephalus in HamstersDENNIS F. KOHN,* NARUMOL CHINOOKOSWONG, AND JO WANG
Department of Comparative Medicine, Medical School, University of Texas Health Science Center, Houston, Texas 77030
Received 13 August 1984/Accepted 17 August 1984
Hydrocephalus was induced in neonatal hamsters after intracerebral inoculation of Mycoplasma pneumo-
niae. Examination of the ependyma from affected animals by electron microscopy did not reveal mycoplasma.However, in an ependymal organ culture system, M. pneumoniae cytadsorbed to ependymal cells.
The pathogenesis of congenital hydrocephalus in humansis often obscuire. In utero infection has been suggested as acause in some instances (1, 11). Animals models havedemonstrated that several viruses and Mycoplasma pul-monis can induce the disease in neonatal rodents (6, 7, 10,12).The pathogenesis in several of the viral models is associat-
ed with gliosis and subsequent obliteration of the aqueductof Sylvius; however, M. pulmonis does not induce aninflammatory response within the central nervous system(CNS). Although the pathogenesis of the M. pulmonis-induced hydrocephalus is not defined, electron microscopyhas revealed that M. pulmonis cytadsorbs to the ependymalcells in hydrocephalic rats and in organ culture (8, 9). It hasbeen postulated that this localization is associated with thepathogenesis of the disease in neonatal rats.
In this study, we used neonatal hamsters and hamsterependymal organ culture to determine whether the humanpathogen, Mycoplasma pneumoniae, was capable of induc-ing hydrocephalus. The hamster was chosen since it hasbeen used by others to study host-M. pneumoniae interac-tions in the trachea and lung (2, 5, 14).
Pregnant hamsters, Eng:ELA (SYR), were purchasedfrom Engle Laboratory Animals, Inc., Farmersburg, Ind.Their 2- to 4-day-old litters were inoculated intracerebrallywith either 0.02 ml of an inoculum containing M. pneumo-niae or sterile saline. Neonates were killed at 7 to 14 dayspost-inoculation. M. pneumoniae M-129-B14, obtained fromJ. B. Baseman, University of Texas Health Science Centerat San Anto'nio, was used for animal and organ culturestudies. Mycoplasma were grown in Hayflick medium con-taining PPLO broth (Difco Laboratories), 20% gamma globu-lin-free horse serum (GIBCO Laboratories), 10% fresh yeastextract, 0.002% phenol red, 1% glucose, and 1,000 U ofpenicillin G per ml. Broth cultures were centrifuged at 10,000x g for 30 min and suspended in phosphate-buffered saline toyield 106 to 108 CFU/ml.Ependymal explants were prepared from the cerebella of
2- to 3-day-old hamsters and placed onto cover slips aspreviously described (8). Explants were maintained in tubescontaining Ham F12 medium supplemented with 20% fetalcalf serum (GIBCO) and 1% glutamine. Cultures exhibitingareas of vigorous ciliary activity were selected for use.Cultures were inoculated with 0.1 ml of an M. pneumoniaesuspension, and specimens were examined by electron mi-croscopy at 2 to 4 days.For light microscopy, brains intact within skulls were
fixed in 10% buffered Formalin, decalcified, embedded in
* Corresponding author.
paraffin, and stained with hematoxylin-eosin. Serial sectionsof brains were cut every 100 ,um in four hamsters.Specimens for scanning electron microscopy (SEM) were
fixed in 3% Millonig buffered glutaraldehyde (pH 7.4),dehydrated in ethanol series, critical point dried with liquidcarbon dioxide, and coated with palladium gold. Epe'ndymalorgan explants for transmission electron microscopy (TEM)were grown on plastic cover slips (Thermanox Flow Labora-tories, Inc.), fixed in 3% buffered glutaraldehyde (pH 7.4) for1 h, and postfixed in 1% buffered 'osmium tetraoxide for 1 h.They were then dehydrated in graded ethanols and propyl-ene oxide and embedded in Araldite. Sections were cut on anMT2-B ultramicrotome and stained with uranyl acetate andReynolds lead citrate.Of 117 hamsters inoculated intracerebrally with M. pneu-
moniae, 46 developed hydrocephalus. Doming of the skullsoccurred in those hamsters most severely affected. Hydro-cephalus was limited to dilatation of the lateral ventricles inall cases (Fig. la). The dilatation was mild in 24 hamsters,moderate in 16, and severe in 6. Histological examination ofbrains intact within skulls revealed no inflammatory re-sponse within the meninges, ventricles, or brain parenchy-ma. The aqueducts and foraminae of Monro were patent. Apyogenic inflammatory response occurred in the middle earand Eustachian tube in one of four hamsters that wereexamined (Fig. lb).Hydrocephalus occurred in both male and female neo-
nates. The occurrence according to sex was examined in 41animals. Within this sample, 9 of 18 males and 7 of 23females became hydrocephalic.Although M. pneumoniae-like bodies were occasionally
observed on the ependyma of the lateral ventricles by SEM,their presence could not be confirmed by TEM. SEM ofinfected organ culture specimens demonstrated large num-bers of M. pneumoniae cells cytadsorbed to ependymalcells. Whereas uninfected cultures had a luxuriant coveringof cilia and microvilli (Fig. 2a), infected cultures had variousdegrees of denuded surfaces (Fig. 2b). TEM revealed M.pneumoniae cytadsorbed to ependymal cells in the organculture system (Fig. 3). M. pneumoniae was observed byTEM to be cytadsorbed to both ciliated and nonciliatedepithelial cells of the middle ear (Fig. 4).To our knowledge, this is the first demonstration of M.
pneumoniae-induced disease within the CNS of an animalmodel. The hydrocephalus produced in these hamsters wascomparable to that induced by M. pulmonis in rats. Lightmicroscopy indicated that there was not an inflammatoryresponse within the CNS or meninges and that stenosis ofthe aqueduct and foraminae was not present. Ependymalorgan culture demonstrated that M. pneumoniae cytadsorbsto ependymal cells; however, this tropism could not beconfirmed by TEM in ependymal specimens from hamsters.
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FIG. 1. (a) Coronal section through the brain of a hydrocephalic hamster. Note the patent aqueduct (arrow) and dilated lateral ventricles.Otitis media is present in one ear (double arrow). Staining was with hematoxylin and eosin. Magnification. x18. (b) Photomicrograph ofmiddle ear, showing impaction of bulla with neutrophilic polymorphonuclear cells. Magnification. x54.
INFECT. IMMUN.
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i_w L- - -iw I.F X% 4..W~ jFIG. 2. SEM of noninfected ependymal organ culture. Note the numerous cilia and dense lawn of microvilli. Bar = 2.3 ,um. (b) SEM of M.
pneumoniae-infected organ culture. Note the loss of microvilli and the few short cilia among the mycoplasma. Bar = 0.74 ,um.
VOL. 46, 1984
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FIG. 3. TEM of a ciliated cell from an ependymal organ culture with several M. pneumoniae cells cytadsorbed (arrows) and two M.pneumoniae cells lying among microvilli and cilia. Bar = 0.3 iim.
Confirmation of a tropism for the ependyma must awaitmore sensitive methods such as peroxidase-antiperoxidaseimmunocytochemical detection of M. pneumoniae antigen.The CNS sequellae in humans may be associated withmycoplasmal infection of the CNS, to autoimmune mecha-nisms, or to toxin elicited from mycoplasmas located in non-
CNS sites (3, 4, 13). Although it is most probable that thehydrocephalus in this hamster model is due to a tropism ofM. pneutmoniae for a site within the CNS, it is possible thatthe pathogenesis is associated with dysfunction of cerebro-spinal fluid secretion or absorption mediated by toxic prod-
ucts of the organism. The otitis media observed was proba-bly associated with an initial infection of the nasopharynxsince Eustachian tubes were affected. M. pneumoniae-in-duced disease was dissimilar from M. pulmonis-associatedhydrocephalus: hydrocephalus occurred in only 40% of theneonates, and there was no arthrogenic effect in the verte-brae and the joints of the limbs.
Further studies will be required to delineate more ade-quately the pathogenesis of this hydrocephalus model and toevaluate the usefulness of the hamster as a model of CNSdysfunction associated with M. pneumoniae.
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FIG. 4. TEM of middle ear from hamster with otitis media. Note M. pnei/noniae cell cytadsorbed to the ciliated epithelial cell. Bar = 0.5±m.
LITERATURE CITED
1. Baumann, B., L. Danon, R. Weitz, R. Blumensohn, T. Schanfeld,and M. Nitzan. 1982. Unilateral hydrocephalus due to obstruc-tion of the foramen of Monro: another complication of intrauter-ine mumps infection? J. Pediatr. 139:158-159.
2. Collier, A. M., W. A. Clyde, and F. W. Denny. 1969. Biologiceffects of Mycoplassma pneuinonirae and other mycoplasmasfrom man on hamster tracheal organ culture. Proc. Soc. Exp.Biol. Med. 132:1153-1158.
3. Decaux, G., M. Szyper, M. Ectors, A. Cornil, and L. Franken.1980. Central nervous system complications of MycoplasInapneumnoniae. J. Neurol. Neurosurg. Psychiatry 43:883-887.
4. Fisher, R. S., A. W. Clark, J. S. Wolinsky, I. M. Parhad, H.Moses, and M. R. Mardiney. 1983. Postinfectious leukoencepha-litis complicating Mycoplasma pneumoniae infection. Arch.Neurol. 40:109-113.
5. Jemski, J. V., C. M. Hetsko, C. M. Helms, M. B. Grizzard, J. S.Walker, and R. M. Chanock. 1977. Immunoprophylaxis ofexperimental Mycoplasmna plleil)oniae disease: effect of aero-sol particle size and site of deposition of M. pneiurnoniae on thepattern of respiratory infection, disease, and immunity in ham-sters. Infect. Immun. 16:93-98.
6. Johnson, R. T., and K. P. Johnson. 1968. Hydrocephalusfollowing viral infection: the pathology of aqueductal stenosisdeveloping after experimental mumps virus infection. J. Neuro-
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pathol. Exp. Neurol. 27:591-606.7. Johnson, R. T., and K. P. Johnson. 1969. Hydrocephalus as a
sequela of experimental myxovirus infectious. Exp. Mol.Pathol. 10:68-80.
8. Kohn, D. F., and N. Chinookoswong. 1980. Pathogenicity ofMcycoplasIna pilinioniis in ependymal organ culture. Infect.Immun. 28:601-609.
9. Kohn, D. F., and N. Chinookoswong. 1981. Cytadsorption ofMvycoplasina plilmonis to rat ependyma. Infect. Immun. 34:292-295.
10. Kohn, D. F., B. E. Kirk, and S. M. Chou. 1977. Mvoplasina-induced hydrocephalus in rats and hamsters. Infect. Immun.16:680-689.
11. Norman, M. G., L. A. Thurber, and H. E. Woolley. 1981.Abnormal leptomeninges and vessels causing fetal hydrocepha-lus: diagnosis of hydrocephalus at 19 weeks gestation by ultra-sound. Acta Neuropathol. 54:283-285.
12. Phillips, P. A., M. P. Alpers, and N. F. Stanley. 1970. Hydro-cephalus in mice inoculated neonatally by the oronasal routewith reovirus type I. Science 168:858-859.
13. Ponka, A. 1980. Central nervous system manifestations associ-ated with serologically verified Mycoplasma pneurnoniae infec-tion. Scand. J. Infect. Dis. 12:175-184.
14. Powell, D. A., P. C. Hu, M. Wilson, A. M. Collier, and J. B.Baseman. 1976. Attachment of Mvc oplasmna pne,lnoniiae torespiratory epithelium. Infect. Immun. 13:959-966.
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