Effects of Haemophilus influenzae meningitis in infant rats on neuronal growth and synaptogenesis

EXI'EHIMENTAL

~XEUROLOGY

50,337-345

Effects of Haemophilus Rats on Neuronal R. AVERILL,

DAMON Departments Medical

(1976)

influenzae Meningitis in Infant Growth and Synaptogenesis

JR., E. RICHARD

MOXON,

AND ARNOLD L. SMITH

1

of Pathology (Neuropathology), and Medicine, The Children’s Hospital Ceflter; and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received

Augzlst

20,1975

The growth of neocortical pyramidal cell dendrites and the number of ethanolic-phosphotungstic acid-stained synapses in the rat brain was studied at various times following survival from experimentally produced neonatal Haemophilus influensae meningitis with bacteremia. The mean number of dendrites and the branching complexity of 30 neurons in rats 7, 20, 40 and 67 days of age following intranasal inoculation of bacteria on day 5 was less than age-matched controls. The mean number of synapses was reduced in three postmeningitic and three control rats at 40 and 67 days of age. This data suggests that reduced neuronal connectivity resulting from neonatal meningitis with bacteremia may be an anatomic correlate of the postmeningitic learning disorders.

INTRODUCTION The sequelae of H. Gzfluenzae type b meningitis may be devastating. Children surviving the infection have a 30% risk of sustaining serious residua including blindness, deafness, seizures and mental retardation (10, 22). Ten percent of grossly normal survivors may have subtle deficits detected by sophisticated psychological testing ( 18, 19). The mechanismsfor these sequelaeare unknown but have generally been presumed to result from widespread histopathologic destruction of central nervous tissue associated with meningeal vasculitis and cerebral microinfarction. This conclusion is based on the histopathologic descriptions of autopsy material : a population significantly different from individuals which survive neonatal meningitis. 1 Supported in part by the Children’s Hospital Medical Center Mental Retardation and Human Development Research Program (HD-06276-03), NICHD. The authors thank Ms. R. Fogelis and Mr. H. Hall without whose technical support this work could not have been carried out.

Copyright All rights

by AcademicPress,Inc. 9 1976 reproduction in any form reserved.

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We have chosen to study an experimental model of neonatal M. influenzae meningitis in the rat for several reasons: (a) the infection is readily produced by noninvasive intranasal inoculation of bacteria, the probable natural route of entry; (b) the occurrence of meningitis may be easily predicted by a 48 hr postinoculation coccygeal vein bacteremia of > lo4 bacteria/ml ; (c) more than 80% of animals inoculated with pathogenic H. in@en~ue survive without treatment ; and (d) we have already demonstrated that postmeningitic animals show a decreased rate of acquisition of operant conditioning (21). We have been unable to detect correlative histopathologic lesions for this learning deficit where the only abnormality within the central nervous system is focal plasma cell infiltrates in the meninges of about 40% of the adults (12). Since it has been shown that other manipulations of the developing brain such as hypothyroidism (7)) decreased rearing complexity (23)) sensory deprivation (S), and corticosteroid administration (14) can decrease the growth of neuronal processes and suggested that the retarded growth of neurons may be an anatomic substrate for mental subnormality (8, 9, 11, 15) we studied the growth of cerebral cortical pyramidal cell dendrites and the number of synapses in the cortex of rats surviving neonatal meningitis. This report demonstrates that H. in@enzue type b meningitis and bacteremia results in a reduction in dendritic numbers, reduced dendritic complexity, and decreasednumber of synapseswhich persists to early adulthood. MATERIALS

AND METHODS

Six Sprague-Dawley COBS/CD rats were studied at each of four ages following intranasal inoculation with bacteria at 5 days of age. The animals were bred and housed under fluorescent lighting of 12 hr on, 12 hr off. Food and water were available ad lib. Litters from eight mothers were cross-fostered on postnatal day 1 and the litters reduced to 10 pups. Four litters were inoculated with type b H. inflztensae strain Eagen (E,) and four with untypeable Ramirez strain (U 11 nonpathogenic) as previously described (12). Litters were housed separately to avoid cross-contamination. Three animals from each type b inoculated litter were selected for anatomic study on the basis of having > lo4 organisms/ml blood 48 hr after inoculations at the following ages: 7 days, 20 days, 40 days and 67 days. Three rats from the litters inoculated with U11 type bacteria were randomly chosen as a control group at each of the sametime points. Animals were weighed and killed by intracardiac perfusion with 1% paraformaldehyde 1.25% glutaraldehyde in 0.1 p phosphate buffer pH 7.4 while under deep ether anesthesia. The brains were carefully removed, weighed, and rinsed overnight in 0.1 M phosphate buffer. The left half of

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the brain was then sliced into coronal blocks embedded in paraffin, sectioned, and stained with hematoxylin and eosin-luxol fast blue for light microscopy. TWO 0.1 X 0.1 cm blocks 0.1 cm lateral to the dorsal longitudinal fissure and 0.5 cm from the right frontal pole were prepared for electron microscopy. The medial block was stained with ethanolic phosphotungstic acid (E-PTA) by the method of Agajanian and Bloom (2) in the 40 and 67 day old rats only. Samples were then embedded in epon and sectioned with glass knives to silver interference color for electron microscopy. The EPTA blocks were trimmed to layer I of cerebral cortex. Three random photographs taken from immediately below the pial surface at 12,000 x were made from each E-PTA stained block and the number of synapses identifiable by the presence of preterminal dense projections, intracleft line and postsynaptic band were counted directly off cut film negatives. Abercrombie’s correction factor (1) for calculating synaptic density was used assuming the sections were 700 A thick and a mean synaptic diameter of 2500 A. The remaining right cerebral hemisphere was impregnated with a rapid Golgi technique ( 16)) embedded in low-viscosity nitrocellulose and serially sectioned at 150 p. The images of ten undamaged layer V pyramidal neurons were traced onto paper with the aid of a Zeiss drawing tube. The neuron soma was drawn in the center of a grid of concentric rings with each interval representing 20 pm calibrated with a stage micrometer (7, 20). No attempt was made to correct for the error produced by converting three-dimensional images to two-dimensional images. The number of primary dendrites, number of dendritic intersections with each ring, and the total number of dendritic endings were tabulated without knowledge of previous treatment. Somatic tissues were embedded in paraffin, prepared for light microscopic examination by standard methods, and stained with hematoxylin and eosin. RESULTS There was no significant difference between the mean body weight or fixed brain weight at any age (Table 1). Significant histologic findings were limited to the meninges in seven of the 12 type b inoculated rats. All three rats examined on postinoculation day 2 had moderate to severe purulent and mononuclear meningitis as described previously (12, 21). One 20 day old, two 40 day old, and one 67 day old rat had rare focal mononuclear meningitis. There were no significant histologic findings in the Urr inoculated animals. Somatic tissues including lung, liver, spleen, kidney, myocardium, urinary bladder, lymph node, skeletal muscle, trachea, nasal mucosa, and skin were free of histologic lesions in all animals.

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SNITII

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A.

RADIAL DISTANCE FROM NEURON WI

B.

D

RADIAL DISTANCE FROM NEURON ()rMl

RADIAL DISlANCC

FROM NEURON

W.4)

FIG. 1. The mean number of dendrites intersecting circular rings at 20 pm intervals from the center of pyramidal cell soma. Each point represents 30 layer V frontal cortex neurons.

The mean number of dendrites is illustrated in Fig. 1. At every point counted, the mean number of dendritic intersections with the circular grid was larger in control animals than in postmeningitics. As a measure of dendritic complexity the mean dendritic branching index was calculated from a ratio of total dendritic endings to primary somal dendrites (7). The fi nd’mg s are presented in Fig. 2 where. there is a sig-

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(days)

FIG. 2. The ratio of primary dendrites to total dendritic endings as a function of time following meningeal infection occurring between 5 and 21 days of age. Each point represents 30 neurons. Twenty, 40 and 67 day indices are significantly different by f-test P < 0.01. Seven-day animals are not different.

nificant difference in the mean dendritic branching index at 20, 40 and 67 days. At each of these points the postmeningitic dendritic branching index was smaller than controls. There was a significant reduction in the total number of layer I E-PTAstained synapses in the postmeningitic rats at 40 days which was no longer significant at 67 days of age (Fig. 3).

FIG. 3. The mean number of E-PTA cantly different (P < 0.01) at 40 days the slight variance at 67 days is not represents nine cortical samples,

synapses in frontal cortex layer of age but “catch up” apparently significant (X 0,2 > P > 0.1).

I is signifioccurs since Each point

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DISCUSSION Decreased numbers of dendritic intersections are already apparent in the rat pyramidal neurons 2 days after inoculation (7 days of age). The degree of alteration is slight, representing a 10% decrease at all levels. The dendritic field diameter is not reduced, however, and at 20 days of age both experimental and control neurons continue to enlarge to adult-sized dendritic fields, but the postmeningitic animals have fewer dendrites in all positions within the dendritic field. There is little increase in dendritic numbers from 20 to 40 days of age and the difference between the two groups at 40 days persists. The 67 day old postmeningitic pyramidal cells have fewer dendrites than the 40 day old treated animals, suggesting an increased loss of dendrites at distances greater than 60 pm from the cell soma. We interpret these findings are representing a decrease in growth and premature loss of dendritic processes in postmeningitic animals. The divergence in dendritic complexity is best seen in alterations of the dendritic branching index where between 7 and 20 days of age differences appear and persist in the 40 and 67 day old animals. Since the neuronal dendritic processes represent at least 90% of the available area for synaptic influence (13)) this reduction in dendritic numbers and dendritic complexity is a significant decrease in cortical neuron connectivity. Similarly, since postsynaptic membranes are reduced, synaptic terminals do not form as illustrated by the reduction of E-PTA-stainable synapses at 40 days of age. The absolute numbers of E-PTA synapses in the normal rats correspond well with those available in the literature (3, 4), except that they apparently increase between 40 and 67 days of age in both normal and postmeningitic animals. This did not occur in Bloom’s series but may represent differences in animal strain or the variation in region studied in both postmeningitic and control rats. The insignificant difference in synaptic numbers at 67 days of age may represent a “catch up” phenomenon perhaps to accommodate for dendritic loss. Similar catch up phenomena have been recognized for brain weight acquisition in nutritional deprivation experiments (6). It is unlikely that undernutrition contributed to decreased cortical connectivity in these animals since the brain and body weights do not differ significantly. We cannot exclude the possibility that local undernutrition of the brain occurs in meningitis however, and it seems plausible that reduced availability of substrates or structural material is the basic mechanism for reduced numbers of membranous components in the cerebral cortex. Whether meningitis with bacteremia affects these mechanisms transport mechanisms, or through decreased blood flow, diminished

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increased corticosteroid elaboration which has already been shown to reduce rat pyramidal cell complexity (14) is yet to be determined. Other authors have suggested that the richness of neonatal interconnections made available by dendritic complexity represents the neuroanatomical substrate of behavior (8, 16). \h’e believe that decreased neuronal complexity and neocortical synapses resulted in decreased performance in the operant conditioning experiment in postmeningitic rats. This model of H. inflzlen,-ae meningitis parallels the human infection with respect to age at onset, pathogenesis, and histopathologic changes. It is significant, however, that the duration of infection in the rat straddles the entire period of acquisition of adult numbers of dendrites (17) and synapses (4). The analogous human development period is the first 3-4 postnatal years. Therefore, the effects of this infection upon the rat brain, though they qualitatively represent the consequences of the human disease, do not characterize the extent of damage expected in H. infizben=ae meningitis of childhood. These observations serve to illustrate the concept that the mental disabilities resulting from neonatal meningeal infection may be related to abnormalities of neuronal maturation rather than diffuse tissue destruction. The latter consequence is fixed and untreatable. The former may be modified by pharmacologic, nutritional and environmental means. The mechanism of this effect on brain growth must be sought so that appropriate therapeutic manipulations may be studied. REFERENCES 1. ABERCROMBIE, Anat. Rec. 2. AGHAJANIAN,

M. 1946. Estimation 94:

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M., and R. JOHNSON. 1970. Quantitative studies of postnatal changes in synapses in rat superficial motor cortex. 2. Zcllforaclz. 110 : 559-568. BLOOM, F. E. 1972. The formation of synaptic junctions in the developing rat brain, pp. 101-119. I>t : “Structure and Function of Synapses.” C. D. Pappas and D. P. Purpura (Eds.), Ranen Press, New York. COLEMAN, P. D., and A. H. RIESEN. 1968. Environmental effects on cortical dendritic fields. I. Rearing in the dark. J. Anat. 102: 363-374. DAVISON, A. N., and J. DOBBING. 1968. The developing brain. 111: “Applied Neurochemistry.” A. N. Davison, and J. Dobbing [Eds.], F. A. Davis Co., Philadelphia, Pa. EAYERS, J. T., and B. GOODHEAD. 1959. Postnatal development of the cerebral cortex in the rat. J. Aunt. 93 : 385-402. HEBB, D. 0. 1949. “The Organization of Behavior,” Wiley, New York. HUTTENLOCHITX, P. R. 1974. Dendritic development in neocortex of children \vitll mental deficit and infantile spasms. i!re~~ro/ogy 24: 203-210.

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B., S. BUCHBENDER, and I. M. GREENBERG. 1962. The incidence of neuroresidua in children after recovery from bacterial meningitis. Arch. Pediatr. 79: 63-71. 11. MARIN-PADILLA, M. 1974. Structural organization of the cerebral cortex (motor area) in human chromosomal aberrations. A Golgi study. I Dl (13-15) Trisomy, Patau syndrome. Brain Res. 66: 375-391. 12. MOXON, E. R., A. L. SMITH, D. R. AVERILL, and D. H. SMITH. 1974. Haewwphillus in&ensue meningitis in infant rats after intranasal inoculation. J. Infect. Dis. 129 : 154-162. 13. MUNGAI, J. M. 1967. Dendritic patterns in the somatic sensory cortex of the cat. J. Anat. 101: 403-418. 14. ADA, M. A. S., and P. R. HUTTENLOCHER. 1974. The effect of corticosteroids on dendritic development in the rat brain. Yale J. Biol. Med. 47 : 155-166. 15. PURPURA, D. P. 1974. Dendritic spine “dysgenesis” and mental retardation. Science. 186 : 11261128. 16. RAKIC, P. 1972. Mode of cell migation to the superficial layers of fetal monkey neocortex. J. Cow@. Neural. 145: 61-84. 17. SARKISOV, S. A., E. N. PAPOVA, and M. N. BOGOLENOV. 1966. Structure of synapses in evolutionary aspect, pp. 225-237. In: “Evaluation of the Forebrain,” R. Hassler and H. Stephan [Eds.]. G. T. Verlag, Stuttgard. 18. SELL, S. H. W., R. E. MERRILL, E. 0. DOYNE, and E. P. ZEMSKY, JR. 1972. Long term sequelae of Haemophillus influenzae meningitis. Pediatrics. 49: 206211. 19. SELL, S. H. W., W. W. WEBB, J. E. PATE, and E. 0. DOYNE. 1972. Psychological sequelae to bacterial meningitis. Two controlled studies. Pediatrics. 49: 212-217. 20. SHOLL, D. A. 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Amt. 87 : 387-406. 21. SMITH, A. L., D. H. SMITH, D. R. AVERILL, J. MARINO, and E. R. MOXON. 1973. Production of Haemophillus in&en,-ae b meningitis in infant rats by intraperitoneal inoculation. Infect. and Immun. 8: 275-290. 22. SPROLES, E. T., J. AZERRAD, C. WILLIAMSON, and R. E. MERRILL. 1969. Meningitis due to Haemophillus influenzae: long term sequelae. f. Pediafr. 75: 782-788. 23. VOLKMAN, F. R., and W. T. GREENOUGH. 1972. Rearing complexity effects branching dendrites in visual cortex of the rat. Science. 176: 1445-1447. 10. KRESKY,

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