Journal of the neurological Sciences, 1975, 26:167-178
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© ElsevierScientificPublishingCompany, Amsterdam- Printed in The Netherlands
Experimental P n e u m o c o c c a l Meningitis Effects of Neuraminidase and other Pneumococcal Constituents on Cerebrospinal Fluid in the Intact Dog R I C H A R D D. O'TOOLE* AND W I L L I A M L. STAHL** Departments of Medicine and Physiology and Biophysics, University of Washington School of Medicine, and the Veterans Administration Hospital, Seattle, Wash. (U.S.A.)
(Received 15 February, 1975)
INTRODUCTION Pneumococci isolated from infected tissues have been shown to elaborate a neuraminidase which cleaves terminal molecules of N-acetylneuraminic acid (NANA) from glycoproteins and gangliosides (Rafelson 1963;Hughes and Jeanloz 1964a, b; Lee and Howe 1966; Kelly, Farmer and Greiff 1967). Intracerebral injection of neuraminidase in mice results in release of N A N A from exposed tissues and also in death of the animal (Kelly and Greiff 1970). In patients with pneumococcal meningitis elevated concentrations of cerebrospinal fluid (CSF) free N A N A have been observed (O'Toole, Goode and Howe 1971 ). Elevated CSF-free N A N A levels have been shown to be correlated with an adverse prognosis in man, and O'Toole et al. (1971) and Muller (1969) proposed that the cleavage of N A N A from the glycoproteins and gangliosides of cerebral tissue might contribute to morbidity and mortality in pneumococcal meningitis. This study was undertaken to study the role of neuraminidase activity in experimentally infected dogs by assessing the effect of intrathecal administration of neuraminidase from pneumococci on morbidity and CSF components of dogs. MATERIALSAND METHODS A type I pneumococcus isolated from the cerebrospinal fluid of a patient with meningitis was employed in this study (O'Toole et al. 1971 ). It was passaged in mice every 6 weeks, and stored in defibrinated rabbit blood at - 7 0 ° C until used. Dr. O'Toolewas supported by the Veterans AdministrationResearchAssociateProgram. *Present address: The MedfordClinic, 1025E. Main Street, Medford,Ore. 97501. **Reprint requests to this author at: NeurochemistryLaboratory,VA Hospital, 4435 BeaconAvenueS., Seattle, Wash. 98108, U.S.A.
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Enzyme assays In the neuraminidase assay, 0.2 ml of spinal fluid or enzyme preparation was incubated at 37°C with 2.5 mg human a-l-acid glycoprotein (Calbiochem), dissolved in 0.8 ml of 0.15 M NaCI and buffered to pH 6.5 with 0.15 M phosphate, as substrate. After 60 min incubation, duplicate 0.4 ml samples were taken from the reaction mixture and analyzed for free NANA by the thiobarbituric acid assay of Warren (1959) with saline-containing substrate as a control and crystalline NANA (KochLight Labs, Ltd., Colnbrook, England) as the standard. One unit was defined as the amount of neuraminidase required to liberate 1 nmole of NANA in 60 min tinder assay conditions. NANA-aldolase activity was assayed according to the method of Brunetti, Jourdian and Roseman (1962). A unit of activity is defined as the quantity of enzyme that will cleave 1.0 ~Lmole of NANA in 15 min under the standard assay conditions, fl-Galactosidase was assayed by a modification of the procedure of Hughes and Jeanloz (1964a). Proteolytic enzyme activity was assayed by the method of Lin, Means and Feeney (1969). Acetylcholinesterase was assayed using the method of McCamen, Tomey and McCamen (1968). Monoamine oxidase was assayed using the procedure of McCamen, McCamen, Hunt and Smith (1965). Succinate dehydrogenase was assayed using the method of Slater, Sawyer and Strauli (1963). Other analyses Protein concentrations of preparations were established by the method of Lowry, Rosebrough, Farr and Randall (1951). Free and total NANA concentrations were determined according to the method of Jakoby and Warren (1961). Spinal fluid leucocyte counts were determined by the standard counting chamber method. Glucose concentrations on CSF and serum (venous blood sampled daily at the time of cisternal puncture) were measured by the Auto-analyzer ferricyanide method. Preparation of crude neuraminidase This was essentially the procedure of Tanenbaum, Gulbinsky, Katz and Sun (l 970). Thawed pneumococci were inoculated into Todd-Hewitt broth (Difco) and incubated at 37~C for 48 hr. Purity of growth was confirmed by culture on to rabbit blood agar plates, and pneumococci were removed by centrifugation at 700 x g for 2 hr at 4°C and discarded. Protein was precipitated from the supernatant with ammonium sulfate at 7 5 ~ saturation. After standing overnight at 4°C, precipitated proteins were centrifugated at 700 x 9 for I hr in the cold, the supernatant was discarded, and the precipitate was taken up in 0.9~o NaCI and stored at -20°C. All samples were pooled, dialyzed against repeated changes of deionized water followed by dialysis against 0.9~o NaC1 (adjusted to pH 7.3 with NaHCO3)and were centrifuged at 95,000 x 9 for 60 min in the cold. The supernatant was concentrated through an XM50 Diaflow membrane filter (Amicon Corporation, Lexington, Mass.), passed through a millipore filter (Millipore Corporation, Bedford, Mass.), pore size 0.45 ~m, and cultured to confirm sterility. The final suspension was adjusted with 0.9~ NaC1 so that 1.0 ml contained 29.3 mg protein and possessed 760 units of neuraminidase activity, no detectable fl-galactosidase activity and, when assayed for proteolytlc
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enzyme activity, hydrolyzed 0.039/~moles bonds/mg protein/0.5 hr. Previous work (Stahl and O'Toole 1971 ) showed that this preparation had many protein components. This preparation contained no detectable neuraminidase activity following exposure to a temperature of 75°C for 15 min.
Preparation of partially purified neuraminidase The procedure of Stahl and O'Toole (1971) was used. Crude enzyme was dialyzed against 0.1 M sodium acetate and applied to columns packed with DEAE-Sephadex in the acetate form (Pharmacia Fine Chemicals, Piscataway, N.J.). Stepwise elution with 0.1M sodium acetate containing sodium chloride (0.14).4 M) yielded a peak of neuraminidase activity in the eluate between 0.2 and 0.3 M sodium chloride. These fractions were pooled, dialyzed against 0.9~ sodium chloride, concentrated against a Diaflo XM-50 filter, passed through a 0.45 ~tm pore-size filter, and stored at - 2 0 ° C until used. This preparation was bacteriologically sterile, and contained 0.028 units of NANA-aldolase activity, no detectable fl-galactosidase activity, and, when assayed for proteolytic enzyme activity, cleaved 0.428 #moles bonds/mg protein/0.5 hr. As shown previously (Stahl and O'Toole 1971) the preparation had only one major protein component with an estimated molecular weight of 69,800. Neuraminidase dose determination Neuraminidase activity and free NANA concentrations were determined for 17 patients previously reported, who had pneumococcal meningitis and elevated CSF-free N A N A levels (O'Toole et al. 1971). Animal studies Mongrel dogs weighing between 15 and 30 kg were used in all experiments. Dogs were anesthetized intravenously with sodium pentobarbital, and all test preparations were administered intracisternally via sterile, disposable 22 gauge needles. A normal saline-5 ~ glucose solution was administered (22.5 ml/kg) by clysis at the end of each day's experiment, and the animals were given water, but no food. Experiment I. 2.3 x 105 to 9.8 x 106 viable pneumococci were suspended in 1.0 ml of normal saline and injected into the cisterna magna of 11 animals in order to establish values for neuraminidase activity and for free NANA concentrations in the infected CSF of dogs. Cisternal fluid was sampled daily for as long as the dogs survived or remained infected and was tested for free NANA and neuraminidase activity. The number of viable pneumococci/ml of infected CSF was estimated with pour plates containing agar and defibrinated rabbit blood. Experiment 2. Crude neuraminidase was administered daily for 5 days to 13 test dogs and heat-inactivated crude neuraminidase was given to 6 dogs. 0.4 ml of active or heat-inactivated enzyme preparation was administered intracisternally to dogs on days 1 and 2, and 0.8 ml was given on days 3, 4 and 5, 2.0 ml CSF was withdrawn daily for analysis prior to administration of the test material. On day 6, 1.0 ml lumbar CSF was obtained, followed by removal of 2.0 ml cisternal CSF. Experiment 3. Four dogs received 2 × 108 heat-killed pneumococci intracisternally daily for 5 days. Pneumococci were grown for 16 hr in Todd-Hewitt broth, washed
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twice in normal saline (pH 7.3), resuspended in 1.0 ml saline, and heated lor 5 rain at 100°C. This suspension contained no enzyme activity and 1.22 mg protein/ml. Seven additional dogs were given 2 x l0 s heat-killed pneumococci: 5 of these animals were simultaneously treated with crude neuraminidase as in experiment I. and 2 control dogs were given the inactivated enzyme. Experiment 4. Three dogs were treated with a suspension of partially purified neuraminidase with total enzyme activity identical to the crude enzyme preparation used in experiments 1 and 2. The enzyme was suspended in saline, buffered to pH 7.3 with bicarbonate. Two animals received the heat-inactivated crude enzyme preparation which was adjusted by dilution in buffered saline to the same protein concentration (1.53 mg/ml) as the partially purified active enzyme. In addition to these experiments CSF was drawn from 24-58 normal healthy dogs for neuraminidase and NANA determinations. Subcellular fractions These were obtained from the cerebral cortex grey matter of dogs using the method of Gray and Whittaker (1962). On experimental day 6, 10 dogs were sacrificed. This was 24 hr following the last intracisternal injection. Three animals had been treated with heat-inactivated crude neuraminidase (expt. 2), 4 were treated with active crude neuraminidase (expt. 2), and 3 had been treated with active partially purified neuraminidase (expt. 4). The brains were isolated immediately after sacrifice, and pia-arachnoid was stripped from the cerebral hemispheres, and 5-7 g of cortical grey matter were removed and placed in ice, then stored at - 70°C. The fractionation was carried out within 1 week. Succinate dehydrogenase was assayed on the same day as the fractionation procedure, whereas monoamine oxidase and acetylcholinesterase were assayed in aliquots stored at-20°C and tested within 2 to 3 weeks of the fractionation. For determination of total NANA, portions of each subcellular fraction were dialyzed against deionized water for several days and were hydrolyzed and assayed for total NANA according to the method of Jakoby and Warren (1961 ).
RESULTS
CSF was obtained from 35 patients with culture-proven pneumococcal meningitis prior to therapy (O'Toole et al. 1971). The CSF of 17 of these patients contained abnormally elevated concentrations of free NANA. The CSF of these 17 patients possessed 23.29 _ 24.35 (SD) units of neuraminidase activity per 0.2 ml (Fig. 1) and 2.49_ 2.19 mg free NANA/100 ml (Fig. 2). Based on these observations dogs were treated with pneumococci or neuraminidase preparations to achieve CSF levels of neuraminidase and NANA at least as high as those found in patients. Clinical observations Four experiments were carried out with dogs. In experiment 1 the CSF of all 11 dogs administered viable pneumoeocci became infected for 4-6 days or until death. Two animals died at 48 hr, 2 died at 96 hr, 2 survived without detectable residua, and
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Fig. 1. Mean CSF neuraminidase activity in experimental pneumococcal meningitis: 17 patients with naturally acquired disease had 23.29 ___24.35 (SD) units per 0.2 ml of CSF (A), 5 experimentally infected dogs developing elevated CSF enzyme concentrations in experiment 1 (23.48+__11.35 units/0.2 ml CSF) (B). These values are compared with mean levelsof CSF enzyme activity in 13 dogs in experiment 2 treated with crude neuraminidase (C), and 3 dogs in experiment 4 treated with purified neuraminidase (D), also 24 normal, untreated dogs had 1.28+ 0.44 units of activity per 0.2 ml CSF (E). All animal CSF samples were obtained by cisternal puncture and therefore have been corrected according to cisternal/lumbar ratios observed in dogs (see RESULTS)for comparison with values found in human lumbar CSF. Fig. 2. Mean CSF concentrations of free NANA in pneumococcal meningitis: 17 patients with naturally acquired disease had 2.49-I-2.19 mg free NANA/100 ml CSF (A) and 3 experimentally infected dogs developing abnormally elevated CSF free NANA concentrations in ~:xperiment 1 (B). These values are compared with the mean free NANA levels detected in the 13 dogs in experiment 2 treated with crude neuraminidase (C), in 3 dogs in experiment 4 treated with purified neuraminidase (D) and in 58 normal, untreated dogs (E, 0.670+0.216 mg/100 ml). CSF samples have been corrected according to cisternal/lumbar ratios as in Fig. 1.
5 continued to show signs o f a residual vestibu|o-cerebellar s y n d r o m e prior to sacrifice, 16-68 days after infection. One o f 6 dogs in experiment 2, which were treated with inactivated crude p n e u m o coccal enzyme, showed extensor rigidity o f all limbs, and total loss o f balance, righting reflex, and integrated m o t o r m o v e m e n t s for 24 hr on day 5 o f the experiment; however, he was entirely well 24 hr later. The other 5 dogs remained clinically healthy t h r o u g h o u t the experiment. Thirteen dogs were also treated with active crude neuraminidase in experiment 2, and 11 remained healthy t h r o u g h o u t the study. The 2 remaining dogs developed symmetrical weakness and decreased pain sensation in their hind legs on day 4 and were unable to stand. This progressed to involvement o f all four limbs in 1 animal 24 hr later; however, following gradual i m p r o v e m e n t during the next week no neurologic deficit was detectable 15 days after the experiment began. The other dog i m p r o v e d rapidly and was j u d g e d to be n o r m a l by the eighth day. Neither animal showed i m p a i r m e n t o f the righting reflex, nystagmus, a b n o r m a l deep t e n d o n reflexes or defective bladder function. In experiment 3, 2 o f 4 dogs treated intrathecally with killed p n e u m o c o c c i remained
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normal ; one transiently showed an unsteady gait on days 4 and 5. and one had'weak hindquarters on day 5 only. The 2 dogs given both killed pneumococci and inactivated crude enzyme showed no ill effects. Three of the 5 animals treated with killed pneucocci and active crude enzyme remained well, 1 showed profound weakness and impaired pain sensation in both rear legs on days 4 and 5 but was normal by day 6. One animal died on day 4 with a rectal temperature of 108°F and at autopsy the only abnormal finding was focal myocardial necrosis. In experiment 4 each of the dogs treated with active or inactivated purified neuraminidase or saline remained clinically healthy throughout the experiment. C S F analysis" - N e u r a m i n i d a s e and N A N A
The CSF of 5 of the 11 dogs administered viable pneumococci in experiment 1 exhibited measurable neuraminidase activity (Fig. 1B) which was higher than values derived from 24 normal, untreated dogs (Fig. 1E). At the time that increased neuraminidase activity was detected, 3 of these 5 animals had free NANA levels (Fig. 2B) higher than values derived from 58 normal control dogs (Fig. 2E). The level of CSF neuraminidase activity in these infected animals was apparently unrelated to simultaneous CSF pneumococcal colony counts, CSF glucose concentrations, or clinical status (Fig. 3). Seven dogs treated with crude neuraminidase (expt. 2) were subjected to lumbar and then cisternal puncture on day 6. Analysis of paired samples from each of the 7 animals revealed that neuraminidase activity was 2.55 times greater in lumbar than in cisternal fluid. Analysis of 6 paired samples from these animals indicated that lumbar NANA concentrations exceeded those of cisternal CSF by a factor of 1.41. CSF neuraminidase activity of crude and purified enzyme treated animals (expts. 2 and 4) may be compared to normal control animals and infected dogs (expt. 1) in Fig. 1. These values were corrected for cisternal/lumbar ratios detected in experimental animals so that comparisons might be made with values derived from lumbar 107.
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Fig. 3. Mean concentrations of protein, glucose and pneumococcal colony-forming units in CSF of 11 dogs experimentally infected with Type I pneumococcus in experiment 1. Values given are for 11 dogs on day 2. 10 dogs on day 3, 9 dogs on day 4, and 7 survivors on day 5.
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spinal fluid samples obtained from infected humans. Spinal fluid levels of enzyme activity in experimentally infected dogs were similar to those observed in infected human and animal CSF, and the levels for crude enzyme-treated dogs were 2 to 5 times higher than those observed for other groups. CSF concentrations of free NANA for crude and partially-purified enzyme-treated animals are compared with values obtained in control dogs and infected dogs in Fig. 2. These values were also corrected for observed differences in lumbar and cisternal free NANA concentrations in order to compare them with values observed in patients with pneumococcal meningitis. Free CSF NANA concentrations for crude and pure enzyme-treated dogs were similar to, or slightly in excess of concentrations in human CSF, and significantly higher than those in infected dogs. C S F analysis - Leucocytes, protein and 9lucose levels The CSF of the 11 infected dogs (expt. 1) reflected changes similar to those observed in man and in other experimentally-infected animals. A brisk leucocytosis accompanied infection, CSF protein concentrations became abnormally elevated, and CSF glucose concentrations fell (Fig. 3). Pneumococcal CSF colony counts peaked on day 3 and declined gradually thereafter. No apparent relationship was observed between pneumoccoccal colony-forming units and CSF protein concentrations; however, an inverse relationship between the CSF glucose and pneumococcal concentrations is suggested by the data. CSF protein and glucose concentrations were not related to outcome; however, by day 3 the remaining dogs were readily divisible into 2 groups according to concentrations of pneumococci in the CSF. Four dogs had less than 6 x 102 and 6 animals had more than 1.5 x 10 5 pneumococcal colony-forming units/ml CSF. None of the 4 animals with low counts died, whereas 4 of the 6 dogs with higher counts died. Intrathecal administration of pneumococci or of extracts from the organisms resulted in an elevation of the CSF protein concentration which varied in degree
1
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Fig. 4. Mean CSF protein concentrations in dogs. The animals were infected with pneumococci, or treated with various bacteriological sterile preparations of the same pneumococci. Values for infected animals (expt. 1) were taken in part from Fig. 3. Values for animals treated with sterile preparations include 6 dogs in expt. 2 treated with heat treated crude ,caraminidase (pneumococcal protein) (A), 13 dogs treated in expt. 2 with active crude pneumococcM ~/,,,raminidase (B), 4 dogs treated in expt. 3 with heat-killed pneumococci (C) and 3 dogs treated in expt. 4 with active partially purified neuraminidase (D).
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Fig. 5. Mean CSF glucose concentrations in dogs infected with a Type 1 pneumococcus, or treated with bacteriologically sterile preparations from the same pneumococcus, Values for infected animals are those given in Fig. 3, and designations for the sterile preparations are the same as those described for Fig. 4. Simultaneous blood glucose concentrations (mean + SD) lor all dogs treated with sterile pneumococcal derivations are shown above the CSF concentrations.
according to the preparation given (Fig. 4). On day 2 the CSF protein concentrations observed in animals treated with heat-killed pneumococci and with both crude and partially purified neuraminidase preparations were only slightly lower than values found in infected dogs. However, CSF protein levels noted in animals treated with crude neuraminidase (pneumococcal protein) (Fig. 4a) were significantly lower than those in CSF of infected dogs on day 2. On day 3 the CSF protein concentrations of infected animals and those treated with whole, killed pneumococci or crude neuraminidase were elevated (Fig. 4) and by days 4 and 5 the CSF protein concentration of animals treated with whole, killed pneumococci and the crude neuraminidase preparation were not significantly different from values observed in infected dogs. Spinal fluid glucose concentrations for dogs treated with partially purified neuramidase and pneumococcal proteins (inactivated neuraminidase preparation) were similar (Fig. 5). CSF glucose levels for animals treated with killed pneumococci or the active crude enzyme preparation, however, were significantly lower than the preceding groups of animals on days 3, 4 and 5 and were not significantly different from values observed in infected animals at any time. Each of the test materials elicited a CSF leucocytosis; however, the magnitude of change was greatest for animals treated with the active crude enzyme preparation or killed pneumococci. Both elicited significantly higher concentrations of leucocytes on days 3, 4 and 5 while only the animals treated with killed pneumococci had significantly higher levels on the second day of study (Fig. 6). Insufficient CSF was obtained for leucocyte counts on the infected animals.
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Cerebral cortex analysis Dog brains were subjected to subcellular fractionations to determine the presence and effects of neuraminidase on total N A N A levels and on the levels of 3 enzymes associated with membranes of nervous tissue. Distributions of monoamine oxidase, acetylcholinesterase, and succinate dehydrogenase closely approximated the distributions of these enzymes in subcellular fractions of nervous tissue from other species (DeRobertis 1967; Swanson, Harvey and Stahl 1973) (data not shown). The intrathecal administration of exogenous neuraminidase had no detectable major effect on the activities of the three marker enzymes of dog brain membranes. It is widely held that 2 of these enzymes, monoamine oxidase and acetylcholinesterase, are important in synaptic function (DeRobertis 1967). The injected neuraminidase has brought about decreases in the NANA content of brain subcellular structures (Table 1). These changes were evident in subcellular fractions obtained from animals treated with either crude or partially purified neuraminidase and decreases were as follows : homogenate, 29 44 ~ ; myelin, 20-35 ~ ; purified mitochondria, 15-34~; and supernatant, 24-34~, when compared to fractions TABLE 1 EFFECT OF N E U R A M I N I D A S E T R E A T M E N T O N T O T A L
Subcellular fraction
Homogenate Nuclear Crude mitochondrial myelin synaptosomal mitochondrial Microsomal Supernatant
NANA
OF D O G B R A I N S U B C E L L U L A R F R A C T I O N S
Total N A N d (p~/mg protein) heat-inactivated crude neuraminidase (3)"
active crude neuraminidase (4)
active partially purified neuraminidase (3)
14.66 _ 2.06 b 8.45 + 1.02 7.56 + 1.63 19.18+0.46 10.54 + 0.80 7.67 + 0.81 21.92 ___0.71 14.05 _+0.32
8.23 + 0.05 7.51 _ 0.29 10.43 __+1.41 12.33+__0.51 11.17 +__1.30 5.07 ___0.70 18.15 + 1.17 9.31 + 0.49
10.35 + 0.61 8.97 + 1.12 9.58 _ 0.51 15.26___0.91 10.25 _ 0.66 6.49 + 0.99 23.99 ___2.21 10.66 + 2.22
a The number of dog brains used is shown in parenthesis. b SD.
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obtained from animals injected with heat inactivated crude neuraminidase. These differences were all significant at the P < 0.001 level. No consistent correlation was evident in the other fractions studied. It would appear that this lowered NANA content does not appreciably affect the absolute levels (and presumably the function) of the marker enzymes studied in the dog brain tissue.
DISCUSSION
Infected dogs showed a poor prognosis in this study and only 2 of 11 dogs survived without detectable neurologic residua. These results are similar to those observed in untreated humans and in the studies which established the dog as a valid experimental model for study of pneumococcal meningitis (Petersdorf and Luttrell 1962: Carpenter and Petersdorf 1962). In contrast, intrathecal administration of whole killed pneumococci and crude and partially-purified pneumococcal neuraminidase resulted in neither death nor permanent morbidity in dogs. General malaise, ataxia and/or hind leg weakness were observed in some animals but these deficits were transient. They were distributed in an apparently random fashion among the experimental groups, suggesting a non-specific effect which may have been mediated by sterile meningeal inflammation. Spinal fluid concentrations of neuraminidase and free NANA observed 24 hr after enzyme administration were comparable to, or exceeded, levels detected in patients and infected animals, indicating that the dosage of enzyme administered was appropriate. The enzyme was administered over a 5-day period, a time similar to that when elevated levels were found in human pneumococcal meningitis (Carpenter and Petersdorf 1962). Enzyme-treated and control animals did not differ with respect to cerebral cortical synaptosomal content of NANA nor did the specific enzyme activities of monoamine oaidase or acetylcholinesterase change, indicating that cortical synaptic structures remained unaffected, at least with reference to these synaptosomal components. However, myelin-rich and mitochondrial fractions of cortical tissue had decreased NANA as compared to control animals. This is presumed to be a direct effect of neuraminidase activity and suggests that the enzyme crossed the pia-arachnoid membranes to act on surface and intracellular cortical substrate. This action resulted in no significant detectable alteration of function in the intact animal. These observations suggest that pneumococcal neuraminidase plays no significant role in the pathogenesis of neurologic dysfunction or death of dogs and, to the extent that findings in this model are applicable, in the death of man. The effects of the neuraminidase on the intact animal were minimal, when compared to the extensive morbidity and mortality noted in infected dogs. In contrast, both killed pneumococci and the active crude enzyme preparation elicited profound changes in CSF protein and glucose, which paralleled the changes seen in infected dogs. It is probable that (an) active principle(s), perhaps an enzyme, present in the crude enzyme preparation and protected against heat inactivation by the intact bacterial cell wall and capsule was responsible for these effects. At the present time the active principle remains unknown.
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Even though the crude enzyme preparation and killed pneumococci brought about profound alterations in CSF protein and glucose concentrations, these effects resulted in no significant morbidity or mortality for the animals. The reciprocal observation has been recorded in human tuberculous meningitis where the administration of dexamethasone to patients with tuberculous meningitis resulted in a significant change toward normal of CSF glucose and protein concentrations without significant clinical benefits (O'Toole, Thornton, Mukherjee and Nath 1969). These observations suggest that abnormal levels of CSF glucose and protein may not be related to brain damage and dysfunction in bacterial meningitis. ACKNOWLEDGEMENTS We wish to thank Drs. John Brunzell and Robert Petersdorf for critical comments in preparation of the manuscript. The excellent technical assistance of Mrs. Elaine Nyuha Loomis is appreciated. SUMMARY The intracisternal administration of a Type I pneumococcus to mongrel dogs resulted in spinal fluid abnormalities and clinical course and outcome similar to those of meningitis in humans. In order to define the pathogenic role of pneumococcal neuraminidase and other pneumococcal extracts in experimental meningitis, the following preparations were derived from the same Type I pneumococcus: crude neuraminidase, partially purified neuraminidase, heat-inactivated crude neuraminidase, and heat-killed pneumococci. These preparations were administered intracisternally daily for 5 days to a series of mongrel dogs. These substances did not produce clinical morbidity similar to that observed in infected animals, even though there was a significant decrease in the NANA content of cortical brain subcellular structures in the neuraminidase-treated dogs. It was concluded that the substances investigated were not responsible for morbidity and mortality in experimental pneumococcal meningitis. Both heat-killed pneumococci and the crude neuraminidase preparation elicited significant alterations in spinal fluid glucose and protein concentrations which were similar to those recorded in infected animals; however these abnormalities were not associated with significant morbidity. It is proposed that spinal fluid glucose and protein abnormalities may not be directly linked to brain damage or dysfunction in this experimental model, or in man.
REFERENCES BRtJNETTI,P., G. W. JOURDIANANDS. ROSEMAN(1962)The sialicacids,Part 3 (Distributionand properties of animal N-acetylneuraminicacid aldolase),J. biol. Chem., 237: 2447-2453. CARPENTER, R. R. ANDR. G. PETERSDORF(1962) The clinicalspectrumof bacterial meningitis,Amer. J. Med., 33: 262-275. DEROBERTIS, E. (1967) Ultrastructure and cytochemistryof the synapticregion, Science, 156: 907-914.
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GRAY, E. G. AND V. P. WHITTAKER (1962) The isolation of nerve endings from brain An electron-micr~, scopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.;. 9t~: 79 -88. HUGHES, R. C. AND R. W. JEANLOZ (1964a) The extracellular glycosidases of Diplococcus pneumomae, Part 1 (Purification and properties of a neuraminidase and a beta-galactosidase), Biochemistry, 3: 1535-1543. HUGHES, R. C. AND R. W. JEANLOZ (t964b) The extracellular glycosidases of Diploeoccus pneumoniae, Part 2 (Purification and properties of a beta-N-acetylglucosaminidase), Biochemistry, 3: 1543-1548. JAKOBY, R. K. AND L. WARREN (1961) Identification and quantitation of N-acetylneuraminic acid in human cerebrospinal fluid, Neurolo.qv (Minneap.), 11 : 232-238. KELLY, R. AND D. GREIFF (1970) Toxicity of pneumococcal neuraminidase, Injection and Immunity. 2: 115-117. KELLY, R. T., S. FARMERAND D. GREIFF (1967) Neuraminidase activities of clinical isolates of Diplococcus pneumoniae, J. Baet., 94: 272-273. LEE, L. T. AND C. HOWE (1966) Pneumococcal neuraminidase, J. Bact., 91: 1418-1426. LIN, Y., G. E. MEANS AND R. E. FEENEY (1969) The action of proteolytic enzymes on N,N-dimethyl proteins, J. biol. Chem., 244: 789-793. LOWRY, O. H., N. J. ROSEBROUGH,A. L. FARR AND R. J. RANDALL (1951 ) Protein measurement with the Folin phenol reagent, J. biol. Chem., 193: 265-275. MCCAMEN, M. W., L. E. TOMEY AND R. E. McCAMEN (1968) Radiomimetric assay of acetylcholinesterase activity in submicrogram amounts of tissue, Life Sci., 7 : 233-244. MCCAMEN, R. E., M. W. MCCAMEN, J. M. HUNT AND M. S. SMITH (1965) Microdetermination of monoamineoxidase in 5-hydroxytryptophan decarboxylase activities in nervous tissues, J. Neurochem,, 12: 15-23. MULLER, H. E. (1969) Die Neuraminidase als pathogenetischer Faktor bei Pneumokokken-Infection, Dtsch. med. Wschr., 94: 2149-2154. O'TOOLE, R. D., L. GOODE AND C. HOWE (1971) Neuraminidase activity in bacterial meningitis, J. clin. Invest., 50: 979-985. O'TOOLE, R. D., G. F. THORNTON, M. K. MUKHERJEEAND R. L. NATH (1969) Dexamethasone in tuberculous meningitis, Ann. Intern. Med., 70 : 39-48. PETERSDORF, R. G. AND C. N. LUTTRELL (1962) Studies on the pathogenesis of meningitis. J. c/in, lnvest.. 41: 311-319. RAEELSON, M. E. (1963) The neuraminidases and their action on glycoproteins and other sialic acid containing compounds, Expos. ann. Biochim. m~d., 24: 121-132. SLATER,T. F., B. SAWYER AND O. STRAULI (1963) Studies of succinate tetrazolium reductase systems, Part 3 (Points of coupling of four different tetrazolium salts), Biochim. biophys. Acta, 77: 383-393. STAHL, W. L. AND R. D. O'TOOLE (1971) Pneumococcal neuraminidase Purification and properties, Biochim. biophys. Acta, 268:480-487. SWANSON, P. D., F. H. HARVEY AND W. L. STAHL (t973) Subcellular fractionation of postmortem brain. J. Neurochem., 20: 465-475. TANENBAUM, S. W., J. GULBINSKY, M. KATZ AND 25. (5. ~SUN ~1~/0} Separation, purification and some properties of pneumococcal neuraminidase isoenzymes, Biochim. biophys. Acta, 198: 242-254. WARREN, L. (1959) The thiobarbituric acid assay of sialic acids, J. biol. Chem., 234:1971 1975.
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Experimental P n e u m o c o c c a l Meningitis Effects of Neuraminidase and other Pneumococcal Constituents on Cerebrospinal Fluid in the Intact Dog R I C H A R D D. O'TOOLE* AND W I L L I A M L. STAHL** Departments of Medicine and Physiology and Biophysics, University of Washington School of Medicine, and the Veterans Administration Hospital, Seattle, Wash. (U.S.A.)
(Received 15 February, 1975)
INTRODUCTION Pneumococci isolated from infected tissues have been shown to elaborate a neuraminidase which cleaves terminal molecules of N-acetylneuraminic acid (NANA) from glycoproteins and gangliosides (Rafelson 1963;Hughes and Jeanloz 1964a, b; Lee and Howe 1966; Kelly, Farmer and Greiff 1967). Intracerebral injection of neuraminidase in mice results in release of N A N A from exposed tissues and also in death of the animal (Kelly and Greiff 1970). In patients with pneumococcal meningitis elevated concentrations of cerebrospinal fluid (CSF) free N A N A have been observed (O'Toole, Goode and Howe 1971 ). Elevated CSF-free N A N A levels have been shown to be correlated with an adverse prognosis in man, and O'Toole et al. (1971) and Muller (1969) proposed that the cleavage of N A N A from the glycoproteins and gangliosides of cerebral tissue might contribute to morbidity and mortality in pneumococcal meningitis. This study was undertaken to study the role of neuraminidase activity in experimentally infected dogs by assessing the effect of intrathecal administration of neuraminidase from pneumococci on morbidity and CSF components of dogs. MATERIALSAND METHODS A type I pneumococcus isolated from the cerebrospinal fluid of a patient with meningitis was employed in this study (O'Toole et al. 1971 ). It was passaged in mice every 6 weeks, and stored in defibrinated rabbit blood at - 7 0 ° C until used. Dr. O'Toolewas supported by the Veterans AdministrationResearchAssociateProgram. *Present address: The MedfordClinic, 1025E. Main Street, Medford,Ore. 97501. **Reprint requests to this author at: NeurochemistryLaboratory,VA Hospital, 4435 BeaconAvenueS., Seattle, Wash. 98108, U.S.A.
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Enzyme assays In the neuraminidase assay, 0.2 ml of spinal fluid or enzyme preparation was incubated at 37°C with 2.5 mg human a-l-acid glycoprotein (Calbiochem), dissolved in 0.8 ml of 0.15 M NaCI and buffered to pH 6.5 with 0.15 M phosphate, as substrate. After 60 min incubation, duplicate 0.4 ml samples were taken from the reaction mixture and analyzed for free NANA by the thiobarbituric acid assay of Warren (1959) with saline-containing substrate as a control and crystalline NANA (KochLight Labs, Ltd., Colnbrook, England) as the standard. One unit was defined as the amount of neuraminidase required to liberate 1 nmole of NANA in 60 min tinder assay conditions. NANA-aldolase activity was assayed according to the method of Brunetti, Jourdian and Roseman (1962). A unit of activity is defined as the quantity of enzyme that will cleave 1.0 ~Lmole of NANA in 15 min under the standard assay conditions, fl-Galactosidase was assayed by a modification of the procedure of Hughes and Jeanloz (1964a). Proteolytic enzyme activity was assayed by the method of Lin, Means and Feeney (1969). Acetylcholinesterase was assayed using the method of McCamen, Tomey and McCamen (1968). Monoamine oxidase was assayed using the procedure of McCamen, McCamen, Hunt and Smith (1965). Succinate dehydrogenase was assayed using the method of Slater, Sawyer and Strauli (1963). Other analyses Protein concentrations of preparations were established by the method of Lowry, Rosebrough, Farr and Randall (1951). Free and total NANA concentrations were determined according to the method of Jakoby and Warren (1961). Spinal fluid leucocyte counts were determined by the standard counting chamber method. Glucose concentrations on CSF and serum (venous blood sampled daily at the time of cisternal puncture) were measured by the Auto-analyzer ferricyanide method. Preparation of crude neuraminidase This was essentially the procedure of Tanenbaum, Gulbinsky, Katz and Sun (l 970). Thawed pneumococci were inoculated into Todd-Hewitt broth (Difco) and incubated at 37~C for 48 hr. Purity of growth was confirmed by culture on to rabbit blood agar plates, and pneumococci were removed by centrifugation at 700 x g for 2 hr at 4°C and discarded. Protein was precipitated from the supernatant with ammonium sulfate at 7 5 ~ saturation. After standing overnight at 4°C, precipitated proteins were centrifugated at 700 x 9 for I hr in the cold, the supernatant was discarded, and the precipitate was taken up in 0.9~o NaCI and stored at -20°C. All samples were pooled, dialyzed against repeated changes of deionized water followed by dialysis against 0.9~o NaC1 (adjusted to pH 7.3 with NaHCO3)and were centrifuged at 95,000 x 9 for 60 min in the cold. The supernatant was concentrated through an XM50 Diaflow membrane filter (Amicon Corporation, Lexington, Mass.), passed through a millipore filter (Millipore Corporation, Bedford, Mass.), pore size 0.45 ~m, and cultured to confirm sterility. The final suspension was adjusted with 0.9~ NaC1 so that 1.0 ml contained 29.3 mg protein and possessed 760 units of neuraminidase activity, no detectable fl-galactosidase activity and, when assayed for proteolytlc
EXPERIMENTAL PNEUMOCOCCAL MENINGITIS
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enzyme activity, hydrolyzed 0.039/~moles bonds/mg protein/0.5 hr. Previous work (Stahl and O'Toole 1971 ) showed that this preparation had many protein components. This preparation contained no detectable neuraminidase activity following exposure to a temperature of 75°C for 15 min.
Preparation of partially purified neuraminidase The procedure of Stahl and O'Toole (1971) was used. Crude enzyme was dialyzed against 0.1 M sodium acetate and applied to columns packed with DEAE-Sephadex in the acetate form (Pharmacia Fine Chemicals, Piscataway, N.J.). Stepwise elution with 0.1M sodium acetate containing sodium chloride (0.14).4 M) yielded a peak of neuraminidase activity in the eluate between 0.2 and 0.3 M sodium chloride. These fractions were pooled, dialyzed against 0.9~ sodium chloride, concentrated against a Diaflo XM-50 filter, passed through a 0.45 ~tm pore-size filter, and stored at - 2 0 ° C until used. This preparation was bacteriologically sterile, and contained 0.028 units of NANA-aldolase activity, no detectable fl-galactosidase activity, and, when assayed for proteolytic enzyme activity, cleaved 0.428 #moles bonds/mg protein/0.5 hr. As shown previously (Stahl and O'Toole 1971) the preparation had only one major protein component with an estimated molecular weight of 69,800. Neuraminidase dose determination Neuraminidase activity and free NANA concentrations were determined for 17 patients previously reported, who had pneumococcal meningitis and elevated CSF-free N A N A levels (O'Toole et al. 1971). Animal studies Mongrel dogs weighing between 15 and 30 kg were used in all experiments. Dogs were anesthetized intravenously with sodium pentobarbital, and all test preparations were administered intracisternally via sterile, disposable 22 gauge needles. A normal saline-5 ~ glucose solution was administered (22.5 ml/kg) by clysis at the end of each day's experiment, and the animals were given water, but no food. Experiment I. 2.3 x 105 to 9.8 x 106 viable pneumococci were suspended in 1.0 ml of normal saline and injected into the cisterna magna of 11 animals in order to establish values for neuraminidase activity and for free NANA concentrations in the infected CSF of dogs. Cisternal fluid was sampled daily for as long as the dogs survived or remained infected and was tested for free NANA and neuraminidase activity. The number of viable pneumococci/ml of infected CSF was estimated with pour plates containing agar and defibrinated rabbit blood. Experiment 2. Crude neuraminidase was administered daily for 5 days to 13 test dogs and heat-inactivated crude neuraminidase was given to 6 dogs. 0.4 ml of active or heat-inactivated enzyme preparation was administered intracisternally to dogs on days 1 and 2, and 0.8 ml was given on days 3, 4 and 5, 2.0 ml CSF was withdrawn daily for analysis prior to administration of the test material. On day 6, 1.0 ml lumbar CSF was obtained, followed by removal of 2.0 ml cisternal CSF. Experiment 3. Four dogs received 2 × 108 heat-killed pneumococci intracisternally daily for 5 days. Pneumococci were grown for 16 hr in Todd-Hewitt broth, washed
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twice in normal saline (pH 7.3), resuspended in 1.0 ml saline, and heated lor 5 rain at 100°C. This suspension contained no enzyme activity and 1.22 mg protein/ml. Seven additional dogs were given 2 x l0 s heat-killed pneumococci: 5 of these animals were simultaneously treated with crude neuraminidase as in experiment I. and 2 control dogs were given the inactivated enzyme. Experiment 4. Three dogs were treated with a suspension of partially purified neuraminidase with total enzyme activity identical to the crude enzyme preparation used in experiments 1 and 2. The enzyme was suspended in saline, buffered to pH 7.3 with bicarbonate. Two animals received the heat-inactivated crude enzyme preparation which was adjusted by dilution in buffered saline to the same protein concentration (1.53 mg/ml) as the partially purified active enzyme. In addition to these experiments CSF was drawn from 24-58 normal healthy dogs for neuraminidase and NANA determinations. Subcellular fractions These were obtained from the cerebral cortex grey matter of dogs using the method of Gray and Whittaker (1962). On experimental day 6, 10 dogs were sacrificed. This was 24 hr following the last intracisternal injection. Three animals had been treated with heat-inactivated crude neuraminidase (expt. 2), 4 were treated with active crude neuraminidase (expt. 2), and 3 had been treated with active partially purified neuraminidase (expt. 4). The brains were isolated immediately after sacrifice, and pia-arachnoid was stripped from the cerebral hemispheres, and 5-7 g of cortical grey matter were removed and placed in ice, then stored at - 70°C. The fractionation was carried out within 1 week. Succinate dehydrogenase was assayed on the same day as the fractionation procedure, whereas monoamine oxidase and acetylcholinesterase were assayed in aliquots stored at-20°C and tested within 2 to 3 weeks of the fractionation. For determination of total NANA, portions of each subcellular fraction were dialyzed against deionized water for several days and were hydrolyzed and assayed for total NANA according to the method of Jakoby and Warren (1961 ).
RESULTS
CSF was obtained from 35 patients with culture-proven pneumococcal meningitis prior to therapy (O'Toole et al. 1971). The CSF of 17 of these patients contained abnormally elevated concentrations of free NANA. The CSF of these 17 patients possessed 23.29 _ 24.35 (SD) units of neuraminidase activity per 0.2 ml (Fig. 1) and 2.49_ 2.19 mg free NANA/100 ml (Fig. 2). Based on these observations dogs were treated with pneumococci or neuraminidase preparations to achieve CSF levels of neuraminidase and NANA at least as high as those found in patients. Clinical observations Four experiments were carried out with dogs. In experiment 1 the CSF of all 11 dogs administered viable pneumoeocci became infected for 4-6 days or until death. Two animals died at 48 hr, 2 died at 96 hr, 2 survived without detectable residua, and
EXPERIMENTALPNEUMOCOCCALMENINGITIS
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DAY OF EXPERIMENT
Fig. 1. Mean CSF neuraminidase activity in experimental pneumococcal meningitis: 17 patients with naturally acquired disease had 23.29 ___24.35 (SD) units per 0.2 ml of CSF (A), 5 experimentally infected dogs developing elevated CSF enzyme concentrations in experiment 1 (23.48+__11.35 units/0.2 ml CSF) (B). These values are compared with mean levelsof CSF enzyme activity in 13 dogs in experiment 2 treated with crude neuraminidase (C), and 3 dogs in experiment 4 treated with purified neuraminidase (D), also 24 normal, untreated dogs had 1.28+ 0.44 units of activity per 0.2 ml CSF (E). All animal CSF samples were obtained by cisternal puncture and therefore have been corrected according to cisternal/lumbar ratios observed in dogs (see RESULTS)for comparison with values found in human lumbar CSF. Fig. 2. Mean CSF concentrations of free NANA in pneumococcal meningitis: 17 patients with naturally acquired disease had 2.49-I-2.19 mg free NANA/100 ml CSF (A) and 3 experimentally infected dogs developing abnormally elevated CSF free NANA concentrations in ~:xperiment 1 (B). These values are compared with the mean free NANA levels detected in the 13 dogs in experiment 2 treated with crude neuraminidase (C), in 3 dogs in experiment 4 treated with purified neuraminidase (D) and in 58 normal, untreated dogs (E, 0.670+0.216 mg/100 ml). CSF samples have been corrected according to cisternal/lumbar ratios as in Fig. 1.
5 continued to show signs o f a residual vestibu|o-cerebellar s y n d r o m e prior to sacrifice, 16-68 days after infection. One o f 6 dogs in experiment 2, which were treated with inactivated crude p n e u m o coccal enzyme, showed extensor rigidity o f all limbs, and total loss o f balance, righting reflex, and integrated m o t o r m o v e m e n t s for 24 hr on day 5 o f the experiment; however, he was entirely well 24 hr later. The other 5 dogs remained clinically healthy t h r o u g h o u t the experiment. Thirteen dogs were also treated with active crude neuraminidase in experiment 2, and 11 remained healthy t h r o u g h o u t the study. The 2 remaining dogs developed symmetrical weakness and decreased pain sensation in their hind legs on day 4 and were unable to stand. This progressed to involvement o f all four limbs in 1 animal 24 hr later; however, following gradual i m p r o v e m e n t during the next week no neurologic deficit was detectable 15 days after the experiment began. The other dog i m p r o v e d rapidly and was j u d g e d to be n o r m a l by the eighth day. Neither animal showed i m p a i r m e n t o f the righting reflex, nystagmus, a b n o r m a l deep t e n d o n reflexes or defective bladder function. In experiment 3, 2 o f 4 dogs treated intrathecally with killed p n e u m o c o c c i remained
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R. D. O'TOOLE, W. L. STAHL
normal ; one transiently showed an unsteady gait on days 4 and 5. and one had'weak hindquarters on day 5 only. The 2 dogs given both killed pneumococci and inactivated crude enzyme showed no ill effects. Three of the 5 animals treated with killed pneucocci and active crude enzyme remained well, 1 showed profound weakness and impaired pain sensation in both rear legs on days 4 and 5 but was normal by day 6. One animal died on day 4 with a rectal temperature of 108°F and at autopsy the only abnormal finding was focal myocardial necrosis. In experiment 4 each of the dogs treated with active or inactivated purified neuraminidase or saline remained clinically healthy throughout the experiment. C S F analysis" - N e u r a m i n i d a s e and N A N A
The CSF of 5 of the 11 dogs administered viable pneumococci in experiment 1 exhibited measurable neuraminidase activity (Fig. 1B) which was higher than values derived from 24 normal, untreated dogs (Fig. 1E). At the time that increased neuraminidase activity was detected, 3 of these 5 animals had free NANA levels (Fig. 2B) higher than values derived from 58 normal control dogs (Fig. 2E). The level of CSF neuraminidase activity in these infected animals was apparently unrelated to simultaneous CSF pneumococcal colony counts, CSF glucose concentrations, or clinical status (Fig. 3). Seven dogs treated with crude neuraminidase (expt. 2) were subjected to lumbar and then cisternal puncture on day 6. Analysis of paired samples from each of the 7 animals revealed that neuraminidase activity was 2.55 times greater in lumbar than in cisternal fluid. Analysis of 6 paired samples from these animals indicated that lumbar NANA concentrations exceeded those of cisternal CSF by a factor of 1.41. CSF neuraminidase activity of crude and purified enzyme treated animals (expts. 2 and 4) may be compared to normal control animals and infected dogs (expt. 1) in Fig. 1. These values were corrected for cisternal/lumbar ratios detected in experimental animals so that comparisons might be made with values derived from lumbar 107.
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Fig. 3. Mean concentrations of protein, glucose and pneumococcal colony-forming units in CSF of 11 dogs experimentally infected with Type I pneumococcus in experiment 1. Values given are for 11 dogs on day 2. 10 dogs on day 3, 9 dogs on day 4, and 7 survivors on day 5.
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spinal fluid samples obtained from infected humans. Spinal fluid levels of enzyme activity in experimentally infected dogs were similar to those observed in infected human and animal CSF, and the levels for crude enzyme-treated dogs were 2 to 5 times higher than those observed for other groups. CSF concentrations of free NANA for crude and partially-purified enzyme-treated animals are compared with values obtained in control dogs and infected dogs in Fig. 2. These values were also corrected for observed differences in lumbar and cisternal free NANA concentrations in order to compare them with values observed in patients with pneumococcal meningitis. Free CSF NANA concentrations for crude and pure enzyme-treated dogs were similar to, or slightly in excess of concentrations in human CSF, and significantly higher than those in infected dogs. C S F analysis - Leucocytes, protein and 9lucose levels The CSF of the 11 infected dogs (expt. 1) reflected changes similar to those observed in man and in other experimentally-infected animals. A brisk leucocytosis accompanied infection, CSF protein concentrations became abnormally elevated, and CSF glucose concentrations fell (Fig. 3). Pneumococcal CSF colony counts peaked on day 3 and declined gradually thereafter. No apparent relationship was observed between pneumoccoccal colony-forming units and CSF protein concentrations; however, an inverse relationship between the CSF glucose and pneumococcal concentrations is suggested by the data. CSF protein and glucose concentrations were not related to outcome; however, by day 3 the remaining dogs were readily divisible into 2 groups according to concentrations of pneumococci in the CSF. Four dogs had less than 6 x 102 and 6 animals had more than 1.5 x 10 5 pneumococcal colony-forming units/ml CSF. None of the 4 animals with low counts died, whereas 4 of the 6 dogs with higher counts died. Intrathecal administration of pneumococci or of extracts from the organisms resulted in an elevation of the CSF protein concentration which varied in degree
1
22] DAY OF EXPERIMENT
Fig. 4. Mean CSF protein concentrations in dogs. The animals were infected with pneumococci, or treated with various bacteriological sterile preparations of the same pneumococci. Values for infected animals (expt. 1) were taken in part from Fig. 3. Values for animals treated with sterile preparations include 6 dogs in expt. 2 treated with heat treated crude ,caraminidase (pneumococcal protein) (A), 13 dogs treated in expt. 2 with active crude pneumococcM ~/,,,raminidase (B), 4 dogs treated in expt. 3 with heat-killed pneumococci (C) and 3 dogs treated in expt. 4 with active partially purified neuraminidase (D).
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Fig. 5. Mean CSF glucose concentrations in dogs infected with a Type 1 pneumococcus, or treated with bacteriologically sterile preparations from the same pneumococcus, Values for infected animals are those given in Fig. 3, and designations for the sterile preparations are the same as those described for Fig. 4. Simultaneous blood glucose concentrations (mean + SD) lor all dogs treated with sterile pneumococcal derivations are shown above the CSF concentrations.
according to the preparation given (Fig. 4). On day 2 the CSF protein concentrations observed in animals treated with heat-killed pneumococci and with both crude and partially purified neuraminidase preparations were only slightly lower than values found in infected dogs. However, CSF protein levels noted in animals treated with crude neuraminidase (pneumococcal protein) (Fig. 4a) were significantly lower than those in CSF of infected dogs on day 2. On day 3 the CSF protein concentrations of infected animals and those treated with whole, killed pneumococci or crude neuraminidase were elevated (Fig. 4) and by days 4 and 5 the CSF protein concentration of animals treated with whole, killed pneumococci and the crude neuraminidase preparation were not significantly different from values observed in infected dogs. Spinal fluid glucose concentrations for dogs treated with partially purified neuramidase and pneumococcal proteins (inactivated neuraminidase preparation) were similar (Fig. 5). CSF glucose levels for animals treated with killed pneumococci or the active crude enzyme preparation, however, were significantly lower than the preceding groups of animals on days 3, 4 and 5 and were not significantly different from values observed in infected animals at any time. Each of the test materials elicited a CSF leucocytosis; however, the magnitude of change was greatest for animals treated with the active crude enzyme preparation or killed pneumococci. Both elicited significantly higher concentrations of leucocytes on days 3, 4 and 5 while only the animals treated with killed pneumococci had significantly higher levels on the second day of study (Fig. 6). Insufficient CSF was obtained for leucocyte counts on the infected animals.
175
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2
3
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DAY OF EXPERIMENT Fig. 6. Mean CSF leucocyte counts for all dogs treated with sterile preparations from Type I pneumococcus. Letter designations are identical to those for Fig. 4.
Cerebral cortex analysis Dog brains were subjected to subcellular fractionations to determine the presence and effects of neuraminidase on total N A N A levels and on the levels of 3 enzymes associated with membranes of nervous tissue. Distributions of monoamine oxidase, acetylcholinesterase, and succinate dehydrogenase closely approximated the distributions of these enzymes in subcellular fractions of nervous tissue from other species (DeRobertis 1967; Swanson, Harvey and Stahl 1973) (data not shown). The intrathecal administration of exogenous neuraminidase had no detectable major effect on the activities of the three marker enzymes of dog brain membranes. It is widely held that 2 of these enzymes, monoamine oxidase and acetylcholinesterase, are important in synaptic function (DeRobertis 1967). The injected neuraminidase has brought about decreases in the NANA content of brain subcellular structures (Table 1). These changes were evident in subcellular fractions obtained from animals treated with either crude or partially purified neuraminidase and decreases were as follows : homogenate, 29 44 ~ ; myelin, 20-35 ~ ; purified mitochondria, 15-34~; and supernatant, 24-34~, when compared to fractions TABLE 1 EFFECT OF N E U R A M I N I D A S E T R E A T M E N T O N T O T A L
Subcellular fraction
Homogenate Nuclear Crude mitochondrial myelin synaptosomal mitochondrial Microsomal Supernatant
NANA
OF D O G B R A I N S U B C E L L U L A R F R A C T I O N S
Total N A N d (p~/mg protein) heat-inactivated crude neuraminidase (3)"
active crude neuraminidase (4)
active partially purified neuraminidase (3)
14.66 _ 2.06 b 8.45 + 1.02 7.56 + 1.63 19.18+0.46 10.54 + 0.80 7.67 + 0.81 21.92 ___0.71 14.05 _+0.32
8.23 + 0.05 7.51 _ 0.29 10.43 __+1.41 12.33+__0.51 11.17 +__1.30 5.07 ___0.70 18.15 + 1.17 9.31 + 0.49
10.35 + 0.61 8.97 + 1.12 9.58 _ 0.51 15.26___0.91 10.25 _ 0.66 6.49 + 0.99 23.99 ___2.21 10.66 + 2.22
a The number of dog brains used is shown in parenthesis. b SD.
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obtained from animals injected with heat inactivated crude neuraminidase. These differences were all significant at the P < 0.001 level. No consistent correlation was evident in the other fractions studied. It would appear that this lowered NANA content does not appreciably affect the absolute levels (and presumably the function) of the marker enzymes studied in the dog brain tissue.
DISCUSSION
Infected dogs showed a poor prognosis in this study and only 2 of 11 dogs survived without detectable neurologic residua. These results are similar to those observed in untreated humans and in the studies which established the dog as a valid experimental model for study of pneumococcal meningitis (Petersdorf and Luttrell 1962: Carpenter and Petersdorf 1962). In contrast, intrathecal administration of whole killed pneumococci and crude and partially-purified pneumococcal neuraminidase resulted in neither death nor permanent morbidity in dogs. General malaise, ataxia and/or hind leg weakness were observed in some animals but these deficits were transient. They were distributed in an apparently random fashion among the experimental groups, suggesting a non-specific effect which may have been mediated by sterile meningeal inflammation. Spinal fluid concentrations of neuraminidase and free NANA observed 24 hr after enzyme administration were comparable to, or exceeded, levels detected in patients and infected animals, indicating that the dosage of enzyme administered was appropriate. The enzyme was administered over a 5-day period, a time similar to that when elevated levels were found in human pneumococcal meningitis (Carpenter and Petersdorf 1962). Enzyme-treated and control animals did not differ with respect to cerebral cortical synaptosomal content of NANA nor did the specific enzyme activities of monoamine oaidase or acetylcholinesterase change, indicating that cortical synaptic structures remained unaffected, at least with reference to these synaptosomal components. However, myelin-rich and mitochondrial fractions of cortical tissue had decreased NANA as compared to control animals. This is presumed to be a direct effect of neuraminidase activity and suggests that the enzyme crossed the pia-arachnoid membranes to act on surface and intracellular cortical substrate. This action resulted in no significant detectable alteration of function in the intact animal. These observations suggest that pneumococcal neuraminidase plays no significant role in the pathogenesis of neurologic dysfunction or death of dogs and, to the extent that findings in this model are applicable, in the death of man. The effects of the neuraminidase on the intact animal were minimal, when compared to the extensive morbidity and mortality noted in infected dogs. In contrast, both killed pneumococci and the active crude enzyme preparation elicited profound changes in CSF protein and glucose, which paralleled the changes seen in infected dogs. It is probable that (an) active principle(s), perhaps an enzyme, present in the crude enzyme preparation and protected against heat inactivation by the intact bacterial cell wall and capsule was responsible for these effects. At the present time the active principle remains unknown.
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Even though the crude enzyme preparation and killed pneumococci brought about profound alterations in CSF protein and glucose concentrations, these effects resulted in no significant morbidity or mortality for the animals. The reciprocal observation has been recorded in human tuberculous meningitis where the administration of dexamethasone to patients with tuberculous meningitis resulted in a significant change toward normal of CSF glucose and protein concentrations without significant clinical benefits (O'Toole, Thornton, Mukherjee and Nath 1969). These observations suggest that abnormal levels of CSF glucose and protein may not be related to brain damage and dysfunction in bacterial meningitis. ACKNOWLEDGEMENTS We wish to thank Drs. John Brunzell and Robert Petersdorf for critical comments in preparation of the manuscript. The excellent technical assistance of Mrs. Elaine Nyuha Loomis is appreciated. SUMMARY The intracisternal administration of a Type I pneumococcus to mongrel dogs resulted in spinal fluid abnormalities and clinical course and outcome similar to those of meningitis in humans. In order to define the pathogenic role of pneumococcal neuraminidase and other pneumococcal extracts in experimental meningitis, the following preparations were derived from the same Type I pneumococcus: crude neuraminidase, partially purified neuraminidase, heat-inactivated crude neuraminidase, and heat-killed pneumococci. These preparations were administered intracisternally daily for 5 days to a series of mongrel dogs. These substances did not produce clinical morbidity similar to that observed in infected animals, even though there was a significant decrease in the NANA content of cortical brain subcellular structures in the neuraminidase-treated dogs. It was concluded that the substances investigated were not responsible for morbidity and mortality in experimental pneumococcal meningitis. Both heat-killed pneumococci and the crude neuraminidase preparation elicited significant alterations in spinal fluid glucose and protein concentrations which were similar to those recorded in infected animals; however these abnormalities were not associated with significant morbidity. It is proposed that spinal fluid glucose and protein abnormalities may not be directly linked to brain damage or dysfunction in this experimental model, or in man.
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