Phase Of Experimental Pneumococcal Meningitis Research Articles

Tracking the transcriptional host response from the acute to the regenerative phase of experimental pneumococcal meningitis

BackgroundDespite the availability of effective antibiotic therapies, pneumococcal meningitis (PM) has a case fatality rate of up to 30% and causes neurological sequelae in up to half of the surviving patients. The underlying brain damage includes apoptosis of neurons in the hippocampus and necrosis in the cortex. Therapeutic options to reduce acute injury and to improve outcome from PM are severely limited.With the aim to develop new therapies a number of pharmacologic interventions have been evaluated. However, the often unpredictable outcome of interventional studies suggests that the current concept of the pathophysiologic events during bacterial meningitis is fragmentary. The aim of this work is to describe the transcriptomic changes underlying the complex mechanisms of the host response to pneumococcal meningitis in a temporal and spatial context using a well characterized infant rat model.MethodsEleven days old nursing Wistar rats were infected by direct intracisternal injection of 2 × 106cfu/ml of Streptococcus pneumoniae. Animals were sacrificed at 1, 3, 10 and 26 days after infection, the brain harvested and the cortex and hippocampus were sampled. The first two time points represent the acute and sub-acute phase of bacterial meningitis, whereas the latter represent the recovery phase of the disease.ResultsThe major events in the regulation of the host response on a transcriptional level occur within the first 3 days after infection. Beyond this time, no differences in global gene expression in infected and control animals were detectable by microarray analysis. Whereas in the acute phase of the disease immunoregulatory processes prevail in the hippocampus and the cortex, we observed a strong activation of neurogenic processes in the hippocampal dentate gyrus, both by gene expression and immunohistology starting as early as 3 days after infection.ConclusionsHere we describe the cellular pathways involved in the host response to experimental pneumococcal meningitis in specified disease states and brain regions. With these results we hope to provide the scientific basis for the development of new treatment strategies which take the temporal aspects of the disease into account.

Heparin inhibits leukocyte rolling in pial vessels and attenuates inflammatory changes in a rat model of experimental bacterial meningitis.

Heparin is a natural proteoglycan that was first described in 1916. In addition to its well characterized effect on blood coagulation, it is becoming clear that heparin also modulates inflammatory processes on several levels, including the interference with leukocyte-endothelium interaction. Anecdotal observations suggest a better clinical outcome of heparin-treated patients with bacterial meningitis. The authors demonstrate that heparin, a glycosaminoglycan, inhibits significantly in the early phase of experimental pneumococcal meningitis the increase of 1) regional cerebral blood flow (125 +/- 18 versus 247 +/- 42%), 2) intracranial pressure (4.5 +/- 2.0 versus 12.1 +/- 2.2 mm Hg), 3) brain edema (brain water content: 78.23 +/- 0.33 versus 79.49 +/- 0.46%), and 4) influx of leukocytes (571 +/- 397 versus 2400 +/- 875 cells/microL) to the cerebrospinal fluid compared with untreated rats. To elucidate the possible mechanism of this observation, the authors investigated for the first time leukocyte rolling in an inflammatory model in brain venules by confocal laser scanning microscopy in vivo. Heparin significantly attenuates leukocyte rolling at 2, 3, and 4 hours (2.8 +/- 1.3 versus 7.9 +/- 3.2/100 microm/min), as well as leukocyte sticking at 4 hours (2.1 +/- 0.4 versus 3.5 +/- 1.0/100 microm/min) after meningitis induction compared with untreated animals. The authors conclude that heparin can modulate acute central nervous system inflammation and, in particular, leukocyte-endothelium interaction, a key process in the cascade of injury in bacterial meningitis. They propose to evaluate further the potential of heparin in central nervous system inflammation in basic and clinical studies.

7-Nitroindazole inhibits pial arteriolar vasodilation in a rat model of pneumococcal meningitis.

This study investigates how the neuronal and inducible nitric oxide synthase (NOS) pathways contribute to the cerebrovascular changes in the early phase of experimental pneumococcal meningitis in rats. Using a closed cranial window preparation, the diameters of pial arterioles were measured during 4 hours after intracisternal injection of heat-killed pneumococci and compared with controls (n = 6). Injection of pneumococci (n = 7) caused a significant increase in pial arteriolar diameter (157 +/- 22% after 4 hours; P < 0.05, compared with 104 +/- 11% in controls), intracranial pressure, CSF white blood cell counts, and brain water content. Treatment with the neuronal NOS inhibitor 7-nitroindazole (50 mg/kg given intraperitoneally, n = 5) prevented pneumococci-induced vasodilation (107 +/- 20% at 4 hours), whereas S-methylisothiourea (SMT; 0.1 mg/kg given intraperitoneally, n = 5), which predominantly inhibits the inducible NOS, did not influence pneumococci-induced vasodilation (154 +/- 38% at 4 hours). S-methylisothiourea at a dose of 1.0 mg/kg (n = 5), attenuated the vasodilation (124 +/- 18% at 4 hours). However, the increase in mean arterial blood pressure after SMT at 1.0 mg/kg, but not at 0.1 mg/kg, suggests that the higher dose of SMT influenced the constitutive NOS activity, causing inhibition of the pneumococci-induced vasodilation. Neither SMT (at both doses) nor 7-nitroindazole influenced the increase in brain water content, intracranial pressure, and CSF white blood cell counts in pneumococci-challenged rats. Our study suggests that pial arteriolar vasodilation in the early phase of experimental pneumococcal meningitis is mediated by the neuronal NOS pathway.

Protective effect of the antioxidant N-acetyl- l-cysteine in pneumococcal meningitis in the rat

We investigated whether the antioxidant N-acetyl- l-cysteine or S-methylisothiourea, an inhibitor of the inducible nitric oxide (NO) synthase, can modulate pathophysiological alterations in an advanced phase of experimental pneumococcal meningitis in the rat. N-acetyl- l-cysteine significantly attenuated the increase in brain water content, intracranial pressure and cerebrospinal fluid white blood cell count 24 h after infection as compared to untreated, infected rats. However, it had no effect on meningitis-associated disturbances in cerebrovascular autoregulation and CO 2 reactivity of cerebral vessels. Treatment with S-methylisothiourea had no effect on the pathophysiological parameters measured in this model. These data suggest that reactive oxygen species, but not NO generated by the inducible NO synthase pathway, are key mediators of changes in brain water content, intracranial pressure and meningeal inflammation in an advanced stage of pneumococcal meningitis in the rat.

Effect of the bradykinin B 2 receptor antagonist Hoe140 in experimental pneumococcal meningitis in the rat

To elucidate the role of bradykinin in the complex pathophysiology of bacterial meningitis we investigated the effect of the bradykinin B 2 receptor antagonist Hoe140, incatibant ( d-Arg[Hyp 3-Thi 5- d-Tic 7-Oic 8]-bradykinin), on pathophysiological alterations in experimental pneumococcal meningitis. Untreated rats injected intracisternally (i.c.) with heat-killed pneumococci developed an increase of regional cerebral blood flow (185.4 ± 27.4%, baseline 100%, mean ± S.D.), brain water content (79.16 ± 0.23%), intracranial pressure (21.4 ± 6.0 mm Hg), and white blood cell count in the cerebrospinal fluid (CSF) (4621 ± 1894 cells/μl) within 6 h after i.c. challenge. Treatment with Hoe140 (0.1 mg/kg i.v. at baseline and 0.05 mg/kg s.c. at 2 h after i.c. challenge) attenuated the increase of brain water content (78.53 ± 0.28%; P < 0.05), intracranial pressure (7.5 ± 2.2 mm Hg; P < 0.05), and regional cerebral blood flow (128.6 ± 23.1%; P < 0.05), and reduced CSF pleocytosis (2690 ± 1898 cells/μl, N.S.). When treatment was started 4 h after i.c. challenge Hoe140 reduced intracranial pressure ( P < 0.05), but was no more capable to significantly influence the other pathophysiological parameters. Treatment with lower (0.01 mg/kg i.v. at baseline, followed by 0.005 mg/kg s.c. at 2 h) and higher (2 mg/kg i.v., followed by 1 mg/kg s.c. at 2 h) concentrations of Hoe140 was ineffective. Likewise, i.c. injection of Hoe140, at different dosages (4 nmol, 40 nmol, 400 nmol) did not significantly alter the pathophysiological parameters in pneumococci-induced meningitis, but caused changes in mean arterial blood pressure at dosages greater than 4 nmol. We conclude that bradykinin is involved as an inflammatory mediator of microvascular changes, brain edema, and increased intracranial pressure during the early phase of experimental pneumococcal meningitis.

Pneumococcal cell wall components induce nitric oxide synthase and TNF-alpha in astroglial-enriched cultures.

Astroglia and microglia, the most numerous cells in the central nervous system (CNS), have been shown to produce the inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) upon stimulation with the cytokines IFN-gamma, IL-1-beta, or bacterial lipopolysaccharides (LPS). However, it is not known whether gram-positive bacteria like Streptococcus pneumoniae cause astroglial cells to release nitric oxide (NO) and TNF-alpha. S. pneumoniae meningitis still has a high incidence and mortality in spite of antibiotic therapy. Cell wall components from S. pneumoniae (pneumococcal cell-wall components, PCW) and TNF-alpha have been shown to cause meningeal inflammation and cerebrovascular changes in experimental meningitis. Addition of PCW to cultured rat astroglial cells increased nitrite in the supernatant significantly after 24 h, from 17 +/- 21 to 133 +/- 62 nM/micrograms protein. Nitrite release was dose-dependent in a range shown to cause meningeal inflammation in vivo and was inhibited by the competitive NO synthase inhibitor NW-nitro-L-arginine (L-NA 10(-4 M)) and dexamethasone (10(-6 M)), with transcriptional and translational inhibition by actinomycin D and cycloheximide, respectively. PCW caused a significant increase in the release of TNF-alpha from astroglial cells after 4 h, from 2 +/- 3.5 pg/ml to 102 +/- 13.5 pg/ml, which was inhibited by dexamethasone (10(-6 M)). Our results suggest a role for astroglial-derived NO and TNF-alpha as mediators of vascular and inflammatory response in the early phase of experimental pneumococcal meningitis.

Experimental pneumococcal meningitis: Cerebrovascular alterations, brain edema, and meningeal inflammation are linked to the production of nitric oxide

We investigated whether treatment with the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA) and the free radical scavenger superoxide dismutase influences cerebral blood flow changes, brain edema, and cerebrospinal fluid pleocytosis in early experimental pneumococcal meningitis. Compared to untreated infected rats, superoxide dismutase given 3 hours after infection significantly attenuated the increase of brain water content, intracranial pressure, and cerebrospinal fluid white blood cell count, but did not modulate the increase in regional cerebral blood flow. N-Nitro-L-arginine treatment (5 mg/kg intravenously, followed by 5 mg/kg/hour) reversed the increase in regional cerebral blood flow; prevented an increase in brain water content, intracranial pressure, and cerebrospinal fluid nitrite concentrations; and reduced cerebrospinal fluid white blood cell count. With a closed cranial window preparation, N-nitro-L-arginine prevented pneumococci-induced dilatation of pial arterioles. When the effective dose was increased twofold, the effects of N-nitro-L-arginine became more pronounced but resulted in the death of 4 of 5 rats, probably due to hemodynamic side effects. In primary cultures of rat cerebral endothelial cells, nitrite concentrations increased after pneumococcal stimulation, which could be prevented by N-nitro-L-arginine and cycloheximide. These data suggest that (a) nitric oxide accounts for regional cerebral blood flow changes and pial arteriolar dilatation in the early phase of experimental pneumococcal meningitis; (b) both superoxide radical and nitric oxide are involved as mediators of brain edema and meningeal inflammation; and (c) cerebral endothelial cells can be stimulated by pneumococci to release nitric oxide presumably via the inducible nitric oxide synthase.

Is nitric oxide involved as a mediator of cerebrovascular changes in the early phase of experimental pneumococcal meningitis?

Nitric oxide (NO) is a mediator of haemodynamic changes and cytotoxicity in in vivo models of inflammation such as endotoxaemic shock. The purpose of this study was to investigate whether NO may be involved in the increase of cerebral blood flow (CBF), intracranial pressure (ICP) and brain water content, known to occur in the early phase of pneumococcal meningitis. Rats injected intracisternally with live pneumococci were either untreated or received 5 mg kg-1 i.v. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Pretreatment with L-NAME prevented the increase in CBF, ICP and brain water content, as seen in untreated animals. CBF tended to return towards baseline when rats were treated with L-NAME 2 h after pneumococcal injection. Whereas none of the untreated and L-NAME-pretreated animals died during the 6 h observation period, 3 out of 9 rats treated with L-NAME and 7 out of 9 rats with simultaneous i.v. injection of L-NAME and L-arginine died. Our results provide preliminary evidence that NO may be involved as a mediator of CBF changes and oedema formation in the early phase of pneumococcal meningitis in the rat. NO inhibition, however, may have detrimental effects of still unidentified cause, as indicated by the increased mortality in treated animals. Further studies with analysis of the causes of mortality, structurally different NO synthase inhibitors and direct evaluation of NO synthase induction are needed to further support this hypothesis.

Methylprednisolone attenuates inflammation, increase of brain water content and intracranial pressure, but does not influence cerebral blood flow changes in experimental pneumococcal meningitis

We investigated the effect of methylprednisolone on pathophysiological alterations in experimental pneumococcal meningitis. Untreated rats injected with pneumococcal cell wall components after hydrolization with M1 muramidase (PCW-M) developed an increase of regional cerebral blood flow (rCBF;165.0 ± 12.8%, baseline 100%,mean±S.E.M.), brain water content(79.23 ± 0.10%), intracranial pressure (ICP;11.9 ± 1.0mmHg) and white blood cell (WBC) count in the cerebrospinal fluid (CSF)(2,709 ± 482cells/μl) within 8 h after intracisternal (i.c.) challenge. Pretreatment with methylprednisolone or administration of methylprednisolone 4 h after i.c. challenge significantly attenuated the increase of brain water content (78.88 ±0.08%and78.82 ± 0.05%,resp.), ICP (7.7 ± 1.1mmHg and4.9 ± 0.8mmHg, resp.) and CSF WBC count (1,257 ± 168cells/μl an976 ± 105cells/μl, resp.). However, methylprednisolone did not inhibit the increae of rCBF (163.5 ± 13.7%and160.9 ± 6.8%, resp.), whereas dexamethasone significantly attenuated microvascular changes. Hypercapnia-induced reactivity of cerebral vessels tested 8 h after i.c. injection was preserved in all groups. In conclusion, we found that methylprednisolone significantly attenuated the increase of brain water content, ICP and CSF WBC count, bu thad no effect on microvascular changes during the early phase of experimental pneumococcal meningitis.

Imaging of Leukocyte-Endothelium Interaction Using In Vivo Confocal Laser Scanning Microscopy during the Early Phase of Experimental Pneumococcal Meningitis

Confocal laser scanning microscopy (CLSM) was used to investigate dynamic aspects of rhodamine 6G-labeled leukocytes in the pial microcirculation during the early phase of pneumococcal meningitis. Closed cranial windows were implanted into anesthetized rats without removing the dura mater. Leukocyte behavior was studied every hour after intracisternal (ic) injection. The number of adherent and extravasated leukocytes was determined during playback of videotaped images. Compared with results in controls, the number of adherent leukocytes increased significantly (P < .05) within 1 h after ic pneumococcal challenge, followed by a further increase up to 6 h after infection. In untreated infected rats, the number of extravasated leukocytes progressively increased from 3 to 6 h after infection. Leukocyte adherence to microvascular endothelium occurred in pial venules but not in arterioles. Dexamethasone pretreatment significantly (P < .05) attenuated leukocyte adherence and transendothelial passage of leukocytes.

Effect of catalase on regional cerebral blood flow and brain edema during the early phase of experimental pneumococcal meningitis.

Previous studies have demonstrated that the radical scavenger superoxide dismutase completely blocked the increase of regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain water content during the early phase of experimental pneumococcal meningitis in the rat. To obtain information on the nature of the reactive oxygen species involved, the effect of catalase, a hydrogen peroxide scavenger, was tested. Rats injected intracisternally with live pneumococci were either untreated or received intravenous catalase. The increase of rCBF and brain water content in infected untreated rats was significantly attenuated by catalase 6 h after intracisternal challenge. ICP increased in both infected groups, with a trend toward lower ICP with catalase treatment. Cerebrospinal fluid white blood cell counts were not significantly different between infected groups. These results and previous experiments using superoxide dismutase suggest that the increase of rCBF, ICP, and brain water content is mainly caused by superoxide or superoxide reaction products.

Antioxidants attenuate microvascular changes in the early phase of experimental pneumococcal meningitis in rats.

We tested in a rat meningitis model 1) whether pneumococcal cell wall components are capable of producing changes in regional cerebral blood flow, brain water content, and intracranial pressure similar to those we have already observed after intracisternal inoculation of live pneumococci and 2) whether antioxidants would modulate these alterations in the early phase of meningitis. Regional cerebral blood flow as measured by laser Doppler flowmetry and intracranial pressure were monitored continuously for 4 hours after intracisternal challenge. Brain edema formation was assessed by brain water content determinations. We investigated the following groups: rats challenged intracisternally with the whole intact pneumococcal cell wall (n = 7) or the pneumococcal cell wall hydrolyzed by the M1-muramidase (n = 7); rats injected intracisternally with phosphate-buffered saline (n = 6); rats pretreated intravenously with superoxide dismutase conjugated with polyethylene glycol (10,000 units/kg) and injected intracisternally with cell wall components (n = 5) or phosphate-buffered saline (n = 6); rats injected intracisternally with phosphate-buffered saline and pretreated intravenously with polyethylene glycol (10% solution, 1.2 ml/kg, n = 5) or continuously treated with intravenous free superoxide dismutase (22,000 units/kg per hour, n = 6); and rats continuously treated intravenously with deferoxamine mesylate (10 mg/kg per hour) and injected intracisternally with cell wall components (n = 6) or phosphate-buffered saline (n = 7). Both pneumococcal cell wall preparations produced a significant increase in regional cerebral blood flow, intracranial pressure, and brain water content. Conjugated superoxide dismutase as well as deferoxamine prevented the increase in intracranial pressure and brain water content. In addition, the increase in regional cerebral blood flow as observed in untreated, cell wall-challenged rats (baseline, 100%; 183.1 +/- 12.3% after 4 hours, mean +/- SEM) was significantly attenuated by administration of both conjugated superoxide dismutase (136.6 +/- 14.1%) and deferoxamine (149.8 +/- 8.2%) (p < 0.05). Polyethylene glycol-conjugated superoxide dismutase alone produced an increase in regional cerebral blood flow (125.6 +/- 8.7% after 4 hours). We found that polyethylene glycol per se accounts for this action. These data show that pneumococcal cell wall components containing teichoic acid produce changes in regional cerebral blood flow, intracranial pressure, and brain water content and that oxygen radicals contribute to these pathophysiological alterations in the early phase of experimental pneumococcal meningitis.

Transforming growth factor beta 2 inhibits cerebrovascular changes and brain edema formation in the tumor necrosis factor alpha-independent early phase of experimental pneumococcal meningitis.

Macrophages and granulocytes seem to play a key role in the pathogenesis of bacterial meningitis. Transforming growth factor beta (TGF-beta) leads to macrophage deactivation, as well as to inhibition of cytokine production and of endothelial granulocyte adhesion. We have investigated the influence of TGF-beta on regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain edema formation during the early phase of experimental meningitis. Rats which were inoculated intracisternally with live pneumococci or with pneumococcal cell wall hydrolyzed by the M1 muramidase (PCW-M) developed an increase of rCBF and ICP within 4 h postintracisternal challenge. A single intraperitoneal injection of TGF-beta 2 but not of TGF-beta 2 vehicle-control prevented the changes of rCBF. Furthermore, TGF-beta 2 significantly reduced the increase of ICP in rats inoculated with PCW-M. Likewise, the elevation of brain water content after intracisternal injection of pneumococci or PCW-M was blocked by pretreatment of rats with TGF-beta 2. TGF-beta 1 exhibited similar inhibitory effects in PCW-M-injected rats. The beneficial effects of TGF-beta 2 on the initial phase after pneumococcal inoculation seem to be tumor necrosis factor alpha- (TNF-alpha) independent since (a) intracisternal or intraperitoneal injection of neutralizing anti-TNF-alpha antibodies did not significantly influence rCBF, ICP, and brain water content in PCW-M-induced meningitis; and (b) TNF-alpha was only occasionally detected at low levels in cerebrospinal fluid at 4 h after PCW-M application.