Evidence for cyclooxygenase activation by nitric oxide in astrocytes

We have evaluated the role of nitric oxide (NO) on the cyclooxygenase pathway in mouse glial cells. Exposure of primary cultures of neonatal mouse cortical astrocytes to bacterial lipopolysaccharide (LPS; 1 μg/ml, 18 h) caused an increase in the release of both nitrite (NO⁻₂) and prostaglandin E₂ (PGE₂2), products of NO synthase (NOS) and cyclooxygenase, respectively. Production of both, NO⁻₂ and PGE₂ by astrocytes, was inhibited by the exposure of the NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME: 1, 10, and 100 μM) in a dose related manner. Besides, other NOS inhibitors such as Nitro L-arginine (NNA: 10⁻³ M) prevented the increase in PGE₂ release from LPS-stimulated astrocytes. Sodium nitroprusside (SNP; 100–200 μM) used as a NO donor caused a dose-related enhancement in the accumulation of PGE₂ induced by LPS and the presence of hemoglobin blocked the SNP effects. The exposure to SNP counteracted the decrease of PGE₂ production in LPS-treated astrocytes in which NO synthesis was blocked by L-NAME. In addition, SNP also enhanced the synthesis of PGE₂ following exogenous arachidonic acid astrocytes exposure. Interestingly, this effect was blocked by indomethacin. Treatment of astrocytes cultures with dexamethasone (0.1, 1 μM) blocked dose-relatedly the LPS-induced release of both NO⁻₂ and PGE₂. As expected, the presence of indomethacin (1, 10, and 20 μM) prevented in a dose related fashion, PGE₂ production by astrocytes following exposure to LPS. These results strongly indicate that in astroglial cells, NO is able to activate the cyclooxygenase pathway. © 1995 Wiley-Liss, Inc.     

Temperature-Related Effects of Adenosine Triphosphate-Activated Microglia on Pro-Inflammatory Factors

Background

Therapeutic hypothermia protects neurons after severe brain injury. Activated microglia produce several neurotoxic factors, such as pro-inflammatory cytokines and nitric oxide (NO), during neuron destruction. Hence, suppression of microglial release of these factors is thought to contribute partly to the neuroprotective effects of hypothermia. After brain insults, adenosine triphosphate (ATP) is released from injured cells and activates microglia. Here, we examined the acute effects of temperature on ATP-activated microglial production of inflammatory factors, and the possible involvement of p38 mitogen-activated protein kinase (p38) underlying such effects.

Methods

Microglia were cultured with ATP at 33, 37, and 39°C, or with ATP in the presence of a p38 inhibitor, SB203580, at 37°C. Cytokine and NO levels, and p38 activation were measured.

Results

Compared to 37°C, TNF-α was reduced at 33°C and augmented at 39°C for 1.5 h. IL-6 was reduced at 33°C for 6 h. NO was reduced at 33°C, but augmented at 39°C for 6 h. p38 was reduced at 33°C for 1 min. SB203580 inhibited ATP-induced TNF-α, IL-6, and NO production.

Conclusion

Lowering temperature rapidly reduced p38 activation and the subsequent p38-regulated production of pro-inflammatory cytokines and NO in ATP-activated microglia, suggesting that attenuation of early phase inflammatory responses via suppression of p38 in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Temperature elevation increased TNF-α and NO production in these cells. These temperature-dependent changes imply that monitoring of TNF-α and NO in the cerebrospinal fluid during the early phase might be useful as biomarkers for responses to therapeutic hypothermia and hyperthermia.

     

Granulocytes in the subarachnoid space of humans and rabbits with bacterial meningitis undergo apoptosis and are eliminated by macrophages

The contribution of leukocyte apoptosis to the resolution of meningeal inflammation in bacterial meningitis was studied in the cerebrospinal fluid (CSF) and in meningeal infiltrates of humans and rabbits by in situ tailing, flow cytometry, agarose gel electrophoresis, and electron microscopy. In humans, the rate of apoptotic granulocytes was 21.0 ± 20.8% (n = 11) in cytocentrifuge preparations and 3.3 ± 3.4 (n = 14) in putride infiltrates of autopsy cases (P = 0.02). In rabbits, CSF pleocytosis peaked 8 h after the initiation of antibiotic treatment (5311 ± 3122/μl). At this time, the rate of apoptotic granulocytes was 15.2 ± 7.3% in CSF and 1.8 ± 1.4% in the meningeal infiltrates (each group n = 6, P = 0.007). Thereafter, the rate of apoptotic granulocytes in CSF declined below 10%. In humans and rabbits, bands representing internucleosomal fragments of approximately 180 base pairs and multiples thereof were documented on agarose gels. Phagocytosis of apoptotic granulocytes by macrophages was visualized by light and electron microscopy. In conclusion, during resolution of subarachnoid space inflammation in bacterial meningitis, a substantial fraction of granulocytes undergoes apoptosis. These granulocytes are removed by phagocytosis by macrophages. Apoptosis is more frequent in granulocytes floating in the CSF than in adherent cells. Received: 12 February 1998 / Accepted: 29 April 1998     

Effect of GABA‐ergic mechanisms on synaptosomal NO synthesis and the nitrergic component of NANC relaxation in rat ileum

Background  γ‐Aminobutyric acid (GABA) acts on specific neural receptors [A, B and C(Aρ)] to modulate gastrointestinal function. The precise role of GABA receptor activation in the regulation of presynaptic nitric oxide (NO) synthesis in nerve terminals is unknown. Methods  Rat ileal nerve terminals were isolated by differential centrifugation. Nitric oxide synthesis was analysed using a L‐[³H]arginine assay. In vitro studies were performed under non‐adrenergic non‐cholinergic (NANC) conditions on isolated ileal segments. Key Results  γ‐Aminobutyric acid inhibited NO synthesis significantly (n = 6, P < 0.05) [(fmol mg⁻¹ min⁻¹) control: 27.7 ± 1.5, 10⁻⁶ mol L⁻¹: 19.7 ± 1.3; 10⁻⁵ mol L⁻¹: 17.5 ± 3.0]. This effect was antagonized by the GABA A receptor antagonist bicuculline and the GABA C receptor antagonist (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA), but not by the GABA B receptor antagonist SCH 50911. The GABA A receptor agonist muscimol [(fmol mg⁻¹ min⁻¹) control: 27.6 ± 1.0, 10⁻⁶ mol L⁻¹: 19.1 ± 1.7, n = 5, P < 0.05] and the GABA C receptor agonist cis‐4‐aminocrotonic acid (CACA) [(fmol mg⁻¹ min⁻¹) control: 29.5 ± 3.2, 10⁻³ mol L⁻¹: 20.3 ± 2.5, n = 6, P < 0.05], mimicked the GABA‐effect, whereas the GABA B agonist baclofen was ineffective. Bicuculline reversed the inhibitory effect of muscimol, TPMPA antagonized the effect of CACA. In functional experiments the GABA A and C receptor agonists reduced the NANC relaxation induced by electrical field stimulation in rat ileum by about 40%. After NOS‐inhibition by Nε‐nitro‐l ‐arginine methyl ester (l ‐NAME) the GABA A receptor agonist had no effect, whereas the GABA C receptor agonist still showed a residual response. Conclusions & Inferences  γ‐Aminobutyric acid inhibits neural NO synthesis in rat ileum by GABA A and GABA C(Aρ) receptor‐mediated mechanisms.     

Inhibition of TNF-α protects in vitro brain barrier from ischaemic damage

Cerebral ischaemia, associated with neuroinflammation and oxidative stress, is known to perturb blood–brain barrier (BBB) integrity and promote brain oedema formation. Using an in vitro model of human BBB composed of brain microvascular endothelial cells and astrocytes, this study examined whether suppression of TNF-α, a potent pro-inflammatory cytokine, might attenuate ischaemia-mediated cerebral barrier damage. Radical decreases in transendothelial electrical resistance and concomitant increases in paracellular flux across co-cultures exposed to increasing periods of oxygen-glucose deprivation alone (0.5–20 h) or followed by 20 h of reperfusion (OGD ± R) confirmed the deleterious effects of ischaemic injury on cerebral barrier integrity and function which concurred with reductions in tight junction protein (claudin-5 and occludin) expressions. OGD ± R elevated TNF-α secretion, NADPH oxidase activity, O₂⁻ production, actin stress fibre formation, MMP-2/9 activities and apoptosis in both endothelial cells and astrocytes. Increases in MMP-2 activity were confined to its extracellular isoform and treatments with OGD + R in astrocytes where MMP-9 could not be detected at all. Co-exposure of individual cell lines or co-cultures to an anti-TNF-α antibody dramatically diminished the extent of OGD ± R-evoked oxidative stress, morphological changes, apoptosis, MMP-2/9 activities while improving the barrier function through upregulation of tight junction protein expressions. In conclusion, vitiation of the exaggerated release of TNF-α may be an important therapeutic strategy in preserving cerebral integrity and function during and following a cerebral ischaemic attack.     

Anti ICAM-1 (CD 54) monoclonal antibody reduces inflammatory changes in experimental bacterial meningitis

We investigated whether monoclonal antibodies directed against intracellular adhesion molecule 1 (ICAM-1 mAb) inhibit brain edema, increase of intracranial pressure (ICP), regional cerebral blood flow (rCBF) and recruitment of white blood cells (WBC) into the cerebrospinal fluid (CSF) in the rat model of the early phase of bacterial meningitis. Brain edema was assessed by brain water content determinations. rCBF measured by laser Doppler flowmetry and ICP were recorded continuously for 6 h after intracisternal challenge. Meningitis was induced with pneumococcal cell walls (PCW). Increase of ICP and brain water content were significantly inhibited (P < 0.05) by intravenous treatment with ICAM-1 mAb (TM-8, 1 mg/kg). Furthermore, ICAM-1 mAb treatment profoundly attenuated (P < 0.05) rCBF increase and WBC invasion into the CSF. These results suggest that the ICAM-1 pathway is critically involved in the early phase of bacterial meningitis.     

In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate

The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of ³H-L-glutamate, ³H-L-aspartate, and ³H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm², and ¹⁴C-D-mannitol permeability values of 0.88 ± 0.13 × 10⁻⁶ cm s⁻¹. Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K_m of 69 ± 15 μM and a J_max of 44 ± 3.1 pmol min⁻¹ cm⁻² for L-aspartate and a K_m of 138 ± 49 μM and J_max of 28 ± 3.1 pmol min⁻¹ cm⁻² for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), −2 (Slc1a2), and −3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and −3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. © 2012 Wiley Periodicals, Inc.     

Tumor necrosis factor-α, interleukin-1β and nitric oxide induce calcium transients in distinct populations of cells cultured from the rat area postrema

The area postrema (AP) represents the medullary sensory circumventricular organ lacking endothelial blood–brain barrier function at the base of the 4th cerebral ventricle. Administration of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or the nitric oxide (NO) donor diethylamino-diazenolate-2-oxide (DEA) caused fast transient rises in intracellular calcium concentrations ([Ca²⁺]i) in distinct populations of cells investigated in a primary microculture of the rat AP. TNF-α caused rapid elevations of [Ca²⁺]i in 8% of all neurons and astrocytes investigated, with limited responses of microglial cells and no responses of oligodendrocytes. 15% of all neurons investigated responded to IL-1β, while only 5–7% of the other cell types showed rises in [Ca²⁺]i. The most pronounced effects were caused by treatment with DEA with some 20% responsive astrocytes and oligodendrocytes, 15% neurons and 10% microglial cells. Evidently, the AP can act as a sensor for circulating TNF-α and IL-1β, or for locally produced cytokines and NO during infection and inflammation.     

Enriched environment fails to increase meningitis‐induced neurogenesis and spatial memory in a mouse model of pneumococcal meningitis

An increase in adult neurogenesis was observed after exposure to enriched environment (EE) and during reconvalescence from experimental pneumococcal meningitis. This study investigated neurogenesis and spatial learning performance 5 weeks after bacterial meningitis and exposure to EE. C57BL/6 mice were infected by intracerebral injection of Streptococcus pneumoniae and treated with ceftriaxone for 5 days. Forty‐eight hours after infection, one group (n = 22) was exposed to EE and the other group (n = 23) housed under standard conditions. Another set of mice was kept under either enriched (n = 16) or standard (n = 15) conditions without bacterial meningitis. Five weeks later, the Morris water maze was performed, and neurogenesis was evaluated by means of immunohistochemistry. Mice housed in EE without prior bacterial infection displayed both increased neurogenesis and improved water maze performance in comparison with uninfected control animals. Bacterial meningitis stimulated neurogenesis in the granular cell layer of the dentate gyrus: with standard housing conditions, we observed a higher density of BrdU‐immunolabeled and TUC‐4‐expressing cells 5 weeks after induction of bacterial meningitis than in the noninfected control group. EE did not further increase progenitor cell proliferation and neuronal differentiation in the subgranular cell layer of the dentate gyrus after bacterial meningitis in comparison with infected mice housed under standard conditions. Moreover, the Morris water maze showed no significant differences between survivors of meningitis exposed to EE and animals kept in standard housing. In summary, exposure to EE after pneumococcal meningitis did not further increase meningitis‐induced neurogenesis or improve spatial learning. © 2009 Wiley‐Liss, Inc.     

Effects of Toll-like receptor 2 agonist Pam3CysSK4 on inflammation and brain damage in experimental pneumococcal meningitis

TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam₃CysSK₄ was applied intracisternally (0.5 μg in 10 μl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam₃CysSK₄ on apoptosis was investigated by intracisternal application of 0.5 μg in 10 μl saline and 40 μg in 20 μl saline. Pam₃CysSK₄ neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam₃CysSK₄ induced pleocytosis, TNF-α and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam₃CysSK₄ at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.     

Hypothermia at 35 °C Reduces the Time-Dependent Microglial Production of Pro-inflammatory and Anti-inflammatory Factors that Mediate Neuronal Cell Death

Background

Therapeutic hypothermia protects neurons after severe brain damage. This effect has been mainly achieved at the core temperatures of 32–34 °C; however, the optimum temperature of therapeutic hypothermia is not fully defined. Here we studied whether hypothermic culture at 35 °C had the same effects on the decrease of time-dependent expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, and nitric oxide (NO) by stimuli-activated microglia as that at 33 °C, as determined in our previous reports, and whether these factors directly induced neuronal cell death.

Methods

We determined the levels of cytokines and NO produced by microglia cultured with adenosine triphosphate (ATP), a toll-like receptor (TLR)2 agonist (N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine, Pam₃CSK₄), or a TLR4 agonist (lipopolysaccharide) under mild hypothermic (33 °C), minimal hypothermic (35 °C), and normothermic (37 °C) conditions. We also determined the viability of rat neuronal pheochromocytoma PC12 cells treated with recombinant TNF-α or IL-10 or (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3, an NO donor).

Results

Production of TNF-α, as well as that of IL-10 and NO were decreased by minimal hypothermia at 1.5–6, and 24–48 h, respectively, compared with normothermia, although some effects were diminished as compared with those by mild hypothermia. Exposure to TNF-α, IL-10, and NOR3 caused the death of PC12 cells in a concentration-dependent manner after 24 h.

Conclusion

Hypothermic culture at 35 °C decreased the production of early-phase TNF-α and late-phase IL-10 and NO from ATP- and TLR-activated microglia as observed at 33 °C, albeit with diminished effects. Moreover, these factors caused the death of neuronal cells in a concentration-dependent manner. These results suggest that the attenuation of microglial production of TNF-α, IL-10, and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death. Minimal hypothermia at 35 °C may be sufficient to elicit neuroprotective effect.     

Cognitive outcome in adults after bacterial meningitis

Objective

To evaluate cognitive outcome in adult survivors of bacterial meningitis.

Methods

Data from three prospective multicentre studies were pooled and reanalysed, involving 155 adults surviving bacterial meningitis (79 after pneumococcal and 76 after meningococcal meningitis) and 72 healthy controls.

Results

Cognitive impairment was found in 32% of patients and this proportion was similar for survivors of pneumococcal and meningococcal meningitis. Survivors of pneumococcal meningitis performed worse on memory tasks (p<0.001) and tended to be cognitively slower than survivors of meningococcal meningitis (p = 0.08). We found a diffuse pattern of cognitive impairment in which cognitive speed played the most important role. Cognitive performance was not related to time since meningitis; however, there was a positive association between time since meningitis and self‐reported physical impairment (p<0.01). The frequency of cognitive impairment and the numbers of abnormal test results for patients with and without adjunctive dexamethasone were similar.

Conclusions

Adult survivors of bacterial meningitis are at risk of cognitive impairment, which consists mainly of cognitive slowness. The loss of cognitive speed is stable over time after bacterial meningitis; however, there is a significant improvement in subjective physical impairment in the years after bacterial meningitis. The use of dexamethasone was not associated with cognitive impairment.The estimated annual incidence of bacterial meningitis is 4–6 per 100 000 adults and Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis (meningococcus) are the causative bacteria in 80% of cases.^1,2 Fatality rates in patients with pneumococcal meningitis (26%) and meningococcal meningitis (7%) are significant.^1,2,3 Even in patients with apparent good recovery, cognitive impairment occurs frequently,⁴ especially after pneumococcal meningitis.^4,5,6 The cognitive functions affected by bacterial meningitis differ between studies, most likely because of the limited numbers of patients examined, and the lack of uniformity across studies in assessment methods and in the definition of cognitive impairment.^{4,5,6,7,8,9,10} We therefore pooled data on cognitive outcome after bacterial meningitis from three of our previous studies to more clearly determine which cognitive functions are affected by bacterial meningitis and to identify which patients are at risk of developing cognitive impairment.     

An infant mouse model of brain damage in pneumococcal meningitis

Bacterial meningitis due to Streptococcus pneumoniae is associated with an significant mortality rate and persisting neurologic sequelae including sensory-motor deficits, seizures, and impairments of learning and memory. The histomorphological correlate of these sequelae is a pattern of brain damage characterized by necrotic tissue damage in the cerebral cortex and apoptosis of neurons in the hippocampal dentate gyrus. Different animal models of pneumococcal meningitis have been developed to study the pathogenesis of the disease. To date, the infant rat model is unique in mimicking both forms of brain damage documented in the human disease. In the present study, we established an infant mouse model of pneumococcal meningitis. Eleven-days-old C57BL/6 (n = 299), CD1 (n = 42) and BALB/c (n = 14) mice were infected by intracisternal injection of live Streptococcus pneumoniae. Sixteen hours after infection, all mice developed meningitis as documented by positive bacterial cultures of the cerebrospinal fluid. Sixty percent of infected C57BL/6 mice survived more than 40 h after infection (50% of CD1, 0% of BALB/c). Histological evaluations of brain sections revealed apoptosis in the dentate gyrus of the hippocampus in 27% of infected C57BL/6 and in 5% of infected CD1 mice. Apoptosis was confirmed by immunoassaying for active caspase-3 and by TUNEL staining. Other forms of brain damage were found exclusively in C57BL/6, i.e. caspase-3 independent (pyknotic) cell death in the dentate gyrus in 2% and cortical damage in 11% of infected mice. This model may prove useful for studies on the pathogenesis of brain injury in childhood bacterial meningitis.     

Modulation of lipopolysaccharide-induced tumor necrosis factor-α production by selective α- and β-adrenergic drugs in mice

In a previous study we demonstrated that mice pretreated with the highly selective α₂-adrenoceptor antagonist CH-38083 showed blunting of the tumor necrosis factor-α (TNF-α) response induced by bacterial lipopolysaccharide (LPS). In the present study, the effect of a selective block of α₂-adrenoreceptor and the role of the sympathetic nervous system (SNS) in the regulation of LPS-induced TNF-α production was explored further using different selective adrenoceptor antagonists and agonists. While adrenalectomy did not prevent the effect of CH-38083, the block of the sympathetic transmission by chlorisondamine fully abolished the inhibitory effect of CH-38083 on LPS-induced TNF-α production, suggesting that the effect of the α₂-adrenoceptor blocking agent is corticosteroid-independent, but it requires intact sympathetic activity. Since the selective block of α₂-adrenoceptors results in an increased sympathetic activity and an increase of the release of noradrenaline (NA) in both the central and the peripheral nervous systems, and in our experiments propranolol, a non-selective β-adrenoceptor antagonist, and atenolol, a selective antagonist of β₁-adrenoceptors, prevented the effect of α₂-adrenoceptor blockade by CH-38083 of the TNF-α response induced by LPS, it seems likely that the excessive stimulation by NA of β₁-adrenoceptors is responsible for this action. The role of β-adrenoceptors and endogenous catecholamines is further substantiated by the finding that pretreatment of animals with propranolol alone resulted in a dose-dependent increase of the TNF-α response induced by LPS, and that isoproterenol, a non-selective β-adrenoceptor agonist, decreased it. Additionally, it was shown that prazosin, an α₁-and α_2B-adrenoceptor antagonist, reduced LPS-induced TNF-α production. However, l-phenylephrine, a selective α₁-adrenoceptor agonist, was not able to modulate the TNF-α response following LPS challenge. Our findings that α₂-and β-adrenoceptor antagonists are able to decrease or increase, respectively, the TNF-α response elicited by LPS indicate that SNS, through release of endogenous catecholamines, is involved in vivo in the regulation of LPS-induced TNF-α production. In this process, the β-adrenoceptor-mediated events seems to play a pivotal role. Since the blockade of sympathetic activity by chlorisondamine failed to affect LPS-induced TNF-α release, it seems likely that, in vivo, the inhibitory effect of SNS on TNF-α production, mediated via β-adrenoceptors, is opposed by an effect of catecholamines on α₂-adrenoceptors. It is suggested that the fine-tuning of TNF-α release exerted by SNS in vivo might be particularly important during immunological and non-immunological stress, when the concentration of catecholamines is increased in the close proximity of TNF-α-secreting cells, which are known to possess adrenoceptors.     

Lipopolysaccharide and pneumococcal cell wall components activate the mitogen activated protein kinases (MAPK) erk-1, erk-2, and p38 in astrocytes

Cell wall compounds of gram-positive bacteria are capable of inducing the biosynthesis of proinflammatory cytokines in CNS cells in a similar way as lipopolysaccharide (LPS) of gram-negative bacteria does. Astrocytes, which lack the CD14 LPS receptor, have also been shown to respond to LPS-stimulation by increased cytokine synthesis. However, almost nothing is known about signaling steps involved in this process. We have therefore examined signaling events in primary cultures of rat astrocytes and the human astrocytoma cell line U373MG, brought about by LPS and pneumococcal cell walls (PCW). Of particular interest to us was the tyrosine phosphorylation patterns and activation states of three members of the mitogen activated protein kinase (MAPK) family, i.e., extracellular signal-regulated protein kinase (erk)-1, erk-2, and the recently identified p38. We show that LPS and PCW initiate tyrosine phosphorylation and activation of erk-1, erk-2, and p38 in a dose-dependent fashion. Inhibitors of tyrosine phosphorylation were able to alleviate this effect and also blocked cytokine production of astrocytes. Both, LPS- and PCW-induced responses of astrocytic cells required the presence of soluble CD14 (sCD14) present in serum. Unraveling the signaling steps induced by bacterial compounds in cells of the CNS may potentially help to elucidate the pathomechanisms of meningitis and central nervous complications of sepsis and may offer options for novel treatment strategies. GLIA 22:295–305, 1998. © 1998 Wiley-Liss, Inc.     

Nitric oxide suppresses L-type calcium currents in basilar artery smooth muscle cells in rabbits

Abstract

Objectives: Nitric oxide (NO) is well known to be a vasodilator, and NO donor compounds are currently used for treating vasospasm following subarachnoid hemorrhage. However, the action mechanism of cerebral vascular relaxation is not yet clear. L-type calcium channels have been determined to play an essential role in smooth muscle contraction. To investigate the role of L-type calcium channels in NO-induced relaxation of basilar smooth muscle cells, we examined the effect of the NO donor, sodium nitroprusside (SNP) on calcium (Ca²⁺) currents using smooth muscle cells isolated from a rabbit basilar artery.

Method: The smooth muscle cells were isolated from rabbit basilar artery by enzyme treatment. To identify L-type Ca²⁺ currents, we used cesium chloride, a potassium channel blocker and Bay K8644, an activator of L-type Ca²⁺ channel.

Results: The L-type calcium currents (91±13·0 pA; n?=?11) were significantly reduced by SNP (32±5 pA; n?=?11; P<0·05). 1H-[1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one, a 3',5'-cyclic guanosine monophosphate inhibitor, blocked the effect of SNP on L-type Ca²⁺ currents, and similar results were obtained after the application of 7-nitroindazole, a specific NO synthase inhibitor. Furthermore, inward currents were enhanced by Bay K8644 (170±22 pA; n?=?5) and were suppressed by SNP (54±13 pA; n?=?5; P<0·05).

Discussion: These results demonstrate that NO suppresses the L-type Ca²⁺ currents in rabbit basilar smooth muscle cells, and suggest that L-type Ca²⁺ channels may play a pivotal role in NO-induced vascular relaxation.     

Imipramine reverses depressive-like parameters in pneumococcal meningitis survivor rats

Pneumococcal meningitis is a severe infectious disease of the central nervous system, associated with acute inflammation and might cause damage to the host, such as deafness, blindness, seizure, and learning deficits. However, infectious diseases can play a significant role in the etiology of neuropsychiatric disturbances. In this context, we evaluated depressive-like parameters; corticosterone and ACTH levels in pneumococcal meningitis surviving rats. Wistar rats underwent a magna cistern tap receiving either 10 μL sterile saline or a Streptococcus pneumoniae suspension at the concentration of 5 × 10(9) cfu/mL. After 3 days of meningitis induction procedure, the animals were treated with imipramine at 10 mg/kg or saline for 14 days (3rd-17th day). The consumption of sweet food was measured for 7 days (10th-17th day). The meningitis group decreased the sucrose intake and increased the levels of corticosterone and ACTH levels in the serum and TNF-α in the cortex; however, the treatment with imipramine reverted the reduction of sweet food consumption, normalized hormonal levels and TNF-α in the cortex. Our results supported the hypothesis that the pneumococcal meningitis surviving rats showed depressive-like behavior and alterations in the hypothalamus-pituitary-adrenal axis.     

A kinetic study of the cytokine/chemokines levels and disruption of blood-brain barrier in infant rats after pneumococcal meningitis

Bacterial meningitis is an inflammation of the meninges and subarachnoid space that occurs in response of bacteria. Young children are particularly vulnerable to bacterial meningitis, two thirds of meningitis deaths in low-income countries occur among children under the age of fifteen. The main bacterial pathogens causing meningitis beyond the neonatal period are Streptococcus pneumoniae, Haemophilus influenza type b and Neisseria meningitidis. Therefore, the aim of this study is to evaluate the kinetic and the levels of TNF-α, IL-1β, IL-6, IL-10 and CINC-1 in different brain regions as well as the blood-brain barrier permeability after meningitis induced by S. pneumoniae in infant Wistar rats. The animals underwent a magna cistern tap receiving either 10μL sterile saline as a placebo or an equivalent volume of a S. pneumoniae suspension at the concentration 1×10(6)CFU/mL. The animals were killed at different times after induction. The brain was removed and the hippocampus and the cortex were isolated and used for the determination of cytokine/chemokine levels and blood-brain barrier permeability. The cerebrospinal fluid was obtained by puncture of the cisterna magna to TNF-α and IL-1β analysis. In the hippocampus, the CINC-1 and IL-1β levels were found increased at 6h, 12h and 24h after pneumococcal meningitis induction. In the cortex the levels of the CINC-1 were increased at 6h, 12h and 24h. The IL-1β and TNF-α were increased at 12h and 24h. The level of IL-6 was increased only after 24h after pneumococcal meningitis induction. In cerebrospinal fluid, the TNF-α was increased at 12h, 24h and IL-1 was increased at 24h after S. pneumoniae induction. The blood-brain barrier breakdown in hippocampus and cortex were observed at 12h until 24h during meningitis. In conclusion, a peak of pro-inflammatory cytokine/chemokine is associated with disruption of the blood-brain barrier in infants with pneumococcal meningitis.     

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

Abstract

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. N^G-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. [Neurol Res 1994; 16: 108-112]