Inge Bauer

Apoptosis induction by different local anaesthetics in a neuroblastoma cell line.

BACKGROUNDnLocal anaesthetics are known to induce apoptosis in clinically relevant concentrations. Hitherto, it is unknown what determines the apoptotic potency of local anaesthetics. Therefore, we compared apoptosis induction by local anaesthetics related to their physicochemical properties in human neuronal cells.nnnMETHODSnNeuroblastoma cells (SHEP) were incubated with eight local anaesthetics, two of the ester and six of the amide types. At least, five concentrations of each local anaesthetic were evaluated. After incubation for 24 h, rates of cells in early apoptotic stages and overall cell death were evaluated by annexin V and 7-amino-actinomycin D double staining by flow cytometry. The concentrations that led to half-maximal neurotoxic effects (LD50) were calculated and compared for all local anaesthetics.nnnRESULTSnAll local anaesthetics were neurotoxic in a concentration-dependent manner. All drugs induced similar rates of early apoptotic cell formation at low concentrations, whereas at high concentrations, late apoptotic or necrotic cell death predominated. Comparison of LD50 values of the different local anaesthetics resulted in the following order of apoptotic potency from high to low toxicity: tetracaine>bupivacaine>prilocaine=mepivacaine=ropivacaine>lidocaine>procaine=articaine. The toxicity correlated with octanol/buffer coefficients and also with experimental potency of the local anaesthetic, but was unrelated to the structure (ester or amide type).nnnCONCLUSIONSnAll commonly used local anaesthetics induce neuronal apoptosis in clinically used concentrations. The neurotoxicity correlates with lipid solubility and thus with the conduction blocking potency of the local anaesthetic, but is independent of the chemical class (ester/amide).

Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells

BACKGROUNDnKetamine has been shown to have neurotoxic properties, when administered neuraxially. The mechanism of this local toxicity is still unknown. Therefore, we investigated the mechanism of cytotoxicity in different human cell lines in vitro.nnnMETHODSnWe incubated the following cell types for 24 h with increasing concentrations of S(+)-ketamine and racemic ketamine: (i) human Jurkat T-lymphoma cells overexpressing the antiapoptotic B-cell lymphoma 2 protein, (ii) cells deficient of caspase-9, caspase-8, or Fas-associated protein with death domain and parental cells, and (iii) neuroblastoma cells (SHEP). N-Methyl-d-aspartate (NMDA) receptors and caspase-3 cleavage were identified by immunoblotting. Cell viability and apoptotic cell death were evaluated flowcytometrically by Annexin V and 7-aminoactinomycin D double staining. Mitochondrial metabolic activity and caspase-3 activation were measured.nnnRESULTSnKetamine, in a concentration-dependent manner, induced apoptosis in lymphocytes and neuroblastoma cell lines. Cell lines with alterations of the mitochondrial pathway of apoptosis were protected against ketamine-induced apoptosis, whereas alterations of the death receptor pathway did not reduce apoptosis. S(+)-Ketamine and racemic ketamine induced the same percentage of cell death in Jurkat cells, whereas in neuroblastoma cells, S(+)-ketamine was slightly less toxic.nnnCONCLUSIONSnKetamine at millimolar concentrations induces apoptosis via the mitochondrial pathway, independent of death receptor signalling. At higher concentrations necrosis is the predominant mechanism. Less toxicity of S(+)-ketamine was observed in neuroblastoma cells, but this difference was minor and therefore unlikely to be mediated via the NMDA receptor.

Antinociceptive effects of systemic lidocaine: involvement of the spinal glycinergic system.

Beside their action on voltage-gated Na(+) channels, local anesthetics are known to exert a variety of effects via alternative mechanisms. The antinociceptive effect of lidocaine is well documented, yet the exact mechanism is not fully understood. Whether glycinergic mechanisms, which play a pivotal role in pain modulation, are involved in lidocaine-induced antinociception is hitherto unclear. In the present study, lidocaine was injected intravenously in rats using the formalin test for acute pain and the chronic constriction injury model for neuropathic pain. The effect of intrathecally administered d-serine (an agonist at the glycine-binding site at the NMDA-receptor), its inactive isomer l-serine, CGP 78608 (antagonist at the glycineB-site of the NMDA-receptor) and strychnine (antagonist at inhibitory glycine-receptors) on lidocaine-induced antinociception was examined. Systemically administered lidocaine was antinociceptive in both acute and chronic pain model. In the formalin test, the effect of lidocaine was antagonized by d-serine, but not by l-serine or strychnine. In the chronic constriction injury model, antinociception evoked by lidocaine was reduced by d-serine, strychnine and CGP 78608, while l-serine had no effect. These results indicate a modulatory effect of lidocaine on the NMDA-receptor. Additionally, since in our study lidocaine-induced antinociception was antagonized by both glycineB-site modulators and strychnine our results may favor the hypothesis of a general glycine-like action of lidocaine or some of its metabolites on inhibitory strychnine-sensitive receptors and on strychnine-insensitive glycine receptors.

The Influence of Adjuvants Used in Regional Anesthesia on Lidocaine-Induced Neurotoxicity In Vitro

Background: Neurotoxic properties of local anesthetics can rarely lead to irreversible neuronal damage as in cauda equina syndrome. Clinically, local anesthetics are often combined with adjuvants to improve or prolong the anesthetic effect, whereas the impact of such adjuvants on lidocaine-induced apoptosis is unclear. Therefore, we investigated the influence of different adjuvants on the neurotoxicity of lidocaine. Methods: Human neuroblastoma cells and primary rat astrocytes were incubated for 24 hrs with lidocaine at a toxic concentration alone and in combination with morphine, sufentanil, clonidine, epinephrine, neostigmine, ketamine, and midazolam. Subsequently, the rates of cell death and early apoptosis were measured by flow cytometry in neuroblastoma cells, whereas astrocyte viability was analyzed by mitochondrial activity assay. In addition, isobolograms were calculated to describe the additive effects of lidocaine with ketamine or midazolam, respectively. Results: Coadministration of lidocaine with sufentanil, clonidine, epinephrine, and neostigmine did not alter the rates of cell death compared with cells treated with lidocaine alone. Morphine improved the viability of astrocytes only at concentrations beyond those occurring clinically. In contrast, coincubation of lidocaine with ketamine or midazolam led to significantly increased rates of cell death. The combined toxicity of ketamine and lidocaine was additive, whereas the combined toxicity of midazolam and lidocaine was subadditive. Conclusions: Sufentanil, clonidine, epinephrine, and neostigmine do not influence the neurotoxicity of lidocaine in vitro. Morphine may have some cytoprotective effect at concentrations greater than those seen intrathecally in humans. In contrast, ketamine and midazolam increase the neurotoxicity of lidocaine in vitro, presumably by additive induction of mitochondrial apoptosis.

Cardiovascular stability and unchanged muscle sympathetic activity during xenon anaesthesia: role of norepinephrine uptake inhibition

BACKGROUNDnIntraoperative hypotension is associated with increased risk of perioperative complications. The N-methyl-d-aspartate (NMDA) receptor (NMDA-R) antagonist xenon (Xe) induces general anaesthesia without impairment of cardiac output and vascular resistance. Mechanisms involved in cardiovascular stability have not been identified.nnnMETHODSnMuscle sympathetic activity (MSA) (microneurography), sympathetic baroreflex gain, norepinephrine (NE) plasma concentration (high-performance liquid chromatography), anaesthetic depth (Narcotrend(®) EEG monitoring), and vital parameters were analysed in vivo during Xe mono-anaesthesia in human volunteers (n=8). In vitro, NE transporter (NET) expressing HEK293 cells and SH-SY5Y neuroblastoma cells were pre-treated with ketamine, MK-801, NMDA/glycine, or vehicle. Subsequently, cells were incubated with or without Xe (65%). NE uptake was measured by using a fluorescent NET substrate (n=4) or [(3)H]NE (n=6).nnnRESULTSnIn vivo, Xe anaesthesia increased mean (standard deviation) arterial pressure from 93 (4) to 107 (6) mm Hg and NE plasma concentration from 156 (55) to 292 (106) pg ml(-1), P<0.01. MSA and baroreflex gain were unaltered. In vitro, ketamine decreased NET activity (P<0.01) in NET-expressing HEK293 cells, while Xe, MK-801, and NMDA/glycine did not. Xe reduced uptake in SH-SY5Y cells expressing NET and NMDA-Rs (P<0.01). MK-801 (P<0.01) and ketamine (P<0.01) also reduced NET activity, but NMDA/glycine blocked the effect of Xe on [(3)H]NE uptake.nnnCONCLUSIONSnIn vivo, Xe anaesthesia does not alter sympathetic activity and baroreflex gain, despite increased mean arterial pressure. In vitro, Xe decreases the uptake of NE in neuronal cells by the inhibition of NET. This inhibition might be related to NMDA-R antagonism and explain increased NE concentrations at the synaptic cleft and in plasma, contributing to cardiovascular stability during Xe anaesthesia.

Hypoxia induces late preconditioning in the rat heart in vivo.

Background:Although hypoxic late preconditioning (LPC) limits ischemia-reperfusion injury in vitro, its cardioprotective effect is not established in vivo. Methods:In part 1, rats were exposed to 4 h of hypoxia (16%, 12%, 8% oxygen) before 24 h of reoxygenation. In part 2, normoxic rats received early preconditioning with sevoflurane (1 minimum alveolar concentration [MAC] for 3 × 5 min), continuous administration of 1 MAC sevoflurane, or 11 mg · kg · h propofol. Thereafter, all rats underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. After reperfusion, hearts were excised for infarct staining. The expression of protein kinase C (PKC)&agr; and PKC&egr; was assessed by Western blot analysis and the expression of heme oxygenase-1 and vascular endothelial growth factor by reverse transcriptase polymerase chain reaction. Results:In normoxic control rats, infarct size was 62 ± 6% of the area at risk. Hypoxic LPC reduced infarct size (LPC16: 36 ± 11%, LPC12: 38 ± 10%, LPC8: 39 ± 11%; each P < 0.001) to approximately the same magnitude as sevoflurane-preconditioning (40 ± 8%; P < 0.001). Combined LPC16 and sevoflurane preconditioning was not superior to either substance alone. Continuous sevoflurane or propofol was not protective. The PKC inhibitor calphostin C abolished the cardioprotective effects of LPC16. PKC&egr;, but not PKC&agr;, expression was increased 6 and 28 h after hypoxic LPC. Heme oxygenase-1 and vascular endothelial growth factor were transiently up-regulated after 6 h. Conclusion:Hypoxic LPC at 8%, 12%, and 16% oxygen reduces infarct size in the rat heart in vivo. This effect is as powerful as sevoflurane-preconditioning. PKC&egr; is a key player in mediating hypoxic LPC.

Acute, short-term hypercapnia improves microvascular oxygenation of the colon in an animal model of sepsis.

INTRODUCTIONnThe deterioration of microcirculatory oxygenation of the gut plays a vital role in the development of sepsis. Acute hypercapnia enhances the microcirculatory oxygenation of the splanchnic region under physiological conditions, while the effect of hypercapnia under sepsis is unknown. The aim of this study was to investigate the effects of acute hypercapnia and hypercapnic acidosis on the colonic microcirculation and early cytokine response in polymicrobial sepsis.nnnMETHODSnExperiments were performed on 103 male Wistar rats. Colon ascendens stent peritonitis (CASP) surgery with varying stent diameters was conducted to establish a moderate polymicrobial sepsis model. In a second series, 24h of sepsis development induced by CASP surgery was followed by 120min of volume-controlled and pressure-limited ventilation with either normocapnic (pCO2 45±5mmHg) or moderate hypercapnic ventilation targets (pCO2 75±5mmHg) via exogenous carbon dioxide application. The effect of acidosis was investigated by metabolically buffering the hypercapnic acidosis with tromethamine. Microcirculatory oxygenation of the colon wall (tissue reflectance spectrophotometry) and hemodynamic variables were recorded continuously and arterial blood gas and cytokine (TNF-α, IL-6, IL-10) levels were analyzed intermittently.nnnRESULTSnIn septic animals the microcirculatory oxygenation of the colon deteriorated under normocapnia (-7.0±7.6% at 90min) but was maintained under hypercapnic acidosis (+3.6±7.6%) and buffered hypercapnia (+1.5±4.4%). Cytokine levels were significantly higher in septic animals as opposed to sham animals but did not differ between normocapnic and hypercapnic groups.nnnCONCLUSIONSnAcute hypercapnic acidosis and buffered hypercapnia both improve splanchnic microcirculatory oxygenation in a septic animal model, thereby counteracting the adverse effect induced by sepsis. The circulating pro- and anti-inflammatory cytokine levels are not modulated after 120min of hypercapnia.

Vasopressin V1A receptors mediate the increase in gastric mucosal oxygenation during hypercapnia

Hypercapnia (HC) improves systemic oxygen delivery (DO₂) and microvascular hemoglobin oxygenation of the mucosa (μHbO₂). Simultaneously, HC increases plasma levels of vasopressin. Although vasopressin is generally regarded a potent vasoconstrictor particularly in the splanchnic region, its effects on splanchnic microcirculation during HC is unclear. The aim of this study was to evaluate the role of endogenous vasopressin on gastric mucosal oxygenation and hemodynamic variables during physiological (normocapnia) and hypercapnic conditions. Five dogs were repeatedly anesthetized to study the effect of vasopressin V(1A) receptor blockade ([Pmp¹,Tyr(Me)²]-Arg⁸-Vasopressin, 35u200a μg/kg) on hemodynamic variables and μHbO₂ during normocapnia or HC (end-tidal CO₂ 70u200a mmHg). In a control group, animals were subjected to HC alone. μHbO₂ was measured by reflectance spectrophotometry, systemic DO₂ was calculated from intermittent blood gas analysis, and cardiac output was measured by transpulmonary thermodilution. Data are presented as mean±s.e.m. for n=5 animals. During HC alone, DO₂ increased from 12±1 to 16±1u200aml/kg per min and μHbO₂ from 70±4 to 80±2%. By contrast, additional vasopressin V(1A) receptor blockade abolished the increase in μHbO₂ (80±2 vs. 69±2%) without altering the increase in DO₂ (16±1 vs. 19±2u200a ml/kg per min). Vasopressin V1A receptor blockade (VB) during normocapnia neither affected DO₂ (13±1 vs. 14±1 u200aml/kg per min) nor μHbO₂ (75±3 vs. 71±5%). Vasopressin V(1A) receptor blockade abolished the increase in μHbO₂ during HC independent of DO₂. Thus, in contrast to its generally vasoconstrictive properties, the vasopressin V1A receptors seem to mediate the increase in gastric microcirculatory mucosal oxygenation induced by acute HC.

Glycine transporter GlyT1, but not GlyT2, is expressed in rat dorsal root ganglion--Possible implications for neuropathic pain

Glycinergic inhibitory neurotransmission plays a pivotal role in the development of neuropathic pain. The glycine concentration in the synaptic cleft is controlled by the glycine transporters GlyT1 and GlyT2. GlyT1 is expressed throughout the central nervous system, while GlyT2 is exclusively located in glycinergic neurons. Aim of the present study was to investigate whether GlyTs are also expressed in the peripheral sensory nervous system and whether their expression is modulated in experimental neuropathic pain. Neuropathic pain was induced in male Wistar rats by Chronic Constriction Injury (CCI) and verified by assessment of mechanical allodynia (von Frey method). Expression patterns of GlyTs and the glycine binding subunit NR1 of the N-methyl-d-aspartate (NMDA) receptor in the spinal cord and dorsal root ganglia (DRG) were analyzed by Western blot analysis, PCR and immunohistochemistry. While both GlyT1 and GlyT2 were detected in the spinal cord, only GlyT1, but not GlyT2, was detected in DRG. Immunofluorescence revealed a strictly neuronal localization of GlyT1 and a co-localization of GlyT1 and NR1 in DRG. Compared to sham procedure, spinal cord and DRG expression of GlyT1 was not altered and NR1 was unchanged in DRG 12 days after CCI. GlyT1, but not GlyT2, is expressed in the peripheral sensory nervous system. The co-expression of GlyT1 and NMDA receptors in DRG suggests that GlyT1 regulates glycine concentration at the glycine binding site of the NMDA receptor. Differential regulation of GlyT1 expression in the spinal cord or DRG, however, does not seem to be associated with the development of neuropathic pain.

MicroRNA-1-associated effects of neuron-specific brain-derived neurotrophic factor gene deletion in dorsal root ganglia

BACKGROUNDnMicroRNAs (miRNAs) regulate gene expression in physiological as well as in pathological processes, including chronic pain. Whether deletion of a gene can affect expression of the miRNAs that associate with the deleted gene mRNA remains elusive. We investigated the effects of brain-derived neurotrophic factor (Bdnf) gene deletion on the expression of miR-1 in dorsal root ganglion (DRG) neurons and its pain-associated downstream targets heat shock protein 60 (Hsp60) and connexin 43 (Cx43) in tamoxifen-inducible conditional knockout mice, Bdnf(fl/fl); Advillin-CreER(T2) (Bdnf cKO).nnnRESULTSnEfficient Bdnf gene deletion was confirmed in DRG of Bdnf cKO mice by Real-Time qRT-PCR and ELISA 10days after completed tamoxifen treatment. In DRG, miR-1 expression was reduced 0.44-fold (p<0.05; Real-time qRT-PCR) in Bdnf cKO compared to floxed wildtype littermate control Bdnf(fl/fl) mice (WT). While Hsp60 protein expression was increased 1.85-fold (p<0.05; Western blot analysis), expression levels of Cx43 and the miR-1-associated transcription factors MEF2a and SRF remained unchanged. When analyzing Bdnf cKO mice 32days after complete tamoxifen treatment to investigate whether observed expression alterations remain permanently, we found no significant differences between Bdnf cKO and WT mice. However, miRNA microarray analysis revealed that 167 miRNAs altered (p<0.05) in DRG of these mice following Bdnf gene deletion.nnnCONCLUSIONSnOur results indicate that deletion of Bdnf in DRG neurons leads to a temporary dysregulation of miR-1, suggesting an impairment of a presumable feedback loop between BDNF protein and its targeting miR-1. This appears to affect its downstream protein Hsp60 and as a consequence might influence the phenotype after inducible Bdnf gene deletion. While this appears to be a MEF2a-/SRF-independent and transient effect, expression levels of various other miRNAs may remain permanently altered.