In Vivo Effects of Neostigmine and Physostigmine on Neutrophil Functions and Evaluation of Acetylcholinesterase and Butyrylcholinesterase as Inflammatory Markers during Experimental Sepsis in Rats.

INTRODUCTION: Recent studies have shown that acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may serve as important diagnostic and therapeutic targets in sepsis. Since polymorphonuclear neutrophils (PMNs) play a pivotal role in the early phase of sepsis, we evaluated the potential therapeutic effects of cholinesterase inhibitors on PMN functions during cecal ligation and puncture- (CLP-) induced sepsis and investigated the roles of AChE and BChE as inflammatory markers under standardized experimental conditions. METHODS: Sham surgery or CLP was performed in male Wistar rats (n = 60). Animals were randomized into four groups: physostigmine, 100 µg/kg; neostigmine, 75 µg/kg; 0.9% saline (control group); and sham group, each applied four times over 24 h. The levels of reactive oxygen species (ROS) production and CD11b/CD62l expression were quantified by flow cytometry at t = 0, 6, 15, 20, and 24 h. Blood gas analysis as well as AChE and BChE activity levels was measured by validated point-of-care measurements. Clinical scores and survival times were determined. RESULTS: CLP induced a significant increase in ROS production and CD11b upregulation by rat PMNs. Treatment with physostigmine or neostigmine significantly reduced ROS production and CD11b upregulation by PMNs 20 h after CLP induction. In physostigmine-treated animals, survival times were significantly improved compared to the control animals, but not in neostigmine-treated animals. While AChE activity significantly decreased in the control animals at t > 6 h, AChE activity did not change in the sham group. BChE activity decreased at t > 20 h in the control animals. CONCLUSION: While AChE activity may serve as an acute inflammatory marker, BChE activity shows a delayed decrease. Administration of centrally acting physostigmine in CLP-induced sepsis in rats has protective effects on PMN functions and improves survival times, which may be of interest in clinical practice.

Flow-cytometric measurement of respiratory burst in rat polymorphonuclear granulocytes: Comparison of four cell preparation procedures, and concentration-response evaluation of soluble stimulants.

Polymorphonuclear neutrophils (PMNs) contribute to organ injury in sepsis, stroke, and other diseases. Evaluation of the oxidative burst by flow cytometry (FCM) is frequently applied to examine PMN status in humans, but rarely in rats. We established a method to assess granulocyte activation in rats by means of FCM analysis of oxidative burst. Two methods for PMN isolation involving Histopaque separation were investigated, and additionally two whole blood techniques. In addition, the concentration-response relation of the stimulants fMLP, PMA, TNF-alpha, and LPS has been determined, both as sole stimulants and for priming. A novel technique with diluted rat whole blood proved to be most appropriate for PMN preparation. One micromolar PMA and fMLP, respectively, are effective concentrations for PMN stimulation in rat whole blood. Priming with 0.1 mug/ml TNF-alpha and 1 mug/ml LPS, respectively, resulted in optimal additional stimulation. This study defined the appropriate conditions for evaluating the reactive oxygen derivate production in rat PMNs by flow cytometry. The rapid, simple, and reliable cell preparation procedure of whole blood dilution that preserves cell integrity and requires only small sample quantities. This is the first systematic dose-response evaluation of soluble stimulants of neutrophil respiratory burst in rats.

Effects of Lidocaine on HT-29 and SW480 Colon Cancer Cells In Vitro.

BACKGROUND: Evidence is growing that the risk of cancer dissemination may be enhanced during the perioperative period. Whether particular anesthetic techniques influence oncological outcome is still under discussion. For pain management, lidocaine can be administered perioperatively by intravenous, intraperitoneal or epidural infusion. Here we investigated the effect of lidocaine on colon carcinoma cell lines (HT-29 and SW480) in vitro. MATERIALS AND METHODS: ELISA BrdU (Roche) for cell proliferation and FITC Annexin V detection kit (BD Pharming) for apoptosis analysis were applied. Cell-cycle profiles were investigated by flow cytometry. RESULTS: Cell-cycle arrest was induced in both cell lines by 1000 µM lidocaine, while no inhibition of cell proliferation was detected. Apoptosis decreased in SW480 but not in HT-29 cells. CONCLUSION: Lidocaine induces cell-cycle arrest in both colon carcinoma cell lines in vitro. The effective drug concentration can be obtained by local infiltration.

Species- and concentration-dependent differences of acetyl- and butyrylcholinesterase sensitivity to physostigmine and neostigmine.

Previous and more recent studies show that cholinesterase inhibitors (ChE-Is) are an important possibility for therapeutic intervention in Alzheimer's Disease, sepsis and other inflammatory syndromes. ChE-Is maintain high levels of acetylcholine (ACh) determining beneficial effects on the disease process. Despite numerous efforts to identify the appropriate choice of agents and dose of ChE-Is, a common protocol regarding concentration- and species-dependent differences in inhibitory potency (IC 50) of clinical relevant ChE-Is is still not available. To evaluate the in vitro sensitivity of Acetyl- and Butyrylcholinesterase (AChE, BChE), we compared the concentration-response effects of physostigmine and neostigmine on cholinesterases in whole blood from rat and human. A spectrophotometrical test system based on in vitro Ellman's reagent has been used to determine the kinetic properties of clinical relevant ChE-Is. In vitro, the enzyme activity of human AChE and BChE was inhibited in a concentration-dependent manner until a residual activity of 4-6% for AChE and 20-30% for BChE (IC 50 human AChE: 0.117 ± 0.007 µM physostigmine, 0.062 ± 0.003 µM neostigmine; IC 50 human BChE: 0.373 ± 0.089 µM neostigmine; 0.059 ± 0.012 µM physostigmine). The inhibition curve of rat BChE in contrast showed no concentration-dependency for physostigmine and neostigmine (87% residual activity even at high inhibitor concentrations). Rat AChE was inhibited in a concentration-dependent manner until a residual activity of 53%. The results suggest that cholinesterases from human and rat show marked species- and inhibitor-dependent differences in sensitivity to physostigmine and neostigmine. Knowledge of such differences may be critical in assessing the possible therapeutic effects of ChE-Is in both species and may guide researchers in the optimal design of future experiments regarding the application of ChE-Is.

Modulation of immune functions in polymorphonuclear neutrophils induced by physostigmine, but not neostigmine, independent of cholinergic neurons.

BACKGROUND: Cholinesterase inhibitors (Ch-I) improve survival in experimental sepsis consistent with activation of the cholinergic-anti-inflammatory-pathway. So far, less is known about whether Ch-I have a direct immunomodulatory effect on immune cells (polymorphonuclear neutrophils, PMN) in the absence of cholinergic neurons. We investigated the concentration-response-effects of physostigmine and neostigmine on the oxidative burst activity (human and rat PMN) and the expression of adhesion molecules on the surface of human PMN under in vitro conditions. METHODS: PMN from 10 healthy humans or 10 rats were pretreated with 2, 10, 24, 97 µM physostigmine or 3, 15, 30, 150 µM neostigmine, primed with tumor-necrosis-factor-alpha (TNF-alpha) followed by stimulation with n-formyl-methionyl-leucylphenylalanine (fMLP) or stimulated with phorbol-12-myristate-13-acetate (PMA). Human and rat samples were assessed by flow cytometry for the generation of oxidative free radicals. Stimulated human PMN were additionally incubated with antibodies against Mac-1 (CD11b) or L-selectin (CD62l). RESULTS: Physostigmine and neostigmine did not alter oxidative burst activity or the expression of adhesion molecules of PMN induced by receptor-dependent activators like fMLP or TNF-alpha/fMLP (rat and human PMN, p=n.s.). Physostigmine, but not neostigmine, inhibited the protein-kinase-C-mediated oxidative burst activity by PMA in a dose-dependent manner (rat and human PMN, p<0.05). Physostigmine, in the concentration range tested, suppressed the expression of CD11b following stimulation with PMA not significantly (human PMN: control: 63.1±10.7 vs. 97 µM physostigmine: 49.9±12.8 MESF, p=n.s.). CONCLUSION: While neostigmine has no effect on functional and phenotypic changes of PMN, the lipid soluble Ch-I physostigmine causes a dose dependent reduction in PMA-induced oxidative burst, independent of neuronal released acetylcholine.

Bosentan reduces oxidative burst in acid aspiration-induced lung injury in rats.

BACKGROUND: Acid aspiration induces lung injury by causing an intense inflammatory reaction. Neutrophils are attracted by various cytokines, such as TNFbeta, and release reactive oxygen species, which then cause acute lung injury. Endothelin antagonists, such as bosentan, have been found to possess anti-inflammatory properties. MATERIALS AND METHODS: We performed a prospective, randomised, controlled study to evaluate the effects of bosentan in a rat model of acid-induced lung injury. Sprague-Dawley rats underwent sevoflurane anaesthesia; lung injury was then induced by instillation of 1.2mL/kg, 0.1M hydrochloric acid. The lungs were ventilated for 6h and then randomised into three groups: bosentan 30mg/kg body weight, 90mg/kg body weight or sodium chloride, each applied immediately after acid aspiration via a gastric tube. RESULTS: After induction of acute lung inflammation, the production of reactive oxygen species by PMN following stimulation with FMLP increased significantly. Comparison of pre-treatment and post-treatment in the 90mg/kg bosentan treatment group did not show a significant increase of reactive oxygen species following stimulation with FMLP. A comparison of the absolute difference of the MESF demonstrated a significant difference between the control group and the group treated with 90mg/kg bosentan. CONCLUSIONS: Bosentan administration at 90mg/kg body weight reduced the release of reactive oxygen species after 360min in acid aspiration-induced lung injury in rats.