Establishment of an animal model of sciatic nerve injury induced by local anesthetics.

Peripheral neurotoxicity injury caused by local anesthetics is a common complication of clinical anesthesia. The study of its mechanism is helpful to prevent and treat the neurotoxic injury of local anesthetics. Previous studies on peripheral neurotoxicity injury caused by local anesthetics have mainly focused on in vitro cell experiments. Due to the lack of an animal model of peripheral neurotoxicity damage caused by local anesthetics, there are few in vivo experimental studies regarding this topic. Herein, 1% ropivacaine hydrochloride was injected into the sciatic nerve by direct incision and exposure of the sciatic nerve to create a local anesthetic neurotoxic injury model. The results showed that 1% ropivacaine hydrochloride could reduce the lower limb motor score and mechanical paw withdrawal threshold in mice 48 hours after injection. Pathological sections showed that 48 hours after treatment with 1% ropivacaine hydrochloride, the sciatic nerve showed increased axonal edema and degeneration, edema between nerve fiber bundles, increased degeneration of axon and myelin sheath vacuoles, edema of nerve bundle membrane and local degeneration and necrosis, and a large number of inflammatory cells around the nerve adventitia were soaked. The above results show that under open vision, 1% ropivacaine hydrochloride can cause injury to the sciatic nerve after 48 h of treatment, which can simulate the neurotoxic damage of local anesthetics. This animal model provides a research tool for studying the mechanism of neurotoxic injury caused by local anesthetics.

In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Local Anesthetic Cardiac Toxicity Is Mediated by Cardiomyocyte Calcium Dynamics.

BACKGROUND: Long-lasting local anesthetic use for perioperative pain control is limited by possible cardiotoxicity (e.g., arrhythmias and contractile depression), potentially leading to cardiac arrest. Off-target cardiac sodium channel blockade is considered the canonical mechanism behind cardiotoxicity; however, it does not fully explain the observed toxicity variability between anesthetics. The authors hypothesize that more cardiotoxic anesthetics (e.g., bupivacaine) differentially perturb other important cardiomyocyte functions (e.g., calcium dynamics), which may be exploited to mitigate drug toxicity. METHODS: The authors investigated the effects of clinically relevant concentrations of racemic bupivacaine, levobupivacaine, or ropivacaine on human stem cell-derived cardiomyocyte tissue function. Contractility, rhythm, electromechanical coupling, field potential profile, and intracellular calcium dynamics were quantified using multielectrode arrays and optical imaging. Calcium flux differences between bupivacaine and ropivacaine were probed with pharmacologic calcium supplementation or blockade. In vitro findings were correlated in vivo using an anesthetic cardiotoxicity rat model (females; n = 5 per group). RESULTS: Bupivacaine more severely dysregulated calcium dynamics than ropivacaine in vitro (e.g., contraction calcium amplitude to 52 ± 11% and calcium-mediated repolarization duration to 122 ± 7% of ropivacaine effects, model estimate ± standard error). Calcium supplementation improved tissue contractility and restored normal beating rhythm (to 101 ± 6%, and 101 ± 26% of control, respectively) for bupivacaine-treated tissues, but not ropivacaine (e.g., contractility at 80 ± 6% of control). Similarly, calcium pretreatment mitigated anesthetic-induced arrhythmias and cardiac depression in rats, improving animal survival for bupivacaine by 8.3 ± 2.4 min, but exacerbating ropivacaine adverse effects (reduced survival by 13.8 ± 3.4 min and time to first arrhythmia by 12.0 ± 2.9 min). Calcium channel blocker nifedipine coadministration with bupivacaine, but not ropivacaine, exacerbated cardiotoxicity, supporting the role of calcium flux in differentiating toxicity. CONCLUSIONS: Our data illustrate differences in calcium dynamics between anesthetics and how calcium may mitigate bupivacaine cardiotoxicity. Moreover, our findings suggest that bupivacaine cardiotoxicity risk may be higher than for ropivacaine in a calcium deficiency context.

Dexmedetomidine pretreatment alleviates ropivacaine-induced neurotoxicity via the miR-10b-5p/BDNF axis.

BACKGROUND: Ropivacaine is commonly applied for local anesthesia and may cause neurotoxicity. Dexmedetomidine (DEX) exhibits neuroprotective effects on multiple neurological disorders. This study investigated the mechanism of DEX pretreatment in ropivacaine-induced neurotoxicity. METHODS: Mouse hippocampal neuronal cells (HT22) and human neuroblastoma cells (SH-SY5Y) were treated with 0.5 mM, 1 mM, 2.5 mM, and 5 mM ropivacaine. Then the cells were pretreated with different concentrations of DEX (0.01 µM, 0.1 µM, 1 µM, 10 µM, and 100 µM) before ropivacaine treatment. Proliferative activity of cells, lactate dehydrogenase (LDH) release, and apoptosis rate were measured using CCK-8 assay, LDH detection kit, and flow cytometry, respectively. miR-10b-5p and BDNF expressions were determined using RT-qPCR or Western blot. The binding of miR-10b-5p and BDNF was validated using dual-luciferase assay. Functional rescue experiments were conducted to verify the role of miR-10b-5p and BDNF in the protective mechanism of DEX on ropivacaine-induced neurotoxicity. RESULTS: Treatment of HT22 or SH-SY5Y cells with ropivacaine led to the increased miR-10b-5p expression (about 1.7 times), decreased BDNF expression (about 2.2 times), reduced cell viability (about 2.5 times), elevated intracellular LDH level (about 2.0-2.5 times), and enhanced apoptosis rate (about 3.0-4.0 times). DEX pretreatment relieved ropivacaine-induced neurotoxicity, as evidenced by enhanced cell viability (about 1.7-2.0 times), reduced LDH release (about 1.7-1.8 times), and suppressed apoptosis rate (about 1.8-1.9 times). DEX pretreatment repressed miR-10b-5p expression (about 2.5 times). miR-10b-5p targeted BDNF. miR-10b-5p overexpression or BDNF silencing reversed the protective effect of DEX pretreatment on ropivacaine-induced neurotoxicity, manifested as reduced cell viability (about 1.3-1.6 times), increased intracellular LDH level (about 1.4-1.7 times), and elevated apoptosis rate (about 1.4-1.6 times). CONCLUSIONS: DEX pretreatment elevated BDNF expression by reducing miR-10b-5p expression, thereby alleviating ropivacaine-induced neurotoxicity.

Effects of intravenous lipid emulsions on the reversal of pacing-induced ventricular arrhythmias and electrophysiological alterations in an animal model of ropivacaine toxicity.

INTRODUCTION: Ropivacaine is considered to have a wider margin of cardiovascular safety. However, several reports of ventricular arrhythmias (VA) due to ropivacaine toxicity have been documented. Intravenous lipid emulsions (ILEs) have recently been used successfully in the treatment of local anesthetic intoxication. The main objective of the present study was to evaluate the efficacy of the ILEs in the prevention of pacing-induced-VA and electrophysiological alterations in an animal model of ropivacaine toxicity. METHODS: Nineteen pigs were anesthetized and instrumentalized. A baseline programmed electrical ventricular stimulation protocol (PEVSP) to induce VA was performed. Ropivacaine (5 mg·kg-1 + 100 µg·kg-1·min-1) followed by normal saline infusion (control group n = 8) or intralipid 20% (1.5 mL·kg-1 + 0.25 mL·kg-1·min-1) for the ILE group (n = 8), were administered three minutes after the ropivacaine bolus. PEVSP was repeated 25 min after the onset of ropivacaine infusion. Pacing-induced VA and electrophysiological abnormalities were assessed in both groups. A sham-control group (n = 3) without ropivacaine infusion was included. RESULTS: Most of the electrophysiological parameters evaluated were affected by ropivacaine: PR interval by 28% (p = 0.001), AV interval by 40% (p = 0.001), sinus QRS by 101% (p = 0.001), paced QRS at a rate of 150 bpm by 258% (p = 0.001), and at 120 bpm by 241% (p = 0.001). Seven animals (87.5%) in the control group and eight animals (100%) in the ILE group developed sustained-VA (p = 0.30). Successful resuscitation occurred in 100% of animals in the ILE group vs. 57% of animals in the control group, p = 0.038. Pacing-induced-VA terminated at the first defibrillation attempt in 75% of the animals in the ILE group vs. 0% in the control group, p = 0.01. CONCLUSION: Ropivacaine strongly altered the parameters of ventricular conduction, thus facilitating the induction of VA. ILEs did not prevent pacing-induced VA. However, facilitated resuscitation and termination of VA were delivered at the first defibrillation attempt compared to the control group.

Pharmacodynamics, toxicology and toxicokinetics of ropivacaine oil delivery depot.

BACKGROUND: Ropivacaine oil delivery depot (RODD) can be used to treat postoperative incision pain. The aim was to study pharmacodynamics, toxicity and toxicokinetics of RODD. METHODS: The base research of RODD were conducted. Thirty rabbits were randomly divided into saline, solvent, ropivacaine aqueous injection (RAI) 0.9 mg, RODD 0.9 mg and RODD 3 mg groups. The sciatic nerve of rabbits were isolated, dripped with RODD and the effect of nerve block were observed. In toxicity study, the rats were divided into saline, solvent and RODD 75, 150 and 300 mg/kg groups, 30 rats per group. In toxicokinetics, rats were divided into RODD 75, 150 and 300 mg/kg groups, 18 rats per group. The rats were subcutaneously injected drugs. RESULTS: The analgesic duration of RODD 3 mg and RAI 0.9 mg blocking ischiadic nerve lasted about 20 h and 2 h, respectively, and their blocking intensity was similar. The rats in RODD 75 mg/kg did not show any toxicity. Compared with saline group, in RODD 150 mg/kg group neutrophils and mononuclear cells increased, lymphocytes decreased and albumin decreased(P < 0.05), and pathological examination showed some abnormals. In RODD 300 mg/kg group, 10 rats died and showed some abnormalities in central nerve system, hematologic indexes, part of biochemical indexes, and the weights of spleen, liver, and thymus. However, these abnormal was largely recovered on 14 days after the dosing. The results of toxicokinetics of RODD 75 mg/kg group showed that the Cmax was 1.24 ± 0.59 µg/mL and the AUC(0-24 h) was 11.65 ± 1.58 h·µg/mL. CONCLUSIONS: Subcutaneous injection RODD releases ropivacaine slowly, and shows a stable and longer analgesic effect with a large safety range.

Effects of nalbuphine on the cardiotoxicity of ropivacaine in rats.

When combined with nalbuphine, local anesthetics show a longer duration of nerve block without increasing complications. However, no evidence is available concerning the effect of nalbuphine on the cardiotoxicity of local anesthetics. The objective of this work is to investigate whether nalbuphine pretreatment can increase the lethal dose threshold of ropivacaine in rats. Anesthetized Sprague Dawley rats were pretreated with different doses of nalbuphine (0.4, 0.8, 1.5, 3.0, 5.0 mg/kg) or NS (normal saline, negative control) or 30% LE (lipid emulsion, positive control) 2 ml/kg/min for 5 min (n = 6). Then 0.5% ropivacaine was infused at a rate of 2.5 mg/kg/min until asystole occurs. Time of arrhythmia, 50% mean arterial pressure- and 50% heart rate-reduction, and asystole were recorded, and ropivacaine doses were calculated. Nalbuphine (0.4-5.0 mg/kg) did not affect ropivacaine-induced arrhythmia, 50% mean arterial pressure-reduction and 50% heart rate-reduction, and asystole in rats compared with NS pre-treatment. The asystole dose threshold (in milligrams per kilogram) of group LE was higher than that of group NS (NS 28.25(6.32) vs. LE, 41.58(10.65); P = 0.04; 95% confidence interval 0.23 to 26.45), while thresholds of arrhythmia, 50% mean arterial pressure-reduction, and 50% heart rate-reduction were not affected by LE. Nalbuphine doses of 0.4-5.0 mg/kg pretreatment did not increase the threshold of ropivacaine cardiotoxicity compared with NS control; 30% LE increases the lethal dose threshold of ropivacaine in rats.

Lipid emulsions attenuate the inhibition of carnitine acylcarnitine translocase induced by toxic doses of local anesthetics in rat cardiomyoblasts.

The aim of this study was to examine the effects of lipid emulsions on carnitine palmitoyltransferase I (CPT-I), carnitine acylcarnitine translocase (CACT), carnitine palmitoyltransferase II (CPT-II), and the mitochondrial dysfunctions induced by toxic doses of local anesthetics in H9c2 rat cardiomyoblasts. The effects of local anesthetics and lipid emulsions on the activities of CPT-I, CACT, and CPT-II, and concentrations of local anesthetics were examined. The effects of lipid emulsions, N-acetyl-L-cysteine (NAC), and mitotempo on the bupivacaine-induced changes in cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and intracellular calcium levels were examined. CACT, without significantly altering CPT-I and CPT-II, was inhibited by toxic concentration of local anesthetics. The levobupivacaine- and bupivacaine-induced inhibition of CACT was attenuated by all concentrations of lipid emulsion, whereas the ropivacaine-induced inhibition of CACT was attenuated by medium and high concentrations of lipid emulsion. The concentration of levobupivacaine was slightly attenuated by lipid emulsion. The bupivacaine-induced increase of ROS and calcium and the bupivacaine-induced decrease of MMP were attenuated by ROS scavengers NAC and mitotempo, and the lipid emulsion. Collectively, these results suggested that the lipid emulsion attenuated the levobupivacaine-induced inhibition of CACT, probably through the lipid emulsion-mediated sequestration of levobupivacaine.

Ropivacaine Induces Cell Cycle Arrest in the G0/G1 Phase and Apoptosis of PC12 Cells via Inhibiting Mitochondrial STAT3 Translocation.

STAT3 has neuroprotective effect via non-canonical activation and mitochondrial translocation, but its effect on ropivacaine-induced neurotoxicity remains unclear. Our previous study revealed that apoptosis was an important mechanism of ropivacaine-induced neurotoxicity; this study is to illustrate the relationship between STAT3 with ropivacaine-induced apoptosis. Those results showed that ropivacaine treatment decreased cell viability, induced cell cycle arrest in the G0/G1 phase, apoptosis, oxidative stress, and mitochondrial dysfunction in PC12 cells. Moreover, ropivacaine decreased the phosphorylated levels of STAT3 at Ser727 and downregulated the expression of STAT3 upstream gene IL-6. The mitochondrial translocation of STAT3 was also hindered by ropivacaine. To further illustrate the connection of STAT3 protein structure with ropivacaine, the autodock-vina was used to examine the interaction between STAT3 and ropivacaine, and the results showed that ropivacaine could bind to STAT3's proline site and other sites. In addition, the activator and inhibitor of mitoSTAT3 translocation were used to demonstrate it was involved in ropivacaine-induced apoptosis; the results showed that enhancing the mitochondrial STAT3 translocation could prevent ropivacaine-induced apoptosis. Finally, the expression of p-STAT3 and the levels of apoptosis in the spinal cord were also detected; the results were consistent with the cell experiment; ropivacaine decreased the expression of p-STAT3 protein and increased the levels of apoptosis in the spinal cord. We demonstrated that ropivacaine induced apoptosis by inhibiting the phosphorylation of STAT3 at Ser727 and the mitochondrial STAT3 translocation. This effect was reversed by the activation of the mitochondrial STAT3 translocation.

Novel insights on the encapsulation mechanism of PLGA terminal groups on ropivacaine.

Currently, the influences of free terminal groups (hydroxyl, carboxyl and ester) of PLGA on encapsulating active pharmaceutical ingredient are relatively ambiguous even though PLGA types were defined as critical quality attributes in vast majority of design of experiment process. In this study, emulsion method combined with premix membrane emulsification technique has been used to encapsulate ropivacaine (RVC), a small molecule local anesthetic in clinical. Based on the narrow particle size distribution, the influences and mechanisms of the terminal groups on properties of ropivacaine loaded microspheres have been investigated in detail. It was found that microspheres prepared by PLGA with hydroxyl or ester groups exhibited lower encapsulation efficiency but faster in vitro release rate than that of carboxyl groups. In the meanwhile, on microcosmic level analysis by quartz crystal microbalance with dissipation, atomic force microscope and confocal laser scanning microscopy, we attributed this distinction to the specific interaction between ropivacaine and different terminal groups. Subsequently, the reaction activation centers were verified by density functional simulation calculation and frontier molecular orbital theory at molecular level. Additionally, pharmacokinetics and pharmacodynamic research of infiltration anesthesia model were performed to compare sustained release ability, duration and intensity of the anesthetic effect in vivo. Finally, potential safety and toxicity were evaluated by the biochemical analysis. This study not only provides a novel mechanism of drug encapsulation process but also potential flexible selections in terms of various anesthesia indications in clinical.

Differences in Cytotoxicity of Lidocaine, Ropivacaine, and Bupivacaine on the Viability and Metabolic Activity of Human Adipose-Derived Mesenchymal Stem Cells.

PURPOSE: We evaluated biological effects of distinct local anesthetics on human adipose-derived mesenchymal stem cells when applied to reduce periprocedural pain during mesenchymal stem cell injections. METHODS AND MATERIALS: Metabolic activity (MTS assay), viability (Live/Dead stain), and gene expression (quantitative real-time reverse-transcriptase polymerase chain reaction) were measured in mesenchymal stem cells incubated with various concentrations of lidocaine, ropivacaine, or bupivacaine during a 12-hr time course. RESULTS: Cell viability and metabolic activity decreased in a dose, time, and substance-specific manner after exposure to lidocaine, ropivacaine, and bupivacaine, with ropivacaine being the least cytotoxic. Cell viability decreases after brief exposure (<1.5 hrs) at clinically relevant concentrations (eg, 8 mg/ml of lidocaine, 2.5 mg/ml of ropivacaine or bupivacaine). Mesenchymal stem cells exposed to local anesthetics change their expression of mRNA biomarkers for stress response (EGR1, EGR2), proliferation (MKI67, HIST2H4A), ECM (COL1A1, COL3A1), and cell surface marker (CD105). CONCLUSIONS: Local anesthetics are cytotoxic to clinical-grade human mesenchymal stem cells in a dose-, time-, and agent-dependent manner and change expression of ECM, proliferation, and cell surface markers. Lidocaine and bupivacaine are more cytotoxic than ropivacaine. Single-dose injections of local anesthetics may affect the biological properties of mesenchymal stem cells in vitro but may not affect the effective dose of MSCs in a clinical setting.

Brain-Derived Neurotrophic Factor Alleviates Ropivacaine-Induced Neuronal Damage by Enhancing the Akt Signaling Pathway.

BACKGROUND Brain-derived neurotrophic factor (BDNF) is one of the neurotrophic factors that modulate critical metabolic activities, including apoptosis, proliferation, and differentiation modulation. Although numerous studies have focused on the damaging effects of BDNF on neurons, the underlying relationship between these effects remains unclear. In the present study, we investigated the protective effect of BDNF on neuronal injury induced by ropivacaine and assessed whether it is related to the Akt signaling pathway. MATERIAL AND METHODS Human neuroblastoma cell line SH-SY5Y cells were stimulated with ropivacaine at different concentrations to induce neuronal injury. MTT analysis, flow cytometry, immunohistochemistry, qRT-PCR, and Western blot were used to investigate the proliferation activity, apoptotic level, and expression of Akt, PCNA, Bax, Bcl-2, and cleaved caspase-3, collectively demonstrating the underlying regulatory mechanisms. RESULTS Compared with the control group, the morphological damage and proliferation inhibition of SH-SY5Y cells induced by ropivacaine were dose-dependent and time-dependent, accompanied by a significant decrease in Akt expression. We treated cells with BDNF or SC79, which is a selective cell-permeable small molecule Akt activator. The results showed that, compared to the ropivacaine group, the morphological damage of neurons was alleviated; cell proliferation activity was enhanced; apoptotic rate was reduced; PCNA, Bcl-2, and phosphorylated Akt expression levels were increased; and Bax and caspase-3 gene and protein expression were decreased. We were able to reverse these effects by administering API-2, an Akt inhibitor. CONCLUSIONS BDNF can alleviate ropivacaine-induced neuronal injury by activating Akt signaling pathway, consequently modulating the proliferation and apoptosis of neurons.

[P38 MAPK inhibitor SB203580 attenuates the toxicity of ropivacaine on PC12 cells].

OBJECTIVE: To investigate the effect of SB203580, a p38MAPK specific inhibitor, on ropivacaine-induced cytotoxicity in PC12 cells.
 Methods: PC12 cells were divided into three groups: the normal group (Group N), cells were cultured for 48 h; the ropivacaine group (Group R), cells were cultured with 15 mmol/L ropivacaine hydrochloride for 48 h; the ropivacaine+SB203580 group (Group R+S), cells were cultured with 15 mmol/L ropivacaine hydrochloride plus 10 µmol/L SB203580 for 48 h. The cell survival rates were detected by MTT assay. The protein levels of cleaved caspase-3, phosphor-p38 (p-p38) and cystolic cytochrome C (Cyt C) were detected by Western blotting.
 Results: Compared with the Group N, the number and survival rate of PC12 cells in the Group R and the Group R+S were significantly reduced (all P<0.05); the number and survival rate of PC12 cells in the Group R+S were significantly higher than those in the Group R (both P<0.05). Compared with the Group N, the levels of p-p38 and cleaved caspase-3, and the content of cytoplasmic Cyt C in the PC12 cells from the Group R and the Group R+S were significantly enhanced (all P<0.05); compared with the Group R, the levels of p-p38 and cleaved caspase-3, and the content of cytoplasmic Cyt C in the PC12 cells from the Group R+S were decreased (all P<0.05).
 Conclusion: The ropivacaine-induced cytotoxicity can be attenuated via inhibition of p38MAPK; which is related to decrease in Cyt C content and cleaved caspase-3 expression.

Roles of CaMKIIß in the neurotoxicity induced by ropivacaine hydrochloride in dorsal root ganglion.

Neurotoxicity of local anesthetics is often reported in the clinic, more and more people pay attention to them. CaMKIIß, a subtype of CaMKII, is detected in the central nervous system. Previous study found that CaMKIIß mRNA are up-regulated in DRG neurons treated with ropivacaine hydrochloride, as well as inhibition of Cav3.2 and Cav3.3 expression can improve the local anesthetics neurotoxicity. In this study, we observed the effect of CaMKIIß on neurotoxicity injury induced by ropivacaine hydrochloride with DRG cell in vitro. We first constructed the pAd-shRNA-CaMKIIß-DRG to inhibit CaMKIIß mRNA expression and detected the cell viability, cell apoptosis rate, CaMKIIß, Cav3.2 and Cav3.3 expression. The results showed that ropivacaine hydrochloride caused the DRG cell injury with cell viability decreased and cell apoptosis rate increased, CaMKIIß, Cav3.2 and Cav3.3 expression up-regulated. Interestingly, inhibition of CaMKIIß expression protected the DRG cell from the neurotoxicity injury induced by ropivacaine hydrochloride, increased the cell viability and decreased the apoptosis rate, as well as inhibition of CaMKIIß expression down-regulated Cav3.2 and Cav3.3 expression. In other words, CaMKIIß is involved with the DRG injury induced by ropivacaine hydrochloride. Inhibition CaMKIIß expression improved DRG injury, increased the cell viability and decreased cell apoptosis rate.

Comparison of myotoxic effects of levobupivacaine, bupivacaine and ropivacaine: apoptotic activity and acute effect on pro-inflammatory cytokines.

We investigated the myotoxic effects of bupivacaine, ropivacaine and levobupivacaine on rat skeletal muscle and compared its apoptotic activity and acute effects on pro-nflammatory cytokines. We divided 40 Wistar albino rats into four equal groups. Rats were injected intramuscularly with 0.5% bupivacaine (group B), 0.5% ropivacaine (group R), 0.5% levobupivacaine (group L) or 0.9% normal saline (group SF). Animals were sacrificed on the second day after the injection. TNF-α, IL-1 and IL-6 levels were examined in muscle tissue using immunohistochemistry and immunofluorescence. Apoptotic cells were visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. We found that levobupivacaine caused the lowest TNF-α, IL-1 and IL-6 expression levels, while the bupivacaine group caused the highest level compared to the other two agents. The greatest number of apoptotic cells was found in the bupivacaine group. Bupivacaine was more myotoxic than other anesthetic agents and increased apoptosis. The number of TUNEL positive apoptotic cells was lowest in the SF group. The greatest IL-1 immunoreactivity was found in the bupivacaine group. Bupivacaine and ropivacaine produced greater IL-6 expression than the SF group. Bupivacaine and ropivacaine caused greater TNF-α expression than the SF group, whereas the immunoreactivity of TNF-α was similar in the bupivacaine and ropivacaine groups.

Local Anesthetics' Toxicity toward Human Cultured Chondrocytes: A Comparative Study between Lidocaine, Bupivacaine, and Ropivacaine.

OBJECTIVE: In orthopedic joint injection, the most frequently used local anesthetics are ropivacaine, bupivacaine, and 1% or 2% lidocaine. The aim of this study was to examine effects of these various anesthetics on the viability of human chondrocytes. Our hypothesis was that all local anesthetics tested damage human chondrocytes in vitro. METHODS: Primary human chondrocytes were isolated and cultured from 6 donated human knee joints (mean age of donors 61.2 years). Local anesthetics were added to these cultures. Toxicity analysis was performed by visualization of cell structure using light microscopy. Determination of vital chondrocytes was performed by use of a Casy cell counter. Chondrocytes' cell death was examined by fluorescence microscopy and an XTT ELISA assay. RESULTS: Light microscope and fluorescence microscope data revealed a defect cell structure and increased number of dead cells after addition of 1% or 2% lidocaine and bupivacaine but not ropivacaine. We were able to show an increased level of XTT activity after treatment with bupivacaine, 2% lidocaine or ropivacaine. The count of vital chondrocytes was significantly decreased after treatment with bupivacaine, 1% or 2% lidocaine, and ropivacaine. CONCLUSIONS: The data show that treatment with local anesthetics induces cell damage of human chondrocytes in vitro. Ropivacaine seems to be a local anesthetic with the lowest toxic potential on human chondrocytes, a feature that may favor its preference for use in joint injection.

Dexmedetomidine enhances ropivacaine-induced sciatic nerve injury in diabetic rats.

BACKGROUND: Previous studies suggest that dexmedetomidine has a protective effect against local anaesthetic-induced nerve injury in regional nerve blocks. Whether this potentially protective effect exists in the context of diabetes mellitus is unknown. METHODS: A diabetic state was established in adult male Sprague-Dawley rats with intraperitoneal injection of streptozotocin. Injections of ropivacaine 0.5%, dexmedetomidine 20 µg kg-1 (alone and in combination), or normal saline (all in 0.2 ml) were made around the sciatic nerve in control and diabetic rats (n=8 per group). The duration of sensory and motor nerve block and the motor nerve conduction velocity (MNCV) were determined. Sciatic nerves were harvested at post-injection day 7 and assessed with light and electron microscopy or used for pro-inflammatory cytokine measurements. RESULTS: Ropivacaine and dexmedetomidine alone or in combination did not produce nerve fibre damage in control non-diabetic rats. In diabetic rats, ropivacaine induced significant nerve fibre damage, which was enhanced by dexmedetomidine. This manifested with slowed MNCV, decreased axon density, and decreased ratio of inner to outer diameter of the myelin sheath (G ratio). Demyelination, axon disappearance, and empty vacuoles were also found using electron microscopy. An associated increase in nerve interleukin-1ß and tumour necrosis factor-α was also seen. CONCLUSIONS: Ropivacaine 0.5% causes significant sciatic nerve injury in diabetic rats that is greatly potentiated by high-dose dexmedetomidine. Although the dose of dexmedetomidine used in this study is considerably higher than that used in clinical practice, our data suggest that further studies to assess ropivacaine (alone and in combination with dexmedetomidine) use for peripheral nerve blockade in diabetic patients are warranted.

CaMK II γ down regulation protects dorsal root ganglion neurons from ropivacaine hydrochloride neurotoxicity.

T-type calcium channels are intimately involved in the local anesthetics neurotoxicity. Does CaMKIIγ regulate T-type calcium currents in local anesthetics neurotoxicity? This study generated pAd-CaMKIIγ and pAd-shRNA adenovirus vectors to up- and down-regulate CaMKIIγ mRNA expression in dorsal root ganglion neurons (DRG). Normal DRG (Normal group), empty vector DRG (Empty vector group), pAd-CaMKIIγ DRG (pAd-CaMKIIγ group) and pAd-shRNA DRG (pAd-shRNA group) were treated or untreated with 3 mM ropivacaine hydrochloride for 4 h. Cell viability, apoptosis rate, CaMKIIγ, pCaMKIIγ, Cav3.2, and Cav3.3 expression were detected. Ultrastructural changes in DRG were observed under a transmission electron microscope. The results demonstrated that the cell viability of DRG treated with ropivacaine hydrochloride decreased markedly, the apoptosis rate, CaMKIIγ, pCaMKIIγ, Cav3.2, Cav3.3 expression increased significantly. CaMKIIγ up-regulation aggravated ropivacaine hydrochloride-induced cell damage and increased Cav3.2 and Cav3.3 expression. In conclusion, CaMKIIγ regulated Cav3.2 and Cav3.3 expression in DRG, which was involved with ropivacaine hydrochloride-induced cell injury.