Comparison of Two Experimental Mouse Dry Eye Models through Inflammatory Gene Set Enrichment Analysis Based on a Multiplexed Transcriptomic Approach.

The goal of this study was to explore the specific signaling pathways related to inflammation in two experimental mouse dry eye (EDE) models. Female C57BL/6 mice housed for 10 days in a controlled desiccative environment were either treated with scopolamine (EDE-1; n = 18) or subjected to extraorbital lacrimal gland excision bilaterally (EDE-2; n = 10). Non-induced mice (n = 20) served as healthy controls. A corneal fluorescein staining (CFS) scoring was used at baseline through to day (D) 10 to evaluate epitheliopathy. At D10, corneas and conjunctivas were collected for multiplexed transcriptomic analysis with the NanoString® mouse inflammatory CodeSet. Both EDE-1 and EDE-2 mice presented a change in corneal integrity, with a significant increase in CFS scores at D10. More gene transcripts were identified in EDE-2 compared with EDE-1 (116 vs. 96, respectively), and only a few were common to both models, 13 for the cornea and 6 for the conjunctiva. The gene functional annotation analysis revealed that the same inflammatory pathways were involved in both models. Comparative profiling of gene expression in the two EDE models leads to the identification of various targets and signaling pathways, which can be extrapolated to and confirmed in human disease.

Comparison of N-methyl-2-pyrrolidone (NMP) and the "date rape" drug GHB: behavioral toxicology in the mouse model.

N-methyl-2-pyrrolidone (NMP) and γ-hydroxybutyrate acid (GHB) are synthetic solvents detected in the recreational drug market. GHB has sedative/hypnotic properties and is used for criminal purposes to compromise reaction ability and commit drug-facilitated sexual assaults and other crimes. NMP is a strong solubilizing solvent that has been used alone or mixed with GHB in case of abuse and robberies. The aim of this experimental study is to compare the acute pharmaco-toxicological effects of NMP and GHB on neurological signs (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, and motor activity (bar, drag, and accelerod test) in CD-1 male mice. Moreover, since cardiorespiratory depression is one of the main adverse effects related to GHB intake, we investigated the effect of NMP and GHB on cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in mice. The present study demonstrates that NMP inhibited sensorimotor and motor responses and induced cardiorespiratory depression, with a lower potency and efficacy compared to GHB. These results suggest that NMP can hardly be used alone as a substance to perpetrate sexual assault or robberies.

Lacidipine Prevents Scopolamine-Induced Memory Impairment by Reducing Brain Oxido-nitrosative Stress in Mice.

Cholinergic deficits and oxido-nitrosative stress are consistently associated with Alzheimer's disease (AD). Previous findings indicate that acetylcholine subdues Ca2+ current in the brain. Cholinergic antagonists (e.g., scopolamine) can instigate Ca2+-induced redox imbalance, inflammation, and cell-death pathways leading to AD-type memory impairment. Earlier, several Ca2+-channel blockers (CCB, e.g., dihydropyridine type) or cholinergic enhancers showed promising results in animal models of AD. In the present research, pretreatment effects of lacidipine (L-type CCB) on learning and memory functions were investigated using the scopolamine mouse model of AD. Swiss albino mice (20-25 g) were administered lacidipine (1 and 3 mg/kg) for 14 days. Scopolamine, an anti-muscarinic drug, was given (1 mg/kg) from days 8 to 14. The mice were subjected to elevated plus maze (EPM) and passive-avoidance (PA) paradigms. Bay-K8644 (a Ca2+-channel agonist) was administered before behavioral studies on days 13 and 14. Biochemical parameters of oxidative stress and acetylcholinesterase (AChE) activity were quantified using the whole brain. Behavioral studies showed an increase in transfer latency (TL) in the EPM test and a decrease in step-through latency (STL) in the PA test in scopolamine-administered mice. Scopolamine enhanced the AChE activity and oxidative stress in the brain of mice which resulted in memory impairment. Lacidipine prevented the amnesia against scopolamine and reduced the oxidative stress and AChE activity in the brain of mice. Bay-K8644 attenuated the lacidipine-induced improvement in memory and redox balance in scopolamine-administered mice. Lacidipine can prevent the oxidative stress and improve the cholinergic function in the brain. These properties of lacidipine can mitigate the pathogenesis of AD-type dementia.

Protective effects of vitamin D on learning and memory deficit induced by scopolamine in male rats: the roles of brain-derived neurotrophic factor and oxidative stress.

The beneficial effects of vitamin D (vit D) on central nervous system disorders have been suggested. In the current research, the protective effects of vit D on learning and memory deficit induced by scopolamine, oxidative stress criteria, brain-derived neurotrophic factor (BDNF), and nitric oxide (NO) in the brain were investigated. Rats were divided into five groups, including (1) Control, (2) Scopolamine (2 mg/kg), (3-5) Scopolamine + Vit D (100, 1000, and 10,000 IU/kg) groups. Vit D administrated for 2 weeks and in the third week scopolamine co-administrated with vit D and behavioral tests, including Morris water maze (MWM) and passive avoidance (PA) tests, were carried out. The cortical and hippocampal tissues were analyzed for BDNF, catalase (CAT), and superoxide dismutase (SOD) activities, thiol content, NO metabolites, and malondialdehyde (MDA) concentration. Scopolamine injection significantly impaired rats' performance on the MWM and PA test. It further enhanced the MDA and nitrite level while decreased thiol content and BDNF levels and SOD and CAT activities in the brain. Administration of both 1000 and 10,000 IU/kg vit D improved cognitive outcome in MWM and PA tests. In addition, vit D elevated thiol content, SOD and CAT activities, and BDNF levels, while reduced nitrite and MDA concentration. Vit D also increased the levels of vit D and calcium in the serum. The results demonstrated that vit D has protective effects on scopolamine-associated learning and memory impairment by improving BDNF levels and attenuating NO and brain tissue oxidative damage.

Discovery of 4-arylquinoline-2-carboxamides, highly potent and selective class of mGluR2 negative allosteric modulators: From HTS to activity in animal models.

Antagonism of the mGluR2 receptor has the potential to provide therapeutic benefit to cognitive disorders by elevating synaptic glutamate, the primary excitatory neurotransmitter in the brain. Selective antagonism of the mGluR2 receptor, however, has so far been elusive, given the very high homology of this receptor with mGluR3, particularly at the orthosteric binding site. Given that inhibition of mGluR3 has been implicated in undesired effects, we sought to identify selective mGluR2 negative allosteric modulators. Herein we describe the discovery of the highly potent and selective class of mGluR2 negative allosteric modulators, 4-arylquinoline-2-carboxamides, following a successful HTS campaign and medicinal chemistry optimization, showing potent in vivo efficacy in rodent.

Search for multifunctional agents against Alzheimer's disease among non-imidazole histamine H3 receptor ligands. In vitro and in vivo pharmacological evaluation and computational studies of piperazine derivatives.

In the search for new treatments for complex disorders such as Alzheimer's disease the Multi-Target-Directed Ligands represent a very promising approach. The aim of the present study was to identify multifunctional compounds among several series of non-imidazole histamine H3 receptor ligands, derivatives of 1-[2-thiazol-5-yl-(2-aminoethyl)]-4-n-propylpiperazine, 1-[2-thiazol-4-yl-(2-aminoethyl)]-4-n-propylpiperazine and 1-phenoxyalkyl-4-(amino)alkylopiperazine using in vitro and in vivo pharmacological evaluation and computational studies. Performed in vitro assays showed moderate potency of tested compounds against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Molecular modeling studies have revealed possible interactions between the active compounds and both AChE and BuChE as well as the human H3 histamine receptor. Computational studies showed the high drug-likeness of selected compounds with very good physicochemical profiles. The parallel artificial membrane permeation assay proved outstanding blood-brain barrier penetration in test conditions. The most promising compound, A12, chemically methyl(4-phenylbutyl){2-[2-(4-propylpiperazin-1-yl)-1,3-thiazol-5-yl]ethyl}amine, possesses good balanced multifunctional profile with potency toward studied targets - H3 antagonist activity (pA2 = 8.27), inhibitory activity against both AChE (IC50 = 13.96 µM), and BuChE (IC50 = 14.62 µM). The in vivo pharmacological studies revealed the anti-amnestic properties of compound A12 in the passive avoidance test on mice.

Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer's disease.

Cholinergic hypothesis of Alzheimer's disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ±â€¯0.066 µM; BuChE = 1.166 ±â€¯0.088 µM; hAChE = 1.758 ±â€¯0.095 µM; Pe = 9.491 ±â€¯0.34 × 10-6 cm s1) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ±â€¯0.166 µM; BuChE = 0.754 ±â€¯0.121 µM; hAChE = 0.472 ±â€¯0.042 µM; Pe = 13.92 ±â€¯0.022 × 10-6 cm s1). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aß1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.

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.

Profiling of hepatic metabolizing enzymes and nuclear receptors in rats with adjuvant arthritis by targeted proteomics.

Inflammatory conditions alter the expression and activity of factors influencing pharmacokinetics, such as metabolizing enzymes. The study examined alterations of hepatic protein levels of cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and nuclear receptors in rats with adjuvant-induced arthritis (AA rats), an inflammatory animal model, by liquid chromatography-tandem mass spectrometry-based targeted proteomics. The protein levels of CYP1A1, CYP1A2, CYP2A1, CYP2A3, CYP2C6, CYP2C12, CYP2D3, CYP2E1, CYP3A9, UGT1A1 and UGT1A2/3 in liver microsomes of AA rats were significantly lower than those in control rats. The protein levels of constitutive androstane receptor (CAR) and retinoid X receptor α (RXRα) in the cytoplasm and nucleus were also significantly decreased, to approximately 60% of the control levels. The decreased protein levels of CYP1A2, CYP2C6, CYP2D3, CYP2E1 and UGT1A1 were potentially associated with downregulation of CAR or RXRα expression in the nucleus.

Transcriptional co-repressor SIN3A silencing rescues decline in memory consolidation during scopolamine-induced amnesia.

Epigenetic modifications through methylation of DNA and acetylation of histones modulate neuronal gene expression and regulate long-term memory. Earlier we demonstrated that scopolamine-induced decrease in memory consolidation is correlated with enhanced expression of hippocampal DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) in mice. DNMT1 and HDAC2 act together by recruiting a co-repressor complex and deacetylating the chromatin. The catalytic activity of HDACs is mainly dependent on its incorporation into multiprotein co-repressor complexes, among which SIN3A-HDAC2 co-repressor is widely studied to regulate synaptic plasticity. However, the involvement of co-repressor complex in regulating memory loss or amnesia is unexplored. This study examines the role of co-repressor SIN3A in scopolamine-induced amnesia through epigenetic changes in the hippocampus. Scopolamine treatment remarkably enhanced hippocampal SIN3A expression in mice. To prevent such increase in SIN3A expression, we used hippocampal infusion of SIN3A-siRNA and assessed the effect of SIN3A silencing on scopolamine-induced amnesia. Silencing of SIN3A in amnesic mice reduced the binding of HDAC2 at neuronal immediate early genes (IEGs) promoter, but did not change the expression of HDAC2. Furthermore, it increased acetylation of H3K9 and H3K14 at neuronal IEGs (Arc, Egr1, Homer1 and Narp) promoter, prevented scopolamine-induced down-regulation of IEGs and improved consolidation of memory during novel object recognition task. These findings together suggest that SIN3A has a critical role in regulation of synaptic plasticity and might act as a potential therapeutic target to rescue memory decline during amnesia and other neuropsychiatric pathologies.

Prolonged Duration Local Anesthesia Using Liposomal Bupivacaine Combined With Liposomal Dexamethasone and Dexmedetomidine.

BACKGROUND: The relatively short duration of effect of local anesthetics has been addressed by encapsulation in drug delivery systems. Codelivery with a single compound that produces an adjuvant effect on nerve block but without intrinsic local anesthetic properties can further prolong the nerve block effect. Here, we investigated whether codelivery of more than 1 encapsulated adjuvant compound can further enhance nerve blockade. METHODS: Liposomes loaded with bupivacaine (Bup), dexamethasone phosphate (DexP), or dexmedetomidine (DMED) were synthesized and its in vitro drug release profiles were determined. Animals (Sprague-Dawley rats) were injected with liposomal Bup (Lipo-Bup) and adjuvants at the sciatic nerve and underwent a modified hot plate test to assess the degree of nerve block. The duration of block was monitored and the tissue reaction was assessed. RESULTS: Coinjection of Lipo-Bup with liposomal DexP (Lipo-DexP) and liposomal DMED (Lipo-DMED) prolonged the duration of sciatic nerve block 2.9-fold compared to Lipo-Bup alone (95% confidence interval, 1.9- to 3.9-fold). The duration of the block using this combination was significantly increased to 16.2 ± 3.5 hours compared to Lipo-Bup with a single liposomal adjuvant (8.7 ± 2.4 hours with Lipo-DMED, P = .006 and 9.9 ± 5.9 hours with Lipo-DexP, P = .008). The coinjection of Lipo-Bup with liposomal adjuvants decreased tissue inflammation (P = .014) but did not have a significant effect on myotoxicity when compared to Lipo-Bup alone. Coinjection of Lipo-Bup with unencapsulated adjuvants prolonged the duration of nerve block as well (25.0 ± 6.3 hours; P < .001) however was accompanied by systemic side effects. CONCLUSIONS: Codelivery of Lipo-DexP and Lipo-DMED enhanced the efficacy of Lipo-Bup. This benefit was also seen with codelivery of both adjuvant molecules in the unencapsulated state, but with marked systemic toxicity.

Early Exposure to General Anesthesia Disrupts Spatial Organization of Presynaptic Vesicles in Nerve Terminals of the Developing Rat Subiculum.

Exposure to general anesthesia (GA) during critical stages of brain development induces widespread neuronal apoptosis and causes long-lasting behavioral deficits in numerous animal species. Although several studies have focused on the morphological fate of neurons dying acutely by GA-induced developmental neuroapoptosis, the effects of an early exposure to GA on the surviving synapses remain unclear. The aim of this study is to study whether exposure to GA disrupts the fine regulation of the dynamic spatial organization and trafficking of synaptic vesicles in presynaptic terminals. We exposed postnatal day 7 (PND7) rat pups to a clinically relevant anesthetic combination of midazolam, nitrous oxide, and isoflurane and performed a detailed ultrastructural analysis of the synaptic vesicle architecture at presynaptic terminals in the subiculum of rats at PND 12. In addition to a significant decrease in the density of presynaptic vesicles, we observed a reduction of docked vesicles, as well as a reduction of vesicles located within 100 nm from the active zone, in animals 5 days after an initial exposure to GA. We also found that the synaptic vesicles of animals exposed to GA are located more distally with respect to the plasma membrane than those of sham control animals and that the distance between presynaptic vesicles is increased in GA-exposed animals compared to sham controls. We report that exposure of immature rats to GA during critical stages of brain development causes significant disruption of the strategic topography of presynaptic vesicles within the nerve terminals of the subiculum.

Beta and gamma carboline derivatives as potential anti-Alzheimer agents: A comparison.

Nine novel ß- and γ-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M1 receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-γ-carboline (6) was the most potent (0.25 µmol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs.

Lycium barbarum polysaccharides prevent memory and neurogenesis impairments in scopolamine-treated rats.

Lycium barbarum is used both as a food additive and as a medicinal herb in many countries, and L. barbarum polysaccharides (LBPs), a major cell component, are reported to have a wide range of beneficial effects including neuroprotection, anti-aging and anticancer properties, and immune modulation. The effects of LBPs on neuronal function, neurogenesis, and drug-induced learning and memory deficits have not been assessed. We report the therapeutic effects of LBPs on learning and memory and neurogenesis in scopolamine (SCO)-treated rats. LBPs were administered via gastric perfusion for 2 weeks before the onset of subcutaneous SCO treatment for a further 4 weeks. As expected, SCO impaired performance in novel object and object location recognition tasks, and Morris water maze. However, dual SCO- and LBP-treated rats spent significantly more time exploring the novel object or location in the recognition tasks and had significant shorter escape latency in the water maze. SCO administration led to a decrease in Ki67- or DCX-immunoreactive cells in the dentate gyrus and damage of dendritic development of the new neurons; LBP prevented these SCO-induced reductions in cell proliferation and neuroblast differentiation. LBP also protected SCO-induced loss of neuronal processes in DCX-immunoreactive neurons. Biochemical investigation indicated that LBP decreased the SCO-induced oxidative stress in hippocampus and reversed the ratio Bax/Bcl-2 that exhibited increase after SCO treatment. However, decrease of BDNF and increase of AChE induced by SCO showed no response to LBP administration. These results suggest that LBPs can prevent SCO-induced cognitive and memory deficits and reductions in cell proliferation and neuroblast differentiation. Suppression of oxidative stress and apoptosis may be involved in the above effects of LBPs that may be a promising candidate to restore memory functions and neurogenesis.

The effect of chronic toxicity of pethidine on the spinal cord: an experimental model in rabbits.

The aim of this study was to evaluate the toxicity of chronic spinal analgesia with pethidine in a rabbit model. We introduced epidural catheters in twenty New Zealand white rabbits, divided into two groups, and we administered 0.5 mg/kg pethidine or the same volume of normal saline through the catheters, for three consecutive days. Throughout the experiment, the animals were evaluated in terms of neurological status using the Tarlov score. After the rabbit's euthanasia, 4 µm sections of spinal cord stained with Hematoxylin-Eosin were analyzed by a pathologist blinded to the study for neurohistopathological changes. The results were statistically analyzed with Prism 5 software for Windows. No significant differences were noticed between the two groups in as far as body temperature (p=0.295) and weight (p=0.139) were concerned. In the group of animals, which received epidural pethidine, nine rabbits showed histological changes suggestive for neurotoxicity at the lumbar level of the spinal cord. These findings were significantly different compared with the control group which received only saline (no microscopic lesions revealed; p=0.0006). When combining the data from both groups or using the pethidine group alone, there was a significant correlation between the presence of neurological injury (Tarlov score) and the presence of the histopathological lesions in the spinal cord (r=-0.709, p=0.0002 and r=-0.635, p=0.013, respectively). Based on our findings, the chronic epidural administration of pethidine in rabbits induces moderate to severe histological changes on the spinal cord, but further investigations are needed to make a definitive statement about the histological effect of pethidine on the neurological tissue.

The effects of gabapentin on acute opioid tolerance to remifentanil under sevoflurane anesthesia in rats.

BACKGROUND: Tolerance to remifentanil during sevoflurane anesthesia may blunt the ability of this drug to reduce anesthetic requirements. Gabapentin has been shown to be effective in reducing postoperative narcotic usage, a reduction that may be associated with a reduction in opioid-induced tolerance and hyperalgesia. We sought to determine whether gabapentin might prevent the observed acute opioid tolerance (AOT) produced by remifentanil in sevoflurane minimum alveolar concentration (MAC). METHODS: Wistar rats were anesthetized with sevoflurane and the effects of gabapentin alone on sevoflurane MAC were determined at doses of 150 and 300 mg · kg(-1). In a second experiment, gabapentin 300 mg · kg(-1) was administered before remifentanil (120 and 240 µg · kg(-1) · h(-1)). The MAC was determined before gabapentin administration and 3 more times at 1.5-hour intervals after drug administration to assess AOT. MAC was determined from intratracheal gas samples using a sidestream gas analyzer; tail clamping was used as a supramaximal stimulus. Statistical analysis was performed with the 1-way analysis of variance test. RESULTS: Remifentanil reduced MAC (2.5 ± 0.2%) by 16% ± 5% and 36% ± 6% (120 and 240 µg · kg(-1) · h(-1), respectively, P < 0.01) with a further reduction produced by coadministration with gabapentin 300 mg · kg(-1) to 39% ± 12% and 62% ± 14%, respectively (P < 0.01 versus remifentanil alone). Gabapentin given alone at 150 and 300 mg · kg(-1) reduced MAC by 26% (both doses, P < 0.01). AOT was observed with remifentanil and characterized by a lower degree of MAC reduction, approximately 1.5 hours later (P < 0.05). However, when remifentanil was administered with gabapentin, the AOT to remifentanil was not observed (P > 0.05). CONCLUSIONS: Gabapentin reduced the sevoflurane MAC and enhanced the MAC reduction produced by remifentanil. This enhancement may limit AOT in rats.

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.

Neurotoxicity of adjuvants used in perineural anesthesia and analgesia in comparison with ropivacaine.

BACKGROUND AND OBJECTIVES: Clonidine, buprenorphine, dexamethasone, and midazolam (C, B, D, M) have been used to prolong perineural local anesthesia in the absence of data on the influence of these adjuvants on local anesthetic-induced neurotoxicity. Therefore, the impact of these adjuvants on ropivacaine (R)-induced death of isolated sensory neurons was assessed. METHODS: The trypan blue exclusion assay was used to assess death of sensory neurons isolated from adult male Sprague-Dawley rats. Drugs were applied, alone or in combination, for 2 or 24 hrs at 37°C. RESULTS: Neuronal viability was halved by 24-hr exposure to R (2.5 mg/mL), far exceeding the neurotoxicity of C, B, D, or M (at 2-100 times estimated clinical concentrations). Plain M at twice the estimated clinical concentration produced a small but significant increase in neurotoxicity at 24 hrs. After 2-hr exposure, high concentrations of B, C, and M increased the neurotoxicity of R; the combination of R + M killed more than 90% of neurons. Estimated clinical concentrations of C + B (plus 66 µg/mL D) had no influence on (i) R-induced neurotoxicity, (ii) the increased neurotoxicity associated with the combination of R + M, or (iii) the neurotoxicity associated with estimated clinical concentrations of M. There was increased neurotoxicity with 133 µg/mL D combined with R + C + B. CONCLUSIONS: Results with R reaffirm the need to identify ways to mitigate local anesthetic-induced neurotoxicity. While having no protective effect on R-induced neurotoxicity in vitro, future research with adjuvants should address if the C + B + D combination can enable reducing R concentrations needed to achieve equianalgesia (and/or provide equal or superior duration, in preclinical in vivo models).

Suspected acute meperidine toxicity in a dog.

OBSERVATIONS: A 22-month-old male neutered Coton De Tulear dog was presented for upper gastrointestinal endoscopy under general anesthesia. The anesthetic plan included premedication with intramuscular meperidine (4 mg kg(-1)) but meperidine was inadvertently administered at ten-fold this dose. Within 5 minutes, the dog was unresponsive to external stimulation, and by 10 minutes post-injection developed generalized signs of central nervous system (CNS) excitement. Initial therapy included inspired oxygen supplementation, and single intravenous (IV) doses of diazepam (0.68 mg kg(-1)) and naloxone (0.03 mg kg(-1)) to no effect. A second dose of diazepam (0.46 mg kg(-1), IV) abolished most of the signs of CNS excitement. General anesthesia was induced and the endoscopy performed. Time to extubation was initially prolonged, but administering naloxone (final dose 0.1 mg kg(-1), IV) to effect enabled extubation. After naloxone, the dog became agitated, noise sensitive, and had leg and trunk muscle twitches. Diazepam (0.30 mg kg(-1), IV) abolished these signs and the dog became heavily sedated and laterally recumbent. Naloxone administration was continued as a constant rate infusion (0.02 mg kg(-1) hour(-1), IV) until approximately 280 minutes post-meperidine injection, at which time the dog suddenly sat up. Occasional twitches of the leg and trunk muscles were observed during the night. The dog was discharged the next day appearing clinically normal. CONCLUSIONS: Given that the CNS excitatory effects of normeperidine are not a mu opioid receptor effect, the use of naloxone should be considered carefully when normeperidine excitotoxicity is suspected. Benzodiazepines may be beneficial in ameliorating clinical signs of normeperidine excitotoxicity.