M. Bansinath

Tail immersion test for the evaluation of a nociceptive reaction in mice: Methodological considerations☆☆☆

The effect of two commonly used methods to immobilize the animals, viz. tube restrainer and wrapping in a diaper (chux) on the tail flick latency in immersion test, was evaluated in mice using a stimulus temperature of 50 degrees C. The animals were immobilized either in the tube or chux briefly (25-30 sec) during the tail flick measurements. The basal tail flick latency was 2.8 +/- 0.2 in the tube restrained and 5.5 +/- 0.3 sec in chux restrained groups (p less than 0.001). The analgesic effect of morphine (1, 3, 4, 7, and 10 mg/kg) was significantly higher in the chux-restrained animals as indicated by the dose ratio of 2.16 for the 50% analgesic response in the chux versus tube restrained mice. The tail flick latency, 15 min after naloxone injection (1 and 3 mg/kg), expressed as % of predrug latency was significantly reduced in the chux- but not the tube-restrained group. The hyperalgesic effect of naloxone could not be detected in chux-restrained animals, when the water temperature was increased to 55 degrees C. The results demonstrate that the restraining procedure will influence the analgesic effects of test drugs in tail immersion test. Furthermore, the stimulus temperature appears to be an important variant that could influence the results in this test. The present results demonstrate the hyperalgesic effect of naloxone after systemic administration in the tail immersion test and supports the concept that tail flick response is tonically inhibited by endogenous opioid systems.

Chronic administration of a nitric oxide synthase inhibitor, Nω-nitro-l-arginine, and drug-induced increase in cerebellar cyclic GMP in vivo

Nω-nitro-l-arginine (NG-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of Nω-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 μg/10 μl/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in Nω-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of Nω-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic Nω-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.

DRV Liposomal Bupivacaine: Preparation, Characterization, and In Vivo Evaluation in Mice

AbstractPurpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy. Methods. Bupivacaine hydrochloride (BUP) was encapsulated into dehydration-rehydration vesicles (DRV) of varying phospholipid (PL) compositions. Two bilayer-forming phospholipids were used, the “fluid” dimyristoyl-phosphatidylcholine and the “solid” dis- tearoyl-phosphatidylcholine (DSPC), with 20 or 40 mol% cholesterol, in the presence of bupivacaine at a 1.28 or 0.64 BUP/PL mole ratio. After rehydration, drug/lipid ratios were determined. The formulation with the highest drug/lipid ratio (DSPC/cholesterol in an 8:2 mole ratio prepared in the presence of bupivacaine in a 1.28 BUP/PL mole ratio) was adjusted to a final bupivacaine concentration of 3.5% or 0.5%. The duration of skin analgesia after subcutaneous injection in mice produced by these formulations was compared with the conventional administration of a plain 0.5% solution of BUP. In addition, the concentration of residual bupivacaine at the injection site was followed for 96 h. Results. The relatively low organic solvent/aqueous phase and membrane/aqueous phase partition coefficients, together with liposomal trapped volume and BUP/PL mole ratio, indicated that most of the drug was encapsulated in the intraliposome aqueous phase of the DRV. The DSPC/cholesterol 8:2 mole ratio had the best drug encapsulation (BUP/PL = 0.36). Compared to plain BUP, these BUP-DRV produced significant prolongation of analgesia, which is explained by longer residence time of the drug at the site of injection. Conclusions. Bupivacaine-DRV may have a role in achieving safe, effective, and prolonged analgesia in humans.

Liposomal delivery systems for local anesthetics

The use of local anesthetics to treat pain has many potential advantages compared to the systemic administration of opioid analgesics. Local infiltration of an analgesic at the painful site avoids possible opioid side effects, including respiratory depression, sedation, nausea, vomiting, pruritus, urinary retention, impaired bowel motility, and development of tolerance. However, the results of clinical trials of local anesthetic infiltration to manage postoperative pain have often been disappointing, primarily due to local pharmacokinetic factors. Being relatively small-sized molecules, local anesthetics readily traverse blood vessel walls and are thus removed from the injection site. This rapid redistribution from the site limits the duration of effective analgesia, and the usefulness of this approach to pain control. Despite this limitation, the concept of local anesthetic infiltration to manage postoperative or posttraumatic pain remains appealing. Often in the past, research efforts to prolong local anesthetic duration centered on structural alterations of the local anesthetic molecule and identification of new agents with local anesthetic action. More recently, the focus has shifted to drug delivery systems that act as reservoirs for local anesthetics. Two essential criteria for an effective drug delivery system are residence time at the injection site and drug release rate. The carrier vehicle must be of sufficient size to resist rapid redistribution from the injection site. Furthermore, slow and sustained drug release from the carrier vehicle is needed to produce significant prolongation of analgesia. Investigators have been experimenting with a variety of matrices as carrier vehicles for local anesthetics. This review is limited to examining one type of carrier vehicle, liposomes, which have already been shown to effectively prolong analgesia in an animal-wound model.1 Liposomes, microscopic lipid vesicles formed when dry lipids are suspended in an excess of water, were first described in 1965. Since then, many types of liposomes have been elaborated. When amphipathic lipid molecules with a polar “head” and 2 hydrophobic hydrocarbon “tails” are suspended in aqueous medium, they spontaneously associate into bilayers. The structure of each bilayer resembles that of animal cell membranes, with the hydrocarbon chains oriented toward one another and the polar head group moieties in contact with the surrounding aqueous phase. The resulting vesicle structure consists of an aqueous compartment surrounded by one or more lipid bilayer(s). The lipid bilayer is relatively impermeable to entrapped substances. Liposome architecture is determined by the nature of the interactions between the lipids and the aqueous medium that occur during the preparation process. Liposomes with a single bilayer, or lamella, are known as unilammellar vesicles, and liposomes with many concentric bilayers are known as multilamellar vesicles. Other liposomes, composed of many smaller vesicles within larger vesicles, are known as multivesicular vesicles. In addition to differing structures, liposome size may vary from less than 20 nm to many microns in diameter. Both water-soluble and lipid-soluble drugs may be incorporated into the aqueous and lipid phases of liposomes, respectively. The liposomes function as vehicles to deliver drugs in high concentrations to specific targets while avoiding systemic drug toxicity, because only a fraction of the drug is bioavailable at any time. Liposomal behavior in vivo and drug release characteristics are dictated by liposome size, structure, and composition. Liposomal size affects in vivo distribution. For example, after subcutaneous administration, liposomes less than 120 nm in diameter readily gain access to capillaries and are thus rapidly redistributed from the site of injection, whereas relatively large liposomes tend to remain From the Department of Anesthesiology, New York University School of Medicine, New York, New York. Accepted for publication August 12, 2000. Reprint requests: Gilbert J. Grant, M.D., Associate Professor, Department of Anesthesiology, New York University School of Medicine, 550 First Ave, New York, NY 10016. E-mail: gilbert.grant@med.nyu.edu

The hyperalgesic effect of naloxone is attenuated in streptozotocin-diabetic mice

In mice with streptozotocin-induced hyperglycemia, nociception was tested after naloxone administration in hot plate and tail immersion tests. The choice of these two tests was to include a supra-spinal nociceptive reflex indicative of higher cognitive process (hot-plate test) as well as a reflex which predominantly represents lower spinal motor mechanisms (tail immersion test). Naloxone-induced hyperalgesia was attenuated in both tests in mice with streptozotocin-induced diabetes. In mice with hyperglycemia induced by intraperitoneal dextrose administration, naloxone hyperalgesia was significantly enhanced in the hot plate test. The basal nociceptive threshold in streptozotocintreated animals was decreased in the immersion but not in the hot plate test. These results indicate that hyperglycemia per se does not adequately explain the changes in naloxone hyperalgesia in experimental models of diabetes. They also suggest that acute hyperglycemia may modify the interaction of endogenous opioid peptides with their receptors only at supra-spinal sites. However, chronic hyperglycemia appears to affect endogenous opioid peptides both at spinal and supra-spinal levels and their interaction with the opiate receptors.

Propofol Modulates the Effects of Chemoconvulsants Acting at GABAergic, Glycinergic, and Glutamate Receptor Subtypes

Background Propofol has been used to treat status epilepticus, but its use in patients with seizure disorders remains controversial, because of concerns that it produces paroxysmal motor phenomenon. Chemoconvulsants act by known discrete mechanisms and neurotransmitters, and therefore, they are useful tools for screening anticonvulsant activity. The main objective of this study was to characterize the effect of propofol pretreatment on convulsions induced by picrotoxin, bicuculline, and strychnine, all which decrease inhibitory neurotransmission, and by N-methyl-D-aspartic acid, kainic acid, and quisqualic acid, which enhance excitatory neurotransmission.

Effect of nitric oxide on mitogenesis and proliferation of cerebellar glial cells

In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesis and cell proliferation of the cerebellar glial cells were assessed. NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO-generating agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernatants. However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitochondrial activity in the glial cells. These results demonstrate that NO inhibits serum-induced mitogenesis and cell proliferation of cultured rat cerebellar glial cells.

Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro.

Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.

Influence of hypo and hyperthermia on disposition of morphine.

Using morphine as a prototype opiate anesthetic, the dispositional changes and cardiovascular effects during hypothermia (30°C) and hyperthermia (40°C) in dogs under isoflurane anesthesia was assessed. Single intravenous bolus injection of 1 mg/kg morphine resulted in a significant and sustained decrease in mean arterial pressure in hypothermic, but not in hyperthermic or normothermic (37°C) conditions. Hypothermic dogs showed significantly higher levels of morphine both in plasma and in cerebrospinal fluid. In contrast, hyperthermia did not affect these levels. Body temperature did not affect the t1/2α, however t1/2β and mean residence time were significantly increased while volume of distribution at steady state and total body clearance were decreased during hypothermia. The results provide evidence that hypothermia is likely to be associated with a sustained increase in opiate levels and might be associated with a enhanced side effects. The results suggests the need for a controlled clinical trial to assess the dose of opiate anesthetics during hypothermia.

Analgesic duration and kinetics of liposomal bupivacaine after subcutaneous injection in mice.

1. The objective of the present study was to assess the time‐course profile of analgesia and bupivacaine concentrations at the site of injection after subcutaneous administration of a single dose of standard bupivacaine or a novel controlled‐release liposomal bupivacaine formulation.