Hot plate versus tail flick: evaluation of acute tolerance to continuous morphine infusion in the rat model.

The development of tolerance to continuous morphine infusion (2, 4 and 6 mg x kg(-1) x hr(-1) was assessed in rats using two different methods for evaluation of nociception, tail flick (TF) and hot plate (HP). The influence of repeated testing on nociception was evaluated using two regimens; series 1 was tested repeatedly 1, 2, 4, 6, and 8 hr after initiating the morphine infusion and series 2 was tested only twice, at maximum morphine effect and at 8 hr. Both, TF and HP showed pain threshold elevation after the morphine administration of 4 or 6 mg x kg(-1) x hr(-1), which reached a maximum at 2 hr after the start of the infusion. HP: reduction of the effect was found in group 4 mg x kg(-1) x hr(-1) in the series subjected to repeated testing; group 6 mg x kg(-1) x hr(-1) showed reduced effect in both sides. TF: the response latencies did not show reduction at 8 hr. Since TF is predominantly a spinal response and HP is predominantly supraspinal, the results suggest that tolerance during the first 8 hr of morphine infusion develops mainly at supraspinal level.

A method of reducing the opioid withdrawal intensity using progressively increasing doses of naloxone.

We assessed the withdrawal intensity in acutely morphine-dependent mice using a pretreatment with escalating doses of naloxone. All animals received a single dose of morphine (100 mg/kg) for the induction of acute opioid dependency. Group 1 (control) received three injections of normal saline and then naloxone 0.8 mg/kg. Group 2 received increasing pretreatment doses of naloxone (0.1, 0.2, and 0.4 mg/kg) and a challenge dose of 0.8 mg/kg. Group 3 received three injections of naloxone 0.1 mg/kg and a challenge dose of 0.8 mg/kg. Groups 4 and 5 were used to verify whether ED(50) found in previous studies was comparable with values obtained in the current experiments. The withdrawal intensity was determined by the number of jumps. The mice of group 1 exhibited significantly more jumps after 0.8 mg/kg of naloxone as compared with group 2. The number of jumps in response to naloxone between groups 1 and 2 and groups 2 and 3 was not significantly different. The results show that pretreatment with increasing naloxone doses significantly reduced the withdrawal intensity as compared with the control group; whereas pretreatment with repeated low antagonist did not reduce it significantly.

Assessing local anesthetic effect using the mouse tail flick test.

We used the tail flick test to quantify duration of local anesthetic-induced conduction block in the mouse. Using a baseline tail flick latency (TFL) between 1.0 and 2.5 sec, sensory block was considered present if TFL was > or = 4 sec. Two 20-microL local anesthetic injections were made on opposite sides of the tail base. TFL was tested every 10 min, and local block duration was interpreted as the time to return of TFL to < 4 sec. We tested three different concentrations of procaine (1%, 2%, and 4%), tetracaine (0.125%, 0.5%, and 1%), and lidocaine (0.5%, 1%, and 2%) with and without epinephrine. The testing method could discriminate between the duration of the various local anesthetic concentrations used. For the 1% concentrations, the duration of sensory block was 2 +/- 4 min (S.D.) for procaine, 20 +/- 10 min for lidocaine, 40 +/- 10 min for tetracaine, and 66 +/- 15 min for lidocaine with epinephrine. We found this to be a simple and reliable means of assessing local anesthetic conduction block in the mouse.

Preliminary evaluation of Vipera palaestinae snake bite treatment in accordance to the severity of the clinical syndrome.

The intravenous administration of a 60 ml dose of Vipera palaestinae antivenin was the suggested standard treatment of every bitten patient. In this study 85 Vipera palaestinae bitten patients where selectively treated with antivenin depending on the severity of the clinical picture. Patients who developed systemic or severe local signs received 20 ml of antivenin over 30 min. If symptoms were still present, an additional 10 ml of antivenin was given until systemic signs subsided. Repeated doses of 10 ml of antivenin was administered in each case of systemic symptom relapse. 49% of patients did not exhibit any systemic symptoms and did not receive antivenin treatment. In 63% of antivenin treated cases symptoms were aborted by a single dose of 20 ml of antivenin. 23% of the antivenin treated patients needed 30-40 ml, 19 needed 50-60 ml and only 1 patient (2%) received 80 ml of drug. Serum sickness complications were found in 44% of antivenin treated patients. The results of this study show that antivenin treatment based on systemic symptoms is effective, required less antivenin than the treatment with fixed dose for each patient and reduces the incidence of serum sickness.

Quantifiable dose-dependent withdrawal after morphine discontinuation in a rat model.

We evaluated the intensity of the withdrawal symptoms after the discontinuation of the morphine infusion in rats. Opiate addiction was induced by progressively increasing intraperitoneal morphine infusion rates. The control group (Group 1) received normal saline. The initial morphine rates were 1, 4, and 16 mg kg(-1) h for Groups 2, 3, and 4, respectively. Infusion rates were gradually increased by a factor of 1.4, 2, 2.8, and 4 on the second, third, fourth, and fifth days, respectively. The last rate was used for 48 h and then infusions were disconnected. Weight reduction, food consumption, and water intake were used for evaluation of withdrawal. All morphine groups showed a significant reduction of body weight during the 4 postdiscontinuation days and a decline in food and water intake on the first postdiscontinuation day. All changes were dependent on the morphine infusion concentration. No changes were observed in the control group. We suggest that the rat model used in this study may be utilized for quantification of spontaneous withdrawal.

Preliminary evaluation of epidural morphine for treatment of heroin withdrawal.

OBJECTIVE: To evaluate the efficacy of epidural morphine in treating heroin withdrawal in patients who failed to detoxify by the other methods. DESIGN: Prospective study. SETTING: Department of Psychiatry of a general hospital. PATIENTS: 8 ASA physical status I patients, aged 26 to 42 years, not having concurrent diseases requiring medication, and who had previously failed other methods of detoxification. INTERVENTIONS: Epidural catheters were inserted at the L(3)-L(4) interspace. Bolus injections of morphine sulfate, 3.0 mg in normal saline, were administered epidurally at 24-hour intervals. Treatment continued for 10 to 12 days. MEASUREMENTS: Withdrawal symptoms, such as mydriasis, insomnia, rhinorrhea, arthralgia, muscular pain, tooth pain, vomiting, diarrhea, dysphoria, and drug craving were monitored. MAIN RESULTS: Withdrawal symptoms ceased within 10 days. Withdrawal symptoms were diminished or entirely abolished by the treatment, and no patient requested to drop out of the program. Discontinuation of the epidural injections did not cause relapse of withdrawal. All patients reported that withdrawal with epidural morphine was considerably easier compared to other methods that they had previously experienced. CONCLUSIONS: A preliminary evaluation of epidural morphine in addicts that failed previous detoxifications showed high effectiveness of this method in reducing withdrawal symptoms.

Spinal anesthesia: significant prolongation of the pharmacologic effect of tetracaine with lipid solution of the agent.

A novel approach for increasing the duration of anesthesia after a single subarachnoid injection of a local anesthetic is presented. Tetracaine 1% 0.5 mg/kg was administered in 10% glucose or in lipid solution (iophendylate) in two groups of rabbits via catheters chronically implanted in the subarachnoid space. The pharmacologic effect was assessed by evaluation of the intensity and duration of the motor blockade according to a three-stage scale. Significant prolongation (447 +/- 13 min vs. 130 +/- 7 min, P less than 0.01) with a moderate decrease in the intensity of the motor blockade was observed with lipid solution as compared to the aqueous solution. This effect is attributed to the slow release of the agent from the lipid phase, which actually functions as a drug depot in the cerebrospinal fluid. The proposed method is suggested as an additional approach for the development of drug delivery system intended for prolongation of spinal anesthesia.

Prolongation of the pharmacologic effect of intrathecal meperidine by the use of a lipid solution of it.

The possibility of prolonging the effect of intrathecally injected meperidine by the use of a lipid solution was examined in this study. An aqueous solution of 5% meperidine HCl, 250 micrograms/kg, and an equimolar solution of meperidine dissolved in iophendylate (Pantopaque) were injected subarachnoidally in two groups of rabbits (n = 9 in each) with chronically implanted catheters in the subarachnoid space at the level of L7-8. The effect of each injection was assessed by evaluation of the pain threshold in the animal's hind limbs and of the degree of motor blockade produced. The duration of analgesia and of motor blockade were significantly longer when the lipid solution was used. Six of nine animals that received the aqueous solution of 5% meperidine HCl exhibited temporary signs of agitation (i.e., biting of hind limbs). None of the animals given the lipid solution of the drug did. The findings are attributed to the slow release of meperidine from the lipid phase that serves as a drug depot in the cerebrospinal fluid. The approach presented is suggested as a basis for the development of lipid solutions that might prolong the duration of spinal analgesia produced by a single intrathecal injection.

Discrepancy between the development of tolerance to bupivacaine in extradural and spinal anaesthesia in rabbits.

We gave equal groups of rabbits seven extradural (500 micrograms kg-1) or intrathecal (250 micrograms kg-1) injections of bupivacaine, at 24-h intervals. A decrease in the duration of motor block was observed after the extradural injections. The intrathecal injections exerted a reproducible effect. An additional regimen was tested in which five doses of bupivacaine 125 micrograms kg-1 were administered intrathecally after a loading dose of 250 micrograms kg-1, when the animals showed partial recovery from the previous dose; there was no decrease in the effect. The absence of tolerance to intrathecal bupivacaine implies that tachyphylaxis to extradural local anaesthetics results from a decrease in availability of the drug to the neural target, rather than a diminution in effect at the site of action.

The partition coefficient as a predictor of local anesthetic potency for spinal anesthesia: evaluation of five local anesthetics in a mouse model.

Local anesthetic partition coefficients correlate with drug potencies in vitro, but in vivo data have not always complimented in vitro results. Despite extensive studies on intrathecal anesthetic action, whether there is correlation between the partition coefficient and local anesthetic potency has not been addressed. Mice (n = 150) were randomly allocated into 15 groups. Intrathecal injections of etidocaine (E), tetracaine (T), bupivacaine (B), lidocaine (L), or procaine (P) were administered and analgetic effect was measured using tail-flick (TF) test. Concentration-response regressions were constructed for each drug; EC50 values were calculated and compared at 95% confidence intervals. The EC50 values between E (0.017%), T (0.019%), and B (0.012%) were not significantly variant. The EC50 of L (0.098%) and P (0.229%) were significantly different from each other and from E, T, and B. The EC50 values were converted to ED50 in nmols. Relative anesthetic potency, defined as the inverse value of ED50 of drug was 23:16:15:2.4:1 for B, E, T, L, and P, respectively. ED50 showed high correlation (R = 0.978) with partition coefficients of local anesthetics. This study implies that the partition coefficient is a predictor of intrathecal local anesthetic potency. We suggest that the mouse model is reliable for evaluation of intrathecal local anesthetic action.

Duration of spinal anaesthesia is determined by the partition coefficient of local anaesthetic.

We have compared the duration of motor block produced by four local anaesthetics administered into a chronically implanted subarachnoid catheter in rabbits. Each group (n = 6) received four different doses of amethocaine, bupivacaine, lignocaine or procaine, and the duration of the resulting motor block was assessed. Dose-response curves were plotted for each drug. As a measure of activity of the anaesthetics, we used the dose of each drug required to produce block of 60-min duration (D60 min) and the correlation between D60 min and different drug properties was examined. An inverse linear correlation (r = 0.995; P < 0.01) was observed between log D60 min and the log of the partition coefficient of the local anaesthetics. No correlation was found between the effect and degree of protein binding, pKa or molecular weight. These results suggest that, in spinal anaesthesia, the partition coefficient could be used as a predictor of the duration of anaesthetic action.

Prolongation of epidural anesthesia using a lipid drug carrier with procaine, lidocaine, and tetracaine.

This study evaluated the effect of a lipid drug carrier (iophendylate) on epidural anesthesia. The intensity and duration of motor blockade produced by aqueous and lipid preparations of local anesthetics were assessed in rabbits with long-term indwelling catheters in the epidural space. Motor blockades produced by procaine (1%, 2%, and 4%), lidocaine (1%, 2%, and 4%), and tetracaine (0.5%, 1%, and 2%) in normal saline solution were compared with the effects produced by equimolar amounts of the drug solutions in iophendylate. Procaine (4%) in aqueous solution produced motor blockade lasting 30 +/- 3.54 min (mean +/- SD) versus 84 +/- 4.18 min in lipid solution. Lidocaine (2% and 4%) in aqueous solution produced motor blockade lasting 41 +/- 4.18 and 65 +/- 6.12 min versus 39 +/- 4.18 and 118 +/- 10.1 min, respectively, in lipid solution. Aqueous tetracaine (0.5%, 1%, and 2%) produced motor blockade of 106 +/- 9.62, 189 +/- 6.52, and 273 +/- 26.8 min versus 284 +/- 14.7, 335 +/- 15.8, and 365 +/- 26.9 min, respectively, in their lipid counterparts. A control group of animals that received normal saline solution or iophendylate alone did not exhibit motor blockade. These results may be attributed to sustained release of local anesthetics from the lipid vehicle. Hence, lipid drug carriers may be effective in prolonging epidural anesthesia.

Prolongation of spinal anesthesia. Differential action of a lipid drug carrier on tetracaine, lidocaine, and procaine.

This study evaluates prolongation of spinal anesthesia by incorporating local anesthetics in lipid formulation. The duration and intensity of local anesthetic effect produced by different concentrations of procaine (1%, 2%, 4%), lidocaine (1%, 2%, 4%), or tetracaine (0.5%, 1%, 2%) dissolved in normal saline were compared to those produced by the same concentration of drugs in lipid (iophendylate) solution. Fifty rabbits with chronic indwelling subarachnoid catheters were divided into ten equal groups. Three days after the operation the catheters were injected with aqueous solutions of the anesthetics, and 24 h later each animal received an equivalent dose of the corresponding drug in free-base form dissolved in iophendylate. The duration and intensity of motor blockade were assessed using a modified Bromage scale. A separate group of animals received plain normal saline and, 24 h later, iophendylate alone. The Kruskal-Wallis test followed by the Tukey-type test for nonparametric multiple comparisons and the Mann-Whitney and Friedman tests were used for statistical analysis at P less than 0.05. Normal saline or iophendylate alone did not produce any motor blockade. Our data show that iophendylate preparations of local anesthetics produce prolonged but less intense motor blockade than the aqueous solutions, except for tetracaine 0.5% in iophendylate, which produced shorter duration of motor blockade. The reduced intensity of motor blockade may be explained by decreased availability of local anesthetic at the nerve tissue due to storage of drug in the lipid depot. The increased duration of blockade signifies a sustained release of drug from the depot.

Intrathecal administration of liposomal morphine in a mouse model.

The authors determined the duration of analgesia, toxicity, and neuraxial distribution of liposomal morphine after intrathecal administration in the mouse. Analgesic duration was determined using the tail-flick test after intrathecal injection of 12.5, 25, or 50 micrograms of plain or liposomal morphine (n = 6 mice/dose/formulation). Toxicity of the formulations was compared by estimating LD50. Neuraxial morphine distribution was determined after 20 micrograms of plain or liposomal morphine. The excised spinal cord and brain were divided into five segments at 1 min, and at 1, 4, and 8 h after injection for both formulations. In addition, for the liposomal morphine, similar sections were obtained at 24 h (n = 6 mice/formulation/time point). Segmental morphine concentration was quantified using radioimmunoassay. Liposomal encapsulation significantly prolonged duration of analgesia for the 25-micrograms (13.4 +/- 1.64 [SE] vs 4.1 +/- 0.5 h) and 50-micrograms doses (16.8 +/- 4.0 vs 4.6 +/- 1.0 h). The estimated LD50 was 200 (confidence interval 151-257 micrograms) for plain morphine, but was not determinable for the liposomal formulation, since no deaths occurred at the largest dose level which could be tested (371 micrograms). For plain morphine, the drug was not confined to a specific neuraxial segment, and segmental levels declined rapidly. After liposomal morphine, the most morphine was concentrated and persisted in the low spinal cord segment at each time interval. These results show that a single dose of liposomal morphine produces prolonged analgesia with decreased toxicity compared to the plain formulation.

Prolonged analgesia with liposomal bupivacaine in a mouse model.

BACKGROUND AND OBJECTIVES: Currently available local anesthetics have relatively limited duration of action and some may cause severe systemic toxicity. An ultralong lasting local anesthetic would be useful to produce prolonged intraoperative anesthesia and extended postoperative analgesia. The goal of this study was to synthesize a sustained release local anesthetic formulation that would produce prolonged sensory block and decrease the possibility of systemic toxicity. METHODS: The effect of liposomes containing 1.1% bupivacaine on duration of sensory block of the mouse tail was compared with equivalent concentrations of bupivacaine with and without epinephrine. Analgesia was assessed using the tail flick test. Systemic toxicity (LD50) was assessed after intraperitoneal injection and in vitro release rates were compared by dialysis technique for liposomal and plain bupivacaine. RESULTS: Sensory block was significantly prolonged with liposomal bupivacaine (130 +/- 38 minutes) compared to plain bupivacaine (46 +/- 11 minutes, P < .01) or bupivacaine with epinephrine (81 +/- 28 minutes, P < .05). The LD50 was significantly lower for plain bupivacaine (61 mg/kg, 95% confidence intervals 47-79) than for liposomal bupivacaine (291 mg/kg, 95% confidence intervals 201-422). The time to 50% in vitro release through a dialysis membrane for liposomal bupivacaine (28 +/- 9 minutes) was markedly prolonged compared to plain bupivacaine (7 +/- 1 minutes). CONCLUSIONS: This study shows that liposomal encapsulation of bupivacaine significantly prolongs duration of action and greatly decreases systemic toxicity of the drug. These findings may be promising for the future production of formulations of ultralong lasting local anesthetics with enhanced efficacy and safety.

Prolonged analgesia and decreased toxicity with liposomal morphine in a mouse model.

Inadequate control of postoperative pain remains a major clinical problem. A reliable method of providing long-lasting postoperative analgesia with a single dose would be very useful. We synthesized a liposomal morphine formulation and compared it to free morphine with regard to duration of analgesia in the mouse. Analgesia was assessed after intraperitoneal injection using the tail-flick test. The systemic toxicity after administration of liposomal and free morphine was compared. The release rate of morphine from liposomes in vitro was also evaluated. The lethal intraperitoneal dose of free morphine in 50% of mice (LD50) was 400 mg/kg. The maximum safe (non-lethal) dose of free morphine was 130 mg/kg. The highest dose of liposomal morphine administered (1650 mg/kg) did not cause death in any animal. Duration of analgesia was significantly prolonged with the highest dose of liposomal morphine (21.5 +/- 5.3 h) compared to the maximum safe dose of free morphine (3.7 +/- 0.75 h), P < 0.01. In vitro experiments showed a slow release rate of morphine from the liposome depot. Prolonged analgesia and decreased systemic toxicity for liposomal morphine are explained by sustained release of morphine from the liposomal depot. These results suggest that liposomal narcotic formulations may provide prolonged analgesia with single-dose administration.

Side-effect evaluation of a new diazepam formulation: venous sequela reduction following intravenous (i.v.) injection of a diazepam emulsion in rabbits.

Diazepam has been incorporated into a stable, submicronized injectable emulsion. Venous sequela induction in rabbits following iv administration of diazepam in a marketed hydroalcoholic solution and in the emulsion were determined and compared over a 5-day period. There was a marked difference in the local reactions induced by the iv administration of the marketed diazepam hydroalcoholic solution and the diazepam emulsion, even on the first postinjection day. This difference was confirmed by pathological analysis. The highest mean venous sequela score was reached by the rabbit group injected with the marketed diazepam solution. It should be noted that no statistical difference was observed between the saline and the diazepam emulsion rabbit groups during the 5 days of the observation period. The moderate increase in the venous sequela score values compared to that for the saline solution should be attributed to the intrinsic effect produced by diazepam itself, and not to the emulsion vehicle, which was shown not to induce any vascular reaction in the present study.

A rabbit model for evaluation of spinal anesthesia: chronic cannulation of the subarachnoid space.

A rabbit model for evaluation of spinal anesthesia is presented. Chronic cannulation of the subarachnoid space was performed in 44 rabbits using the translumbar approach. An autopsy was performed 24 h after the operation on four of the animals. Intrathecal injections of methylene blue did not reveal any leakage from the spinal space. X-ray examination performed on the second and 30th days after the implantation indicated free spread of the injected solution in the subarachnoid space without any obstruction. Repeated injections of four identical doses of bupivacaine at 3-day intervals showed reproducible pharmacologic effects. Administration of different doses of the anesthetic produced a clear dose-response relationship. The relative activity of the anesthetic agents was found to be identical to that previously obtained in humans. No significant complications after the implantation have been recorded. We suggest the current model as an additional appropriate tool for the investigation of spinal anesthesia.

Perineural antinociceptive effect of opioids in a rat model.

BACKGROUND: The research on conductive analgesia induced by perineural opioids generated a large body of conflicting data. In this study we reassessed the antinociceptive response to perineural administration of morphine, fentanyl or meperidine in a rat model. METHODS: Analgesia was assessed using the hind paw withdrawal latency (HPWL) response to radiant heat. The opioid dose producing 20% of maximal possible effect (20%MPE) for systemic analgesia was calculated for each drug. Then sciatic blockade was performed with the dose corresponding to 20%MPE. The injected hind paw was used to measure direct perineural effect and the contralateral hind paw was used as an indicator of systemic effect. RESULTS: The response latency produced by morphine or fentanyl was not significantly different for ipsilateral (perineural effect) or contralateral (systemic effect) paw (27+/-11 vs. 28+/-16 and 3l+/-16 vs. 23+/-16 s, respectively). However, the meperidine group showed significantly higher %MPE for the ipsilateral paw (79+/-32 s) than for the contralateral paw (27+/-22 s). CONCLUSIONS: The results indicate that perineural fentanyl or morphine do not produce analgesia. Perineural block produced by meperidine was attributed to local anesthetic-like effect, rather than to drug interaction with opioid receptor.

A rat sciatic nerve model for independent assessment of sensory and motor block induced by local anesthetics.

The purpose of this study was to develop a reliable model to independently quantify motor and sensory block produced by local anesthetics. The sciatic nerve was blocked in 52 rats by injecting 0.2 mL of 0.125%, 0.25%, 0.5%, or 0.75% bupivacaine (n = 13 for each concentration). Accurate needle placement was achieved using a nerve stimulator at 0.2 mA and 1 Hz. Ten control rats received 0.9% saline (n = 5) or sham nerve stimulation (n = 5). Motor block was assessed by measuring hindpaw grip strength with a dynamometer. Sensory block was determined by measuring hindpaw withdrawal latency from radiant heat. The intensity of both motor and sensory block measured at 30-min intervals was plotted against time until full recovery to obtain the area under the curve. Intergroup comparisons using analysis of variance showed increasing area under the curve with increasing concentrations of bupivacaine for motor blocks (P < 0.05 for all intergroup comparisons except 0.5% vs 0.75%) and sensory blocks (P < 0.05 for all intergroup comparisons). Normal saline or sham nerve stimulation did not result in any motor or sensory block.