Faculty Bibliography

We describe a mouse model for evaluation of skin anesthesia after infiltration of local anesthetic. The method involves subcutaneous injection of the anesthetic over the abdomen, and monitoring the vocalization response to electrical stimulus as a measure of analgesia. Prior to drug injection, the vocalization threshold was determined. Mice that vocalized at < or = 8 mA were included in the study. The model was tested using representative agents of the two classes of local anesthetics, bupivacaine, an amide, and chloroprocaine, an ester. The time course and dose response were assessed after injection. The median analgesic time was 15, 40, and 55 min for 0.015%, 0.0625%, and 0.25% bupivacaine and 30, 50, and 55 min for 0.125%, 0.25%, and 2.0% chloroprocaine, respectively. Statistical analysis of the data showed that this method is sufficiently sensitive to detect differences between the dose and duration of local anesthesia (p<0.05, by log rank test of the survival curves). To further validate the model, we compared the duration of anesthesia between the 0.5% bupivacaine and a new long-acting liposomal formulation of 2% bupivacaine. The results showed that the new formulation significantly prolonged the duration of anesthesia (p<0.05). This simple and reliable method may facilitate research on the pharmacology of infiltration anesthesia and the development of new local anesthetics and/or formulations

PURPOSE: Sufentanil, a lipophilic opioid, is used to provide analgesia for labour and Caesarean section, but may cause neonatal depression. Factors affecting placental transfer of sufentanil were investigated using human placentas. STUDY DESIGN: Transfer and uptake of sufentanil by the human placenta were studied using a single pass (open) in vitro perfusion model. The effects of change in sufentanil concentration (1-100 ng.ml-1) and change in fetal pH (range 7.4-6.8) on placental transfer were studied. Placental metabolism of sufentanil and the effects of maternal protein content (fresh human plasma, albumin 4%, Media 199) on placental transfer were also investigated utilizing a closed (recirculated) in vitro perfusion system. RESULTS: Sufentanil transfer was 2% at five minutes and plateaued at 12% by 45 min. The clearance index (CI = sufentanil clearance/antipyrine clearance) was 0.56 +/- 0.2 for maternal to fetal (MTF) and 0.44 +/- 0.2 in the fetal to maternal (FTM) directions (P = NS). The CI was 0.5 +/- 0.2 for 1 ng.ml-1 and 0.61 +/- 0.3 for 100 ng.ml-1 sufentanil concentration (P = N.S.). The placenta contained 7.1 +/- 2 and 9.8 +/- 3 ng.g-1 sufentanil following MTF and FTM perfusions for 90 min at 1 ng.ml-1. The placenta did not metabolize sufentanil. After one hour MTF washout, placental sufentanil content was 2.3 +/- .5 ng.g-1 with 0.08 ng.ml-1 sufentanil in the umbilical vein. Maternal plasma decreased MTF CI from 0.41 +/- 0.1 for albumin and 0.4 +/- 0.1 for Media 199 to 0.17 +/- .06 for plasma (P < 0.05). Decreasing fetal pH increased MTF CI from 0.57 +/- .13 at pH 7.4 to 1.6 +/- .4 at pH 6.8 (P < 0.05). CONCLUSION: Sufentanil crossed the placenta by passive diffusion and accumulated in placental tissue, which acted as a drug depot, slowing the initial transfer. Placental transfer was decreased by maternal plasma proteins, but not by albumin. Fetal acidosis increased placental transfer. Due to its low initial umbilical vein concentration, sufentanil may be the opioid of choice when delivery is imminent (< 45 min)

To elucidate the biochemical mechanisms of spinal anesthesia, we studied the effects of procaine and tetracaine on protein phosphorylation in the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/approximately 2%, 10 microL, N = 5/drug), or saline (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm cord segment was homogenized and centrifuged, and an aliquot of the supernatant was used for phosphorylation assays. Calcium-dependent phosphorylation was initiated by incubating the samples in buffer containing [gamma-32P]ATP at 37 degrees for 30 min. The proteins were electrophoresed using slab gel and two-dimensional electrophoresis, and phosphorylated proteins were visualized by autoradiography. The data demonstrated that spinal anesthesia changes the phosphorylation state of five endogenous substrate proteins with apparent molecular masses of 130 (protein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (protein-e) kDa. In two-dimensional electrophoresis, protein-a resolved into two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F(2,16) 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, using the Newman-Keuls test, indicated that local anesthetics significantly affected phosphoproteins (P < 0.05) except for protein-al in the tetracaine-treated group. Further characterization of these phosphoproteins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia

BACKGROUND: Wound infiltration with local anesthetics does not reliably produce satisfactory postoperative analgesia, and the dose of local anesthetic which may be safely administered is limited by the potential for systemic toxicity. This study evaluated the efficacy of a slow-release liposomal bupivacaine formulation on duration of wound analgesia. METHODS: Multilammelar liposomes containing bupivacaine were assessed using a rat paw wound model. Twenty-four hours after surgical incision, paw wounds determined to be hyperalgesic to graded force testing with von Frey hairs were infiltrated with 0.3 ml of 2% liposomal bupivacaine, 0.5% plain bupivacaine, saline, or 'empty' (normal saline) liposomes (n = 6/group). The duration of analgesia was measured. The 0.5% plain concentration was chosen because, in preliminary experiments, larger doses were often fatal. Analgesia duration was compared using Mann-Whitney U test at P 0.05. In other rats, plasma bupivacaine levels after wound infiltration with either 2% liposomal formulation or 0.5% plain formulation were assessed (n = 8/group). RESULTS: The mean duration of analgesia was 23 +/- 3 (SD) min for plain bupivacaine and 180 +/- 30 min for liposomal bupivacaine. No wound analgesia was detected in animals given normal saline or 'empty' liposomes. Plasma bupivacaine levels tended to be lower after liposomal than plain bupivacaine. CONCLUSIONS: The 8-fold increase in duration of wound analgesia and the lower plasma levels seen with the liposomal formulation are explained by gradual drug release from the liposomal depot. These results may have important implications for achieving safe and effective analgesia with wound infiltration techniques in humans

To understand the biochemical mechanisms involved in spinal anesthesia, we measured protein kinase C (PKC) activity and expression of immediate early oncogene protein, c-Fos, in the spinal cord. Spinal anesthesia was induced in mice using intrathecal injection of either 10 microL procaine or tetracaine (0.067 M/approximately 2%). Control groups were treated with either saline or ethanol. Animals were killed at 1, 5, and 15 min after the injection and the caudal 3 cm of the spinal cord was processed for biochemical analysis. PKC activity was measured by the transfer of a phosphate group from [gamma-32P]adenosine 5'-triphosphate to the threonine group on a synthetic peptide specific for PKC. Western blot analysis was used to detect changes in c-Fos protein expression. When compared to saline-treated controls, PKC activity was increased significantly (P < 0.0005) in procaine- and tetracaine-treated groups whereas ethanol decreased PKC activity. The less lipid-soluble procaine produced a larger increase in PKC activity than did the more lipid-soluble tetracaine. Moreover, parallel to the effect on PKC activity, procaine was more potent than tetracaine as a c-Fos inducer. These results implicate some role for a PKC- and c-Fos-dependent pathway in the mechanism of spinal anesthesia. However, these results also demonstrate a lack of correlation between an increase in PKC levels and either potency or lipid solubility of the anesthetics. The increased PKC activity may not be the sole mechanism for spinal anesthesia. These data on the effects of local anesthetics on PKC activity and c-Fos in vivo are of relevance for studies aimed at delineating the biochemical basis of spinal and epidural anesthesia

STUDY OBJECTIVE: To determine the effect of intrathecal fentanyl on maternal hemodynamics. DESIGN: Prospective. SETTING: Labor and delivery suite of a university medical center. PATIENTS: 23 ASA status I nonlaboring term parturients presenting for elective cesarean section. INTERVENTIONS: Patients were given either 1,200 ml lactated Ringer's Solution (Group 1, n = 12) or no intravenous (i.v.) fluid (Group 2, n = 11). A combined spinal-epidural technique was then performed. Fentanyl 25 micrograms was administered intrathecally through a 24-gauge Sprotte or 25-gauge Whitacre spinal needle. After completion of the hemodynamic study, a catheter was threaded into the epidural space for local anesthetic administration. MEASUREMENTS AND MAIN RESULTS: Baseline hemodynamic data [systolic (SBP), diastolic, and mean arterial pressure, heart rate, stroke volume, cardiac output, end-diastolic volume, and ejection fraction] were obtained in triplicate using noninvasive blood pressure monitoring and impedance cardiography. After administration of intrathecal fentanyl, hemodynamic measurements were recorded at 3-minute intervals for 30 minutes. These values were compared with baseline for both groups. Ten patients in each group completed the study. Intrathecal fentanyl administration did not result in any maternal hemodynamic changes in Group 1, and a few small statistically significant changes in Group 2. Measured SBP was always greater than 100 mmHg in all patients during the study. CONCLUSION: Intrathecal administration of fentanyl 25 micrograms in nonlaboring term parturients does not produce clinically important maternal hemodynamic changes

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

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