Histomorphometric analysis of the effects of creatine on rat myometrium

Objective: To analyze the myometrial thickness of rats subjected to creatine (Cr) ingestion. Study design: A total of 14 rats was equally divided into the control group (ConGr) receiving 1 ml potable water and the creatine group (CrGr) subjected to the ingestion of 1.6 g/kg Cr diluted in 1 ml potable water. At the end of 8 weeks, the animals were anesthetized (xylazine and ketamine) and sacrificed, the uteri and ovaries stained with hematoxylin and eosin, the thickness of both the myometrium and the epithelium measured and the follicles counted. Results: Analysis revealed a significant increase in thickness of the myometrium in the CrGr (272.26 +/- 66.71 mu m) contrasted with that from the ConGr (160.76 +/- 35.65 mu m), CrGr > ConGr (p < 0001). Conclusion: Our data suggest that Cr changed myometrial morphology in rats by enhancing myometrial thickness, but its action mechanism in the smooth muscle is still unclear.

The effect of premedication with ketamine, alone or with diazepam, on anaesthesia with sevoflurane in parrots (Amazona aestiva)

Abstract Background Premedication is rarely used in avian species. The aim of this study was to evaluate the effect of premedication on the quality of sevoflurane induction and anaesthesia in parrots. We hypothesised that premedication would facilitate handling and decrease the minimum anaesthetic dose (MAD). Thirty-six adult parrots were randomly distributed in three groups: group S (n = 12) was premedicated with NaCl 0.9%; group KS (n = 12) was premedicated with 10 mg.kg-1 ketamine; and group KDS (n = 12) was premedicated with 10 mg.kg-1 ketamine and 0.5 mg.kg-1 diazepam, delivered intramuscularly. After induction using 4.5% sevoflurane introduced through a facemask, the MAD was determined for each animal. The heart rate (HR), respiratory rate (RR), systolic arterial blood pressure (SAP), and cloacal temperature (CT) were recorded before premedication (T0), 15 minutes after premedication (T1), and after MAD determination (T2). Arterial blood gas analyses were performed at T0 and T2. The quality of anaesthesia was evaluated using subjective scales based on animal behaviour and handling during induction, maintenance, and recovery. Statistical analyses were performed using analysis of variance or Kruskal-Wallis tests followed by Tukey’s or Dunn’s tests. Results The minimal anaesthetic doses obtained were 2.4 ± 0.37%, 1.7 ± 0.39%, and 1.3 ± 0.32% for groups S, KS, and KDS, respectively. There were no differences in HR, RR, or CT among groups, but SAP was significantly lower in group S. Sedation was observed in both the premedicated S-KS and S-KDS groups. There were no differences in the quality of intubation and recovery from anaesthesia among the three groups, although the induction time was significantly shorter in the pre-medicated groups, and the KS group showed less muscle relaxation. Conclusions Ketamine alone or the ketamine/diazepam combination decreased the MAD of sevoflurane in parrots (Amazona aestiva). Ketamine alone or in combination with diazepam promoted a good quality of sedation, which improved handling and reduced the stress of the birds. All protocols provided safe anaesthesia in this avian species.

Influence of chest wall on lung mechanics during inspiration

RATIONALE: The interaction between lungs and chest wall influences lung volume, that determines lung history during respiration cycle. In this study, the influence of chest wall mechanics on respiratory system is assessed by the evaluation of inspiration pressure-volume curve (PV curve) under three different situations: closed-chest, open-chest and isolated lung. The PV curve parameters in each situation allow us to further understand the role played by different chest wall elements in the respiratory function. Methods: Twenty-four male Wistar rats (236 ± 29 g) were used. The animals were weighted and then anesthetized with xylazine 2% (O,SmL/kg) and ketamine 10% (0,9mL/kg), exsanguinated and later tracheostomies with a metallic cannula (14 gauge).The cannula was connected to an automatic small animal insufflator. This setup was connected to a pressure transducer (32 samples/s). The 24 animals were randomly separated in three groups:(i) closed chest,(ii) open chest and (iii) isolated lung. The rats were insufflated with 20mL quasi-statically (constant speed of 0,1mUs). lnsufflated volume and measured pressure data were kept and PV curves were obtained for all animals. The PV curves were fitted (non-linear least squares) against the sigmoid equation (1) to obtain the sigmoid equation parameters (a,b,c,d). Elastance measurements were obtained from linear regression of pressure/volume measurements in a 0,8s interval before and after the calculated point. Results: The parameters a,b and c showed no significant change, but the parameter d showed a significant variation among the three groups. The initial elastance also varied between open and closed chest, indicating the need of a higher pressure for the lung expansion, as can be seen in Table 1. Conclusion: A supporting effect of the chest wall was observed at the initial moments of inspiration, observed as a higher initial elastance in open chest situations than in closed chest situations (p=0,00001). The similar initial elastance for the isolated lung and closed chest may be explained by the specific method used for the isolated lung experiment. As the isolated lung is supported by the trachea vertically, the weight of the tissue may have a similar effect of the residual negative pressure in the thorax, responsible for maintaining the residual volume.

Influence of chest wall on lung mechanics during inspiration.

RATIONALE: The interaction between lungs and chest wall influences lung volume, that determines lung history during respiration cycle. In this study, the influence of chest wall mechanics on respiratory system is assessed by the evaluation of inspiration pressure-volume curve (PV curve) under three different situations: closed-chest, open-chest and isolated lung. The PV curve parameters in each situation allow us to further understand the role played by different chest wall elements in the respiratory function. Methods: Twenty-four male Wistar rats (236 ± 29 g) were used. The animals were weighted and then anesthetized with xylazine 2% (0,5mL/kg) and ketamine 10% (0,9mL/kg), exsanguinated and later tracheostomized with a metallic cannula (14 gauge). The cannula was connected to an automatic small animal insufflator. This setup was connected to a pressure transducer (32 samples/s). The 24 animals were randomly separated in three groups: (i) closed chest, (ii) open chest and (iii) isolated lung. The rats were insufflated with 20mL quasi-statically (constant speed of 0,1mL/s). Insufflated volume and measured pressure data were kept and PV curves were obtained for all animals. The PV curves were fitted (non-linear least squares) against the sigmoid equation (1) to obtain the sigmoid equation parameters (a,b,c,d). Elastance measurements were obtained from linear regression of pressure/volume measurements in a 0,8s interval before and after the calculated point. Results: The parameters a, b and c showed no significant change, but the parameter d showed a significant variation among the three groups. The initial elastance also varied between open and closed chest, indicating the need of a higher pressure for the lung expansion, as can be seen in Table 1. Table 1: Mean and Standard Deviation of parameters obtained for each protocol. Protocol: Closed Chest – a (mL) -0.35±0.33; b (mL) 13.93±0.89; c (cm H2O) 21.28±2.37; d (cm H2O) 6.17±0.84; r²** (%) 99.4±0.14; Initial Elastance* (cm H2)/mL) 12.72±6.66; Weight (g) 232.33±5.72. Open Chest - a (mL) 0.01±0.28; b (mL) 14.79±0.54; c (cm H2O) 19.47±1.41; d (cm H2O) 3.50±0.28; r²** (%) 98.8±0.34; Initial Elastance* (cm H2)/mL) 28.68±2.36; Weight (g) 217.33±7.97. Isolated Lung - a (mL) -0.09±0.46; b (mL) 14.22±0.75; c (cm H2O) 21.76±1.43; d (cm H2O) 4.24±0.50; r²** (%) 98.9±0.19; Initial Elastance* (cm H2)/mL) 7.13±8.85; Weight (g) 224.33±16.66. * Elastance measures in the 0-0,1 mL range. ** Goodness of sigmoid fit versus measured data Conclusion: A supporting effect of the chest wall was observed at the initial moments of inspiration, observed as a higher initial elastance in open chest situations than in closed chest situations (p=0,00001). The similar initial elastance for the isolated lung and closed chest may be explained by the specific method used for the isolated lung experiment. As the isolated lung is supported by the trachea vertically, the weight of the tissue may have a similar effect of the residual negative pressure in the thorax, responsible for maintaining the residual volume.

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated -cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis-Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.

Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro

Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine (NK), dehydronorketamine and three hydroxylated NK metabolites in all three species. The metabolic patterns formed differ in production rates of the metabolites and in stereoselectivity of the hydroxylated NK metabolites. In vitro pharmacokinetics of ketamine N-demethylation were established by incubating ten different concentrations of racemic ketamine and the single enantiomers of ketamine for 8 min and data modeling was based on Michaelis-Menten kinetics. These data revealed a reduced intrinsic clearance of the S-enantiomer in the racemic mixture compared with the single S-enantiomer in human microsomes, no difference in equine microsomes and the opposite effect in canine microsomes. The findings indicate species differences with possible relevance for the use of single S-ketamine versus racemic ketamine in the clinic.

Assessing the efficiency of a pharmacokinetic-based algorithm for target-controlled infusion of ketamine in ponies

The objective of this study was to assess a pharmacokinetic algorithm to predict ketamine plasma concentration and drive a target-controlled infusion (TCI) in ponies. Firstly, the algorithm was used to simulate the course of ketamine enantiomers plasma concentrations after the administration of an intravenous bolus in six ponies based on individual pharmacokinetic parameters obtained from a previous experiment. Using the same pharmacokinetic parameters, a TCI of S-ketamine was then performed over 120 min to maintain a concentration of 1 microg/mL in plasma. The actual plasma concentrations of S-ketamine were measured from arterial samples using capillary electrophoresis. The performance of the simulation for the administration of a single bolus was very good. During the TCI, the S-ketamine plasma concentrations were maintained within the limit of acceptance (wobble and divergence <20%) at a median of 79% (IQR, 71-90) of the peak concentration reached after the initial bolus. However, in three ponies the steady concentrations were significantly higher than targeted. It is hypothesized that an inaccurate estimation of the volume of the central compartment is partly responsible for that difference. The algorithm allowed good predictions for the single bolus administration and an appropriate maintenance of constant plasma concentrations.

Effect of ketamine on the limb withdrawal reflex evoked by transcutaneous electrical stimulation in ponies anaesthetised with isoflurane

The purpose of this study was to evaluate the anti-nociceptive activity of ketamine and isoflurane in horses using a limb withdrawal reflex (WR) model. Single and repeated stimulations were applied to the digital nerve of the left forelimb in ponies anaesthetised with isoflurane before, during and after intravenous administration of racemic ketamine. Surface electromyographic activity was recorded from the deltoid muscle. Higher stimulation intensity was required to evoke a reflex during ketamine administration. Furthermore, the amplitudes of response to stimulations were significantly and dose-dependently depressed and a flattening of the stimulus-response curves was observed. The reflex activity recovered partially once the ketamine infusion finished. The results demonstrated that the limb WR can be used to quantify the temporal effect of ketamine on the sensory-motor processing in ponies anaesthetised with isoflurane.

Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans

To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies.

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.

Evaluation of the isoflurane-sparing effects of lidocaine infusion during umbilical surgery in calves

OBJECTIVE: To evaluate the isoflurane-sparing effects of lidocaine administered by constant rate infusion (CRI) during umbilical surgery in calves. STUDY DESIGN: Randomized 'blinded' prospective clinical study. ANIMALS: Thirty calves (mean 4.7 +/- SD 2.5 weeks old) undergoing umbilical surgery. METHODS: After premedication with xylazine (0.1 mg kg(-1) , IM), anaesthesia was induced with ketamine (4 mg kg(-1) , IV) and maintained with isoflurane in O(2) administered through a circle breathing system. The calves were assigned randomly to receive a bolus of 2 mg kg(-1) lidocaine IV after induction of anaesthesia, followed by CRI of 50 mug kg(-1) minute(-1) (group L, n=15) or a bolus and CRI of 0.9% sodium chloride (NaCl, group S, n=15). End-tidal isoflurane was adjusted to achieve adequate depth of anaesthesia. Heart rate, direct arterial blood pressure and body temperature were measured intraoperatively. Groups were compared by t- tests, anova or Mann-Whitney rank sum test as appropriate. RESULTS: The end-tidal concentration of isoflurane (median, IQR) was significantly lower in group L [1.0% (0.94-1.1)] compared to group S [1.2% (1.1-1.5)], indicating a 16.7% reduction in anaesthetic requirement during lidocaine CRI. Cardiopulmonary parameters and recovery times did not differ significantly between groups. CONCLUSION AND CLINICAL RELEVANCE: Lidocaine CRI may be used as a supplement to inhalation anaesthesia during umbilical surgery in calves in countries where such a protocol would be within the legal requirements for veterinary use in food animals. This study did not show any measurable benefit to the calves other than a reduction in isoflurane requirement.

Comparison of the effects of racemic ketamine and S-ketamine for anesthesia in Rheem gazelles (Gazella subgutturosa marica) and Subgutturosa gazelles (Gazella subgutturosa subgutturosa)

OBJECTIVE: To evaluate effects of racemic ketamine and S-ketamine in gazelles. ANIMALS: 21 male gazelles (10 Rheem gazelles [Gazella subgutturosa marica] and 11 Subgutturosa gazelles [Gazella subgutturosa subgutturosa]), 6 to 67 months old and weighing (mean+/-SD) 19 +/- 3 kg. PROCEDURES: In a randomized, blinded crossover study, a combination of medetomidine (80 mug/kg) with racemic ketamine (5 mg/kg) or S-ketamine (3 mg/kg) was administered i.m.. Heart rate, blood pressure, respiratory rate, rectal temperature, and oxygen saturation (determined by means of pulse oximetry) were measured. An evaluator timed and scored induction of, maintenance of, and recovery from anesthesia. Medetomidine was reversed with atipamezole. The alternate combination was used after a 4-day interval. Comparisons between groups were performed with Wilcoxon signed rank and paired t tests. RESULTS: Anesthesia induction was poor in 2 gazelles receiving S-ketamine, but other phases of anesthesia were uneventful. A dominant male required an additional dose of S-ketamine (0.75 mg/kg, i.m.). After administration of atipamezole, gazelles were uncoordinated for a significantly shorter period with S-ketamine than with racemic ketamine. Recovery quality was poor in 3 gazelles with racemic ketamine. No significant differences between treatments were found for any other variables. Time from drug administration to antagonism was similar between racemic ketamine (44.5 to 53.0 minutes) and S-ketamine (44.0 to 50.0 minutes). CONCLUSIONS AND CLINICAL RELEVANCE: Administration of S-ketamine at a dose 60% that of racemic ketamine resulted in poorer induction of anesthesia, an analogous degree of sedation, and better recovery from anesthesia in gazelles with unremarkable alterations in physiologic variables, compared with racemic ketamine.

Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 microg/ml S-ketamine over 120 min under isoflurane anesthesia was performed in Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses).

Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated beta-CD

CE with multiple isomer sulfated beta-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of norketamine in liquid/liquid extracts of the two body fluids. Ketamine, norketamine, and dehydronorketamine could be unambiguously identified via HPLC fractionation of urinary extracts and using LC-MS and LC-MS/MS with 1 mmu mass discrimination. The CE assay was used to characterize the stereoselectivity of the compounds' enantiomers in the samples of five ponies anesthetized with isoflurane in oxygen and treated with intravenous continuous infusion of racemic ketamine. The concentrations of the ketamine enantiomers in plasma are equal, whereas the urinary amount of R-ketamine is larger than that of S-ketamine. Plasma and urine contain higher S- than R-norketamine levels and the mean S-/R-enantiomer ratios of dehydronorketamine in plasma and urine are lower than unity and similar.