Conscious Rabbit Model Research Articles

Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits.

Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the epsilon isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKCepsilon, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKCepsilon but also that of Src and Lck, indicating that Src and Lck are downstream of PKCepsilon in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.

Arterial Remodeling in the Rabbit: Structural Changes in Response to Chronic Hindlimb Ischemia

Background The structure of arteries changes in response to changes in flow. Most studies of flow-induced arterial remodeling either focus on the microcirculation, employ nonphysiologic flow models (AV fistula), or use acute measurements of flow. We used a chronic, conscious rabbit model of remodeling of the internal iliac artery (IIA) after external iliac (EIA) ligation to study the interaction of flow and structural changes in a large conduit artery collateral. Methods Thirty-four adult male NZW rabbits underwent distal right EIA ligation or sham operation. Animals had aortography at 0, 1, 2, 4, or 8 weeks, were killed, and perfusion fixed, and the IIAs were excised. Luminal diameter (LD) and medial thickness (MT) and medial smooth muscle nuclear number (NN) were measured in the IIA by computer digital image analysis. Transit time flow probes were placed on the common iliac artery (CIA) of 28 additional rabbits 1 week before EIA ligation. Flow data were measured daily. Data (mean ± SEM) were analyzed by ANOVA (*P Results Right IIA LD increased more than 20% at 2 weeks and more than 40% at 8 weeks. Right IIA MT increased in a bimodal fashion at 1 and 4 to 8 weeks. Right IIA wall area increased progressively from 20% at 1 week to 80% at 8 weeks (P P Conclusion This model of modestly increased collateral flow demonstrates arterial remodeling with increased arterial diameter, luminal area, wall thickness, and nuclear number. These structural changes are first seen at 1 to 2 weeks and are complete at 8 weeks. Because the early increase in medial thickness is not associated with an increase in cell nuclei, early remodeling may involve changes in extracellular matrix. Late remodeling may involve smooth muscle proliferation. The temporal discordance between structural change and shear normalization suggests that factors other than shear normalization may either initiate or perpetuate the arterial remodeling in this model.

Enhancement of nasal absorption of insulin using chitosan nanoparticles.

To investigate the potential of chitosan nanoparticles as a system for improving the systemic absorption of insulin following nasal instillation. Insulin-loaded chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate anions. They were characterized for their size and zeta potential by photon correlation spectroscopy and laser Doppler anemometry, respectively. Insulin loading and release was determined by the microBCA protein assay. The ability of chitosan nanoparticles to enhance the nasal absorption of insulin was investigated in a conscious rabbit model by monitoring the plasma glucose levels. Chitosan nanoparticles had a size in the range of 300-400 nm, a positive surface charge and their insulin loading can be modulated reaching values up to 55% [insulin/nanoparticles (w/w): 55/100]. Insulin association was found to be highly mediated by an ionic interaction mechanism and its release in vitro occurred rapidly in sink conditions. Chitosan nanoparticles enhanced the nasal absorption of insulin to a greater extent than an aqueous solution of chitosan. The amount and molecular weight of chitosan did not have a significant effect on insulin response. Chitosan nanoparticles are efficient vehicles for the transport of insulin through the nasal mucosa.

The Standard Peritoneal Permeability Analysis in the Rabbit: A Longitudinal Model for Peritoneal Dialysis

The development of an experimental peritoneal dialysis (PD) model in rabbits to investigate peritoneal transport characteristics during a longitudinal follow-up and to assess normal values of these peritoneal transport parameters. Peritoneal transport parameters were determined in conscious, unrestrained rabbits by standard peritoneal permeability analysis adjusted for rabbits (SPAR). In this test a 1-hour dwell with 3.86% glucose dialysate is used. Dextran 70 (1g/L) was added to the dialysate to allow calculation of fluid kinetics. Dialysate samples were taken before, 10, and 40 minutes after instillation and at the end of the dwell. Blood was drawn at the end of the dwell. Eighteen female New Zealand White rabbits (2565 g) were included for catheter implantation. SPARs were performed in 15 animals; the other 3 were excluded due to complications. The mass transfer area coefficients (MTACs) of the low molecular weight solutes urea (MTAC(urea)) and creatinine (MTACcr) were calculated. The clearances of albumin (CIalb) and IgG (CI(IgG)), glucose absorption, and fluid transport were computed. Coefficients of intraindividual variation (Vc) were calculated for these parameters. The main complications were catheter obstruction and/or dislocation. Five rabbits underwent uncomplicated PD during a 4-week period. Fifteen SPARs in 15 stable rabbits were performed and analyzed to obtain normal values. Means and standard deviations of the transport parameters were as follows: MTAC(urea) 2.24+/-0.57 mL/min, MTACcr 1.61+/-0.30 mU/min, CI(alb) 52.9+/-17.2 microL/min, CI(IgG) 44.5+/-22.9 UL/min. The transcapillary ultrafiltration rate was 0.66+/-0.13 mL/min and the lymphatic absorption rate 0.47+/-0.26 mL/min. The parameters of solute transport were upscaled to those in humans using two different methods. MTACs of low molecular weight solutes in rabbits and patients were of the same order of magnitude, but the clearance of albumin was approximately four times higher in rabbits than in patients, and that of IgG eight times. In all rabbits sieving of sodium was observed. The dialysate/plasma (D/P) of sodium decreased to a minimum at 40 min (p<0.003 vs the initial value), followed by a rise to 60 min. The minimal value was 0.884+/-0.002. The coefficients of variation calculated on 7 rabbits that underwent two or more SPARs were similar to those assessed from the patient data. This indicates stability of the model and reproducibility of the SPAR. The conscious rabbit model for PD can be used for repeated studies on peritoneal transport.

Role of adenosine receptors in late preconditioning against myocardial stunning in conscious rabbits.

Conscious rabbits underwent six 4-min coronary occlusions interspersed with 4-min periods of reperfusion for 2 consecutive days (days 1 and 2 of stage I); 2 wk later, they underwent the same protocol (days 1 and 2 of stage II) except that they received either 8-(p-sulfophenyl)theophylline (SPT) on day 1 (group I, n = 5) or 2-chloro-N6-cyclopentyl-adenosine (CCPA) on the day before day 1 (group II, n = 6). In both groups I and II, on day 1 of stage I, systolic wall thickening (WTh) remained significantly depressed for several hours, indicating myocardial stunning; on day 2, however, the total deficit of WTh was approximately 50% less than on day 1 (P < 0.01), indicating the development of late preconditioning (PC) against myocardial stunning. Despite administration of SPT, in group I the deficit of WTh during stage II was 55% less on day 2 than on day 1 (P < 0.05). Similar results were obtained in three other rabbits treated with PD-115199 on day 1. In group II, pretreatment with CCPA during stage II failed to decrease the deficit of WTh on day 1. This study presents a new conscious rabbit model for studying myocardial stunning that is relatively inexpensive and technically less demanding than larger animal models. In this model, the development of late PC against myocardial stunning is not blocked by nonselective blockade of adenosine receptors with either SPT or PD-115199, nor is it induced by activation of adenosine A1 receptors with CCPA, indicating that adenosine receptors are not involved in the pathogenesis of this phenomenon.

Increased cardiac workload by adrenoceptor agonists for the estimation of potential antiischemic activity in a conscious rabbit model

The antiischemic effect of drugs can be detected at a lower dose range if the cardiac workload is increased. A brief period of frequency-loading (ventricular overpacing = VOP) results in well-defined, reproducible changes in cardiac parameters in the conscious, chronically instrumented rabbit; however, rapid pacing frequently evoked ventricular tachycardia or even fatal ventricular fibrillation. Therefore, cardiac workload has been increased by i.v. administration of adrenoceptor agonists, such as isoproterenol (ISO), phenylephrine (PHE), and their combination, respectively. The doses applied (especially the combination of 2 μg/kg ISO and 16 μg/kg PHE, giving optimal changes) were sufficient to produce a marked elevation of both the ST segment in the intracavital electrogram and the left ventricular end-diastolic pressure, without evoking cardiac arrhythmias. We compared the effect of this adrenergic “test” stimulus with that of VOP on hemodynamic and electrophysiological parameters of the heart, and furthermore, on the modification of responses to both “test” stimuli by oral administration of the coronary vasodilator: Isosorbide-5-mononitrate (IS-5-N), given in a dose of 40 mg/kg. Both VOP- and ISO+PHE-induced changes were significantly attenuated by IS-5-N, and a temporal coincidence of the maximal effects was found as well. We reached the following conclusion: The combined administration of ISO and PHE not evoking fatal arrhythmias in the dose range applied can replace the more risky VOP as a “test” workload in the estimation of antiischemic action.

Infarct limitation of the second window of protection in a conscious rabbit model.

Myocardial protection associated with ischemic preconditioning (PC) wanes within an hour or two. It has recently been observed, however, that a delayed phase of protection appears about 24 h after ischemic PC in anesthetized rabbits and dogs which might be related to synthesis of cytoprotective proteins. We tested whether a second window of protection could be induced in conscious rabbits. Rabbits chronically instrumented with a coronary artery occluder and ECG electrodes experienced a 30-min coronary occlusion followed by 3 h reperfusion. Infarct size was measured with triphenyltetrazolium chloride. 35.7 +/- 2.3% of the risk zone infarcted in control animals. PC with 4 cycles of 5-min coronary occlusion/10-min reperfusion 24 h prior to the 30-min ischemia decreased infarction to 24.1 +/- 1.4% of the risk zone (P < 0.01). During the 30-min occlusion 3 of 7 non-PC rabbits developed ventricular fibrillation, while this arrhythmia did not occur in the 7 PC animals (P < 0.1). Myocardial hsp70 content in PC rabbits was twice that in controls. Collateral blood flow was not different in the two groups. A second window of protection exists in conscious rabbits which minimizes both infarction and arrhythmias, and cytoprotective protein content is increased in the myocardium of protected animals.

Infarct limitation of the second window of protection in a conscious rabbit model

Myocardial protection associated with ischemic preconditioning (PC) wanes within an hour or two. It has recently been observed, however, that a delayed phase of protection appears about 24 h after ischemic PC in anesthetized rabbits and dogs which might be related to synthesis of cytoprotective proteins. We tested whether a second window of protection could be induced in conscious rabbits. Rabbits chronically instrumented with a coronary artery occluder and ECG electrodes experienced a 30-min coronary occlusion followed by 3 h reperfusion. Infarct size was measured with triphenyltetrazolium chloride. 35.7 ± 2.3% of the risk zone infarcted in control animals. PC with 4 cycles of 5-min coronary occlusion/10-min reperfusion 24 h prior to the 30-min ischemia decreased infarction to 24.1 ± 1.4% of the risk zone (P < 0.01). During the 30-min occlusion 3 of 7 non-PC rabbits developed ventricular fibrillation, while this arrhythmia did not occur in the 7 PC animals (P < 0.1). Myocardial hsp70 content in PC rabbits was twice that in controls. Collateral blood flow was not different in the two groups. A second window of protection exists in conscious rabbits which minimizes both infarction and arrhythmias, and cytoprotective protein content is increased in the myocardium of protected animals.

Prolongation of lidocaine-induced epidural anesthesia by medium molecular weight hyaluronic acid formulations: pharmacodynamic and pharmacokinetic studies in the rabbit.

We evaluated the utility of medium molecular weight hyaluronic acid for prolonging the local anesthetic activity of lidocaine in a rabbit model of epidural analgesia. Equiviscous formulations were prepared as either a physical mixture of lidocaine hydrochloride and sodium hyaluronate (where drug release occurred via diffusion) or as a lidocaine-hyaluronate complex (where drug release occurred via diffusional and electrostatic processes). The novel hyaluronic acid formulations were functionally evaluated, relative to lidocaine solution, in an intact, conscious rabbit model. The hyaluronate formulations were well tolerated. The duration of sensory block and loss of weight-bearing was prolonged twofold by the lidocaine-hyaluronate complex relative to the solution (P < 0.05). In terms of motor block, flaccid paresis occurred after administration of the solution formulation, whereas only partial motor block was evident after administration of the viscous formulations. Pharmacokinetic modeling of the lidocaine plasma concentration-time data indicated that the rate of drug absorption from the lidocaine-hyaluronate complex was decreased fourfold relative to the solution (P < 0.05). These observations indicate that ionic complexes of local anesthetics with medium molecular weight hyaluronic acid may offer advantages for the prolongation of epidural analgesia.

Prolongation of Lidocaine-Induced Epidural Anesthesia by Medium Molecular Weight Hyaluronic Acid Formulations

We evaluated the utility of medium molecular weight hyaluronic acid for prolonging the local anesthetic activity of lidocaine in a rabbit model of epidural analgesia.Equiviscous formulations were prepared as either a physical mixture of lidocaine hydrochloride and sodium hyaluronate (where drug release occurred via diffusion) or as a lidocaine-hyaluronate complex (where drug release occurred via diffusional and electrostatic processes). The novel hyaluronic acid formulations were functionally evaluated, relative to lidocaine solution, in an intact, conscious rabbit model. The hyaluronate formulations were well tolerated. The duration of sensory block and loss of weight-bearing was prolonged twofold by the lidocaine-hyaluronate complex relative to the solution (P < 0.05). In terms of motor block, flaccid paresis occurred after administration of the solution formulation, whereas only partial motor block was evident after administration of the viscous formulations. Pharmacokinetic modeling of the lidocaine plasma concentration-time data indicated that the rate of drug absorption from the lidocaine-hyaluronate complex was decreased fourfold relative to the solution (P < 0.05). These observations indicate that ionic complexes of local anesthetics with medium molecular weight hyaluronic acid may offer advantages for the prolongation of epidural analgesia. (Anesth Analg 1995;80:740-6)

Ventricular overdrive pacing-induced anti-ischemic effect: a conscious rabbit model of preconditioning

To study whether ventricular overdrive pacing (VOP) induces preconditioning, rabbits were equipped with right ventricular electrode catheters for pacing and intracavital recording and polyethylene cannulas in the left ventricle and right carotid artery to measure intraventricular pressure and blood pressure. One week after surgery in conscious animals, VOP at 500 beats/min over 2, 5, or 10 min resulted in an intracavital S-T segment elevation, shortening of ventricular effective refractory period, decrease in maximum rate of pressure development and blood pressure, and increase in left ventricular end-diastolic pressure proportional to the duration of stimulation. A 5-min preconditioning VOP applied 5 or 30 min before a 10-min VOP markedly attenuated ischemic changes, whereas a 2-min VOP had no effect. In anesthetized rabbits, a 5-min VOP slightly increased guanosine 3',5'-cyclic monophosphate (cGMP) and profoundly elevated adenosine 3',5'-cyclic monophosphate (cAMP) content in left ventricular samples. When this VOP was preceded (5 or 30 min) by a preconditioning VOP, the cAMP increase was significantly attenuated, whereas the cGMP increase was amplified. We conclude that a single 5-min VOP induces preconditioning in association with alterations in cardiac cyclic nucleotide contents.