Jean-Marie Juan

Comparison of the diagnostic value of cardiac troponin I and T determinations for detecting early myocardial damage and the relationship with histological findings after isoprenaline-induced cardiac injury in rats

Cardiac troponins I (cTnI) and T (cTnT) have been shown to be highly sensitive and specific markers of myocardial cell injury. The purpose of this study was to investigate the diagnostic value of cTnI and cTnT with regard to creatine kinase (CK) and lactate dehydrogenase (LD) and to determine whether they can be used for early diagnosis of myocardial damage in rats, and to examine the relationship between cTnl and cTnT release with histological examinations, using isoprenaline-induced cardiac muscle damage as an experimental model in the rat. Eighteen Wistar rats per group were treated with a single dose of either isoprenaline (iso) or with normal saline as a control group. The anti-cTnI and cTnT monoclonal antibodies (mAbs) employed in the cTnI (Access) and cTnT (Elecsys) assays cross-react with cTnI and cTnT of the rat. A highly significant rise of cTnl or cTnT was found already 2 h after iso. The time-courses of cTnI and cTnT were monophasic in form. The highest cTnI (mean+/-S.D., 1.1+/-2.3 ng/ml) and cTnT (mean+/-S.D. 3.6+/-30 ng/ml) were found 4 h after iso. cTnI and cTnT significantly increased in iso-treated rats in comparison with controls whether the differences between 2-, 4- and 6-h levels and basal levels were considered or not. The areas under cTnl and cTnT curves (AUC) (0-6 h) and the maximal cTnI and cTnT (0-6 h) after iso were significantly different from the controls. For CK and LD, no elevation in comparison with controls could be detected (except a trend for LD whether or not the difference between 6-h levels and basal levels were considered (P=0.08) and for LD AUC (0-6 h) (P=0. 059)). Correlations between maximal cTnI and cTnT and AUC were 0.69 (P=0.0001) and 0.60 (P=0.0066), respectively. Histological examinations of iso-treated rats revealed acute focal or multifocal myofibrillar degeneration of the myocardial tissue in ten out of 14 rats and showed the earliest alterations 4 h after iso in one treated rat. Only four of the controls exhibited evidence of mild changes and slight mononuclear cell infiltration. cTnl and cTnT peak values to at least 0.35 and 1.3 ng/ml, respectively, were necessary to detect histological myocardial cell injury after iso. cTnI and cTnT were found to be early markers for diagnosing iso-induced myocardial damage in comparison with CK and LD. Elevations of cTnI and cTnT appeared to relate to the severity of histologic changes after myocardial injury. Although there was a difference in the absolute concentration of results between cTnI and cTnT assays, due to a lack of standardization and heterogeneity in the cross-reactivities of mAbs to various troponin I and T forms, cTnI and cTnT can be used as easily measurable target parameters for detection of cardiotoxic and/or cardiodegenerative effects in rats.

Evaluation of cardiac troponin I and T levels as markers of myocardial damage in doxorubicin-induced cardiomyopathy rats, and their relationship with echocardiographic and histological findings

BACKGROUND Cardiac troponins I (cTnI) and T (cTnT) have been shown to be highly sensitive and specific markers of myocardial cell injury. We investigated the diagnostic value of cTnI and cTnT for the diagnosis of myocardial damage in a rat model of doxorubicin (DOX)-induced cardiomyopathy, and we examined the relationship between serial cTnI and cTnT with the development of cardiac disorders monitored by echocardiography and histological examinations in this model. METHODS Thirty-five Wistar rats were given 1.5 mg/kg DOX, i.v., weekly for up to 8 weeks for a total cumulative dose of 12 mg/kg BW. Ten rats received saline as a control group. cTnI was measured with Access(R) (ng/ml) and a research immunoassay (pg/ml), and compared with cTnT, CK-MB mass and CK. By using transthoracic echocardiography, anterior and posterior wall thickness, LV diameters and LV fractional shortening (FS) were measured in all rats before DOX or saline, and at weeks 6 and 9 after treatment in all surviving rats. Histology was performed in DOX-rats at 6 and 9 weeks after the last DOX dose and in all controls. RESULTS Eighteen of the DOX rats died prematurely of general toxicity during the 9-week period. End-diastolic (ED) and end-systolic (ES) LV diameters/BW significantly increased, whereas LV FS was decreased after 9 weeks in the DOX group (p<0.001). These parameters remained unchanged in controls. Histological evaluation of hearts from all rats given DOX revealed significant slight degrees of perivascular and interstitial fibrosis. In 7 of the 18 rats, degeneration and myocyte vacuolisation were found. Only five of the controls exhibited evidence of very slight perivascular fibrosis. A significant rise in cTnT was found in DOX rats after cumulative doses of 7.5 and 12 mg/kg in comparison with baseline (p<0.05). cTnT found in rats after 12 mg/kg were significantly greater than that found after 7.5 mg/kg DOX. Maximal cTnI (pg/ml) and cTnT levels were significantly increased in DOX rats compared with controls (p=0.006, 0.007). cTnI (ng/ml), CK-MB mass and CK remained unchanged in DOX rats compared with controls. All markers remained stable in controls. Analysis of data revealed a significant correlation between maximal cTnT and ED and ES LV diameters/BW (r=0.81 and 0.65; p<0.0001). A significant relationship was observed between maximal cTnT and the extent of myocardial morphological changes, and between LV diameters/BW and histological findings. CONCLUSIONS Among markers of ischemic injury after DOX in rats, cTnT showed the greatest ability to detect myocardial damage assessed by echocardiographic detection and histological changes. Although there was a discrepancy between the amount of cTnI and cTnT after DOX, probably due to heterogeneity in cross-reactivities of mAbs to various cTnI and cTnT forms, it is likely that cTnT in rats after DOX indicates cell damage determined by the magnitude of injury induced and that cTnT should be a useful marker for the prediction of experimentally induced cardiotoxicity and possibly for cardioprotective experiments.

Dispersion-based reentry: mechanism of initiation of ventricular tachycardia in isolated rabbit hearts

The aim of the study was to determine whether facilitation of reentry by potassium-channel openers is related to dispersion of refractoriness and/or modification of anisotropic properties of ventricular myocardium. The dispersion of ventricular effective refractory period (VERP), longitudinal and transverse ventricular conduction velocities (θL and θT, respectively), and wavelength [λ = VERP × θ(L or T)] were studied in Langendorff-perfused left ventricular epicardium in 20 rabbits during infusion of incremental doses of levcromakalim or nicorandil. Dispersion of refractoriness was assessed using standard deviation of VERP mean (SD-VERP), dispersion index (DI; SD-VERP/mean VERP), and maximum dispersion (Dmax = VERPmax - VERPmin). Ventricular conduction velocities and anisotropic ratio were not modified, whatever the dose used. VERP and λ were significantly shortened at high concentrations of levcromakalim and nicorandil. At these doses, SD-VERP, DI, and Dmax were increased significantly. Analysis of ventricular tachycardia induction, performed using a high-resolution ventricular mapping system, confirmed that heterogeneity and shortening of VERP were factors inducing functional conduction block. Our data suggest that, in rabbit left ventricular epicardium, functional conduction block facilitating the occurrence of reentry could be initiated by shortening and, especially, by dispersion of refractoriness during infusion of potassium-channel openers.The aim of the study was to determine whether facilitation of reentry by potassium-channel openers is related to dispersion of refractoriness and/or modification of anisotropic properties of ventricular myocardium. The dispersion of ventricular effective refractory period (VERP), longitudinal and transverse ventricular conduction velocities (thetaL and thetaT, respectively), and wavelength [lambda = VERP x theta(L or T)] were studied in Langendorff-perfused left ventricular epicardium in 20 rabbits during infusion of incremental doses of levcromakalim or nicorandil. Dispersion of refractoriness was assessed using standard deviation of VERP mean (SD-VERP), dispersion index (DI; SD-VERP/mean VERP), and maximum dispersion (Dmax = VERPmax - VERPmin). Ventricular conduction velocities and anisotropic ratio were not modified, whatever the dose used. VERP and lambda were significantly shortened at high concentrations of levcromakalim and nicorandil. At these doses, SD-VERP, DI, and Dmax were increased significantly. Analysis of ventricular tachycardia induction, performed using a high-resolution ventricular mapping system, confirmed that heterogeneity and shortening of VERP were factors inducing functional conduction block. Our data suggest that, in rabbit left ventricular epicardium, functional conduction block facilitating the occurrence of reentry could be initiated by shortening and, especially, by dispersion of refractoriness during infusion of potassium-channel openers.

Effects of Ketamine on Ventricular Conduction, Refractoriness, and Wavelength Potential Antiarrhythmic Effects

Background: The aims of the study were to verify the effects of ketamine on ventricular conduction velocity and on the ventricular effective refractory period, to determine its effects on anisotropy and on homogeneity of refractoriness, and to use wavelength to determine whether ketamine has antiarrhythmic or arrhythmogenic properties. Methods: A high‐resolution epicardial mapping system was used to study the effects of 50, 100, 150, and 200 micro Meter racemic ketamine in 15 isolated, Langendorff‐perfused rabbit hearts. Five hearts were kept intact to study the effects of ketamine on spontaneous sinus cycle length (RR) interval and its putative arrhythmogenic effects. In 10 other hearts, a thin epicardial layer was obtained by an endocardial cryoprocedure (frozen hearts) to study ventricular conduction velocity, ventricular effective refractory periods (five sites), and ventricular wavelength. Results: Ketamine induced a concentration‐dependent lengthening of the RR interval. Ketamine slowed longitudinal and transverse ventricular conduction velocity with no anisotropic change, and it prolonged the ventricular effective refractory period with no significant increase in dispersion. Ventricular longitudinal and transverse wavelengths tend to increase, but this was not statistically significant. Finally, no arrhythmia could be induced regardless of the ketamine concentration. Conclusion: Ketamine slowed ventricular conduction and prolonged refractoriness without changing anisotropy or increasing dispersion of refractoriness. Although these effects should result in significant antiarrhythmic effects of ketamine, this should not be construed to suggest a protective effect in ischemic or other abnormal myocardium.

Acute changes in aortic wall mechanical properties after stent placement in rabbits.

PURPOSE To evaluate mechanical property changes after endovascular stent placement in small-diameter arteries. MATERIALS AND METHODS Self-expanding stents (Wallstent) were placed in the infrarenal aorta of five New Zealand White rabbits via a surgical right femoral approach. Blood pressure changes (deltaP) were monitored in the aorta. Blood flow velocity was measured with a 20-MHz, pulsed Doppler probe (n = 4) to calculate the pulsatility index. Aortic diameter (dA) and diameter changes (delta(d)) were measured with a 20-MHz probe in echo-tracking mode. Diameter compliance (Cd) and distensibility coefficient (DC) were calculated as Cd = 2(delta)d/(delta)P and DC = 2delta(d)/delta(P)/dA. RESULTS Aortic diameter increased from 3.360 +/- 0.4033 mm to 4.020 +/- 0.3033 mm after stent placement at the stent level only. Compliance decreased from 77.644 +/- 24.306 mm kPa(-1) to 31.150 +/-8.245 x 10(-3) mm kPa(-1) at the stent level, and was then significantly lower than upstream (98.500 +/- 53.196 mm kPa(-1)) and down-stream (59.047 +/- 13.833 mm kPa(-1)). There was no significant change in pulsatility index. CONCLUSIONS Endovascular stent placement produces a significant decrease in arterial wall compliance of the rabbit abdominal aorta.

Time-course of cardiac troponin I release from isolated perfused rat hearts during hypoxia/reoxygenation and ischemia/reperfusion

The study was designed to determine the time-course of cardiac troponin I (cTn-I) release in isolated and Langendorff-perfused rat hearts during hypoxia and reoxygenation (H/Reox), and after various durations of total ischemia and subsequent reperfusion (I/R). For this purpose, in H/Reox, cTn-I was measured with the conventional Access immunoassay (ng/ml) and a new immunoassay which operates at pg/ml, and compared with creatine kinase (CK), lactate dehydrogenase (LD) and cardiac troponin T (cTn-T). In I/R, cTn-I was compared with CK and LD. The anti-Tn-I mAbs used in cTn-I assays cross-react with cTn-I of the rat. A clear difference between time-courses and concentration levels of cTn-I in I/R and H/Reox models was found. In I/R, maximum release of cTn-I, CK and LD similarly occurred within minutes following reperfusion; however cTn-I did not return to baseline values. cTn-I levels were not linked to the duration of ischemia. In I/R, we were only able to detect small cTn-I concentrations. In H/Reox experiments, cTn-I, CK and LD increased time-dependently. We found higher cTn-I maximal peak levels detected with the Access immunoassay than with the new assay (median, 0.346 ng/ml per min/g dry wt vs 132 pg/ml per min/g dry wt). cTn-T maximal concentrations were lower than maximal cTn-I levels (median, 0.117 ng/ml per min/g dry wt). Time-courses of cTn-I release were roughly similar with both assays in the H/Reox model (r = 0.90). These data indicate that the cTn-I time-course is related to experimental model (I/R or H/Reox), but also likely depends on the sensitivity of cTn-I assays in such experimental conditions.

Experimental model for comparative evaluation of pharmacologically induced vasodilation of arterial wall mechanical properties.

&NA; Arterial wall compliance (C) and distensibility coefficient (DC) are key factors of pathologic physiology, especially in arteries less than 2 mm in diameter. The aim of this study was to design an experimental model allowing comparative measurement of C and DC during pharmacologically induced vasodilation on small‐diameter arteries. Both femoral arteries were exposed in eight New Zealand White rabbits. Diameter (d) and systolic/diastolic diameter changes (&dgr;d) were measured simultaneously, and C and DC were calculated before and after topical application of 1 mL of 4% papaverine on the right side and topical application of 1 mL of 1% lidocaine on the left side. Diameter measurements were performed by echo tracking with 20‐MHz implanted microprobes. After papaverine and lidocaine application, respectively, d increased from 1.36 mm to 2.23 mm (P < 0.0001) and from 1.45 mm to 2.4 mm (P < 0.0001), &dgr;d increased from 0.0568 mm to 0.0571 mm (P = 0.34) and from 0.064 mm to 0.077 mm (P < 0.01), C increased from 5.7 × 10‐3 mm/mm Hg to 6 × 10‐3 mm/mm Hg (P < 0.02) and from 6.23 × 10‐3 mm/mm Hg to 8.49 × 10‐3 mm/mm Hg (P < 0.01), and DC decreased from 4.22 × 10‐3 mm Hg‐1 to 2.61 × 10‐3 mm Hg ‐1 (P < 0.0004) and from 4.36 × 10‐3 mm/mm Hg to 3.46 × 10‐3 mm/mm Hg (P < 0.005). Papaverine‐ and lidocaine‐induced changes were significantly different for &dgr;d, C, and DC (P < 0.01). These results suggest that, unlike that with papaverine, lidocaine‐induced vasodilation leads the artery up to the nonlinear part of its pressure/diameter relationship, with decreased distensibility contrasting with increased diameter and compliance. Our experimental model may be useful to compare the effects of different vasoactive drugs at different concentrations on the mechanical properties of the arterial wall.

Regional blood flows are affected differently by PEEP when the abdomen is open or closed: An experimental rabbit model

ObjectiveThe study of induced circulatory changes requires simultaneous assessment of multiple regional circulations because of interactions and compensatory mechanisms. Positive end expiratory pressure mechanical ventilation (PEEP) is known to cause marked, and potentially deleterious, cardiovascular changes. Our aim was to use a comprehensive approach to assess PEEP-induced circulatory changes in openvs closed abdomen animals.Material and methodsIn the anesthetized rabbit, we used implantable Doppler micro-probes to measure blood flow simultaneously in the ascending aorta, inferior vena cava, portal vein, hepatic artery, common carotid artery, and renal artery. We studied spontaneously breathing animals (Group A), and open (Group B) and closed abdomen (Group C) animals mechanically ventilated at 0 (ZEEP) and 12 cm H2O PEEP.ResultsIn Group A, all biological and hemodynamic variables remained unchanged for three hours at the end of the surgical procedure. In Groups B and C, ZEEP produced no significant hemodynamic change. PEEP induced a decrease in carotid, hepatic, and renal artery blood flow in Groups B and C, a decrease in heart rate and mean arterial blood pressure in Group B, and a decrease in aorta blood flow in Group C.ConclusionsThese experimental results demonstrate the usefulness of the comprehensive approach of circulatory changes, and confirm that PEEP may have deleterious effects on regional blood flow, even without significant change in cardiac output, especially when the abdomen is open.RésuméObjectifL’étude de changements circulatoires induits exige une évaluation simultanée de multiples circulations régionales à cause des interactions et des mécanismes compensateurs. La ventilation mécanique à pression télé-expiratoire positive (PTEP) cause des modifications cardio-vasculaires marquées, potentiellement nocives. Nous évaluons, selon une démarche globale, les changements circulatoires induits par la PTEP dans un abdomen animal ouvert vs fermé.MéthodeNous avons utilisé, chez un lapin anesthésié, des microsondes Doppler implantables pour mesurer le débit sanguin simultanément dans l’aorte ascendante, la veine cave inférieure, la veine porte, l’artère hépatique, l’artère carotide commune et l’artère rénale. Nous avons observé les animaux en respiration spontanée (Groupe A), et des animaux à l’abdomen ouvert (Groupe B) ou fermé (Groupe C) sous ventilation mécanique à 0 (ZEP) et 12 cm H2OPTEP.RésultatsDans le Groupe A, toutes les variables biologiques et hémodynamiques sont demeurées inchangées pendant trois heures après la fin de l’intervention chirurgicale. Dans les Groupes B et C, la ZEP n’a produit aucun changement hémodynamique significatif. La PTEP a provoqué une baisse du débit sanguin artériel carotide, hépatique et rénal chez les lapins des Groupes B et C, une baisse de la fréquence cardiaque et de la tension artérielle moyenne chez ceux du Groupe B et une baisse du débit sanguin aortique chez ceux du Groupe C.ConclusionCes résultats expérimentaux démontrent l’utilité d’aborder globalement les changements circulatoires et confirment que la PTEP peut provoquer des effets nocifs sur le débit sanguin régional, même sans modification significative du débit cardiaque, surtout lorsque l’abdomen est ouvert.

Diode laser and microvascular anastomosis-long term follow up

Direct diode laser assisted carotid end-to-end microanastomosis (LAMA) versus contralateral manual sutures microanastomosis (CMA) were performed in 70 Wistar rats. The laser beam- wavelength 830 nm, maximum power 3 W and continuous wave was transmitted through a micromanipulator and provided a focused spot of 300 micrometers in diameter. After placement of three 10.0 stitches for edge coaptation, LAMA was achieved on left common carotid (0.8 - 1.2 mm) using laser shots (average 3) of 500 mW power, 4.5 s duration and 700 W/cm2 irradiance each. CMA was performed on right carotid by means of six 10.0 stitches. Light and scanning electron microscopy (n equals 82) showed in LAMA that re-endothelialization was complete on day 10 while collagenous fusion of media and adventitia was evident. The patency rate was 93% after LAMA versus 93% after CMA. The intra operative advantages of LAMA versus CMA: shorter operating time minimizing organ ischemia (13 min versus 22 min) and reduced endothelial trauma were evidenced. The technical advantages of diode laser were pointed out: small size of device, absence of water cooling system, no maintenance and decreasing price of diodes.

Compliance of laser-assisted microvascular anastomosis: a comparative study with manual anastomosis (preliminary results)

The compliance of microvascular anastomosis is an important predictive factor for long term patency of graft or vascular reconstruction. This experimental study compare the compliance of manual suture and laser assisted end to end microvascular anastomosis. In nine New-Zealand white rabbits we performed manual end-to-end suture anastomosis on the left femoral artery and laser assisted anastomosis on the right femoral artery, with a diode laser (wavelength 988 nm, power output 500 mW). Compliance was obtained by echotracking (CBI 8000 sonomicrometry system with 20 MHz implantable microprobe from Crystal-Biotech, USA) on the anastomosis site as well as upstream, and downstream from the anastomosis. Vessel compliance was lower on the manual suture side compared to the laser assisted anastomosis side, especially downstream from the anastomosis.