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THE CARDIOPROTECTION MODEL IN RATS INDUCED  BY ISCHEMIA-REPERFUSION IN VIVO BY POSTCONDITIONING WITH ISOFLURANE ANESTHESIA


Authors: L. Vasiluta, Gabriel Gheorghiu, V. Ordodi, Dorel Sandesc, Lucian Petrescu



Received for publication: 11th of June, 2013
Revised: 21st of July, 2013



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SUMMARY: (Hide the summary)

Isoflurane is a halogenated anesthetic volatile type commonly used in experimental and clinical practice, with possible cardioprotective in postconditioning and preconditioning of pharmacological ischaemia. This paper aims to compare the effects of isoflurane during reperfusion in postischemic postconditioning in rat hearts in vivo. A total of 20 anaesthetized rats (n = 10/lot) was subjected to ischemia for a period over 30 min and 2 hours of reperfusion , respectively . Postconditioning with isoflurane anesthesia (iso -post ) has been carried out only on one of the groups consisting of 10 rats, and the single administration of isoflurane (2.1 % ) 3 minutes prior to reperfusion up to 2 minutes after reperfusion, with a total episode administration of reperfusion for 5 minutes. Cardioprotective was monitored using two primary indicators: reduction of infarct size expressed as a percentage as the ratio AN / AR ( necrosis area / risk area ) and decreased incidence of arrhythmias arrhythmogenic estimated by score arrhythmogenic . Were also monitored and evaluated and two  secondary indicators: heart rate ( HR ) and mean arterial  pressure  (MAP).  Our results obtained  on the model of I / R ( ischaemia / reperfusion )regional   in vivo at rat shows that anesthesia with Isofluran   on postconditioning protocol did not lead to significant anti -infarct protection and not was reduced nor significantly arrhythmias that were induced by I/ R(ischaemia/reperfusion).


Key Words:

Isoflurane, preconditioning, postconditioning, I/R(ischemia/reperfusion), rats heart.

 


 

OBJECTIVES

Standardization of the experimental model of ischemia / reperfusion  regional  in vivo at rat hearts  -  similar reproduce human clinical conditions in acute coronary syndromes.

Isofluran an Halogen-volatile anesthetic is  widely used in the clinic and are known for the tissue protective properties in terms of their administration protocols as pre - respectively, in  postconditioning at various animal models of ischemia / the experimental reperfusion, as well as in humans. The purpose of this study was to test Isofluran in vivo if had the effect on  postconditioning  . Protocol postconditioning    studied was the one who, according to the literature, the maximum was induced on cardioprotection in isolated administration.

 

THE STUDY MATERIAL

He was represented by 20 adult Sprague -Dawley rats weighing 370-450 g, divided into 2 groups (n = 10 rats / group, first  lot=control-lot and second-lot=postconditioning with isoflurane  (ISO-POST lot) who received  food and  water ad  libitum , solid food is suppressed  by 12 hours  prior to the experiment.  All experiments  were  performed in accordance  with the Guide for the Care and Use of Animals ( U.S. National Institutes of Health No. 85-23 , 1996) and the Committee of Ethics and Conduct and Pharmacy “ Victor Babes ” Timisoara , which approved methodology working on the basis of a draft drawn up in advance.

 

EXPERIMENTAL PROTOCOL

Anesthesia was   obtained by   intraperitoneal administration of a mixture of ketamine ( 80 mg / kg) with xylazine ( 5 mg / kg) . Both groups of 10 rats were subjected  to  ischemia  lasting period  of 30 min , respectively  , for 2 h  reperfusion  . Anesthesia postconditioning was carried out only one of the groups consisting of 10 rats , and the single administration  of isoflurane (2.1 % ) 3 minutes prior to reperfusion up to 2 minutes after reperfusion , with a total administration reperfusion episode lasting 5 minutes. The experimental design is schematized in Fig . 1 .

 

STUDY RESULTS AND DISCUSSION.

Cardioprotection was monitored by using 2 primary indicators :

A) reduced infarct size expressed as a percentage as the ratio AN / AR and

B) reduce the incidence of arrhythmias estimated by arrhythmogenic score .

Analysis of infarct area was made with image processing software ( Image  J software ) that allows individual selection and calculation of areas ischemic  areas (AR ) and of necrotic areas (AN ) by computerized planimetry . By multiplying  each  of these  sections  analyzed aryl weight and summing the results obtained were the final dimensions AR , AN for each heart.

Analysis was performed according to a score arrhythmogenic after Curtis and Walker modified , as follows : 0 - no arrhythmias ; 1 - between 1- 30 ventricular extrasystoles, 2 - more than 30 ventricular extrasystoles ; 3 - less than 3 episodes  of tachycardia and / or fibrillation ventricular, 4 - 3 to 5episoade tachycardia  and / or fibrillation and 5 - more than 5 episodes of tachycardia and / or fibrillation .

Cardioprotection as secondary indicators of hemodynamic variables were used: (i) heart rate (HR) and (ii) mean arterial pressure (MAP). Hemodynamic Data (peripheral  derivation  DII and peripheral channel pressure) were continuously acquired throughout the duration of the experiment  and the interpretation, is measured at certain points in time during ischemia and respectively reperfusion.

 

 

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Fig. 1. The experimental study of the effects of the anesthetic isoflurane postconditioning.

 

 

Primary indicators of cardioprotection:

A. The size of experimental myocardial

In the Fig. 2 and 3 show the effects of isoflurane administration  in  the group of  rats subjected postconditionarii   anesthesia  (Iso-Post),  effects were compared with control-lot subjected to ischemia / reperfusion (I / R)n    in the absence of protective interventions.

Expression of the area at risk (AR) as a percentage from  left ventricle  (AR /% of LV area)  do  not show differences   between groups, which attest to   the reproducibility of the experimental model, the fact that the placement of the wire was performed at the same level of coronary artery occlusion is quasi-identical (see picture below).

 

 

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Fig. 2. Area at risk (AR) in groups conditioned with isoflurane

 

For both batches, analyzing experimental data (Table

1) showed low values    of standard error mean (SEM) obtained   for risk areas   studied, indicating   a  high homogenity in   the   studied  groups  confirming reproducibility in terms of experimental conditions.

 

 

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Table 1. Average percentage values   of areas at risk (AR)

 

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Fig. 3.   Infarcted  area in control  group comparative  with isoflurane anesthesia group.

 

In the Fig. 3 is observed that the postconditioning with isoflurane  (Iso Post) was not associated  with the cardioprotective  size infarcted area (AR + AN) although slightly lower showed no significant difference compared to control group.

 

 

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Table 2. Mean percentage values  of areas of necrosis (AN)

 

In the case of areas of necrosis (AN), on the one hand it can be observed a significant  reduction  of the percentage value suggesting that this strategy medium applied to exert a measurable biological and secondly to obtain low values  of average standard errors for each of the groups studied (Table 2) is a further confirmation of the reproducibility of the experimental method used and the fact that it was used a sufficient number of animals to achieve statistical significance.

The  figures  4-5 are  representative  examples  of staining with TTC (2,3,5 trifeniltetrazol  Chloride = TTC) for the groups under study.

 

B. Episodes of ventricular arrhythmias

Analysis was   performed  according  to  a   score arrhythmogenic note 1 to 5 where:

 

 

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Fig. 4. Example of heart in the control group (I / R) after staining with TTC.

 

 

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Fig. 5. Example of heart in group Iso-Post after TTC staining.

 

 

  • 0 - no arrhythmias;
  • 1 - ventricular extrasystoles from 1 to 30;
  • 2 - more than 30 ventricular extrasystoles;
  • 3 -  less  than 3 episodes  of tachycardia  and / or fibrillation;
  • 4 - 3 to in 5 episodes of tachycardia / or fibrillatio
  • 5 - more than 5 episodes of tachycardia  and / or fibrillation.

 

The  data  presented  below  are  the occurrence  of arrhythmias caused by the 30 minutes of ischemia test (index ischemia). Fig.6.

 

 

 

 

Fig. 6.   The incidence  of arrhythmias  induced by ischemic postconditioning with isoflurane

 

The figures 7-10 are shown examples of arrhythmias recorded during the experiment studied groups.

 

 

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Fig. 7. Extrasystoles systematized in control group

 

 

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Fig. 8. Example of ventricular tachycardia in control group

 

 

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Fig. 9. Example of systematic  extrasystoles (bigeminy) in group Iso-POST

 

 

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Fig. 10. Example of ventricular tachycardia installed during reperfusion in group Iso-POST

 

 

Secondary indicators of cardioprotection:

As we mentioned to standardize experimental model, the two secondary indicators, heart rate (FC) and mean arterial  pressure (MAP)  were continuously monitored during the experiments, the data being collected but only at certain times according to a predetermined protocol. The average  values of hemodynamic  parameters at predetermined points of time are presented in Table 3.

As shown in the table 3, there were no significant differences  in  the recovery  of functional  parameters reperfusion  in the group treated  with isoflurane  at a certain time of reperfusion  comparativ  with control group.  However  the IZO-POST group is highlighted slight reduction, but not statistically on significant arrhythmias, induced by I/R.

 

 

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Table 3. Mean ± SEM absolute hemodynamic parameters.

 

 

CONCLUSIONS

Isoflurane  used  for anesthesia  postconditioning protocol  did not result in  significant   anti-infarction myocardial protection, nor reduced arrhythmias induced by I / R (ischaemia  / reperfusion).

 

References:

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Correspondence to:
Correspondence to: Lucian Vasiluță, e-mail: l.vasiluta@yahoo.com