Identificación Taxonómica de pequeños mamíferos

Electron microscopy may detect areas of necrosis as early as 20 minutes (Fishbein et al. 1981, Nachlas and Shnitka, et al 1963) after coronary occlusion, however, due to sampling problems and high cost, this method does not lead itself readily to the quantitation of infarct size.

The present methodology using Triphenyl tétrazolium chloride represents a technique whereby infarct size can be reliably determined with relatively low variability.

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Triphenyl tétrazolium chloride (TTC) and nitroblue tétrazolium (NET) are used for the gross histochemical staining technique. Infarct size determination using these histochemical stains have shown to be reliable and simple to use (Lie, et.al 1975, Klein, et.al 1981 and Warltier, et al 1981). The colourless tétrazolium salts form coloured precipitates (TTC-red and NBT- blue) in the presence of intact dehydrogenase enzyme systems, requiring a short incubation.

2.3.2 Mechanism for Staining

Tétrazolium salts are chemically characterised by a ring structure, which contains one carbon and four nitrogen atoms, one o f which is quartemary. The reduction is combined with opening of the ring, which leads to the coloured formazan. Tétrazolium salts are reduced by redox systems having a lower redox potential than the dye itself.

In the areas o f necrosis where reflow has been established after ischaemia the intracellular enzymes are rapidly washed out (Klein, et.al 1985). Therefore the areas of necrosis lack dehydrogenase activity and fail to stain (Vivaldi, et al 1985, and Fishbein, et al 1981). The biochemical mechanism for tétrazolium staining has clearly been explained by Klein et.al (1981), who have shown that the viable myocardium reduces the TTC salts to a formazan pigment. This is achieved by the oxidation of NADH by diaphorases to form the formazan stain. Normal and risk areas of the myocardium will react with the TTC salt to form the formazan pigment this will stain the myocardium brick red, while the infarcted myocardium will not take up the stain, as a result of washout of the cofactors, dehydrogenase and diaphorases, on reperfiision.

A number of studies have been carried out to assess the accuracy of the TTC technique for quantitating and comparison of infarct size with other techniques (Kloner, et.al 1981, Warltier, et al 1981 and Vivaldi, et.al 1985). The accuracy of staining with TTC as a macroscopic means for identification and quantitation of myocardial infarcts after coronary occlusion in the dog was assessed by Fishbein et.al (1981) where they reported TTC staining to correlate well with histological techniques.

2.3.3 Advantages of TTC Staining

Shorter experiments can be designed for specific studies, avoiding some complications of long-term anaesthesia and instrumentation, and in avoidable spontaneous changes in heart rate and blood pressure or arrhythmias, which could complicate interpretation of results. Infarct size determination using TTC staining is accurate and reliable, since it is easier to planimeter the infarct in one complete section.

Other methods utilising enzymatic alterations in tissue or morphological studies by light and electron microscopy pose technical problems such as rapid freezing in liquid nitrogen and /or careful preparation of numerous specimens. While TTC is economical and infarct size can be quantitated as early as 3-6 hours after coronary occlusion (Fishbein, et.al 1981).

2.3.4 Analysis of Infarct and Risk Areas

Once the infarct is delineated grossly a number of techniques are available for estimating infarct size. All have advantages and disadvantages but seem to be o f acceptable reproducibility and accuracy. The only difficulties that may arise when measuring infact size may be due to patchy infarction, such infarcts are only known to occur in the global ischaemic model (Fishbein, et al 1981). However, more sophisticated methods of quantitation such as those based on electronic image analysis would probably be helpful in such circumstances.

2.4 Discussion

There is a wide spectrum of collateral flow in many different species that are used in the laboratory. Collateral flow can be determined using radiolabelled microsphere technique during myocardial ischaemia. In animal experiments collateral flow is divided into four different groups these are high (guinea pig), significant (dog and cat), minimal (rat) and

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zero collateral flow (ferret, baboon, rabbit and pig). Therefore differences in collateral flow found in the species may explain some of the contradictory results obtained with studies of the pharmacological limitation of myocardial infarction.

Pathologic demonstration of myocardial infarction is necessary for validating newly developed radioisotopic and computed axial tomographic techniques of infarct imaging, for assessing the effects of pharmacologic interventions on myocardial infarct size (Kloner, et al 1978 and Rude, et al 1979) and for diagnosing myocardial infarction in cases of sudden death (Fallon, 1979). Therefore reliable techniques for assessing the presence and extent of myocardial infarction shortly after coronary occlusion are important. Histologic methods are useful, but the myocardium must be ischaemic for 12 to 24 fours before definite signs of necrosis are present (Jennings, 1969 Jennings, et al 1973). The TTC staining has recently been used to delineate infarcted myocardium ( Knight, 1965, Lie, et al 1975, Kloner, et al 1978 and Rude, et al 1979) at times earlier than the development of histologic necrosis (Lie, et al 1975 and Fallon, 1979). TTC technique is an accurate method for determining infarcts grossly. Results of TTC staining compete well with histologic evidence of necrosis 6 hours after occlusion and with ultra structural evidence of necrosis as early as 3 hours after occlusion. Also the areas that were shown to be necrotic by light and electron microscope failed to stain with TTC, these regions unstained by TTC always showed numerous mitochondrial amorphous dense bodies, marked intracellular edema, and disrupted sarcolemmal membranes; features that have been associated with iireversible myocardial cell damage (Jennings, et al 1969, Jennings, et al 1973 and Kloner, et al 1974). The loss of TTC staining depends on the time course of irreversible myocardial injury, as well as the kinetics o f cofactor diffusion from necrotic myocardium. For example because o f potential differences in collaterals, (Vivaldi, et al 1985) myocardial damage following coronary artery occlusion in the rat probably develops more rapidly than that in man, and like wise, the time course of TTC staining defects evolution is probably more rapid in rat.

coronary occlusion and is of considerable value. Time consuming and costly histologic or ultra structural studies are not necessary as a routine. Shorter experiments can be designed for specific studies, avoiding some complications which could complicate interpretation of results. Early quantitation with TTC should be helpful in experimental studies of the progressive events of infarct evolution, as well as in evaluation of the effects of interventions during the very early phases of ischaemic injury.