Influence of incubation time and rinsing buffer on the distribution of the tracer lanthanum in canine heart muscle.

Abstract

Lanthanum (La) is an extracellular tracer, which stains the interstitial space and the cell surface. This study investigates to what extent the distribution of lanthanum in the myocardium of cardioplegically arrested non-ischaemic hearts was influenced by (a) different incubation times in La containing fixative, (b) different kinds of buffer for rinsing and postfixation dilution and (c) different degree of cellular oedema. Myocytes exhibiting La surface staining, with and without intracellular La, were quantified and the volume density of myofibrils (VVMf) as a parameter for the degree of cellular oedema was determined morphometrically. Samples were taken immediately after cardiac arrest induced by coronary perfusion with a cardioplegic solution. Tissue blocks 1 mm3 in size were fixed by immersion for different time periods in a fixation solution containing 1.1% La(NO3)3. Fixation was followed by rinsing in cacodylate or phosphate buffer. The postfixation solution also contained either cacodylate or phosphate buffer. For La detection electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS), was used. Our results show: (i) the volume densities do not differ significantly in specimens rinsed and postfixed in cacodylate or phosphate buffers; (ii) the percentage of myocytes with La surface staining depends on the incubation time in La containing fixative, independent of the rinsing buffer; (iii) the percentage of myocytes with intracellular La correlates significantly with the VVMf; (iv) the incubation time with La containing fixative does not significantly affect the intracellular La staining of slightly swollen cells; and (v) intracellular La distribution patterns differ in cacodylate- and phosphate-buffered specimens. Thus, La tracer methods in conjunction with microanalysis are valuable tools to detect alterations in membrane permeability not visible by conventional transmission electron microscopy (cTEM) in non-ischaemic hearts exhibiting a well preserved ultrastructure.