Uptake of lodinated Contrast Material in lschemic ... - Europe PMC

Uptake of lodinated Contrast Material in lschemic Myocardium as an Indicator of Loss of Cellular Membrane Integrity Jerrold L. Abraham, MD, Charles B. Higgins, MD, and John D. Newell, MD

Differential uptake of iodine containing radiographic contrast medium (I) in myocardial infarcts compared with normal mycardium has been detected by computerized transmission tomography (CTT). In this study the histologic and cellular distribution of I in ischemically damaged canine myocardium after intravenous administration of contrast material was examined by the use of scanning electron microscopy and energy dispersive X-ray microanalysis of fresh frozen cryosections. Analysis of individual cells in 6-t thick sections mounted on carbon substrates showed that I was detectable in the ischemically damaged but not the normal myocardial cells. A decline in the potassium-to-sodium ratio confirmed the loss of membrane integrity in the ischemically damaged cells that accumulated I. These results indicate that I enters ischemically damaged but not normal myocardial cells suggesting that CTT scans after intravenous administration of contrast material may be capable of defining the area of the myocardium in which cells have lost membrane integrity after an ischemic injury. (Am J Pathol 1980, 101:319-330)

]RECENT STUDIES in our laboratory have shown that areas of myocardial ischemic damage in experimental animals may be detected on computerized transmission tomography (CTT) as a result of greater accumulation of contrast material in damaged compared with normal myocardium."2 A preliminary report has indicated that this phenomenon had also been observed in CTT scans of the beating hearts of patients in whom a myocardial infarction had occurred.3 It has not yet been determined whether the iodine accumulation is intracellular or extracellular. The importance of intracellular accumulation is that it may be a marker of loss of cellular integrity. In the current study straightforward sample preparation techniques allowed preservation of the myocardium in a state close to that in vivo with respect to water-soluble components, for the determination of whether iodinated water-soluble contrast material enters the myocardial cell after ischemic injury. The use of unfixed fresh frozen cryosections for scanning electron microscopic (SEM) examination and energy-dispersive X-ray analysis (EDXA) allowed direct correlation of histologic appearance with the relative cellular concentration of the various elements of interest (Na, P, S, Cl, K, Ca, and I). From the Departments of Pathology and Radiology, University of California, San Diego, School of Medicine, La Jolla, California. Supported in part by USPHS Grant HL-00201. Accepted for publication May 22, 1980. Address reprint requests to Jerrold L. Abraham, MD, Department of Pathology, M-012, University of California, La Jolla, CA 92093.

0002-9440/80/1110-0319$01 .00

© American Association of Pathologists

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Material and Methods Three anesthetized (pentobarbital sodium, 25 mg/kg) male mongrel dogs, weighing 25 to 28 kilograms, underwent operative ligation of the obtuse marginal branch of the circumflex coronary artery, which produces an infarct between the papillary muscles, occasionally extending into the posterior papillary muscle.' After 48 hours the animal was given an intravenous bolus injection of iodinated contrast material (meglumine/sodium diatrizoate - 370 mg iodine/ml) at a dose of 2 ml/kg. Ten minutes after administration of contrast material the dogs were anesthetized (pentobarbital Na, 25 mg/kg), and the beating heart was excised. The heart was immediately sectioned transversely from base to apex. Tissue samples from the area of the visually apparent myocardial infarction and from normal myocardium were immediately quench-frozen in a beaker of thawing isopentane cooled in liquid nitrogen. The size of the frozen portion of tissue (razor-cut) was approximately 1.2 cm in diameter and 2-3 mm in thickness. The total time from excision of the heart to frozen tissue was less than 60 seconds. Contiguous portions of infarcted and normal tissue were fixed in standard formalin fixative (10% phosphate-buffered formalin) for paraffin embedding and sectioning. After freezing, the tissue was stored in liquid nitrogen for transportation to the laboratory. The frozen tissue was attached to a cryostat mounting support with an organic adhesive (O.C.T.) in the chamber of a cryostat maintained at -20 C. The tissue itself was not allowed to visibly thaw, and was only transferred from a Dewar containing liquid nitrogen to the cryostat chamber as the adhesive began to freeze (became opaque). Sections 6 u thick were cut for light-microscopic study (hematoxylin and eosin [H & E] stain) and for microanalysis (serial sections). Sections for microanalysis were picked up off the microtome blade by, and allowed to adhere to the surface of, a dry, new, polished pure carbon stub (Ted Pella Co.). Within 2-3 seconds after contact with the section, the stub was immersed in liquid nitrogen for storage until SEM examination. In some cases, the sections were stored in a freezer at -70 C for up to a month before SEM examination. We observed no adverse effects of such storage. Each frozen block of tissue was stored at -70 C for future use if necessary. For SEM examination the stubs were taken directly from immersion storage in liquid nitrogen to the vacuum chamber of an ETEC Autoscan SEM. The time from removal from liquid nitrogen to evacuation of the SEM specimen chamber was approximately 5 seconds. Observation of several sections on stubs for this length of time in the normal room air revealed no signs of melting. The earliest change in appearance of the section (loss of the white color seen in the frozen state) was noted after 15 seconds at room temperature (22 C). In this manner the SEM itself was used to dry the sections, which remained frozen under the high vacuum (1 torr reached within 15 seconds). The thickness of the sections allowed them to dry within a few seconds as they warmed up in the specimen chamber of the SEM. After the usual 90 seconds for complete pump-down to 10-5 to 10' torr, the specimen was ready for examination in the SEM. No diffusion of elements from the edge of the tissue onto the contiguous carbon surface was noted. Observation and EDXA were done under standardized SEM operating conditions-20 kV accelerating voltage, a beam current of approximately 3.0 na (adjusted to give a constant background count rate from the carbon stub contiguous to the tissue section), the specimen tilted 45 degrees from the horizontal, and the working distance (the distance from the final aperture to the specimen) 14 mm. The EDXA system used was a 30 sq mm Kevex Si(Li) detector (3.5 cm from the specimen) with a resolution of 152 ev (full width, half maximum) and a Kevex 5100 Quantex analytic system with processing of spectral data, printout and disk storage controlled by a PDP 11/05 minicomputer. The spectrometer was operated with 1024 channels between 0 and 10 keV, with a pulse processing time of 8 microseconds. The following "windows," or spectral regions of interest, were examined for each raster analyzed (in keV): Na (1.01-1.09), Mg (1.22-1.30), P (1.98-2.06), S (2.27-2.35), Cl (2.59-2.67), K (3.28-3.36), Ca (3.66-3.74), I(La) (3.90-3.98), I

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(L/.) (4.19-4.27), and Fe (6.36-6.44). The presence of iodine in conjunction with calcium would be difficult to determine if only the La line of iodine were examined. Use of the L,B line in addition to the La eliminates the problem of the overlap of the Ca K,B line with the I La line. The relative intensities observed for the I La and L,/ lines, respectively, were 1.0 and 0.5. Therefore, only a twofold loss in sensitivity for iodine would be observed in the presence of calcium. In the SEM, both secondary electron (SE) and backscattered electron (BSE) images were observed,4 the SE image for surface morphology and the BSE image (in which contrast is dependent on atomic number variations) for compositional differences within the section. A standard ETEC solid state BSE detector was used.4 Sections were examined at low magnification first (10 diameters) to observe any defects in the section (eg, folds, holes, distortions) and for comparison with the light-microscopic serial section. The specimen was rotated in the SEM such that the final orientation allowed a selected straight line to be drawn across the section in the horizontal plane (the X plane of stage motion). For normal tissue as determined by light-microscopic examination, a line traversing the greatest length was chosen. For infarcted tissue, a line passing through both the most normal and most severely damaged myocardium was chosen. Having selected a line for analysis, we analyzed the section from one edge to the opposite edge, using the stage micrometer to move the specimen in the X direction in increments of 0.025 inch. Fifteen to 25 regions along this line were analyzed. The actual area analyzed at each increment was a raster that measured 10 x 10 IL (a 4 X 4-cm raster on the viewing screen at 4000X magnification), visually observed to ensure the analysis of parenchymal cells and not other tissue or spaces. The use of only the X stage movement eliminated any significant movement of the specimen in the vertical, or Z direction, thus maintaining a constant working distance in the easiest manner. Under the conditions specified above, sufficent X-ray counts were obtained at each raster within a 20-second period. Net Count Rates

We determined net count rates for the various elements by collecting and storing a spectrum from the supporting carbon disk under conditions identical to those used for analyzing the tissue. We subtracted this stored "background" spectrum from each acquired spectrum prior to calculating the counts for each element. This background intensity on subsequent analytic runs was reproduced within 1% of the preceding run.

Results Limitation and Accuracy of Methods

Light Microscopy

When we compared the images from the light microscope and SEM, the foreshortening of the latter was evident (a result of the 45-degree tilt of the section in the SEM) but did not interfere with histologic correlation between light-microscopic and SEM examination. SEM and EDXA

Although the rasters selected for analysis could be identified as being in myocardial cells, the ultrastructural features of the cells could not be reliably identified in either the SE or BSE modes. The limitation in recognition of subcellular detail was not due to specimen charging. These un-

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coated 6-,t sections exhibited no conductivity problems and yielded 10-nm SE resolution if necessary. The freezing technique and the thickness of the section were the determining factors in limiting the resolution. For true ultrastructural studies, thinner sections and transmission imaging are necessary.'5,6 To test the reproducibility of the data collection from these frozen sections, we used three procedures. The first consisted of repetitively analyzing the same raster. With these thick sections, no deterioration in the Xray signal was observed. This finding is important because of the significant problem of mass loss in thin sections.7 In 10 consecutive analyses of the same raster, the phosphorus count rate varied from 64 to 70 cps (mean 68.0 + 2.58, s.d.). At the same time the potassium count rate varied from 73 to 80 cps (mean 76.5 ± 2.32). The second involved analyzing multiple areas of a section. In fourteen rasters within histologically normal tissue analyzed under identical conditions, the phosphorus count rate varied from 60 to 97 cps (mean 78.3 ± 8.8), and the potassium count rate varied from 60 to 103 cps (mean 78.2 ± 12.2). These ranges reflect possible micro differences in composition and/or thickness of the section. A third study involved storing the frozen tissue block at -70 C for 2 weeks and then cutting an additional cryosection for SEM and EDXA examination. The values obtained from the second analyses were indistinguishable from the first. The elements, phosphorus and potassium, used above were chosen only as examples; all the elements examined showed similar consistency. With sodium, for example, repetitive analyses of the same raster revaled a range of 5-9 cps (mean 7.3 ± 1.3). The second analysis test showed a range of 2 to 14 cps (mean 8.5 ± 4.1). The poorer accuracy for sodium than for P and K is the result of its lower concentration, and most of all its lower energy X-ray emission, which is readily absorbed by the tissue and the X-ray detector itself. This makes Na determination more susceptible to variation than elements with higher energy X-rays and higher concentrations in the tissue. The absolute detection limits with the use of our technique are not yet known. Comparison of reported concentrations of elements in normal and abnormal myocardium (determined by quantitative microprobe 8 or bulk chemical 9 analysis) with our observed count rates allows some estimation. Potassium is detected in normal tissue at its concentration of 1.67% dry weight 8 and could be detected at one-tenth or less of this concentration, ie, approximately 0.1% dry weight. The normal Na concentration of 0.43% dry weight is near the detection limits under the conditions we were using, but the increased concentration with ischemia (1.31% dry weight)8 is easily detectable. Although Ca is present in physiologically im-



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portant concentrations in normal myocardium (0.003% dry weight 9), these levels are below the detection limits of this preparation and analytic system. We easily identified the discrete calcifications observed in ischemic myocardium.'0 Our detection limit for iodine is on the order of 0.1% dry weight, based on the reported values of bulk analysis of similar tissues 2 and on observed count rates for a pure specimen of contrast medium. Analysis of Normal Myocardium

The count rates for the elements analyzed are shown in Table 1. In the histologically normal myocardium, the K/Na ratio, as well as other elements, showed only small variation from one edge of the section to the other. We observed no I or Ca peaks above background in the normal sections. Analysis of Ischemic Myocardium

Elemental analysis of histologically infarcted myocardium showed dramatic differences from the normal. The results are shown in Figure 1 and Table 2. As the border of the infarct was reached in the sequence of rasters analyzed, the K/Na ratio fell precipitously at the margin between normal and infarcted myocardium. As the region of infarction was reached in the sequence of analyses, iodine was detectable at the margin of the infarcted myocardium. The iodine count rate rose to a maximum level as the K/Na ratio decreased. The congruence of these changes strongly implies that the iodine is selectively present within the ischemic myocardial cells. Cells with marked calcification (easily detected in the BSE image, Figure 2) were not often the same cells as those containing the iodine. Cells containing iodine were located adjacent to and interspersed with cells having high calcium content. In a few instances, iodine and high calcium Table 1-Results of X-Ray Microanalysis of Normal Myocardium *

Na P S CI K Ca I(La) I (Lfi)

Mean + SD of 14 rasters

Left edge

Center

Right edge

2 77 64 31 73 4 6 7

3 60 57

13

8.5±4.1

80

78.3 +8.8 74.4 ± 16.8 32.5 ± 6.7 78.9 + 12.2 6.2 + 2.8

27 67 6 10 8

69 29 77 4 6 8

7.2±1.4 6.1 ± 1.9

* Uncorrected net counts per second of some regions analyzed in a single section (see text).

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Table 2-Results of X-Ray Microanalysis of lschemic Myocardium

Na P S Cl K Ca I (La) I (L,O)

Histologically most normal

Border of infarcted and normal

Infarcted

11 75 64 43

23 84 86 86 65 11 18 14

33 70 129 129 48 14 36 24

77 11

9 9

* Uncorrected net X-ray counts per second for 3 selected areas of section.

content were detected in the same myocardial cells. The inclusions containing calcium and phosphorous were identical to those that developed in mitochondria as a response to ischemia.5 8,10 All 3 dogs showed similar changes from normal to ischemic regions of myocardium. The distribution of ischemic changes varied with the choice of section and plane of section, making the codistribution of I and Na, K, and so forth, a most useful determination. Discussion

The current study revealed that systemically administered iodinated contrast material enters ischemically damaged myocardial cells, while it is virtually excluded from the normal myocardial cell. The myocardial cell with high iodine content showed a distinctly lower K/Na than normal myocardial cells. This finding implies that these cells have been sufficiently damaged to disrupt membrane function or integrity. Since iodine increases the X-ray attenuation value of tissues, it facilitates detection of myocardial infarction on computerized transmission tomography.1-3 The results of the current study suggest that the recognition of differential accumulation of iodine, and by inference contrast material, indicates and is a marker on CTT scans of loss of myocardial cellular membrane integrity. The approach we used for elemental analysis of iodine content and other elements employing unfixed cryosections of light-microscopic dimensions and SEM plus EDXA analysis is much simpler than some previously described techniques,5'6"10"'1 although it does not permit ultrastructural resolution. In studies requiring only the cellular level of resolution in correlation of tissue morphology with soluble elements, this approach should be readily applicable, eg, in forensic studies. The factors involved in producing and interpreting changes in the ratio of potassium to sodium in ischemic tissue have been discussed in detail by McVie and others.'2"-8 Bulk tissue levels of K and Na were quite stable de-



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spite what would be considered undesirable tissue handling, such as storage at room temperature or even 37 C for a few days.'2 The recent report by Hagler, Sherwin, and Buja " provides some data with which to compare our results, even though the methods are not identical. Hagler et al compared different methods of tissue preparation in an analytic electron-micioscopic study of plastic embedded and ultracryotomy sections. They confirmed that most calcium, phosphorous, and other ions are lost from conventionally fixed and embedded tissue. With non aqueous fixation such as alcoholic aldehyde (80% ethanol, 20% aldehyde fixative), there is better retention of calcium and phosphorous even in sections embedded in plastic. Of course, the high content of chlorine in conventional epoxy resin precludes analysis for chlorine. No sodium or potassium was detectable in the plastic embedded sections even with alcohol fixation. In contrast, quick-frozen ultracryotomy sections showed detectable potassium, sodium, chlorine, and phosphorous even in the absence of detectable calcium. Sodium was not evaluated, since their sections were mounted on copper grids (in EDXA the copper L X-ray line overlaps the sodium K X-ray line). From Figures 5 and 7 of Hagler et al " we have calculated the net X-ray count rate for several elements for comparison with our results. For example, the potassium X-ray intensity in their control cytoplasm was 4.0 cps and 0.75 cps in the ischemic cytoplasm. The differences in section thickness (our 6 ,u vs their 100-200 nm) and in instrument operating conditions account for our higher observed X-ray intensities. What we gain is this increased sensitivity, and what we lose by our technique is spatial resolution. Earlier studies 2 showed a small amount of I in the normal myocardium. This finding is consistent with the absence of detectable I in the normal myocardium in this study for two reasons: 1) the previous work used 1-cm cubes of tissue, and 2) it used X-ray-induced fluorescence (XRF) rather than electron probe microanalysis (EPMA). XRF produces much less background radiation than EPMA and thus has a much higher signal-tonoise ratio, with greater sensitivity by 2 or more orders of magnitude. However, XRF does not permit the high spatial resolution possible with a focused beam of electrons. Since we analyzed only parenchymal myocardial fibers, this would suggest that the little amount of I present in the normal myocardium by bulk XRF analysis2 is largely in the extracellular space. Previous studies have shown a greater increase in X-ray attenuation of ischemically damaged compared to normal myocardium in CTT scans of the ex situ ' and in situ3 beating heart. This greater contrast enhancement

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of ischemically damaged tissue correlates with the several times higher content of iodine in the damaged compared with normal myocardium.2 Thus, iodinated contrast material serves the purpose on CTT scans that 99'l1Tc-pyrophosphate does for scintigraphic scans 16-18; it results in a positive image of the area of ischemic injury, in contrast to the negative image seen with thallium scintigraphy. While it is clearly established that `mTcpyrophosphate is accumulated within the irreversibly damaged myocardial cell,'7 the concentration of iodine in the damaged myocardium has not been known. The current study has clearly indicated that the iodine is present within the ischemically damaged myocardial cells and that these cells have lost normal cellular membrane function. The marked decline in the K/Na of the cells containing iodine suggests but does not prove that these cells are irreversibly damaged. It is not clear at this time whether reversibly injured cells accumulate iodine. References 1. Higgins CB, Siemers PT, Schmidt W, Newell JD: Evaluation of myocardial ischemic damage of varying ages by computerized transmission tomography: Time dependent effects of contrast material. Circulation 1979, 60:284-291 2. Higgins CB, Sovak M, Schmidt W, Siemers PT: Differential accumulation of radiopaque contrast material in acute myocardial infarction. Am J Cardiol 1979, 43:47-51 3. Lipton M, Brundage B, Doherty P, Berninger W, Redington R, Carlsson E: Cardiac computed tomography in patients with ischemic heart disease. Circulation 1978, 58(Suppl II): 194 4. Abraham JL, DeNee PB: Biomedical applications of backscattered electron imaging-One year's experience with SEM histochemistry, Scanning Electron Microscopy. Edited by 0 Johari. Chicago, IIT Research Institute, 1974, pp 251-258 5. Trump BF, Mergner WJ, Kahng MW, Saladino AJ: Studies on the subcellular pathophysiology of ischemia. Circulation 1976, 53(Suppl I):17-26 6. Gupta BL, Hall TA, Naftalin RJ: Microprobe measurement of Na, K and Cl concentration profiles in epithelial cells and intercellular spaces of rabbit ileum. Nature 1978, 272:70-73 7. Hall TA, Gupta BL: Beam-induced loss of organic mass under electron microprobe conditions. J Microscopy 1974, 100:177-188 8. Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klofman S, Reimer KA: Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog. Am J Pathol 1978, 92:187-214 9. Dittmer DS, Grebe RM (eds): Handbook of Ciruclation. Philadelphia, W. B. Saunders, 1959, p 38 10. Ashraf M, Bloor CM: X-ray microanalysis of mitochondrial deposits in ischemic myocardium. Virchows Arch [Cell Pathol] 1976, 22:287-297 11. Hagler HK, Sherwin L, Buja LM: Effect of different methods of tissue preparation on mitochondrial inclusions of ischemic and infarcted canine myocardium. Lab Invest 1979, 40:529-544 12. McVie JG: Postmortem detection of inapparent myocardial infarction. J Clin Pathol 1970, 23:203-209 13. Dennis J, Moore RM: Potassium changes in functioning heart under conditions of ischemia and of congestion Am J Physiol 1938, 123:443-447



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14. Crout JR, Jennings RB: An improved histochemical method for the demonstration of potassium. J Histochem Cytochem 1957, 5:170-177 15. Raiskina ME, Opalyeva-Stegantseva VA, Ratovskaya VI, Ostapova VN, Veber OP: Postmortem diagnosis of ventricular fibrillation by K and Na distribution in the myocardium and skeletal muscle in out-of-hospital sudden death from acute ischemic heart disease. Am Heart J 1977, 94:154-162 16. Shen AC, Jennings RB: Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 1972, 67:441-452 17. Buja LM, Parkey RW, Stokely EM, Bonte FJ, Willerson JT: Pathophysiology of technetium-99m stannous pyrophosphate and thallium-201 scintigraphy of acute anterior myocardial infarcts in dogs. J Clin Invest 1976, 57:1508-1522 18. Buja LM, Parkey RW, Dees JH, Stokely EM, Harris RA, Bonte FJ, Willerson JT: Morphologic correlates of technetium-99m stannous pyrophosphate imaging of acute myocardial infarcts in dogs. Circulation 1975, 52:596-607

Acknowledgments Mrs. Jaen Douglass provided excellent technical assistance with sample preparation.

A

Figure 1A-Light micrograph of cryosection of myocardium showing normal and infarcted myocardium (see text). B-SEM, secondary electron image of serial section to that shown in A. Line across which areas were analyzed is superimposed. CDistribution of X-ray intensities for Na, K, and I along line shown in B. Note drop in K and rise in Na and I associated with the infarcted tissue vs normal. DEDXA spectrum (prior to background subtraction) from ischemic myocardium showing labeled peaks for elements and also displaying background (dotted line ). E-EDXA spectrum from normal myocardium.

Figure 2-4nfarcted myocardium, SEM, backscattered elec-

tron (BSE) image showing intracellular granules containing calcium phosphate (dark in this negative BSE image).

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