Morphometric evaluation of volume shifts between intra- and extra-cellular space before and during global ischemia.код для вставкиСкачать
THE ANATOMICAL RECORD 241:319-327 (1995) Morphometric Evaluation of Volume Shifts Between Intra- and Extra-Cellular Space Before and During Global Ischemia A. SCHMIEDL, G. HAASIS, PH.A. SCHNABEL, M.M. GEBHARD, AND J. RICHTER Department of Anatomy, Division of Electron Microscopy (A.S., J.R.), Department of Physiology, Division of Vegetative Physiology and Pathophysiology (G.H., M.M.G.), University of Gottingen, Gottingen, and Departments of Pathology (PH.A.S.)and Experimental Surgery (M.M.G.), University of Heidelberg, Heidelberg, Germany ABSTRACT Background: It is well known that all forms of cardiac arrest lead to global ischemia combined with alterations in cellular and interstitial volume. The aim of this study was to investigate the nature of these alterations with respect to different methods of cardiac arrest and establish the extent of their mutual influence at the onset as well as during the course of global ischemia. Methods: Three tested clinical methods were employed to induce cardiac arrest by a) aortic cross clamping, b) coronary perfusion with the cardioplegic solution St. Thomas, and c ) coronary perfusion with the cardioplegic solution histidine-tryptophane-ketoglutarate(HTK).The arrested hearts were subjected to global ischemia at 25°C. The size of the myocytes, as well as the interstitial space of myocytes, was determined morphometrically. The contraction state of myocytes was evaluated according to a score. Results: We found that the degree of contraction, as well as nature of alterations in the cellular and interstitial volumes, depended both on the form of cardiac arrest and on the duration of ischemia. The following relationships were established. High contraction at the onset of ischemia leads to expulsion of fluid from the interstitium between bundles of myocytes into the tissue clefts increasing their size. The decrease in contraction during ischemia leads to narrower tissue clefts. Cellular swelling at the onset of and during ischemia is caused by volume shifts between intracellular and interstitial space. An increase in cellular volume during global ischemia and/or additional contraction reduce the interstitium within bundles of myocytes. Sufficient relaxation and/or interstitial edema enlarge the interstitium. Conclusions: Cellular and interstitial alterations seen at the onset and during the course of ischemia are dependent upon the method of cardiac arrest. Furthermore, a considerable mutual influence is exerted by the alterations in cellular and interstitial spaces. o 1995 WiIey-Liss, Inc. Key words: Heart, Dog, Cardioplegia, Cardiac arrest, Ischemia, Morphometry, Interstitial space, Contraction state Myocardial cells show physiological alterations of their volume and contraction state during the cardiac cycle (Caulfield and Borg 1979; Borg and Caulfield 1981). On the other hand, cardiac arrest and the resulting global ischemia lead to defined pathological cellular alterations. Mitochondria show loss of their matrix and cristae, as well a s swelling, cellular edema, and alterations in the contraction state (Katz, 1973; Hearse et al., 1977; Pine et al., 1979; Poole-Wilson et al., 1979; Jennings and Reimer, 1981; Tranum-Jensen et al., 1981; Vanderwee et al., 1981; Schmiedl et al., 1989; Schnabel et al., 1990).The occurrence and degree of alterations depend on the method used for cardiac arrest (Bretschneider, 1964; Paulussen et al., 1968; 0 1995 WILEY-LISS, INC Schnabel et al., 1987, 1990; Gebhard et al., 1987; Schmiedl et al., 1990b). Different procedures can be performed to introduce cardiac arrest, resulting in a n interruption of blood flow, and, therefore, in a global myocardial ischemia. After inflow occlusion by cross clamping of the venae cavae and additionally the aorta, the heart begins to fibrillate. The reduction of its creatine phosphate level Received January 31, 1994; accepted September 15, 1994. Address reprint requests to Dr. rer. nat. Andreas Schmiedl, Zentrum Anatomie, Kreuzbergring 36, 3400 Gottingen, Germany. 320 A. SCHMIEDL E T AL. leads to cardiac arrest in systole, combined with a modest ischemic tolerance and a n early occurrence of cellular alterations (Bretschneider, 1964; Paulussen et al., 1968). Hypothermia and introduction of cardiac arrest by coronary perfusion with cardioplegic electrolyte solutions leads to a cardiac arrest in diastole. The specific electrolyte composition of these cardioplegic solutions (see Materials and Methods) leads to a cardiac arrest within seconds without a phase of fibrillating. Here, creatine phosphate is not used up. The decay of energy rich phosphates and the cellular alterations during ischemia take place in slow motion (Paulussen et al., 1968). Therefore, myocardial protection with cardioplegia leads to significantly better tolerance to ischemic stress (Bretschneider, 1964; Gebhard et al., 1987). The volume of the interstitial space after cardiac arrest and during global ischemia may be influenced in a variety of ways. Not only a n interstitial edema but also a n increase in contraction, as well as swelling of myocytes, for example, can alter the interstitium (PooleWilson et al., 1979; Borg and Caulfield, 1981; Schmiedl e t al., 1989). Therefore, for a precise evaluation of interstitial and intracellular alterations and possible mutual inf luences, both compartments should be investigated in the same sample. Morphometric methods allow a reproducible, exact definition of all parameters necessary to evaluate these alterations. Whereas the use of extracellular tracers leads to a remarkable variability of values (Polimeni, 1974; Reil et al., 1987), the size of the interstitial space should be defined morphometrically as discussed earlier (Schmiedl et al., 1991). For morphometry, i t was useful to separate the myocardial interstitial space into two types of reaction spaces, which were evaluated at different magnifications using a light microscope (Schmiedl et al., 1990a; Haasis et al., 1991). The first type, known as a tissue cleft (Henle, 1876), serves as a disposal sink during relaxation-contraction cycles (Feneis, 1944). The second one, called the interstitium within bundles of myocytes, contains connective tissue and capillaries. The aim of this study was to i n v e s t i g a t e gos. The vena cava superior and inferior, as well as the aorta and the arteria pulmonalis, were taped with tourniquet bands. Cardiac arrest was introduced by aortic cross clamping (ACC), in which the taped tourniquet bands were drawn together. For surface cooling, Tutofusin solution (comp. in mmol/L: 140 N a + , 5 K + , 2.5 C a + + , 1.5 M g + + , 153 C1-; Pfrimmer, Erlangen, Germany) was used. After a phase of fibrillation the lack of creatine phosphate led to a halt of contractile activity. For coronary perfusion with cardioplegic solutions, a polyethylene catheter was advanced up to the aortic valve via the arteria subclavia. Immediately after tightening of the taped vessels, coronary perfusion was started using a roller pump. With the onset of the perfusion the aortic valve closed and the perfusat ran into the coronary system. For aspiration, the right atrium was opened. Volume relief of the left ventricle followed by opening the left atrium and by a n incision near the apex. The following cardioplegic solutions were used: 1. The cardioplegic solution St. Thomas (composition in mmol/L: 91.1 NaC1, 14.8 KC1, 15 MgCl,, 1.2 CaCl,, 25 NaHCO,, 1.2 procaine.HC1, 1.2 MgSO,, 1.2 KH,PO,; Dr. F. Kohler, Alsbach, Germany). 2. The cardioplegic HTK solution (com. in mmol/L: 15 NaC1, 9 KC1, 4 MgCl,, 180 histidine, 18 histidine.HC1, 30 mannitol, 2 tryptophane, 1 K- Ketoglutarate [Custodial@]; Dr. F. Kohler, Alsbach, Germany). The coronary perfusion with St. Thomas solution lasted 4 min; the HTK solution, 11min. After this time complete equilibration of the interstitial space was achieved. The high K + content of the St. Thomas solution led to a continued depolarisation of the myocytes and therefore to a n arrest in diastole within seconds. The reduction of Na+ and Ca2+ to cytoplasmic values in the HTK solution led to a halt of electrical and mechanical activity and, therefore, to a n arrest in diastole within seconds. After excision, the hearts were incubated at 25°C in the solutions used for cardiac arrest. Samples from the subendocardial third of the free ventricular wall were taken a t the onset of global ischemia and at predefined 1. the interstitial and cellular alterations after dif- times during global ischemia at 25°C. During tissue ferent forms of cardiac arrest and following global isch- processing, the incubated ventricle was reduced from emia, the apex to the base, until the samples reached a tissue 2. in what way cellular alterations influence the in- concentration of 2 pmol/gww ATP-the theoretical terstitial space a t the onset and during the course of limit of resuscitation (Bretschneider, 1964). Lower ischemia, and ATP levels did not lead to a resumption of heart func3. the degree of volume shifts in the arrested and tion during reperfusion. ischemic ventricular myocardium of the heart. Samples were taken from the aortic cross clamped hearts over 3 hours every 15 min, from the St. Thomas MATERIALS AND METHODS arrested hearts over 4 hours, and from the HTK arThe experiments were carried out on the left ventric- rested hearts over 6 hours in each case, every 30 min. ular myocardium of canine hearts. Composition and Each method of cardiac arrest was carried out on 6 application of the cardioplegic solutions corresponded canines, respectively. to the original clinically accepted prescriptions (Hearse Immersion fixation of the samples followed in a fixet al., 1981; Gebhard et al., 1987). Anesthesia and ex- ation solution containing 1.5% glutaraldehyde and perimental procedure were performed as described 1.5% paraformaldehyde in 0.1 M sodium cacodylate elsewhere (Preusse et al., 1981).In brief, after premed- buffer a t pH 7.4 (half strength-Karnovsky) (Karication the anaesthesia was introduced with Trapanal@ novsky, 1967) with a n effective osmolarity of 440 mosand then continued with a combination of Dipidolor@ mol/L (Hayat, 1989). Postfixation, dehydration, and and N 2 0 / 0 2(3:l).Respiration was maintained with a n embedding were carried out continuously and automatendotracheal tube connected to a respirator. Median ically (Histomat, Bio-Med, Theres, Germany). Non-orithoracotomy was followed by ligation of the vena azy- ented semithin sections were stained with 1%azure 32 1 VOLUME SHIFTS IN CELLULAR SPACE Pathological Contraction TABLE 1. Multistage sampling technique Parameters Interstitial space Tissue clefts between bundles of myocytes (VVTJ Interstitium within bundles of myocytes (VVIS) Cellular space Bundles of myocytes contraction state Myocytes Cellular compartments Myofibrills (VvMf) Sarcoplasm (VVsJ Mitochondria (VVMf) Nuclei (VNc) Magnification x 100 x 1,000 x 20000 x 50000 and methylene blue. Ultrathin sections were stained with uranyl acetate and lead citrate. Quantitative investigations according to the point counting method (Weibel, 1979) were carried out on line. Light microscopical evaluation was carried out by using a n ocular integrated 100-point lattice test system (Integrationsplatte, Zeiss, Oberkochen, Germany). For electron microscopical evaluation a n EM 10 (Zeiss, Oberkochen) equipped with a TV camera was used. A 72-point lattice test system was mounted on the monitor-screen. Three samples per canine heart and time point were evaluated. Parameters were determined according to a multistage sampling technique (Table 1). The volume densities of the interstitial space were determined in 30 test fields per sample. The cellular volume densities were determined in 50 test fields per sample. The cellular volume densities were related to the myocardial cells as reference unit. The cellular volume was determined by the fraction V V M ~+ VvsPl VVMf, which is regarded as a parameter of cellular edema, independent of the reference space (DiBona and Powell, 1980). VvMf can be related to the bundles of myocytes of the left ventricular myocardium. Therefore, VVMf was related to l-VvTe, which corresponds to the bundles of myocytes as reference space. If this parameter remains constant during ischemia, the relative decrease of VVMf is caused by cell swelling and VVMf is a n additional parameter for cellular edema. The contraction state of sarcomeres was determined in 30 test fields according to a classification, described elsewhere (Schnabel et al. 1990, Schmiedl et al. 1993). Physiological Contraction Relaxation I-bands are always visible. Contraction I-bands have generally disappeared; Z-band spacing is slightly wider than the A-band, visible under physiological conditions. Relaxationkontraction (incomplete relaxation) Relaxed and contracted sarcomeres are found in one test field. Overcontraction Submaximal contractions resulting in A-band compression and apparent thickening of Z-bands. Hypercontraction Submaximal to maximal contractions, which lead to A-band compression and additionally to clumping of actin and myosin filaments. Contraction band Maximal shortening of sarcomeres which, in addition to actidmyosin clumping, leads to distortion of the contractile system. This formation is regarded as irreversible damage (Ganote, 1983). All results are given as mean values 2 SD. Significant differences were noted for P values of 0.05 or less using the WilcoxonMann-Whitney test for unpaired samples and the Wilcoxon matched paired signed rank test for paired samples. RESULTS In the following, alterations of the cellular and interstitial morphometric parameters after different types of cardiac arrest at the onset as well as during ischemia are described. Additionally, the values obtained by morphometry of interstitium and myocytes are compared with each other in order to obtain informations on possible mutual influences. Onset of Ischemia In hearts without ischemic stress, the cellular volume and the degree of contraction, as well as the size of the interstitial space, depend on the form of cardiac arrest (Figs. 1-4). Cellular parameters The quotient VVMi+ Vvsp.NVMfused as a parameter for cellular edema differed in the cardioplegically arrested hearts. Myocytes of HTK arrested hearts were significantly less swollen than myocytes of St. Thomas arrested hearts. The values for the cellular edema of St. Thomas arrested hearts corresponded to those of cross clamped hearts (Fig. 2a). The percentage of test fields with relaxed sarcomeres was significantly higher after perfusion with HTK solution than after ACC. The relaxation in St. Thomas arrested hearts was negligible (Fig. 2b). ACC and cardiac arrest with St. Thomas solution led to a remarkable degree of pathological contractions, e.g., over- and hypercontractions (Fig. 3). Interstitial parameters and the influence of cellular parameters The size of tissue clefts (VVTc)after aortic cross clamping (ACC) and after coronary perfusion with cardioplegic solutions differed significantly (Fig. 4a). ACC resulted in a significantly higher VVTccompared with hearts arrested by coronary perfusion. In St. Thomas arrested hearts the values for VVTc were somewhat higher compared with HTK arrested hearts. The size of the interstitium within bundles of myocytes (VvIs) in cross clamped hearts (Fig. 4b) was significantly reduced by swelling and contraction of myo- 322 A. SCHMIEDL ET AL. Fig. 1. Interstitium within bundles of myocytes (a; x 960) and myocytes surrounding a capillary (b; x 5,000) after aortic cross clamping. Contracted or overcontracted (arrow) and more or less swollen myocytes due to, in part, very small interstitial spaces. Interstitium within bundles of myocytes fc; x 960) and myocytes (d; x 5,000) after coronary perfusion with St. Thomas solution and 60 min of global ischemia. Contracted and swollen myocytes do not lead to a significant decrease of the interstitium within bundles of myocytes because of interstitial edema. Interstitium within bundles of myocytes (e; x 960) and myocytes (f; X 5,000) after coronary perfusion with HTK solution and 120 min of ischemia. The swollen, predominantly relaxed myocytes do not significantly influence the size of the interstitium. 323 VOLUME SHIFTS IN CELLULAR SPACE O'" CELLULAR EDEMA T T St. THOMAS 0.6 HTK 0.1 0.2 0 10 0 a 120 60 180 GLOBAL ISCHEMIA AT 2 5 'C (min) t RELAXATION (%) le3 4 I , THOMAS 80 I T 68 0 HTK 18 20 0 b 10 m GLOBAL ISCHEMIA AT 25 'C (min) Fig. 2. a: Cellular swelling determined by , V + vvsp / V, after different forms of cardiac arrest and global ischemia a t 25°C. b Contraction state of sarcomeres (% of test fields) after different forms of cardiac arrest and global ischemia at 25°C. *Significant alterations (P< 0.05) compared to the onset of ischemia cytes compared to that of cardioplegically arrested hearts (Fig. 2b). In HTK arrested hearts the significantly less pronounced cellular edema and the high degree of relaxation led to the highest VvIs (Figs. 2a,b; 4b). After St. Thomas perfusion, myocytes showed high contraction and swelling. However, a n interstitial edema led to values of VvIs comparable to those after HTK perfusion (Fig. 4b) During Ischemia Cellular parameters After ACC, the parameter for cellular swelling, the quotient VVMi + Vvsp /VVMf,increased continuously during ischemia. After 60 min the cell volume increased significantly compared with the onset of ischemia. After St. Thomas perfusion, as well as after HTK perfusion, cell volume after 120 min of ischemia was significantly higher compared to the onset of ischemia (Fig. 2a). After ACC, test fields with relaxed sarcomeres increased significantly after 60 min of ischemia, remained constant between 60 and 120 min, and decreased after 180 min of ischemia. After St. Thomas perfusion, relaxation was significantly pronounced after 60 min of ischemia. However, the percentage of test fields exhibiting relaxation was significantly lower compared with the other methods of cardiac arrest in- 324 A. SCHMIEDL ET AL. PATHOLOGIC CONTRACTION - ACC ... S t . Thomas HTK creased after 10 min of ischemia. During the following time VvIs did not alter significantly, because the swelling of myocytes was compensated for by the almost complete relaxation (Figs. 2a,b; 4b). Thus, the decrease and constancy of VvIs during global ischemia also depends on the method used for cardiac arrest and even more on the cellular alterations. Degree of volume shifts at the onset and during ischemia 0 80 120 [min] Global Ischemia a t 2 5 ° C Fig. 3. Pathological contraction after different forms of cardiac arrest and global ischemia at 25°C. *Significant decrease (P < 0.05) compared to the onset of ischemia. vestigated (Fig. 2b). After HTK perfusion, myocytes showed a higher degree of relaxation compared with the other methods of cardiac arrest. In comparison to the onset of ischemia, relaxation increased significantly after 180 min (Fig. 2b). Interstitial parameters and the influence of cellular parameters After ACC, the decrease of VvTc over 60 min was related to the significant relaxation (Fig. 2b, 4a). During further ischemic stress the decrease of relaxation combined with a renewed occurrence of contraction led to a n increase of VVTowhich was significant after 180 min, compared to the onset of ischemia (Fig. 4a). After St. Thomas perfusion, the significant decrease of VvTc over 10 min was related to a decrease of pathological contraction (Figs. 3,4a) and a decrease of fluid concentration occurring during the equilibration process. The slight increase of VvTc during the following time of ischemia was related to a continued high contraction of myocytes (Figs. 2b, 4a). After HTK perfusion, VvTcexhibited a significant decrease after 60 min, which was caused by the significant increase of relaxation (Fig. 2b, 4a). In the following ischemic period only slight alterations occurred. Thus, the time course of alterations of the size of tissue clefts differed from method to method used for cardiac arrest and was influenced by the occurrence of cellular alterations. The size of the interstitium within bundles of myocytes (VvI,) after ACC decreased significantly over 60 min and was related to the significant increase in cell volume (Figs. 2a, 4b). The constancy of VvIs up to 120 min was related to a simultaneous increase in cellular volume and relaxation. After 180 min, VvIs remained almost constant, although swelling and contraction of myocytes significantly increased (Figs. 2a,b; 4b). After St. Thomas perfusion, VvIs did not significantly alter during 120 min of ischemia. The significant swelling combined with a n insufficient relaxation of myocytes (cf. Fig. 2b) led only after 180 min of ischemia to a significantly lower VvIs compared to the onset of ischemia. After HTK perfusion, VvIs significantly de- The volume density of myofibrils (VVMf)was comparable after ACC and St. Thomas perfusion, but significantly lower than after HTK perfusion. During ischemia VVMf significantly decreased after 60 min in the aortic cross clamped hearts and after 120 min in the St. Thomas arrested hearts and the HTK arrested hearts (Fig. 5a). The relation of VVMf to the bundles of myocytes (VvM,/mb) of the left ventricular myocardium additionally included the interstitial space as reference unit. This parameter showed no alterations after ACC during 120 min of ischemia, but showed a significant decrease following 180 min. After St. Thomas and HTK perfusion and following ischemia no significant alterations occurred. The approximate constancy of VVMfrelated to bundles of myocytes during a longer time of ischemia verifies that the relative decrease of VVMfwas caused by fluid movements from the interstitial to the intracellular space and not at all by loss of myofibrils. DISCUSSION Our results show that the size of the interstitial space of left ventricular myocardium a t the onset of ischemia as well as the way i t acts during ischemia varies among the different types of cardiac arrest. The multistage sampling method for the morphometrical determination of interstitial and cellular parameters has never been described before. With our methods, information on the reactions of interstitial and intracellular space can be obtained. Further, their mutual influence at the onset and during ischemia after different types of cardiac arrest can be investigated. Fixation Technique Recent investigations have shown that the technique of fixation (physical fixation, chemical fixation by immersion or perfusion) influences only the size of tissue clefts but not the interstitium between bundles of myocytes (Schmiedl e t al., 1991). Because of this, the application of immersion fixation for the investigation of different time points is possible without loss of information. The size of tissue clefts after immersion fixation is overestimated compared with physical fixation (Schmiedl e t al., 1991), but this applies to all methods of cardiac arrest investigated. The osmolarity of our fixation and postfixation solution (Hayat, 1989) prevent artificial shrinkage or swelling of the compact tissue (Tranum-Jensen et al., 1981). The volume changes during dehydration apply to the whole tissue block (Bone and Denton, 1971) independent of the type of cardiac arrest and does not lead to fluid shifts between interstitial and intracellular space. During embedding, no volume shifts occur (Gerdes et al., 1992). . 325 VOLUME SHIFTS IN CELLULAR SPACE 21 TISSUE CLEFTS ( X ) T T zz 20 i3 St. THOMAS 10 I6 11 1% 10 n b I 2 0 0 a 10 60 120 188 GLOBAL ISCHEMIA AT 25 'C (rnin) INTERSTITIAL SPACE ( X ) H ACC El St. THOMAS 0 HTK 10 b 60 120 180 GLOBAL ISCHEMIA AT 25 'C (rnin) Fig. 4. a: Size of tissue clefts (VVTJafter different forms of cardiac arrest and global ischemia a t 25°C. b Size of the interstitium within bundles of myocytes (Vvls)after different forms of cardiac arrest and global ischemia a t 25°C. *Significant inrease ( P < 0.05) compared to the onset of ischemia. Interstitial Space and Contractile Activity At the onset of ischemia the ATP concentrations are comparable in the differently arrested hearts (Schnabe1 et al. 1987; Schmiedl et al., 1993). Sufficient ATP levels in combination with physiological Ca2+ or in combination with the Ca2+ content in the St. Thomas solution leads to a high degree of contraction and pathological contraction in cross clamped or St. Thomas arrested hearts (cf. Figs. Zb, 3). Therefore, the size of tissue clefts is influenced by the greater or lesser remaining activity of the hearts and their ability to contract during contact with the fixation solution (Billingham, 1983; Schnabel et al., 1990; Schmiedl et al., 1993). This results in fluid shifts into the tissue clefts and a higher level of VVTc(cf. Fig. 4a). Using the cardioplegic solution HTK for coronary perfusion, the reduced values of calcium result in a significantly higher degree of relaxation without expulsion of fluid into the tissue clefts leading to the lowest values of VVTc(cf. Figs. 2c, 3, 4a). In aortic cross clamped and HTK arrested hearts, the more or less pronounced relaxation during ischemia minimizes the volume of fluid in the tissue clefts and leads to a decrease in VVTc(cf. Figs. Zb, 3, 4a). The increase of this parameter after ACC and 180 min of ischemia is the result of the onset of rigor mortis associated with the renewed occurrence of contraction (Hearse et al., 1977; Vanderwee et al., 1981). After St. Thomas perfusion, the insufficient relaxation of myocytes and a possible interstitial edema lead to continued high values of tissue clefts (cf. Fig. 4a). 326 A. SCHMIEDL ET AL. lnterstitium and Cellular Alterations VOLUME DENSITY O F MYOFIBRILS ' , * Y ~ .A 70 - -ACT #St H T Thomas K ~~ 80 50 40 v 2 > 30 20 10 0 0 a 80 Global I s c h e m i a a t 2 5 ° C VOLUME DENSITY O F YYOFIBRILS RELATED TO BUNDLES O F YYOCYTES - 70 8o - 80 El 40 c Bp Y 50 P 30 > P 20 10 0 h - 0 80 120 180 Global I s c h e m i a at 2 5 ° C Fig. 5. a: Volume density of myofibrils (V,,) after different forms of cardiac arrest and global ischemia a t 25°C. b: VVMfrelated to bundles of myocytes (V,,,,,) after different froms of cardiac arrest and following global ischemia a t 25°C. *Significant alterations ( P < 0.05) compared to the onset of ischemia. The size of the interstitium between bundles of myocytes (VvIs) was influenced by cellular edema, and by the different degree of contraction (cf. Figs. 2-4). Our results confirm earlier observations, which lead to the conclusion that a higher cell radius of contracting myocytes during systole results in a decrease in the size of the interstitial space (Pine et al., 1979; Borg and Caulfield 1981), and that diastole or mechanisms, which reduce contractility, such as acidosis expand the interstitial space (Poole-Wilson et al., 1979). However, the interstitium within bundles of myocytes can be also modified by additional alterations occurring during ischemia. The VvIs increased after ACC and 180 min in spite of additional increase of cellular volume and renewed occurrence of contraction compared to 120 min of ischemia (cf. Figs. 2a,b; 4b). The cause may be disturbances within the connective tissue leading to a disintegration of the collagen struts combined with a n insufficient connection to adjacent myocytes (Caulfield and Borg, 1979; Robinson et al., 1983). This would result in slippage of cells in either transverse or lateral directions and therefore to a n increase of the interstitium within bundles of myocytes (Borg and Caulfield, 1981). After St. Thomas perfusion and ischemia a n increase in cellular edema and a n insufficient relaxation occur. Both alterations do not significantly influence the size of the interstitium within bundles of myocytes, because the restriction is partly compensated for by the interstitial edema. After HTK perfusion and ischemia, VvIs remains almost constant, because the swelling of myocytes is compensated for by the almost complete relaxation. Therefore, the size of the interstitium within bundles of myocytes does not alter. Interstitial Space and Volume Shifts The constancy of the relation of VVMf to bundles of myocytes during a pronounced ischemic period has shown that the swelling of cells (decrease in VVMf)and the restriction of interstitium (decrease in VVIJ are the result of a shift of fluid from the interstitial to the intracellular space and not of myofibrillolysis. This disintegration of myofibrils seems to occur after ACC and Size of Tissue Clefts and Orientation of Myocytes Statements about the alteration in the size of tissue 180 rnin of ischemia, because the VVMf related to bunclefts after immersion fixation should always take into dles of myoctes decrease significantly compared to the consideration the different orientation of myocytes at onset of ischemia. Thus, the multistage sampling technique used the base, equator, and apex of the heart. The orientation of the myocytes a t the base and apex is almost allows investigation of interstitial and cellular alteroblique to epi- and endocardium and at the equator ations during ischemia. With this method, addialmost vertical. This different orientation leads to a tionally, relations between interstitial and cellular pagreater size of the tissue clefts at the apex and at the rameters are possible and volume shifts between base than at the equator of the heart (Henle, 1876). interstitial and cellular compartments are recorded. This does not apply to our samples taken from the apex DEDICATION of the ventricle only at the onset of ischemia. During We should like to dedicate this publication to the ischemia, however, samples were taken from the incubated hearts by continuous diminishing of the ventricle former director of the Department of Physiology, Prof. from the apex to the equator and to the base (cf. Ma- Dr. Dr. h.c. H.J. Bretschneider, whose sudden death in terial and Methods). The oblique orientation of myo- December 1993 shocked and saddened us all. cytes in samples taken from the base may additionally ACKNOWLEDGMENTS enlarge the tissue clefts. This may explain the more or less pronounced increase of VVTcafter ACC and 180 We are indebted to Dr. H. Fehrenbach for encouragmin of ischemia, after St. Thomas perfusion and 120 ing discussions. We thank Ms. S. Freese, Ms. A. min of ischemia, as well as after HTK perfusion and Gerken, Ms. H. Hiihn, and Ms. M. Scheumann for skill180 min of ischemia (Fig. 4a). ful technical assistance; Ms. C. Maelicke, B.Sc., for the VOLUME SHIFTS IN CELLULAR SPACE English correction; and Ms. H. Altmann for support in preparing the manuscript. Parts of the results were presented at the 55th meeting of the Deutsche Gesellschaft fur Herz- und Kreislaufforschung in Mannheim (1989) and at the 86th meeting of the Anatomische Gesellschaft in Szeged (1991). 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