two steps i n t h e research. contain i c e (Photo. 2 ). In t h e second case (rapid freezing), i c e develops

Transcription

1 210. E F F E C T S OF F R E E Z I N G ON MUSCLE T I S S U E 8. J. LUYET A s announced i n t h e program of t h i s meeting, I was t o treat, i n my commmication, t h e e f f e c t s of freezing and of freeze-drying on muscle t i s s u e. But, on account of t h e limited t i m e a t my disposal, I s h a l l limit myself t o t h e e f f e c t s of freezing. Our mode of approach i n t h a t study has been t o examine: (I) t h e process of invasion and occupation of t h e t i s s u e s by ice, and (11) t h e changes brought about i n them as a r e s u l t of such invasion and occupation. The present paper will be divided i n t o two p a r t s corresponding t o these two steps i n t h e research. A basic d i s t i n c t i o n t o make, at t h e st&, i s whether t h e muscle i s a l i v e o r dead when frozen. The two s t a t e s i n t e r e s t two groups of professionals: t h e physiologists and the food technologists. The physiologists may w a n t t o preserve t h e muscle a l i v e by freezing, t h e food technologists are satisfied with t h e preservation of dead t i s s u e, which they designate as meat. (One may foresee a t i m e when we w i l l freeze e n t i r e animals and preserve them a l i v e u n t i l we are ready t o k i l l them.) The terms l i v e o r dead, o r muscle and meat, c a l l f o r qualificat i o n s. The terms muscle i n prerigor and i n r i g o r are, i n a sense, more specific, and I s h a l l use them, though t h e i d e a l would be t o know, i n each case, which properties and which functions are preserved i n a p a r t i c u l a r state. I. MODE OF INVASION AND OCCUPATION OF MUSCLF: BY ICE W e should distinguish here three main c s e s : (1) When freezing i s slow, t h e temperature remaining at t h e plateau of t h e freezing curve f o r a t i m e of t h e order of an hour; ( 2 ) When freezing i s rapid, the termperature remaining at t h e freezing plateau f o r a t i m e of t h e order of a second o r a f r a c t i o n of a second; (3) When freezing i s extremely rapid, as when one immrses a single muscle f i b e r i n a bath of isopentane at -150 C; t h e temperature, then, does not remain at t h e freezing point f o r any e a s u r a b l e t i m e. The f i r s t method i s t h e one generally used i n t h e meat-freezing industry under t h e name of rapid freezing; t h e second and t h i r d methods are, at present, used only i n laboratory experiments. I n t h e f i r s t instance (slow freezing), i c e forms large masses (C i n Photograph 1 of Fig. 1) between bundles of fibers (B) which it pushes a p a r t ; t h e fibers, dehydrated i n t h e course of freezing, do not contain i c e (Photo. 2 ). In t h e second case (rapid freezing), i c e develops within t h e fibers, where it forms t h i n and long spears oriented longit u d i n a l l y (they are seen i n cross sections i n Photos. 3 and 4 ). I n t h e

2 211. t h i r d (very rapid freezing), t h e i n t r a c e l l u l a r ice spears are gradually thinner f r o m t h e center t o t h e periphery; at the periphery where the cooling rate i s t h e highest, the formation of i c e might be e n t i r e l y impeded (Figs. 2 and ZA), but we have no way of ascertaining it, f o r p a r t i c l e s of ice less than 100 angstroms across may not be detectable. (The growth of i c e spears i n the longitudinal direction, within single fibers, i s i l l u s trated i n the motion picture film.) (For mre d e t a i l s, c f. Luyet, 1961, 1962, Rapatz and wet, 1959, and Menz and Luyet, 1961). There are, of course, other conditions than t h e cooling rates which affect t h e node of i c e formation and expansion, but I won't have time t o discuss them. 11. CHANGES IN S T R U C m AND FUNCTIONS OF MUSCLl3 CAUSED BY FFEEZING The p r i n c i p a l changes caused i n muscle by freezing and/or thawing are: (a) a dehydration and shrinkage, (b) an impairment of t h e physiologic a l functions, (c) a disturbance of the s t r u c t u r a l framework, (d) chemical alterations, (e) a dramatic breakdown designated as "thaw-rigor" phenomenon which takes place upon thawing. Since the e s s e n t i a l s of dehydration and shrinkage have been i l l u s t r a t e d i n Fig. 1, and the chemical changes are extensively covered i n t h e literature, I shall confine myself here t o some observations on t h e other three categories of changes. Impairment of Physiological Functions (8) Freezing, by p r a c t i c a l l y any method, causes some damage t o l i v i n g muscle. L i t t l e i s known on the relationship between t h e damage produced and (a) t h e amount of i c e formed, and (b) the mde of freezing. I shall mention here two series of experiments which we made t o answer these questions: one on isolated muscle of rats, t h e other on the e n t i r e foot of mice. After freezing at -8 and exposure f o r 10 minutes i n t h e frozen state at t h e specimen temperatures recorded i n Graph C of Fig. 3, the rat muscle d i d not resgond t o stimulation. Neither did it when, after being supercooled t o -10 it suddenly froze and was kept i n t h e frozen state f o r only one minute. After freezing according t o the schedule recorded i n Curves A and By when t h e temperature did not drop below t h e freezing plateau (except f o r a short supercooling), and when t h e t i m e i n t h e frozen state did not exceed 10 t o 15 minutes, the muscles responded t o e l e c t r i c stimulation. The amount of ice formed i n t h e l a t t e r case exceeded 50% of t h e w a t e r. (Luyet and collaborators, reports i n press.), Thus, muscle can support t h e formation of q u i t e a l a r g e m u n t of i c e i n it, provided it i s not maintained i n the frozen state f o r mre than a f e w minutes. Similar r e s u l t s were obtained i n preliminary experiments with muscles i n s i t u i n t h e rat's f o o t. (For mre d e t a i l s, see Luyet, 1964.) (b) Msturbance of Structural Framework Slow freezing may permit t h e fibers t o be dehydrated and t o shrink without disturbance of t h e f i b r i l l a r and filamentous meshwork; however, we

3 212. have no experimental evidence for that possibility. Rapid freezing, resulting in intracellular ice, causes an aggregation of the fibrils and the filaments which are compressed between the ice spears, but we do not know whether this condition is reversible or not. Our studies, by light and electron microscopy, of the disturbance of the striations and the lgyofilaments in prerigor and in rigor muscle, nonfrozen and frozen-thawed, led to the following okfervations, which I present as preliminary: Whereas non-frozen, prerigor and in rigor muscles can hardly be distinguished, except in the degree of contraction, and possibly in the thickness of the z line (which seem thicker in the muscle in rigor), there is a marked difference between the frozen-and-thawed prerigor and in rigor muscle. The tissue frozen while in rigor resisted the disturbing action of freezing and thadng, that frozen in prerigor generally shows the enomus disturbance designated as "thaw-rigor" phenomenon now to be described. (Other differences between prerigor and in rigor muscles are illustrated in Fig. 8.) (c) Thaw Rigor The thaw-rigor phenomenon is characterized, in particular, by the following features: (1) an enomus shortening of the muscle upon thawing (compare photographs 1 and 5 in Fig. 4); (2) a considerable widening (same photographs); (3) a release of a pool of fluid; (4) the obliteration of the striations; (5) a disintegration of the fibrillar structure, the contents of the fibers being transformed into a floculent mass (Fig. 5); (6) the formation of large irregular transversal bands (Fig. 6); (7) intense contortional movements; (8) contraction of freeze-dried muscle upon being rehydrated after having been stored in the freeze-dried state. We shall now examine more closely some of these changes. 1. The Obliteration of the Striations: If a muscle is permitted to shorten fully during thawing, most of the striations disappear. Those remaining in a few places have very short sarcomeres, as one would expect to find in cases of strong contraction. If the muscle is not permitted to contract freely, as when a small bundle of fibers is allowed to thaw between a slide and a coverslip, the striations may be maintained for some time (15 minutes or mre) before they disintegrate. What they finally leave behind is the floculent mass mentioned above (Fig. 5). 2. The Irregular Bands. We are giving particular attention, in our work, to the mechanism of formation of the irregular bands mentioned by several investigators and examined in a recent study by Cassens, Briskey and Hoekstra (1963). (Figs. 6 and 7 are reproductions of photographs from that study.) The authors point out that one can trace remnants of fibrillar structure in the bands (Fig. 7) and propose that the latter results from the severe contraction involved in thaw-rigor. Bendall and Wismer-Pedersen (1962) had attributed them to a precipitation of sarcosplasmic proteins. The preliminary information that we have from both light and electron microscopy suggests that the irregular bands result fromthe juxtaposition of distorted striations occurring after the thaw-rigor contraction (for details see paper in press by Menz and Luyet). 3. Contortional Movements. These particularly striking movements are illustrated in the motion picture film.

4 Contraction of Freeze-Dried Muscle upon Rehydration. Also illustrated in the film is the remarkable property of freeze-dried muscle to undergo the "thaw-rigor" process, when it is rehydrated, after it had been stored, for possibly long periods of time, in the dry state. Appendix Conmarison of Preriaor and In-Rigor. Freeze-Thawed and Cooked Muscle The interesting pictures showed by Dr. Paul on the effects of cooking on the structure of muscle should be compared with the electronmicrographs that we obtained in a study of the effects of (a) freeze-thawing in prerigor and in-rigor fibers (Fig. 8, Photos. 1 and 2, respectively), and (b) cooking in prerigor and in-rigor fibers (Fig. 8, Photos. 3 and 4, respectively). Whereas the structure of the in-rigor muscle "stays put," so to say, and is little affected by freeze-thawing, or by cooking (Photos. 2 and 4, respectively), prerigor muscle undergoes the great turmoil described as the thaw-rigor phenomenon (Photo. l), and the no-lesser turmoil resulting from cooking (Photo. 2). There are interesting points of comparison between Dr. Paul's photographs and ours. REFERENCES Bendall, J. R. and J. Wismer-Pedersen. J. Food Sci. 27, 144, Cassens, R. G., E. J. Briskey and W. G. Hoekstra. Nature 197, 1119, 19638; and 1004, 1963b. Luyet, B. Ibw Temp. Microbiology Symposium. Campbell Soup Co., Camden, New Jersey 63, Lu>-et, B. J. Freeze-Drying of Foods Natl. Acad. of Sci. Natl. Res. Council 194, Luyet, B. Annals Finnish Acad. Sci., (Paper presented at the Symposium on Hibernation in Helsinki, in 1962.) Menz, L. J. and B. Luyet. Biodynamica 5 261, Rapatz, G. and B. Luyet. Biodynamica l3, 121, 1959.

5 214. Legends f o r t h e Figures of Paper by B. Luyet Fig. 1. Effects of slow and rapid freezing on muscle t i s s u e. -Photo. 1: Cross section showing large i c e masses C between t h e fibers B, i n slowly frozen t i s s u e. Photo. 2: Portion of t h e same preparations at a higher magnification. The dehydrated f i b e r s contain no i c e. Photos. 3 and 4: Cross sections of fibers frozen rapidly (Photo. 3) and very rapidly (Photo. 4), showing i n t r a c e l l u l a r i c e ( t h e white areas o r d o t s ). The fibers are not shrunken. Magn.: Photo. 1: 130x; Photo. 2: 1000~; Photo. 3: 440x; Photo. 4: looox. (Selected from Luyet's and from Rapatz and Luyet's f i l e s and publications.)

6 215. Fig. 2. Electron micrograph of cross section of muscle fiber after ultra-rapid freezing (by immersion of single fiber in isopentane at -150') followed by vacuum sublimation of the ice. In the lower part of the photograph, that is, in the mre central regions of the fiber, there are ice cavities (white spots) msasuring 2300 angstroms. The size of the cavities decreases gradually fromthe central to the peripheral regions until, near the edge (upper part of photograph), there is no evidence of cavities larger than the space normally separating the myofilaments (about 150 angstroms.) Magn: 17500~. (From Menz and Luyet, 1961; reproduced by permission of Biodynamica.)

7 216. Fig. 2A. Magni- f i e d view of an area of t h e upper part of electron micrograph shown i n F i g. 2. Magn.: 56,000~. (From Menz and Luyet, 1961; reproduced by permission of Biodynamic a. ) - Fig. 3. Tracings of temperature-time curves when a muscle from t h e l e g of a rat i s frozen i n baths at -3OC. (Curve A), o r at -5O (Curve B), or at -100 (Curve C ), then kept i n t h e frozen state f o r t h e time indicated i n abscissa and thawed. (The t i m e zero is, f o r each curve, t h e t i m e of (Selected from L u y e t ' s files; Work supported i n i t i a t i o n of freezing.) U. S. Air Force.) OC I 2 0"- IO0-0"- A - I

8 217. Fig. 4. Size and shape of t h e frogs sartorius after various successive treertments. Photo. 1: Stretched t o i n s i t u length. photo. 2: After excision. Photo. 3: After stimulation t o tetanus. Photo. 4: I n t h e frozen state. Photo. 5: After thawing, i n "thaw-rigor I' - M a g n. : Zx. (Selected from Menz and Luyet's f i l e s ; work supported by Campbell Soup Co.) Fig. 5. Obliteration of t h e s t r i a t i o n s following t h e "thawrigor" contraction. The striat i o n s s t i l l c l e a r immediately a f t e r thawing, i n a restrained specimen (photograph at l e f t ), are obliterated 15 minutes later (photograph at r i g h t ). Magn. 970x. (Selected from Rapatz and Luyet's f i l e s ; work supported by Campbell Soup Co )..

9 218. Fig. 6. Phase-contrast photograph of "Irregular Ban formed i n muscle which has undergone t h e thaw-rigor (From Cassens, process. e t al., 1963; Reproduced by permission of Biodynamic a. ) Fig. 7. Electromicrograph of "Irregular Bands" formed i n muscle which has undergone t h e thaw-rigor process. Magn.: 2 0, ~. (From Cassens e t al., 1963; Reproduced by permission of Biodynamica.)

10 219. Fig. 8. Electron micrographs of longitudinal sections of chicken muscle ( p e c t o r a l i s s u p e r f i c i a l i s ) showing t h e e f f e c t of various treatments on t h e s t r i a t i o n s. Photo. 1: Prerigor muscle after rapid freezing and thawing. (Note t h e " i r r e g u l a r band" (black a r e a ). Photo. 2: In-rigor muscle treated as t h a t shown i n Photo. 1. Photo. 3: Prerigor muscle after heating (cooking). Photo. 4. In-rigor muscle t r e a t e d as t h a t shown i n Photo. 3. Magn.: 14,000~. (Selected from Menz and Luyet's f i l e s ; work supported by USDA.)