FROM THE CAHDIOLOGICAL LABORATORY, DEPARTMENT OF MEDICINE, 4 N I ) T H E DEPARTMENT OF PATHOLOGY, ALLMANNA SJUKHUSET, MALMO, SWEDEN

THE INFLUENCE OF HYPOTHERMIA ON THE MICROSCOPICAL PICTURE OF DIFFERENT

ORGANS IN RABBITS

By

P. HAIL, G. H J i j R K M A N , B. JOHANSSON, L. JUHLIN and G. TIBBLIN

Received 6.ii.60

Hypothermia was used some 20 years ago in the treatment of ma- lignant tumours. During the last decade it has also been found useful in operations on the heart and on the brain. Methods now available for lowering the body temperature are regarded as safe. Nevertheless, some questions call for further investigation, such as: Does hypo- thermia produce morphological tissue changes?

Microscopical changes after hypothermia have been observed in organs from various animals, but opinions differ in respect to both the extent and severity of the changes, and some authors even deny the occurrance of such changes.

Artificial hypothermia, i.e. relatively rapid lowering of the general body temperature of anaesthetised human beings, found increasingly wide use after the fundamental investigations by Bigelow ( 1 ) . The microscopical changes described in these cases of hypothermia vary widely. Some authors, for example, found an extracellular accumula- tion of fluid in different organs and degenerative changes and accu- mulation of inflammatory cells. Some investigators such as Sarajas ( 1 3 ) and Knocker ( 7 ) claim that these changes, a t least in dogs, are severe even if the organism is not cooled for any length of time, and even if the reduction in temperature is only small. On the other hand, other authors have not found any changes a t all or a t most slight changes (3 , 6 ) .

M A T E R I A L A N D M E T H O D

Nineteen full-grown rahhits were used. The animals anaesthetised with allyliso- hutylmalonylthiocarhamidesodium (Baytinal, Bayer) and afterwards cooled in a re- frigerator (2) to $28" C. The temperature was maintained as steady as possihle at this level. Six rabbits were cooled for 48 hours, 2 for 34 and 2 for 38 and the remainder for 2-3 hours. Artificial respiration was not induced. The animals were killed immediately after the experiment. After careful gross inspection various organs (heart, liver, spleen, lungs and kidneys) were removed and fixed in 1 0 per cent formalin.

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One thick slice was removed from the left and another from the right half of the heart. The slices were emhedded and were cut f rom various levels, so that several representative sections were obtained from each half of the heart . The lungs were sliced from the apex down towards the base, and several sections were prepared. Sections were cut from the left and from the right liver lobes. The direction of the slices o f the kidneys and spleen were such a s to secure the largest possible sections.

411 o f the specimens were stained with van Gieson, haematoxylin eosin and scarlet red, and sometimes also for iron pigment.

A l l specimens were studied independently hy two examiners, and in no instance were discrepancies noted.

The control material consisted o f 10 rabbits, which were only anaesthetised. The results were also compared with those obtained in a series of 80 rabbits, sensitised according to Rich & Grryorg (12) hy varying doses of horse serum. In the latter series histopathological changes were ohservcd in the heart , lungs, spleen and kidneys (no other organs studied) o f 10-20 per cent of the animals (depending on the different experimental conditions and which organs were examined). Aschoff- granulomalikc changes and alterations rcsemhling periarteritis nodosa were ohserved in the heart a s well a s partial muscle necrosis and an accumulation o f inflammatory cells. The spleen and the kidneys also showed vessel changes. In addition amyloid deposits were seen in the spleen.

I t E S I' I, T S A N D D I S C I' S S I 0 h'

Heart. According to Sarnjns et nl . ( 1 3 ) , the most typical finding in dogs after hypothermia is necrotic muscle fibres in the myocardium with signs of necrosis, mainly hyalinisation of the cytoplasm with simultaneous nuclear pyknosis in single fibres or groups. Early re- active cellular infiltrates consisting mainly of lymphocytes or lympho- cyte-like cells also appear. Patchy calcified spots are seen in animals killed four days or more after hypothermia. Though the animals in the present investigation were under hypothermia for a much longer period than those in Sarajas's investigation, no such changes were observed. Our experience with microscopy of organs from dogs in which the general body temperature had been lowered-2 hours a t + 30" C-is limited, but in the 8 animals which we examined thor- oughly, we were not able to find any changes resembling those de- scribed by Sarajas. Neither were we able to demonstrate any such changes in the hearts of rabbits, mice, guinea-pigs and hedgehogs that had been submitted to hypothermial.

Slight dilatation of the venous system was, however, noted. This re- action had also been observed by Knocker ( 7 ) , but we were not able to confirm her other observations. IVhether this dilatation was due to obstruction or to a toxic effect, is difficult to say. In this connection it might be of interest to mention that Muirhead et al . ( 1 1 ) found histological changes in most of the organs of the body when only part of the dog's body (one leg) had been cooled.

1 In this connection i t is of interest that no pathologic changes were found in the heart muscle f rom the right and left ventricle in a hoy aged seven, who had been in a hypothermic state for six days. The patient had been operated upon hecause of a pulmonary stcnosis with thc aid of a heart-lung-machine. Just af ter the opera- tion the heart stopped and s o long t ime elapsed lwfore it hcgan to heat again that irreversihlc hrain damage appcarcd. The patient was left a t a temperature varying hetwern 81.5" (; and 27" (: (mean 28.8" (;) for six days hefiirc death.

Cellular infiltrates were occasionally seen in the rabbits, but they were limited in extent and could not he ascribed to the hypothermia, because similar changes were also found in the control animals. Cellu- lar infiltrates in the heart muscles from apparently healthy rabbits have also been demonstrated by Miller ( 9 ) and Mc Keown ( l o ) , for example.

The lungs generally showed hypercmia and vascular dilatation. Oc- casionally there was pronounced venous congestion and dilatation of the capillary bed. No cellular infiltrates or necrotic foci were seen and no atelectasis was observed with certainty.

Hyperernia and vascular dilatation were also seen in the liver. Rut the staining methods used failed to show any signs of hepatocellular damage.

of congestion. No other changes were observed. It is believed that during hypothermia the total amount of circulating blood is decreased and that in the liver and spleen increased. This might explain the hyperemia observed. Histo- pathological examination of renal tissue revealed no pathological al- terations.

The spleen also showed a certain degree

C 0 XI A1 E S T S

A s stated above, opinions differ concerning the effect of hypothermia on the microscopical picture, some authors claiming that there is a definitely pathological picture in the hypothermic state, while other authors have not been able to find such changes. The present investiga- tion supports the latter view.

One reason for the varying results might he the use of different kinds of experimental animals. Knocker ( 7 ) and Sarajas ( 1 3 ) used dogs cooled down to a temperature of 28" C and 27.5" C. Both found seri- ous changes. Falkmer & Kjellyren ( 3 ) studied rabbits cooled down to 28-30' C and Kaufman ( 6 ) examined rats a t a temperature of 18-20" C. These lastmentioned authors found only slight changes. This, however, does not necessarily mean that dogs tolerate hypothermia less than other experimental animals; no changes were found by us in the hearts of dogs that had been kept a t 30" C for 2 hours. In Sarajns's & Knocker's (13, 7 ) investigations on dogs, the hypothermia was deeper than that induced in the present investigation and Sarajas kept the body tempera- ture of the dogs low for a longer period. These factors cannot he de- cisive, however, because Fisher et al. (1) were unable to demonstrate any significant morphological changes in the lungs, heart, liver or kidneys of a large series of dogs that had been kept a t 23-24" C for up to 21 hours. In this connection it should be observed that Sarajas's series included 2 dogs that had been kept at 22" C for 4 hours and showed no myocardial changes 11 days later. Hypotherniia as such need not produce any persisting microscopical changes.

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The type of anaesthetic used might be of importance. However, the changes observed did not appear to be linked to any certain anaesthetic. Fisher et al. ( 4 ) used ether as well as nembutal without finding any difference between the effect of the two anaesthetics.

Another factor difficult to judge but possibly of some significance is ventilation. Fisher et al. ( 4 ) pointed out that the difference in ex- perimental technique, inter alia methods of ventilation of the animals, might help to explain the differences in the results reported by different authors.

A very important factor is the control material, which was missing in some of the investigations referred to above. It has long been known that rabbits hearts sometimes show spontaneous cellular infiltrates in the myocardium.-Falkmer & Kjellgren ( 3 ) stressed the extremely common occurrance of mycotic and bacterial diseases in rabbits. Juvenelle et al. (5 ) found small subendocardial petechiae in the heart of an apparently healthy dog, and in a large series of dogs I,ann,ek (8) showed that thallium poisoning can produce profound myocardial changes.

S Ll M M A R Y

19 rabbits were cooled to 28" C and kept at this temperature between 2 and 48 hours. Various organs were examined with the naked eye and microscopically. No microscopic changes definitely ascribable to hypothermia were observed.

The lack of agreement in the literature on microscopical changes following hypothermia might be due to different experimental condi- tions. The importance of such factors as duration and depth of hypo- thermia, the influence of anaesthesia and the ventilation of the animals, is stressed.

H E F E R E N C E S

1. Higelow, W. G . : Hypothermia. Its possihle roll in cardiac surgery; an investiga- tion of factors governing survival in dogs at low hodv temncrature. Ann. Surg. 132: 849,1950.

2. Biorck. G . : Darnaaard-Nielsen. M . . Rutl. H.. Haeqer. K . & W u l f f . H . B.: Box for ~, . . . refrigeration and rewarming in animal experimentation and human sur- gery. Scand. J. Clin. Lah. Invest. 6 : 277,1954.

3. F a l k m r r , S. & Kje l lgren , S.: An experimental study on rahhits with special re- ferenee to the liver. Aeta Soc. med. upsaliens. 60: 199, 1955.

4. Fishrr , E . R. , Fcdor, E . J . & Fisher, B . : Pathologic and Histochemieal ohserva- tions in experimental hypothermia. Archiv. Surg. 75: 817,1957.

5. J u v e n e l k , A . 4 . , Lind, J . & Wcgelius, C.: A new method of extraeorporeal circu- lation. Deep hypothermia comhined with artificial circulation. Am. Heart J. 47 : 692,1954.

6. Koufrnan, N., Gaonn, T . L . & Hil l , R . W.: The effects of prolonged hypothermia on the rat . Arch. Pathol. 66: 96, 1958.

7. Knocker, Ph.: Effects of experimental hypothermia on vital organs. The Laneet 11: 837,1955.

8. Lannrk, N.: A clinical and experimental study on the electrocardiogram in dogs. Stockholm 1949.

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9. hfi l ler , C . I’h. Jr.: Spontaneous interstitial myocarditis in rahhits. J . Exp. Mcd.

10. Mc K e o w n , Fl . : Personal communication. 11. Muirheatl, E . E . , Ashworth , C . T. , Kregel , L . A . & Hill , J . M . : Experimental freez-

ing shock. Arch. Surg. 45: 863,1942. 12. Rich, A . R . & Gregory, J . E.: Further experimental cardiac lesions of rheumatic

type produced by anaphylactic hypersensitivity. Hull. Johns Hopkins Hosp. 75: 115,1944.

13. Sarajns, H . S. S . : Evidence for heart damage in association with systemic hypo- thermia in dogs. Am. Heart J. 51 : 298,1956.

40 : 543,1924.