616-001 . 19-005 THE IMMEDIATE VASCULAR CHANGES IN

TRUE FROSTBITE

RAYMOND GREENE From the Pathologkd Laboratory of Tind0l House

Emergency Ho8pita1, Aylesbury

(PLATES XL AND XLI)

THERE is general agreement on the physiological reactions of the body to cold, which were summarised by Sir Thomas Lewis in his Holme Lectures (1941). This unanimity is dissipated when we consider the exact nature of the breakdown in adaptation which occurs in trench foot and immersion foot (chilling) and in frostbite (freezing). Some previous investigators, for instance Lorrain Smith, Ritchie and Dawson (1915-16), have claimed that the effects of prolonged exposure to mild cold are similar in kind to, though they may differ in degree from, those of short exposure. Others, like Lake (1917), have taken the view that the changes evoked by severe cold are different in kind from those produced by lesser degrees of cold.

Chilling

The first serious attempt to study the effects of mild cold was made by Lorrain Smith et al., whose work was inspired by the epidemic of trench foot at that time causing many thousands of casualties in the British Army.

Their experimental animals-rabbits-stood on a floor of mud which could be made dry or wet a t will, in a chamber the atmosphere of which was kept at about zero centigrade. At this temperature little effect was produced when the mud was dry and the circulation free, only slight transient swelling showing to the naked eye. When, however, tho animal's foot was plunged into water a t 37' C. at the end of the period of freezing, swelling was far greater. Histological examination showed great edema, with separation and some destruction of the collagen bundles. The smaller arteries and veins showed dilatation, enlargement of the intimal endothelial cells, vacuolation of the muscle fibres of the media and infiltration of the perivascular tissue with polymorphonuclear and mononuclear cells. The capillaries showed similar changes and often the surrounding tissues were infiltrated with red cells. The general subcutaneous connective tissue showed considerable edematous swelling and in the spaces between the fibres there was a plentiful deposit of

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fibrin granules and threads which stained clearly with eosin. The arterioles which lay near the nerves showed cellular changes but here there was no great mdema and no fibrin deposit. Whereas in other experiments (without rapid warming) no changes were seen in the nerves themselves, in this experiment the nerves showed signs of edema and the axis cylinders were irregularly thickened and stained diffusely with hrematoxylin. There was however no evidence of degeneration.

The same changes were observed in different degrees in experiments with wet cold mud, with and without constriction of the limb, with and without subsequent rapid warming ; also in experiments with dry cold mud with constriction and with rapid warming. Unfortunately no histological examination was made of the effects of dry cold mud alone. Macroscopically the only effect was transient cedema, which was greatly increased by damp or constriction of the limb. In the presence of these complicating factors, the full histological picture described above was produced. The main changes were in the walls of the blood vessels, whence an excess of transudate and even blood poured forth into the surrounding tissues. The effects on nerve and muscle appeared to be slight. In no case was severe damage produced by mild cold unless damp, constriction or rapid warming were added as complicating factors.

In their summary the authors stress the absence of any evidence of thrombosis or of serious damage to nervous tissue, such as demyelination. They conclude (p. 185) that the essential change in frostbite consists in damage to the blood vessels. " There is probably an initial constriction of the vessels, but when this passes off it is because of their damaged condition that the swelling of the feet occurs while the circulation is being restored to the n o m l ".

Two years later Lake performed a series of experiments with an immense variety of tissue cultures, obtained from men, rabbits, mice and frogs, as well as with the intact human skin. Like Smith et d. he found it impossible to produce any effect other than slight transient swelling by subjecting the skin to mild degrees of dry cold. Only when the tissues were frozen solid were permanent results produced. He ascribed the severe results recorded by Smith et al. to the complicating factors of wet (which increased heat loss), sudden warming (which increased the cedema by increasing arterial supply) and constriction of the limb (which increased cedema by decreasing venous return). The real importance of Lake's work rests, however, on his clear differentiation between the effects of chilling short of solidification, and true frostbite with solidification, a phenomenon which in his experiments occurred at a critical temperature of about -6OC. The work of Lewis and Love (1926) was afterwards to show that, owing to the effecta of supercooling (a phenomenon which varies in different individuals, in part at least with the oil content of the skin), this critical temperature may lie anywhere between -2.2 and -25" C. Lake was aware of this phenomenon, which he mentioned in a confidential report to the War Office in 1918.

Tissue cultures in nutrient media frequently changed are capable of survival for an indefinite period at blood heat. If subjected to the toxic action of their own metabolites, however, the survival time falls slowly as the temperature is reduced, owing to the fact that, while anabolism is inhibited, katabolism is unaffected. At 15-20' C. (Le. about room tempera- ture), the survival time reaches a low figure. At this point katabolism is also affected and the production of toxic metabolites begins to lessen. The survival time therefore improves until, a t about +So C., katabolism also ceases and the tissues are in a state of suspended animation, requiring neither oxygen nor nutriment for their survival. In practice, the survival time is not

VASCULAR CHANGES I N FROSTBITE 26 1

indefinite, though Lake succeeded in freezing a live kitten until all evidence of life had ceased and afterwards restoring it to health and ultimate reproductive activity (personal communication). This happy state is ended suddenly at the point at which solidifkation occurs, in his experiments at about -6’ C., below which the survival time is nil (see below).

The importance of the “‘excessive and injurious ” exudation of fluid also attracted the attention of Lewis and Love, who studied the effects of cold on the human forearm and came likewise to the conclusion that permanent damage was rarely produced by cold directly unless the skin was actually frozen. With this may be compared the observation of Brahdy (1935) that, in New York, where frostbite is common amongst street workers, it does not occur until the temperature falls to -4.4’ C., even in a high wind.

During several journeys in the high Himalaya I have had ample opportunity of observing the effects of cold. Chilling of the feet of both porters and climbers was common. Their feet often became swollen, but gentle warming between the hands was suffi- cient to restore them to normal. It was obvious in such minor cases that no structural damage had been done. The symptoms which followed in more serious cases were clearly dependent upon the amount of swelling.

It would appear then that all observers are in substantial agreement upon the immediate effects of cold short of solidification. Chilled tissues need little nutriment or oxygen and the greater damage occurs during the thaw. At ordinary temperatures the capillary walls are protected by the strongly muscular arterioles. At low temperatures the arterioles are constricted almost to obliteration, the less muscular arteries contain blood at great pressure, the capillaries and veins ax? almost empty. As the temperature rises the arterioles relax and the arterial pressure comes suddenly on the capillaries, whose permeability has already been increased by anoxia. Transudation of plasma and even of blood occurs and the part becomes adematous. If, owing to excessive arterial supply (as when the part is rapidly warmed) or deficient venous drainage (as when the limb is constricted) the transudation is very great, pressure may be exerted from without on the venous end of the capillaries, still further increasing the capillary pressure and initiating a vicious circle. Finally the circulation to the affected extremity is so seriously disorganised that necrosis occurs. The action of water in increasing the damage probably resides chiefly in its conductivity. Imbibition at low temperatures is negligible (Lewis, 1939-42).

The present war has drawn attention to the existence of a condition sometimes known as immersion foot. This occurs in shipwrecked mariners whose feet are subjected to prolonged exposure to cold insufficient to freeze them. Clinically immersion foot is apparently identical with trench foot. Ungley and Blackwood (19459, who have given the first detailed description of the condition,

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262 R. GREENE

have concentrated chiefly on the neurological changes. That such changes occur in chilling has been mentioned by various authors (e .g . Greene, 1940, 1941). Ungley and Blackwood found demyelina- tion. Why this occurs is not yet clear. There are indications that it may be the direct result of cold. It seems to me more probable that it has the same causation as the similar peripheral neuritis of arteriosclerosis and diabetes and that it is due to the prolonged anoxia of the stage of vascular constriction. That it is not due to mdema seems clear from Ungley and Blackwood’s observation that no correlation exists between the degree of swelling and the neurological prognosis.

True frostbite

More severe degrees of freezing if sufficiently prolonged produce gangrene.

The histological changes have been studied by a number of German workers, who have been impressed by the great importance of the subject in war. These authors (Kriege, 1889; Uschinsky, 1893; Hodara, 1896 a and b ; Rischpler, 1900 ; Marchand, 1908 ; Ricker, 1927) agree that an initial vasoconstriction is followed by vasodilatation, with damage to the vessel walls. They differ in respect of the significance which they attach to the primary constriction and the secondary dilatation, and on the question of the occurrence of thrombosis, which was observed by Kriege and Hodara but only rarely or not at all by the others. Lake observed the rarity of tissue destruction by chilling and its frequency by freezing to solidification and assumed the production in the latter of ‘‘ intravenous clotting ” but cited no evidence. Lewis and Love,. who observed no thrombosis, assumed that the injury in severe freezing was due to the formation of ice crystals, but did not explain the fact that the formation of ice crystals is often followed by complete recovery.

It seems probable that many factors may be involved, including, perhaps, subtle changes in intracellular chemistry resulting directly from cold, but it is obviously important from the point of view of treatment that any doubt as to the immediate vascuIar changes in true frostbite should be cleared up.

EXPERIMENTAL INVESTIUATION

Technique. The experimental animal chosen was the albino mouse. This animal is convenient because it is easy to handle and to breed and can be produced in a constant size, a point the importance of which will afterwards be seen. The tail is but sparsely covered with hair, through which the colour of the skin can easily be observed. Depilation, with the risk of damage to the epithelium, is therefore unnecessary. If the tail only is used the heaIth of the animal is not impaired, nor is its future breeding capacity reduced. The apparatus used is shown in fig. 1.

The mouse lies in tube A, which is narrow enough to prevent it from turning, but wide enough to leave its respiration unimpeded. The tail lies in the narrow tube B. A diaphragm made of a finger oot over the proximal

VASCULAR CHANGES IN FROSTBITE 263

end, with a hole for the tail, adds to the comfort of the mouse by separating the cold atmosphere of B from contact with its body. Mice die easily and such precautions are worth while. A mouse will usually enter the tube with alacrity, apparently mistaking it for a mouse hole. If it struggles it is best to return it to its cage. If forced against its inclination to take part in the experiment, a mouse usually dies. C is a jacket which can be packed with carbon dioxide snow. D is a thermometer. The temperature recorded in the central tube varies between -62 and -67" C.

D

A C FIG. 1.-Freezing apparatus. x $.

(A) Wide bore glass tube in which mouse lies.

(B) Narrow glass tube for mouse's tail. (C) Wide bore glass tube packed with solid carbon dioxide. (D) Thermometer inserted through small cork of inner tube B.

Cork on left i s perforated to allow circulation of air.

The production of standard frostbite (8.F.B.) In a large number of preliminary experiments it was shown

that, provided a mouse of constant weight with a tail of constant length be employed, exposure of the tail to a constant temperature for a constant time results in an almost constant degree of damage. The changes observed in the tail are the same and, though these changes may occupy, relatively, slightly different times, the ultimate degree of necrosis at the end of 7-10 days is almost identical. Thus when tails 10-11 em. in length belonging to five mice each weighing 30-40 g. were frozen for 15 minutes, the whole of that part of each tail which was surrounded by CO, snow suffered from dry gangrene and waB ultimately lost. The changes observed were as follows.

Immediately after freezing the tails were white and solid. In the course of a few minutes, as the freezing passed off, the tails became soft and of a bright pink colour. In the course of an hour they became slightly swollen and slightly cyanosed. After 24 hours they were very edematous and mauve in colour. The swelling persisted and petechiae OccuITed in 2-3 days. The swelling then gradually passed off. Between the 4th and 6th days gangrene of the tip occurred (on the 4th day in one mouse, the 5th day in three and the 6th day in one) and spread rapidly, the affected area being first a deep purple, then brown and finally dark purplish brown and dry. All tails separated on the 7th day except one, which did so on the 10th day.

When the tails were frozen for only five minutes the results were the same, except that petechiae were not observed and the

264 R. GREENE

final loss was less, a stump 2.5 cm. in length remaining after separa- tion. The effect of freezing for three minutes was substantially the same. Though dry gangrene was established in 8 days, separa- tion did not occur till the 11th.

When the tail was frozen for one minute the changes were similar. They are described in greater detail, as this was the freezing time adopted in later experiments. Immediately after freezing the whole tail was pale and hard. Thawing was complete in 10 minutes, by which time there was obvious hyperaemia, especially of the tip. (Edema was not so obvious as after more severe freezing, but occurred to a very slight extent, the swelling appearing after the hyperamia, which faded in five hours. At one day the terminaI cm. was a brownish purple. At two days the terminal 2 mm. were dark brown. In the course of the next 6 days the dark brown area gradually invaded the purple area and dried from the tip upwards. At 8 days the terminal em. was in a state of dry gangrene, the rest of the tail, despite the fact thatsit had been frozen stiff, being normal. Separation occurred at various times up to 18 days. Probably the actual separation depended on trauma, often self-imposed. The other effects were constant. It will be convenient to refer to the effects of freezing for one minute at -62' C. a tail 10-11 cm. in length belonging to a mouse weighing 30-40 g. as standard frostbite (S.F.B.).

Histological changes

Ten mice were subjected to S.F.B. Their tails were amputated at varying times afterwards and segments examined at distances of 1, 4 and 8 em. from the tip.

1. Two tails amputated immediately after freezing, when they were pale and hard. The only change is a probable constriction of the caudal artery, an observation which confirms the macroscopic appearance. It is difficult to demonstrate microscopically with certainty.

2. Tail amputated 5 minutes after freezing, when thawing was almost complete and colour returning. No significant changes are observed in the tail except in the distal om., that is to say the part of the tail doomed to ultimate gangrene, where the blood in the vessels forms a shrunken pigmented mass in which the outlines of red blood corpuscles are seen (figs. 2 and 3). There is also slight swelling of the vascular endothelium.

3. Tail amputated 30 minutes after freezing, when hyperamia, especially of the tip, and slight swelling were visible. The contracted mass is again seen in the caudal artery of the distal segment, where badly stained nuclei point to early necrosis. The contraction of arteries throughout the whole tail has passed off.

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VASCIJLAR OHANQES IN FROSTBITE

PLATE XL

PIG. 2.-Caudal artery of mouse. S.F.B. Tail FIG. 3.--Caudalartery of mouse. S.F.B. Tail amputated after 5 minutes. There is slight amputated after 2 days. Similar but less swelling of the intima and the centre of the marked changes in longitudinal section. lumen is occupied by a dense clump of H. and van Gieson. x 290. corpuscles. H. and E. x 350.

FIG. 4.-Caudal artery of mouse following FIG. 5.-Caudal vein. S.F.B. Tail amputated immersion in cold salt water for 24 hours. after 2 days. Vessel full of hemolysed Amputation immediately afterwards. The blood. H. and E. x220. artery is blocked by hyaline thrombus. H. and E. x 350.

VASCULAR CHANGES IiV PBOSTBITE 265

4. Tail amputated one hour after freezing. There is very little further change, though intimal damage of an artery is well shown.

5. Two tails amputated one day after freezing, when the terminal cm. h a assumed a brownish colour. Despite the obvious change in the macroscopic appearance of the tail, there is little further microscopical change, except for massive infiltration of the dermis with polymorphonuclear leucocytes.

6. Three tails amputated 2 days after freezing, when the extreme tips had become dark brown. The sections show early generalised necrosis of the distal cm., which does not stain with haematoxylin and only diffusely with eosin. The only cells showing any nuclear staining are those of the cartilaginous intervertebral discs. In the middle of one tail the sirlusoids of the bone marrow are hyperaemic and there is diffuse infiltration with lymphocytes

In order to make clearer the effects of true freezing, further experiments were performed with the same degree of cold over longer periods, two mice being used in each experiment.

Freezing for 2 minutes, amputation after one hour. Here the tissue spaces are distended by oedema (fig. 6).

Freezing for 3 minutes, amputation after one hour. Here there is obvious edema of the dermis and arterial damage can be seen

Freezing for 15 minutes, amputation after one day. (Edema of the dermis ; pyknosis of nuclei in bone marrow cells ; hsmolysis in superficial blood vessels (fig. 5 ) ; blood vessels grossly dilated and filled with clumped corpuscles ; sub-epidermal and intradermal infiltration with staphylococci. The nuclei of the epidermis and of the superficial segments of hair follicles are in some places badly stained.

Freezing for 15 minutes, amputation immediately. The appear- ances are indistinguishable from those of the unfrozen controls.

An almost constant change is the presence in the moribund but never in the viable tissues of clumps of blood corpuscles occupying the whole or almost the whole lumen of the vessels. It is curious that they have not been mentioned by previous workers, and it seems possible that they have been mistaken for thrombi, which are often mentioned but in my experience rarely seen. Haemolysis is frequent but not constant in the vessels of both viable and moribund tissues.

CONCLUSIONS

(fig. 7).

(fig. 8).

Freezing to solidification does not, in the particular tissue used in these experiments, result of necessity in necrosir5. The complete recovery of the frozen proximal part of the tail may be compared with the complete recovery seen when an area of the human skin

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266 R. UREENE

(usually the nose) becomes suddenly white and hard in a very cold wind. If a warm hand is rapidly clapped on to the affected part it recovers. If it is allowed to remain cold for a minute, or even less, blistering and loss of tissue occur. Microscopical examination of the part of the tail destined to recover shows no constant change other than an initial vasoconstriction (corresponding to the pale macroscopic stage) followed by vasodilatation (corresponding to the bright pink macroscopic stage). On the other hand that part of the tail which is doomed to necrosis shows damage to the arteries and " silting up '' of vessels with corpuscles. !J%ough cedema follows freezing and is greater when freezing is more prolonged, it cannot be correlated exactly with the final damage. In S.F.B. death of the tip occurs though the cedema is very slight. When frozen for five minutes the whole tail is more edematous, but the proximal 2.5 cm. survive despite this.

Like Rischpler and Lewis and Love I found no sign of throm- bosis in tails subjected to dry cold. It occurred only once in my experiments, in a tail suspended for 24 hours in sea-water at 1-5" C. (fig. 4). It is clearly a secondary and unessential occurrence. The essential change is the loss of fluid from the blood vessels. Occasionally damage may be so severe that whole blood is lost. Usually it is plasma only which escapes from the vessels, leaving the corpuscles stranded. The silting up of the vessels with corpuscles may be sufficient to cut off the blood supply to distal tissues. Thereafter there occur those changes which, often superficially regarded as the signs of death itself, are in reality the reactions of a living animal to the presence in its midst of what has become a foreign body and a source of irritation.

SUMMARY The physiological adaptation to cold may break down when

the tissues are subjected to prolonged degrees of mild cold, especially in the presence of water and of subsidiary factors which increase the capillary pressure or permeability. In such circumstances damage is done by excessive trksudation from the blood vessels into the surrounding tissues. A still more serious breakdown may occur in the presence of severe cold. In these circumstances the blood supply to distal tissues is cut off by the silting up of the afferent vessels by stranded red cells. True thrombosis, though it may occur as a secondary phenomenon, has no part in the process.

After the completion of this paper I had the opportunity of discussing my findings with Dr Leiv Kreyberg of Oslo. He drew my attention to a short paper (Rotnes and Kreyberg, 1932), not previously seen by me, in which he had arrived at similar conclusions by a different experimental method.

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VASUULAR CHANGES IN FROSTBITE

PLATE XLI

FIG. 6.-Caudal artery. Tail frozen for 2 minutes and amputated after one hour. Groat perivascular mdema. H. and E. x 290.

FIG. 7.-Caudal tissues. S.F.B. Tail am- putated after two days. Lymphocytic infiltration. H. and van Gieson. x 290.

FIG. 8.-Caudal artery. Tail FIG. 9.-Caudal artery of normal FIG. 10.-Caudal artery of frozen for 3 minutes and mouse. H. and E. x 265. normal mouse. H. and E. amputated after one hour. x 265. Swelling and vacuolation of t>he media. H. and van Gieson. x 125.

VASCULAR CHANGES IN PROSTBITE 267

I wish to express my thanks to the Medical Research Council for a grant in aid of this work ; to Professor J. Machtosh, in whose laboratory it was carried out ; to Dr H. M. Carleton for his advice in the interpretation of the microscopical preparations and for taking the photomicrographs ; and to Mr A. C . Welsford for much technical assistance.

REFERENCES

BRAHDY, L. . . . . . . 1935. J . Amer. Med. AWOC., civ, 529. GEEENE, R. . . . . . . 1940. Lancet, i, 303.

HODARA, M. . . . . . , 1896a. Milnch. med. Wsohr., xliii, 341.

KREWE, H. . . . . . . 1889. Arch. path. Anat., cxvi, 64. LAKE, N. C. . . . . . . 1917. Lancet, ii, 557. LEWIS, T. . . . . . . 1941. Brit. Med. J . , ii. 795, 837 and 869.

LEWIS, T., AND LOVE, W. s. . 1926. Heart, xi& 27. MARCHAND, F. . . . . . 1908. InKrehlandMarchand’sHandbuch

der allgemeinen Pathologie, vol. i, Allgemeine Aetiologie. Leipig.

RICKES, G. . . . . . . 1927. Sklerose und Hypertonie der inner- vierten Arterien. Berlin.

RISCHPLER, A. . . . . . 1900. Beitr. path. Anat., xxviii, 541. ROTNES, P. L., AND KREYBERQ,

SMITH, J. L., RITCHIE, J., AND

UNULEY, C. C., AND BLACK- 1942. Lancet, ii, 447.

USCHINSKY, N. . . . . . 1893. Beitr. path. Anat., xii, 115.

1, . . . . . , 1941. Ibid, ii, 689.

9 , . . . . . . 18966. Monatschr. prakt. Dem. , xxii, 445.

,, . . . . . . 1939-42. Clin. Sci., iv, 349.

1932. Acta path. et microbial. Scand., Supp.

1915-16. This Journal, xx, 159. L. xi, 162.

DAWSON, J.

WOOD, w.