STUDIES ON GANGRENE FOLLOWING COLDINJURY. IX. THE EFFECT OF RUTIN ANDOTHER CHEMICAL AGENTS ON THE COURSEOF EXPERIMENTAL FROSTBITE IN RABBITS

Frederick A. Fuhrman, J. M. Crismon

J Clin Invest. 1948;27(3):364-371. https://doi.org/10.1172/JCI101967.

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STUDIES ON GANGRENEFOLLOWINGCOLD INJURY. IX. THEEFFECTOF RUTIN ANDOTHERCHEMICALAGENTSONTHE

COURSEOF EXPERIMENTALFROSTBITE IN RABBITS 1, 2

By FREDERICKA. FUHRMANAND J. M. CRISMON(From the Department of Physiology, Stanford University School of Medicine,

Stanford, California)

(Received for publication August 27, 1947)

Methods for the production of controlled coldinjury and the course of events following untreatedinjury of this type in rabbits have been describedearlier in this series of papers (1, 2). Such in-jury leads rapidly to massive edema of the frost-bitten foot, drying and subsequent separation atthe line to which the part was immersed in afreezing mixture. An invariable early feature ofthis controlled cold injury is loss into the injuredregion of large amounts of protein-containing fluid(3). The reversibility of this type of injury hasbeen demonstrated by experiments in which theextent of tissue loss was greatly reduced or pre-vented by physical measures: rapid warming inwater at + 420 C. (4), rigid plaster or plasticcasts or elastic pressure dressings (5). This re-versibility emphasizes the importance of events'subsequent to thawing as causal factors in theproduction of gangrene and suggests that the pos-sible beneficial effects of chemical agents shouldbe considered.

Treatment of frostbite by means of drugs hasbeen generally unsuccessful (6 to 8). RecentlyLange and Boyd (9) reported that tissue lossfollowing experimental frostbite was reduced bytreatment with heparin intravenously, but Quin-tanilla, Krusen and Essex (10) were unable todemonstrate the effectiveness of this anticoagulant.This paper reports some attempts to influence thecourse of controlled experimental cold injuryin rabbits by means of a number of chemicalagents. The flavonol glycoside, rutin, was found

1 A part of the work described in this paper was doneunder a contract, recommended by the Committee onMedical Research, between the Office of Scientific Re-search and Development and Stanford University. Thestudy was also aided by grants from the John and MaryR. Markle Foundation and' the Fluid Research Fund ofthe Stanford University School of Medicine.

2An abstract has appeared in Federation Proceedings,1948, 7, 38.

to reduce the extent of tissue loss following frost-bite of rabbit feet; the major part of this report istherefore devoted to this substance.

METHODS

Albino rabbits maintained on a diet of Albers' FamilyRation, a dry commercial food in pellet form, were usedin all experiments. The animals were anesthetized bythe administration intraperitoneally of dial, 90 mgm. perkgm., supplemented with ether immediately before frost-bite. The hair was removed from the ear or from onehind leg by close clipping. The distal 4 to 5 cm. ofthe ear, or the distal portion of the foot to the level ofthe tuberosity at the base of the fifth metatarsal, wasfrozen by immersion in a mixture of alcohol-water-ethylene glycol, cooled with solid CO to - 550 C.The ears were immersed for 60 to 90 seconds, the feetfor three minutes. Complete details of this procedurehave been given previously (1).

EXPERIMENTALRESULTS

1. Rutin.8 Substances with Vitamin P activityhave been reported to influence capillary perme-ability and fragility (11, 12). One of these sub-stances, rutin, has become available recently inpure form. It has been reported to be effectivein the treatment of capillary bleeding associatedwith hypertension in man (13, 14), and a recentreport (15) indicates that it decreases the inci-dence of widespread petechiae and ecchymoses indogs following X-irradiation. Because capillaryinjury is such a prominent feature in the lesionsproduced by exposure to severe cold, the effectof rutin was studied on standardized frostbite inrabbits in order to determine the possible prophy-lactic and/or therapeutic usefulness of this agent.In the following experiments rutin was adminis-

8 The rutin used in these experiments was supplied bythe Western Regional Research Laboratory, UnitedStates Department of Agriculture, through the courtesyof Dr. Floyd DeEds.

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TABLE I

Frostbite of rabbitfeetClosely clipped foot immersed to level of tuberosity at

the base of fifth metatarsal in freezing mixture at -54 to-56° C. for three minutes. Foot thawed in air.

Rutin administered by stomach tube daily. Dose 100mgm. per kgm. in rabbits No. 243, 244, 245 and 246.Dose 50 mgm. per kgm. in others.

Doses of Appearancerutin of gangrene,

Ani- __ days aftermal frostbite Resultnum-Be-Afe

iury - Wet Dry

222 5 17 4 7 Lost toes; dorsum scarred224 10 6 3 6 Lost toes; dorsum scarred225 6 6 3 (toes) 5 Lost toes only230 1 5 5 (toes) 6 Lost toes only229 1 5 - - Lost 2 phalanges of 1 toe231 1 5 5 (toes) 6 Lost toes only243 0 3 4 (toes) 4 Lost toes only244 0 3 3 4 Lost toes and 1 cm. of foot245 0 3 3 (toes) 5 Lost distal 2 phalanges246 0 3 3 (toes) 4 Lost toes only

15 controls* 2 (9)t 4(3) Complete loss (11); loss of all3 (6) 5(9) but narrow plantar pad (4)

6(2)7(1)

* These animals are the same as those used in a previousstudy (1).

t The numbers in parentheses indicate the number ofanimals.

tered daily by stomach tube as a 10 mgm. per ml.suspension in 1 per cent gelatin.

The extent of tissue loss was studied follow-ing controlled cold injury of the feet of rabbitsin six cases in which rutin, 50 mgm. per kgm.daily, was administered both before and afterinjury, and in four additional cases in which rutinwas administered only during the period afterfrostbite. The results are given in Table I. Ob-servations on the development of gangrene andthe extent of tissue loss in 15 control animals areincluded for comparison. In untreated animalsthree-minute immersion of the foot into a freezingmixture at - 550 C. led to loss of the entire in-jured region in 11 of the animals and to loss ofall but a small amount of tissue on the plantar sur-face of the foot in four animals. In nose of therutin-treated animals was the tissue loss as greatas that observed in the controls. In most of thetreated animals tissue loss was confined to the toesand in two instances involved only parts of thetoes. The time of appearance of wet gangrene, al-though it occurred only in the toes of treated ani-

mals, was delayed as compared to the controls.The difference between the times required for thedevelopment of wet gangrene in the two series ofanimals was statistically significant (P < 0.001).

Doses of rutin similar to those used above failedto influence the course of cold injury to rabbitears. Frostbite, produced by immersing the distalhalf of the ear in a freezing mixture at - 550 C.for one minute, led to gangrene and complete lossof the injured part in 12 control animals. Fiveanimals treated with rutin both before and afterinjury developed gangrene and lost all of thefrostbitten parts of the ears. In one animal whichreceived rutin the appearance of wet and drygangrene was delayed one and three days, re-spectively, as compared to control animals. Nodelay was observed, however, when the experimentwas later repeated on the remaining ear of thesame animal.

Direct observation of the circulation (16) inthe ear of a rabbit treated with rutin before frost-bite showed that the local circulatory changes weresimilar to those seen in frostbitten rabbit ears fol-lowing procaine block of the stellate ganglion(cf. section 4 below). Stasis, ordinarily com-plete in true capillaries within 10 minutes afterthawing, was delayed up to 80 minutes in animalstreated with rutin. Carbon particles were seenmoving in large vessels and thoroughfare chan-nels, but none was observed in true capillaries.Subsequently carbon accumulated in true capil-laries during the growth of massive edema, butthe amount was less than in untreated ears at thesame time after frostbite.

Since the process of stasis following cold in-jury appears to depend upon the loss of plasmafrom damaged capillaries (3) the effect of rutinupon loss of trypan blue from the blood wasstudied. Adult rats, anesthetized with pento-barbital (37.5 mgm. per kgm. intraperitoneally),were subjected to cold injury by placing upon theshaved abdomen 5-ml. beakers, 18 mm. in diam-eter, filled with solid carbon dioxide. Seven ani-mals were used as controls and five were given 200mgm. of rutin per kgm. body weight by stomachtube one hour before injury. Two beakers wereapplied to each animal; one was left in place forone minute and one was left two minutes. Tenminutes after application of the beakers 0.5 ml. perkgm. of 1.5 per cent trypan blue was injected into

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the lateral tail vein. The interval between in-jection and the first appearance of blue in the in-jured areas was timed. Since the duration of in-jury did not alter the time of appearance of the dyein the injured areas the values have been grouped.The mean interval for appearance of dye in thecontrols was 32 seconds (S.E. + 1.49), while thatin animals treated with rutin was 68 seconds (S.E.± 3.41). These times were significantly differ-ent (P < 0.001).

In order to determine the extent to which lossof trypan blue into frostbitten regions is influencedby vasoconstriction, Privine (naphazoline hydro-chloride), 10 mgm. per kgm., was injected intra-peritoneally into three rats 10 minutes before in-jury with solid carbon dioxide as described above.

The interval between trypan blue injection andappearance of blue in the injured regions was 35seconds; this is not significantly different fromthe interval observed in control animals. It there-fore seems unlikely that delayed penetration oftrypan blue into frostbitten areas in rutin-treatedanimals is due to vasoconstriction of the type pro-duced by Privine.

An increased peripheral vasoconstriction assoc-iated with fatal muscle trauma in dogs has beenshown by Wang et al. (17) to result in prolonga-tion of fluorescein circulation time. It was con-sidered possible that rutin produces a type of vaso-constriction which may prolong the fluoresceincirculation time. Consequently the circulationtime from lateral tail vein to eyes and oral mucosawas measured with this dye in five control ratsand in five rats given 200 mgm. per kgm. of rutinby stomach tube one hour before the fluorescein.The mean circulation times were 5.18 and 5.41seconds, respectively. These times are not signifi-cantly different. Failure to observe a prolongedfluorescein circulation time in rutin-treated ratsis further evidence against the view that increasedperipheral vasoconstriction occurs after rutin ad-ministration. These observations do not excludethe possibility that redistribution of local bloodflow by closure of precapillary sphincters withoutmajor effect upon the arterioles may account forthe results obtained with rutin.

2. Vasodilator drugs. Carbon dioxide, nitro-glycerine and acetyl-fl-methyl choline chloride(Mecholyl) were administered to a limited num-ber of animals in order to observe the effect of

vasodilatation on the course of severe cold in-jury.

Carbon dioxide, 7 per cent or 10 per cent inoxygen, was administered by inhalation to fouranimals after injury of the ear at - 550 C. for 90seconds (three animals) or 60 seconds (one ani-mal). Inhalation of the CO2 for 15 minutes fol-lowing thawing resulted in an increase in respira-tory rate and hyperemia of the uninjured ear.This treatment was repeated two or three times at30-minute intervals. Complete loss of the partof the ear immersed in the freezing liquid oc-curred as it did in controls. Comparison of thetime of appearance of gangrene in the frostbittenanimals treated with CO2 with the time of ap-pearance of gangrene in control animals (1) indi-cates that gangrene was delayed about one day inthe treated animals. Carbon dioxide administeredby means of iontophoresis (one hour daily forfour days) to the injured ear of two animalsfrostbitten for 90 seconds at - 520 C. was noteffective in accomplishing retention of frostbittentissue. Wet gangrene appeared on the fourth andfifth days and dry gangrene on the fifth and sixthdays after injury; these times are about one daylater than in control animals.

Nitroglycerine was administered to two animalsfollowing frostbite of the ears. After 90-secondimmersion at - 520 C., there were given intra-venously 0.15 mgm. per kgm. nitroglycerine, 14and 42 minutes after injury. After 60-second im-merson at - 550 C. this dose of nitroglycerinewas given six times during the first hour afterthawing. In these animals wet gangrene appearedon the fifth and fourth days and dry gangrene onthe seventh and fifth days, respectively, after in-jury. Complete loss of the frostbitten portion ofthe ears occurred.

Mecholyl was administered by iontophoresisto one animal following frostbite of the foot forthree minutes at - 540 C. The treatment was re-peated on the second day. Wet and dry gangreneappeared on the third and fifth days after in-jury. Tissue loss involved the entire injuredportion of the foot except for a small amount oftissue on the plantar surface; such tissue loss isnot different from that in untreated animals (1).

3. Anticoagulants. Dicumarol (3,3'-methylene-bis [4-hydroxycoumarin] ) was used in order to as-certain the effect of prolonged clotting time upon

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the course of experimental frostbite. It is un-suitable as a possible therapeutic agent becauseof the slow onset of action. Dicumarol was ad-ministered by stomach tube in doses of 5 or 10mgm. per kgm. (18). The mean prothrombintime, measured by the one-stage method of Quickwith brain thromboplastin (19), was 17 secondsbefore treatment and 87 seconds at the time offrostbite. In four of six animals treated withdicumarol, fatal hemorrhages occurred on the dayof frostbite or on the following day so that resultscould not be determined. In two animals, follow-ing frostbite of one foot at - 550 C. for threeminutes, tissue loss involved the entire injuredpart of the foot with the exception of a tongueof tissue in the plantar surface; similar resultswere obtained in four of 15 control animals (1).

Heparin was administered intravenously tofive animals after frostbite of one foot at - 550 C.for three minutes (three animals) or two minutes(two animals). The treatment with heparin, 10mgm. per kgm. intravenously, was continued forthree days; at this time wet gangrene had ap-peared in all animals and further administration ofheparin was deemed useless. This dose of heparinwas given to one animal daily, to a second every12 hours and to a third (two-minute frostbite)every eight hours. In two animals (one frost-bitten three minutes and one two minutes) ex-ternal pressure dressings were applied two hoursafter injury and left in place for six days; heparin,10 mgm. per kgm., was administered to these an-imals every 12 hours for three days. The tissueloss in the last animal mentioned above involvedthe toes only; this was the result to be expectedfrom this duration of injury without treatment(1). All other animals treated with heparin, orwith heparin plus pressure dressing, lost theentire frostbitten portion of the foot to the levelof immersion. In these experiments heparin ap-peared to be completely without benefit, and theaddition of heparin administration appeared tovitiate completely the beneficial results ordinarilyobtained with pressure dressings alone (5).

4. Procaine block of the stellate ganglion. In-terruption of the sympathetic nerve supply to ex-tremities injured by cold has been described asan important aid in the treatment of frostbite(20, 21). An experimental approach to thisproblem was reported by Orlov (22). Rat tails

exposed to ethyl chloride spray for two minutesbecame necrotic if left untreated, but if nerve blockwas produced by procaine infiltration of the tis-sues at the base of the tail during the first fewhours after injury necrosis was prevented. Theprocedure employed by Orlov does not permitclear-cut distinction between effects which maybe the result of sensory paralysis and those whichare the result of interfering with the sympatheticsupply.

In the present study both ears of six rabbitswere frostbitten by immersion for one minute at-550 C. This degree of injury produced a uni-form series of changes followed by gangrene andloss of tissue in control animals (1). Procaineblock of the stellate ganglion on one side wasproduced from three to 125 minutes after freezing,while the animal was still under the anesthetic(Dial). In four of these animals a second pro-caine injection was made on the day followinginjury. Usually 1.0 ml. of 2 per cent procainesolution with 1: 30,000 epinephrine was intro-duced, but in some cases as much as 2.0 ml. wereused because of failure to obtain prompt vasodila-tion. The preparation was judged to be a satis-factory one only if a marked and persistent in-crease of 40 to 50 C. in skin temperature could bedemonstrated in the ear on the blocked side. Theeffect of procaine block of the stellate ganglionon the local circulation of the ear following coldinjury has already been presented (16). Com-plete loss of the distal part of the ear to the levelof immersion occurred. The only difference whichappeared between the ears subjected to procaineblock and those which were untreated was a de-lay of about one day in the development of wetand dry stages of gangrene. Wet gangrene ap-peared on the second day in one animal, on thethird day in two and on the fourth day in three.Dry gangrene appeared on the fourth day in twoanimals, on the fifth in three and on the sixth dayin one.

5. Miscellaneous. The possible usefulness ofa number of other drugs and hormones in thetreatment of experimental frostbite, with the aimof prevention of gangrene, was briefly exploredin rabbits after injury of the ear for one minuteat - 550 C. or the foot for three minutes at - 550C. Vasoconstrictors (epinephrine and/or syn-ephrine), suggested by the work of Lake (23),

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did not influence the development of gangrene.Desoxycorticosterone and progesterone, selectedfor their possible effect on capillary permeability(cf. 24) and water retention (25), were also with-out benefit. In addition a limited number ofexperiments were carried out to test the effect ofincreasing blood volume by whole blood transfu-sion, or increasing the colloidal osmotic pressureof the plasma by the administration of 25 per centhuman plasma albumin 4 and of increasing the so-dium ion concentration in the extracellular phase.Because of the small number of experiments andthe uniform failure of any of these agents to de-lay or prevent gangrene following standard coldinjury, these results are not presented in detail.

DISCUSSION

In view of the generally unfavorable results ob-tained in the experimental treatment of severe coldinjury by means of chemical agents, the responseof such injuries to treatment with rutin representsa singularly interesting exception. It is of interestthat during the last war Greene noted that the in-cidence of "shelter foot" was high among thosedeficient in Vitamins C and P (26) and Lake (7)has recommended the use of these vitamins in thetreatment of cold injury. Not only is the favorableresponse of frostbite to treatment with' rutin oftherapeutic interest but it may serve also as a pos-sible means of elucidating the mechanism of ac-tion of substances having Vitamin P activity aswell as the nature of cold injury itself.

Most of the literature on rutin and related sub-stances has thus far consisted of clinical reportsand has emphasized the use of flavonol glycosidesin conditions characterized by spontaneous capil-lary bleeding, believed to be the consequence ofincreased capillary fragility. Nearly all of the testsused as a basis for judging responses to treatmenthave involved some measure of the tendency ofcapillaries to rupture under increased internalpressure. This preoccupation with reduced in-cidence of capillary bleeding as a manifestationof rutin action has directed attention toward a phe-nomenon which is relatively non-specific. Capil-lary rupture may be the consequence of a wide va-riety of injurious influences, only some of which

'The human plasma albumin was kindly supplied byCutter Laboratories, Berkeley, California.

are similar to those encountered in scurvy andrelated deficiency states.

Among those reports in which Vitamin P-likesubstances are said to influence the permeability ofcapillaries as well as their tendency to rupture, weare aware of only one which presents evidenceindicating specific study of this point (11). Whileinjuries produced by cold have been reported to beaccompanied by extravasation of blood, presum-ably from capillaries (8), the progress of severecold injury to gangrene and loss of tissue is notalways associated with rupture of small bloodvessels. On the other hand, massive edema pro-duced by loss of protein-rich fluid from the bloodstream is an invariable consequence of such in-juries. To the extent that the natural history ofcapillary hemorrhage may be described correctlyin terms of a process having increased permeabilityas an early feature and complete loss of continuityas a late manifestation, the favorable action ofrutin in reducing the amount of tissue lost fromgangrene after cold injury and in reducing theincidence of capillary bleeding in other conditions(cf. 15) may be fundamentally similar.

A recent paper by Lee and Lee (27) presentsexcellent evidence that the stage of bleeding inexperimental scurvy is preceded by marked en-gorgement of the true capillaries and venules.These authors have shown also that this engorge-ment is associated with a remarkable decrease inthe sensitivity of metarterioles and precapillarysphincters to the application of epinephrine.

The extent of gangrene after cold injury hasbeen reduced greatly by measures designed tocontrol the amount of edema (5). Padded non-distensible dressings applied before swelling orpressure bandages applied after maximal swelling,were effective in saving about the same amount oftissue saved by treatment with rutin. Sinceneither of the physical methods of treatment pre-vented stasis in true capillaries or early loss ofplasma from these damaged vessels, the preserva-tion of injured tissue appeared to depend uponother factors.

Stasis in true capillaries of the ear was delayed,but not completely prevented, after frostbite inan animal treated with rutin. Gangrene and lossof tissue occurred in these frostbitten ears as wellas in those of animals receiving no rutin. Pre-vention of gangrene by rutin was observed only in

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rabbit feet injured by cold. The successful resultsobtained following the use of rutin by mouth aslate as five minutes after injury suggest that themechanism of its action is not the prevention ofprimary leakage and stasis in the true capillaries.It is more likely that rutin delays or prevents thegradual extension of vascular occlusion, ordinarilynot complete in untreated frostbite in rabbits un-til about 50 hours after injury (2).

In any capillary system responding to injuryby loss of the usual ability to retain plasma pro-tein, leakage of protein continues only as long asblood flows through injured capillaries. Accumu-lation of packed red blood cells or occlusion ofcapillaries by contraction of precapillary sphinc-ters (28) halts the flow of blood into these vesselsand therefore stops the loss of protein-containingfluid. It is probable that closure of true capil-laries by packed red cells after frostbite and pos-sibly after prolonged application of tourniquets(29, 30) is responsible for progressive reductionin the rate of protein loss into the injured regionwith time after these injuries. In both these typesof injury the protein concentration of edema fluidis high initially and is later progressively reduced.The data of Glenn et al. (31) show that the appli-cation of plaster casts to burned feet of animalssharply reduces the rate of loss of protein fromthe blood in the injured region.

The examples cited in the preceding paragraphpresent some circumstances in which there is re-duction in rate of protein loss from injured capil-laries, yet for none of these examples is there anyconvincing evidence that the reduced rate of lossis the result of decreased capillary permeability.In the case of frostbite, direct observations ofcapillaries (16), as well as indirect evidence basedupon tests with fluorescein (32), indicate that theslowed rate of protein loss in the later stages of in-jury depends upon reduction of total area availablefor filtration of material from the capillaries.

The above considerations serve to emphasizethe need for recognizing in phenomena, such asincreased loss of protein from capillaries and per-haps even increased capillary fragility, the possiblerole of changes in the pattern of local blood flow.The experiments presented in this report on ratsinjected with trypan blue showed that the dye wasslower to appear in frostbitten skin areas of rutin-treated animals than in controls. A similar effect

of rutin in delaying the loss of dye following othertypes of injury has been reported by Ambrose andDeEds (33). Such differences could be producedeither by decrease in the permeability of individualcapillaries or by reduction of the total capillaryarea available for exchange in treated rats. Di-rect evidence permitting a decision between thesetwo mechanisms is now available.. Soluble prepa-rations of various materials having Vitamin P ac-tivity have been reported to produce closure ofprecapillary sphincters and metarterioles upontopical application to the exposed rat mesoap-pendix (34). Intravenously administered rutincauses a 2.5-fold increase in sensitivity of vesselsin the rat mesoappendix to topically applied epi-nephrine (35). This evidence, together with theresponse of cold injury to various forms of treat-ment (5), suggests that reduction of the size ofthe capillary bed is the dominant factor.

In the experiments reported here, heparin wasineffective in preventing tissue loss following frost-bite of rabbit feet at - 550 C. Our data are notdirectly comparable to those of Lange et at. (9,36), who reported beneficial effects of heparintherapy in experimental frostbite, since they usedtemperatures of -120 to - 200 C. and studied ab-dominal skin as well as rabbit feet. Frozen areasof abdominal skin overlie muscle and viscerathrough which blood flow continues during freez-ing, and the pattern of local blood flow and verticaltemperature gradients in the abdomen are dis-similar to those in the extremities. Furthermore,in our experience (1), the temperature range of- 120 to - 200 C. used by Lange is one in whichfreezing of rabbit feet does not invariably occureven after exposure for periods as long as onehour. Gangrene does not occur without actualfreezing if the period of exposure is as brief asone hour. The recent experiments of Quintanillaet al. (10), who were also unable to demonstratethe effectiveness of heparin in the treatment offrostbite of rabbit feet, illustrate the variability inthe severity of injury in this temperature range.

SUMMARY

The effectiveness of a number of chemicalagents in preventing gangrene following stand-ardized severe cold injury to the feet and ears ofrabbits has been studied. Rutin, 50 to 100 mgm.per kgm. per day by stomach tube, was effective in

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restricting the loss of tissue from gangrene follow-ing frostbite of rabbit feet. In 15 control animals,immersion of the foot to the level of the tuberosityon the fifth metatarsal in liquid at - 550 C. re-sulted in complete loss of the exposed part in 11cases and loss of all but a narrow portion of theplantar pad in four cases. Of ten animals simi-larly exposed but treated with rutin, nine lostonly toes and one lost toes plus about 1 cm. offoot. Rutin was ineffective in preventing loss oftissue following frostbite of rabbit ears.

Rutin-treated animals did not develop stasis inthe true capillaries of frostbitten ears as early asdid untreated animals. The delay in onset ofstasis was similar to that observed after procaineblock of the stellate ganglion or rapid thawing ofthe frozen ear in water at + 42° C.

Rutin was found to delay the escape of trypanblue dye from the blood stream into frozen areas ofabdominal skin in rats. The time from injectionof the dye to first appearance of blue in the frost-bitten areas of animals receiving rutin was ap-proximately twice as long as that in controls.Vasoconstriction induced by intraperitoneal in-jection of Privine did not delay the appearance oftrypan blue in frostbitten areas in spite of themarked blanching produced in normal skin areas,and the circulation time as measured by fluoresceinwas not increased by treatment with rutin. Ittherefore seems doubtful that rutin exerts its ef-fect through peripheral vasoconstriction involvingarterioles. Evidence is presented for the view thatalteration in the pattern of blood flow through thecapillary bed is a consequence of the administrationof rutin.

Procaine block of the stellate ganglion delayedbut did not prevent the development of gangrenein frostbitten ears.

The following drugs and hormones were foundto be ineffective in the prevention of gangrenefollowing frostbite.

1. Vasodilator agents: carbon dioxide, nitro-glycerine and acetyl-,/-methyl choline chloride(Mecholyl).

2. Anticoagulants: heparin and dicumarol.3. Vasoconstrictors: epinephrine and/or syn-

ephrine.4. Steroid hormones: desoxycorticosterone and

progesterone.Alterations of blood volume, plasma colloidal

osmotic pressure and extracellular phase volumeby the use of whole blood transfusion, administra-tion of concentrated human plasma albumin andintravenous injection of sodium chloride solutions,all failed to prevent gangrene following standardcold injury.

ACKNOWLEDGMENT

The valued assistance of Miss Ruth L. Dryer in theexperimental work reported above and the advice andstatistical treatment of the data by Dr. F. W. Weymouthare gratefully acknowledged.

BIBLIOGRAPHY

1. Fuhrman, F. A., and Crismon, J. M., Studies ongangrene following cold injury. I. A method forproducing gangrene by means of controlled injuryby cold. J. Clin. Invest., 1947, 26, 229.

2. Fuhrman, F. A., and Crismon, J. M., Studies ongangrene following cold injury. II. General courseof events in rabbit feet and ears following un-treated cold injury. J. Clin. Invest., 1947, 26, 236.

3. Fuhrman, F. A., and Crismon, J. M., Studies ongangrene following cold injury. III. Edema fol-lowing cold injury: its magnitude and the com-position and source of edema fluid. 3. Clin.Invest., 1947, 26, 245.

4. Fuhrman, F. A., and Crismon, J. M., Studies ongangrene following cold injury. VII. Treatmentof cold injury by means of immediate rapid warm-ing. J. Clin. Invest., 1947, 26, 476.

5. Crismon, J. M., and Fuhrman, F. A., Studies ongangrene following cold injury. VIII. The useof casts and pressure dressings in the treatment ofsevere frostbite. J. Clin. Invest., 1947, 26, 486.

6. Bigelow, W. G., Modern conception and treatmentof frostbite. Canad. M. A. J., 1942, 47, 529.

7. Lake, N. C., Frostbite and trench foot; in surgeryof Modern Warfare, Edited by H. Bailey. Wil-liams and Wilkins, Baltimore, 1942, Ed. 2, Vol. 2,p. 530.

8. Davis, L., Scarff, J. E., Rogers, N., and Dickinson,M., High altitude frostbite. Surg., Gynec. &Obst., 1943, 77, 561.

9. Lange, K., and Boyd, L. F., Functional pathology ofexperimental frostbite and prevention of subse-quent gangrene. Surg., Gynec. & Obst., 1945, 80,346.

10. Quintanilla, R., Krusen, F. H., and Essex, H. E.,Studies on frost-bite with special reference to treat-ment and the effect on minute blood vessels. Am.J. Physiol., 1947, 149, 149.

11. Armentano, L., Bentsath, A., Beres, T., Rusznyik,St., and Szent-Gyorgyi, A., Ueber den Einflussvon Substanzen der Flavongruppe auf die Permea-bilitit der Kapillaren. Vitamin P. Deutsche Med.Wchnschr., 1936, 62, 1325.

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12. Lavollay, L., I. L'autoxydation des diphenols en

particulier de l'adrenaline. II. Structure et rolefonctionnel de la vitamin P. (Actualites Scien-tifiques et Industrielles, 943.) Paris, 1942, Li-brairie Scientifique, Hermann et Cie.

13. Griffith, J. Q., Jr., Couch, J. F., and Lindauer, M. A.,Effect of rutin on increased capillary fragility inman. Proc. Soc. Exper. Biol. & Med., 1944, 55,228.

14. Shanno, R. L., Rutin: A new drug for treatment ofincreased capillary fragility. Am. J. M. Sc., 1946,211, 539.

15. Rekers, P. E., and Field, J. B., Control of hemor-rhagic syndrome and reduction in X-irradiationmortality with a flavanone. Science, 1947, 107, 16.

16. Crismon, J. M., and Fuhrman, F. A., Studies on

gangrene following cold injury. VI. Capillaryblood flow after cold injury, the effects of rapidwarming, and sympathetic block. J. Clin. Invest.,1947, 26, 468.

17. Wang, S. C., Painter, E. E., and Overman, R. R.,The mechanism of prolonged fluorescein circula-tion time in experimental traumatic shock. Am.J. Physiol., 1947, 148, 69.

18. Lehmann, J., Hypo-prothrombinemia produced by3-3'-methylene-bis (4-Hydroxycoumarin) and itsuse in the treatment of thrombosis. Science, 1942,96, 345.

19. Quick, A. J., The nature of the bleeding in jaundice.J. A. M. A., 1938, 110, 1658.

20. Ducuing, J., D'Harcourt, J., Folch, A., and Bofill,J., Les troubles trophiques des extremitis produitspar le froid sec en pathologic de guerre. J. deChir., 1940, 55, 385.

21. Burdenko, N. N., The effect of frostbite on thesympathetic nervous system. Am. Rev. SovietMed., 1943, 1, 15. From Khirurgia, 1942, No.5-6, p. 3.

22. Orlov, G. A., Experimental studies on the effect ofnovocaine block on the healing of frozen tissues.Khirurgia (Moscow), 1937, No. 10, p. 15.

23. Lake, N. C., An investigation into the effects of coldupon the body. Lancet, 1917, 2, 557.

24. Abramson, D. I., Vascular Responses in the Ex-tremities of Man in Health and Disease. Univ.Chicago Press, Chicago, 1944.

25. Hisaw, F. L., and Astwood, E. B., The physiologyof reproduction. Ann. Rev. Physiol., 1942, 4, 503.

26. Greene, R., Frostbite and kindred ills. Lancet, 1941,2, 689.

27. Lee, R. C., and Lee, N. Z., The peripheral vascularsystem and its reactions in scurvy: an experi-mental study. Am. J. Physiol., 1947, 149, 465.

28. Chambers, R., and Zweifach, B. W., Topographyand function of the mesenteric capillary circula-tion. Am. J. Anat., 1944, 75, 173.

29. Pochin, E. E., Oedema following ischaemia in therabbit's ear. Clin. Sci., 1942, 4, 341.

30. Harman, J. W., Local vascular phenomena inducedin skeletal muscle by acute ischemia. FederationProc., 1947, 6, 393.

31. Glenn, Wm. W. L., Gilbert, H. H., and Drinker,C. K., The treatment of burns by the closed-plaster method, with certain physiological consid-erations implicit in the success of this technique.J. Chin. Invest., 1943, 22, 609.

32. Crismon, J. M., and Fuhrman, F. A., Studies ongangrene following cold injury. V. The use offluorescein as an indicator of local blood flow:fluorescein tests in experimental frostbite. J. Clin.Invest., 1947, 26, 268.

33. Ambrose, A. M., and DeEds, Floyd, Effect of rutinon permeability of cutaneous capillaries. J.Pharmacol. & Exper. Therap., 1947, 90, 359.

34. Haley, T. J., Clark, W. G., and Geissman, T. A.,Studies on "Vitamin P." I. Topically applied"Vitamin P"-lcke substances on the mammaliancapillary bed. Proc. Soc. Exper. Biol. & Med.,1947, 65, 202.

35. Berez, R., Crismon, J. M., and Fuhrman, F. A.,Unpublished.

36. Lange, K., Boyd, L. J., and Loewe, L., The func-tional pathology of frostbite and the prevention ofgangrene in experimental animals and humans.Science, 1945, 102, 151.

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