STUDIES ONGANGRENEFOLLOWINGCOLDINJURY. V. THEUSEOFFLUORESCEINAS AN INDICATOR OFLOCALBLOODFLOW:

FLUORESCEINTESTS IN EXPERIMENTALFROSTBITE

By J. M. CRISMONAND FREDERICKA. FUHRMAN(Fromn the Department of Physiology, Stanford University School of Medicine,

Stanford University, California)(Received for publication September 1, 1946)

The application of the fluorescein test to thestudy of frostbite has been described recently byLange and Boyd (1). In the course of studiesof experimental frostbite carried on in this lab-oratory, certain of the results obtained with thefluorescein test have been difficult to explaih onthe basis of interpretations suggested by theproposers of the method. Two groups of ex-periments were undertaken in order to studycertain aspects of fluorescein exchange not al-ready reported in the literature. The first groupof experiments consisted of studies on normalrabbits of skin fluorescence and fluorescein dis-tribution in body fluids: these have been presentedpreviously (2). The present paper reports thesecond group of studies carried out on normalrabbits and on rabbits subjected to severe coldinjury.

METHOD

The techniques employed in the fluorescein test, themeasurement of skin fluorescence after the injection offluorescein, and the analysis of body fluids for fluoresceinwere described previously (2). Skin fluorescence underultraviolet light was photographed with a Kodak Recomarhaving a Kodak anastigmat lens, f. 4.5 of 105 mm. focallength. Black and white photographs were taken at f.8on Ansco Superpan Press film with exposure times of 15seconds through a Wratten No. 15 filter or 90 secondsthrough a Wratten No. 62 filter. The animals used inall of the experiments were adult New Zealand whiterabbits from a uniform strain.

RESULTS

1. FJuorescence in normal skinWhen the intravenous injection of fluorescein

was made while the animals were exposed toultraviolet light, bright greenish-yellow fluores-

1 The work described in this paper was done under acontract, recommended by the Committee on MedicalResearch, between the Office of Scientific Research andDevelopment and Stanford University.

cence was detectable in hair-free areas of theskin within a few seconds after the beginningof injection. The intensity of color became max-imal in about 15 minutes. The decline of in-tensity was, at first, rapid and then progressivelymore gradual over a period of 2 to, 2Y2 hours.In the rabbit ear, the regions which glowed mostintensely were those relatively free of large bloodvessels. The larger blood vessels stood out asdark streaks which could be obliterated by pres-cure with a glass slide. The areas renderedischemic by pressure showed no decrease offluorescence.

2. Fuorescence in frostbitten ears of rabbitsThe influence of injury by frostbite upon the

distribution of fluorescein after intravenous in-jection was studied on rabbits' ears. Frostbitewas produced by immersing the distal 3 to 4cm. of the ears in a freezing mixture consistingof alcohol, ethylene glycol, and water with suffi-cient solid carbon dioxide added to bring thetemperature between - 520 C. and - 70° C.The details of the method of producing standardcold injury and the general course of changesfollowing such injuries have been described inprevious reports (3, 4).

Abnormalities in the distribution of fluores-cein in the frostbitten portion of rabbits' earsincluded decreased rate of entrance of dye,slower rate of removal, and lower maximum in-tensity of fluorescence than was observed in thenormal ear. Observations made at varying in-tervals after injury showed that the local changes,which were responsible for the abnormalities offluorescein distribution, undergo modificationwith time. The photographs in Figure 1 illus-trate the delayed penetration and exit of the dyeas well as the differences in results of identicaltests made immediately after inquiry and repeated

268

STUDIES ON GANGRENEFOLLOWINGCOLD INJURY. V

some 18 hours later. Figure 1A shows a photo-graph of the normal ear of a rabbit taken underultraviolet light before the injection of fluoresceinand before the ear was frostbitten. The small

bright spot near the tip of the ear is the fluores-cence from a drop of lubricating oil which wassmeared on the skin from the electric clipper. Fig-ure 1B was taken 15 minutes after the ear was re-

FIG. 1. DISTRIBUTION OF FLUORESCEIN IN A FROSTBITTEN RABBIT EAR AT VARYING TIMES AFTER INJURYAND AFTER FLUORESCEIN INJECTION

A. Normal ear under ultraviolet light before frostbite, and before injection of fluorescein.B. 15 minutes after frostbite (1-minute immersion at - 70° C.) and 10 minutes after injection of 75 mgm.

per kgm. sodium fluorescein into the marginal ear vein of the opposite ear.C. 30 minutes after frostbite and 25 minutes after fluorescein.D. 18 hours 30 minutes after frostbite. Fluorescein completely removed.E. 18 hours 50 minutes after frostbite and 10 minutes after a second intravenous injection of fluorescein.F. 19 hours 5 minutes after frostbite and 25 minutes after the second fluorescein injection.

Photographs taken with ultraviolet light from an HGCH4lamp. Lamp filters consisted of a heat re-sisting red-purple and a UV blue-purple glass filter. Lens filter was Wratten No. 15. Exposure 15 sec-onds at f.8 on Ansco Superpan Press film.

269

I

J. M. CRIS'MON AND FREDERICK A. FUHRMAtN.-

8

z 6 1

LL4

0

z:

-O 1 ~~~23 4 5 6TIME IN HOURS

Fi(;. 2. IN TEN S1 ()OF FLUORESCENCEOF RABBITS' EARS AT DIFFERENT TI3IFAFTER FROSTBITE

Ears expose(d at - 550 C. for 90 seconds. Fluorescein injected at zero time.x Normal ears (6 animals)

Immediately after frostbite (5 animals)* 1 hour after frostbite (4 animals)

1 day after frostbite (3 animals)1 2 days after frostbite (3 animals)

3 days after frostbite (2 animals)

moved from the freezing mixture. The exposureto cold was 1 minute at - 70° C.2 Fluorescein wasinijected intravenously in the opposite ear 10 min-utes before the photograplh as taken. The depthof immiersion in the freezing mixture is clearly indi-cated by the virtual absence of fluorescence in thedistal 3 cm. of the ear. The larger vessels of thedistal part of the ear are faintly fluorescent at thistime, with an al)preciable spread of dye about thecentral artery for a distance of about 8 mm. intothe injured area. Some lack of slharpness in thejunction between the brightly fluorescent, normalpart and the injured region suggests that limitedp)enetration of fluorescein was taking place at thatpoint. Figure 1C is a photograph of the same

animal taken 30 minutes after frostbite and 25minutes after the injection of dye. It will be

2 The results were the same when the temperature ofthe freezing mixture was - 52' to - 56° C.

noted that the intensity of fluorescence had dim-inished to a considerable degree in the lnormiialpart of the ear, while that in the injured area hadreached a brightness almost equal to that observedat 10 minutes in the uninjured areas, and that thefluorescence almost obscures vascular detail. Fig-ure 1D, photographed 18'2 hours after the firstinjection of fluorescein, shows the complete re-moval of dye from all parts of the ear at that time.Figure 1E illustrates the appearance of the earat 18 hours and 50 minutes after frostbite and 10minutes after the second injection of fluorescein.In this photograph the vascular detail, which waseasily made out in the normal part of the ear at10 minutes after the first injection of dye, is al-most completely obscured by the massive edemaand the distribution of fluorescein in the skin. Thepenetration of dye into the injured region can beseen only faintly outlining the major vascular

E PERIODS

270-

STUDIES ON GANGRENEFOLLOWINGCOLD INJURY. V

channels. Figure 1F, photographed 25 minutesafter the second injection of fluorescein, shows a

striking reduction in the amount of dye in theinjured region as compared with the amount whichaccumulated during an identical interval after in-jection when the injury was only 30 minutes old.

3. Time intensity relationships of fluorescence innormal and frostbitten ears of rabbits

Photoelectric measurements were made of thechange in intensity with time of fluorescence in 6normal rabbit ears and in 17 ears at various timesafter frostbite. Figure 2 shows the results ofthese measurements in curves plotted with theintensity of fluorescence on the axis of ordinates interms of galvanometer units and time in hours afterthe injection of fluorescein on the axis of abscissas.In normal ears, the change in itensity of fluor-escence with time was characterized by a very

rapid rise to a maximum within 15 minutes and a

somewhat less rapid decline in intensity, with finaldisappearance of glow occurring between 2 and 3hours after injection of dye. The rate of increasein intensity measured in frostbitten ears when thedye was injected immediately or at 1 hour afterinjury is slower than that observed in normal ears.

When the injection of fluorescein was made at suc-

sessively longer intervals after frostbite, furtherslowing of both the entrance and exit of dye oc-

curred. At intervals of 1 and 2 days after frost-bite the maximum intensity reached was only a

little over a third of that reached in normal ears.

The tendency for exit of fluorescein from the&frost-bitten region to be slower than its entrance, a phe-nomenon noticeable in the comparison betweenmeasurements made when the dye was injected im-mediately after frostbite with those made at 1 hour,was greatly exaggerated in the tests made at in-tervals of 1 and 2 days. While no striking differ-ences were noticeable between the measurementsmade on the first and second days after injury,.thetests on the third day showed only a feeble fluor-escence. The maximum occurred about 2 hoursafter the injection of dye and was followed by a

greatly prolonged course of removal. In some

cases fluorescence was detected by direct obser-vation as long as 24 hours after dye injection.

Since the rates of entrance and removal of dyewere shown to become progressively slower as

the interval between injury and the test was in-creased, only the earliest tests were subjected tostatistical comparison. The significance of differ-ence in rates of fluorescein exchange between thatin normal ears and that in ears tested immediatelyafter frostbite was calculated (5). Table I pre-sents measurements of fluorescence at 10 minutesafter the injection of dye and at 2 hours after in-jection in 5 normal ears and in 5 frostbitten earsof rabbits receiving fluorescein immediately afterfrostbite. Comparison of the intensity of fluor-escence in both series at 10 minutes after injectionshows that the brilliance is significantly greater innormal ears. Analysis at this time interval wasconsidered to be free of differences which mightbe attributed to gross deficiencies of blood flowin the frostbitten ears because skin temperaturemeasurements taken at this time showed that thetemperature was rising very rapidly. The pinkcolor of the skin and the large size of major blood

TABLE I

Measurements of intensity of fluorescence in normal andfrostbitten ears of rabbits at intervals after injection

of fluoresceinData from 5 animals

NormalearsFrostbitten ears 90-second- Normal ears ecxposure at -55° C.

1 2 3 4 5 6 7 8

ColumnFluorescence in Fluorescence in

galvanometer units Time galvanometer units Timeto to

maxi- maxi-10 Maxi- Decr. mum 10 Maxi- Decr. mum

min. mum 2 hrs.* min. mum 2 hrs.*

min.. min.8.5 10.4 9.5 15 0.6 7.5 2.7 707.5 9.0 9.0 19 1.5 5.5 2.0 584.5 4.5 4.5 10 1.5 4.2 0.8 547.1 7.1 7.0 9 0.3 3.5 2.5 604.0 4.8 3.7 20 0.2 2.0 1.5 84

=6.78 6.74 0.82 1.90SEk =0.752 1.163 0.285 0.344

= Mean.SEx = Standard error of mean.* Difference in galvanometer units between maximum

readings and readings taken two hours after injection offluorescein. Comparison of mean values at the base ofcolumns 1 and 5 indicates difference in rate of entrance ofdye in normal and frostbitten ears. Standard error of thedifference between means of columns 1 and 5 is 1.213 andP< 0.01 Comparison of columns 3 and 7 indicates differ-ence in rate of exit of the dye Standard error of the differ-ence between the two means is 0.804 and P < 0.001.P expresses the probability that such differences would beencountered by errors of random sampling.

271

J. M. CRISMONAND FREDERICK A. FUHRMAN

vessels also furnished evidence of rapid bloodflow.

The fluorescence at 10 minutes, at the maxi-mum, and the decrease in 2 hours following theinjection of dye (see Table I) was greater innormal ears than in those injured by cold. Sta-tistical comparison of the mean values for col-umns 1 and 5 showed a significant difference.Therefore, since the most rapid rate of entry ofdye into frostbitten ears is significantly slowerthan the rate of entry into normal ears, the stillslower rates of entry encountered at longer in-tervals after injury must also be lower than therate in normal ears. The slowing of removal ofdye from frostbitten ears as compared with therates of removal from normal ears was found tobe significant by means of a similar assessmentof the difference between the means of valuesgiven in columns 3 and 7. The data in columns3 and 7 were obtained by subtracting from thegalvanometer deflection measured at the peak offluorescence, the galvanometer deflection meas-

ured 2 hours after the maximum was reached.The difference between these amounts of changeover a period of 2 hours in the two groups is con-

sistent with the marked difference in the generalslopes of the curves of declining intensity of fluor-escence. Progressive flattening of the curves isnoted with increase in the interval between injuryby cold and the injection of dye. Hence, thevalidity of the difference established betweenmeasurements on normal ears and on frostbittenears tested immediately after frostbite may be pre-sumed to extend to the greater differences ob-served at longer time intervals.

4. The concentration of fluorescein in edema fluidand in blood

In order to determine whether the delayed re-

moval of fluorescein from frostbitten regions was

a phenomenon restricted to layers of the skin near

the surface or one involving the deeper layers also,analyses of blood and of edema fluid from frost-bitten feet were made in three animals. One hindfoot of each of three rabbits was frostbitten by im-mersion for 3 minutes in the freezing mixture at- 550 C. (3). Intravenous injection of fluores-cein, 75 mgm. per kgm. body weight, was given at10 minutes after frostbite. At intervals from 120

TABLE II

Comparison of fluorescein concentrationin blood and edema fluid

75 mgm. per kgm. fluorescein given intravenously 10minutes after exposure of one foot at - 550 C. for 3 minutes

Blood Plasma Edema fluidAnimal Time after fluorescein fluorescein* fluoresceinnumber fluorescein concen- concen- concen-

tration tration tration

mi. mgm. per mgm. Per mgm. per100 ml. 100 ml. 100 mi.

78 120 1.78 2.92 S.1074 140 3.20 5.25 10.7881 175 0.20 0.33 8.25

* Plasma fluorescein concentration calculated from wholeblood fluorescein concentration assuming a hematocrit of 39per cent (mean of 9 animals) and no penetration of fluores-cein into red blood cells.

to 175 minutes after the injection of dye, samplesof blood and of edema fluid were taken for fluor-escein analysis. Data from these experimentsare presented in Table II. The low concentra-tions of dye found in the blood of these animalsis similar to that observed in normal animals atcomparable times after fluorescein administration(2). Edema fluid, withdrawn from the frost-bitten feet at the same time as the blood sampleswere obtained, contained dye at several times theconcentration found in the blood. On the basisof an estimated hematocrit value of 39 per centin rabbit No. 78, where the smallest difference isshowq, the calculated plasma concentration of dyewas 2.92 mgm. per 100 ml., only a little more thanhalf of that in the edema fluid from the foot of thesame animal.

5. The influence of increased blood flow uponfluorescein exchanges in frostbitten rabbitears

Measurements of skin temperature in frostbittenears and feet of rabbits have shown that the bloodvessels of the injured regions do not participatein thermovascular reflex changes in diameter (4).However, the vessels of parts of ears proximalto the injured region show both tonic constrictionand the ability to dilate in response to local appli-cation of heat or following procaine block of thestellate ganglions (6). The flow of blood into theinjured area of frostbitten ears is, in part, de-termined by the degree of dilatation of the majorarteries proximal to the line of frostbite. Directobservation of blood flow in frostbitten ears

272

STUDIES ON GANGRENEFOLLOWINGCOLD INJURY. V

treated by rapid thawing in warm water or byprocaine block of the stellate ganglion showedthat the irreversible stage of stasis was delayedwhen blood flow was increased, whether the in-crease was brought about by heat or by sympa-thetic paralysis (6). If stasis was allowed toprogress without treatment for 20 to 50 minutes,the subsequent increase of blood flow was ineffec-tive in restoring movement of blood in true capil-laries as determined by direct observation.

The influence of increased blood flow upon thedistribution of fluorescein in the frostbitten earsof rabbits was tested in the following manner.Both ears of each of three rabbits were frost-bitten by immersion for 1 minute in the freezingmixture at - 550 C. At intervals varying from54 to 125 minutes after frostbite, 1 to 2 ml. of 2per cent procaine hydrochloride solution were in-jected into the region of the stellate ganglion onone side. Five minutes later fluorescein was in-

6

ua- 2 ~

D0

ir 6X

w~~~~

2.

001 2 3 4 5 6

TIME IN lIQIRSFIG. 3. INTENSITY OF FLUORESCENCEOF RABBITS' EARS

AFTER FROSThITE AND PROCAINE BLOCK OF RIGHT STEL-LATE GANGLION

Both ears of each rabbit exposed at -55° C. for oneminute. Procaine injected 2 hours after frostbite. Fluor-escein injected 5 minutes after procaine (zero time onfigure). Upper curves in each figure-right frostbittenear, ganglion blocked. Lower curves in each figure-left frostbitten ear. Each figure represents one animal.

jected by vein in the usual dose of 75 mgm. perkgm. body weight. Over the ensuing 2 to 6hours, the intensity of fluorescence was measuredin both ears. The curves presented in Figure 3show that in all three animals the fluorescence ofthe ear on the side subjected to sympathetic blockreached a higher maximum intensity than thatmeasured on the injured, untreated ear. Whilethe rate of increase in fluorescence was higher onthe blocked side, the rate of removal was about thesame for both.

The increase in fluorescence, observed in frost-bitten ears under the circumstances describedabove, was measured at a time after injury whenstasis in true capillaries was completely developed.Paralysis of the sympathetic nerve supply to theear, at comparable times after injury in other ex-periments, had been found to be incapable of re-storing blood flow in these vessels (6), eventhough the rise in skin temperature indicated anincreased total blood flow. Since the exposureto cold of both ears in each animal was the same,the difference in intensity of fluorescence follow-ing procaine block of the stellate ganglion shouldbe attributed to alteration of the minute volumeor local pattern of blood flow rather than todifferences in capillary permeability.

DISCUSSION

Observations of skin color, skin temperature,arterial pulsation, and movement of blood watchedunder the microscope, at least in some vascularchannels, in the frostbitten ears of rabbits showthat total blood flow is for a time greater thanthat in normal ears and does not become com-pletely arrested until more than 50 hours afterinjury (4, 6). During the intervals characterizedby rapid blood flow, tests with fluorescein showboth delayed entrance and delayed exit of dyefrom the skin of the injured region. Lange's re-cently reported observations in which similarfluorescein tests were used upon accidental andexperimental cold injury also point out the para-dox of impaired dye exchange in the presence ofgood blood flow (1). He observed diminishedfluorescence in regions injured by mild cold andwas able to show that capillary blood obtainedfrom poorly staining regions contained as muchfluorescein as that from normal, brightly fluores-

273

J. M. CRISMONAND FREDERICK A. FUHRMAN

cent skin. Two possible explanations were of-fered to account for the differences observed:namely, that capillary permeability had decreasedas the result of cold injury and that local vaso-constriction resulted in reduction of filtration pres-sure. The second of these explanations was con-sidered to be a local change probably not involvingmore than axon reflexes, since the difference couldnot be abolished by local anesthesia or by sympa-thetic block. Severe cold, sufficient to freeze thetissues, resulted in delayed entrance of fluoresceinand apparent hyperfluorescence when observationswere made at successively longer intervals afterdye injection.

The present studies on gangrene resulting fromcold injury include several observations which aredifficult to reconcile with Lange's assumptionthat reduced capillary permeability was the factorresponsible for the abnormal distribution of fluor-escein in tissues injured by cold. The rapidswelling of frostbitten parts, which begins virtuallywith the return of blood flow after thawing, andthe high protein concentration of the edema fluidare changes not consistent with decreased capil-lary permeability. The delayed penetration offluorescein into frostbitten regions also seemsdifficult to explain upon the basis of Lange's sec-ond suggestion, decreased filtration pressure, sincethis would have to coincide with the appearanceof massive edema and the rise of subcutaneoustissue pressure to levels as high as 25 cm. ofwater (7).

An attempt to explain the abnormal distributionof dye upon the basis of simple changes in capil-lary permeability would require the additional as-sumption that the permeability changes be selectivewith respect to the plasma constituents as well asto the direction of their free passage. If de-creased capillary permeability were responsible forthe delayed entrance of dye into the skin of thefrostbitten region, the capillaries would have tobe capable of passing large quantities of plasmaprotein into the interstitial fluid during the timethat the loss of fluorescein from the blood in thesame region was being restrained. Data presentedin a previous report (7) furnish ample evidenceof protein loss from the blood into the frostbittenears and feet of rabbits.

Comparison between the fluorescence of frost-bitten regions and the adjacent, uninjured areas

at about 1 hour after the injection of dye showsthat the glow from the injured region is muchbrighter than that from the normal area. This isan example of the "hyperfluorescence" of skinsubjected to severe cold injury, described byLange and Boyd (1). However, serial measure-ments made from the time of injection of dyefurnish evidence on two important points: (a)the maximum brightness of the two areas doesnot coincide in time; and (b) the intensity offluorescence at maximum for injured areas islower than the maximum intensity reached innormal skin. Thus, what, on first inspection,might seem to be a hyperfluorescence of the in-jured area is actually less than the maximum in-tensity reached af an earlier time in normal skin.The appearance of hyperfluorescence is the resultof removal of dye from adjacent uninjured areasat a faster rate than from the frostbitten area.This circumstance again poses the problem of ap-parently selective changes in permeability. If itbe assumed that the hyperfluorescence is an ex-pression of increased capillary permeability (1),then it must be a selective form of change inpermeability, manifesting a decreased restraint ofdye as it leaves the blood stream in the injuredregion and then failure of fluorescein to moveinto the capillaries when the diffusion gradienthas become reversed by the decline of concentra-tion of dye in the blood. Some form of permeabil-ity change, which is peculiarly selective with re-spect to particle size and which operates only inone direction, must be postulated to account forthe facts. Such a postulate departs widely fromthe accumulated experience of investigators whohave studied the nature of exchanges across capil-lary membranes (8, 9, 10).

Both theoretical considerations (10) and meas-urements on normal animals (2) indicate thatfluorescein should become distributed in a mannersimilar to that observed for other relatively smallmolecules, i.e., largely by diffusion. The majorfactors involved in such transfers of dissolvedmaterial include the diffusion gradient, the perme-ability of capillaries, the blood flow rate, and thesurface area available for diffusion. The evidencepresented above shows that the dye is slow inentering and still slower in leaving areas injuredby severe cold. At the same time, other evidenceindicates that the diffusion gradients, the minute

274

STUDIES ON GANGRENEFOLLOWINGCOLD INJURY. V

volume blood flow, and the degree of capillarypermeability are all such that faster rather thanslower transfer of dye would be expected. As topossible alteration of the surface area availablefor diffusion, direct observation of blood flow infrostbitten, transilluminated ears of rabbits hasshown that stasis begins early in the true capil-laries and is complete within 10 minutes of thereturn of blood flow after thawing. Flow persistsin arteriovenous anastomoses and arteriolarvenu-lar capillaries. Since protein loss into the edemafluid is high in the early period and lower atlater stages, it is not difficult to conceive of theearly loss as occurring via abnormal leakage fromthese capillaries, which later are prevented fromparticipating as exchange surfaces by having beenblocked by silting red blood cells. Thus, bloodflow persists at large minute volume and relativelyhigh pressure in the channels remaining patent.This circumstance serves to explain both therapid production of edema fluid by filtration andthe interference with fluorescein distribution;there is a sharp reduction in the area available fordiffusion. The dye, which is filtered out fromthe arteriorlar-venular capillaries, finds its wayback into the blood stream, not by the usual re-versal of gradient as the concentration of dye inthe blood declines, but by slower removal vialymphatics and by movement of dye into adjacentuninjured regions where stasis has not occurred.

The above interpretation of phenomena ob-served in the aplication of fluorescein tests to thestudy of frostbite is consistent with other mani-festations of altered blood flow and fluid exchangeoccurring after severe cold injury. Informationabout the degree of involvement of capillaries bystasis and its resolution may provide helpfulguides in treatment and prognosis. Thus, inaddition to the use of fluorescence as a clear-cutindicator of the presence or absence of blood flow,the estimation of rate of dye exchange in injuredtissues may be of clinical value.

SUMMARY

Analyses were made of the distribution of intra-venously injected fluorescein in normal ears ofrabbits and in ears severely injured by cold. Theresults showed that both the dye's entrance intoand its exit from the injured regions were slowerthan the exchanges observed in uninjured tissues.

The maximum intensity of fluorescence reachedin frostbitten skin was lower than the maximumrecorded in normal skin. Repeated tests on thesame animal showed that the exchanges of dyebecame progressively slower with time after in-jury.

When both ears of rabbits were frostbitten andprocaine block of the stellate ganglion was pro-duced on one side, the tests with fluoresceinshowed earlier and higher maximum intensity offluorescence in the frostbitten ear on the sideblocked. This maneuver had been shown in otherexperiments to be followed by delay in the onsetof stasis. The greater intensity of fluorescenceon the side having increased blood flow as theresult of blockage of the stellate ganglion wasattributed to the longer persistence of blood flowthrough true capillaries and, hence, greater oppor-tunity for exchange of dye between the blood andinterstitial fluid.

Edema fluid was taken from frostbitten feet ofrabbits at a sufficiently long interval after theintravenous injection of fluorescein to permit theconcentration of dye in the blood to fall to verylow levels. Analysis of the edema fluid showedthat the fluorescein concentration was at leasttwice as great as that in the blood.

The mechanisms of impaired dye exchange infrostbitten tissues are discussed, and a hypothesisis presented in which the impairment of dye ex-change is attributed to reduction of available sur-face for diffusion by the development of stasis inthe true capillaries following cold injury.

BIBLIOGRAPHY

1. Lange, K., and Boyd, L. J., The functional pathologyof experimental frostbite and the prevention ofsubsequent gangrene. Surg., Gynec. & Obstet.,1945, 80, 346.

2. Crismon, J. M., and Fuhrman, F. A., Studies on gan-grene following cold injury. IV. The use of fluor-escein as an indicator of local blood flow: distri-bution of fluorescein in body fluids after intravenousinjection. J. Clin. Invest., 1947, 26, 259.

3. Fuhrman, F. A., and Crismon, J. M., Studies on gan-grene following cold injury. I. A method for pro-ducing gangrene by means of controlled injury bycold. J. Clin. Invest., 1947, 26, 229.

4. Fuhrman, F. A., and Crismon, J. M., Studies on gan-grene following cold injury. II. General course ofevents in rabbit feet and ears following untreatedcold injury. J. Clin. Invest., 1947, 26, 236.

275

J. M. CRISMONAND FREDERICK A. FUHRMAN

5. Fisher, R. A., Statistical Methods for Research Work-ers. Ed. 6, Oliver and Boyd, London, 1936.

6. Crismon, J. M., and Fuhrman, F. A., Studies on gan-

grene following cold injury. VI. Capillary bloodflow after cold injury. J. Clin. Invest., 1947, 26.(In press.)

7. Fuhrman, F. A., and Crismon, J. M., Studies on gan-

grene following cold injury. III. Edema follow-ing cold injury: its magnitude and the compositionand source of edema fluid. J. Clin. Invest., 1947,26, 245.

8. Zweifach, B. W., The structural basis of permeabilityand other functions of blood capillaries. ColdSpring Harbor Symp. Quant. Biol., 1940, 8, 216.

9. Danielli, J. R., and Stock, A., The structure andpermeability of blood capillaries. Biol. Rev., 1944,19, 81.

10. Teorell, T., Kinetics of distribution of substances ad-ministered to the body. II. The intravascularmodes of administration. Arch. Internat. de Phar-macodyn. et de Therap., 1937, 57, 226.

276