MICROVASCULAR
Effects
RESEARCH
lib,
360-372 (1975)
of Subzero Temperatures in the Oral Mucous
on the Microcirculation Membrane
JOYCE H. LEFEBVRE AND LARS
E. A. FOLKE
Division of Periodontology, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455 Received April 25,1975 To determine the nature of circulatory impairment upon tissue exposure to freezing temperatures, a standardized reproducible technique was devised to correlate microcirculatory changes to tissue survival. Everted intact hamster cheek pouches were immersed in 40% propylene glycol at subzero temperatures of -15, -16, -17, and -18” for 1 min actual freezing time, followed by rapid rewarming in 37” Ringer’s solution. Microvascular function was continuously monitored with vital microscopy prior to freezing and for 60 min after freezing. Subsequent observations and recordings were made at daily intervals up to 30 days. Macroscopic tissue changes were documented concomitantly. The critical temperature for tissue recovery in the hamster cheek pouch was -15’. One-minute exposures to -15, -16, -17 and -18” produced predictable gross tissue changes ranging from no damage to complete necrosis. Each macroscopic tissue change was associated with a certain microvascular impairment after thawing. Blood flow ceased during freezing but was reestablished during the first 5 min of thawing. Thereafter, the microvascular vessels demonstrated characteristic differences in vascular flow, stasis, and intravascular occlusion that seemed specifically related to each of the subzero temperatures chosen.
INTRODUCTION Although the therapeutic value of freezing is being empirically accepted, little is known about the extent, selectivity, and controllability of tissue destruction and recovery following such injury. The indications for using this form of therapy are thus uncertain. Although it is acceptedthat physiological tissue changesoccur in responseto subzero temperatures, the exact interrelationships between temperature, exposure time, thawing rates, and the final tissue damage are not well established. Some tissues do survive primary physicochemical injuries, which implies that additional mechanisms such as anoxia due to vascular damageare necessaryfor final injury. Therefore, restoration of local circulation may be a determining factor for survival of frozen tissue. The present study was undertaken primarily to find a sequenceof freezing temperatures at a standardized exposure to produce quantitatively different but predictable injuries.
The intent was to document,
by means of vital microscopy,
the microvascular
changesrelated to well-defined tissue injuries induced by specific freezing temperatures and exposure times in the intact hamster cheek pouch. Various vascular manifestations such as hemoconcentrations, decreased flow rates, increased permeability, stasis, hemolysis, Copyright 0 1975 by Academic Press, Inc. 360 All rights of reproduction Printed in Great Britain
in any form reserved.
increased
viscosity,
and thrombosis have
SUBZERO TEMPERATURES ON MICROCIRCULATION
361
been observed in freeze-injury studies by Lynch and Adolph (1957), Sullivan and Towle (1957), Wymann and Drapeau (1959), Kulka (1965), Suzuki and Penn (1965), Weatherley-White et al. (1969), and Zacarian (1970). Brief exposures to freezing temperatures have also been reported to reduce the flexibility of red cells (Isselhard, 1968),aggregateplatelets (Isselhard, 1968; Sullivan and Towle, 1957), and cause disproportional venular dilatation with peripheral arterial constriction (Mundth, 1964; Kulka, 1965). Much controversy remains, however, in regard to the initiation, sequence,interaction, and significance of these factors. MATERIALS
AND METHODS
Experimental Animals
All observations in this investigation were made of the vascular bed in the cheek pouch of male and nonpregnant female golden hamsters (Mesocricetus auratus) varying in weight from 100-I 50 g. The animals were maintained on Purina laboratory chow and on tap water ad libitum, kept in individual cages,and housed in a temperature and light-controlled room with 12 hr each of light and darkness. Anesthesia
Animals initially received an intraperitoneal injection of 0.22 cc/100 g body weight of Diabutal (Pentobarbital sodium 60 mg/cc). Supplemental 0.05-cc doses were administered when the animal showed signs of awakening, approximately every 45-60 min. Preparation of the Cheek Pouch for Transillumination
The cheek pouch was prepared according to the method of Folke (1970). Following eversion and elimination of debris, the cheek pouch was checked for transparency and carefully placed in contact with a plate-glass floor of a stainless steel receptacle. Two outlets facilitated continuous perfusion of fluids through the trough (Fig. 1). The everted cheek pouch was continuously immersed in Ringer’s solution (37 + 1”) prior to injury and during postfreeze observations. Microvascular function was observed and recorded for a minimum of 30 min prior to injury. Cheek poucheswith circulatory abnormalities were eliminated from the study at this time. Freezing and Thawing Procedure
The freezing solution consisted of 40 % propylene glycol in Ringer’s solution. In the control experiments no vascular changes were observed following immersion of the cheek pouch in this solution for 2 hr at 37”. The cheek pouch was irrigated and frozen with propylene glycol, which waskept in a 2-liter reservoir submergedin an alcohol-dryice bath. The bath temperature was maintained within f 0.5” by adding or withdrawing dry ice contained within a wire-mesh basket. This freezing solution was used in ranges above -21” to avoid crystallization. Just prior to introducing the cold solution, the 37” Ringer’s solution was quickly drained from the trough, and cold propylene glycol was then circulated by means of a combined forced-air-vacuum system. This assured a rapid introduction and evacuation of this viscous fluid and maintained a constant temperature in the trough once the desired temperature had been attained. The immersion optic was raised to preclude a variable temperature transfer. Temperatures of the
362
LEFEBVRE AND FOLKE
FIG. 1. The anesthetized hamster with the cheek pouch stretched, immobilized, and immersed in the trough. Perfusion solutions are circulated by means of outlets A and B. Photographed during freezing procedure; note that pouch is white, opaque, and frozen to line of immersion.
solution in the trough were measured with a YSI rectal probe having a range of -40 to 100” and registered on a YSI telethermometer, model 42SC, with a temperature range of -40 to 150” and a reproducibility of + 0.05”. The actual time the tissues were frozen was the criterion for a reproducible injury. The frozen cheek pouch was thawed by quickly replacing the cold immersion fluid in the receptacle with 37” Ringer’s solution. A uniform trough temperature of 37” was established within 5 min. Gross Observations
The cheek pouches were examined up to 30 days after freezing. Four degrees of grossinjury were distinguished : (1) no damage(no apparent gross permanent changes), (2) minimum damage(temporary sloughing of epithelium), (3) partial necrosis of frozen tissue, and (4) complete necrosis of frozen tissue. Vital Microscopic Observations
The selectedvascular bed was monitored visually and recorded photographically to document vascular patterns, directions of vascular flow, stasis, hemorrhage, and other intravascular phenomena associatedwith arterioles, metarterioles, capillaries, venules, and small veins. Vascular functions were continuously observed for a minimum of 1 hr after freezing at 6, 12, and 24-hr intervals and at varying times up to 30 days. All postfreezetimes referred to in the text begin at the time the cheek pouch was irrigated
SUBZERO TEMPERATURES ON MICROCIRCULATION
363
with 37” Ringer’s solution. Microvascular behavior was recorded cinemicroscopically using a modified Leitz Ortholux microscope equipped with an electrically driven Paillard-Bolex H-16 M-4 camera adjusted for time-lapse photography. Time-lapse observations were recorded with a Leica 35-mm single-lens reflex camera. All cinerecordings were made on Eastman Kodak type 7242 Ektachrome EF film. Vascular Carbon Labeling The carbon suspension used contained about 100 mgjcc of 200-500 A carbon particles stabilized with 4.5 % fish glue preserved in 1.3 % phenol (Guenther-Wagner Pelikan-Werke, Hannover, Germany [Batch # C 1l/143 la]). An initial dose of 0.05 cc/100 g body weight was introduced via the cannulated femoral vein 15 min prior to freezing to check for possible prefreezing labeling. A subsequent dose of 0.10 cc/100 g body weight was injected during thawing. RESULTS
AND
OBSERVATIONS
Our observations are based on the study of 36 hamster cheek pouches exposed for 1 min to 2 hr to temperatures ranging from -5 to -20” (Table 1). The cheek pouch demonstrated a remarkable recovery even when frozen solid for 1 hr at -10” (Fig. 2). TABLE EFFECT OF VARIOUS
SUBZERO TEMPERATURES
-5”
-10 -14
-15
Number of animals
temperature (min : set)
Tissue response (number of animals)
1 1 2 1 1 1 1 4
62:00 lo:OO 60:00b 12o:OO 3:00 I:00 9:30 l:OOb
No damage No damage Minimum damage Partial necrosis Minimum damage Partial necrosis Partial necrosis No damage (3) Minimum damage (1) Minimum damage Partial necrosis Complete necrosis Complete necrosis Minimum damage (4) Minimum damage (1) Partial necrosis (5) Complete necrosis Complete necrosis (6) Complete necrosis (2) Complete necrosis
-16 -17
4 6
2:30 lo:OO 17:00 58:OO : 56b 1:lOb
-18
1 6 2 1
:30 : 57 2:OOb 1:00
1 1
-20 (1Not frozen. b Average.
AND EXPOSURE TIMES
Exposureto specific
Temperatureof immersion fluid (“C)
1
364
LEFEBVRE
181 0.5
I
’
’
’
1”
2
3
4
5
AND
’ “lo
FOLKE
’
’ 20
” 30
‘%F%
Time (minutes) FIG. 2. Effect of various subzero temperature-time exposures on the hamster cheek pouch.
However, freezing for 2 hr at -10” induced partial necrosis. The pilot experiments indicated that -15” was a critical temperature for the cheek pouch as an exposure for 1, 2.5, 10, and 17 min induced no damage, minimum damage, partial necrosis, and complete necrosis, respectively (Fig. 2). It is apparent from these experiments that a critical temperature exists, below which severedamagecan be expectedeven with minimum exposure; above this temperature, damage will be less severe or nonexistent. In this experimental model, the critical temperature was -15” at an exposure of 1 min. Macroscopic Tissue Changes Following Exposure to -15, -16, -17, and -18” for 1 min
All cheekpoucheswerefrozen instantaneously at -15, -16, -17, and -18”. The tissues becamewhite, opaque, and solid (hard) to the line of immersion (Fig. 1). But unlike the instant uniform freezing, thawing usually occurs within 30 set and in a proximal direction. No macroscopic alterations of the cheek pouches were noted at initial thaw. However, at 60 min after freezing, edemawas noted in all pouches but one. Exposure to -15”
Generally, no observable macroscopic changes beyond minimal edema occurred when the cheek pouches were subjected to -15” (Table 1). This edema was maximally manifested by 6 hr and was largely resolved by 24 hr. The pouchesappearedcompletely recovered after 34 days. Subsequent periodic examinations revealed no further changes. Although -15” induced few macroscopic alterations, the pouches were fragile and easily damaged during the first 24 hr. If bruised during this time, the tissue manifested epithelial sloughing and tackiness. Exposure to -16”
Three of four pouches demonstrated considerable edema following an exposure to -16”. The tissue was edematousfor 24 days. There was evidenceof epithelial sloughing and tackiness in all pouches in 3-5 days. The tissue recovered, however, in 7-9 days (Table 1). Exposure to -17 and -18”
The tissue responsejust described was quite different from that seen in pouches subjected to -17 and -18”. All but one demonstrated signs of necrosis in 24 hr. Wet
SUBZERO TEMPERATURES ON MICROCIRCULATION
365
gangrenedeveloped in 3-4 days. Five of six pouches exposedto -17” manifested 3 to 3 tissue loss (Fig. 3), and all pouches subjectedto -18” necrosedto the line of immersion (Fig. 4). The necrosed tissue subsequently sloughed, and the injured base healed in 15-20 days. Comparative Vascular ChangesFollowing Exposure to -15, -16, -17, and -18” for 1 min All cheek pouches manifested vascular changes after being frozen at these four temperatures (Fig. 5). During freezing the circulation was obscured, and on thawing all vesselsdemonstrated stasis of light-colored red blood cells. The blood cells did not regain normal color in situ on thawing, but were replaced by normally colored erythrocytes with the return of blood flow. This occurred suddenly in the arterial system, but a different exchangewas noted in the venous system.As blood entered a venule or vein, it was less forceful and displayed individual corpuscular flow along the vessel walls. This characteristic laminated flow was a consistent finding in all venous vessels(Fig. 6). At no time were thrombi, hemorrhage, or extravasation of white blood cells or carbon black particles detected during microscopic analysis. Small light-colored masseswere observed in arterioles and venules usually within 1 min of resumption of blood flow. Thesegradually disappeared in IO min. Such masseswere predominantly noted following thermal insults at -15 and -16”. On initial thawing, resumption of arterial flow occurred before venular flow. The arterial system was less subject to dysfunction than the venous system and continued
FIG.
3. Hamstercheekpouch 4 daysafter exposureto -17” for 1 min, exhibiting partial necrosis.
366
LEFEBVRE AND FOLKE
Fig. 4. Hamster cheek pouch 6 days after exposure to -18” for 1 min, exhibiting complete necrosis.
MICROVASCULAR
ALTERATIONS
MICROVASCULAR
RECOVERY
Merial Stasis
Vascular Dilatation
VIWJS Stork
\x IO&n
’
S&nin.
lhdur
6tkur
”
’ 24hour
.
46 hour
TIME
FIG.
5. Microvascular response following freezing (1 min).
\.
.
60 hour
367
SUBZERO TEMPERATURES ON MICROCIRCULATION
FIG. 6. Laminated venous flow (arrow) 3 min after freezing, -15”, x250.
TABLE 2 MICROVASCULAR RESPONSEFOLLOWING FREEZING (1 MIN) -15°C
A. 1 hr after freezing Initial flow Reduced flow Sticking of W.B.C. Hemoconcentration Venous stasis B. 6 hr after freezing Sticking of W.B.C. Venous stasis Arterial stasis
-17°C
-18°C
30 set + -IO Transient
30 set ++ Sf 0 30 min
3-4 min ++ ++ ++ 30 min
34 min
+ +
++ ++
++ ++
+++ +++ +++
++
++
+++
+++
C. 24 hr after freezing Vascular dilatation Microvascular necrosis D. 48 hr after freezing Microvascular recovery Partial microvascular distortion Microvascular necrosis
-16°C
++ l ++
10 min
++
+++
+ ++
368
LEFEBVRE
AND
FOLKE
to function hours after apparent venous stasis.None of the arterial vesselsdemonstrated carbon labeling. Both uniform and hourglass-type constrictions of somearterial vessels were noted and recorded at all temperatures but not consistently in all animals. Major differences in vascular functions were noted between pouches exposed to -15 and -16” and pouches subjected to -17 and -18”. Only temporary vascular changes occurred at -15”, whereas at -16” minor stasis of some venules and small veins occurred approximately 30 min after injury (Table 2). The arterial systems in both pouches, however, continued to function. Pouches exposed to -17 and -18” exhibited substantial venous stasis within 30 and 10 min respectively, whereas arterial flow continued for several hours. Exposure to -1.5”
The microvasculature manifested a variety of temporary abnormalities. Initial blood flow usually started within 30 set of thawing and was characterized by localized reversed, intermittent, and reduced flow in vesselsof all types. The pattern of flow returned to normal in all vesselswithin 5-10 min. Few changeswere noted thereafter (Table 2). Occasionally, capillaries and small venules exhibited irregular luminal walls. Temporary rouleaux formation and stasis with concomitant plasma skimming were observed frequently in these vessels. However, these phenomena did not precede a general stasis. Sticking of white blood cells was seenon occasion in venules and small veins within the 60-min postfreezeperiod and commonly observed 6 hr later (Table 2). There were no visual indications of abnormal permeability. Intravenously injected carbon particles continued to circulate freely in all functioning vesselsthroughout the experimental period of 4 hr. However, carbon particles did accumulate in capillaries and small venules during temporary stasis,but they dispersedon resumption of flow. Exposure to -16”
Vascular changesat -16” were basically similar to those described at -15”. However, the changeswere protracted rather than transient in nature (Table 2). Thirty minutes after thawing, blood flow was slower, and hemoconcentration and stasis followed. These manifestations remained unchanged during the remainder of the 60-min postfreezeobservation period. Approximately 30 min after freezing, isolated carbon deposits were observedalong the luminal wall of somevenules and small veins. In no instance was extravasation of carbon noted. Stasis was present within 6 hr in several microvessels throughout the edematous pouch. A few sticking leukocytes were observed in some venules and small veins (Table 2B). Macroscopically, the large vessels remained engorged and distorted for 2-5 days (Table 2D). By 7 days, the cheek pouch appeared normal. Exposure to -17”
Unlike the previous gradual changes,-17” produced a dramatic irreversible vascular injury in 5-10 min (Table 2). Within 334 min after thawing, somevascular function was initiated in the arterial vessels (Fig. 7) and followed by a characteristic laminated corpuscular flow through the venules (Fig. 7). The venular flow resumed initially (Fig.
SUBZERO TEMPERATURESON MICROCIRCULATION
369
FIG. 7. (a) Venous and arterial vessels prior to freezing in -17” fluid, x250. (b) Arterial vessels functioning well, venules, and small veins beginning to function (arrows). Larger veins are static. Two minutes after freezing, -17”, x250. (c)Laminated venous flow (arrow), 3 min after freezing, -17”, x250. (d) Venules and small veins are developing stasis (arrows), 59 min after freezing, -17”, x250.
7), but it succumbed to a subsequent irreversible hemoconcentration and stasis in 5-10 min. Stasis was preceded by spastic, reversed blood flow without dimensional changes of vascular diameter. Much of the venous system exhibited stasis within 30 min (Table 2). Six hours after freezing, only plasma flow and occasional corpuscular movement were detected in the largest veins. The arterial systemcontinued to function for 6 hr or more but was static for 24 hr (Fig. 5). Extensive carbon labeling occurred in the smaller venous vesselswithin 10 min after freezing. The deposits were seenas individual irregular massesalong the luminal walls. Exposure to -18
Vascular changeswere fundamentally similar to those demonstrated at -17”; however, the vascular functions immediately after freezing were seldomasfully coordinated or as rapidly reinstated. In 3-4 min, most vesselsexhibited someflow, but shortly thereafter the venules manifested hemoconcentration and stasis (Table 2). Few venous vesselsfunctioned more than four times. All arterial blood flow ceasedwithin 6 hr (Table 2B). Carbon accumulations in the venous vesselswere characteristically heavy, but at no time were extravasations of carbon particles or erythrocytes detected. 13
370
LEFEBVRE AND FOLKE
DISCUSSION From this study it is evident that the incidence and extent of tissue (cheek pouch) necrosis following freezing are dependent upon, among other factors, the temperature and duration of exposure. Little is known of the relationship between thesetwo factors and the resultant injury in the living mammal. Consequently, the principle portion of this study was designed to keep duration constant at 1 min and investigate variable temperatures as they affect a single parameter, the microcirculation. The critical temperature for the hamster cheek pouch in this experimental model is -15”. Total recovery is predictable above this temperature, and exposures to -16, -17, and -18” produce various degrees of tissue damage. These observations coincide with those of Taylor (1957) and Kreyberg (1957). The latter had speculatedthat the critical temperature for mammalian skin was somewhere between -10 and -20”. Taylor later found it to be -15” for rodent skin. He reported that excised skin of fetal mice and rats, cooled to -15”, remained undamaged and survived as autogenous transplants. However, only portions survived when cooled to -18”, whereasall necrosed at -20”. Taylor (1957)concluded that it was the temperature rather than duration of exposure that was responsible for cold-induced injury at the critical temperature. Such complete independencefrom duration of exposure was not experienced in this study since cheek pouches exposed to -15” for 1, 2.5, 10, and 17 min manifested complete recovery, minimum damage,partial necrosis, and complete necrosis, respectively. The difference between the results of Taylor’s work and those of the present study could have been related to the experimental circumstances. It is evident from this study that various manifestations after a freezing insult are related to different microcirculatory responsesthat occur shortly after thawing. The rapid irreversible changes seen after exposure to -18” are in sharp contrast to the essential negative findings at -15” (Fig. 2). The occurrence of lessextensive rheological changes at -16 and -17” demonstrate the temperature dependency. The biological explanation for thesevariations would require extensive investigations. Although many details are unclear, it appears that some change in the microvesselsaffects the blood components, especially the erythrocytes. It is unlikely that freezing induced a direct injury to the erythrocytes since unaffected RBCs entering the insulted microvascular bed upon resumption of blood flow also lose their integrity. The fact that circulating blood manifests thesechangeswithin minutes after entering the injured vesselssuggests that local factors are operative. It is not prolonged stasispev se that seemsdetrimental to the erythrocytes since static venous blood at -15” does not resemblethe hemolyzed massesobserved at -18”. There is considerable speculation about the cause of stasis. Knisely et al. (1945) attribute stasisto a sludging of blood, which they believe is a transitional phase before agglutination. They postulated that a precipitation of the protein coat renders cells sticky. Initial erythrostasis in the hamster cheek pouch cannot, at this time, be attributed to such agglutination since erythrocytic aggregates beyond the simple rouleaux formations were not observed. Cells simply becamepacked in nonfunctioning vessels.The fact that administration of anticoagulants has not consistently prevented stasis in frozen tissue refutes the theory that the coagulation mechanism is involved
SUBZERO TEMPERATURESON MICROCIRCULATION
371
(Lange and Boyd, 1945, Quintanilla et al., 1947; Bellman and Adams-Ray, 1956; Sullivan and Towle, 1957; Kulka, 1965; Reite, 1965; and Arturson, 1966). Thrombosis was not encountered in the cheek pouch experiment, nor was it reported by Sullivan and Towle (1957). However, thrombosis may be a later manifestation at a time when vital microscopic resolution of damagedtissue is no longer possible. Investigators reporting thrombosis generally have failed both to examine them histochemically and to correlate them with the earliest time of occurrence in an injured tissue. However, Zacarian et al. (1970) did observe emboli, particularly in venular vessels, almost instantaneously after thawing. Other factors contributing to circulation impairment have been suggested. The phenomenon of arteriovenous shunting has been proposed as a contributing factor to early capillary stasisby diverting blood from the capillaries (Crismon and Fuhrman, 1947; Weatherley-White et al., 1969). Although arteriovenous anastomoses do not occur in the cheek pouch (Poor and Lutz, 1958; Folke, 1969),vein-to-vein and arteryto-artery anastomoseswere present. The shunting of blood flow by means of artery-toartery anastomoses was well demonstrated in severely injured pouches exposed to -17 and -18”. During venous stasis, arterial function was maintained for hours by means of these shunts. Although shunting is an interesting sequel to freeze-injury, it is probably not the causeof stasis but rather the result of the altered blood pressure differential following venous stasis. It is difficult to evaluate the role of arterial constriction in circulatory impairment. In this study, arterial constrictions were observed in the hamster cheek pouch, but the presenceof this phenomenon cannot be correlated to temperature, time of observation, size of vessels,or degree of general vascular dysfunction. The significance of vascular constriction in freeze-injury is, therefore, uncertain, which tends to agree with previous conclusions by Sullivan and Towle (1957), Lynch and Adolph (1957), and Mundth (1964). ACKNOWLEDGMENT This investigation was supported by Grant No. DE 03174-01 from the National Institute of Dental Research. REFERENCES ARTURSON, G. (1966). Capillary permeability in experimental rapid freezing with rapid and slow
rewarming. Acru Chir. &and. 131,402. BELLMAN,S., AND ADAMS-RAY,J. (1956). Vascular reactions after experimental cold injury. Angiology 7, 339. CRISMON, J. M., AND FUHRMAN,F. A. (1947). Studies on gangrene following cold injury. VI. Capillary blood flow after cold injury, the effects of rapid warming, and sympathetic block. J. C/in. Inuest., 26,468. FOLKE, L. E. A. (1969). Anatomical considerations and functional behavior of the microvasculature in the oral mucous membrane. Ph.D. thesis, University of Minnesota. FOLKE, L. E. A. (1970). Revascularization in the oral mucous membrane following microvascular injury. Acra Odontol. &and. 28, 785. ISSELHARD,W. (1968). Influence of plasma volume expanders on microcirculatory disturbances. Bibl. Haematol. 29, 889. KNISELY, M. J., ELIOT, T. S., AND BLOCK, E. H. (1945). Sludged blood in traumatic shock. I. Microscopic observations of the precipitation and agglutination of blood flowing through vesselsin crushed tissue. Arch. Surg. 51, 220.
372
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L. (1957). Local freezing. Proc. Roy. Sot. Ser. B. London. 147,546. B. (1965). Cold injury of the skin: The pathogenic role of microcirculatory impairment. Arch Environ. Health 11,484. LANGE, K., AND B~YD, L. J. (1945). The functional pathology of experimental frostbite and the prevention of subsequent gangrene. Surg. Gynecol. Obstet. 80,346. LEFEBVRE, J. (1970). Effect of low temperatures on the oral mucous membrane. M.S.D. thesis, University of Minnesota. LYNCH, H. F., AND ADOLPH,E. F. (1957). Blood flow in small blood vessels during deep hypothermia. J. Appl. Physiol. 11. 192. MUNDTH, E. D. (1964). Studies on the pathogenesis of cold injury microcirculatory changes in tissue by freezing. In “Proceedings Symposia on Arctic Medicine and Biology” (E. Viereck, ed.), Vol. IV, p. 51. Arctic Aeromedical Laboratory, Fort Wainwright, Alaska. MUNDTH, E. D., LONG, D. M., AND BROWN,R. D. (1964). Treatment of experimental frostbite with low molecular weight dextran. J. Trauma 4,246. POOR,E., AND Lurz, B. R. (1958). Functional anastomotic vessels of the cheek pouch of the hamster. Anat. Rec. 132, 121. QUINTANILLA,R., DRUSEN,F. H., AND ESSEX, H. E. (1947). Studies on frostbite with special reference to treatment and the effect of minute blood vessels. Amer. J. Physiol. 149, 149. REITE,0. B. (1965). Functional qualities of small blood vesselsin tissue injured by freezing and thawing. Acta Physiol. Stand. 63, 111. SULLIVAN, B. J., AND TOWLE, L. B. (1957). Vascular responses to local cold injury. Amer. J. Physiol. KREYBERG, KULKA, J.
189,498
M., AND PENN,I. (1965). A reappraisal of the microcirculation during general hypothermia. Surgery 58,105l. TAYLOR, A. C. (1957). The effect of rate of cooling on survival of frozen tissues. Proc. Roy. Sot. Ser. B. London 147,466. WEATHERLY-WHITE, R. C. A,, KNIZE, D. F., GEISTERFER, D. J., AND PATON, B. C. (1969). Experimental studies in cold injury. V. Circulatory hemodynamics. Surgery 66,208. WYMAN, L. C., AND DRAPEAU L. L. (1959). Vascular reactions to cold in the cheek pouch of intact and adrenalectomized hamsters. Amer. J. Pbysiol. 194,799. ZACARIAN, S. E., STONE, D., AND CLATER, M. (1970). Effects of cryogenic temperatures on microcirculation in the golden hamster cheek pouch. Cryobiology 7,127. SUZUKI,
Effects
RESEARCH
lib,
360-372 (1975)
of Subzero Temperatures in the Oral Mucous
on the Microcirculation Membrane
JOYCE H. LEFEBVRE AND LARS
E. A. FOLKE
Division of Periodontology, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455 Received April 25,1975 To determine the nature of circulatory impairment upon tissue exposure to freezing temperatures, a standardized reproducible technique was devised to correlate microcirculatory changes to tissue survival. Everted intact hamster cheek pouches were immersed in 40% propylene glycol at subzero temperatures of -15, -16, -17, and -18” for 1 min actual freezing time, followed by rapid rewarming in 37” Ringer’s solution. Microvascular function was continuously monitored with vital microscopy prior to freezing and for 60 min after freezing. Subsequent observations and recordings were made at daily intervals up to 30 days. Macroscopic tissue changes were documented concomitantly. The critical temperature for tissue recovery in the hamster cheek pouch was -15’. One-minute exposures to -15, -16, -17 and -18” produced predictable gross tissue changes ranging from no damage to complete necrosis. Each macroscopic tissue change was associated with a certain microvascular impairment after thawing. Blood flow ceased during freezing but was reestablished during the first 5 min of thawing. Thereafter, the microvascular vessels demonstrated characteristic differences in vascular flow, stasis, and intravascular occlusion that seemed specifically related to each of the subzero temperatures chosen.
INTRODUCTION Although the therapeutic value of freezing is being empirically accepted, little is known about the extent, selectivity, and controllability of tissue destruction and recovery following such injury. The indications for using this form of therapy are thus uncertain. Although it is acceptedthat physiological tissue changesoccur in responseto subzero temperatures, the exact interrelationships between temperature, exposure time, thawing rates, and the final tissue damage are not well established. Some tissues do survive primary physicochemical injuries, which implies that additional mechanisms such as anoxia due to vascular damageare necessaryfor final injury. Therefore, restoration of local circulation may be a determining factor for survival of frozen tissue. The present study was undertaken primarily to find a sequenceof freezing temperatures at a standardized exposure to produce quantitatively different but predictable injuries.
The intent was to document,
by means of vital microscopy,
the microvascular
changesrelated to well-defined tissue injuries induced by specific freezing temperatures and exposure times in the intact hamster cheek pouch. Various vascular manifestations such as hemoconcentrations, decreased flow rates, increased permeability, stasis, hemolysis, Copyright 0 1975 by Academic Press, Inc. 360 All rights of reproduction Printed in Great Britain
in any form reserved.
increased
viscosity,
and thrombosis have
SUBZERO TEMPERATURES ON MICROCIRCULATION
361
been observed in freeze-injury studies by Lynch and Adolph (1957), Sullivan and Towle (1957), Wymann and Drapeau (1959), Kulka (1965), Suzuki and Penn (1965), Weatherley-White et al. (1969), and Zacarian (1970). Brief exposures to freezing temperatures have also been reported to reduce the flexibility of red cells (Isselhard, 1968),aggregateplatelets (Isselhard, 1968; Sullivan and Towle, 1957), and cause disproportional venular dilatation with peripheral arterial constriction (Mundth, 1964; Kulka, 1965). Much controversy remains, however, in regard to the initiation, sequence,interaction, and significance of these factors. MATERIALS
AND METHODS
Experimental Animals
All observations in this investigation were made of the vascular bed in the cheek pouch of male and nonpregnant female golden hamsters (Mesocricetus auratus) varying in weight from 100-I 50 g. The animals were maintained on Purina laboratory chow and on tap water ad libitum, kept in individual cages,and housed in a temperature and light-controlled room with 12 hr each of light and darkness. Anesthesia
Animals initially received an intraperitoneal injection of 0.22 cc/100 g body weight of Diabutal (Pentobarbital sodium 60 mg/cc). Supplemental 0.05-cc doses were administered when the animal showed signs of awakening, approximately every 45-60 min. Preparation of the Cheek Pouch for Transillumination
The cheek pouch was prepared according to the method of Folke (1970). Following eversion and elimination of debris, the cheek pouch was checked for transparency and carefully placed in contact with a plate-glass floor of a stainless steel receptacle. Two outlets facilitated continuous perfusion of fluids through the trough (Fig. 1). The everted cheek pouch was continuously immersed in Ringer’s solution (37 + 1”) prior to injury and during postfreeze observations. Microvascular function was observed and recorded for a minimum of 30 min prior to injury. Cheek poucheswith circulatory abnormalities were eliminated from the study at this time. Freezing and Thawing Procedure
The freezing solution consisted of 40 % propylene glycol in Ringer’s solution. In the control experiments no vascular changes were observed following immersion of the cheek pouch in this solution for 2 hr at 37”. The cheek pouch was irrigated and frozen with propylene glycol, which waskept in a 2-liter reservoir submergedin an alcohol-dryice bath. The bath temperature was maintained within f 0.5” by adding or withdrawing dry ice contained within a wire-mesh basket. This freezing solution was used in ranges above -21” to avoid crystallization. Just prior to introducing the cold solution, the 37” Ringer’s solution was quickly drained from the trough, and cold propylene glycol was then circulated by means of a combined forced-air-vacuum system. This assured a rapid introduction and evacuation of this viscous fluid and maintained a constant temperature in the trough once the desired temperature had been attained. The immersion optic was raised to preclude a variable temperature transfer. Temperatures of the
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FIG. 1. The anesthetized hamster with the cheek pouch stretched, immobilized, and immersed in the trough. Perfusion solutions are circulated by means of outlets A and B. Photographed during freezing procedure; note that pouch is white, opaque, and frozen to line of immersion.
solution in the trough were measured with a YSI rectal probe having a range of -40 to 100” and registered on a YSI telethermometer, model 42SC, with a temperature range of -40 to 150” and a reproducibility of + 0.05”. The actual time the tissues were frozen was the criterion for a reproducible injury. The frozen cheek pouch was thawed by quickly replacing the cold immersion fluid in the receptacle with 37” Ringer’s solution. A uniform trough temperature of 37” was established within 5 min. Gross Observations
The cheek pouches were examined up to 30 days after freezing. Four degrees of grossinjury were distinguished : (1) no damage(no apparent gross permanent changes), (2) minimum damage(temporary sloughing of epithelium), (3) partial necrosis of frozen tissue, and (4) complete necrosis of frozen tissue. Vital Microscopic Observations
The selectedvascular bed was monitored visually and recorded photographically to document vascular patterns, directions of vascular flow, stasis, hemorrhage, and other intravascular phenomena associatedwith arterioles, metarterioles, capillaries, venules, and small veins. Vascular functions were continuously observed for a minimum of 1 hr after freezing at 6, 12, and 24-hr intervals and at varying times up to 30 days. All postfreezetimes referred to in the text begin at the time the cheek pouch was irrigated
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363
with 37” Ringer’s solution. Microvascular behavior was recorded cinemicroscopically using a modified Leitz Ortholux microscope equipped with an electrically driven Paillard-Bolex H-16 M-4 camera adjusted for time-lapse photography. Time-lapse observations were recorded with a Leica 35-mm single-lens reflex camera. All cinerecordings were made on Eastman Kodak type 7242 Ektachrome EF film. Vascular Carbon Labeling The carbon suspension used contained about 100 mgjcc of 200-500 A carbon particles stabilized with 4.5 % fish glue preserved in 1.3 % phenol (Guenther-Wagner Pelikan-Werke, Hannover, Germany [Batch # C 1l/143 la]). An initial dose of 0.05 cc/100 g body weight was introduced via the cannulated femoral vein 15 min prior to freezing to check for possible prefreezing labeling. A subsequent dose of 0.10 cc/100 g body weight was injected during thawing. RESULTS
AND
OBSERVATIONS
Our observations are based on the study of 36 hamster cheek pouches exposed for 1 min to 2 hr to temperatures ranging from -5 to -20” (Table 1). The cheek pouch demonstrated a remarkable recovery even when frozen solid for 1 hr at -10” (Fig. 2). TABLE EFFECT OF VARIOUS
SUBZERO TEMPERATURES
-5”
-10 -14
-15
Number of animals
temperature (min : set)
Tissue response (number of animals)
1 1 2 1 1 1 1 4
62:00 lo:OO 60:00b 12o:OO 3:00 I:00 9:30 l:OOb
No damage No damage Minimum damage Partial necrosis Minimum damage Partial necrosis Partial necrosis No damage (3) Minimum damage (1) Minimum damage Partial necrosis Complete necrosis Complete necrosis Minimum damage (4) Minimum damage (1) Partial necrosis (5) Complete necrosis Complete necrosis (6) Complete necrosis (2) Complete necrosis
-16 -17
4 6
2:30 lo:OO 17:00 58:OO : 56b 1:lOb
-18
1 6 2 1
:30 : 57 2:OOb 1:00
1 1
-20 (1Not frozen. b Average.
AND EXPOSURE TIMES
Exposureto specific
Temperatureof immersion fluid (“C)
1
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181 0.5
I
’
’
’
1”
2
3
4
5
AND
’ “lo
FOLKE
’
’ 20
” 30
‘%F%
Time (minutes) FIG. 2. Effect of various subzero temperature-time exposures on the hamster cheek pouch.
However, freezing for 2 hr at -10” induced partial necrosis. The pilot experiments indicated that -15” was a critical temperature for the cheek pouch as an exposure for 1, 2.5, 10, and 17 min induced no damage, minimum damage, partial necrosis, and complete necrosis, respectively (Fig. 2). It is apparent from these experiments that a critical temperature exists, below which severedamagecan be expectedeven with minimum exposure; above this temperature, damage will be less severe or nonexistent. In this experimental model, the critical temperature was -15” at an exposure of 1 min. Macroscopic Tissue Changes Following Exposure to -15, -16, -17, and -18” for 1 min
All cheekpoucheswerefrozen instantaneously at -15, -16, -17, and -18”. The tissues becamewhite, opaque, and solid (hard) to the line of immersion (Fig. 1). But unlike the instant uniform freezing, thawing usually occurs within 30 set and in a proximal direction. No macroscopic alterations of the cheek pouches were noted at initial thaw. However, at 60 min after freezing, edemawas noted in all pouches but one. Exposure to -15”
Generally, no observable macroscopic changes beyond minimal edema occurred when the cheek pouches were subjected to -15” (Table 1). This edema was maximally manifested by 6 hr and was largely resolved by 24 hr. The pouchesappearedcompletely recovered after 34 days. Subsequent periodic examinations revealed no further changes. Although -15” induced few macroscopic alterations, the pouches were fragile and easily damaged during the first 24 hr. If bruised during this time, the tissue manifested epithelial sloughing and tackiness. Exposure to -16”
Three of four pouches demonstrated considerable edema following an exposure to -16”. The tissue was edematousfor 24 days. There was evidenceof epithelial sloughing and tackiness in all pouches in 3-5 days. The tissue recovered, however, in 7-9 days (Table 1). Exposure to -17 and -18”
The tissue responsejust described was quite different from that seen in pouches subjected to -17 and -18”. All but one demonstrated signs of necrosis in 24 hr. Wet
SUBZERO TEMPERATURES ON MICROCIRCULATION
365
gangrenedeveloped in 3-4 days. Five of six pouches exposedto -17” manifested 3 to 3 tissue loss (Fig. 3), and all pouches subjectedto -18” necrosedto the line of immersion (Fig. 4). The necrosed tissue subsequently sloughed, and the injured base healed in 15-20 days. Comparative Vascular ChangesFollowing Exposure to -15, -16, -17, and -18” for 1 min All cheek pouches manifested vascular changes after being frozen at these four temperatures (Fig. 5). During freezing the circulation was obscured, and on thawing all vesselsdemonstrated stasis of light-colored red blood cells. The blood cells did not regain normal color in situ on thawing, but were replaced by normally colored erythrocytes with the return of blood flow. This occurred suddenly in the arterial system, but a different exchangewas noted in the venous system.As blood entered a venule or vein, it was less forceful and displayed individual corpuscular flow along the vessel walls. This characteristic laminated flow was a consistent finding in all venous vessels(Fig. 6). At no time were thrombi, hemorrhage, or extravasation of white blood cells or carbon black particles detected during microscopic analysis. Small light-colored masseswere observed in arterioles and venules usually within 1 min of resumption of blood flow. Thesegradually disappeared in IO min. Such masseswere predominantly noted following thermal insults at -15 and -16”. On initial thawing, resumption of arterial flow occurred before venular flow. The arterial system was less subject to dysfunction than the venous system and continued
FIG.
3. Hamstercheekpouch 4 daysafter exposureto -17” for 1 min, exhibiting partial necrosis.
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LEFEBVRE AND FOLKE
Fig. 4. Hamster cheek pouch 6 days after exposure to -18” for 1 min, exhibiting complete necrosis.
MICROVASCULAR
ALTERATIONS
MICROVASCULAR
RECOVERY
Merial Stasis
Vascular Dilatation
VIWJS Stork
\x IO&n
’
S&nin.
lhdur
6tkur
”
’ 24hour
.
46 hour
TIME
FIG.
5. Microvascular response following freezing (1 min).
\.
.
60 hour
367
SUBZERO TEMPERATURES ON MICROCIRCULATION
FIG. 6. Laminated venous flow (arrow) 3 min after freezing, -15”, x250.
TABLE 2 MICROVASCULAR RESPONSEFOLLOWING FREEZING (1 MIN) -15°C
A. 1 hr after freezing Initial flow Reduced flow Sticking of W.B.C. Hemoconcentration Venous stasis B. 6 hr after freezing Sticking of W.B.C. Venous stasis Arterial stasis
-17°C
-18°C
30 set + -IO Transient
30 set ++ Sf 0 30 min
3-4 min ++ ++ ++ 30 min
34 min
+ +
++ ++
++ ++
+++ +++ +++
++
++
+++
+++
C. 24 hr after freezing Vascular dilatation Microvascular necrosis D. 48 hr after freezing Microvascular recovery Partial microvascular distortion Microvascular necrosis
-16°C
++ l ++
10 min
++
+++
+ ++
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to function hours after apparent venous stasis.None of the arterial vesselsdemonstrated carbon labeling. Both uniform and hourglass-type constrictions of somearterial vessels were noted and recorded at all temperatures but not consistently in all animals. Major differences in vascular functions were noted between pouches exposed to -15 and -16” and pouches subjected to -17 and -18”. Only temporary vascular changes occurred at -15”, whereas at -16” minor stasis of some venules and small veins occurred approximately 30 min after injury (Table 2). The arterial systems in both pouches, however, continued to function. Pouches exposed to -17 and -18” exhibited substantial venous stasis within 30 and 10 min respectively, whereas arterial flow continued for several hours. Exposure to -1.5”
The microvasculature manifested a variety of temporary abnormalities. Initial blood flow usually started within 30 set of thawing and was characterized by localized reversed, intermittent, and reduced flow in vesselsof all types. The pattern of flow returned to normal in all vesselswithin 5-10 min. Few changeswere noted thereafter (Table 2). Occasionally, capillaries and small venules exhibited irregular luminal walls. Temporary rouleaux formation and stasis with concomitant plasma skimming were observed frequently in these vessels. However, these phenomena did not precede a general stasis. Sticking of white blood cells was seenon occasion in venules and small veins within the 60-min postfreezeperiod and commonly observed 6 hr later (Table 2). There were no visual indications of abnormal permeability. Intravenously injected carbon particles continued to circulate freely in all functioning vesselsthroughout the experimental period of 4 hr. However, carbon particles did accumulate in capillaries and small venules during temporary stasis,but they dispersedon resumption of flow. Exposure to -16”
Vascular changesat -16” were basically similar to those described at -15”. However, the changeswere protracted rather than transient in nature (Table 2). Thirty minutes after thawing, blood flow was slower, and hemoconcentration and stasis followed. These manifestations remained unchanged during the remainder of the 60-min postfreezeobservation period. Approximately 30 min after freezing, isolated carbon deposits were observedalong the luminal wall of somevenules and small veins. In no instance was extravasation of carbon noted. Stasis was present within 6 hr in several microvessels throughout the edematous pouch. A few sticking leukocytes were observed in some venules and small veins (Table 2B). Macroscopically, the large vessels remained engorged and distorted for 2-5 days (Table 2D). By 7 days, the cheek pouch appeared normal. Exposure to -17”
Unlike the previous gradual changes,-17” produced a dramatic irreversible vascular injury in 5-10 min (Table 2). Within 334 min after thawing, somevascular function was initiated in the arterial vessels (Fig. 7) and followed by a characteristic laminated corpuscular flow through the venules (Fig. 7). The venular flow resumed initially (Fig.
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369
FIG. 7. (a) Venous and arterial vessels prior to freezing in -17” fluid, x250. (b) Arterial vessels functioning well, venules, and small veins beginning to function (arrows). Larger veins are static. Two minutes after freezing, -17”, x250. (c)Laminated venous flow (arrow), 3 min after freezing, -17”, x250. (d) Venules and small veins are developing stasis (arrows), 59 min after freezing, -17”, x250.
7), but it succumbed to a subsequent irreversible hemoconcentration and stasis in 5-10 min. Stasis was preceded by spastic, reversed blood flow without dimensional changes of vascular diameter. Much of the venous system exhibited stasis within 30 min (Table 2). Six hours after freezing, only plasma flow and occasional corpuscular movement were detected in the largest veins. The arterial systemcontinued to function for 6 hr or more but was static for 24 hr (Fig. 5). Extensive carbon labeling occurred in the smaller venous vesselswithin 10 min after freezing. The deposits were seenas individual irregular massesalong the luminal walls. Exposure to -18
Vascular changeswere fundamentally similar to those demonstrated at -17”; however, the vascular functions immediately after freezing were seldomasfully coordinated or as rapidly reinstated. In 3-4 min, most vesselsexhibited someflow, but shortly thereafter the venules manifested hemoconcentration and stasis (Table 2). Few venous vesselsfunctioned more than four times. All arterial blood flow ceasedwithin 6 hr (Table 2B). Carbon accumulations in the venous vesselswere characteristically heavy, but at no time were extravasations of carbon particles or erythrocytes detected. 13
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DISCUSSION From this study it is evident that the incidence and extent of tissue (cheek pouch) necrosis following freezing are dependent upon, among other factors, the temperature and duration of exposure. Little is known of the relationship between thesetwo factors and the resultant injury in the living mammal. Consequently, the principle portion of this study was designed to keep duration constant at 1 min and investigate variable temperatures as they affect a single parameter, the microcirculation. The critical temperature for the hamster cheek pouch in this experimental model is -15”. Total recovery is predictable above this temperature, and exposures to -16, -17, and -18” produce various degrees of tissue damage. These observations coincide with those of Taylor (1957) and Kreyberg (1957). The latter had speculatedthat the critical temperature for mammalian skin was somewhere between -10 and -20”. Taylor later found it to be -15” for rodent skin. He reported that excised skin of fetal mice and rats, cooled to -15”, remained undamaged and survived as autogenous transplants. However, only portions survived when cooled to -18”, whereasall necrosed at -20”. Taylor (1957)concluded that it was the temperature rather than duration of exposure that was responsible for cold-induced injury at the critical temperature. Such complete independencefrom duration of exposure was not experienced in this study since cheek pouches exposed to -15” for 1, 2.5, 10, and 17 min manifested complete recovery, minimum damage,partial necrosis, and complete necrosis, respectively. The difference between the results of Taylor’s work and those of the present study could have been related to the experimental circumstances. It is evident from this study that various manifestations after a freezing insult are related to different microcirculatory responsesthat occur shortly after thawing. The rapid irreversible changes seen after exposure to -18” are in sharp contrast to the essential negative findings at -15” (Fig. 2). The occurrence of lessextensive rheological changes at -16 and -17” demonstrate the temperature dependency. The biological explanation for thesevariations would require extensive investigations. Although many details are unclear, it appears that some change in the microvesselsaffects the blood components, especially the erythrocytes. It is unlikely that freezing induced a direct injury to the erythrocytes since unaffected RBCs entering the insulted microvascular bed upon resumption of blood flow also lose their integrity. The fact that circulating blood manifests thesechangeswithin minutes after entering the injured vesselssuggests that local factors are operative. It is not prolonged stasispev se that seemsdetrimental to the erythrocytes since static venous blood at -15” does not resemblethe hemolyzed massesobserved at -18”. There is considerable speculation about the cause of stasis. Knisely et al. (1945) attribute stasisto a sludging of blood, which they believe is a transitional phase before agglutination. They postulated that a precipitation of the protein coat renders cells sticky. Initial erythrostasis in the hamster cheek pouch cannot, at this time, be attributed to such agglutination since erythrocytic aggregates beyond the simple rouleaux formations were not observed. Cells simply becamepacked in nonfunctioning vessels.The fact that administration of anticoagulants has not consistently prevented stasis in frozen tissue refutes the theory that the coagulation mechanism is involved
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(Lange and Boyd, 1945, Quintanilla et al., 1947; Bellman and Adams-Ray, 1956; Sullivan and Towle, 1957; Kulka, 1965; Reite, 1965; and Arturson, 1966). Thrombosis was not encountered in the cheek pouch experiment, nor was it reported by Sullivan and Towle (1957). However, thrombosis may be a later manifestation at a time when vital microscopic resolution of damagedtissue is no longer possible. Investigators reporting thrombosis generally have failed both to examine them histochemically and to correlate them with the earliest time of occurrence in an injured tissue. However, Zacarian et al. (1970) did observe emboli, particularly in venular vessels, almost instantaneously after thawing. Other factors contributing to circulation impairment have been suggested. The phenomenon of arteriovenous shunting has been proposed as a contributing factor to early capillary stasisby diverting blood from the capillaries (Crismon and Fuhrman, 1947; Weatherley-White et al., 1969). Although arteriovenous anastomoses do not occur in the cheek pouch (Poor and Lutz, 1958; Folke, 1969),vein-to-vein and arteryto-artery anastomoseswere present. The shunting of blood flow by means of artery-toartery anastomoses was well demonstrated in severely injured pouches exposed to -17 and -18”. During venous stasis, arterial function was maintained for hours by means of these shunts. Although shunting is an interesting sequel to freeze-injury, it is probably not the causeof stasis but rather the result of the altered blood pressure differential following venous stasis. It is difficult to evaluate the role of arterial constriction in circulatory impairment. In this study, arterial constrictions were observed in the hamster cheek pouch, but the presenceof this phenomenon cannot be correlated to temperature, time of observation, size of vessels,or degree of general vascular dysfunction. The significance of vascular constriction in freeze-injury is, therefore, uncertain, which tends to agree with previous conclusions by Sullivan and Towle (1957), Lynch and Adolph (1957), and Mundth (1964). ACKNOWLEDGMENT This investigation was supported by Grant No. DE 03174-01 from the National Institute of Dental Research. REFERENCES ARTURSON, G. (1966). Capillary permeability in experimental rapid freezing with rapid and slow
rewarming. Acru Chir. &and. 131,402. BELLMAN,S., AND ADAMS-RAY,J. (1956). Vascular reactions after experimental cold injury. Angiology 7, 339. CRISMON, J. M., AND FUHRMAN,F. A. (1947). Studies on gangrene following cold injury. VI. Capillary blood flow after cold injury, the effects of rapid warming, and sympathetic block. J. C/in. Inuest., 26,468. FOLKE, L. E. A. (1969). Anatomical considerations and functional behavior of the microvasculature in the oral mucous membrane. Ph.D. thesis, University of Minnesota. FOLKE, L. E. A. (1970). Revascularization in the oral mucous membrane following microvascular injury. Acra Odontol. &and. 28, 785. ISSELHARD,W. (1968). Influence of plasma volume expanders on microcirculatory disturbances. Bibl. Haematol. 29, 889. KNISELY, M. J., ELIOT, T. S., AND BLOCK, E. H. (1945). Sludged blood in traumatic shock. I. Microscopic observations of the precipitation and agglutination of blood flowing through vesselsin crushed tissue. Arch. Surg. 51, 220.
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L. (1957). Local freezing. Proc. Roy. Sot. Ser. B. London. 147,546. B. (1965). Cold injury of the skin: The pathogenic role of microcirculatory impairment. Arch Environ. Health 11,484. LANGE, K., AND B~YD, L. J. (1945). The functional pathology of experimental frostbite and the prevention of subsequent gangrene. Surg. Gynecol. Obstet. 80,346. LEFEBVRE, J. (1970). Effect of low temperatures on the oral mucous membrane. M.S.D. thesis, University of Minnesota. LYNCH, H. F., AND ADOLPH,E. F. (1957). Blood flow in small blood vessels during deep hypothermia. J. Appl. Physiol. 11. 192. MUNDTH, E. D. (1964). Studies on the pathogenesis of cold injury microcirculatory changes in tissue by freezing. In “Proceedings Symposia on Arctic Medicine and Biology” (E. Viereck, ed.), Vol. IV, p. 51. Arctic Aeromedical Laboratory, Fort Wainwright, Alaska. MUNDTH, E. D., LONG, D. M., AND BROWN,R. D. (1964). Treatment of experimental frostbite with low molecular weight dextran. J. Trauma 4,246. POOR,E., AND Lurz, B. R. (1958). Functional anastomotic vessels of the cheek pouch of the hamster. Anat. Rec. 132, 121. QUINTANILLA,R., DRUSEN,F. H., AND ESSEX, H. E. (1947). Studies on frostbite with special reference to treatment and the effect of minute blood vessels. Amer. J. Physiol. 149, 149. REITE,0. B. (1965). Functional qualities of small blood vesselsin tissue injured by freezing and thawing. Acta Physiol. Stand. 63, 111. SULLIVAN, B. J., AND TOWLE, L. B. (1957). Vascular responses to local cold injury. Amer. J. Physiol. KREYBERG, KULKA, J.
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M., AND PENN,I. (1965). A reappraisal of the microcirculation during general hypothermia. Surgery 58,105l. TAYLOR, A. C. (1957). The effect of rate of cooling on survival of frozen tissues. Proc. Roy. Sot. Ser. B. London 147,466. WEATHERLY-WHITE, R. C. A,, KNIZE, D. F., GEISTERFER, D. J., AND PATON, B. C. (1969). Experimental studies in cold injury. V. Circulatory hemodynamics. Surgery 66,208. WYMAN, L. C., AND DRAPEAU L. L. (1959). Vascular reactions to cold in the cheek pouch of intact and adrenalectomized hamsters. Amer. J. Pbysiol. 194,799. ZACARIAN, S. E., STONE, D., AND CLATER, M. (1970). Effects of cryogenic temperatures on microcirculation in the golden hamster cheek pouch. Cryobiology 7,127. SUZUKI,
