Trental

Background & objectives: Frostbite, the severest form of cold injury is a serious medical problem for our Armed Forces operating in the snow bound areas at high altitude. Effects of treatment by rapid rewarming in tea decoction followed by combined therapy of pentoxifylline, aspirin and vitamin C were evaluated in amelioration of tissue damage due to experimentally induced frostbite in rats.

Methods: Experiments were conducted in 2 groups (25 each) of albino rats (control i.e., untreated and experimental i.e., treated). Frostbite was produced experimentally in all the animals by exposing one of the hind limbs at -12+/-1 deg C with wind flow 25-30 lit/min for 30 min in a freezing-machine, with simultaneous recordings of rectal and ambient temperatures. The degree of tissue damage was assessed after 10 days. Following cold exposure, neither external thawing nor any medication was given to the animals of the control group; while the exposed limb of the experimental animals was rewarmed in tea decoction maintained at 37-39 deg C for 30 min immediately after cold exposure, with simultaneous oral ingestion of warm tea decoction. These animals were also given pentoxifylline (40 mg/kg), aspirin (5 mg/kg) and vitamin C (50 mg/kg) twice daily orally for the next 7 days.

Results. In the control group, 68 per cent animals suffered from severe (56%) to very severe (12%) frostbite, while the remaining 32 per cent had moderate frostbite. No animals of this group could escape injury or suffered anything less than moderate frostbite; whereas 52 per cent of experimental animals escaped injury (no frostbite) and 32 and 16 per cent suffered only with primary and moderate degree of injury, respectively. None from this group suffered from severe or very severe frostbite.

Interpretation & conclusion: It is evident from the study that this combined therapy resulted in significant improvement in the degree of tissue preservation and proved to be highly beneficial as an immediate treatment of frostbite in rats. The combined pharmacological properties of these drugs might have altered the haemorrheologic status of blood and produced curative beneficial effect in improving tissue survival. Clinical studies are required for confirmation of these beneficial effects in humans, which has already been taken up.

Key words Armed forces - cold injury - high altitude - rats - therapeutic treatment

Frostbite, the severest form of cold injury, generally occurs on prolonged exposure to freezing temperature or even on brief exposure to a very severe cold and windy environment. Among civilians frostbite is not very common except for occasional instances seen among mountaineers, but it is a major health hazard and a serious medical problem for our Armed Forces, operating in severe cold and hazardous weather conditions at high altitude. In spite of extensive studies conducted on the prevention and treatments1-10, frostbite continues to be of serious concern. Further, the treatment of frostbite once it occurs is not very satisfactory, particularly when there is a delay in starting therapy; and in extreme field situations, delays in the evacuation of frostbite victims to hospital do occur due to serious logistic problems. The time lag between occurrence of frostbite and the initiation of therapy plays a crucial role in its curative process3,4. Our previous studies3,4 included evaluation of the use of pentoxifylline, aspirin and vitamin C along with the simple procedure of rewarming in warm water, tea decoction etc., singly as well as in different combinations in the treatment of frostbite.

Based on the beneficial effects observed with rapid rewarming in a decoction (37 deg C) of Indian black tea followed by high dose vitamin C^sup 3^, and the promising results of a pentoxifylline and aspirin4, a simple treatment procedure applied in the field situation was formulated and the present study was conducted to evaluate the synergistic effect, if any, of rapid rewarming in tea decoction maintained at 37 deg C immediately after induction of frostbite followed by combined therapy with pentoxifylline, aspirin and vitamin C as a measure of immediate treatment of frostbite in rats.

Material & Methods

Experiments were conducted during non-summer months (October-March) at the Defence Institute of Physiology and Allied Sciences (DIPAS), Delhi using a new method which more or less simulates the condition in which frostbite occurs in humans at high altitude cold areas, where a combination of wind and cold (wind-chill factor) contributes to freezing of the tissues. Fifty albino rats (SpragueDawley) of either sex with body weight ranging between 175-200 g were taken from the animal house of DIPAS. The care of the animals and the maintenance of animal house were done by a qualified veterinarian. The study was approved by the Ethical Committee of the Institute. Animals were maintained on Hind-Lever diet and tap water ad-libitum. They were divided randomly into two equal groups, keeping the sex ratio and body weight similar in both the groups.

The animal was placed on a thin wooden platform and semi-restrained with the help of a perspex harness, while one of the hind limbs was protruded outside. Frostbite was produced experimentally in all the animals by exposing the protruded hind limb in a freezing-machine by maintaining the temperature at -12+/-1 deg C with wind flow of 25-30 I/min for 30 min. Each animal was exposed to frostbite once only. During the exposure, the body of the animal was not exposed to the cold and remained at room temperature (26-28 deg C) of the airconditioned laboratory. The freezing machine was designed and fabricated at the DIPAS workshop. A stream of air from an air-compressor, which has a pressure regulator and a moisture filter, was passed through a series of copper coils, submerged in freezing mixture consisting of ice and salt. The cool air then entered a small glass receptacle in which the limb of the rats was exposed. The three parameters of exposure viz., temperature, duration and wind velocity were independently as well as simultaneously controlled and measured. Thus it was feasible to expose the animal's limb for a predetermined duration, temperature and wind velocity. The degree of exact tissue loss was dependent primarily on the above three factors.

The present method of experimentally induced frostbite in animals has certain advantages. The temperature, wind speed and duration of exposure can be kept at a constant level or any one of them can be individually controlled, so that injury of different types and severity can be produced which is useful in physiological and pharmacological studies. The rectal temperature and the temperature inside the glass receptacle were monitored constantly during the exposure by YSI Telethermometer, model 46 TUC (Yellow Spring Instruments Co,., USA) in conjunction with appropriate probes. At the end of 30 min exposure, the rat's limb was removed from the freezing-machine and the immediate apparent changes in the paw, which was generally hard, were noted. On the basis of tissue necrosis which is evident by 10-15 days3, the nature and severity of the degree of tissue damage were assessed and classified after 10 days of exposure as very severe frostbite (VSFB), severe frostbite (SFB), moderate frostbite (MFB), primary/mild frostbite (PFB) and no frostbite (NFB) according to the criteria published earlier3. Utmost care was exercised (i) in handling the delicate injured parts during and after rewarming, so that no further damage occurred; (ii) for prevention of infection as secondary infection is a major contributory factor in determining the severity of cold injury and; (iii) that the frozen tissues once rewarmed be never re-exposed to cold.

While standardizing the method, it was found that with this degree of cold, wind and duration of exposure, about 50 per cent animals develop SFB, 30-40 per cent MFB, and 10-20 per cent VSFB.

In the present experiment, after the cold exposure, external rewarming agents were not applied to the exposed limb of the animal of the control group nor any medication was given to them. However, they were kept under constant observation with hygienic care in an air-conditioned animal house. The exposed limb of the animals of experimental group was rewarmed rapidly in a decoction of tea leaves maintained at 37-39 deg C for 30 min immediately after cold exposure, with simultaneous oral ingestion of about 5 ml of warm tea decoction. Ten g of tea leaves (Brook Bond Red Label) per litre of warm water was boiled for 5 min, then bringing the temperature down to 37-39 deg C without adding cold water or ice. Rewarming of the exposed parts was done in a wide mouth vacuum flask (3 1 capacity) with regular stirring. During the rewarming period the temperature was maintained by the addition of warm tea decoction from time to time. The animals were also administered orally pentoxifylline, 40 mg/kg (Trental(C) - 400; Hoechst India Ltd.), aspirin, 5 mg/kg (acetyl salicylic acid) and vitamin C, 50 mg/kg (Glaxo, India) as single intake each time, twice daily4 for the next 7 days. Rewarming in tea decoction was done for two successive days, once daily for 30-35 min in the forenoon between 1000 - 1200 h. From day 2 onwards silver sulphadiazine cream was applied externally on the affected skin surface of all the animals of both the groups to prevent secondary infection.

Hind paw volume was measured before cold exposure (day 0) and thereafter on days 3, 6 and 10 of cold exposure in all the animals of both groups. Paw volumes were measured by simple volume displacement method upto,a pre-marked position using a well graduated glass centrifuge tube11.

Student's T test was used to test the significance of the degree of frostbite as well as the paw volume changes on different days after cold exposure and also between the two groups.

Results

The initial rectal temperatures (Tre) of all the animals of both groups were similar (37.3-38.2 deg C). There was no appreciable change in Tre during cold exposure of the limb. Recording of Tre could not be continued during the rewarming phase and thereafter.

SFB was seen in 14 of the 25 animals (56%) in the control group, VSFB in three (12%) and MFB in 8 (32%). None of the animal of this group escaped frostbite or had PFB. In the experimental group, the limb of 13 animals (52%) remained completely free from any type of tissue injury, 8 (32%) had PFB and 4 animals (16%) had MFB. None from this group suffered with SFB or VSFB, demonstrating the beneficial (P

The changes in paw volume are depicted in the Fig. On day 3 post cold exposure, both groups showed a significant (P

Discussion

Cold injury is believed to occur due to anoxia resulting from circulatory insufficiency as well as the direct freezing effect of cold on the exposed parts. The purpose of treatment should be to correct the sluggish circulation which occurs as a result of cold injury as quickly as possible to establish even a slight increase in blood flow in the extremities and prevent tissue damage. This can be done with rapid rewarming immediately on recognition of the cold injury. During the rewarming process ischaemia is relieved and re-perfusion takes place due to improvement in sluggish circulation in the affected organs, coupled with severe pain. Rewarming of frozen tissues is associated with generation of oxygen-derived free radicals, which may lead to additional tissue injury12. At this stage particularly during the rewarming phase use of pentoxifylline, a haemorrheologic agent effective in increasing blood flow to the affected micro circulation and enhancing tissue oxygenation and use of aspirin, an inhibitor of thrombus formation and also an analgesic along with vitamin C, an antioxidant may provide adequate protection by scavenging singlet oxygen and free radicals13 and help in preventing tissue damage to a great extent.

The findings of the present experiments suggest that the immediate rewarming of the exposed area possibly acts by restoring blood flow rapidly, so that there is inadequate time for the plasma to be lost and the red cells to sludge3. The exact mechanism for the beneficial effect of rewarming in tea decoction is not clearly understood. However, this may be attributed...to the possible role of the constituents of tea leaves, which contain volatile oil, tannic and gallic acids, quercentin, theme, xanthine, theophylline and theobromine. Quercentin is known to reduce abnormal capillary fragility and thereby maintain its integrity14. Theine relaxes smooth muscle and is a powerful nerve stimulant15. The role of aminophylline/theophylline (constituents of tea leaves) in improving thermogenesis, cold tolerance and prevention of hypothermia has also been reported16,17. The oral ingestion of the same decoction may have improved cold tolerance due to its thermogenic effect.

Although the precise mechanism of action is not very clear, pentoxifylline improves RBC flexibility and deformability and helps in the reduction of platelet aggregation and increase in blood fibrinolytic activity18. Clinical responses to longterm oral pentoxifylline resulted primarily from improved flexibility of erythrocytes and reduced blood viscosity; a decreased concentration of fibrinogen may have contributed to the latter. Reduced functions of platelets and granulocytes may also be involved19-21. Further, pentoxifylline plays a vital role in reducing the associated morbidity and limits the extent of amputation.

It appears that both the antiplatelet aggregation activity as well as the analgesic action of aspirin 22 potentiates the effect of pentoxifylline. Hastening of recovery due to the administration of vitamin C is possibly due to its antioxidant effect12-23 as well as the increased metabolism and thermogenic properties24. Vitamin C rapidly enters into leukocytes and potentiates cellular immunity, thus protecting against subsequent infection, which is a major contributory factor in determining the severity of injury23. Vitamin C also helps in collagen synthesis and is known for its use in accelerating the wound healing process3,23. The protective effect of vitamin C against cold and frostbite has also been demonstrated successfully by many investigators2,3,23-25.

The increase in paw volume due to oedema is attributed in part to vasodilatation and much more to accumulation of exudate in the tissues9. This is perhaps due to the beneficial effect of combined therapy which might have helped in preventing tissue damage. In the experimental group, paw volume reduced to near normal levels with mild or no injuries of the limb in 84 per cent of the animal. Aspirin acts as an effective pain reliever during the post exposure rewarming phase22 which might have helped in preventing the animals of experimental group from damaging their own paws. The untreated controls were unable to bear the severe pain and damaged their paws by scratching and biting.

In conclusion, it can be said that administration of pentoxifylline, a haemorrheologic agent and aspirin, an inhibitor of prostaglandin along with a high dose of vitamin C, an antioxidant preceded by rapid rewarming in tea decoction maintained at 37-39 deg C, was highly beneficial as an immediate treatment of frostbite in rats. The combined pharmacological effect of these drugs, preceded by rapid rewarming might have altered the haernorrheologic status of blood and resulted in a protective role in treating experimentally induced frostbite.

The findings call for clinical studies for confirmation of the beneficial effects of the combination therapy in man. Positive findings in human subjects, if established, will help to formulate a simple treatment procedure, which will be self-applicable in the field by the victim himself, immediately on recognition of cold injury.

Acknowledgment

The authors thank Dr S.S. Verma and Ms Sikha Arora for statistical treatment of the data and Dr Rameshwar Singh for the care and maintenance of the animals throughout the study.

References

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Reprint requests : Shri G. Bhaumik, Scientist C, Defence Institute of Physiology & Allied Sciences, Defence Research & Development Organization, Lucknow Road, Timarpur, Delhi 110054, India

S.S. Purkayastha, G. Bhaumik, S.K.S. Chauhan, P.K. Banerjee & W. Selvamurthy

Cold Physiology Department, Defence Institute of Physiology & Allied Sciences Defence Research & Development Organization, Delhi, India

Received December 18, 2001

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