Secondary Logo

Journal Logo

Protective Effect of Extract of Ginkgo biloba 761 against Frostbite Injury in Rats

Aizawa, Tetsushi M.D.; Kuwabara, Masahiro M.D.; Kubo, Satoshi M.D.; Aoki, Shimpo M.D., Ph.D.; Azuma, Ryuichi M.D., Ph.D.; Kiyosawa, Tomoharu M.D., Ph.D.

Plastic and Reconstructive Surgery: June 2019 - Volume 143 - Issue 6 - p 1657-1664
doi: 10.1097/PRS.0000000000005648
Experimental
Free

Background: When frostbite thaws, reperfusion injury has a crucial impact on tissue injury, and production of free radicals induces further tissue damage. This study examined whether extract of Ginkgo biloba 761 could ameliorate frostbite injury as a free radical scavenger.

Methods: Seventy-five Fisher 344 rats were divided into five groups of 15, and frostbite injury was created in each animal by sandwiching the left hind foot between a frozen magnet (−78.5°C) and a room-temperature magnet. Group I received saline; groups II, III, and IV received extract of Ginkgo biloba 761 (200, 100, and 50 mg/kg, respectively); and group V received superoxide dismutase (12 mg/kg). All drugs were injected intraperitoneally three times at 24-hour intervals. The wound surface area was measured throughout the wound healing period. Wounds were also harvested at various times to count cells stained by monoclonal antibodies for 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine.

Results: Compared to group I, the wound surface area was significantly smaller in groups II and III on days 1 and 3 after wound creation. Histologic examination revealed significantly more 4-hydroxy-2-nonenal–stained cells and 8-hydroxy-2′-deoxyguanosine–stained cells in group I compared to other groups on day 1. However, there was no difference in the total healing period among the groups. A higher dose test of extract of Ginkgo biloba 761 (300 mg/kg daily) induced animal death, probably because of toxicity.

Conclusion: Extract of Ginkgo biloba 761 demonstrated a protective effect against frostbite in the present model and probably alleviated reperfusion injury by reducing tissue peroxidation.

Tokorozawa, Saitama, Japan

From the Department of Plastic and Reconstructive Surgery, National Defense Medical College.

Received for publication November 9, 2017; accepted October 15, 2018.

Disclosure:The authors have no financial interest to declare in relation to the content of this article. There was no funding for this study.

Tetsushi Aizawa, M.D., 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan, nagareyamatsudo@live.jp

Frostbite occurs when exposure to low temperature causes tissue to freeze, generally on the extremities, and it can lead to extensive amputation loss and disability. Injury to tissues can occur with both freezing and thawing. Intracellular formation of ice crystals at the time of freezing results in damage to proteins and lipids, which affects cell membrane stability.1 Crystallization of extracellular water in the tissues causes dehydration of cells and alters intracellular electrolyte concentrations, leading to modifications of protein structure.2 Both dehydration of cells and occlusion of microvessels cause tissue ischemia and eventual necrosis. However, tissue damage progresses further with thawing. Intravascular platelet aggregation occurs in the arterioles almost immediately after thawing, and the entire microcirculation suffers endothelial damage in less than 60 minutes.3 Damage to endothelial cells by the freeze-thaw process leads to leakage efflux of intravascular fluid and promotes interstitial edema that results in compression of capillaries and vascular stasis.4

Tissue damage after thawing is exacerbated by reperfusion injury. Because of the deleterious effects of the freezing process, endothelial cells release various products of the arachidonic acid cascade5 and free radicals,6,7 after which the free radicals cause further endothelial cell damage. Generation of free radicals during reperfusion after normothermic ischemia has been reported to induce tissue necrosis.8–11 In addition, it was reported that depletion of free radicals at the time of reperfusion can protect skin flaps from ischemic necrosis.3,12–19 Free radical scavengers also protect against skin necrosis induced by frostbite, with scavengers such as superoxide dismutase and deferoxamine lessening tissue necrosis after freezing.20,21 These observations suggest that protection against free radical–induced injury during reperfusion in the postthaw phase of frostbite might possibly reduce tissue damage.

An extract made from dried Ginkgo biloba leaves (i.e., extract of Ginkgo biloba 761) has antioxidant properties and has been used for cerebrovascular disease, cardiovascular disease, sensory disorders, respiratory disorders, dementia, and inflammation. Extract of Ginkgo biloba 761 has demonstrated a protective effect against ischemic injury to skin flaps in a rat model,22,23 and improvement of flap viability was suggested to be attributable to removal of free radicals. We hypothesized that extract of Ginkgo biloba 761 would have a similar effect of reducing tissue damage caused by reperfusion injury after frostbite. Accordingly, this study was performed to examine the efficacy of extract of Ginkgo biloba 761 in a rat model of frostbite.

Back to Top | Article Outline

MATERIALS AND METHODS

Experimental Model

The protocol of this study was approved by the Animal Research Committee of the National Defense Medical College. Seventy-five healthy Fisher 344 rats weighing 150 to 250 g were divided into five groups of 15 animals, and each animal was subjected to cold injury by using a frozen magnet. Rats were anesthetized by intraperitoneal injection of a mixture of medetomidine (0.375 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg). The dorsal surface of the left hind limb was shaved with electric clippers, and depilatory cream was applied for 3 minutes to remove residual hair. Nickel-plated neodymium magnets (2400 gauss; diameter, 13 mm; thickness, 2 mm; weight, 1.8 g) were cooled in crushed dry ice (−78.5°C) for 15 minutes, and another identical magnet was embedded in polyurethane foam at room temperature (Fig. 1). The left hind limb of the rat was placed on the foam-embedded magnet and immobilized with dressing film (Airwall; Kyowa Co., Ltd., Osaka, Japan). Then, a cooled magnet was placed on the dorsum of the foot using tweezers, resulting in close application to the skin by magnetic force (Fig. 2). After 1 minute, the cooled magnet was removed and a new magnet was placed immediately at the same site. Exchange of the magnets was performed within 3 seconds, which was too rapid for thawing of the tissue to occur. A total of two magnets were applied, resulting in a freezing time of approximately 2 minutes. Subsequently, the frozen skin was rewarmed at room temperature (22°C). Recovery from anesthesia was assisted by intraperitoneal injection of atipamezole (0.75 mg/kg), and the animals were returned to their cages without dressing the wounds. Each animal was housed individually and allowed free access to food and water during the study.

Fig. 1.

Fig. 1.

Fig. 2.

Fig. 2.

Back to Top | Article Outline

Control Model

To investigate whether the magnets could cause pressure sores, a room-temperature magnet was placed on the dorsum of the foot in control rats after preparation in the same manner as in the experimental rats, and the magnet was exchanged once within 3 seconds (positioned for 2 minutes in total) to mimic the frostbite model. The skin at the application site was assessed after 24 and 48 hours.

Back to Top | Article Outline

Study Treatment

Treatment was started immediately before exposure to frostbite and was continued for 2 days; thus, three doses of each agent were administered. Group I received 1 ml of saline intraperitoneally every 24 hours. Groups II, III, and IV received extract of Ginkgo biloba 761 suspended in 1 ml of saline (Tebonin; Dr. Willmar Schwabe Pharmaceuticals GmbH & Co. KG, Karlsruhe, Germany) intraperitoneally at 200, 100, and 50 mg/kg, respectively, every 24 hours. Group V received superoxide dismutase (Radicut; Mitsubishi Tanabe Pharma Corp., Osaka, Japan) intraperitoneally at 12 mg/kg every 24 hours. On the day of wound creation, medication was given immediately before the experimental procedure.

Back to Top | Article Outline

Evaluation

The surface area of the wound was determined by tracing the margin on photographs using a high-resolution computer mouse, with the wound being defined as the region with epidermal defects, black discoloration, and/or blistering. Then, the number of pixels corresponding to the wound surface area was calculated by using ImageJ software (National Institutes of Health, Bethesda, Md.).

Back to Top | Article Outline

Histopathologic Examination

After the animals were killed humanely, the entire frostbite wound was harvested from two rats in each group on days 1, 3, and 5. The nine remaining rats in each group were monitored until the epithelialization of the wound was complete. To detect the effects of reactive oxygen species, tissue sections were stained by a monoclonal antibody for 4-hydroxy-2-nonenal, with cytoplasmic brown staining accepted as indicating lipid peroxidation. Other sections were stained with a monoclonal antibody for 8-hydroxy-2′-deoxyguanosine to detect oxidative DNA damage by stained brown staining of nuclei. Using an optical microscope, stained cells were counted in three high-power fields randomly selected at the marginal region of the wound. Then, the mean number of stained cells was compared between each group.

Back to Top | Article Outline

Statistical Analysis

The wound surface area and the number of positive cells per high-power field in each group were expressed as the mean ± SD and were compared by the Kruskal-Wallis H test. All analyses were performed using StatMate V statistical software (ATMS Co., Ltd., Tokyo, Japan), and significance was accepted at p < 0.05.

Back to Top | Article Outline

RESULTS

Control Model

Control rats did not have any wounds, skin lesions, irritation, swelling, discoloration, or bruising at 24 and 48 hours after removal of the magnets.

Back to Top | Article Outline

Wound Healing in the Experimental Model

Table 1 shows the wound surface area in each group on days 1, 3, 5, 7, 10, 14, and 18 after wound creation. Figure 3 shows the typical course of wound healing in each group. In all groups, no further tissue loss occurred after day 7, and complete healing was seen by day 21 in all animals. Figure 4 displays the wound healing rate calculated on the days when the wound area was measured. The wound area reached its maximum extent on day 3 to day 5 in each group. Compared with group I, the wound size was significantly smaller in groups II and III on day 1 (p < 0.001 and p < 0.01 by the Dunn test) and on day 3 (p < 0.001 by the Dunn test). Similarly, the wound size was obviously smaller in group V on day 3 and day 5 without significant difference. However, the mean duration of wound healing showed no significant difference between the groups.

Table 1.

Table 1.

Fig. 3.

Fig. 3.

Fig. 4.

Fig. 4.

Back to Top | Article Outline

Histology

When sections were harvested on day 1, significantly more epithelial cells, fibroblasts, and vascular endothelial cells were positive for staining by 4-hydroxy-2-nonenal (51 ± 5.3 cells per high-power field) and 8-hydroxy-2′-deoxyguanosine (55 ± 7.2 cells per high-power field) in group I than in group II (0.7 ± 1.6 and 1.0 ± 1.7 cells per high-power field), group III (0.3 ± 0.6 and 0.7 ± 1.2 cells per high-power field), group IV (33.0 ± 6.9 and 30.7 ± 8.5 cells per high-power field), or group V (2.0 ± 1.7 and 1.7 ± 2.1 cells per high-power field) (Figs. 5 and 6). On days 3 and 5, no stained cells were detected in wound sections from any of the groups.

Fig. 5.

Fig. 5.

Fig. 6.

Fig. 6.

Back to Top | Article Outline

DISCUSSION

Administration of extract of Ginkgo biloba 761 reduced the severity of frostbite because the wound surface area was smaller in groups II and III compared to group I on day 1 and day 3 after wound creation. Histologic findings suggested that the antioxidant effect of extract of Ginkgo biloba 761 protected the tissues from reperfusion injury.

Extract of Ginkgo biloba 761 contains 22 to 27 percent flavonoids and 5 to 7 percent terpene lactones. These two important classes of compounds have different properties that are responsible for the pleiotropic effects of extract of Ginkgo biloba 761. The flavonoids include glycosides of quercetin, kaempferol, and isorhamnetin, which show direct antioxidant activity by chelating prooxidant transitional metal ions and also promote expression of antioxidant proteins that increase production of antioxidant molecules such as glutathione.24–26 In contrast, terpene lactones (which include ginkgolides A, B, C, J, and M, and bilobalide) are platelet-activating factor antagonists that reduce platelet activation and aggregation.27,28 Extract of Ginkgo biloba 761 also inhibits vasospasm induced by thromboxanes in the presence of platelet activation, and this effect contributes to alleviation of ischemic tissue injury.29 The tissue-protective effect of extract of Ginkgo biloba 761 in the present frostbite model may be explained by such mechanisms, and it is likely that its antioxidant activity was important in ameliorating frostbite injury because superoxide dismutase (a pure antioxidant) also reduced the wound size in the present model, although the difference was not statistically significant.

Based on pharmacokinetic studies of extract of Ginkgo biloba 761 performed in animals and humans, the time to the peak plasma concentration of the flavonoid components is approximately 6 hours after oral administration and the elimination half-life is approximately 10 hours, whereas the plasma levels of terpene lactones reach a peak within 0.5 to 1.5 hours and the half-life ranges from 2 to 4 hours.30–32 To maintain an effective plasma level, oral administration twice daily (every 12 hours) is recommended for clinical treatment.33 However, the extract of Ginkgo biloba 761 regimens used in previous studies have varied with regard to dosage, interval, and administration method. To ensure consistent drug delivery without technical difficulties, we administered extract of Ginkgo biloba 761 intraperitoneally every 24 hours in the present study. The daily dose was determined by reference to the previous reports about dermal ischemia-reperfusion injury in skin flap models.22,23 Our findings in this study suggested that 50 mg/kg of extract of Ginkgo biloba 761 (the lowest dose tested) was insufficient to improve wound healing when administered once per day. In contrast, many of the animals died at a dose of 300 mg/kg daily (the highest dose), probably because of toxicity. Therefore, the appropriate dose seems to be in the range of 100 to 200 mg/kg every 24 hours, whereas a higher dose was associated with death, suggesting a potential risk if systemic administration of extract of Ginkgo biloba 761 was to be used clinically.

In this frostbite model, the wounds were circular and generally corresponded in size and shape to the magnets placed for wound creation. Even the largest wound did not extend beyond the margin of the contact site with the magnet. Therefore, it was considered that the wound area was determined by the severity of peripheral zone injury, rather than central injury. Accordingly, the current results suggest that extract of Ginkgo biloba 761 is more likely to be effective at the border zone of frostbite where reperfusion injury may have a greater impact than at the central zone where direct damage caused by freezing is more important. The histologic findings supported this concept, revealing numerous cells with lipid peroxidation in group I versus very few in group II. These cells presumably underwent necrosis and phagocytosis within a few days, because no stained cells were found on days 3 and 5 in either group.

Despite our findings that suggested a protective effect of extract of Ginkgo biloba 761 against frostbite injury in the present model, there was no significant difference of the wound healing time among the groups. In a pilot study, even administration of extract of Ginkgo biloba 761 for 14 days after frostbite injury did not significantly promote wound healing (data not shown). These results were probably influenced by wound contraction, which is prominent in rodent models because the loose surrounding skin allows rapid wound shrinkage. In contrast, wound healing in humans occurs mainly because of proliferation of new epithelium from the wound edges.34,35 Although wound contraction could have been prevented by placing a retaining frame at the wound edges, we did not do so because this study was focused on the mechanism of injury rather than the healing process. If extract of Ginkgo biloba 761 was used clinically, it would be likely to shorten the healing period. However, additional studies will be required to investigate the beneficial effect of extract of Ginkgo biloba 761 on wound healing in other experimental models that avoid the influence of wound contraction or by using a model of impaired healing.

Extract of Ginkgo biloba 761 is already used as a dietary supplement for vascular problems, and further clinical application is expected in the future. During a Himalayan expedition, administration of extract of Ginkgo biloba 761 was reported to improve vasomotor disorders of the extremities.36 The results of the present study suggest that it may be possible to extend the clinical application of extract of Ginkgo biloba 761 to treatment of frostbite in the future.

It seems probable that the effect of extract of Ginkgo biloba 761 is not specific for frostbite, and it may also be effective for wounds associated with ischemia-reperfusion injury or other types of free radical–induced tissue damage, such as pressure ulcers, crush injuries, free tissue flap transfer, chemical burns, or radiation-induced injury. Therefore, further studies may be warranted in models of these conditions to investigate the potential clinical applications of extract of Ginkgo biloba 761.

Back to Top | Article Outline

CONCLUSIONS

In the present rat model, extract of Ginkgo biloba 761 showed a protective effect against frostbite injury. Extract of Ginkgo biloba 761 alleviated reperfusion injury by reducing tissue peroxidation through its action as a free radical scavenger. The appropriate dose was in the range of 100 to 200 mg/kg. Although there was no difference of the total healing period, further investigation is required to determine whether extract of Ginkgo biloba 761 accelerates wound healing in both acute and chronic wound models.

Back to Top | Article Outline

REFERENCES

1. Heggers JP, Robson MC, Manavalen K, et al. Experimental and clinical observations on frostbite. Ann Emerg Med. 1987;16:1056–1062.
2. Reamy BV. Frostbite: Review and current concepts. J Am Board Fam Pract. 1998;11:34–40.
3. Marzella L, Jesudass RR, Manson PN, Myers RA, Bulkley GB. Morphologic characterization of acute injury to vascular endothelium of skin after frostbite. Plast Reconstr Surg. 1989;83:67–76.
4. Bourne MH, Piepkorn MW, Clayton F, Leonard LG. Analysis of microvascular changes in frostbite injury. J Surg Res. 1986;40:26–35.
5. McCauley RL, Hing DN, Robson MC, Heggers JP. Frostbite injuries: A rational approach based on the pathophysiology. J Trauma 1983;23:143–147.
6. Su CW, Lohman R, Gottlieb LJ. Frostbite of the upper extremity. Hand Clin. 2000;16:235–247.
7. Vogel JE, Dellon AL. Frostbite injuries of the hand. Clin Plast Surg. 1989;16:565–576.
8. Angel MF, Narayanan K, Swartz WM, et al. The etiologic role of free radicals in hematoma-induced flap necrosis. Plast Reconstr Surg. 1986;77:795–803.
9. Bulkley GB. Free radical-mediated reperfusion injury: A selective review. Br J Cancer Suppl. 1987;8:66–73.
10. Fridovich I. The biology of oxygen radicals. Science 1978;201:875–880.
11. Granger DN, Rutili G, McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981;81:22–29.
12. Im MJ, Manson PN, Bulkley GB, Hoopes JE. Effects of superoxide dismutase and allopurinol on the survival of acute island skin flaps. Ann Surg. 1985;201:357–359.
13. Im MJ, Shen WH, Pak CJ, Manson PN, Bulkley GB, Hoopes JE. Effect of allopurinol on the survival of hyperemic island skin flaps. Plast Reconstr Surg. 1984;73:276–278.
14. Manson PN, Anthenelli RM, Im MJ, Bulkley GB, Hoopes JE. The role of oxygen-free radicals in ischemic tissue injury in island skin flaps. Ann Surg. 1983;198:87–90.
15. McCord JM. The superoxide free radical: Its biochemistry and pathophysiology. Surgery 1983;94:412–414.
16. Hoshino T, Maley WR, Bulkley GB, Williams GM. Ablation of free radical-mediated reperfusion injury for the salvage of kidneys taken from non-heartbeating donors: A quantitative evaluation of the proportion of injury caused by reperfusion following periods of warm, cold, and combined warm and cold ischemia. Transplantation 1988;45:284–289.
17. Ratych RE, Chuknyiska RS, Bulkley GB. The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells. Surgery 1987;102:122–131.
18. Weatherley-White RC, Knize DM, Geisterfer DJ, Paton BC. Experimental studies in cold injury: V. Circulatory hemodynamics. Surgery 1969;66:208–214.
19. Kontos HA, Wei EP, Ellis EF, et al. Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ Res. 1985;57:142–151.
20. Manson PN, Jesudass R, Marzella L, Bulkley GB, Im MJ, Narayan KK. Evidence for an early free radical-mediated reperfusion injury in frostbite. Free Radic Biol Med. 1991;10:7–11.
21. Paramonov BA, Turkovski II, Doroshkevich OS, Taranova VN, Pomorski KP. Effect of local application of superoxide dismutase on dielectric parameters of cooled skin in rats. Bull Exp Biol Med. 2008;146:588–590.
22. Bekerecioğlu M, Tercan M, Ozyazgan I. The effect of Ginkgo biloba extract (Egb 761) as a free radical scavenger on the survival of skin flaps in rats: A comparative study. Scand J Plast Reconstr Surg Hand Surg. 1998;32:135–139.
23. Sambuy MT, Costa AC, Cohen C, Chakkour I. Effect of Ginkgo biloba extract (GbE-761) on the survival of fasciocutaneous flaps in rats. Phytother Res. 2012;26:299–302.
24. Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimer’s disease models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis. 2003;5:287–300.
25. Gohil K, Packer L. Global gene expression analysis identifies cell and tissue specific actions of Ginkgo biloba extract, EGb 761. Cell Mol Biol (Noisy-le-grand) 2002;48:625–631.
26. Oken BS, Storzbach DM, Kaye JA. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease. Arch Neurol. 1998;55:1409–1415.
27. Smith PF, Maclennan K, Darlington CL. The neuroprotective properties of the Ginkgo biloba leaf: A review of the possible relationship to platelet-activating factor (PAF). J Ethnopharmacol. 1996;50:131–139.
28. Oyama Y, Chikahisa L, Ueha T, Kanemaru K, Noda K. Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide. Brain Res. 1996;712:349–352.
29. Stücker O, Pons C, Duverger JP, Drieu K, D’Arbigny P. Effect of Ginkgo biloba extract (EGb 761) on the vasospastic response of mouse cutaneous arterioles to platelet activation. Int J Microcirc Clin Exp. 1997;17:61–66.
30. Zheng B, Teng L, Xing G, et al. Proliposomes containing a bile salt for oral delivery of Ginkgo biloba extract: Formulation optimization, characterization, oral bioavailability and tissue distribution in rats. Eur J Pharm Sci. 2015;77:254–264.
31. Biber A. Pharmacokinetics of Ginkgo biloba extracts. Pharmacopsychiatry 2003;36(Suppl 1):S32–S37.
32. Yan-Yan Z, Li-Li G, Guo-Ming S, Rong R, Jing-Zhen T. Determination of ginkgolides A, B, C, J and bilobalide in plasma by LC-ESI (-)/MS/MS (QQQ) and its application to the pharmacokinetic study of Ginkgo biloba extract in rats. Drug Res (Stuttg.) 2016;66:520–526.
33. Drago F, Floriddia ML, Cro M, Giuffrida S. Pharmacokinetics and bioavailability of a Ginkgo biloba extract. J Ocul Pharmacol Ther. 2002;18:197–202.
34. Davidson JM. Animal models for wound repair. Arch Dermatol Res. 1998;290(Suppl):S1–11.
35. Greenhalgh DG. Models of wound healing. J Burn Care Rehabil. 2005;26:293–305.
36. Roncin JP, Schwartz F, D’Arbigny P. EGb 761 in control of acute mountain sickness and vascular reactivity to cold exposure. Aviat Space Environ Med. 1996;67:445–452.
Copyright © 2019 by the American Society of Plastic Surgeons