Animal Experimental Studies

Many different animal models have been used to investigate the underling mechanisms of transferred tissues survival in order to simulate them with the relevant in humans.

Although Kernahan et al in 1965 reported no beneficial effect of HBO in island skin flaps in pigs, in 1966 Mc Farland and Wermuth69 used some shortcomings from the previous study and examined the HBO action on experimental pedicle flaps and composite skin grafts in rats. Their conclusion was that HBO had a definitive role to prevent necrosis extension in both grafts and flaps. The possible explanation for the opposing results of those studies is that pig flap is a true random flap, while the one of rat is myocutaneous with incorporated blood supply.

In 1967 Champion et al70 exposed pedicle flap in a rabbit model for 2 hrs, twice a day for five days, and achieved survival 100% of HBO flaps versus 50 % in the control group. Flap survival was relevant to alveolar and skin oxygen tension. None of the flaps treated with compressed air survived. HBO was proposed to maintain flaps viability until the restoration of microvasculature.

The following year Shulman and Kron71 showed in a rat model that HBO combined with repeated grafting reduced in half the necessary healing time for partial thickness wounds comparing it with the relevant of control group. In parallel, the possibility to prevent infection through high oxygen levels was demonstrated in transferred tissues, while the rats in the HBO group were not infected, although bacterial contamination occurred in all the studied animals.

In 1968 Wald and Georgiades72 studied intensive HBO therapy in experimental rat skin flaps, demonstrating a 22% increase in flap survival.

Perrins and Winter73 in 1970 used pigs with shallow wounds to test the HBO efficacy in skin covering in order to compare this model with human healing rhythm of the recipient bed after grafts were detached. The results were 80% skin coverage in the HBO group versus 49% in the control group.

In a study74 comparing PO2 in rat skin flaps between HBO at 3 ata and 100% FiO2 in normobaric conditions, oxygen tension in hyperbarism raised up to 600 mmHg whereas in 1 ata no raise occurred.

Niinikoski75 and Hunt in 1970 also used tubed rat skin flaps to compare the size of viable area in a HBO group versus a similar control group breathing normal air. The HBO group showed 51% extension in length of survived flap versus the control.

In a study from Jurrel and Kaisjer76 pedicle rat flaps were compared concerning the survival area according to the onset of HBO. Better results were obtained when HBO was started immediately after the surgery compared to those when HBO was delayed more than 24hrs. Even though the results of the delayed HBO use were still of statistical importance, the prompt onset of HBO was emphasized.

Using a model of provoked and predictable necrosis in rat skin flaps, Arturson et al.77 demonstrated that in the HBO treated group there was a statistical important improvement in flap survival in comparison to the control group.

In 1973 irradiated rats were studied by Greenwood and Gilchrist78 to evaluate the HBO effect in skin flaps 6 months post radiation. The HBO treated group showed a significant flap survival compared with the control group treated with air.

Niinikoski and Kivisaari79 in 1975 performed a controlled study in rats with HBO at 2 ata to evaluate the healing rate in open wounds with intact or disturbed circulation. Improvement has been shown only in the devascularized wounds of HBO group, implying the role of hyperoxic healing enhancement in compromised wounds.

Manson et al.80 in 1980 studying pedicle flaps in Guinea pigs demonstrated that animals treated with HBO had a three fold increase of capillary net when compared to controls.

Caffee and Gallagher81 in 1981, using modern flap designs in pigs have not shown effectiveness of HBO use in improving flap survival.

Tan et al.82 in 1984 examined the HBO effect in island flaps in rats, in comparison with a control group treated with air under pressure and normobaric 100% oxygen. Significant flap survival was noticed only in the HBO group. The authors suggest that as oxygen tension is lower in flaps compared with skin tension, proper tissue oxygenation may be restored only through hyperbaric hyperoxia .

Nemiroff and Lungen83 in 1987 performed a controlled randomized study to examine the possible HBO action in irradiated skin flaps in rats, comparing as well ordering effects of HBO and effects of flap elevation and radiation (in 15 groups). HBO was performed before flap elevation, soon after surgery, (4hrs) or later. Authors reveal that the sooner the HBO is initiated after the "surgery", (in the study "surgery" represents a programmed flaps failure) the better are the results for flap survival. Thus, in case of a diagnosed problematic flap HBO should be administered on time.

In the same study authors used flap histopathologic analysis to show that the number and possibly the size of capillaries were significantly greater in the HBO group. They conclude that HBO enhances flap survival through promotion of microvasculature.

Composite skin grafts in rabbits were used by Rubin et al84 in 1988 to examine the HBO effect. The HBO group was submitted in hyperbaric exposure twice a day for ten days, resulting in significant grafts survival compared with the control group.

A protocol including HBO combined with pentoxyphylline, known to increase red cell flexibility was used by Nemiroff and Ryback85 in 1988 on rats skin flaps. Animal groups included a control group, two HBO groups (one with and the other without pentoxyphylline) and one group treated only with pentoxyphylline. Statistically important results concerning flaps survival were achieved in the group where HBO was combined to pentoxyphylline, although both the remaining groups showed improved results versus the control group.

Zamboni et al.32 in 1989 experimented on axial pattern flap survival in rats with HBO, administered during or just after prolonged flap ischemia of 8 hrs. It was a controlled study comparing the control group versus subgroups, using HBO during ischemia or reperfusion. HBO was shown to increase flaps viability when administered both during or just after ischemia.

Kerwin et al.86 in 1992 used a cat model to evaluate HBO efficacy on pedicled skin flap after having clamped the nourishing artery. HBO was performed once a day at 2 ata, whereas in most experimental studies it was used twice a day. Beyond the improvement in flaps color and exudation after HBO use, survival was similar both in HBO and control group. Pellitteri et al.87 in 1992 performed a controlled study on random skin flaps in swine with the application of intensive and tapering HBO. Flaps were designed to obtain a predictable length of necrosis. Their results demonstrated a diminution of 35% in flap necrosis when HBO was administered.

The HBO effect in preserved free flaps of rats was studied by Tai et al.88 in 1993. After 18hrs of flap preservation HBO administration induced flap survival from 10% (in air) to 60%. Authors presume that HBO application inhibited the role of xanthino-oxydase, improving flap survival.

Zamboni et al.27 in 1993 used Laser Doppler flowmetry to evaluate HBO effect on ischemic skin flaps in rats during reperfusion. They revealed that hyperbaric hyperoxia enhances distal microvascular skin perfusion, responding in improvement of flap survival.

Xenon washout method for measuring blood flow and vital fluorescein microscopy as index of functional capillary density was used by Sirsjo et al.89 in 1993 to detect the efficacy of HBO, to treat postischemic muscle tissue. Their results suggested that HBO enhances the recovery of blood flow and functional capillary density in post-ischemic muscle tissue, indicating attenuation of the microvascular dysfunction or of the damage in the postischemic period.

Stewart et al.90 in 1994 examined the effect of free-radical scavengers combined with HBO on random-pattern skin flaps in rats. 10 treatment groups of rats were randomly assigned. Superoxide dismutase, catalase, alphatocopherol acetate and the inhibitor allopurinol were used to combat or scavenge radicals. HBO was performed daily at 2,5 at a. The goal of the study was to increase the oxygen delivery to the flap and simultaneously reduce or prevent the action of free radicals. The combination of treatments resulted in significantly increased random-pattern flap survival in all HBO groups, except in the allopurinol one, compared to untreated controls.

Recently in 2002 Prada et al.91 used a model of axial pattern skin in rats to evaluate the effect of allopurinol, superoxide-dismutase and hyperbaric oxygen. The percentage of flap necrosis was significantly smaller in all-experimental groups when compared to controls.

Erdmann et al.92 in 1995 used a highly immunogenic skin allograft mouse model to determine the HBO effect as an immunosuppressive agent. The controlled study used three randomized groups with varying dose of HBO. Authors suggest that low dose and intermediate HBO treatment delayed skin allografts rejection, confirmed also histologically.

A controlled study by Zamboni et al.93 in 1996 evaluated in a microcirculation model on a rat gracilis muscle flap the HBO action on flap arterial neutrophil concentration and the relevant pulmonary neutrophil sequestration. Acquired results demonstrated that HBO significantly reduced concentration to sham levels without inducing pulmonary sequestration.

The result of HBO use in free tissue transfer was extensively studied by Stevens et al.94 in 1996 on abdominal adipocutaneous island flaps in rats. Vascular pedicles were clamped twice for 6 hrs each, followed by a 2 hr interval of reperfusion; then ischemia was reapplied for different periods (6,10,14 hrs). After the second period of performed ischemia animals were randomly assigned in groups treated with air, 100% normobaric oxygen, and HBO. A statistically significant increase in flap survival was reaffirmed only in the HBO group.

In a study by Ramon et al.95 in 1998 concerning single-pedicle transverse abdominis myocutaneous flap in a rat model and possible HBO effect, results suggested that in the treated group flap survival differed significantly from the controlled animals.

Bayati et al.96 in 1998 evaluated the HBO effect in the stimulation of angiogenesis to improve the viability of prefabricated flaps. Two modalities, basic fibroplast growth factor (BFGF) and HBO were used separately and together on prefabricated myocutaneous flaps in rats. Laser Doppler skin perfusion and histological examination were performed to evaluate post stimulation vascularity. Both HBO and BFGF used separately, increased significantly the survived area of the prefabricated flap. The combined application of these two modalities induced furthermore the effect, leading to near complete flap survival through improved vascularity.

Zhang et al.97 in 1998 demonstrated in a controlled study that postoperative HBO increases neovascularisation (documented histologically) and the acquired survival of the rat ear composite graft was significantly important (82% versus 26,5% in control group).

The question for the use of HBO to attenuate lipid peroxidation on free skin grafts was posed by Lemarie et al.98 in 1998, as in their study HBO exacerbated the degree of oxidative stress in relevant rat flaps.

Axial skin flaps in rats subjected to total venous occlusion were used by Lozanno et al.99 in 1999 to evaluate the effect of HBO and medicinal leeching. Arterial inflow was left intact. Five animal groups were randomly assigned according to combined or single treatment. The study demonstrated that although HBO alone was not effective, when it was combined with leeching it resulted in significant flap survival compared with leeching alone.

The effect of HBO on microvascular anastomosis healing and patency was studied by Shi et al.100 in 1999 in a rat model. The authors using eighty anastomosed femoral arteries in five groups concluded that HBO might be useful in improving the healing of microvascular anastomoses, mainly in vessels that have undergone crush injury.

Yucel et al.101 in 2000 examined the effects of HBO and heparin, used combined or separately on epigastric venous flap in rats. Their results imply that flap survival rate in the combined modalities group was significantly higher than in the control, or in the heparine group.

Gampper et al.102 in 2002 studied the potential for HBO to decrease the effects of a secondary ischemic event on epigastric flap model in rat. Cross clamping of the vascular pedicles for 2hrs was followed 24 hrs later by flap reelevation and reocclusion for 5hrs. Rats were divided into three groups. The first group received HBO immediately before primary ischemia, the second underwent similar treatment prior to secondary ischemia and the third group used as control. The results showed that all control flaps were nonviable, where complete (20% versus 31%) or partial flap survival (48% versus 55%) occurred respectively in the two treated groups. In conclusion, HBO significantly increased the survival of flaps subjected to a secondary ischemia even if administered before the primary ischemia.

Survival of distal skin paddle through HBO was evaluated by Richards et al.103 in 2003 in a rat tubed pedicle flap model. Their results showed that at 5 days after pedicle division flap survival in the HBO groups was of statistical importance, as compared to the relevant of control groups.

Ischemia reperfusion injury (RI) and HBO action on the mechanism involving the expression of adhesion molecules was studied extensively by

Hong et al.104in 2003 in a rat musculocutaneous flap. The study consisted of gross examination for flap survival, histology, immunihistochemical staining, myeloperoxidase assay, flow cytometric study of CD18, and Northern blot analysis on ICAM-1 messenger RNA expression. Their results suggest that HBO does not alter the expression of CD18, but decreases the expression of ICAM-1. The point of HBO application, whether applied before or after RI, did not show any differences in outcome. HBO increased flap survival against RI through a protective mechanism involving downregulation of ICAM-1 on endothelial cells.

Recently Li et al.105 in 2004 experimented on auricular composite grafts in rabbits with HBO. They suggest that HBO enhanced graft survival mainly in the larger composite grafts.

In conclusion, the last 40 years several experimental studies were performed concerning the possible role of HBO on compromised tissues, using different animal models as also various types of transferred tissues.

Although this variety of studies includes non-homogenous models regarding flat or graft type, blood supply, tissue origin, procedure for programmed "failure", host immune status making comparison not reliable, it is certain that the common denominator is the presence of hypoxia. Statistically important improvement of compromised tissue survival was reported in almost all the controlled studies, thus indicating that HBO counteracts the hypoxic insult.

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