The Bovine Hemoglobin-Based Oyxgen-Carrying Solution (HBOC-201) Improves Flap Survival

The Bovine Hemoglobin-Based Oyxgen-Carrying Solution (HBOC-201) Improves Flap Survival in a Rat Model of Epigastric Flap Failure

Delio P. Ortegon MD, Michael R. Davis MD, James B. Sampson, Edward J. Dick MD, Vikram Kashyap MD, Jeffrey D. Kerby MD, PhD

INTRODUCTION Despite continued improvements in surgical technique and post-operative management of pedicled flaps, partial flap necrosis continues to be a substantial problem in plastic and reconstructive surgery. Partial flap loss has been reported in as many as 10-31% of pedicled musculocutaneous flaps used for head and neck reconstructions (1,2). Several researchers have sought interventions that would decrease the incidence of this complication. Pharmaceutical interventions have been sought to reduce the degree of reperfusion injury, increase vasoactivity, and alter the rheology of blood (3-8). Additionally, mechanical means of ischemic preconditioning have also been studied (9-10). However, few studies have been conducted to determine the effects oxygen therapeutic drugs. In the 1980s, Ramasastry et al. studied the effects of the perfluorocarbon Fluosol-DA (20%) and found that it did not alter flap survival in a model of epigatric skin flap failure and attributed its failure as a treatment regimen to increased blood viscosity in animals treated with Fluosol-DA (11). Recent studies, however, have shown that the bovine-hemoglobin based oxygen-carrying solution Hemopure® (HBOC-201), increased skeletal muscle oxygenation in a model of critcal arterial stenosis (12). It has also been showing to be an effective low volume resuscitation fluid in a procine model of controlled hemorrhage/resuscitation (13). It is these characteristics that have lead us to hypothesize that the administration of HBOC-201 would decrease the area of necrosis in a rat model of planned epigastric flap failure.

METHODS The experimental protocols were approved by the Wilford Hall Medical Center Clinical Investigations Division Scientific Review Board and were performed in accordance with the recommendations of the Good Laboratory Practices. Eight three male Sprague-Dawley rats were randomly assigned to one of four groups all of which had surgically created epigastrc skin flaps. Group one served as control animals. These animals underwent the surgical creation of the epigastric skin flap and had no further interventions. Group two (HBOC-1) received HBOC-201 preoperatively and then on post-operative days 3 and 5 (1gm/dl IV per tail vein injection). Group three (HBOC-2) were also given HBOC-201 preoperatively and on post-operative days 3 and 5 (2 gm/dl IV per tail vein injection). Finally, group four (LR) received intravenous injections of Lactated Ringer’s solution per tail vein preoperatively and on post-operative days 3 and 5. This volume was chosen to correspond to the highest volume of HBOC-201 given and served as a hemodilution control. All animals were housed in the individual cages and given water and standard chow ad libitum. After a seven-day observation period, the animals underwent the surgical procedure and injections, as determined by group.

Surgical Procedure All animals were anesthetized with intraperitoneal injections of a Acepromazine and Ketamine mixture (Ketamine 75mg/kg and Acepromazine 2.5 mg/kg). All animals were then prepped and shaved and a 5cm X 7cm ventral epigastric flap drawn to include only the medial branches of the left epigastric bundle, as described by Padubidri et al. (14). The flap was then raised in a cranial to caudad direction with identification of bilateral epigastric arteries at the epigastric/femoral artery junction. The right epigastric bundle was then ligated and divided. The left epigastric bundle was then examined and the left lateral branch of the epigastric artery ligated and excluded from the flap, leaving only the medial braches of the left epigastric artery. The flap was then replaced and secured with a running, 6-0 nylon. The animals were then recovered and examined daily for 7 days.

Flap Evaluation During the seven day observation period, the animals were inspected daily. Upon completion of the study, all animals were euthanized and digital photographs taken of each animal. Additionally, the entire skin flap was then removed and placed in formalin for histologic examination. All of the images were examined using Image Pro software®. The software was used to calculate the total flap area (cm2) and the area of flap necrosis. Using this data, the percentage of flap necrosis for each animal was then calculated using the following formula: Percent Necrosis= Area of Necrosis X 100 Total Area of Flap Additionally, paraffin imbedded tissue slides were prepared from a representative area of the flap. These slide were used for Hemotoxilin &Eosin (H&E) preparations.

Pathologic Examination The H&E preparations were examined by a board certified pathologist who was unaware of the animal group or treatment. After examining each slide, the pathologist scored each slide for degrees of neovascularization, inflammation, and venous congestion. Each category of evaluation was assigned a numerical score and this data was then used to calculate the median score for each group and used for subsequent statistical analysis.

Statistical Analysis Statistical analysis of the percentage necrosis data was initially completed by one-way analysis of variance (ANOVA) and followed by Tukey Test for multiple comparisons of groups using GraphPad InStat v. 3.05 for Window 95/NT (GraphPad Software, San Diego CA). This data is presented as a mean value +/- standard error of the mean (SEM). The histologic and immunohistochemistry scoring data were analyzed using a nonparametric analysis of variance (Kruskal-Wallis Test) given that this data consisted of non-ordinal values. The median values for each category is presented and used in comparisons. Differences were considered statistically significant at p<0.05.

RESULTS Percentage Flap Necrosis Animals in both HBOC-201 demonstrated less percentage of necrosis than the control and LR groups. Animals in the control group had a mean percentage flap necrosis of 22.24% while the area of necrosis for HBOC 1 and HBOC 2 was 9.14% (p=0.014 v control) and 12.22% (p=0.06 v control). While the animals in the HBOC 2 and LR groups demonstrated decrease percentage necrosis v. controls the differences were not statistically significant (p=0.061 and p=0.096, respectively), Figure 1.

Pathological Scoring Hematoxylin and Eosin staining and examination of the skin flaps revealed several interesting trends. Histologic examination of the slides revealed that specimens from animals in both HBOC groups demonstrated less venous congestion than did the control or LR groups. Levels of inflammation also differed in the groups. Animals from the LR and HBOC 2 groups demonstrated increased inflammation versus the control group. Finally, the levels of neovascularization were also different among the groups. Analysis, of this parameter demonstrated that animals that received 2gm/dl of HBOC-201 had increased levels of neovascularization versus all of the other groups (Figure 2). Statistical analysis of the median score for each group revealed that these trends were not statistically significant.

DISCUSSION This preliminary study of the oxygen therapeutic HBOC-201 (Hemopure ®) sought to determine whether the administration of this agent could alter flap survival in a model of planned epigastric flap failure. This product consists of polymerized bovine hemoglobin in a modified lactated ringers solution (table 2). The oxygen therapeutic HBOC-201 appears to decrease the area of necrosis at a dose of 1gm/dl in a rat model of epigastric flap failure. Comparison of the groups that received HBOC-201 to the hemodilution group (LR) suggests that this effect is not purely explained by the hemodilution and that the intrinsic properties of HBOC-201 (i.e. its oxygen carrying capacity, its resultant decrease in viscosity, or the antioxidant effect of NAC) may lend to improved flap survival. Additionally, investigation of similar products has demonstrated decreased transfusion requirements in subjects treated with HBOCs and thus may greatly impact the care of the patients in many specialties. Further investigation of this drug and its effects on flap survival is warranted.

30.00 28.00 26.00

24.00 *** p=0.0964 22.00 20.00 18.00 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 Figure 1 Histology Results

1.5 Median value 1

0 Congestion Inflammation Neovascularization Control HBOC 1 HBOC 2 LR Figure 2

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