The Effect of Nitroglycerin Treatment Initiation Time on Survival in Partial Flap Necrosis: An Experimental Study

Abstract

Background: Nitroglycerin is suggested to improve flap survival based on promising results; however, there are no data on the effectiveness of treatment initiation time. This study aimed to compare the effect of various nitroglycerin treatment initiation times on partial flap survival. Materials and Methods: The study included 50 Sprague-Dawley rats. Modified McFarlane flaps were elevated on the dorsum of each rat. Group A received placebo treatment. Groups B, C, D, and E received topical nitroglycerin 2% starting 1 day before surgery, on the day of surgery, postoperative d 2, and postoperative d 4, respectively. After 7 days, the flap survival rates were calculated. Afterward, the severity and extent of inflammation and ischemia, and the severity of edema were evaluated histologically. Results: The flap survival rate was highest in group B, followed by groups C, D, E, and A. The difference between groups B and C was not significant, whereas the difference between group B and groups A, D, and E was. In addition, the difference between groups A, D, and E was not significant. Histological analysis showed that inflammation was less severe in groups B and C than in groups A, D, and E. Ischemia was the most severe in groups A and D and was the least severe in group C. Conclusion: Topical nitroglycerin treatment increases flap survival when initiated before or on the day of surgery, but has no benefit when initiated on postsurgery d 2 or 4. Preoperative initiation of nitroglycerin treatment positively affects flap survival.

Introduction

Many pharmacological agents have been suggested to improve the survival of flaps that have a tendency to undergo partial necrosis. ¹ One such agent whose effectiveness has been evaluated is nitroglycerin. Nitroglycerin was predicted to increase flap vascularity due to its vasodilator and antithrombotic effects.^2,3 Rohrich et al.'s ⁴ initial study on rats and pigs showed that there is an increase in flap survival with the use of nitroglycerin. Many subsequent experimental studies confirmed this finding.^4‐7 These experimental studies were followed by clinical studies and small case series that observed the benefit of nitroglycerin treatment for hypospadias treatment and peripheral necrosis.^8,9 Today, topical nitroglycerin treatment is frequently used to increase the survival of mastectomy flaps and relevant studies are ongoing.^10‐12 A recent prospective randomized study by Gdalevitch et al. ¹³ reported that a single dose of topical nitroglycerin applied during the early postoperative period reduces the rate of necrosis in mastectomy flaps by 50%. ¹³ A recent meta-analysis also confirmed the benefit of nitroglycerin use for mastectomy flaps. ¹⁴

Predicting flap survival intraoperatively based on clinical observation alone is not always possible ¹⁵ ; therefore, the use of such imaging tools such as the SPY Elite laser angiographic system (Novadaq Technologies) or ViOptix Intra Ox (ViOptix, Inc.) has been recommended to increase prediction accuracy. ¹⁶ Nonetheless, acquisition and use of these instruments require financial resources that are beyond the reach of some healthcare institutions. In the case of failure to predict flap survival via clinical evaluation or patient follow-up, unexpected partial flap necrosis can become evident 2 to 4 days postsurgery. Unless there is excess tension in the flap, hematoma under the flap, or pressure on the pedicle (in the axial flaps) surgical options are limited. In such cases, successful pharmacological treatment can reduce tissue loss and reduce/eliminate the need for secondary surgery.

Although there is a general consensus concerning the beneficial effect of nitroglycerin, there are no data on the effectiveness of its initiation during the preoperative versus late postoperative period. It is possible to investigate this subject in a clinical study, due to the safe side effect profile of nitroglycerin. However, standardization problems may arise in the planning of these studies in humans. There are many variables (neoadjuvant chemotherapy, flap thickness [oncological surgeon], smoking habits, comorbidities, etc.) that may obscure the results. So, a standardized experimental study is designed to clarify this issue. The aims of the present study were to determine the effect of topical nitroglycerin application on flap survival and to compare the efficacy of different treatment initiation times.

Materials and Methods

The study protocol was approved by the local animal experimentation ethics committee (registration number: 2020-53). All procedures, including care and use of animals, were performed in accordance with the Guide for the Care of the Laboratory Animals published by the National Institutes of Health. ¹⁷

Preparation of Ointments

Placebo and nitroglycerin ointments were prepared by a licensed pharmacist. Both ointments were prepared to be indistinguishable from each other in terms of color, odor, and density. The placebo consisted of Vaseline and emollient cream. Similar to the clinical studies by Gdalevitch et al. ¹³ and Turin et al., ¹¹ nitroglycerin ointment included nitroglycerin 2% as the active ingredient, as well as the 2 placebo ingredients. The sufficient amount of ointment to cover the flap was used.

Data from previous studies investigating nitroglycerine use on the same flap model ¹⁸ was used to calculate the effect size. The calculation of the sample size was conducted using GPower software. ¹⁹ The total sample size of 50 subjects (10 subjects in each group) achieves 80% power to detect statistical differences with a 0.516 effect size among the means of mean percent surviving area of the 5 groups with a .05 significance level.

The study included 50 Sprague-Dawley rats weighing 200 to 250 g. The rats were housed under a 12-hour day/night cycle, with temperature maintained at 22 °C to 24 °C. The rats were fed with standard rat chow and tap water ad libitum. After induction of general anesthesia using ketamine hydrochloride (87.5 mg kg⁻¹) and xylazine hydrochloride 2% (12.5 mg kg⁻¹), caudally based 9 × 3-cm flaps comprising skin, subcutaneous tissue, and panniculus carnosus (modified McFarlane flaps) were elevated on the dorsum of each rat. ¹⁹ To prevent the flap from surviving as a graft, the donor area was closed primarily and the flap was tubularized after elevation, as proposed by Kelly et al. ²⁰ The tip of the tubularized flap was sutured to the cranial end of the donor site (Figure 1). As a modification of Kelly's proposal, intermittent sutures were applied between the resulting longitudinal incisions on the flap and donor area, so as to prevent flap sagging and auto-cannibalism. To prevent cannibalism the rats were kept in separate cages postsurgery.

Figure 1.

View of a tubularized flap after insetting.

The rats were randomly divided into 5 equal groups. Each group received topical treatment every 12 h. Group A received topical placebo treatment starting on the day of surgery. Group B was treated with topical nitroglycerin 2% starting on the day before surgery. Group C was treated with topical nitroglycerin 2% starting on the day of surgery. Group D and group E were treated with topical nitroglycerin 2% starting on postoperative d 2 and 4, respectively (Table 1). On postoperative d 7, the rats were euthanized using CO₂. Digital photographs were taken and the entire flap tissue was harvested for standardized photography and histopathological examination.

Table 1.

Nitroglycerin Treatment Schedules, According to Group.

	One day before the surgery	Surgery	d 1	d 2	d 3	d 4	d 5	d 6	d 7
Group A	-	Placebo	Placebo	Placebo	Placebo	Placebo	Placebo	Placebo	End
Group B	✓	✓	✓	✓	✓	✓	✓	✓	End
Group C	-	✓	✓	✓	✓	✓	✓	✓	End
Group D	-	-	-	✓	✓	✓	✓	✓	End
Group E	-	-	-	-	-	✓	✓	✓	End

Measurement of Flap Survival

After sacrification, the flaps were harvested. The sutures providing tubularization were removed, and then the flaps were spread across a flat surface and a ruler was placed near the specimen for measurement (Figure 2). Digital photographs were taken from a standardized distance of 40 cm using a tripod. Digital image analysis software (Digimizer v4.1.1.0) was used to measure the surviving flap area. The evaluators used the software for calculations and were unaware of the study group of the subjects that were analyzed. The flap survival rate was calculated by dividing the viable flap area by the size of the initial flap (27 cm²), so as to avoid the effect of necrotic contraction postsurgery.

Figure 2.

View of a flap prepared for standardized photography.

Histopathological Examination

Excision specimens were fixed in a 10% buffered formalin solution. Next, 2 strips of 3-mm thick tissue through the necrotic and intact transition area were sampled in each rat. Samples were embedded in paraffin blocks, and then 5-µm thick sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus BX51).

Histopathological evaluation was performed using 4 criteria: the severity of inflammation and ischemia was scored 1 to 3; the extent of inflammation was scored as 1 or 2 according to the presence of inflammation through the tissue layers; the severity of edema was scored as 1 or 2. The evaluators examining the specimens were also blinded.

Statistical Analysis

Data were analyzed using IBM SPSS Statistics for Windows v.23 (IBM Corp.). Descriptive statistics for continuous variables are given as median (range), whereas categorical variables are shown as frequency (percentage). Kruskal–Wallis variance analysis was used to compare the treatment groups. The Dunn’s test or Dunn–Bonferroni test was used for pairwise comparisons. The level of statistical significance was set at P < .05.

Results

Among the 50 rats included in the study, 10 could not complete the study due to auto-cannibalism, as follows: group A: n = 2; group B: n = 1; group C: n = 2; group D: n = 2; and group E: n = 3. Data for the remaining 40 rats were analyzed. Based on the photographic analysis, mean surviving area in the placebo group (group A) was 6.55 cm² (6.19-8.19). Group B had a mean surviving area of 14.97 cm² (13.56-16.83), whereas the mean surviving area in group C was 11.25 cm² (9.59-12.81). Group D and group E had a mean surviving area of 8.97 cm² (6.58-9.45) and 7.71 cm² (5.66-8.43), respectively (Figure 3). There was a significant difference in the flap survival rate between all groups (P < .001). Group B had the highest flap survival rate, which was significantly higher than in all other groups, except group C (P = .135). Group C had a higher flap survival rate than group A (P = .007), yet the difference between group C and group D (P = .613), and between group C and group E (P = .088) was not significant. In addition, the difference in flap survival between group A and group D (P = 0.520), and between group A and group E (P = 0.132) was not significant (Table 2).

Figure 3.

The appearance of rats in groups A, B, C, D, and E at the end of d 7.

Table 2.

Surviving Area and the Survival Rate, According to Group ^a .

	A	B	C	D	E	P
Surviving area (cm2)	6.55 (6.19–8.19)x, z	14.97 (13.56–16.83)y	11.25 (9.59–12.81)y, w	8.97 (6.58–9.45)z, w	7.71 (5.66–8.43)z, w	<.001*
Survival rate (%)	0.24 (0.23–0.30)x, z	0.55 (0.50–0.62)y	0.42 (0.36–0.47)y, w	0.33 (0.24–0.35)z, w	0.29 (0.21–0.31)z, w	<.001*

^a Different superscript letters indicate different groups. *Statistically significant results.

Histopathological analysis showed that the extent of inflammation was similar in all 5 groups (P = .153). The severity of inflammation was lowest in groups B and C (P = .013) (Figure 4A and B). On the other hand, the severity of edema was lowest in groups A and E (P = .001). Ischemia was most severe in groups A and D and was the least severe in group C (P = .003) (Table 3).

Figure 4.

Histological view of specimens (hematoxylin and eosin [H&E], 40 × ). (A) A specimen is taken from a rat in group B. Mild inflammation is found in the subcutaneous tissue, muscle, and fascia. Edema is found in the fascia (inlet, arrow). Epidermis is intact. (B) A specimen is taken from a rat in group D. Whole-thickness shows severe neutrophilic inflammation throughout the dermis, subcutaneous tissue, skeletal muscle, and the fascia that is most severe in between the muscle bundles and in the fascia.

Table 3.

Histopathological Examination Results, According to Group ^a .

	Group A	Group B	Group C	Group D	Group E	P
Severity of inflammation b	3 (2-3)x, y, c	1 (1-3)x, y	2 (1-3)x, y	3 (3-3)z	3 (3-3)z	.013*
Extent of inflammation	2 (2-2)	2 (1-2)	2 (1-2)	2 (2-2)	2 (2-2)	.153
Severity of edema	1 (1-1)x	2 (2-2)y	2 (1-2)x, y	2 (2-2)y	1 (1-2)x, y	.001*
Ischemia	3 (2-3)x	2 (2-2)x, y	1 (1-2)y	3 (2-3)x	2 (2-3)x, y	.003*

^a Different superscript letters indicate different groups.

^b The unadjusted results for pairwise comparisons are given for severity of inflammation, as the adjusted P values are >.05. *Statistically significant results.

Discussion

The present findings show that topical nitroglycerin application 1 d before surgery or the same day as surgery increases flap survival. The flap survival rate did not differ significantly between starting the treatment 1 d before surgery and the same day as surgery, although starting the treatment 1 d before surgery may yield a better outcome. Nitroglycerin treatment initiated during the late period (postsurgery d 2 and d 4) had a similar effect on flap survival as the placebo.

Nitroglycerin is among the most commonly studied pharmacological agents, in terms of its effect on flap survival. Although many agents that are experimentally beneficial don't find clinical use, ¹ nitroglycerin has also been clinically shown to be beneficial^10,11; however, some experimental studies have shown that nitroglycerin is not beneficial.^18,21‐23 One reported argument used to explain nitroglycerin's ineffectiveness is insufficient dosage. ¹³ Gdalevitch et al. ¹³ reported that 1 postoperative dose of nitroglycerin reduces flap necrosis by 50%. Nevertheless, a more recent study reported that the most significant effect can be obtained by administering nitroglycerin every 12 h. ²⁴ Although a single dose can be effective, we think that treatment should be continued regularly for a minimum of 3 d. As it was reported that topical nitroglycerin 2% has an effect duration of 3 to 8 h, ²⁵ discontinuing the treatment after a single dosage can limit its effect. This issue was addressed in a recent meta-analysis that reported serial application, rather than a single dose, is advised. ²⁶ In the present study, topical nitroglycerin was applied every 12 h, which led to successful outcomes.

Increasing vascularity via the use of pharmacological agents for preconditioning before surgery has been studied. ²⁷ Various agents have been used, both successfully^28‐30 and unsuccessfully.^31,32 An important factor associated with successful results is the pharmacodynamic effect of the drug administered. It can be predicted that it would be more beneficial to use late-acting drugs or those that have potentiated effects over time. Nitroglycerin has a rapid effect, and due to the development of tolerance, long-term use is not beneficial. ³³ Accordingly, the benefit of starting nitroglycerin preoperatively is doubtful; however, evidence suggests that nitroglycerin sprays can be used prophylactically to increase exercise tolerance in patients with stable angina pectoris^34,35 and we think it would be useful to further investigate this in clinical series. Although there isn't a significant difference between starting nitroglycerin treatment before and immediately after surgery, flap survival rates are higher when preconditioning is performed.

In cases in which clinical examination is inadequate and there is no access to assistive devices such as SPY, unpredictable partial flap necrosis can occur. In some such cases, it may take a few days for necrosis to be evident or surgeons may fail to recognize the signs indicative of necrosis. When necrosis starts to develop in the distal flap, if the underlying cause is insufficient flap vascularity, surgical options are quite limited, but use of an agent that can increase vascularity can help salvage the flap. In cases in which necrosis is observed late, treatment with a drug the increases vascularity can be started a few days after surgery. Nonetheless, the present findings show that starting topical nitroglycerin on postsurgery d 2 and d 4 does not improve flap survival.

The efficacy of nitroglycerin for treating a flap that has begun to undergo necrosis has been investigated. Vania et al. ²⁶ suggest that in doubtful cases nitroglycerin can be used empirically, as the treatment can be ineffective when started late and nitroglycerin's excellent safety profile allows for its empiric use. Although local application of nitroglycerin reduces the likelihood of systemic side effects, the most common side effects include headache and hypotension. Headache can be treated with such simple analgesics as paracetamol. ³⁶ If hypotension is encountered (systolic blood pressure <100 mm Hg), it is recommended that topical nitroglycerin be diluted 1:2 or 1:4 with Bacitracin cream, so as to facilitate continued nitroglycerin treatment. ¹⁶ These reports indicate that the side effects of nitroglycerin are easy to treat, and as treatment started in the late period is unsuccessful, we think that empirical use before or immediately after surgery is more appropriate, so long as there are no serious contraindications.

The last issue to be considered is the dosage of the drug administered. In previous studies, nitroglycerine was applied in the form of patches^4,5,18,22 and ointments.^7,13,37 While the dosage of the drug in grams was stated in the studies using the patch form, exact weight in grams was not mentioned in studies using the ointment form, probably due to difficulties in calculating the exact amount. Only Turin et al. ¹¹ stated that when they used nitroglycerin 2% in the breast, they applied 35 mg of drug per breast to cover the whole area. In the current study, just like clinical studies, 2% ointment was used and enough application was made to cover the flap but exact weight of the agent was not calculated. However, it is possible to say that the dosage correlates with the amounts used in clinical studies.

Although the current study is the first to investigate the use of nitroglycerin in the late period, it also has limitations. First of all, the time required for the flaps to go to full-thickness necrosis in rats may not be the same as in humans. Delayed treatment may be effective if tissue necrosis is slower in humans and a clinical trial may clarify this situation. Nevertheless, we believe our study can pave the way for such a study. Authors also would like to address high auto-cannibalism rates, which may distort the results of the study. Flaps were tubularized to prevent graft take, however, flaps tend to sag when subjects recover from anesthesia. Although the flaps were suspended with intermittent sutures, minimal sagging occurred and the rate of auto-cannibalism around 20% could not be prevented. We suggest to place these intermittent sutures more frequently to minimize sagging.

Conclusion

Topical nitroglycerin application increases flap survival when started before or immediately after surgery, although no benefit is observed when starting treatment on postsurgery d 2 or d 4. Based on the present findings, we think additional research is warranted to determine the utility of topical nitroglycerine for pharmacological preconditioning before surgery.