Indian Journal of Plastic Surgery
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 Table of Contents    
ORIGINAL ARTICLE
Year : 2018  |  Volume : 51  |  Issue : 2  |  Page : 196-201
 

Comparison of subdermal and perforator delay techniques on a rat flap model


1 Department of Plastic and Reconstructive Surgery, Istanbul Training and Research Hospital, Istanbul, Turkey
2 Department of Histology and Embryology, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey
3 Department of Plastic and Reconstructive Surgery, Bahcelievler Medicana Hospital, Istanbul, Turkey
4 Department of Plastic and Reconstructive Surgery, Istanbul Diabetes Hospital, Istanbul, Turkey

Date of Web Publication29-Oct-2018

Correspondence Address:
Dr. Merdan Serin
Department of Plastic Surgery, Istanbul Research and Teaching Hospital, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijps.IJPS_28_17

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 » Abstract 

Background: In this study, we investigated the subdermal and perforator delay phenomena as a method to improve flap survival. Materials and Methods: In this experimental study, we used 24 rats in three groups. In the control group, the dorsal flaps were elevated and reinserted back to their place. In the experimental groups, we practiced the delay phenomena with two different techniques. In the first experimental group, cranial and lateral side incisions were performed; however, the flaps were not cut-off from the underlying fascia. In the second experimental group, we placed a silicon sheet under the planned flap to cut-off the circulation from the perforator vessels. Four weeks after the delay procedure, the flaps were raised completely and reinserted back to their place. Results: The average of necrotic area in the control group was 21.9% (±7.70). There was no necrosis in both experimental groups (P < 0.0001). Histological examination revealed that collagen density in both of the experimental groups was increased in comparison to the control group, it has only been found a significant first experimental group (P = 0.0315). We have not found any significant difference in lymphocyte density between the groups. Angiographic imaging has showed an increase in the vascular density in the flaps of the first experimental group. Conclusion: We believe that both of these delay techniques can be adapted to clinical applications and used safely to increase flap survival.


Keywords: Flap delay; rat; skin flap


How to cite this article:
Altinel D, Biltekin B, Serin M, Sahin U, Altindas M. Comparison of subdermal and perforator delay techniques on a rat flap model. Indian J Plast Surg 2018;51:196-201

How to cite this URL:
Altinel D, Biltekin B, Serin M, Sahin U, Altindas M. Comparison of subdermal and perforator delay techniques on a rat flap model. Indian J Plast Surg [serial online] 2018 [cited 2018 Dec 14];51:196-201. Available from: http://www.ijps.org/text.asp?2018/51/2/196/244362



 » Introduction Top


Flap survival depends on many internal and external factors.[1],[2],[3],[4],[5] Understanding of flap physiology is crucial for the success in reconstructive surgery. Flap microcirculation is composed of small vessels such as arterioles, capillaries and venules. These can be affected by many biochemical and neurological impulses. Flap circulation can be severed by the impairment of arterial or venous circulation.[6] In this regard, venous flaps have certain advantages to traditional arterial flaps among which are easier design and less donor-site morbidity.[7] Many factors have been shown to have effects on flap circulation such as topical applications,[8] pharmacological agents,[9] lasers,[10] photodynamic therapy,[11] oxygen pressure,[12] and scar penetration of neovascularisation.[13],[14]

Delay procedure has been shown to have significant impact on flap survival.[15] Although the developments in the techniques of axial and free flaps have decreased the need for this procedure, many surgical and non-surgical delay procedures have been defined.

Delay procedure is frequently performed to increase the viability of flaps. Classically, this procedure is performed 2–3 weeks before the surgery. Axial pattern skin flaps can also benefit from the delay procedure. In previous studies, rat skin island flap models have been used as a model for the delay procedure.[16] As a result, the delay procedure has been shown to have positive effects on flap circulation and has been adopted by plastic surgeons.

The delay procedure performed on the TRAM flap is one of the best examples of this application. In the practice of plastic surgery, the delay procedure can be performed with surgical techniques (incisions, saturations and ligations), pharmacological agents (vasoconstrictor and vasodilator agents), lasers[17] and mechanical impulses (extracorporeal shock wave).[18]

Subdermal plexus from the adjacent skin and musculocutaneous/septocutaneous perforators are the two major blood supplies of skin flaps. In this study, our aim was to compare the effectiveness of subdermal and perforator delay procedures with clinical and histological findings in a rat skin flap model.


 » Materials and Methods Top


This study was conducted in the Istanbul University Cerrahpasa Medical School Animal Research Laboratory with the approval of Istanbul University local Ethical Committee. Twenty-four female Sprague-Dawley rats averaging at 225 g weight were used. 9 cm × 3 cm caudally based dorsal flaps were designed on the back skin of each animal [Figure 1].[19] The flaps were raised at the subcutaneous level, and subdermal plexus was preserved during this procedure. The deep fascia was not included in the flap to avoid any vascular supply from the subfascial and suprafascial plexuses.
Figure 1: Mc Farlane dorsal skin flap model

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Animals were divided into three groups with eight in each group.

First experimental group (subdermal delay)

In this group, the first delay procedure was performed in which the flaps were only incised at the cranial edge. One week later, a second delay procedure involving the excision of the lateral edges was performed [Figure 2]. This procedure was performed in two stages to reduce the surgical stress and to enhance the new vessel formation. The connection of the flap with the underlying fascia was kept intact during these procedures. Four weeks later, the first delay procedure, the flaps were raised completely and sutured back to their location with 4.0 polypropylene sutures together with the control group.
Figure 2: (a) Superior and (b) lateral incisions on the first experimental group

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Second experimental group (perforator delay)

In this group, a delay procedure was performed in which the flaps were only incised at the cranial edge, and the flaps were dissected from the underlying fascia. A silicon sheet was placed under the flap. Lateral edges were kept intact [Figure 3]. Four weeks after the delay procedure, the flaps were raised completely and sutured back to their location with 4.0 polypropylene sutures together with the control group.
Figure 3: (a) Silicone sheet used in the second experimental group. (b) Silicone sheet is placed under the flap from a superior incision

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Control group

Caudally based flaps were raised without any delay and sutured back to their location with 4.0 polypropylene sutures along with the other animals from the experimental groups [Figure 4].
Figure 4: Skin flaps raised on caudal pedicle, 4 weeks after the delay

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Post-operative follow-up was performed by photographing of the flaps at 1st, 3rd, 5th and 7th days following the final surgery. At the end of the follow-up period, flap areas were marked and photographed. The marked flap areas were copied to an X-ray film, were photographed. Flap area calculations were performed with the VistaMetrix software (© SkillCrest, LLC).

All the animals were sacrificed with high-dose barbiturate, and the tissue samples were taken for the histological examination. Samples from the proximal and distal portion of the flaps were prepared in 10% formaldehyde. Lymphocyte and collagen densities were evaluated under light microscopy. H and E staining and Masson staining were used, and scoring was performed with a cell density scale.

Kruskal–Wallis and Dunn's multiple comparison tests were used for statistical analysis.

Angiography was performed on two rats on the 7th post-operative day before sacrification.

Inferior vena cava and aorta were cannulated [Figure 5]. A mixture of 40 ml 40% barium sulphate and 10% gelatine was prepared inside saline solution.[20] This solution was injected into the vessels, and the animals were kept in − 20°C overnight. The flaps were removed from the animals on the next day. The flaps were stabilised on plastic plates from corners. The flaps were photographed on high-resolution mammography machines.
Figure 5: (a) Dissection (b) Cannulation for the angiographic studies

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 » Results Top


During the follow-ups, the flaps of the control group started to show discoloration on the first post-operative day. Demarcation of the necrotic areas became visible between on 5th and the 7th day postoperatively. The average of the necrotic area on the flaps was 21.9% (±7.70).

In the experimental groups, two animals on Group 1 and three animals on Group 3 showed some discoloration on the first post-operative day. These findings were all confined in areas which were 1–2 cm in diameter. On the follow-ups, these areas recovered completely; hence, there was no necrosis at the end of the study on both of these groups. These results were significantly different than the control group (P < 0.0001) [Figure 6].
Figure 6: Photographs of the flaps at the end of the study from selected samples. (a) Control group. (b) First experimental group. (c) Second experimental group

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Evaluation of histological findings

Lymphocyte and collagen densities were evaluated under light microscopy [Figure 7]. Seven pairs of samples were taken from each group. Density scoring was performed in accordance with the following scale:
Figure 7: Collagen density shown with the Masson's trichrome staining. (a) Control group, (c) first experimental group, (e) second experimental group. Lymphocyte density shown with haematoxylin and eosin staining. (b) Control group, (d) first experimental group, (f) second experimental group

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  • 0: None
  • 1+: Low density
  • 2+: Medium density
  • 3+: High density.


There was only a statistically significant difference in the collagen density results of the control group and the proximal of the first experimental group (P = 0.0315) [Figure 8].
Figure 8: (a) Average of lymphocyte density scorings. (b) Average of collagen density scorings

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In the samples taken from the distal flap portions of the experimental groups, there was a significant increase in the vascular density; however, this parameter has not been scored.

Evaluation of angiography results

The angiography images were magnified and evaluated. In the subjective evaluation of the images, the experimental groups showed a slight increase in the amount of vascularisation when compared to the control group. This difference was especially visible in the first experimental group [Figure 9].
Figure 9: Angiography results from selected samples. (a) Control group. (b) First experimental group

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 » Discussion Top


Delay procedure on the skin flaps makes larger-sized flaps possible. The exact mechanisms behind this procedure are not clear. Many studies have been conducted to answer this question.[21],[22],[23],[24] The amount of vascularity increases required for an adequate delay still remains as an important question. This will depend on many variables as well as the delay technique. Although we believe that it will not be possible to quantitatively measure this amount, the future studies involving more variables might give us valuable insight into this point.

At the end of the study, none of the experimental groups showed a permanent circulation problem or necrosis. This finding can be further supported by the fact that no distortion on the epidermis was found microscopically. Under light microscopy, there was an increase in the vascular density and neovascularisation. The 4 weeks of delay time used in our study was probably effective in reducing the amount of necrosis for the perforator delay group. There was a significant amount of discoloration after the flap elevation in three animals in this group. This discoloration did not eventually result in permanent necrosis. We think that this might have been different if a shorter duration of delay time was chosen. Although 2 weeks of delay time was shown to be adequate for dermal delay techniques,[25] to the best of our knowledge, there was not any previous study on the adequate amount of delay time for the perforator technique. We thought that the dermal vascularity increase will be much slower in perforator delay group, which in principle will be similar to the expander techniques in which the perforator circulation is similarly interrupted.

In our study, there was not any significant increase in the lymphocyte density in the experimental groups. These results suggest that the flaps were not in an inflammation state. Collagen density has increased in both experimental groups. The collagen formation suggests an increase in neovascularisation and fibroblast activity. Zhou et al. have shown in a study that the injection of fibroblast growth factor and collagen matrix increases neovascularisation on rabbits.[26] Rao et al. have also shown an increase in angiogenesis with collagen matrix and endothelial and mesenchymal stem cells.[27] All of these studies indicate that the importance of collagen matrix for neovascularisation.

The angiographic imaging performed in our study was technically demanding. We believe that angiographic studies on small animals such rats will usually result in a low success rate and are difficult to interpret. A slight increase in vascularisation in experimental groups was detected with these studies. This increase in vascularisation has to be further investigated for venous and arterial components. Lymphatic vessels also seem to play an important role on flap viability. We believe that these can be further investigated with lymphoscintigraphic imaging on larger mammals such as pigs.

It is crucial to point out the fact that regional variations have to be taken into consideration. These results may turn out differently in other flap locations. Differences between human and rat physiologies are another factor to be considered.[28] These differences might be particularly important in neovascularisation and delay durations. There are many unknowns in the equation of flap physiology. Although many of these factors have been clarified by previous studies, it should not be discouraging to see that similar studies reveal different results.

Histological results show that collagen and vascular densities were lower on the second experimental group. Although there was not any permanent necrosis in any of the experimental groups, these results might suggest that necrosis would be more likely in this second experiment group. The incisions that were performed in stages on the first experimental group might have positive effects on flap circulation by decreasing the amount of stress and ischaemia on the tissues. It seems that by gradually increasing the stress we can enhance the circulation of the flap. In a previous study, Callegari et al. have showed that choked vessels can enlarge with the ligation of blood vessels from the adjacent angiosomes.[29] This result is consistent with our findings. Lateral incisions performed in the first experimental group do have a similar effect described in the earlier study of George et al. In this study, an occlusion clamp was used instead of the lateral incisions performed in our study. We believe that both of these techniques would open adjacent choked vessels as described earlier which would yield to similar results regarding the flap survival.


 » Conclusion Top


Our results support the idea that the delay procedure has a positive effect on flap viability. In conclusion, flap viability and histological data at the end of the study suggest that either subdermal or perforator circulation can be interrupted for a successful delay procedure. The papers published in angiosomes,[30] and the perforasomes[31] have changed our understanding of the random flaps in a profound way. These theories actually made it clear that there are actually no random flaps. However, in practice when the surgeon is unable to identify the angiosomes and the perforasomes associated with the skin area where the flap is to be raised the term random flap is used. Circulation problems are common with these types of flaps since they are raised without the knowledge of their vascular supply. These problems usually limit the usage of these of flaps on smaller defects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 » References Top

1.
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2.
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4.
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5.
Earle AS, Fratianne RB, Nunez FD. The relationship of hematocrit levels to skin flap survival in the dog. Plast Reconstr Surg 1974;54:341-44.  Back to cited text no. 5
    
6.
Seyhan T, Deniz M, Borman H, Ulusal Güzel B, Cağlar B. Comparison of two different vascular delay methods in a rat cranial epigastric perforator flap model. Ann Plast Surg 2010;64:89-92.  Back to cited text no. 6
    
7.
Yan H, Brooks D, Jackson WD, Angel MF, Akdemir O, Zhang F, et al. Improvement of prearterialized venous flap survival with delay procedure in rats. J Reconstr Microsurg 2010;26:193-200.  Back to cited text no. 7
    
8.
Huemer GM, Froschauer SM, Pachinger T, Kwasny O, Schoffl H. A comparison of pretreatment with a topical combination of nonivamide and nicoboxil and surgical delay in a random pattern skin flap model. J Plast Reconstr Aesthet Surg 2009;62:914-9.  Back to cited text no. 8
    
9.
Karacaoglu E, Yuksel F, Turan SO, Zienowicz RJ. Chemical delay: An alternative to surgical delay experimental study. Ann Plast Surg 2002;49:73-80.  Back to cited text no. 9
    
10.
Acikel C, Kenkel JM, Nojima K, Hoopman JE, Saboorian MH, Brown SA, et al. Evaluation of flashlamp-pumped pulsed-dye laser (585 nm) in nonsurgical delay of dorsal rat cutaneous flaps. Plast Reconstr Surg 2005;115:2032-41.  Back to cited text no. 10
    
11.
Belmont MJ, Marabelle N, Mang TS, Hall R, Wax MK. Effect of photodynamic therapy on revascularization of fasciocutaneous flaps. Laryngoscope 2000;110:942-5.  Back to cited text no. 11
    
12.
Stewart RJ, Moore T, Bennett B, Easton M, Newton GW, Yamaguchi KT, et al. Effect of free-radical scavengers and hyperbaric oxygen on random-pattern skin flaps. Arch Surg 1994;129:982-7.  Back to cited text no. 12
    
13.
Serin M, Bayramicli M. Evaluation of scar penetrating neovascularisation in a rat epigastric flap model. J Plast Surg Hand Surg 2015;19:1-5.  Back to cited text no. 13
    
14.
Edstrom LE, Robson MC, Hopp D. Survival of skin flaps based on scar. Ann Plast Surg 1981;7:44-7.  Back to cited text no. 14
    
15.
Holzbach T, Neshkova I, Vlaskou D, Konerding MA, Gansbacher B, Biemer E, et al. Searching for the right timing of surgical delay: Angiogenesis, vascular endothelial growth factor and perfusion changes in a skin-flap model. J Plast Reconstr Aesthet Surg 2009;62:1534-42.  Back to cited text no. 15
    
16.
Komorowska-Timek E, Timek TA, Brady S, Buncke HJ. Perfusion, viability, and pedicle dependence in acute and delayed rat island skin flaps. Microsurgery 2007;27:146-51.  Back to cited text no. 16
    
17.
Cohen M, Kattan A, Gat A, Almogi N, Katzir A, Villan A, et al. Comparison of flash lamp pulsed-dye laser (585 nm) and conventional surgery in the delay of random dorsal rat flaps. Lasers Surg Med 1999;25:178-86.  Back to cited text no. 17
    
18.
Reichenberger MA, Keil H, Mueller W, Herold-Mende C, Gebhard MM, Germann G, et al. Comparison of extracorporal shock wave pretreatment to classic surgical delay in a random pattern skin flap model. Plast Reconstr Surg 2011;127:1830-7.  Back to cited text no. 18
    
19.
Mcfarlane RM, Deyoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177-82.  Back to cited text no. 19
    
20.
Quinodoz P, Quinodoz M, Nussbaum JL, Montandon D, Pittet B. Barium sulphate and soft-tissue radiology: Allying the old and the new for the investigation of animal cutaneous microcirculation. Br J Plast Surg 2002;55:664-7.  Back to cited text no. 20
    
21.
Reinisch JF. The pathophysiology of skin flap circulation. The delay phenomenon. Plast Reconstr Surg 1974;54:585-98.  Back to cited text no. 21
    
22.
O'Neill JV, Toomey JM, Snyder GG 3rd. Factors involved in the skin flap delay phenomenon. Surg Forum 1975;26:528-30.  Back to cited text no. 22
    
23.
Cutting C, Bardach J, Rosewall D. Skin flap delay procedures: Proximal delay versus distal delay. Ann Plast Surg 1980;4:293-6.  Back to cited text no. 23
    
24.
Isoda S, Yano Y, Jin Y, Walters HL 3rd, Kondo J, Matsumoto A, et al. Influence of a delay on latissimus dorsi muscle flap blood flow. Ann Thorac Surg 1995;59:632-7.  Back to cited text no. 24
    
25.
Restifo RJ, Ahmed SS, Isenberg JS, Thomson JG. Timing, magnitude, and utility of surgical delay in the TRAM flap: I. Animal studies. Plast Reconstr Surg 1997;99:1211-6.  Back to cited text no. 25
    
26.
Zhou J, Zhao Y, Wang J, Zhang S, Liu Z, Zhen M, et al. Therapeutic angiogenesis using basic fibroblast growth factor in combination with a collagen matrix in chronic hindlimb ischemia. ScientificWorldJournal 2012;2012:652794.  Back to cited text no. 26
    
27.
Rao RR, Peterson AW, Ceccarelli J, Putnam AJ, Stegemann JP. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis 2012;15:253-64.  Back to cited text no. 27
    
28.
Hallock GG. The rat TRAM flap: A human analogue? Plast Reconstr Surg 1995;96:233-4.  Back to cited text no. 28
    
29.
Callegari PR, Taylor GI, Caddy CM, Minabe T. An anatomic review of the delay phenomenon: I. Experimental studies. Plast Reconstr Surg 1992;89:397-407.  Back to cited text no. 29
    
30.
Taylor GI, Palmer JH. The vascular territories (angiosomes) of the body: Experimental study and clinical applications. Br J Plast Surg 1987;40:113-41.  Back to cited text no. 30
    
31.
Saint-Cyr M, Wong C, Schaverien M, Mojallal A, Rohrich RJ. The perforasome theory: Vascular anatomy and clinical implications. Plast Reconstr Surg 2009;124:1529-44.  Back to cited text no. 31
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]



 

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