KEY WORDS
Flap delay - rat - skin flap
INTRODUCTION
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
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
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
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
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
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
RESULTS
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
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
-
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
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
DISCUSSION
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
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.
