Annals of Burns and Fire Disasters - vol. XIII - n. 2 - June 2000

THE EFFECT OF THE DELAY PHENOMENON ON THE ZONE OF STASIS IN BURNS: AN EXPERIMENTAL STUDY IN RABBITS

lsik S, Kopal C., Karacalioglu O., Selmanpakoglu N.

Department of Plastic and Reconstructive Surgery, Gu1hane Military Medical Academy, Ankara, Turkey Department of Nuclear Medicine, G01hane Military Medical Academy

SUMMARY. Twenty-five New Zealand white rabbits (weight 3.0 ± 0.5 kg) were used to study the destiny of skin responding to ischaernia in experimental burns. In the first group, dorsal delay flaps (4 x 10 cm) with the caudal pedicle (N°5) were elevated in two stages and sutured to their donor sites. On day 7 of elevation, dorsal acute flaps the same size were elevated counterlaterally to the delay flaps. The extent of the distal necrotic areas was calculated one week later. In the second group, delay flaps were elevated and rows of burns were created in the distal half of the flap (N°10). A probe (0.5 x 1 cm of each) was heated in boiling water and applied four times on the skin for 20 sec in such a fashion that a cross-shaped interspace area (0.5 cm, in width) was achieved. The same pattern of burn was created on the normal skin next to the flap. In the third group, delay flaps and acute flaps were elevated in the abovementioned fashion and the same pattern of burn was created in the distal halves of the flaps. Tests, including laser Doppler flowmetry, nuclear irnaging, and autoradiography, were performed on days 1 and 7 post-burn. In the first group, the average necrotic area in the acute flaps was 310.4 mm², and all the delayed flaps survived. Burn rows were then created 2 cm proximally to the distal edges of the flaps in groups 2 and 3. In group 3, the distal necrosis of the acute flaps was significantly increased to 774 ± 138 mm² on day 7. The laser Doppler flowmetry measurements revealed decreased distal circulation in the acute flaps, and 4.5 ± 2.2 perfusion units in the delay flaps on day 7 post-burn. The amount of necrotic change in burned areas in group 2, as calculated from autoradiographies, was found to be 316 ± 62 mm² in normal skin and 274 ± 45 mm² in delay flaps on day 7. In group 3 the amount was 288 ± 54 mm² in delay flaps and 680 ± 41 mm² in acute flaps. To conclude, it seems that the mechanism involved in the saving of ischaemic tissues does not significantly save the zone of stasis in burns.

The zone of stasis is the part of the burn wound that includes not only the central area of the burn wound (when present) but also the most peripheral hyperaemic reaction in the surrounding tissues. The metabolic and infectious reactions occurring in this area usually lead to further necrosis, which results in increased burn width and depth. The saving of the zone of stasis and the understanding of the pathophysiological events in this region have been the recent goal of burn research. Many researchers have reported that this zone can be saved.The events occurring in this zone are defined by some authors as "progressive burn ischaemia".
Numerous reports have focused on ways of increasing the tissues' tolerance of ischaemia.' As the understanding of the physiology of tissue ischaemia has progressed, some reports on the use of newly discovered chemical mediators have appeared. However, as these chemical materials have been invented, there have also been reports in burns literature regarding the same chemicals used to save the zone of stasis. The delay phenomenon is the only method that is generally accepted in clinical practice to increase the ischaernic tolerance of the flap tissue. The mechanism most often suggested for the delay phenomenon is the opening and patency of choked vessels which have been demonstrated histologically.
In a recent study, we demonstrated experimentally that the zone of stasis can be saved by the administration of recombinant tissue-type plasminogen activator (r-tPA).Its main effect is on vessels coagulated by burn injury. We suggested that vessel patency might be more important than other mediators released by burn injury. If this is so, the delay phenomenon should completely save the zone of stasis in burns. Also, if the pathophysiologies of the ischaemia zone in flaps and the zone of stasis in burns are the same, there is no need to use the same material in research on the two zones. The present study was designed to study the fate of the zone of stasis in delayed flaps that are responding to ischaemia.

Material and method

The study group consisted of 25 New Zealand white rabbits (weight 3.0 ± 0.5 kg). The animals were anaesthetized by intramuscular injection of ketamine hydrochloride (1 mg/kg). All the back skin was shaved with clippers. Five of the rabbits were used for a standard flap design - the midline of the back was marked and a dorsal skin flap measuring 4 x 5 cin was elevated laterally to the midline with a caudal pedicle. A silicone sheet 0.5 mm thick the same size as the flap was placed beneath the elevated flap, which was then fixed to the recipient bed by means of silk skin sutures. Three days later the sutures were removed and the delayed flap was completed to the size of 4 x 10 cm. by adding the caudal portion to the flap under anaesthesia. The immediate flap of 4 x 10 cin was also elevated on the left side, identically to that on the right. The flaps were sutured to their recipient beds. One week later, the distal necrotic areas of the flaps were drawn on mm-square sheets, and the percentages of the distal necrotic areas were calculated.
The second experimental group consisted of ten rabbits. After the above-described preparation of the back skin, the right-hand delay flap was elevated in two stages. After the first week of delay, the blood flow in the delayed flap and in normal skin was measured by laser Doppler (Laserflo BPM2, Vasemedics, USA) with a skin probe (Vasamedics, cat. N°P440). The measurements were performed in three different points of the distal and proximal flap portions, holding the probe in position for 1 min. The average blood flow in each part was calculated as a perfusion unit (PU) (PU = mI LD/min/100 g tissue). A brass probe with a leg (0.5 x 1 em) was immersed in boiling water until thermal equilibrium was achieved. The probe was then applied for 20 see to the delay flap 2 em from the distal and media] edges of the flap, without any pressure being applied to it. After 5 min, the heated probe was again applied for 20 sec to the flap 0.5 cm laterally to the previously burned area. Another burn row was created in the same fashion 0.5 em proximally to the first burn rows. Four burn rows with a cross-shaped interspace area between them were thus formed (Fig. 1). The same pattern of burn injury was created on the skin on the left side, without any flap. Blood flow measurements were performed in the interspaces 24 h and 7 days post-burn. At each testing time, five rabbits were prepared for nuclear imaging and autoradiography, which are described in the following sections. Tissue specimens including the burned areas were excised. The donor skin sites were repaired primarily and the rabbits were allowed to live.
In the third experimental group (N°= 5), the delay flap on the right side was elevated in two stages (N°= 10). One week later, an acute flap the same size was elevated on the left side (Fig. 2). The same burn patterns were created in both flaps, and cross-shaped interspaces were thus created. The burned flaps were tested using the same parameters 24 h and 7 days post-burn (five at each testing time).
Nuclear imaging and autoradiography. At testing times, the rabbits were injected with 6 mCi of technetium-99m methoxyisobutylisonitrile (99Tcm-MIBI) via the femoral veins under pentobarbital anaesthesia. The specimen, including healthy skin measuring 2 cm around the burned area, was removed after 30 min. The donor areas were closed primarily. The specimen was placed on a translucent film layer (thickness 0.2 min), which was used as a carrier in order to prevent distortion of the specimen and spreading of the radioactive agent by extravasated fluids. It was immediately put under a gamma camera (Starcam 400, General Electrics, USA) and images were obtained with a pinhole collimator for 15 min with a 256 x 256 matrix. The uptake of the radioactive agent was expressed as a percentage of the radioactivity taken up in the interspaces compared with that taken up in the burned areas. After completion of nuclear iinaging, the specimen was used for direct autoradiography. It was put on a film (Hypertihn-MP, Amersham, UK) in a dark room with close contact and both were then placed in an autoradiography cassette fflypercassate, Amersham,UK). After 24 h of exposure at -70 'C, the film was developed using an automatic processor (Cronex T-G Processor, Dupont, USA).
The necrotic/viable tissue borders obtained from the autoradiographies were marked and the total necrotic area was counted with the aid of a semitransparent sheet with Imul 2 grids.
Analysis. The blood flow measurements, radioactive agent uptake percentages, and necrotic areas obtained from the autoradiographies were expressed in terms of mean standard deviation. Significance was assigned at p < 0.05 and the values were statistically compared using the paired twotailed Student's t test.

Fig. 1 - Schematic drawing of delay flap and burn rows applied in phase 2 of the study Fig. 2 - Schematic drawing of delay and acute flaps and burn rows in phase 3

Results

In the first group, the average necrotic area of the acute flaps was 310.4 mm², while nearly all the delayed flaps survived with an average necrotic area of 4 mm².Burn rows were then created 2 cin proximally from the distal edges of the flaps in groups 2 and 3. When the delay flap was compared with normal skin in group 2, the necrotic changes between the burn rows began to be seen at the 24th h and were comparable with the process on day 7 post-burn (Figs. 3, 4).

Fig. 3 - The appearance of interspaces in the delay flap 24 h postburn. Note the progressive necrosis between the rows. Fig. 4 - Comparative appearance of necrosis in delay flap (d) and normal skin (n).

No significant differences were found in the laser Doppler flowmetry measurements and gamma camera counts at test times. When compared with healthy skin, nuclear imaging revealed that the uptakes in the burned areas ranged between 20 and 40% (Fig. 5).

Fig. 5 - Nuclear imaging of the specimen shown in Fig. 4.

The necrotic areas, which were calculated from the autoradiographies in group 2, were found to be 316 ± 62 mat' in normal skin and 274 ± 45 mm² in delay flaps on day 7 post-burn (Figs. 6, 7).

Fig. 6 - Autoradiography of the delay flap (d) versus normal skin (n). Note the marked distinct borders of the necrotic areas. Fig .7 - Grapfic representation of necrotic area (mm2)obtained from autoradiographies.Each line represent one rabbit.

When the delay flaps were compared with the acute flaps in group 3, a significant decrease was observed in the distal circulation of the acute flap, and a rate of 4.5 ± 2.2 PU was found in the delay flaps on day 7 post-burn. Distal necrosis in the acute flaps increased significantly to 774 ± 138 mm² at this stage, while it was 12 ± 4 mm² in the delay flaps (Figs. 8, 9).

Fig .8 - Appearance of necrosis in the delay flap versus the acute flap on day 7 post-burn Fig. 9 - Graphic representation of distal flap necrosis in phase 1 and 3 studies (acute versus delay flaps).

Nuclear imaging revealed 0-10% uptakes of interspaces in the acute flaps (Fig. 10). The area of necrotic change in the burned zones was 288 ± 54 mm² in the delay flaps and 680 ± 41 mm² in the acute flaps (p < 0.023)
(Figs. 7, 11).

Fig.10 - Nuclear imaging of delay (d) versus acute (a) flaps. Note the distal and extensive decreased uptakes in the acute flap. Fig. 11 - Autoradiography of a specimen in the phase 3 study. Note the marked distinct necrosis borders and extensive necrosis of the acute flap (a).

Discussion

The present study describes a dorsal skin flap model in rabbits. This model was used because its width is sufficient for the elevation of two flaps. It is advisable to use two flaps with burn rows in the same animal, since the metabolic conditions in different rabbits may affect flap survival. It was not possible to find any axial vessels in the pedicles of our flaps and all the vessels were positioned perpendicularly to the long axis of the flaps, as shown in Fig. 12.

Fig. 12 - Appearance of undersurface of acute (a) and delay (d) flaps. Note the direction of vascular territories clearly seen in the acute flap. These territories could not be seen so easily under the delay flap because the silicone sheet placed under the delay flap caused thickening of the flap.

We hypothesized that this type of vessel territory might be beneficial for the opening of anastornotic and choked vessels in delayed flaps where blood flow has been maintained. Morris and Taylor showed that the delay phenomenon would have its maximum effect 48-72 h after elevation of the rabbit skin flap. On the basis of these findings, we planned to elevate the delay flap in two Stages. First, half the long axis of the flap was raised and then, three days later, the size of the delayed flap was increased to 4 x 10 cm. A silicone sheet was placed between the delayed flap and its donor area in order to prevent the skin graft effect and to achieve a continuous response to the delay, as shown by Hammond` in the rat with the McFarlane flap model. The present study showed a nearly 100% survival rate in the delayed dorsal flap in the rabbit and an average rate of 86% in the acute flap.
Regas and Ehrlich 14 were the first to describe the "comb burn model" for the evaluation of the zone of stasis in burns. We have made some slight modifications to this model. Each row was burned at different times because we suspected the presence of burn injury in the interspace area between the two legs of the burning device owing to heat convection. In the present study, the interspace areas were largely protected from direct burn injury. A crossshaped interspace area was used. It may be hypothesized that if a tissue responding to ischaernia is spared from burn injury, the direction is less important. Also, the mediators released from the burn rows would be more effective in this cross-shaped pool. We observed that the part of the delay flap distal to the burn can be saved even if the circulation in this area is impaired by burns.
Laser Doppler blood flowmetry and gamma camera images established the circulation status of each area. Our results showed a slightly increased circulation at the base of the flaps. The circulation was impaired in the distal parts of the acute flaps and was nearly normal in the delay flaps, compared with normal skin. These techniques did not however define the exact borders of the necrotic areas. Autoradiography gives objective data for the calculation of survival rates. This simple technique is the method of choice when spatial resolution is more important than absolute sensitivity.` Autoradiography produces quantitative images in which the absorbency of the film image is directly proportional to the amount of radioactivity. If there had been access to a digital autoradiography system, the results would have been quantitated.
With regard to the flaps, the burn injury increased the distal necrotic area of the acute flaps in this study. This can be attributed to a partial decrease in blood flow to. the already ischaemic distal parL Conversely, the burn injury did not further increase the necrofic part of the delayed flap. This may be explained by the rich, dilated anastomotic and choked vessels in delayed flaps, a finding that was confirmed histologically.These vessels probably saved the part distal to the burn rows. The difference between the necrotic areas of the delayed and the acute flaps suggested two different pathophysiological mechanisms that accentuated each other in the acute flaps. Burn oedema and some mediators released from the burned areas are not included in the pathophysiology of ischaemic necrosis. The flap survival rates may suggest that these factors do not affect tissues responding to ischaemia or that they are cleansed from the environment by the blood circulation in delayed flaps.
When the survival rates of the interspaces between burned rows on delayed flap and of normal skin obtained from the autoradiographies were compared, a slight, statistically non-significant difference was found. The delay phenomenon was not capable of saving more interspace area than normal skin. In other words, anatomically dilated choked vessels in delay flaps were unable to save the interspaces. This may be due to additional pathophysiological mechanisms acting on tissues responding to ischaemia. There may be constriction and/or coagulation of these choked vessels or some necrofizing mediators may have been released from the burned areas, e.g. thromboxane A2. These vasoactive agents mostly act on vessels by vasoconstriction and platelet adherence. In our recent study, we showed the effect of a potent fibrinolytic agent (recombinant tissue-type plasminogen activator) on the saving of the zone of stasis in rats.' We suggested that maintenance of vessel patency could save the zone of stasis. In a recent study, Tan et aU showed that superoxide dismutase did not prevent post-burn dermal necrosis, although they found increased levels of malonyl dialdehyde in burned skin tissue. Nevertheless, the effect of the delay phenomenon was apparent when compared with intersPaces in acute flaps where all the interspaces became necrotic. This finding also suggested that an additional mechanism involved in burns leading to ischaemia resulted in total necrosis of the zone of stasis. As a result, the term "progressive burn ischaemia", used to describe the pathophysiological events in the zone of stasis, seems appropriate, where the terms "progressive" and "burn" are used together with ischaemia because the mechanisms involved in burns are apparently different from those involved in flap ischaemia.In conclusion, it seems that the mechanism involved in saving ischaemic tissues does not significantly save the zone of stasis in burns. There would appear to be different pathophysiological events or mediators in burns and ischaernia that accentuate each other's effects. Research on these challenging entities should be undertaken separately, using some recently discovered mediators in each condition.

RESUME. Pour étudier le destin de la peau qui répond à l'ischémie après les brûlures expérimentales, les Auteurs ont utilisé 25 lapins blancs de la Nouvelle-Zélande (poids moyen 3,0-3,5 kg), divisés dans trois groupes. Dans le premier groupe, les Auteurs ont élevé des lambeaux dorsaux retardés (4 x 10 cm) munis de pédicule caudal (n' 5) et suturés aux sites donneurs. Au 7ème jour après l'élévation, des lambeaux aigus dorsaux ont été élevés contre-latéralement aux lambeaux retardés. Après une semaine, la superficie des zones nécrotiques a été calculée. Dans le deuxième groupe, les lambeaux retardés ont été élevés et des rangs de brûlures ont été créés dans la moitié distale du lambeau (n' 10). Une sonde (0,5 cm) a été réchauffée dans de l'eau bouillante et appliquée quatre fois sur la peau pour 20 sec afin de créer un espace interspatial à tonne de croix (largeur 0,5 cm). Le même dessin de brûlure a été créé sur la peau normale près du lambeau. Dans le troisième groupe, les lambeaux retardés et les lambeaux aigus ont été élevés dans la même façon et le même dessin de brûlures a été créé dans les moitiés distales des lambeaux. Les Auteurs ont effectué des tests, y compris la débitmétrie au laser doppler et l'autoradiographie le premier jour et le 7ème jour après la brûlure. Dans le premier groupe, l'aire moyenne nécrotique des lambeaux aigus était de 310,4 mm², et tous les lambeaux retardés ont survécu. Les rangs de brûlures ont été créés à 2 cm proximalement aux bords distaux des lambeaux dans les groupes 2 et 3. Dans le 3ème groupe la nécrose distale des lambeaux aigus augmentait en manière significative à 774 ± 138 mm² au 7ème jour. Le mesurage laser doppler de la débitmétrie a révélé une circulation augmentée distale dans les lambeaux aigus, et une unité de perfusion de 4,5 ± 2,2 dans les lambeaux retardés au 7ème jour après la brûlure. La quantité des changements nécrotiques dans les zones brûlées du groupe 2, calculée sur la base des autoradiographies, a été évaluée comme 316 ± 62 mm² dans la peau normale et 247 ± 45 mm² dans les lambeaux retardés au 7ème jour. Dans le groupe 3 les résultats étaient 288 ± 54 mm² dans les lambeaux retardés et 680 ± 41 mm²dans les lambeaux aigus. En conclusion les Auteurs considèrent que la mécanisme qui préserve les tissus ischémiques ne sauve pas en manière significative la zone de la stase dans les brûlures.

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