the limiting factor in these flaps is the unpredictable blood supply , which , if insufficient , may produce irreversible damages to the microcirculation .
this damage results in partial or complete flap necrosis and renders the wound more susceptible to infection , thereby causing further healing impairment ; thus , improving distal blood supply in the random skin flap is an important goal .
the cellular damage that occurs during tissue reperfusion after ischemia is the result of a cascade of events involving free - radical oxygen and inflammatory mediators .
molecular oxygen plays a central role in the reparative healing process and is one of the critical nutrients of the wound .
hyperbaric oxygen ( hbo ) increases the tolerance of tissue to ischemia and enhances free - radical formation ; however , hyperoxia can increase the biochemical defence mechanisms against free radicals and improve the survival probability of ischemic tissue .
the hbo has a protective effect on microcirculation possibly by interfering with the deleterious action of activated neutrophils on microvascular endothelium .
one of the most important components of the intracellular antioxidant system is glutathione , a powerful active radical scavenger that is depleted in ischemia - reperfusion ( ir ) injury .
n - acetylcysteine ( nac ) is a prodrug that supplies bioavailable cysteine for glutathione replenishment . in the presence of overwhelming active oxygen species ( ros ) ,
the antioxidant nac prevents some of these deleterious effects , indicating an involvement of oxidative stress during hbo exposure .
the effects of hbo in improving the survival of a random skin flap are already known .
thus , it is important to study its combination with other substances , such as enzymes , nac , and so on , to potentiate their effect to be applied to patients .
thus , in this study , our aim is to investigate the role of hbo , nac , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design .
the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) .
all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) .
thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum .
the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo , n = 8) , and g - hn ( hbo plus nac , n = 8) .
after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m .
subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m .
a rectangular area ( 2  8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark .
15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] .
a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] .
an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place .
15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd .
, so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days .
hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization
, 100% medical oxygen was flushed through the chamber for 5 min to displace the room air .
the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere .
all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days .
the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days .
g - nh : flap proceedings and eight daily injection of nac , followed by hbo every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded .
once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day
the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format . under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest . on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment .
the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups .
the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) .
significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p
the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) .
all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) .
thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum .
the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo ,
n = 8) , and g - hn ( hbo plus nac , n = 8) .
after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m .
subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m .
under general anaesthesia , the dorsal regions were shaved and the animals were fixed in the prone position .
a rectangular area ( 2  8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark .
15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] .
a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] .
an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place .
15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles
a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd .
, so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days .
hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization
, 100% medical oxygen was flushed through the chamber for 5 min to displace the room air .
the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere .
all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days .
the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days .
g - nh : flap proceedings and eight daily injection of nac , followed by hbo
every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded .
once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day
the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format .
under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest .
on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment .
the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups .
the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) .
significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p
in figure 4 , the mean of necrosis ( % ) is shown by groups .
the average flap necrosis area was 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn group .
means and standard deviation of the percentage of necrosis on the random skin flaps in g - s , g - nac , g - hbo , and g - hn groups .
the means of necrosis in all the experimental groups were lesser than those of the g - s group . there was a significant difference between g - hbo < g - nac ( p < 0.01 ) and g - hn < g - nac ( p < 0.001 ) ( anova test ) in table 1 ,
the results of macroscopic evaluation ( area in mm ) for each group are presented .
means , standard deviation , and range of necrosis area ( mm ) in the skin flaps of g - s , g - nac , g - hbo , and g - hn on day 8 g - hbo group showed a smaller area of flap necrosis , when compared with g - s , but was not significant ( p = 0.12 ) ; however , when compared with the g - nac group , a significant difference was noted ( p < 0.01 ) . the flap survival in g - hbo was the same as that noted in g - hn , and when compared with the g - nac , the g - hn group showed significant difference ( p < 0.01 ) . in this study ,
however , the combination of nac and hbo , in contrast to the expectations , had no better survival , when compared with administration of hbo alone .
there has been much controversy over the angiogenic properties of hbo . in skin wounds ,
the angiogenic properties induced by hbo are derived from the increase in oxygen tension that may persist for several hours after hbo .
comparison of g - s and g - nac groups revealed that repeated  on - off  exposures produce a favourable environment in random flaps . in this study , hbo administered after surgery improved the survival of random skin flaps .
this finding is in contrast to the original hypothesis that suggests that extra oxygen increases the production of free radicals that would increase flap necrosis .
hong jp showed that when hypoxic tissue is exposed to hbo , there is an increase in po2 in the plasma and a reduction in po2 , and the rate of diffusion of oxygen increases .
nac is a precursor of glutathione , a potent antioxidant that inhibits the induction of pro - inflammatory cytokines .
glutathione also induces the production of nitric oxide synthase ( inos ) as well as adhesion molecule 1 .
this antioxidant also induces the production of a vascular cell adhesion molecule 1[101517 ] and stimulates the production of nitric oxide ( no ) . in this study ,
the flaps treated with hbo alone led to improvement in the average survival than those treated with a combination therapy with both hbo and nac , suggesting that these agents do not potentiate one another .
the groups treated with distilled water and nac alone exhibited the worst results in this experiment .
the doses of nac ( 300 mg / kg / day ) utilized in this study were chosen owing to the reported low toxicity of this drug and favourable results in the protection of random skin flaps in rat model . the plastic barrier interposed between the flap and donor bed
hbo in association with nac may be responsible for a protective effect on the deleterious outcome of nac .
when nac is used alone , the measures of necrotic areas were found to be higher in the g - nac ( 24% ) . on the other hand , with the combination of hbo and nac , the measures of necrotic areas
probably , in this study , the correct concentration of nac could have inhibited angiogenesis and wound - healing response .
this inhibition is considered to occur through an imbalance in the cellular redox state or an undetermined mechanism .
it has been demonstrated that hbo can increase the tolerance of tissue to ischemia , diminish metabolic disturbances , improve tissue microcirculation , and reduce platelet aggregation .
these characteristics , combined with the ability of plasma to carry dissolved oxygen to areas where red blood cells can not reach , have been shown to have a beneficial effect on oxygenation of many hypoxic tissues . in the present study , the combination of hbo and antioxidants therapy did not improve survival above and beyond the effect of hbo administered alone , suggesting that the potential toxic effects of hyperoxia from ros were not minimized by antioxidant therapy with nac .
oxidizing species , such as free radicals and hydrogen peroxide , may serve as cellular messengers mediating complex redox - sensitive processes , such as extracellular matrix formation , cytokine action , angiogenesis , and cell motility .
knight et al . demonstrated in rabbits that high dose of nac has no significant difference in the survival of flaps .
furthermore , ramon et al . showed an increase in the survival of the transverse rectus abdominis myocutaneous flap treated with hbo alone .
demonstrated in rats that hbo associated with vitamin e and c could improve the survival of epigastric island skin flaps , while rocha et al . reported that hbo alone showed a protective effect in the ischemic skin flap , which was associated with reduced expression of apoptosis
however , despite these results , multicenter prospective clinical studies are clearly needed to compare hbo treatment with other mechanical or pharmacologic interventions , to improve wound healing for grafting or to support flap survival .
with the experimental model , it can be concluded that hbo produced an improvement in the distal blood supply of the random skin flap ; however , this improvement was not significant . furthermore , the effect of combination with nac was not significantly different on the survival of flap , when compared with the hbo therapy alone .
 objective : our aim is to investigate the role of hbo ( hyperbaric oxygen ) , nac ( n - acetylcysteine ) , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design.materials and methods : thirty - two male wistar rats were randomly divided into four groups : g - s ( sham : n = 8) , g - nac ( nac : n = 8) , g - hbo ( hbo : n = 8) , and g - hn ( hbo plus nac : n = 8) . 
 a rectangular skin flap ( 2  8 cm2 ) was dissected from the muscular dorsal layer , preserving the cranial pedicle . 
 polyethylene film was placed over the muscular layer and an interrupted 3.0 nylon suture was employed to fix the flap into the original place . on the eighth day , full - thickness biopsy samples ( 2  1 cm2 ) were collected from the proximal , middle , and cranial areas of the skin flap , and in a site away from the flap labelled as the control area.results:the measurements of necrotic areas in the groups were 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn . 
 significant difference was observed between the groups g - hbo and g - hn as well as g-nac.conclusion:hbo is associated with reduced area of necrosis of skin flap . 
 the g - nac group was associated with poor results when examined in isolation . 
 the association between hbo and nac did not produce favourable results with respect to the use of hbo alone . 
 these findings suggest that the diffusion of oxygen through the interstitial space was the determining factor of more favourable results of hbo . 
INTRODUCTION
MATERIALS AND METHODS
Ethical committee
Animal housing and groups
Anaesthesia
Surgical procedure
NAC and distilled water administration procedures
HBO procedure
Sequential daily procedures
Seven days follow-up
Euthanasia
Determination of flap necrosis area
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION