Is HBOT treatment effective in recovering zone of stasis? An experimental immunohistochemical study

doi:10.1016/j.burns.2009.06.210

Burns

Volume 36, Issue 4, June 2010, Pages 539-544

https://doi.org/10.1016/j.burns.2009.06.210 Get rights and content

Abstract

Studies aimed at recovering the zone of stasis are one of the major issues of experimental burn studies. Hypoxia and oedema at that zone may cause irreversible changes. Due to anti-oedematous and antihypoxic effects of hyperbaric oxygen treatment (HBOT), it may be beneficial in recovering the zone of stasis. We performed an experimental study using 20 Sprague–Dawley rats, each weighing 350–450 g. The rats were first divided into two groups as 24 h and 5 days. Subsequently, control and treatment groups (five rats in each group) were formed. For burn wounds, we used the burn comb model, which was described by Regas and Ehrlich. In the treatment group, 2.5 ATA HBOT was applied for 90 min twice daily. A 0.8-cm punch biopsy was performed and samples for histological examination were taken from the centre of burn area. The rats were sacrificed by administering ‘3 mci technetium-99m methoxy butyl nitrite’ (⁹⁹Tc^m MIBI) through the femoral vein. Biopsy materials were evaluated by histological and immunohistochemical methods. Dorsal skin fragment, excised to 1 cm margin, was imaged by scintigraphic measurements with a gamma camera. Wet and dry weight measurements of excised skin fragments were taken. As a result, HBOT showed a positive effect at the cellular level in the first 24 h. It increased recovery potential by augmenting neovascularisation and decreasing oedema in the 5-day group.

Introduction

Severe burn, besides causing direct tissue death with its thermal effect, also causes dermal vascular occlusion and acute thrombosis [1]. Three distinct zones are formed in the burn region. The innermost zone is the place where tissue death occurs. Blood flow is impaired in the first 24 h in the surrounding zone, called the zone of stasis. In this zone, petechial haemorrhages, vascular thrombosis, increased vascular permeability and local oedema are seen [2]. Local oedema increases the severity of hypoxia and burn-specific sludge phenomenon [3]. All these factors contribute to reduced blood flow and lead to progressive ischaemia and necrosis in the zone of stasis, therefore necrotic tissue extends beyond the initial burn site. The outermost surrounding layer, known as the hyperaemic zone, always stays viable [2]. Stopping or modifying progress of the zone of stasis is the major target of burn studies. Many drugs and methods were used for this purpose [4], [5], [6]. Prevention of pathological changes of this zone may prevent progression of partial-thickness burn into a full-thickness burn. Effectiveness of hyperbaric oxygen treatment (HBOT) on acute and chronic wounds was shown in various studies. However, the mode of its effects in the zone of stasis is yet unclear [7], [8]. In this study, we hypothesised that anti-oedematous, anti-inflammatory and antihypoxic effects of HBOT either stop or hinder the progressive pathological changes of the zone of stasis. We evaluated the results by using scintigraphic, histological, immunohistochemical and wet-to-dry weight parameters.

Section snippets

Burn model

This study was performed in the experimental laboratory of the Department of Underwater and Hyperbaric Medicine of the Faculty of Medicine, Istanbul University. Twenty adult Sprague–Dawley rats weighing 350–400 g were used. All animals were fed with rat chow and water ad libitum. Before the operation, the rats were anaesthetised intraperitoneally with ketamine (Ketalar), 50 mg kg^–1 of body weight, and xylazine (Rompun), and 5 mg kg^–1 of body weight. The dosage was repeated when necessary. The dorsum

Macroscopy

None of the rats died. The treatment and control groups of the 24-h subjects (Groups I^1t and I^1c) revealed no difference in the comparison of necrotic and vital zones. Burn zones progressed and occupied all vital interspaces in the control group (Group II^5c). In the 5-day treatment group (Group II^5t), burned and vital interspaces were separated from each other with a distinct border. In this group, skin appendices were also vital.

Nuclear images

There was no statistically significant difference in the

Discussion

Therapeutic use of HBOT in thermal burns was first reported by Wada et al. [11] In 1969, Gruber et al. demonstrated that the area sub-adjacent to a full-thickness injury was hypoxic and could be raised to normal or supra-normal levels through the administration of oxygen under pressure, and this was followed by a series of animal experiments that demonstrated a significant reduction of oedema, improved microcirculation, reduced inflammatory responses, faster epithelisation and improved wound

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This model consists of four rows with dimensions of 1 by 2 cm with interspaces of the half by 2 cm. As stated in previous studies, skin regions that have direct contact with the stump represent third-degree burns, and interspaces represent second-degree burns [17]. The metal stamp was then kept in boiling water for 5 min to reach the same temperature as the boiling water.
Intermittent Fasting (IF) has proved to have various positive effects on life span, diseases, and healing of tissues in rodents. We evaluated the protective effect of fasting in maintaining the ischemic zones in burn wounds.
In this study, 20 rats were divided into two groups where the IF rats were deprived of food for three months. Burn wounds were created by burn comb model on the back of all rats. On days 3 and 21 after injury, five rats in each group were euthanized whereby samples were collected for histopathological, immunohistochemical (Bcl2, P53 and VEGF), and biochemical (MDA, TAC, HP) evaluations.
Histopathological analysis revealed epithelial layer and zone of ischemia remained viable in the intermittent fasting group on day 3. On the 21st day, epithelialization, angiogenesis, inflammation, fibrocyte-fibroblast, and collagen density were different in the ischemic and necrotic zones between the control and intermittent fasting groups (p<0.05). We found no statistical differences in Bcl2, P53, VEGF, MDA, TAC, and HP on day 3 between the intermittent fasting and control groups.
Intermittent fasting before burn wounds reduces tissue damage caused by ischemia and enhanced the viability of cells in zone of stasis. It also accelerated wound healing by increasing epithelialization and collagen production in the skin and regulating inflammatory responses. This intervention appears to be related to better collagen arrangement and angiogenesis.
The effect of Tarantula cubensis D6 on zone of stasis in a rat burn model
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This shows that the temperature of the stasis zone of the TC group was higher and thus the viability of the stasis zone increased with TC. If we look at the evaluation of the histopathological data, vascular density calculation or capillary number are frequently used among the evaluation methods, since the increase in tissue blood flow indicates an increase in the perfusion of the stasis zone [11,43,44]. Eyuboglu et al. in their study examining the effect of adipose tissue-derived stromal vascular fraction (SVF) on the stasis zone, calculated vascular density in the stasis zone as an indicator of angiogenesis, naturally increased blood flow, and rescue of the stasis zone [21].
Burn injuries are one of the most devastating injuries. Saving the zone of stasis decreases burn size, morbidity, and mortality. Tarantula cubensis (TC) increases epithelization of wounds, and increases wound contraction. In this study, we investigated the effects of TC on the stasis zone.
36 Wistar albino female rats were divided into 3 groups. Using brass comb burn model, at days 0 and 3, physiological serum solution in group 2, TC injection in group 3and no injection in group 1 was applied. Thermal and normal images were taken on day 10 and rats were sacrificed for histopathological examination.
We found a statistically significant difference between the 1st and 3rd group, 2nd and 3rd group regarding viable wound areas (p < 0.05), temperature difference (body-stasis zone) and mean body temperature (p < 0.05). We found no statistically significant difference between groups regarding neovascularization, inflammatory density, and vital cutaneous appendages. We found a statistically significant difference in epithelial layer thickness between the1st and 3rd groups, 2nd and 3rd groups(p < 0.05).
TC increases stasis zone viability macroscopically, increases epithelial layer thickness histopathologically, thus it promotes wound healing in burn wounds. This increase in stasis zone viability was also established with thermal imaging.
Is There a Place for Hyperbaric Oxygen Therapy?
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A multitude of animal studies have shown that HBOT can aid in preserving tissues along the zone of stasis by numerous potential mechanisms. These mechanisms include reducing edema at the site of injury [93] , attenuating dermal ischemia [94], prevention of partial to full thickness tissue injury [95], preservation of both cellular and microcirculation integrity [96], enhancing neutrophil killing [36], and maintaining ATP levels for normal endothelial physiology [96,97]. Newer studies also suggest that HBOT can reduce the mechanical allodynia of burn injuries by upregulating opioid and melatonin receptors at multiple sites, further enhancing recovery [98].
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According to the reports by the World Health Organization (WHO), American Burn Association, and Centers for Disease Control and Prevention (CDC), 1.1 million people suffer burns every year in America alone [1,2] and 3500 people die annually due to burns [3]. Thermal damage caused by burn injury results in three different zones from the contact center of the cause towards the periphery [4,5]. The central zone where the damage is irreversible is the zone of coagulation.
Mesenchymal Stem Cells (MSCs) are known to contribute to wound healing by increasing tissue regeneration. This study examined the effect of MSC-Lyophilizate (MSC-L) on the recovery of the zone of stasis in thermal burns.
A comb was used to induce second-degree thermal burns (1 × 2 cm) on the dorsum of the rats. Within 30 min after the burn, MSC-L derived from the umbilical cord was administered to the experimental group and 1.5 ml of 0.9% isotonic sodium chloride to the sham group. The control group did not receive any intervention. Tissue samples were collected on postoperative day 7. Histopathological assessments were made using a microscope with digital camera attachment. SPSS for IBM 25 was used for data analysis.
Epithelial loss and subepidermal bullae were observed in the control and sham groups on day 7. In the experimental group, the MSC-L administration was found to increase epithelial tissue formation and neovascularization in the dermis. We found no significant pathological findings in the epidermis and dermis in the experimental group.
Administration of umbilical cord-derived MSC-L is of potential importance in wound healing. In our study, we observed that MSC-L that contained 1.5 million cells contributed significantly to the recovery of the stasis zone of burn.
Current concepts on burn wound conversion—A review of recent advances in understanding the secondary progressions of burns
2016, Burns
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The majority of articles addressed better studied concepts such as ischemia (9 studies) and inflammation (6 studies), whereas more recently discovered mechanisms such as autophagy (3 articles) had less focus, as expected. Articles grouped under “other” included studies on stem cell therapies [15,16], enzymatic wound debridement [17], burn excision [18] and hyperbaric oxygen treatment (HBOT) [19]. We found that most studies on burn wound progression over the last five years involved experimental treatments in animal models, and very few studies were solely focused on further elucidating the mechanisms behind burn wound conversion (Fig. 2).
Burn wound conversion describes the process by which superficial partial thickness burns convert into deeper burns necessitating surgical intervention. Fully understanding and thus controlling this phenomenon continues to defy burn surgeons. However, potentially guiding burn wound progression so as to obviate the need for surgery while still bringing about healing with limited scarring is the major unmet challenge. Comprehending the pathophysiologic background contributing to deeper progression of these burns is an essential prerequisite to planning any intervention. In this study, a review of articles examining burn wound progression over the last five years was conducted to analyze trends in recent burn progression research, determine changes in understanding of the pathogenesis of burn conversion, and subsequently examine the direction for future research in developing therapies. The majority of recent research focuses on applying therapies from other disease processes to common underlying pathogenic mechanisms in burn conversion. While ischemia, inflammation, and free oxygen radicals continue to demonstrate a critical role in secondary necrosis, novel mechanisms such as autophagy have also been shown to contribute affect significantly burn progression significantly. Further research will have to determine whether multiple mechanisms should be targeted when developing clinical therapies.
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Is HBOT treatment effective in recovering zone of stasis? An experimental immunohistochemical study

Abstract

Introduction

Section snippets

Burn model

Macroscopy

Nuclear images

Discussion

Resuscitation

Burns

Burns

Burns

Burns

Burns

Clin Plast Surg

Burns

Burns

Burns

Elucidating the vascular response to burns by a new rat model

J Trauma

U75412E, a lazoroid, prevents progressive burn ischemia in a rat burn model

Am J Pathol