Gut mucosal cell kinetics change after burn injury. Intestinal ischaemia in the postburn period results in structural alterations of the gastrointestinal tract mucosa [1,2]. Tissue damage after ischaemic insult results largely from the generation of oxygen free radicals and peroxidases . Malondialdehyde (MDA) is an intermediate product of lipid peroxidation and is used for assessment of tissue injury attributable to free radicals produced by ischaemia-reperfusion (IR).
In recent years, tumor necrosis factor-alpha (TNF-α) has been recognized to be closely involved in and related to burn sepsis, postburn multiple organ dysfunction syndrome (MODS) and prognosis [4,5].
Apoptosis is programmed death and removal of senescent or otherwise dysfunctional cells without inflammation. Both apoptosis and proliferation by mitosis are continually ongoing in the gut epithelium to maintain mucosal cellular balance. This delicate balance can be influenced by several factors, such as chronic disease, nutritional depletion, or severe burn injury .
The burned patient may need anaesthesia for the escharectomy in the early postburn period. Propofol is extensively used in anaesthesia of burned patients during operative and nonoperative procedures. It has a potent antioxidant activity in both in vitro and in vivo studies [7,8]. Propofol also attenuates lipid peroxidation in cases of anticipated IR phenomenon in humans [9,10]. Ketamine is another commonly used anaesthetic for burned patients and has antioxidative properties in vitro [11,12].
Previous work in our laboratory demonstrated that propofol as an anaesthetic agent may prevent bacterial translocation in an animal model of burn injury . However, it has not been investigated whether, and to what degree, propofol and ketamine might contribute to gut mucosal epithelial proliferation, apoptosis and serum TNF-α levels in burn injury either clinically or experimentally. The aim of the present study was to determine the effects of propofol and ketamine as anaesthetic agents on intestinal lipid peroxidation, serum TNF-α levels and gut mucosal epithelial apoptosis in relation to proliferation in an animal model of burn injury.
The experimental protocol used for this study was approved by the Animal Investigation Committee of the Ministry of Health Ankara Research and Training Hospital and adhered to National Institutes of Health guidelines for the use of experimental animals. Animals were housed in individual cages in a temperature-controlled room with alternating 12-h light–dark cycles, and acclimatized for a week before the study. Food was removed 8 h prior to the study, but all animals were allowed free access to water. Sixty male Wistar Albino rats (mean body weight 220 ± 12 g) were randomly assigned into four groups of 15 rats per group.
Anaesthesia was induced with 10 mg kg−1 propofol (Abbott Propofol; Abbott Laboratories, Chicago, IL, USA) in Groups 1 and 2, and 30 mg kg−1 ketamine (Ketalar, Parke-Davis, EWL Ezcacibasi Warner Lambert, Istanbul, TR) in Groups 3 and 4 via the ventral tail vein. The left carotid artery and right internal jugular vein were catheterized to continuously monitor mean arterial pressure (MAP) and to maintain anaesthesia and give fluid, respectively. Anaesthesia was maintained with 20–30 mg kg−1 h−1 propofol in Groups 1 and 2, and 50–60 mg kg−1 h−1 ketamine in Groups 3 and 4 for 12 h. After a baseline (before burn injury) measurement of MAP was obtained, it was monitored for the next 12 h.
The burn size for a third-degree burn covering 30% of total body surface area (TBSA) was calculated as previously described . The injury procedure was modified from that described by Walker and Mason . After induction of anaesthesia, the hair was removed from the animal's dorsum, and a 30% TBSA full-thickness burn was created by immersing dorsal skin in 95°C water for 10 s in Groups 2 and 4. This method results in clearly demarcated full-thickness burns that destroyed the cutaneous nerves and rendered the injured areas insensate, and thus not a source of discomfort for the experimental animals. Groups 1 and 3 were anaesthetized as the others but had no burn injury. MAP was maintained within 10% of baseline levels in all animals wih the adjustment of intravenous infusion of lactated Ringer's solution. Burned area was covered with Opsite (Smith & Nephew Medical Ltd, England) and animals were transferred to a warming blanket. At 12 h postburn, animals in all groups were sacrificed by cervical dislocation, and samples were taken as described below.
Determination of intestinal lipid peroxidation
Terminal ileum samples were obtained and weighed at 12 h postburn. The samples were immediately frozen and stored at −70°C until analysis within 2 weeks. Samples subsequently were homogenized in buffer and assayed for MDA content using the thiobarbituric acid (TBA) reaction as described by Uchiyama and Mihara . Briefly, 0.5 mL of homogenate (10% concentration) was mixed with 3 mL of 1% H3 PO4. After addition of 1 mL of 0.67% TBA reagent, the tubes were heated in boiling water for 45 min. The colour formed was extracted with 4 mL of n-butanol and centrifuged. The colour intensity of the butanol layer was estimated by the spectrophotometric absorbance at 532 nm. MDA content was then expressed as nanomoles per gram of tissue (nmol g−1).
Serum TNA-α level
The serum level of this proinflammatory cytokine in each rat was measured by enzyme-linked immunosorbent assay (ELISA; Biosource International, USA) and expressed as picograms per millilitre (pg mL−1).
Immunohistochemistry for apoptosis
The extent of apoptosis was evaluated using a previously described immunohistochemical method that identifies cell death as reflected by DNA fragmentation . We used the TUNEL (terminal deoxyuridine nick-end labelling) method (Apoptag, Intergen, NY, USA) to identify apoptotic cells. Immediately after each rat was sacrificed, a 2 cm proximal segment of the small bowel was fixed in formalin and embedded in paraffin. Two sections of 3 μm were deparaffinized, rehydrated in graded alcohol and washed with deionized water. Protein in prepared section was digested using proteinase K (20 μL mL−1 in phosphate-buffered saline (PBS)), and endogenous peroxidase activity was quenched with 2% H2 O2 in PBS. Seventy-five microlitres of equilibration buffer were placed on each section, and then diluted terminal deoxyribonucleotidyl transferase (TdT) enzyme solution was applied and incubated at 37°C for 1 h. After incubation, the slides were placed in stop/wash buffer and 55 μL of antidigoxigenin peroxidase was added, and then slides were incubated at room temperature for 30 min. The sections were again washed and diaminobenzidine-hydrogen peroxide was applied for colour development. Sections were than counterstained with 2% hematoxylin and mounted for examination. In each section, we selected 10 full-length villi randomly and the total number of TUNEL-positive cells in these villi were counted. Apoptotic cells were identified as those with brown staining of the nucleus, or as apoptotic bodies, which are fragments of apoptotic cells engulfed by neighboring epithelial cells. All epithelial cells within the 10 villi were counted, and apoptosis was expressed as a percentage of apoptotic cells in the total cells counted. The TUNEL index values for the two sections per rat were averaged to give the percentage of apoptosis in the proximal gut of each rat.
Immunohistochemistry for proliferation
Proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) . Deparaffinized histological sections were pretreated with proteases and HCl to decrease background contamination. Then sections were incubated with PCNA-horseradish peroxidase conjugate (Santa Cruz, SC-56) at a 1: 50 dilution overnight at 4°C, washed with PBS, and treated with 3,3′-Diaminobenzidine (3,3′-DAB) peroxide for colour detection. After counterstaining and mounting, PCNA-positive cells (stained red-brown) were counted from the base of the crypt to the villus tip on two sections from each animal and expressed as percentage of the total number of labelled cells divided by the total number of cells counted.
All group data were expressed as mean ± SD where appropriate. Analysis of the group means for MDA, TNF-α, TUNEL index and PCNA labelling index were compared using Kruskall–Wallis one-way analysis of variance (ANOVA). Data for pairs of groups were compared using the Tukey HSD multiple comparisons test. P-values < 0.05 were accepted as significant.
MAP were 85 ± 12 mmHg in Group 1, 93 ± 11 mmHg in Group 2, 90 ± 10 mmHg in Group 3 and 87 ±13 mmHg in Group 4. There were no significant differences in baseline measurement of MAP between the groups (P > 0.05). Also there was no difference in the volume of fluid needed in all groups (P > 0.05).
Ileal content of the lipid peroxidation byproduct MDA is shown in Figure 1. There was no significant difference between groups with no burn injury (Groups 1 and 3) in terms of MDA at 12 h. Ileal level of MDA increased significantly in Group 4 at 12 h postburn by mean 112.4 ± 10.2 nmol g−1 compared with Group 3 (48.4 ± 5.6 nmol g−1) and Group 2 (59.8 ± 3.2 nmol g−1) (P < 0.05). Also MDA level was not significantly different in Group 2 compared to Group 1 (55.2 ± 3.5 nmol g−1) (P > 0.05).
The serum TNF-α findings at 12 h postburn in all groups are shown in Figure 2.
Group 4 had the highest mean TUNEL index of all the groups (265/10) (P < 0.05). Also the mean TUNEL index value in Group 2 (53/10) was higher than that of Group 1 (3/10) and Group 3 (5/10). TUNEL index values of Groups 1 and 3 were similar (Figs 3 and 4).
Small bowel epithelial cell proliferation remained unchanged and the number of PCNA positive stained cells was not different between groups (P > 0.05) (Fig. 5).
The morphological and functional integrity of the gut is maintained by a balance between epithelial cell proliferation and cell death. Both apoptosis and mitosis are continually ongoing in the gut epithelium to maintain mucosal homeostasis. Cutaneous thermal injury has been shown to disrupt this delicate balance [19,20]. As previously shown, the loss of gut epithelial cells after severe burn is due to an increase in apoptotic cell death. The most prominent apoptotic reaction was seen at 12 h postburn [6,21] so this time point was chosen in our study.
There is still controversy in literature regarding the potential mechanisms for the changes in gut epithelium seen after burn injury. It could be due to intestinal ischaemia, with a resultant reperfusion injury, or the systemic effect of inflammatory mediators released immediately following injury [22-24].
Oxygen-derived free radicals have been proposed as important mediators of tissue injury and are known to be released during reperfusion after reversal of ischaemia and may possibly be responsible for the apoptosis of enterocytes [3,25]. Both the highest MDA and TUNEL-assay values in Group 4 among other groups were in the same line with these figures. Consequently, according to our results, propofol seems to have some advantages for the prevention of apoptosis and oxidative stress in comparison with ketamine.
Peroxidative decomposition of membrane lipids that results in production of toxic metabolites such as MDA has been considered as the basis of oxidative cell injury . It is obvious that TBA reagent (TBAR) method for MDA assessment is not very specific. However, it is frequently used as a simple and easy method for assessment of tissue injury attributable to the free radicals that are produced by IR in our laboratory.
Systemic levels of TNF-α have been found to be elevated after burn injury and may exert a deleterious effect on some organs leading to apoptosis [21,27,28]. Triggering of the apoptotic response by TNF-α in gut epithelium may be initiated by either increased systemic levels acting locally in the epithelium or by increased local production of TNF-α in the gut mucosa itself. Both mechanisms are valid options and could explain the observed phenomenon of increased gut epithelial apoptosis. Although, there is some controversy about the specificity of the TUNEL-assay as it may also stain necrotic cells [29,30], in the present study, TUNEL-assay was used for assessment of gut epithelium apoptosis. As other apoptosis assays except TUNEL-assay were not available in our research laboratory. Based on our results, there may be a relationship between TNF-α level and apoptosis. But this requires further investigations.
Therefore, in our experimental model to investigate the effects of propofol and ketamine as anaesthetic agents on gut mucosal epithelial apoptosis in relation to proliferation, we examined both the intestinal MDA content and serum TNF-α level after burn injury.
It has been proposed that extensive and early escharectomy is of critical importance in decreasing the circulating level of endotoxin and bacterial load in the burn site . Also, early excision interrupts the stimulus for further translocation. Hence, burned patients may need anaesthesia in the early postburn period for operative procedures. In this context our study was the first experimental design that investigated the effects of intravenous anaesthetics on gut mucosal epithelial apoptosis after burn injury.
In this study, intestinal MDA content, serum TNF-α level, PCNA index and the TUNEL method were applied. All these procedures confirmed the observation that apoptosis of enterocytes decreased significantly with propofol anaesthesia with a stable MDA content and less increased TNF-α level compared to ketamine anaesthesia after burn injury. The findings also show that apoptosis of enterocytes was more marked when tissue MDA content and serum TNF-α level were increased. Another interesting finding of the study is the similar PCNA values of all groups. This may be due to slower PCNA immunohistochemical reactions. However, this needs further investigations.
Propofol appears to inhibit lipid peroxidation either by reacting with lipid peroxyl radicals to form the relatively stable propofol phenoxyl radical , or by scavenging peroxynitrite which is a potent oxidant formed by a rapid reaction between nitric oxide and superoxide radical [33,34], or both. The free radical scavenging properties of propofol resemble those of the endogenous antioxidant α-tocopherol (vitamin E) that is chemically similar to propofol. Each molecule of propofol could scavenge two radical species and has significant antioxidant activity in vitro . In a previous study, Luo and colleagues showed that propofol attenuates TNF-α-induced apoptosis in cultured human umbilical vein endothelial cells in vitro . Acquaviva and colleagues also confirmed that propofol protects astroglial cells in a dose-dependent manner against peroxynitrite-mediated DNA damage and apoptosis, and also utilizes heme oxygenase-1 in cultured astrocytes [36,37]. In a study by Salgo and Pryor Trolox, a water soluble vitamin E analog, and some phenolic antioxidants like 2,6-diisopropylphenol, similarly inhibit peroxynitrite-mediated oxidative stress and apoptosis in rat thymocytes . Chang and colleagues has demonstrated that propofol, at a therapeutic concentration, could protect mouse macrophages in vitro from nitric oxide-induced cell death and apoptosis .
Ketamine has been recommended for induction of anaesthesia and sedation in patients with circulatory failure because of its sympathomimetic actions . Also it has antioxidative properties in vitro [11,12]. Despite the antioxidant action, ketamine increased apoptotic cell death with morphological changes in cultured rat cortical neurons by activation of glycogen synthase kinase-3 . There is one in vitro study in the literature that showed ketamine decreased endotoxin-stimulated TNF-α production in cultured human blood .
In all of these previous studies, effects of propofol and ketamine on apoptosis were examined in vitro. Our experimental burn injury model is the first in vivo study in this aspect. Also our findings were different in these literature findings. Further in vivo studies will clarify this concordance.
In our study, to prevent the effects of haemodynamic changes on the results, animals were resuscitated to their baseline MAP rather than to their baseline cardiac output. This is a potential criticism of current study. Although techniques are available for cardiac output determination in the rat, these methodologies were not available in our laboratory. It is concluded that either ischaemia with a resultant reperfusion of tissues, or increased inflammatory mediators like TNF-α, or both, may possibly be responsible for the apoptosis of gut mucosal epithelial cells subsequent to serious burn injury. This apoptosis may be one of the contributory factor in the disruption of intestinal barrier integrity, resulting to a translocation of intestinal endotoxin and bacteria [13,43-45].
The antioxidant propofol as an anaesthetic agent protected against apoptosis of enterocytes with a stable tissue MDA and serum TNF-α level compared to ketamine anaesthesia in an animal model of burn injury. The clinical significance of these results must be elucidated by further studies.
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