INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Sepsis, a common and significant complication of acute illness, often leads to a progressive and catastrophic condition known as multiple organ dysfunction syndrome (MODS). Although recognized as a leading cause of morbidity and mortality in critically ill patients (1), MODS pathogenesis remains poorly understood.

Recent studies suggest that the integrity of the gut is an important determinant of clinical outcome in septic patients (2, 3). Under normal physiologic conditions, the intestines provide a critical protective barrier against toxic enteric contents including bacteria and endotoxin (2); however, various acute illnesses are known to disrupt this mucosal barrier (2, 3). The putative mechanism of organ dysfunction during sepsis involves an unregulated systemic inflammatory response in which the host's immune system is inappropriately activated resulting in systemic organ injury and failure (3). This injury may result from immune-mediated cellular necrosis or activation of pathways leading to programmed cell death (apoptosis) (4, 5). Nitric oxide (NO) has been shown to be an important participant in mammalian host defense mechanisms as well as a critical signal transduction agent in many physiologic processes (6). Under normal physiologic conditions, NO plays several roles in the alimentary tract, including sphincter and smooth muscle tone modulation and antibacterial actions (7, 8). In contrast to tightly controlled basal production in normal physiologic states, overproduction of NO (via induction of nitric oxide synthase type 2, NOS 2) has been observed in several gut-related disease states, including enterocolitis (9), and sepsis-induced cardiovascular collapse (10). In these conditions, NO may have pathological consequences via enhancement of epithelial cell permeability (11, 12), promotion of necrosis (9, 13), and/or activation of apoptotic pathways (14).

Recent studies have also shown that the cellular actions of NO (favorable versus pathological) are highly dependent upon controllers of both NO production and consumption (15, 16). For example, NO avidly interacts with superoxide anion to form peroxynitrite, an aggressive reactive oxygen species known to cause cellular injury, selective nitration of protein tyrosine residues, and epithelial cell apoptosis (15). In addition to cellular toxicity induced by NO and peroxynitrite, systemic NOS 2 induction contributes to hemodynamic instability during sepsis, potentially promoting mucosal ischemia, necrosis, and apoptosis (10). Thus, NO dysregulation, via uncontrolled generation through NOS 2 induction or enhanced formation of peroxynitrite, may be a key mediator of epithelial injury and ileal dysfunction during sepsis.

Although both cellular necrosis and apoptosis have been observed during sepsis-related MODS (4, 5), no previous studies have evaluated their time-dependencies during the early phase of gut-related injury. In addition, although NOS 2 induction has been demonstrated in various organs during overt sepsis (10), the role of NO in the early phase of intestinal injury, particularly in relation to epithelial necrosis and apoptosis, has not been evaluated. Thus, the present study was designed to examine the development and relative contribution of epithelial cell necrosis versus apoptosis during the first 4 h of sepsis-induced ileal mucosal injury. Furthermore, we investigated the hypothesis that NO dysregulation (due to NOS 2 induction or peroxynitrite formation) participates in these events. To examine these issues, we employed an in situ autoperfused feline ileal preparation designed to minimize gross tissue hypoxia and the development of mucosal ischemic injury (17). This relevant and established animal model allowed us to serially evaluate ileal tissues, simultaneously examining cellular and mitochondrial ultrastructure, apoptosis, and NO dysregulation in the epithelium over a period of 4 h in animals treated with intravenous endotoxin (lipopolysaccharide [LPS]) and in time-matched control animals.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparation

The animal preparation used for this study has been described previously (17, 18), and all experiments were approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee. Briefly, after an overnight fast, male cats were anesthetized with intramuscular ketamine hydrochloride (25 mg/kg) and intravenous sodium pentobarbital (10 mg/kg), intubated for mechanical ventilation, and had their carotid and femoral arteries and femoral veins cannulated. After a midline abdominal incision, a 40- to 50-g segment of ileum was isolated with its blood and lymph supply intact. The remainder of the small and large intestines were then resected, and the superior mesenteric artery was cannulated. In situ ileal autoperfusion was achieved via blood flow delivered by way of a connection with the carotid artery cannula (17, 18). Venous pressures were maintained at 0 mm Hg throughout the experiment, and superior mesenteric venous blood flow was recorded via an in-line flow probe. The venous effluent was collected in a reservoir and reinfused via the femoral vein using an extracorporeal circuit that was primed with heparinized, white cell-free blood from a donor cat.

Systemic arterial pressure and superior mesenteric venous blood flow were recorded continuously. Fluid loss was replenished by administering buffered isotonic saline via the femoral vein (5 to 10 ml/ kg/h). Arterial and venous blood samples were withdrawn into heparinized syringes and processed immediately for measurement of blood gas parameters using a blood gas analyzer (ABL3; Radiometer America, Westlake, OH), and hemoglobin concentration and oxygen saturation and content using a co-oximeter (OSM3; Radiometer America) that was specifically calibrated for feline blood. Rectal temperature was monitored throughout the experiment and maintained at 39° C.

Experimental Protocol

On completion of the surgical procedures and after a 30- to 40-min period over which the ileal preparation was allowed to stabilize, baseline measurements [Pa_O2, Pa_CO2, arterial pH, total hemoglobin (Hb), oxyhemoglobin (HbO₂), arterial oxygen saturation, oxygen content, arterial pressure, and superior mesenteric venous blood flow] were obtained. Animals were then randomly assigned to receive intravenous LPS (3.0 mg/kg; n = 9), or buffered isotonic saline vehicle (control; n = 5). The LPS used in these investigations was from Escherichia coli, serotype 0127:B8 (Sigma Chemical Company, St. Louis, MO) which was dissolved in buffered isotonic saline and used at a concentration of 1.0 mg/ml. Arterial PCO₂ and PO₂ were kept within normal limits by adjusting the fraction of inspired oxygen (FI_O2) and minute ventilation as needed. Arterial pH was maintained by administering bicarbonate intravenously as needed. The Pa_O2 to FI_O2 ratio was determined at baseline (time 0) and at hourly intervals thereafter with the FI_O2 increased from that of room air (21% O₂) to 1.0 (100% O₂) for approximately 10 min. When necessary, additional donor blood was administered via the extracorporeal circuit, to keep mean arterial pressure above 90 mm Hg.

Evaluation of Tissue Oxygen Availability

Ileal HbO₂ contents were measured at baseline and every 30 min using in vivo reflectance spectrophotometry, as described previously (17, 19, 20). Briefly, relative changes in tissue HbO₂ levels were detected using a diode-array ultraviolet-visible spectrophotometer (Model 8452A; Hewlett-Packard, Waldbronn, Germany) equipped with a remote reflectance fiberoptic probe (Model RSA-HP-84F; Labsphere, North Sutton, NH). The relative HbO₂ content of the ileal tissues was determined from the absorbance of the incident light (deuterium lamp, 190 to 820 nm) measured at a wavelength of 580 nm relative to a nearby, neutral (isosbestic) wavelength (570 nm) (19). Measurements made at 580 nm reflect changes in the relative HbO₂ content as well as changes in relative blood volume. Measurements made at 570 nm reflect changes in relative volume only, as this is an isosbestic wavelength with regard to changes in HbO₂ and is thus unaffected by oxygenation status (19).

Relative HbO₂ contents were expressed as a percentage of the maximal measurable range in each preparation, established by making measurements with the animal breathing an FI_O2 = 1.0 at baseline (most oxygenated condition) and an FI_O2 = 0 (100% nitrogen) at the end of the experiment (least oxygenated condition) before the mean arterial pressure dropped below 60 mm Hg. Relative changes in local tissue blood volume were also determined from absorbance measurements made at a wavelength of 570 nm (19) and expressed as a percentage of the same measurements made at baseline. At least three determinations of relative blood volume were made at each time point by measuring the absorbance at randomly selected locations along the intact ileal segment. Each measurement encompassed approximately 0.6 cm³ of tissue. In addition to these local in vivo tissue assessments, global parameters of oxygen availability to the ileal tissues were examined by collecting blood samples from the arterial line and analyzing them for total Hb concentration and oxygen content, and measuring total ileal blood flow, as described previously.

Electron Microscopy and Mitochondrial Injury Evaluation

Ileal tissue samples were obtained for electron microscopy processing and evaluation at baseline and at 2 and 4 h post-treatment. Each time, approximately 1.0 to 1.5-cm pieces of ileum were taken from either end of the intact segment. Several cross-sectional rings (approximately 2 to 3 mm wide) of ileal tissue were excised from the proximal ends of these pieces (i.e., away from the ligature), diced further along their longitudinal axes, and immediately submerged into isotonic fixative (4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M sucrose in 0.1 M phosphate buffer, pH = 7.4) for approximately 2 h at room temperature. The tissue was then minced such that longitudinal sections through the villi were easily obtained. Tissue pieces were then repeatedly rinsed in isotonic buffer (0.1 M sucrose in 0.1 M phosphate buffer, pH = 7.4), postfixed in 1% osmium tetroxide in rinse buffer for 1 h at room temperature, rinsed again in rinse buffer, and stored overnight at 4° C. The following morning, the tissue pieces were allowed to come to room temperature, then dehydrated through an ascending series of ethanol solutions. After rinsing with propylene oxide, they were infiltrated with and embedded in Spurr media which polymerized overnight at 60° C. The next day, thin sections (80 to 90 nm) were cut on a Reichart Ultracut E microtome, mounted on copper grids, stained with 2% uranyl acetate and Reynolds lead citrate, and then later examined using a Phillips CM-12 transmission electron microscope at 60 kV (17).

Using electron microscopy, mitochondrial ultrastructure was evaluated at two different locations within each ileal sample. Mitochondrial ultrastructure was examined three randomly selected and widely separated areas at each of these two locations on two different grids prepared from samples obtained at each time point. Examinations were made in a blinded fashion by two reviewers, and the severity of ultrastructural injury was quantified by determining a composite score (based upon the scale of 0 to 5 as shown in Table 1) which represented all the mitochondria visualized within the microscopy field. Thus, each reviewer had six total evaluations of mitochondrial injury made at each of the two locations (i.e., villus tips and villus crypts) per time point. The results obtained by the two reviewers were pooled to yield a mean mitochondrial injury score at each of the two locations for each sample.

A strategy designed to consistently grade the severity of mitochondrial ultrastructural injury was established. It was derived from a scoring system based upon the progressive stages of cellular injury, as described by Trump and colleagues (21). This staging system enumerates the characteristic progression of ultrastructural changes that occurs within cells in various models of inflammatory injury (e.g., ischemia- reperfusion). The association between the degree of mitochondrial injury and the defined stages of cellular injury (Table 1) was employed to quantify and standardize the severity of ultrastructural injury to the ileal mitochondria.

Detection of Programmed Cell Death (Apoptosis)

The presence of epithelial apoptosis in ileal tissues was assessed at baseline and at 2 and 4 h post-treatment using terminal uridyl deoxytransferase nick end-labeling (TUNEL) histochemical analysis (22). Ileal tissue samples (approximately 1.0 g) were excised and placed in 10% neutral buffered formalin for a minimum of 72 h. Samples were then washed in 0.2 M phosphate-buffered saline (PBS) and routinely embedded in paraffin. Cross-sections of ileal tissue (5 µm) were cut and mounted onto ProbeOn Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissues were deparaffinized using Hemo-D (Fisher Scientific) and rehydrated through a standard ethanol gradient (100% to 70%).

Cellular apoptosis was detected using the TdT-FragEL DNA Fragmentation Kit (Oncogene Research Products, Cambridge, MA). After a PBS rinse, the tissue sections were then permeabilized with 20 µg/ml proteinase K for 20 min at room temperature. Endogenous peroxidases were deactivated with 3% H₂O₂ in methanol for 5 min, then tissue sections were incubated in the TdT-labeling reaction mixture at 37° C for 1.5 h. After stopping the reaction with 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH = 8) and application of the blocking buffer (4% bovine serum albumin in PBS) for 10 min, peroxidase-streptavidin conjugate was then added and allowed to incubate for 30 min at room temperature, followed by a buffer rinse. Finally, a 3,3'-diaminobenzidine solution was applied to the tissue sections for 10 min followed by methyl green counterstaining. The apoptotic index (the percentage of cells staining positive for apoptosis) was determined at four random locations within the ileal mucosa for each sample. The ileal villus tips were preferentially evaluated because positive staining for apoptosis was not observed within the villus crypts in any of the samples.

Ileal Immunoprevalence of NOS 2 and 3-Nitrotyrosine (3-NT)

Immunolocalization of NOS 2 and 3-NT in ileal tissues was evaluated at baseline and at 2 and 4 h post-treatment using immunohistochemical techniques adapted from Robertson and coworkers (23). Ileal tissue samples (approximately 1.0 g) were excised and placed in 10% neutral buffered formalin for a minimum of 72 h. Samples were then washed, embedded in paraffin, sectioned, and processed for slides as described previously. After blockade of endogenous peroxidase activity, slides were incubated in boiling citrate buffer (10 mM sodium citrate, 10 mM citric acid, pH = 6) for 15 min to enhance antigenicity. Normal goat serum (10% in PBS) was then applied for 30 min at room temperature to prevent nonspecific binding. Tissue sections were incubated for 1.5 h at room temperature with rabbit anti-mouse polyclonal antibodies for NOS 2 (1:100 dilution; Transduction Laboratories, Lexington, KY) and 3-NT (1:100 dilution; Upstate Biotechnology, Lake Placid, NY). Nonimmune rabbit IgG (1:200 Vector Laboratories, Burlingame, CA) was used as the isotypic negative control to account for nonspecific binding. Biotinylated goat anti-rabbit antibody (1:200 dilution; Vector Laboratories) was then applied to the tissues for 20 min, followed by incubation in ABC Elite complex (Vector Laboratories) for 30 min.

Positive immunoreactivity was visualized using 3,3'-diaminobenzidine followed by hematoxylin counterstain. Immunoprevalence of NOS 2 and 3-NT was evaluated by color threshold analysis as described previously (24). Briefly, mucosal images (i.e., ileal villi) observed through an Olympus BX-40 microscope were captured at ×200 through a digital camera (Pixera Corporation, Los Gatos, CA) having a maximal resolution of 1.2 million pixels. Image Pro Plus software (Media Cybernetics, Silver Springs, MD) was used for analysis of the captured images. Briefly, a series of adjacent (but nonoverlapping) 100 × 100 pixel boxes were mapped out overlaying the entire villus region of the ileal mucosa. A red-green-blue color histogram analysis tool was then used to evaluate each box for pixel color intensities in the blue channel, previously demonstrated to best resolve brown (positive immunoreactivity, diaminobenzidine staining) versus blue (negative immunoreactivity, hematoxylin counterstaining) colors (24). For each box area, the percentage of pixels exhibiting brown intensity was determined. Data obtained from at least 4 to 6 boxes, encompassing the entire villus region per ileal section, were averaged to yield an overall immunoreactivity for NOS 2 and 3-NT, respectively, in each ileal sample.

Statistical Analyses

All data are expressed as the mean ± SEM. Comparisons of relative HbO₂ content, relative blood volume, arterial Hb concentration, ileal perfusion, mitochondrial injury score, apoptotic index, and NOS 2 and 3-NT immunoprevalence were made using one-way analyses of variance (treatment) with repeated measures (time). Post hoc analyses were performed using Fisher's test for least significant difference or the Newman-Keuls test, which take into consideration interactions due to multiple comparisons (25). Statistical significance was based on a value of p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Ileal Oxygen Availability

All measured and/or determined data regarding the availability of oxygen to the ileal tissues were relatively unchanged over the 4-h experimental period and did not differ significantly between the two groups (Figure 1). Measurements of relative HbO₂ content and relative tissue blood volume made at multiple distinct locations along the intact ileal segment using in vivo reflectance spectrophotometry demonstrated no significant differences between the two groups at baseline and remained quite stable over time. In fact, relative ileal HbO₂ content remained greater than 85% of the maximal measurable capacity in both groups through 4 h (87 ± 6% versus 87 ± 5%, control versus LPS-treated, respectively; not significant [NS]). In addition, no significant changes were observed in arterial Hb concentration or the total ileal blood flow as measured across the intact vascular bed in either group over the 4-h period (Figure 1). Importantly, total ileal blood flow (362.5 ± 45.0 versus 359.4 ± 39.9 ml/min/kg; control animals versus LPS-treated), relative ileal HbO₂ content (93.3 ± 5.7 versus 92.2 ± 3.9%; controls versus LPS), relative ileal blood volume (116.1 ± 8.8 versus 105.1 ± 4.1%; controls versus LPS), and arterial Hb concentration (16.7 ± 1.2 versus 16.5 ± 0.4 g%; controls versus LPS) were not significantly different between the groups 1 h after LPS administration. Thus, these findings suggest that ileal oxygen availability was quite sufficient and remained relatively stable over time after treatment with LPS.

View larger version (33K): [in this window] [in a new window]   Figure 1.   (Top) Relative HbO2 content in ileal tissues, as a percent of the maximal measurable range, and arterial Hb concentration over time in the control and LPS-treated groups (values are means ± SEM). (Bottom) Relative blood volume in ileal tissues, as a percent of baseline, and total ileal blood flow over time in the control and LPS-treated groups (values are means ± SEM). All measurements were relatively unchanged and stable over time and without any significant differences between the groups, indicating adequate oxygen availability in the ileal tissues through 4 h.

Cellular and Mitochondrial Ultrastructure in the Ileum

Two hours after the administration of LPS, treated animals demonstrated significant cellular and mitochondrial ultrastructural injury in the ileal epithelium relative to time-matched control animals (Figure 2, top). Evaluation of the ultrastructural features provided characteristic evidence confirming that the injury resulted from necrosis rather than apoptosis. These features included rounding of the nuclei with a lack of nuclear chromatin condensation; apoptotic bodies and plasma and nuclear membrane blebbing, typical of apoptosis (Figure 2C); and severe and widespread mitochondrial swelling with disruption of membrane integrity, characteristic of necrosis (Figure 2D). Though mitochondrial injury varied from mild to profoundly severe in the LPS-treated animals, on average, treatment was associated with significant swelling of all intramitochondrial spaces, in particular the cristae. By contrast, epithelial cells and their mitochondria were ultrastructurally normal in all respects in time-matched control ileal samples (Figures 2A and 2B). Given that there was no significant difference in the relative presence or severity of the mitochondrial injury between the ileal villus tips and crypts in either group, the results of theses two locations were pooled for comparison over time (Figure 2, bottom).

View larger version (68K): [in this window] [in a new window]   Figure 2.   (Top) Representative electron photomicrographs of ileal villus epithelial cells (left, original magnification = ×3,500) and corresponding mitochondria (right, original magnification = ×55,000) from control (A and B, respectively) and LPS-treated (C and D, respectively) animals at 2 h post-treatment. Note that nuclei in the epithelial cells from the LPS-treated animal (C, arrow) were rounded without the presence of nuclear chromatin condensation, apoptotic body formation, or nuclear or plasma membrane blebbing. In addition, mitochondria from these cells (D, arrow) demonstrated significant swelling of all compartments with dramatic loss of membrane integrity. By contrast, epithelial cells and their mitochondria from the control animal (A and B, respectively, arrows) were ultrastructurally normal in all respects. (Bottom) Injury scores of ileal villus mitochondria for the control and LPS-treated groups over time (values are means ± SEM). Mitochondrial ultrastructural injury was dramatically evident and quite severe within 2 h after LPS treatment (p < 0.001, compared with baseline* and time-matched controls). In contrast, mitochondrial injury was virtually absent in control animals and changed very little from baseline over time.

Apoptosis in the Ileal Epithelium

Through the use of DNA fragment end-labeling, the extent of cellular apoptosis in the ileal epithelium was found to be rather minimal in both groups through 2 h post-treatment and predominantly confined, when present, to the villus tip regions. Less than 3% of the epithelial cells were positively identified as apoptotic in either group through 2 h (Figure 3, bottom). However, by 4 h post-treatment, a significant increase in nuclear chromatin staining was observed in the epithelium of the villus tips of LPS-treated animals (Figure 3B). The apoptotic index rose to nearly 5× the baseline values (Figure 3, bottom), whereas little change was observed in the epithelium of time-matched controls (Figure 3A). Preexposure of control tissue sections to deoxyribonuclease (DNase) provided extensive detection of epithelial DNA fragmentation (average 98% positive cells) (Figure 3C), demonstrating the reliability of the TUNEL method in our laboratory and the integrity of untreated control samples.

View larger version (70K): [in this window] [in a new window]   Figure 3.   (Top) Representative color photomicrographs of ileal villi from control (A) and LPS-treated (B) animals at 4 h post-treatment and baseline tissues pretreated with DNase (C ) to yield a positive control (original magnification = ×800). Note that there was very little positive staining of epithelial cell nuclei for apoptosis in the control animal (A), whereas considerable staining for apoptosis was evident in the LPS-treated animal (B, arrow) at 4 h. DNase-treated control tissue (C, arrow) demonstrated extensive epithelial cell DNA fragmentation as expected. (Bottom) Apoptotic index (percent of epithelial cells with nuclei staining positive for DNA fragmentation) for the control and LPS-treated groups over time (values are means ± SEM). Note that there was a significant increase in the frequency of epithelial cells staining positive for apoptosis in the LPS-treated group at 4 h (p < 0.05, compared with baseline and 2-h measurements* and time-matched controls).

Ileal Immunoprevalence of NOS 2 and 3-NT

Immunohistochemical staining of ileal tissues demonstrated a significantly increased prevalence of NOS 2, but only after 4 h after the treatment with LPS (Figures 4B and 5, top). This staining was predominantly in the villus tip regions and primarily confined to epithelial cells. Although a trend suggesting greater NOS 2 expression was observed, no significant increase was demonstrated in control animals (Figures 4A and 5, top). Similarly, significant increases in 3-NT (a biomarker of peroxynitrite formation) immunoprevalence were observed after 4 h following LPS treatment (Figures 4D and 5, bottom), but not in time-matched control animals (Figures 4C and 5, bottom).

View larger version (103K): [in this window] [in a new window]   Figure 4.   Representative color photomicrographs of ileal villi from control (A and C ) and LPS-treated (B and D) animals immunostained for the prevalence of NOS 2 (upper panels) and 3-NT (lower panels) at 4 h post-treatment (original magnification = ×800). A significantly increased immunoprevalence for NOS 2 (B) and 3-NT (D) was observed in the epithelium 4 h after LPS treatment, whereas time-matched control tissues demonstrated little or no evidence of immunostaining (A and C ).

	DISCUSSION

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Despite intense research regarding the pathogenic mechanisms of sepsis, this systemic inflammatory response still has an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization (1, 2). It is now clear that the gastrointestinal tract plays an important role in this pathogenesis, as both a site of end-organ injury and a contributor to immune activation via bacterial translocation (2, 3). Thus, the viability and integrity of the barrier epithelial cells are critical aspects of sepsis survival (2, 3). Because delineation of the early events responsible for epithelial dysfunction and death is likely to provide insights for improved therapies, we employed a feline model of sepsis to evaluate epithelial changes within 4 h after systemic LPS exposure. We have previously used this well-established model to examine the development of LPS-induced ileal injury and found that significant increases in ileal vascular permeability(18),oxygen consumption-oxygen delivery (O₂-DO₂) alterations (17, 18), and severe mitochondrial injury (17) occurred within this 4-h timeframe. This preparation uniquely allows for simultaneous time-dependent assessments of ileal tissue characteristics in situ and enables serial tissue procurement for biochemical and molecular evaluations.

It is well known that tissue acidosis, O₂-DO₂ mismatching, and impaired oxygen extraction occur in the setting of systemic organ injury during sepsis (18, 26). This findings have led to the traditionally held hypothesis that sepsis-induced systemic organ injury, such as occurs in the intestines, results from altered tissue perfusion (i.e., ischemia) leading to hypoxia-induced cell injury (29). As such, the standard clinical approach has been to augment tissue DO₂ in patients with established sepsis, often rendering worsened outcomes (27, 28). Interestingly, we have previously observed (17), and demonstrate once again in the present study, that dramatic ileal mucosal injury after LPS treatment (Figure 2) occurs without significant changes in ileal blood flow at either the local (tissue) or global (organ) level and no apparent decrease in oxygen availability in the preparation throughout the 4-h experimental period (Figure 1). These findings are consistent with those of VanderMeer and coworkers (26), who recently showed that sepsis-induced mucosal acidosis (i.e., anaerobic respiration) occurs despite unchanged tissue oxygen content and elevated mucosal oxygen tensions. In addition, other investigators have observed net increases in skeletal muscle oxygen tensions (30) and increased blood flow distribution to the intestinal mucosal layer (31) during sepsis. Thus, although we cannot rule out transient microcirculatory abnormalities and potential microregional tissue hypoxia, the measurements provided herein (in combination with other investigations) suggest that the traditional ischemia hypothesis may not completely explain sepsis-related ileal mucosal injury. Rather than impaired DO₂, abnormal oxygen utilization and/or other mechanisms are likely involved (17).

While cellular necrosis is an established hallmark of sepsis-related organ failure, recent studies suggest that apoptosis may also play a role in evolving organ dysfunction (4, 5, 32, 33). In the present study, we investigated the early time-dependencies of these two pathways of cell death in ileal tissue during sepsis. LPS treatment caused time-dependent epithelial necrosis by 2 h post-treatment (as evidenced by mitochondrial ultrastructural changes), whereas no changes were observed in the control animals (Figure 2). The cellular and subcellular ultrastructural changes observed were consistent with cell necrosis and were not features of apoptotic cell injury (34). This early necrosis occurred without any significant change in ileal blood flow or oxygen availability in the tissues (Figure 1). In contrast, significant increases in apoptosis were not observed until 4 h after LPS treatment (Figure 3). Although the contribution and significance of epithelial apoptosis occurring later in time were not investigated in this study, these findings suggest that both necrotic and apoptotic pathways are involved in early epithelial cell death, but that necrosis precedes (and perhaps promotes) the latter energy-consuming (i.e., programmed) process.

An important aspect of our investigations was to evaluate the contributions of NO-mediated pathways during early intestinal injury, particularly in relation to epithelial necrosis and/or apoptosis. Endogenous NO-mediated pathways of injury during endotoxemia have received intense investigation in the last decade, and induction of NOS 2 is now recognized as a key mechanism of the intense vasodilatation and circulatory collapse that often occurs in this setting (3, 10). Separate from an established influence on cardiovascular stability during sepsis, the roles of NO-mediated pathways in local tissue viability and repair are less clear. For example, previous studies have suggested that intestinal overproduction of NO (via induction of NOS 2) participates in other gut-related disease states, including enterocolitis (9), and elicits both favorable and unfavorable effects (7). These include mucosal injury (13, 14), increased epithelial cell permeability (11, 12) and death (9, 14, 32), and enhanced bacterial defenses in vivo (7, 8). Thus, the roles and effects of NOS 2 induction during sepsis-induced intestinal injury may be time variant and dependent upon local conditions.

In an attempt to determine the potential roles of NOS 2 induction during early evolution of sepsis-induced ileal injury, we used immunohistochemical evaluation for detection and localization of enzyme expression. This approach allowed us to assess relative intensities of expression as well as determination of cell types involved. At 4 h post-LPS, significant cytosolic immunostaining for NOS 2 was observed in intact epithelial cells in both the ileal villus tip and crypt regions (Figures 4 and 5). Positive immunoreactivity was also detected in resident immune cells. Our observations are in general agreement with the findings of others demonstrating NOS 2 induction in the ileal tissues after LPS treatment (11, 12), particularly within the villus tips (35). However, previous reports were based upon measurements made at single or later time points (13) and failed to control for changes in ileal blood flow (11, 12, 35). Our finding of significant increases in NOS 2 immunoprevalence occurring only after 4 h was clearly dissociated from the dramatic evidence of epithelial necrosis present by 2 h. Further analysis using all available data and conducted using nonparametric correlation analyses (Pearson's analysis) to test for a statistically significant association between NOS 2 immunoprevalence and mitochondrial injury score demonstrated no significant relationship (p = 0.4, NS, with a power > 0.85). Obviously, these findings demonstrate that NOS 2 induction neither precedes nor apparently mediates the early ileal epithelial necrosis induced by LPS. Thus, whereas intestinal NOS 2 induction does occur during sepsis-mediated injury, it appears to be unnecessary for early epithelial cell death to occur in vivo (at least in the absence of overt alterations in oxygen availability).

View larger version (25K): [in this window] [in a new window]   Figure 5.   Immunoprevalence of NOS 2 (top) and 3-NT (bottom) over time in the control and LPS-treated groups (values are means ± SEM). Note that NOS 2 and 3-NT immunoprevalences were significantly increased at 4 h in the LPS-treated group (p < 0.05, compared with baseline and 2-h measurements* and time-matched controls).

Separate from altered NO production capacity during sepsis (primarily through NOS 2 induction), products formed from the interaction of NO with other reactive species have become increasingly important. For example, NO interacts with superoxide anion at a diffusion-limited rate to form peroxynitrite (15, 16). In a variety of isolated cell systems, peroxynitrite has been shown to possess aggressive oxidative properties including lipid and protein thiol peroxidation, DNA damage, and selective nitration of protein tyrosine residues (14). This agent is therefore associated with both cellular necrosis and apoptosis in vitro, depending upon the existing conditions and the cell types being studied. Animal and human studies have employed analytical and immunological methods for detection of 3-NT, as a marker of peroxynitrite formation in vivo (33). Increased protein nitration has already been associated with sepsis-related renal failure and pulmonary injury (33).

In the present study, we observed extensive ileal protein nitration 4 h after LPS treatment (Figures 4 and 5). This immunoprevalence was particularly evident in the epithelium, but was not necessarily localized to regions identical to those staining for NOS 2 (as qualitatively evaluated by comparing staining patterns of serial 5-µm tissue sections). This finding is consistent with the bimolecular formation of peroxynitrite, limited by both the rate of NO production and the prevalence of superoxide anion (15, 16), and suggests that endogenous antioxidant defenses (particularly superoxide dismutase) may have become overwhelmed during early LPS-induced ileal injury. Although detection of NOS 2 induction and protein nitration temporally coincide, it should be noted that alternate sources of NO [e.g., constitutive NO production (36)] may contribute to the protein nitration present in the ileal tissues at 4 h. This possibility is supported by recent investigations by Zingarelli and colleagues (36), who have demonstrated tissue tyrosine nitration in NOS 2-deficient mice after treatment with high doses of LPS (50 mg/kg).

Although ischemic injury to the intestines could explain the early necrotic injury to the ileal mucosa observed in the LPS-treated animals (29), global indices of ileal tissue oxygen availability, including ileal blood flow and relative HbO₂ levels, were unchanged in the LPS-treated animals. In addition, using an identical experimental approach, we have recently shown that the distribution of blood flow within the ileum (i.e., the mucosa and muscularis layers) is unaltered by endotoxemia (17). Likewise, other investigators (31) have shown that intestinal mucosal acidosis occurs without a redistribution of blood flow away from the gut mucosa. However, it should be noted that the global measures of tissue oxygenation used in these experiments cannot exclude the existence of microcirculatory disturbances within the ileum (37) or rule out the possibility of hypoxia-induced injury in this model. On the other hand, these findings suggest that nonischemic oxidative processes, including the activation of intracellular enzymes (35, 38) and the immune system (13, 14, 37, 39, 40) and/or inhibition of mitochondrial oxidative phosphorylation (15, 17, 41), may contribute to LPS-induced mucosal injury. For instance, recent investigations have shown that xanthine oxidase activation by LPS results in intestinal mucosal injury within 2 to 4 h (35). Moreover, this enzyme causes acute tissue injury through the formation of superoxide anion (38). Others have shown that mitochondrial oxidant production is accelerated in response to TNF- (41), a mediator of the systemic inflammatory response to LPS. Other potential sources of oxidants include inflammatory cells [e.g., macrophages (42) and neutrophils (39)] and intracellular inflammatory pathways, such as those mediated by arachidonic acid (40).

Regardless of the cause of early ileal injury, the findings of these studies suggest that oxidants other than NO and peroxynitrite [e.g., superoxide anion (38, 42) and hydrogen peroxide (41)] may be responsible for the severe epithelial injury during the early phase (i.e., within the first 2 h) of sepsis. This observation is consistent with recent investigations demonstrating no improvement in outcome using NOS 2 inhibitors in animals or humans with established sepsis (10, 43). Clearly, protein nitration occurred after the early epithelial necrosis observed at 2 h, and coincided with the appearance of increased apoptosis. This is consistent with recent studies demonstrating peroxynitrite-induced apoptosis in cultured epithelial cells (14).

In summary, this study provides several unique insights into the pathogenesis of intestinal injury during endotoxemia. First, epithelial injury occurred within 2 h of LPS administration and under apparent nonhypoxic (given measurable limitations) conditions. Second, cell injury during early sepsis resulted primarily from necrosis rather than apoptosis. Finally, by measuring time-dependent variables of cell morphology and NO production, this study demonstrated a dissociation of LPS-induced ileal epithelial necrosis and NO dysregulation. While systemic induction of NOS 2 has been considered an important phenomenon in the pathogenesis of endotoxemia, the results of employing NOS isoform inhibitors have been variable and controversial (43, 44). Our findings suggest that while NOS 2 induction and NO-related oxidative injury occur during sepsis-induced intestinal dysfunction, these events may not mediate the early epithelial necrosis observed. Thus, the variable success of NOS inhibitors in both humans and animal models may be related to this dissociation. Further definition of the early events that mediate epithelial necrosis (particularly in the apparent measurable absence of mucosal ischemia and tissue hypoxia) and the roles of NO dysregulation in later apoptosis appears warranted to enable rational improvements in clinical treatment options for the critically ill.