Radiation exposure in association with accidents, terror strikes, and war is a small, but serious threat to the general public, as well as to first responders, cleanup and rescue workers, and military personnel. In a radiological emergency scenario, a large number of casualties would be expected, thus requiring triage and optimized management in intensive care departments (1).
Acute radiation sickness is characterized by damage to the hematopoietic system and gastrointestinal tract. Translocation of intestinal microflora by the lymphatic or hematogenous routes with resulting septic complications is a major cause of morbidity and mortality after exposure to moderate to high doses of total or near-total body irradiation (TBI) (1). Similarly, in most animal species, systemic microbial infection due to dysfunction of the intestinal mucosal barrier plays a major role in development of radiation-induced sepsis and lethality (2, 3).
There are obvious interactions between the hematopoietic and gastrointestinal systems after exposure to TBI. Low doses of TBI (3–7.5 Gy in the mouse model) result in temporary injury to the tight junction between epithelial cells of the intestinal mucosal lining. Higher doses of radiation (8–15 Gy) also lead to crypt stem cell death or arrest of mitotic activity, thus resulting in inadequate replacement of the villus epithelium. Mice with a germ-free gut were observed to be more resistant to acute lethality after exposure to moderate to high radiation doses (4). Conversely, mice receiving bone marrow transplantation or irradiation with partial bone marrow shielding have significantly increased LD50/10 (the time point generally viewed as “intestinal”), although the number of surviving crypts at a given dose remains the same (5). Analogously, the benefits of selective decontamination of the digestive tract with poorly absorbable antimicrobials have been well documented in several preclinical studies, and selective decontamination of the digestive tract is recommended in clinical radiological emergency scenarios under certain conditions (1).
Bacteremia-associated endotoxin accumulation in the circulation is the primary cause of inflammation, multiple organ failure, and mortality in septic conditions (6). The early detection of sepsis has received increased attention in recent years because of the high rate of mortality in intensive care units and because timely intervention with antimicrobial and supportive therapy has proven effective in reducing mortality in critically ill patients (7). To that end, the utility of specific and sensitive sepsis biomarkers, i.e., early markers that demonstrate high correlation and predictive value with subsequent development of systemic infection and/or lethality, has been explored (8–10). Several early-onset markers have been proposed and evaluated in animal models and clinically in various conditions associated with septicemia [reviewed in Kibe et al. (11)]. The prohormone, procalcitonin (PCT), is produced only by the thyroid during normal conditions but is produced and secreted by other parenchymal cells during conditions associated with bacteremia. Procalcitonin shows particular promise as an early marker of bacteremia and has thus been proposed as a method for timely detection and intervention in systemic infection (12–14).
Thus far, little is known about the utility of PCT and other markers in radiation-induced bacterial translocation and lethality. Whereas late progression of acute radiation sickness is quite similar to classic sepsis, the early-onset point and cascade of events are poorly understood. The present study assessed the utility of PCT as a biomarker of radiation-induced endotoxemia and bacterial translocation and evaluated the predictive ability of PCT in post-TBI lethality in a mouse model. The results may have implications for the preclinical development of radiation protectors and mitigators and may be relevant to clinical radiological emergency scenarios.
MATERIALS AND METHODS
The animal experimental protocols used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Central Arkansas Veterans Healthcare System and University of Arkansas for Medical Sciences, Little Rock, Ark. Male C57/BL6 mice (Charles River, Wilmington, Mass) were used in this study. Mice were housed in conventional cages under standardized conditions with controlled temperature and humidity and a 12-h/12-h day-night light cycle. Mice were allowed free access to water and chow (Harlan Teklad laboratory diet 7012; Purina Mills, St Louis, Mo) during the entire period of handling.
A total of 177 mice, obtained at 6 to 7 weeks of age and weighing 22 to 25 g at the time of experimentation, were used for the studies reported here. The mice were observed twice daily during the experimental period, and those appearing moribund (exhibiting excessive weight loss, lethargy, huddling, shivering, hunched posture, or vocalization) were killed immediately by CO2 asphyxiation followed by cervical dislocation, in accordance with American Veterinary Medical Association Guidelines on Euthanasia.
Irradiation was performed in a Shepherd Mark I model 25 137Cs irradiator (J. L. Shepherd & Associates, San Fernando, Calif). Unanesthetized mice were held in a well-ventilated custom-made Plexiglas restrainer on a turntable rotating at 5 revolutions/min. The mice were exposed to uniform TBI at a dose rate of 1.35 Gy/min.
Initial survival studies to determine the LD50/10 were carried out in 40 mice exposed to a TBI dose gradient of 8, 8.5, 9, 9.5, and 10 Gy (eight mice per group). The mice were monitored for 30 days after TBI, and the number of dead/moribund mice recorded. Survival data at day 10 were used to estimate the LD50/10 dose for subsequent experiments. The doses TBI doses for the subsequent experiment were chosen so as to straddle the LD50 as closely as possible to achieve the maximum statistical power for the receiver operating characteristic (ROC) analysis (see below).
In vivo intestinal permeability assay
Intestinal permeability was measured in 17 mice (four to five per group) as described by Li et al. (15). Briefly, 4 days after exposure to 0-, 9-, 10-, or 12-Gy TBI, the mice were anesthetized with isoflurane inhalation, a midline laparotomy was performed, and the renal artery and vein were ligated bilaterally. A 10-cm small intestinal segment, located 5 cm distal to the ligament of Treitz, was isolated and tied off. One hundred microliters of 4-kd fluorescein isothiocyanate–conjugated dextran (FITC-dextran 25 mg/mL in phosphate-buffered saline) was injected into the isolated intestine using a 30-gauge needle, and the abdominal incision was closed. After 90 min, blood was collected from the retro-orbital sinus, and plasma was separated by centrifuging at 4°C, 8,000 revolutions/min for 10 min. The concentration of FITC-dextran was determined with a fluorescence spectrophotometer (Synergy HT; Bio-Tek Instruments, Winooski, Vt) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Standard curves to calculate FITC-dextran concentration in the plasma samples were prepared from dilutions of FITC-dextran in phosphate-buffered saline.
Evaluation of parameters of systemic infection
First, the post-TBI time course of plasma PCT levels was established. Seventy-two mice were exposed to 9 or 10 Gy (i.e., closely straddling the LD50/10 estimate) and killed (six per group) at baseline and 4, 6, 8, and 10 days after TBI. Plasma PCT was measured as described below.
To determine the correlation among the various parameters of systemic infection, analysis of bacterial translocation, PCT, lipopolysaccharide (LPS), and LPS-binding protein (LBP) in the same animals (see description of methods below) was performed on 36 mice (four per group), killed on schedule at baseline or on days 3.5, 7, and 10 after exposure to 9- and 10-Gy TBI. Peripheral blood counts were measured on days 0, 3.5, and 10 by collecting whole blood by retro-orbital bleeding using heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) into sodium-EDTA–coated microtubes (Fisher Scientific). Hematology profiles were measured using a HEMAVET 950 instrument (Drew Scientific, Oxford, Calif).
Finally, the value of early plasma PCT levels in predicting post-TBI lethality was assessed with ROC analysis in a separate experiment, performed on 12 mice exposed to 9-Gy TBI.
Bacterial translocation was determined as bacterial load in liver tissue and was quantified by real-time polymerase chain reaction (PCR) using the 16S rRNA gene consensus sequence. The gene sequence of rRNA is highly conserved in bacteria throughout evolution, owing to their central role in protein synthesis (16). Several studies have established the use of the 16S rRNA gene as a standard method for identification and classification of prokaryotes (17). The total load of bacteria in the liver was determined using primer sequences to amplify the highly conserved sequence for a broad species consensus as reported elsewhere (18, 19).
Livers were removed aseptically and homogenized immediately. Bacterial translocation was quantified by real-time PCR (20). Briefly, DNA was isolated from sterile livers harvested at baseline and at 3.5, 7, and 10 days after exposure to TBI (9 or 10 Gy) using a DNA purification kit (Promega, Madison, Wis). Real-time PCR was performed using Power SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and 16S rRNA gene targeted primers, forward (5′-AAC GCG AAG AAC CTT AC-3′) and reverse (5′-CGG TGT GTA CAA GAC CC-3′). Serially diluted bacterial genomic DNA was used to generate the standard curve. Polymerase chain reaction–derived bacterial counts were expressed as nanogram bacterial DNA per gram of mouse liver tissue.
Plasma PCT levels were measured using the mouse PCT enzyme-linked immunosorbent assay (ELISA) kit (CSB-E10371m) from Cusabio Biotech, Carlsbad, Calif. Briefly, blood collected in EDTA-coated tubes was centrifuged at 1,500g at 4°C for 15 min, and plasma used immediately for assay. The 96-well precoated plates were loaded with 100 μL of standard and plasma samples. After incubation for 1 h at 37°C with biotinylated tracer antibody, samples were developed against a horseradish peroxidase–avidin conjugate and absorbance measured at 450 nm. The concentration of PCT in each sample was quantified against the standard curve. Values are expressed as nanogram PCT protein per milliliter of plasma.
Plasma levels of LPS were measured at baseline and 3.5, 7, and 10 days after TBI (9 or 10 Gy) by the PyroGene recombinant factor C endotoxin detection assay kit (50–658 U) from Lonza, Walkersville, Md. The assay is based on the activation of recombinant factor C by endotoxin binding whereby the activated moiety cleaves a synthetic substrate to generate a fluorogenic compound with excitation/emission wavelength of 380/440 nm. Blood was obtained and centrifuged as described above, collected into endotoxin free tubes, and used immediately. Serially diluted Escherichia coli endotoxin standard and samples were loaded on a precoated 96-well plate and incubated for 1 h at 37°C. The difference in fluorescence between time 0 and 1 h was used to calculate endotoxin concentration from the standard curve. Values are expressed as endotoxin units (EU) per milliliter of plasma.
Plasma LBP level was measured using the mouse LBP ELISA kit (HK205) from Hycult Biotech, Uden, the Netherlands. The assay is based on a sandwich ELISA method with biotinylated anti-LBP antibody. Blood was collected and centrifuged as described above, and plasma was used immediately for assay. One hundred microliters of standard and plasma samples was loaded onto precoated 96-well plates, incubated for 2 h at room temperature. Biotinylated tracer antibody was incubated for 1 h at room temperature and developed against a streptavidin-peroxidase conjugate, and absorbance was measured at 450 nm. The concentration of LBP was determined against the standard curve. Values are expressed as nanogram LBP protein per milliliter of plasma.
Data are presented as mean ± SEM. Statistical analysis was performed with NCSS 2007 (NCSS, Kaysville, Utah). The LD50/10 was estimated with probit analysis; comparisons of bacterial translocation, hematologic parameters, LPS, LBP, and PCT among groups according to radiation dose and time point were performed with analysis of variance (ANOVA) and two-sided t test as appropriate, and predictive value was assessed with binormal ROC analysis. Correlation between two variables was estimated by Pearson correlation coefficient. Two-tailed P < 0.05 was considered statistically significant.
The estimated LD50/10 in mice exposed to 8 to 10 Gy with 0.5-Gy intervals was 9.44 ± 0.24 Gy (Fig. 1, A and B). Therefore, the doses selected for investigation of parameters of systemic infection were 9 and 10 Gy, i.e., doses that, because of the steepness of the radiation dose response curve, straddle the LD50/10 closely.
In vivo assessment of intestinal permeability 4 days after irradiation revealed a highly significant dose-dependent (P < 0.0001) increase when all exposure groups were considered (Fig. 2A). Compared with sham-irradiated controls, permeability in mice exposed to 10 Gy was increased 5-fold (P = 0.01), whereas 12-Gy TBI was associated with a 15-fold increase (P = 0.004). Analyzed as a separate group, permeability in the 9-Gy-exposure group was elevated 3-fold, but the difference did not reach statistical significance.
Measurement of plasma PCT levels 4, 6, 8, and 10 days after exposure to 9- or 10-Gy TBI showed a significant increase on day 4 with a highly statistically significant postexposure time dependence (P < 10−6) and radiation dose dependence (P = 0.002) (Fig. 2B).
Radiation-induced bacterial translocation is known to start around day 7 and peaks around 2 weeks after TBI (21, 22). Bacterial translocation was determined on days 0, 3.5, 7, and 10 after exposure to 9- or 10-Gy TBI, as shown in Figure 3A. No bacterial translocation was observed at baseline and 3.5 days after TBI. In contrast, significant bacterial loads were detected on day 7 (P = 0.0006 compared with 3.5 days), and this increased further between days 7 and 10 (P = 0.000005). Translocation was more pronounced after 10-Gy TBI than after 9-Gy TBI (ANOVA P = 0.01).
Changes in peripheral blood count were measured to reflect immune system dysfunction on days 0, 3.5, and 10 (Fig. 3, B–D). White blood cells were significantly diminished on both days 3.5 and 10 (P < 0.0001) compared with baseline. The red blood cell counts also showed a significant decline on days 3.5 (P = 0.02) and 10 (P < 0.0001). Platelet counts did not show a significant difference on day 3.5 but were significantly reduced (P < 0.0001) on day 10.
Plasma endotoxin (LPS) levels and plasma LBP levels, measured by the recombinant factor C limulus amebocyte lysate method and an ELISA method, respectively, are shown in Figure 4, A and B.
Lipopolysaccharide is shed from the cell wall of gram-negative bacteria and generally corresponds with the systemic bacterial load. Similar to bacterial translocation, although there was no increase in LPS levels after exposure to radiation between baseline and 3.5 days, the LPS levels increased significantly from 3.5 to 7 days (P = 0.001) with a further increase from 7 to 10 days (P = 0.009). A significant difference in LPS level was observed as a function of radiation dose (ANOVA P < 0.0003), mainly because of a difference at 10 days (P = 0.008).
After exposure to 9-Gy TBI, the LBP levels exhibited no increase from baseline to 3.5 days, a borderline significant increase (P = 0.03) from 3.5 to 7 days, but a highly significant increase between days 7 and 10 (P = 0.006). Analysis of variance revealed no difference in LBP levels between the two radiation doses.
Plasma PCT levels, measured by an ELISA method, and the results of the ROC analysis of PCT level at day 3.5 as a predictor of 10-day lethality after 9-Gy TBI are shown in Figure 5A. After exposure to 9 Gy, plasma PCT levels were significantly elevated already on day 3.5 (P = 0.002 compared with baseline) and increased further from 3.5 to 7 days (P = 0.003) and from 7 to 10 days (P = 0.00005). Moreover, the difference between the two TBI doses was highly statistically significant (ANOVA P = 0.00005) again mainly because of a difference at 10 days (0.003).
Receiver operating characteristic analysis revealed that the area under the curve (AUC) was 0.88 ± 0.09 (i.e., significantly better than 0.5 as for a random predictor), thus confirming that PCT levels at 3.5 days can serve as an early indicator of post-TBI lethality (Fig. 5B).
The correlation among the various sepsis parameters and bacterial translocation was calculated for the pooled data set. Significant positive correlations were observed between bacterial translocation and LPS (0.91), LBP (0.95), and PCT (0.98), thus providing r2 values of 0.83 for LPS, 0.90 for LBP, and 0.96 for PCT.
Extensive research and data are available from laboratory models and clinical studies on the progression of sepsis. However, there is a paucity of predictive biomarkers for use in preclinical radiation models.
Ionizing radiation, rather than causing massive tissue necrosis, affects primarily actively and rapidly proliferating cells in the bone marrow and the intestinal crypts. The killing of intestinal progenitor cells in the crypts elicits an increase in mucosal permeability through a combination of disruption of epithelial tight junctions and insufficient replacement of the villus epithelium. Intestinal bacteria that penetrate the defective mucosal barrier are an important source of bacteremia after radiation exposure and the main cause of lethality in radiological or nuclear emergencies. Exposure to ionizing radiation causes enhanced intestinal permeability, leading to bacterial and LPS translocation, development of systemic inflammation/sepsis, a so-called “radiation-induced multiple organ dysfunction syndrome.” The present study aimed at determining whether PCT can be used as an early predictive marker of lethality in a mouse model of TBI.
An extensive body of literature exists regarding the importance of gut bacteria for the outcome after exposure to TBI, the types of bacteria encountered, and the recovery of bacteria from the systemic circulation and various organs (21, 23). The kinetics of radiation-induced bacteremia is significantly different from that of general sepsis models. For example, studies using the cecal ligation and puncture model have documented translocation of gut microflora and the rapid progression of sepsis in animal models, with death generally occurring within 48 h of cecal ligation and puncture (24). Although the sequence of events for TBI exposure is similar, the TBI model progresses more gradually, with most deaths occurring between 7 and 21 days. Notably, whereas mucosal injury peaks 3.5 to 4 days after exposure to radiation, bacterial translocation is generally first present at 6 to 7 days and peaks about 2 weeks after TBI, thus highlighting the importance of concomitant mucosal barrier dysfunction and immune system compromise (22). While irradiated mice have reduced oral intake compared with controls and frequently exhibit a substantial loss of body weight, compensating for poor intake of water and/or food has been shown to adversely impact outcomes in radiation studies (25). Therefore, because pair-feeding would likely have had an adverse effect on the outcome in this study, it was not used.
Endotoxins are complex polysaccharides found on the outer membrane of bacteria and are shed naturally into their surroundings. The predominant endotoxin of gram-negative bacteria is LPS, whereas that of gram-positive bacteria is lipoteichoic acid. A gradual buildup of endotoxin level in circulation predates the onset of detectable bacteremia and hence can be used as a reasonably early marker. Clinically, LPS levels of up to 25 pg/mL (≈0.25 EU) are normal, levels of 25 to 700 pg/mL (≈0.25–7 EU) indicate early onset of bacteremia, and values greater than 10 ng/mL (≈100 EU) are associated with severe septicemia (26). In our study, elevated levels of LPS were not observed before 7 days after TBI, thus at an intermediate time that roughly corresponded to the onset of detectable bacterial translocation to the liver. Hence, although LPS can serve as a minimally invasive indicator of bacterial translocation, it is not suitable as a predictor of septicemia.
The high-affinity interaction between LBP, a 65-kd protein, and LPS forms the stimulatory complex for macrophages via CD14 leading to the production of cytokines (27). Hence, LBP-LPS-sCD14 interaction forms the basis for immune cell activation and immune response against bacteremia. Elevated levels of serum LBP were found in critically ill neonates with both gram-positive and gram-negative infections and were reported to be on par with PCT and a significantly better diagnostic marker for bacterial sepsis than interleukin 6 and C-reactive protein (28, 29). In contrast, our study revealed that LBP levels were elevated only 10 days after TBI, at a time when there is already significant lethality. Thus, LBP does not appear to have value as an early predictor of radiation-induced bacterial translocation or lethality.
Procalcitonin has generated substantial interest as a sepsis biomarker since its first use in 1993 to differentiate bacterial infection from viral meningitis (30). Procalcitonin is produced as a prohormone of calcitonin in the C cells of thyroid and is rarely, if not never, secreted into the circulation under normal physiological conditions. During conditions of bacteremia, on the other hand, almost every parenchymal cell in the body has been reported to produce and secrete PCT (31). Hence, circulating PCT is primarily extrathyroid in origin and associated with bacteremia. The unrestrained production of cytokines acts as the primary stimulatory factor of PCT production (32). Plasma PCT levels are normally less than 0.1 ng/mL, increase within 4 h of onset of bacteremia, and often reach 100- to 1,000-fold normal levels in severe sepsis (33). In the clinic, a concentration of greater than 1.2 ng/mL is generally considered as a cutoff for bacteremia confirmation in critically ill patients (33). Procalcitonin is also a fairly specific marker of bacteremia. Procalcitonin levels in plasma are not significantly altered by viral infection and in nonseptic systemic inflammatory response syndrome, and PCT has been proposed as an early biomarker of late mortality in clinical septicemia (34). Although PCT can also be elevated in severe bacterial infection as well as in severe sterile inflammation, these conditions would be unlikely to occur in the early phase after exposure to radiation in the absence of combined injury (35).
In the present study, bacterial translocation to liver tissue was virtually undetectable on day 3.5 after TBI, significantly elevated by day 7, and very high at day 10. An increase in plasma PCT level was evident already at the earliest time point (3.5–4 days) and exhibited a high positive correlation with increase in bacterial load and endotoxin accumulation after irradiation. Moreover, the predictive value of PCT as an early biomarker, as estimated by ROC analysis, showed an AUC of 0.88 ± 0.9, significantly better than the 0.5 value of a random “predictor.” Because antimicrobial therapy is of documented benefit in radiation-induced sepsis, if PCT were to be validated as a predictor of radiation-induced bacterial translocation, appropriate antibiotics could be instituted and thus help rescue some individuals exposed to a potentially lethal dose of TBI. Further studies are needed to establish a cutoff number for various animal models, and a larger number of subjects are needed to construct empirical rather than binormal ROC curves.
In conclusion, an increase in intestinal mucosal permeability and bacterial translocation with a more or less parallel accumulation of endotoxin in plasma was observed after exposure to TBI. Lipopolysaccharide-binding protein, contradictory to expectation, did not exhibit consistent early changes after irradiation, and LPS coincided with, but did not precede, the onset of bacterial translocation. Elevated plasma PCT was observed from day 3.5 onward, i.e., before appreciable bacterial translocation or endotoxemia, but coinciding with the increase in permeability of the intestinal mucosa. Although these findings will have to be extended to other animal models and with a wider range of TBI doses, alone and in combination with other injuries, correlation and ROC analysis of the present data suggest that PCT can be used as an early-onset biomarker for TBI-induced lethality. These findings may have implications for the preclinical development of radiation protectors and mitigators and may also prove relevant to clinical radiological emergency scenarios.
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