Sepsis is predominantly a disease of the aged. Although 660,000 to 750,000 people become septic annually in the United States, the incidence increases more than 100-fold with age (1, 2). Nearly 60% of all cases of sepsis occur in patients older than 65 years of age, and 80% of septic deaths occur in this population. In addition, the mortality of septic patients over age 85 is nearly four times that seen in septic children (1).
Despite the fact that sepsis is generally a disease of elderly patients, the vast majority of animal studies modeling the disease use 6- to 16-week-old mice, which corresponds to a human age of 10 to 17 years (3). Because mortality varies widely with age in septic patients, it is unclear whether young mice recapitulate the pathobiological changes seen in aged patients with overwhelming infection. As an example, we recently demonstrated that age increases mortality and decreases sensitivity to antibiotics in septic mice subjected to cecal ligation and puncture (CLP), a common murine model of ruptured appendicitis (3, 4).
Apoptosis is disproportionately increased in the spleen and gut epithelium in both young septic mice (5–8) and septic patients (9) when compared with cell death in other tissues. Elevated splenic apoptosis appears to be detrimental in sepsis in young animals because either overexpression of the antiapoptotic protein Bcl-2 in lymphocytes of transgenic mice or administration of caspase inhibitors decreases lymphocyte apoptosis and improves survival in mice subjected to CLP (10–12). Similarly, tissue-specific overexpression of Bcl-2 prevents gut epithelial apoptosis and improves survival in both CLP and Pseudomonas aeruginosa pneumonia (8, 13) in young mice.
Aging is independently associated with increased splenic and gut epithelial apoptosis. Aged mice and rats have increased basal and activation-induced lymphocyte (splenic and peripheral) apoptosis, which is associated with increases in Fas, Fas ligand, TNF-α, and TNFR1, and aged human lymphocytes are also more prone to undergo apoptosis (14–19). The effect of age on basal gut epithelial apoptosis is less clear because aging has been reported to increase intestinal cell death in human but not murine small intestine (20, 21). However, studies demonstrating increased gut epithelial apoptosis in aged mice subjected to ionizing radiation or calorie restriction (21, 22) suggest that intestinal cell death may increase when age is combined with an additional perturbation.
Although aging may cause splenic and gut apoptosis, this does not automatically translate to a further increase in cell death in these tissues under stress. For instance, liver apoptosis is mildly increased in aged rats under basal conditions, but young rats treated with a direct-acting genotoxic agent have a 10-fold increase in liver apoptosis while old animals have essentially no apoptotic response to this stress (23).
Because sepsis and aging independently increase lymphocytic and gut epithelial apoptosis but aging decreases or leaves unchanged apoptosis in response to genotoxic stress, the combined effect of age and overwhelming infection is unclear. To address this question, we studied splenic and intestinal cell death in young and aged mice subjected to CLP.
MATERIALS AND METHODS
CLP was performed by the methods of Baker et al. (4) on ND4 mice (Harlan, Indianapolis, IN) or C57BL/6 mice (ordered from the National Institute on Aging, which maintains colonies through Harlan). Anesthesia was induced with 5% halothane followed by maintenance with 2.5% halothane. A small midline abdominal incision was performed, and the cecum was exteriorized and ligated immediately distal to the ileocecal valve, in a fashion that did not result in intestinal obstruction. The cecum was then punctured with a hollow-bore needle (the gauge of the needle and the number of punctures varied with the genetic background of mice and are outlined below). After gently being squeezed to extrude some stool, the cecum was replaced in the abdomen, and the abdominal wall was closed in layers. Each animal then received a subcutaneous injection of 1 mL 0.9% NaCl to compensate for third-space fluid loss. Sham mice were treated identically, except that the cecum was neither ligated nor punctured. All manipulations were conducted in random order with respect to the age of the mouse and to whether an animal was subjected to CLP or sham manipulation. All animals acclimatized for at least 7 days in our vivarium before manipulation and were maintained on 12-h light-dark cycles with free access to food and water at all times. Of note, all studies were performed only on male mice to avoid the confounding effects of estrous cycle (24–26). All experiments were conducted in accordance with the National Institutes of Health guidelines for the use of laboratory animals and with approval of the Washington University Animal Studies Committee.
Animals were sacrificed 24 h after CLP. Spleens were harvested and immediately fixed in 10% formalin. The entire small intestine was removed simultaneously, opened along its cephalocaudal axis, washed with 0.9% NaCl, and fixed in 10% formalin for 24 hours. The intestine was then rolled proximal to distal with the luminal side facing outward.
Apoptotic cells were quantified using two techniques: active caspase 3 staining and hematoxylin and eosin (H&E) staining. Active caspase 3 staining was done as previously described (8, 27). Briefly, paraffin-embedded tissues were heated at 60°C for 10 min, rehydrated, and incubated at 23°C in 3% H2O2 in methanol for 10 min to block endogenous peroxidase activity. Slides were microwaved in citrate buffer (pH 6.0) for 9 min at 89°C to facilitate antigen retrieval and then incubated for 1 h with polyclonal rabbit antiactive caspase-3 (1:100; Cell Signaling, Beverly, MA) at 23°C. Sections were then incubated at 23°C with secondary biotinylated goat antirabbit antibody (1:200; Vector Laboratories, Burlingame, CA) for 30 min followed by Vectastain ABC (Vector Laboratories) for 30 min at 23°C. Slides were developed with nickel-enhanced DAB substrate solution at 23°C for 30 min and then counterstained with hematoxylin.
Apoptosis was determined on H&E-stained sections using morphologic criteria. Cells with characteristic nuclear condensation (pyknosis) and fragmentation (karyorrhexis) were considered to be apoptotic.
Cell death was quantified in the spleen by counting cells staining positive for active caspase 3 in five random high-power (×400) fields. Apoptosis was quantified in the intestinal epithelium by counting cells in the most distal 100 contiguous crypts from well-oriented intact crypt–villus units with Paneth cells at the crypt base and an unbroken epithelial column extending to the villus tip. Apoptosis was quantified by a grader who was blinded to sample identity in all experiments.
Cellular proliferation quantification
A subset of ND4 mice received an intraperitoneal injection of 120 mg/kg 5-bromo-2′-deoxyuridine (BrdU, Sigma, St. Louis, MO) 90 min before sacrifice to label cells in S-phase as has previously been described (27). Immunohistochemical detection of BrdU was performed using a commercially available kit according to manufacturer’s recommendations (PharMingen, San Diego, CA). M-phase cells were identified on sections stained with hematoxylin alone by searching for the presence of cells exhibiting the characteristic morphology of the mitotic spindle. Guts were harvested 24 hours after CLP, and S- and M-phase cells were counted in 100 contiguous crypts as above.
Data comparing apoptosis or proliferation were analyzed using one-way analysis of variance followed by the Newman-Keuls multiple comparison test. Data were analyzed using the statistical program Prism 3.0 (GraphPad Software, San Diego, CA) and are presented as mean ± SEM. P values <0.05 were considered statistically significant.
Splenic apoptosis is disproportionately increased in aged, septic mice
Young (2-month) and aged (22-month) ND4 mice (n = 3–9/group) received either single-puncture CLP with a 23-gauge needle or sham laparotomy and had their spleens removed following sacrifice 24 h later. This injury was chosen based on pilot studies demonstrating that it results in low mortality (10%) in young ND4 mice (data not shown). The mortality of aged ND4 mice is unknown because of lack of availability of these animals. Although all aged ND4 animals survived to the 24-h sacrifice time point, subjectively, most of them appeared severely ill (but not imminently moribund). Splenic apoptosis was similar in young and aged animals subjected to sham laparotomy; however, there was a marked increase in splenic apoptosis in aged septic animals compared with young septic animals or aged sham mice (Figs. 1 and 2A).
To verify that this effect was not strain dependent, similar experiments were performed in young (4-month) and aged (24-month) C57BL/6 mice (n = 4–10/group) that received either double-puncture CLP with a 25-gauge needle or sham laparotomy. Previous studies demonstrated that this injury gives 25% mortality in young mice and 80% mortality in aged mice (3). Similar to results in outbred ND4 mice, splenic apoptosis increased in aged septic C57BL/6 mice compared with either young animals subjected to CLP or aged animals subjected to sham laparotomy (Fig. 2B). Similar qualitative results were seen with H&E staining in both strains (data not shown).
Gut epithelial apoptosis is disproportionately increased in aged, septic mice
The same ND4 animals examined for splenic cell death were also assayed for gut epithelial apoptosis. Because sepsis-induced intestinal apoptosis occurs in a more patchy distribution than observed in the spleen, gut epithelial cell death was quantified by both H&E (Figs. 3 and 4A) and active caspase 3 staining (Fig. 4B) across 100 contiguous crypts. As in previous reports in mice (21), apoptosis was similar in young and aged animals subjected to sham laparotomy. However, there was a marked increase in gut epithelial apoptosis in aged septic animals compared with young septic animals or aged sham mice.
Gut proliferation is unaffected by aging and sepsis
Because critical illness and aging have both been independently reported to alter intestinal proliferation (20, 27–30), we examined whether sepsis and aging affected gut cell division. Neither gut epithelial proliferation as assayed by the incorporation of BrdU (Fig. 5A) nor cell division (Fig. 5B) as assayed by the presence of visible mitotic spindles was altered in a statistically significant fashion by either CLP, aging, or the combination of sepsis and aging in ND4 mice. The ratio of S-phase to M-phase cells, a common method of studying cell cycle kinetics in the intestinal epithelium (27, 31, 32), was similar between young sham and septic animals (186 and 185, respectively), was mildly decreased in aged sham animals (130), and was increased greater than 50% in aged septic mice (309).
This study demonstrates that the combination of aging and sepsis leads to an increase in both splenic and gut epithelial apoptosis, far greater than would be predicted based on the effects of either variable in isolation. Although gut apoptosis is increased, intestinal proliferation is unaltered by the combination of aging and sepsis, suggesting that cellular production and death are not linked in this tissue 24 h after the onset of infection.
Previous studies on various stressors applied to aged animals have demonstrated marked differences in lymphocyte apoptotic response. TNF-α, influenza, or H2O2 all induce increased lymphocyte apoptosis in aged animals compared with genetically identical younger animals. In contrast, T-cell receptor agonist withdrawal, heat shock, ionizing radiation, and staurosporine all lead to a decrease in apoptosis in lymphocytes obtained from aged animals (15–17, 33–35). Because there is a stress-specific response of aged lymphocytes, it was unclear what the response to aging and sepsis would be. This response is potentially of significant clinical interest because sepsis-induced apoptosis has been linked to poor outcomes in young mice. Answering this question in an in vivo fashion was critical because this is the only convincing way to demonstrate an animal’s response to the multiple competing pro- and antiinflammatory signals simultaneously operative in a mouse that undergoes CLP (36, 37). The discrepancy seen between aged, septic animals and young, septic animals serves as an example that using young mice to model a disease that predominantly affects the aged might lead to results that ultimately are not representative of the septic patient.
The mechanism that underlies increased apoptosis in aged, septic mice is unclear. Prior studies have shown that lymphocytes from aged donors are more susceptible to TNF-α-induced apoptosis or antibodies that are agonistic to TNFR1, and circulating mononuclear cells from elderly patients are more susceptible to oxidative stress-induced apoptosis than cells from younger counterparts (14, 33, 38). We have previously demonstrated increased TNF-α in aged mice (3), and it is possible TNF-α plays an important role in sepsis-induced apoptosis in the aged. It is unlikely to be the sole determinant of the elevation of cell death, however, because both lymphocyte and gut epithelial apoptosis occur, at least in part, through the mitochondrial pathway (8, 10, 11, 13). In addition, markedly elevated TNF-α levels in mice injected with LPS do not result in disproportionate increases in splenic or gut apoptosis compared with more clinically relevant models of sepsis. Although the mechanism underlying the increased apoptosis remains to be determined, a comparison of the splenic apoptosis data between ND4 and C57BL/6 mice shows that the combined effects of age and sepsis are strain independent. Genetic background has a considerable impact on both immune response and survival in sepsis (39). The majority of studies on the role of apoptosis in sepsis have been in either C57BL/6 mice or FVB/N mice [although the initial study was in female ND4 mice (5)]. The fact that the apoptosis data were so similar between the outbred ND4 strain and inbred C57BL/6 strain implies that this is likely to be common cellular response in aging. Further studies comparing splenic and gut samples from autopsy specimens previously obtained from young and aged septic patients (9) are warranted to demonstrate if aging and infection lead to the same response in humans.
The data showing no difference in gut epithelial proliferation were surprising. Both sepsis and aging have previously been independently shown to alter intestinal proliferation (20, 27–30). We therefore expected to find that both variables would change the number of S- and M-phase cells, and the combination would possibly have profound effects. Instead, we found no differences between any groups in the number of S- or M-phase cells but instead only found the ratio of S- to M-phase cells increased substantially in aged septic mice, possibly indicating the presence of a block at G2/M similar to that seen in the intestine with ionizing radiation (31). It is possible that this is a strain effect because previous studies on gut proliferation in aging or sepsis have not been done in ND4 mice, but we do not otherwise have an explanation for the discrepancy between our current results and published data.
Although this study provides new insights into the relationships among age, sepsis, and apoptosis, it has a number of limitations. First, the functional significance of the increase in apoptosis in aged, septic mice cannot be ascertained from this study. Results from a number of investigators demonstrate that lymphocyte apoptosis and gut epithelial apoptosis are up-regulated in sepsis (5–9, 40–42), and this appears to be detrimental in young mice. However, it does not inherently follow that a substantial increase in splenic and gut cell death is detrimental to the aged in sepsis. Although it is plausible that apoptosis plays a similar role in aged animals and young mice, it is also possible that cell death is beneficial or an epiphenomenon in aged mice or that it has differential effects depending on the tissue where apoptosis is occurring.
Studies were also limited to male animals. Epidemiologic studies have shown that men are more likely to develop sepsis than women, and most studies show that they are more likely to die of the disease (1, 2, 43). Extensive laboratory studies on this gender dimorphism implicate the immunomodulatory effects of sex hormones as playing a critical role in this survival advantage (25, 26, 44), and the exclusion of female mice from this study precludes an analysis of the relationship between gender and apoptosis in the aged, septic animal. In addition, although increased apoptosis was clearly present in the spleens of aged mice, we did not determine if the population of cells dying was the same in young and aged mice, nor did we distinguish between percentage of apoptosis between the red pulp and white pulp. We also examined the intestine in only a single strain, so we cannot conclude that the pattern of apoptosis seen in ND4 intestines and C57BL/6 and ND4 spleens is similar in the guts of C57BL/6 mice. There was also a discrepancy in the data for gut epithelial apoptosis that was statistically significant by H&E but not active caspase 3 (Fig. 4), and additional quantitative measurements would have been beneficial to verify the results seen with H&E. Finally, we did not include a negative control by examining apoptosis in a tissue that has not been shown to have elevated cell death in young septic mice.
Despite these limitations, these results demonstrate that the combination of aging and sepsis can result in pathological perturbations substantially greater than either alone would be expected to produce. Future studies of aged transgenic animals that overexpress Bcl-2 in a tissue-specific fashion or giving apoptosis inhibitors to aged animals should delineate the physiological significance of our results.
The authors thank the Washington University Digestive Diseases Research Morphology Core.
1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med
2. Martin GS, Mannino DM, Eaton S, Moss M: The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med
3. Turnbull IR, Wlzorek JJ, Osborne D, Hotchkiss RS, Coopersmith CM, Buchman TG: Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture. Shock
4. Baker CC, Chaudry IH, Gaines HO, Baue AE: Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery
5. Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, Karl IE: Apoptosis in lymphoid and parenchymal cells during sepsis: findings in normal and T- and B-cell-deficient mice. Crit Care Med
6. Hiramatsu M, Hotchkiss RS, Karl IE, Buchman TG: Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin- and TNF-independent pathway. Shock
7. Hotchkiss RS, Dunne WM, Swanson PE, Davis CG, Tinsley KW, Chang KC, Buchman TG, Karl IE: Role of apoptosis in Pseudomonas aeruginosa
8. Coopersmith CM, Stromberg PE, Dunne WM, Davis CG, Amiot DM, Buchman TG, Karl IE, Hotchkiss RS: Inhibition of intestinal epithelial apoptosis and survival in a murine model of pneumonia-induced sepsis. JAMA
9. Hotchkiss RS, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Matuschak GM, Buchman TG, Karl IE: Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med
10. Hotchkiss RS, Swanson PE, Knudson CM, Chang KC, Cobb JP, Osborne DF, Zollner KM, Buchman TG, Korsmeyer SJ, Karl IE: Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis. J Immunol
11. Hotchkiss RS, Tinsley KW, Swanson PE, Chang KC, Cobb JP, Buchman TG, Korsmeyer SJ, Karl IE: Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc Natl Acad Sci USA
12. Hotchkiss RS, Chang KC, Swanson PE, Tinsley KW, Hui JJ, Klender P, Xanthoudakis S, Roy S, Black C, Grimm E, Aspiotis R, Han Y, Nicholson DW, Karl IE: Caspase
inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat Immunol
13. Coopersmith CM, Chang KC, Swanson PE, Tinsley KW, Stromberg PE, Buchman TG, Karl IE, Hotchkiss RS: Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice. Crit Care Med
14. Schindowski K, Leutner S, Muller WE, Eckert A: Age-related changes of apoptotic cell death in human lymphocytes. Neurobiol Aging
15. Phelouzat MA, Laforge T, Arbogast A, Quadri RA, Boutet S, Proust JJ: Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo
expression of functional Fas receptors. Mech Ageing Dev
16. Telford WG, Miller RA: Aging increases CD8 T cell apoptosis induced by hyperstimulation but decreases apoptosis induced by agonist withdrawal in mice. Cell Immunol
17. Zhang Y, Wang Y, Gilmore X, Xu K, Chen M, Tebebi P, Mbawuike IN: Apoptosis and reduced influenza A virus specific CD8+
T cells in aging mice. Cell Death Differ
18. Potestio M, Pawelec G, Di Lorenzo G, Candore G, D’Anna C, Gervasi F, Lio D, Tranchida G, Caruso C, Romano GC: Age-related changes in the expression of CD95 (APO1/FAS) on blood lymphocytes. Exp Gerontol
19. Pagliara P, Chionna A, Panzarini E, De Luca A, Caforio S, Serra G, Abbro L, Dini L: Lymphocytes apoptosis: young versus aged and humans versus rats. Tissue Cell
20. Ciccocioppo R, Di Sabatino A, Luinetti O, Rossi M, Cifone MG, Corazza GR: Small bowel enterocyte apoptosis and proliferation are increased in the elderly. Gerontology
21. Holt PR, Moss SF, Heydari AR, Richardson A: Diet restriction increases apoptosis in the gut of aging rats. J Gerontol A Biol Sci Med Sci
22. Martin K, Kirkwood TB, Potten CS: Age changes in stem cells of murine small intestinal crypts. Exp Cell Res
23. Suh Y, Lee KA, Kim WH, Han BG, Vijg J, Park SC: Aging alters the apoptotic response to genotoxic stress. Nat Med
24. George RL, McGwin G, Metzger J, Chaudry IH, Rue LW: The association between gender and mortality among trauma patients as modified by age. J Trauma
25. Zellweger R, Wichmann MW, Ayala A, Stein S, DeMaso CM, Chaudry IH: Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males. Crit Care Med
26. Angele MK, Schwacha MG, Ayala A, Chaudry IH: Effect of gender and sex hormones on immune responses following shock. Shock
27. Coopersmith CM, Stromberg PE, Davis CG, Dunne WM, Amiot DM, Karl IE, Hotchkiss RS, Buchman TG: Sepsis from Pseudomonas aeruginosa
pneumonia decreases intestinal proliferation and induces gut epithelial cell cycle arrest. Crit Care Med
28. Varedi M, Greeley GH, Herndon DN, Englander EW: A thermal injury-induced circulating factor(s) compromises intestinal cell morphology, proliferation, and migration. Am J Physiol
29. Rafferty JF, Noguchi Y, Fischer JE, Hasselgren PO: Sepsis in rats stimulates cellular proliferation in the mucosa of the small intestine. Gastroenterology
30. Saxena SK, Thompson JS, Crouse DA, Sharp JG: Epithelial cell proliferation and biodistribution of radiolabeled urogastrone in the gastrointestinal mucosa of young and old mice. Life Sci
31. Coopersmith CM, Gordon JI: Gamma-ray-induced apoptosis in transgenic mice with proliferative abnormalities in their intestinal epithelium: re-entry of villus enterocytes into the cell cycle does not affect their radioresistance but enhances the radiosensitivity of the crypt by inducing p53. Oncogene
32. Chandrasekaran C, Coopersmith CM, Gordon JI: Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators. J Biol Chem
33. Aggarwal S, Gollapudi S, Gupta S: Increased TNF-alpha-induced apoptosis in lymphocytes from aged humans: changes in TNF-alpha receptor expression and activation of caspases. J Immunol
34. Spaulding CC, Walford RL, Effros RB: The accumulation of non-replicative, non-functional, senescent T cells with age is avoided in calorically restricted mice by an enhancement of T cell apoptosis. Mech Ageing Dev
35. Avula CP, Fernandes G: Inhibition of H2
-induced apoptosis of lymphocytes by calorie restriction during aging. Microsc Res Tech
36. Remick DG, Newcomb DE, Bolgos GL, Call DR: Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock
37. Deitch EA: Animal models of sepsis and shock: a review and lessons learned. Shock
38. Gupta S: Tumor necrosis factor-alpha-induced apoptosis in T cells from aged humans: a role of TNFR-I and downstream signaling molecules. Exp Gerontol
39. Loven L, Larsson L, Nordstrom H, Lennquist S: Serum phosphate and 2,3-diphosphoglycerate in severely burned patients after phosphate supplementation. J Trauma
40. Ayala A, Chung CS, Xu YX, Evans TA, Redmond KM, Chaudry IH: Increased inducible apoptosis in CD4+
T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin. Immunology
41. Chung CS, Wang W, Chaudry IH, Ayala A: Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated. Am J Physiol Gastrointest Liver Physiol
42. Ayala A, Herdon CD, Lehman DL, Ayala CA, Chaudry IH: Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators. Blood
43. Schroder J, Kahlke V, Staubach KH, Zabel P, Stuber F: Gender differences in human sepsis. Arch Surg
44. Diodato MD, Knoferl MW, Schwacha MG, Bland KI, Chaudry IH: Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis. Cytokine