Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis

Abstract

Objective: Zinc deficiency is common among populations at high risk for sepsis mortality, including elderly, alcoholic, and hospitalized patients. Zinc deficiency causes exaggerated inflammatory responses to endotoxin but has not been evaluated during bacterial sepsis. We hypothesized that subacute zinc deficiency would amplify immune responses and oxidant stress during bacterial sepsis {lsqb;i.e., cecal ligation and puncture (CLP){rsqb; resulting in increased mortality and that acute nutritional repletion of zinc would be beneficial.

Design: Prospective, randomized, controlled animal study.

Setting: University medical center research laboratory.

Subjects: Adult male C57BL/6 mice.

Interventions: Ten-week-old, male, C57BL/6 mice were randomized into three dietary groups: 1) control diet, 2) zinc-deficient diet for 3 weeks, and 3) zinc-deficient diet for 3 weeks followed by oral zinc supplementation for 3 days (n = 35 per diet). Mice were then assigned to receive either CLP or sham operation (n = 15 each per diet). CLP and sham-operated treatment groups were further assigned to a 7-day survival study (n = 10 per treatment per diet) or were evaluated at 24 hours (n = 5 per treatment per diet) for signs of vital organ damage.

Measurements and main results: Sepsis mortality was significantly increased with zinc deficiency (90% vs. 30% on control diet). Zinc-deficient animals subject to CLP had higher plasma cytokines, more severe organ injury, including increased oxidative tissue damage and cell death, particularly in the lungs and spleen. None of the sham-operated animals died or developed signs of organ damage. Zinc supplementation normalized the inflammatory response, greatly diminished tissue damage, and significantly reduced mortality.

Conclusions: Subacute zinc deficiency significantly increases systemic inflammation, organ damage, and mortality in a murine polymicrobial sepsis model. Short-term zinc repletion provides significant, but incomplete protection despite normalization of inflammatory and organ damage indices.

Figure 1

Alteration of zinc status following dietary modification. A, plasma zinc levels in mice receiving a control diet (Ctrl), a zinc-deficient diet (Zn−), or a zinc-deficient diet followed by acute zinc supplementation (Zn+) (n = 5 per dietary group) (*p < 0.05). B, Liver and lung tissue homogenates from the same animals analyzed for total tissue metallothionein content.

Figure 2

Zinc deficiency increases sepsis mortality. Time-dependent survival following sham operation or and cecal ligation and puncture (CLP) in animals assigned to control (Ctrl), zinc deficient (Zn−), or zinc deficiency followed by short-term zinc supplementation (Zn+) groups (n = 10 for each group). Survival of 100% was observed in all sham-operated animals (resulting in an overlap of the mortality curves for the three sham-operated groups). A significant increase in mortality following CLP was associated with zinc deficiency (p < 0.05).

Figure 3

Zinc deficiency increases lung pathology. Histologic assessments of H&E-stained lung tissue from representative (A) zinc-deficient sham operation (Zn−/sham), (B) control diet cecal ligation and puncture (Ctrl/CLP), (C) zinc-deficient CLP (Zn−/CLP), and (D) zinc deficient followed by acute zinc supplementation before CLP (Zn+/CLP) animals. Focal areas of inflammatory cell infiltrate along with features consistent with perivascular edema and epithelial attenuation were most pronounced in the Zn−/CLP group, whereas the Zn+/CLP group exhibited lung histology similar to that of sham operation. E, Quantitative analysis of lung air space demonstrated a significant reduction in the Zn−/CLP group compared with the Zn−/sham, Zn+/CLP, and Ctrl/CLP groups (*p < 0.05). F, Relative copy number (RCN) of mRNA transcripts constitutively expressed by lung epithelia and endothelia in the setting of acute lung injury (see text), including the ATP-binding-cassette transporter (ABCA), solute carrier transporter 34A2 (SLC34A2), vascular endothelial growth factor A (VEGFA), and surfactant protein A (SFTPA) (*p < 0.05). KDR, kinase insertion domain receptor; SOX, Syr-related HMG box transcription factor; SFTPB, surfactant associated protein B.

Figure 4

Morphology of the spleen, liver, and ileum. Morphologic evaluation of H&E-stained tissues in each of the treatment groups. Because all sham-operated animals had similar morphologic findings, for the sake of comparison, only the zinc-deficient sham operation (Zn−/sham) group is shown. Representative spleen samples were examined [magnification: ×4 and ×40 (insets)] in each treatment group. Minor hypocellularity of splenic lymphoid nodules (dark blue) was noted in the control diet cecal ligation and puncture (Ctrl/CLP) group. The hypocellularity was more exaggerated in the zinc-deficient CLP (Zn−/CLP) group and was associated with clusters of condensed apoptotic cells (inset, white arrows), whereas the zinc supplementation CLP (Zn+/CLP) group was similar to that of the Ctrl/CLP group. Relative to sham-operated (e.g., Zn−/sham) and Zn+/CLP animals, representative livers of Ctrl/CLP and Zn−/CLP (×10) animals demonstrated mild hepatocellular edema. There were no significant morphologic abnormalities of representative ilea (×10) in any of the treatment groups.

Figure 5

Modulation of cellular tissue injury by zinc status. Assessment of cell apoptosis in representative lung (A, B), spleen (C, D), ileal (E), and liver (F) tissues in each of the treatment groups. A consistent trend was observed in all tissues wherein zinc-deficient cecal ligation and puncture (Zn−/CLP) animals demonstrated an increased frequency of transferase-mediated dUTP-biotin nick end labeling-positive cells compared with the control diet CLP (Ctrl/CLP) group and that zinc supplementation (Zn+/CLP) reduced cell death. The extent of cell death was most apparent in lung and spleen tissues. Although a similar pattern was observed in the liver and ileum, the overall number of transferase-mediated dUTP-biotin nick end labeling-positive cells was notably less by comparison (*p < 0.05).

Figure 6

Increased systemic inflammatory response during sepsis in response to zinc deficiency. Comparisons of plasma interleukin (IL)-6 (A) and IL-10 (B) concentrations in each of the treatment groups (n = 5 per dietary group) demonstrated a significant increase in both cytokines 24 hours after cecal ligation and puncture (CLP) in zinc-deficient animals (Zn−/CLP). By contrast, both cytokine levels were significantly reduced, similar to values observed in the control diet CLP (Ctrl/CLP) group, in animals receiving acute zinc supplementation (Zn+/CLP) (*p < 0.05).