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Infection and Immunity, October 2005, p. 7006-7010, Vol. 73, No. 10
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.10.7006-7010.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Lab Animal Resources,1 Department of Molecular and Integrative Physiology,2 Department of Preventative Medicine and Public Health, University of Kansas Medical Center, Kansas City, Kansas 661603
Received 8 March 2005/ Returned for modification 27 April 2005/ Accepted 19 June 2005
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Pathogen-free male and female, 4- to 12-week-old CAST/EiJ and C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine), which have different sensitivities to LeTx (11), and 3- to 5-week-old F1 (parents, CAST/EiJ x C57BL/6J), F2 CAST/Ei (parents, F1 x CAST/EiJ), and F2 C57BL/6 (possible twi carriers; parents, F1 x C57BL/6J) mice were used for these studies, which were approved by the Institutional Animal Care and Use Committee.
Recombinant PA and LF (List Biological Laboratories, Inc., Campbell, CA) were resuspended in 0.1% bovine serum albumin (BSA), pH 
7.4, and stored in BSA-precoated tubes at –20°C. Purity of toxins was established via sodium dodecyl sulfate-polyacrylamide gels by the manufacturer. LeTx (PA, 4 µg/g of body weight, and LF, 2 µg/g) was administered by intraperitoneal (i.p.) injection, and mice were observed for clinical signs. The dosage was approximately 5 50% lethal doses based on Price et al. (12). Once clinical signs that preceded death by 2 to 3 h were identified, LeTx animals displaying these signs and matched control animals (i.p. injection of saline) were anesthetized via halothane inhalation. Blood was drawn via cardiac puncture and collected in serum tubes or in sodium citrate tubes for plasma isolation. The Veterinary Laboratory Resources, Inc. (Overland Park, Kansas), processed sera for chemistry panels using a Bayer Advia 1650 system (Bayer Diagnostics, Terrytown, New York) and plasma for coagulation panels using an STA Compact system (Diagnostica Stago, Parsippany, N.J.).
In animals that were sacrificed or died, the major organs were examined in situ, harvested, placed in 10% formalin, and embedded in paraffin. Sections, 8 µm thick, were processed for hematoxylin and eosin (H&E) staining or immunohistochemistry.
Immunohistochemical staining of fibrinogen, which cross-reacts with fibrin, was performed as follows: deparaffinization; 3% H2O2, 10 min; Tris-buffered saline (TBS) with 0.05% Tween (TBST); 0.1% proteinase K in TBS, 10 min; TBST; 1:1,000 rabbit anti-human fibrinogen (Dako, Carpinteria, CA), which cross-reacts with mouse fibrinogen, overnight at 4°C; TBST, LSAB+ (Dako), and DAB+ (Dako), 10 min.
Total glutathione was measured in the lungs and livers of LeTx C57BL/6J mice that were sacrificed when they displayed dyspnea and severe lethargy and in control (saline) C57BL/6J mice sacrificed at matching times. The assay was performed as previously described (4).
LeTx animals showed severe lethargy and weakness, hunched posture, and/or severe dyspnea within 2 to 3 h prior to death. Less common clinical signs included ataxia, rear limb paraplegia, bloat, and pale extremities. CAST/EiJ mice died at 26.7 h ± 2.0 (mean ± standard error [SE]; median, 24.9 h) with 1/6 surviving, and C57BL/6J died at 60.9 h ± 14.1 (median, 51.9 h) with 2/5 surviving (Mann-Whitney U test, P = 0.036 for CAST/EiJ versus C57BL/6J LeTx mice that died). All F1 mice succumbed to LeTx (58.2 ± 8.2 h; n = 5), while 4/5 F2 CAST/Ei animals succumbed to LeTx (31.3 ± 1.9 h), and 2/3 F2 C57BL/6 mice survived, with the third animal dying at 78 h.
Blood panels revealed hypoglycemia, elevated blood urea nitrogen (BUN), hypoproteinemia, hypoalbuminemia, and hypoglobulinemia in LeTx C57BL/6J mice (58.6 ± 6.7 h, mean time of advance clinical signs ± SE) compared to vehicle-injected C57BL/6J mice (Table 1). There were no clear differences in the chemistry profile between LeTx (24.4 ± 1.2 h) and control CAST/EiJ mice.
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Histopathologically, the two main findings were hemorrhage into secondary and tertiary bronchi (10/29) (Fig. 1A) and evidence of heart muscle damage (9/29) that sometimes included fibroblast infiltration into the interstitium (Fig. 1B). Other findings included hepatic microabscesses and granulomas (4/29), small intestinal mucosal edema (1/29), and hematoma formation in the cardiac ventricular muscle wall (1/29).
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The blood chemistry of LeTx C57BL/6J animals provides an insight into pathogenesis. Hypoglycemia in LeTx mice was generally not low enough to cause clinical symptoms; thus, the low level is likely a secondary finding due to increased vessel permeability enabling loss of glucose into the extravascular space. LeTx C57BL/6J mice also showed hypoproteinemia, hypoalbuminemia, and hypoglobulinemia compared to control animals, and vessel leakage is the likely cause of these reductions. LeTx was found to induce human endothelial cell apoptosis in vitro, suggesting a direct effect on the endothelial cells (8). However, tissue sections from LeTx mice that were stained for the apoptotic marker cleaved caspase-3 failed to reveal labeled endothelial cells (unpublished observation), but this does not rule out endothelial cell dysfunction, which could account for increased vessel leakage. In support of this possibility, LeTx has been found to alter endothelial barrier function in vitro independently of apoptosis or necrosis (14).
The elevated BUN in LeTx C57BL/6J mice in the presence of normal creatinine levels indicates a prerenal disorder, such as hemorrhage into the peritoneal or thoracic cavities, which was observed in several mice in this study. Intestinal hemorrhage was present in only one animal; however, we have observed hemorrhage in the intestine and in other organs (e.g., spleen and brain) in numerous subsequent LeTx C57BL/6J mice.
LeTx C57BL/6J animals showed a significantly decreased platelet count, decreased fibrinogen, and elevations in PT and APTT compared to controls. The decrease in platelets and fibrinogen is explained by their increased usage, which occurs in a consumption coagulopathy such as disseminated intravascular coagulation (DIC), and/or increased loss due to increased vessel permeability or hemorrhage. Fibrin deposits were observed, albeit inconsistently, in a variety of organs from C57BL/6J, F1, and F2 mice, indicating that DIC was part of the pathophysiology in at least some LeTx mice. The elevations in both PT and APTT suggest a coagulopathy affecting all three (intrinsic, extrinsic, and common) clotting cascade pathways. The most likely cause for these changes would be vessel leakage, hemorrhage, and/or DIC.
There is a high concentration of toxin receptors in the bronchi (3), and lung involvement during LeTx pathogenesis is indicated by dyspnea, bronchial hemorrhage, and glutathione depletion. The latter finding reflects oxidative stress in the lung, although some edema also may be occurring, since glutathione is measured as nmol/mg (wet weight) tissue. In either case, LeTx leads either directly or indirectly to cellular dysfunction in the lung.
LeTx CAST/EiJ mice survived to only 25 h, which was similar to findings in a previous report (11), while C57BL/6J, F1, and F2 C57BL/6 LeTx mice generally survived longer than CAST/Ei or F2 CAST/Ei LeTx mice. There was an absence of clear blood chemistry findings indicating vessel leakage in LeTx CAST/Ei mice, but their blood was extremely difficult to collect in ample quantities and these mice did show evidence of hemorrhage. Furthermore, histopathology was similar to that for C57BL/6J animals, and 2/5 CAST/EiJ had evidence of fibrin staining. Thus, these mice may progress along a pathophysiological pathway similar to that seen with LeTx C57BL/6J mice, but a rapid onset of respiratory distress or circulatory shock may account for early death.
In summary, LeTx C57BL/6J mice suffered from vessel leakage, hemorrhage, and/or DIC based on chemistry profile, coagulation panels, fibrin deposits, and pathology. Since vessel leakage, hemorrhage, and/or DIC has been observed in anthrax cases (1, 2, 6, 7, 9, 10, 13), it suggests that LeTx in mice represents significant components of anthrax pathophysiology.
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ACKNOWLEDGMENTS |
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This work was supported by internal funds from the University of Kansas Medical Center Lab Animal Resources Department and by external funds from the National Institutes of Health grant U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (MRCE).
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FOOTNOTES |
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REFERENCES |
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