Figure 2
Figure 2. In vivo effects of AADH. (A) Twenty-four–hour survival in mice challenged through intravenous injection of 30 mg/kg ConA following intraperitoneal injection of 570 µg AADH (n = 15, dashed line) or saline (n = 16, solid line). Found differences are significant (P = .0005, with hazard ratio = 8.384 and 95% confidence interval (CI) of ratio 2.371-21.79). (B) Plasma of saline- or AADH-treated mice was collected 3 or 6 hours after ConA challenge and pooled (n = 6). Histone H3 content was characterized by western blotting. For comparison, a pooled mouse plasma sample from mice not challenged with ConA is shown. Relative densities as determined by densitometry using ImageJ are indicated. (C) Seventy-two–hour survival in CLP-challenged C57Bl/6 mice treated by intraperitoneal injection with 570 μg AADH 12 hours before, directly after, and 12 hours after CLP (prophylaxis, n = 10, dashed black line) or only 4 hours after CLP (n = 10, dashed gray line). Saline-treated, CLP-challenged mice are shown as a solid black line (n = 15), whereas survival of sham-treated animals is shown as a dotted gray line (n = 15). Differences between AADH-treated and nontreated groups are significant (for both the prophylactic and treatment regimes, with P = .0031 and P = .0275, respectively, and hazard ratios = 5.471 and 3.345, 95% CI of ratio 1.774-16.87 and 1.143-9.787, respectively. (D-H) Effect of AADH in a mouse model of LPS-induced sepsis. C57Bl/6 mice were treated with LPS 20 mg/kg i.p. and 570 µg AADH after 1 hour of the LPS challenge. (D) Survival in mice receiving LPS and vehicle (solid line) and mice receiving LPS plus AADH (dashed line) (n = 9/group). The difference between groups is significant, with P = .0375 and hazard ratio = 5.4328 and 95% CI of ratio 1.088-17.22. (E-H) Mice were euthanized 8 hours after LPS challenge. (E) Representative histological hematoxylin and eosin-stained sections of lungs from mice receiving saline (e1), LPS + vehicle control (e2), or LPS + AADH (e3). (F) Quantification of histological analysis, n = 4 per bar. (G) Quantification of neutrophils in the bronchoalveolar lavage (BAL) (white bars, left y-axis) of adherent, intravascular (black bars, right y-axis), and interstitial (gray bars, right y-axis) neutrophils. Discrimination between neutrophils in either location was made based on an antibody to Ly6G injected 5 minutes prior to euthanasia (n = 6-8 for each bar). (H) Assessment of lung plasma leakage based on the exudation of the plasma tracer FITC dextran (n = 6-8). Statistical significance was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference (P < .05) compared with the LPS-challenged group.

In vivo effects of AADH. (A) Twenty-four–hour survival in mice challenged through intravenous injection of 30 mg/kg ConA following intraperitoneal injection of 570 µg AADH (n = 15, dashed line) or saline (n = 16, solid line). Found differences are significant (P = .0005, with hazard ratio = 8.384 and 95% confidence interval (CI) of ratio 2.371-21.79). (B) Plasma of saline- or AADH-treated mice was collected 3 or 6 hours after ConA challenge and pooled (n = 6). Histone H3 content was characterized by western blotting. For comparison, a pooled mouse plasma sample from mice not challenged with ConA is shown. Relative densities as determined by densitometry using ImageJ are indicated. (C) Seventy-two–hour survival in CLP-challenged C57Bl/6 mice treated by intraperitoneal injection with 570 μg AADH 12 hours before, directly after, and 12 hours after CLP (prophylaxis, n = 10, dashed black line) or only 4 hours after CLP (n = 10, dashed gray line). Saline-treated, CLP-challenged mice are shown as a solid black line (n = 15), whereas survival of sham-treated animals is shown as a dotted gray line (n = 15). Differences between AADH-treated and nontreated groups are significant (for both the prophylactic and treatment regimes, with P = .0031 and P = .0275, respectively, and hazard ratios = 5.471 and 3.345, 95% CI of ratio 1.774-16.87 and 1.143-9.787, respectively. (D-H) Effect of AADH in a mouse model of LPS-induced sepsis. C57Bl/6 mice were treated with LPS 20 mg/kg i.p. and 570 µg AADH after 1 hour of the LPS challenge. (D) Survival in mice receiving LPS and vehicle (solid line) and mice receiving LPS plus AADH (dashed line) (n = 9/group). The difference between groups is significant, with P = .0375 and hazard ratio = 5.4328 and 95% CI of ratio 1.088-17.22. (E-H) Mice were euthanized 8 hours after LPS challenge. (E) Representative histological hematoxylin and eosin-stained sections of lungs from mice receiving saline (e1), LPS + vehicle control (e2), or LPS + AADH (e3). (F) Quantification of histological analysis, n = 4 per bar. (G) Quantification of neutrophils in the bronchoalveolar lavage (BAL) (white bars, left y-axis) of adherent, intravascular (black bars, right y-axis), and interstitial (gray bars, right y-axis) neutrophils. Discrimination between neutrophils in either location was made based on an antibody to Ly6G injected 5 minutes prior to euthanasia (n = 6-8 for each bar). (H) Assessment of lung plasma leakage based on the exudation of the plasma tracer FITC dextran (n = 6-8). Statistical significance was tested using one-way analysis of variance with Dunnett post hoc test. *Indicates significant difference (P < .05) compared with the LPS-challenged group.

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