Figure 2.
Anti-GPVI treatment ameliorates LPS-induced pulmonary inflammation and edema formation. (A) Experimental scheme of LPS-induced ALI in C57BL/6J WT mice treated with either anti-GPVI antibody JAQ1 or nonimmune immunoglobulin G (IgG; control) intraperitoneal (IP), 5 days before experiments. LPS was administered at a dose of 5 mg/kg bw IN. NaCl (0.9%) was given as vehicle control. Samples were collected after 4 hours. (B) Representative micrographs of cytospin preparations of BALF cells stained with DiffQuick staining kit for differential cell count. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DMC4500-0265792920 camera (Leica Microsystems). Scale bar, 50 μm. (C) Quantification of total cell count and neutrophilic granulocytes in the BALF using a Neubauer improved hemocytometer and differential cell staining of cytospin preparations. (D) Spectrophotometric determination of MPO activity in the BALF cell pellet. (E) Quantification of the indicated cytokines (pg/mL) and (F) total protein content (μg/mL) in the cell-free BALF via DuoSet enzyme-linked immunosorbent assay kits (R&D Systems) and Pierce BCA Protein Assay kit (Thermo Scientific), respectively. (G) Pearson correlation between total protein content (μg/mL) in the cell-free BALF and the neutrophil count in the BALF (×104 mL−1) of LPS-treated control (gray) and JAQ1-administered mice (red). (H) Representative macroscopic images of perfused whole lungs of mice with indicated treatment after IV injection of 20 mg/kg bw Evans blue dye (EBD), 30 minutes before tissue sampling. Blue color shows extravascular EBD. Quantification of formamide-extracted EBD was performed via spectrophotometry against a standard curve and depicted as μg EBD per g lung tissue. (I) Quantification of relative Hb content in the supernatant after erythrocyte lysis of the BALF cell pellet at 405 nm via spectrophotometry. (J) Determination of the change in body temperature 4 hours after administration of LPS or NaCl. (K) Blood lactate levels were determined via a blood gas analyzer, using blood taken from the left ventricle. Each data point represents 1 mouse. Bars represent mean ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Anti-GPVI treatment ameliorates LPS-induced pulmonary inflammation and edema formation. (A) Experimental scheme of LPS-induced ALI in C57BL/6J WT mice treated with either anti-GPVI antibody JAQ1 or nonimmune immunoglobulin G (IgG; control) intraperitoneal (IP), 5 days before experiments. LPS was administered at a dose of 5 mg/kg bw IN. NaCl (0.9%) was given as vehicle control. Samples were collected after 4 hours. (B) Representative micrographs of cytospin preparations of BALF cells stained with DiffQuick staining kit for differential cell count. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DMC4500-0265792920 camera (Leica Microsystems). Scale bar, 50 μm. (C) Quantification of total cell count and neutrophilic granulocytes in the BALF using a Neubauer improved hemocytometer and differential cell staining of cytospin preparations. (D) Spectrophotometric determination of MPO activity in the BALF cell pellet. (E) Quantification of the indicated cytokines (pg/mL) and (F) total protein content (μg/mL) in the cell-free BALF via DuoSet enzyme-linked immunosorbent assay kits (R&D Systems) and Pierce BCA Protein Assay kit (Thermo Scientific), respectively. (G) Pearson correlation between total protein content (μg/mL) in the cell-free BALF and the neutrophil count in the BALF (×104 mL−1) of LPS-treated control (gray) and JAQ1-administered mice (red). (H) Representative macroscopic images of perfused whole lungs of mice with indicated treatment after IV injection of 20 mg/kg bw Evans blue dye (EBD), 30 minutes before tissue sampling. Blue color shows extravascular EBD. Quantification of formamide-extracted EBD was performed via spectrophotometry against a standard curve and depicted as μg EBD per g lung tissue. (I) Quantification of relative Hb content in the supernatant after erythrocyte lysis of the BALF cell pellet at 405 nm via spectrophotometry. (J) Determination of the change in body temperature 4 hours after administration of LPS or NaCl. (K) Blood lactate levels were determined via a blood gas analyzer, using blood taken from the left ventricle. Each data point represents 1 mouse. Bars represent mean ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal