Table 1.
Sequence of primer pairs used in semi-quantitative real-time PCR.
Table 2.
Blood glucose levels (mg/dL) in diabetic* and non-diabetic rats and effect of sepsis induced by CLP**.
Figure 1.
Percentage of diabetic and non-diabetic rats submitted to CLP-induced sepsis that died in a given time. Diabetes was induced by i.v. injection of alloxan (42 mg/kg/iv) in Wistar rats and 10 days later CLP was performed (12 punctures with a 20 G needle). n = 5/group, repeated three times with identical results. Data are presented as mean ± SEM. **P<0.01 diabetic vs. non-diabetic.
Figure 2.
Diabetic and non-diabetic rats were submitted to CLP and after 6 h the lungs were removed and processed. (A) Photomicrographs of peribronchovascular axis in lung stained with haematoxylin-eosin; ‘B’ stands for bronchiole and ‘V’ for venule. Note the presence of oedema around the venule (leakage area marked in black bars). Photographs were taken at an original magnification of 200x. (B) Quantification of perivascular oedema by light microscopy with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length).The number of points falling on areas of perivascular oedema and the number of intercepts between the lines of the integrating eyepiece and the basal membrane of the vessels were counted. The oedema index was calculated as follows: number of points1/2/number of intercepts. Ten random non-coincident microscopic fields containing a bronchus and a venule were evaluated for each group, n = 5 per group. (C) Evaluation of lung oedema by total protein content in the BAL after 6 hours of CLP or sham-operated, n = 5/group and scale bar = 50 µm. Data are presented as mean ± SEM. ***P<0.001.
Figure 3.
Inflammatory cell infiltration in lung at 6h after CLP.
Diabetic and non-diabetic rats were submitted to CLP and after 6h the lungs were removed and processed. (A) Photomicrographs of lung parenchyma stained with haematoxylin-eosin. (B) Mononuclear and polymorphonuclear cell index was determined in the parenchyma. The cell index quantification was performed with an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines (known length); cells were evaluated at x1,000 magnification. Points falling on mononuclear or PMN cells were counted and divided by the total number of points falling on tissue areas in each microscopic field. Ten random non-coincident microscopic fields were evaluated for each group, n = 5/group and scale bar = 50 µm. Data are presented as mean ± SEM. * P<0.05; ***P<0.001.
Figure 4.
Inflammatory cell infiltration in BAL fluid and lung COX2 expression at 6h after CLP.
Diabetic and non-diabetic rats were submitted to CLP or SHAM (false surgery) and after 6h bronchoalveolar lavage (BALF) was performed. (A) mononuclear and PMN cell were counted in haematoxilin-eosin stained cytospin preparations of BALF cells after total cell count was performed under light microscopy. (B) Expression of COX2 protein in lung homogenates six hours after CLP analysed by immunoblotting using antibodies to COX-2 and quantified by densitometric analysis of the immunoblot bands. Density values of bands were normalized to the total β-actin present in each lane and expressed as a percentage of control. n = 5/group, data are presented as mean ± SEM; *, P<0.05.
Figure 5.
NFkB activation in alveolar macrophages 6h after CLP.
Alveolar macrophages (AM) were obtained by lung lavage six hours after CLP and allowed to adhere in culture plates for 1 h. Total mRNA or total protein was extracted from AMs. (A) 20 µg of total protein analysed by immunoblotting using antibodies to phosphorylated – IκBα and β-actin.The bands were quantified by densitometric analysis. Density values of bands were normalized to the total β-actin present in each lane and expressed as percentage of control. (B) 50 µg of total protein analysed by immunoblotting using antibodies to phosphorylated – p65 and β-actin. (C) cDNA was synthesized from total mRNA extracted and the expression of SOCS-1. (D) MyD88 mRNA was analysed by RT-PCR. mRNA expression levels were calculated by the comparative Ct method and normalized to GAPDH levels with non-diabetic CLP given an arbitrary value of one. n = 5/group, data were presented as mean ± SEM.* P<0.05; ***, P<0.001.