Confocal laser scanning microscopy of leukocyte adhesion in the microcirculation of the inflamed rat knee joint capsule

Objective: The aim of the study was to develop a model of intravital micros copy of the microcirculation of the rat knee joint capsule in acute inflamm ation with the help of confocal laser scanning microscopy (CLSM). Methods: The microvascular architecture of the joint capsule was investigat ed by the use of corrosion casting techniques. Knee joint capsule microcirc ulation of anesthetized rats could be visualized by dissection of the ligam entum infrapatellare rectum and application of a CLSM. Rhodamine 6C was use d as a leukocyte marker. All acute arthritis was induced by intraarticular injection of 0.1 mL of 1% Carrageenan solution. Each experiment lasted for 3 h. Leukocyte adhesion (LA) was measured in 100 mu m of vessel length. Leu kocyte-rolling velocity (Vwbc), systemic leukocyte count (WBC), and differe ntial blood count (DBC) were monitored in defined intervals. Finally, myelo peroxidase (MPO) activity in frozen-tissue homogenate served as a parameter of neutrophil content. Results: Leukocyte adhesion in carragenan-treated animals was 9.9 +/- 0.2 l eukocytes / 100 mu m vessel (mean +/- SD: n = 6). while sham- treated anima ls had 2.8 +/- 0.2. Monoclonal antibodies (MAb) to alpha 4 integrin (0.2 mg /kg) reduced LA to 2.6 +/- 0.1 (p < 0.01). Vwbc/gamma quotients were 0.37 m u ms/s in control animals and 0.53 mu ms/s in anti-alpha 4-treated animals (p < 0.01). Changes in DBC were marked by lymphopenia and granulocytosis af ter 180 min. At this time point-control animals had 5.1 G/L LC; and 2.6 G/l PMN. Animals treated with unspecific antibody had 4.7 G/L LC and 4.9 G/L P MN, while anti-alpha 4-treated animals had 10.6 G/L LC and 2.4 G/L PMN. Pho tometrically determined extinction of oxidized tetramethylbenzidine (TMB: m easure for MPO content) was 1.058 +/- 0.555 in control animals and 0.245 +/ - 0.093 in sham-treated animals. Tissue homogenate from unspecific IgG trea ted group had 0.776 +/- 0.140 and from anti-alpha 4-group 0.334 +/- 0.155 ( p < 0.05). Conclusions: Confocal laser scanning microscopy is a tool for the study of the microcirculation in nontransparent organs. The microcirculation of the acutely inflamed rat knee joint capsule carl serve as a model of integrin-m ediated LA in vivo. Antiadhesive treatment of animals can reduce the tissue infiltration with inflammatory cells.