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 microscopy of the microcirculation of the rat knee joint capsule in acute inflammation with the help of confocal laser scanning microscopy (CLSM).


METHODS
The microvascular architecture of the joint capsule was investigated by the use of corrosion casting techniques. Knee joint capsule microcirculation of anesthetized rats could be visualized by dissection of the ligamentum infrapatellare rectum and application of a CLSM. Rhodamine 6G was used as a leukocyte marker. An 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 microns of vessel length. Leukocyte-rolling velocity (Vwbc), systemic leukocyte count (WBC), and differential blood count (DBC) were monitored in defined intervals. Finally, myeloperoxidase (MPO) activity in frozen-tissue homogenate served as a parameter of neutrophil content.


RESULTS
Leukocyte adhesion in carrageenan-treated animals was 9.9 +/- 0.2 leukocytes/100 microns vessel (mean +/- SD; n = 6), while sham- treated animals 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 micron s/s in control animals and 0.53 micron s/s in anti-alpha 4-treated animals (p < 0.01). Changes in DBC were marked by lymphopenia and granulocytosis after 180 min. At this time point-control animals had 5.1 G/L LC and 2.6 G/I PMN. Animals treated with unspecific antibody had 4.7 G/L LC and 4.9 G/L PMN, while anti-alpha 4-treated animals had 10.6 G/L LC and 2.4 G/L PMN. Photometrically determined extinction of oxidized tetramethylbenzidine (TMB; measure 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 treated 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 can serve as a model of integrin-mediated LA in vivo. Antiadhesive treatment of animals can reduce the tissue infiltration with inflammatory cells.
Finkenauer
V
V
Department of Rheumatology/Microcirculation, Hoechst Marion Roussel, Frankfurt, Germany.
Bissinger
T
T
Funk
R H
RH
Karbowski
A
A
Seiffge
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology / Q000502:physiology; D001168:Arthritis / Q000139:chemically induced / Q000473:pathology* / Q000503:physiopathology; D001783:Blood Flow Velocity; D002351:Carrageenan / Q000633:toxicity; D002448:Cell Adhesion; D016319:Corrosion Casting; D004195:Disease Models, Animal; D039441:Integrin alpha4; D007719:Knee Joint / Q000098:blood supply* / Q000473:pathology / Q000503:physiopathology; D007962:Leukocytes / Q000473:pathology*; D008297:Male; D051379:Mice; D008833:Microcirculation / Q000473:pathology; D018613:Microscopy, Confocal / Q000379:methods*; D009195:Peroxidase / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D039441:Integrin alpha4; D002351:Carrageenan; D009195:Peroxidase