Claes Lundberg

Polymorphonuclear leukocyte accumulation in inflammatory dermal sites as measured by51Cr-labeled cells and myeloperoxidase

Two methods for quantitating polymorphonuclear leukocyte (PMN) accumulation in inflammatory skin lesions were studied. The lesions were produced in rats by intradermal injections of different dilutions of zymosan-activated plasma (ZAP). PMN accumulation in the skin lesions was estimated by determination of (1) homologous51Cr-labeled PMNs and (2) activity of myeloperoxidase (MPO) in the tissue sample.125I-labeled human serum albumin was used for mesaurement of albumin extravasation. The [51Cr]PMN content and MPO activity in the skin lesions were both proportional to the concentration of ZAP injected. The correlation coefficient (r) between the two methods of measuring PMN accumulation in the inflammatory skin lesions was calculated to be 0.81±0.13 (mean±SD, N=8), The proportionality of the PMN accumulation to the different dilutions of injected ZAP, as measured both by [51Cr]PMN and by MPO activity, and the correlation of the two methods to each other, suggest that these two methods are reliable for measuring PMN accumulation in vivo. The inflammatory reaction also included albumin extravasation, which reached a relatively high level already at the lowest concentration of injected ZAP and did not seem to parallel PMN accumulation.

Effects of homodimeric isoforms of platelet-derived growth factor (PDGF-AA and PDGF-BB) on wound healing in rat.

Platelet-derived growth factor (PDGF) has been suggested to have a significant role in wound healing. The present work was aimed at studying the effects of PDGF-AA and PDGF-BB homodimers on developing granulation tissue in rats. Subcutaneously implanted hollow cylindrical cellulose sponges were used as an inductive matrix for the ingrowth of granulation tissue. Fifty microliters of solutions containing 0, 5, 50, or 500 ng of PDGF-AA or PDGF-BB homodimers was injected daily into the sponges; 7 days after implantation the granulation tissue in the sponge cylinders was analyzed. Five hundred nanograms of PDGF-BB stimulated significantly the accumulation of collagen, indicated by the elevated hydroxyproline content of the sponge (+34%, P < 0.001). Similarly, the amounts of RNA-ribose, nitrogen, hexosamines, and uronic acids were significantly higher, reflecting a PDGF-BB-induced increase in the accumulation of RNA, protein, and glycosaminoglycans. Analyses of wound fluid showed no essential changes in the composition of different cell types after PDGF-BB-treatment. The PDGF-AA-treatment increased significantly the mean amount of RNA but there were no significant changes in other parameters. In vitro both PDGF-AA and PDGF-BB increased significantly the number of rat granulation tissue derived fibroblasts in culture at concentrations of 10 and 30 ng/ml. This proliferative effect resulted in a lowered level of protein synthesis per cell. To conclude, PDGF-BB accelerates granulation tissue formation both in vitro and in vivo, whereas PDGF-AA is effective in vitro but it is clearly less effective in vivo.

The inflammatory reaction in an experimental model of open wounds in the rat. The effect of arachidonic acid metabolites

The study concerned the effect of arachidonic acid metabolites on the inflammatory reaction in granulation tissue of open wounds in rats. Metabolites or inhibitors were applied in a wound chamber attached to circular, open, full-thickness skin wounds 5 days post-wounding. The adjacent wound served as control. Blood flow, albumin extravasation and accumulation of polymorphonuclear leucocytes (PMNLs) were measured in the granulation tissue. Prostaglandin E2 (PGE2 5.7 microM) increased blood flow and albumin extravasation by 95 and 16%, respectively, without affecting PMNLs. Leukotriene B4 (LTB4 2.7 microM) increased PMNL accumulation by 142% without altering albumin extravasation. Indomethacin (28 microM, repeatedly) did not affect blood flow or albumin extravasation, but increased PMNL accumulation by 21%. Methylprednisolone (3.3 mM, repeatedly) reduced blood flow and albumin extravasation by 29 and 31%, respectively, without influencing PMNLs. The granulation tissue obviously responds to exogenous PGE2 and LTB4. Endogenous arachidonic acid metabolites seem to play only a minor role in the inflammatory process in this model.

Quantification of the Inflammatory Reaction and Collagen Accumulation in an Experimental Model of Open Wounds in the Rat: A Methodological Study

An experimental model for studying the early healing of open wounds in the rat is described. With this model, exudate is easily collected, a standardized granulation tissue is achieved at different post-wounding time intervals and local treatment of the wounds is possible. The model involves the use of stainless steel rings with covers, fastened to the edges of two circular full-thickness open wounds, one on each side of the animals back in the thoracic region. Blood flow and water content of the granulation tissue were determined acutely 3, 5, 7 and 10 days after wounding. The collagen accumulation in the granulation tissue was assessed by the amount of hydroxyproline, at the same time intervals. The amount of exudate which accumulated in the chamber was measured daily. Blood flow in the granulation tissue, as measured by radioactive microspheres, reached a maximum of 947 ml X min-1 X (100 g dry weight)-1 7 days after wounding. The water content, as assessed by freeze-drying, also reached its peak on the 7th day (5.5 ml X (g dry weight)-1). Changes in water content were found to be due to changes in interstitial fluid volume, as studied by 59Fe-labelled erythrocytes and 51Cr-EDTA. The amount of hydroxyproline in the granulation tissue increased from 22 micrograms X (mg dry weight)-1 on day 3 to 37 micrograms X (mg dry weight)-1 on day 10. Exudation increased to 28 microliters X h-1 X cm2-1 on day 4 and then declined. The results suggest that the inflammatory reaction, expressed as alterations in blood flow and water content, reaches a maximum on day 7 after wounding. The data also demonstrate a continuous increase in the collagen content of the granulation tissue during the 10-day period of observation. In addition, exudation was found to reach a peak on the 4th to 5th day, and then declined.

Permeability-increasing ability of PAF-acether in rat skin

Platelet-activating factor (PAF-acether), a phospholipid compound with effects on several cells, e.g., platelets and polymorphonuclear leukocytes (PMNs), was examined for its effect on microvascular permeability in rat skin. It was found to increase microvascular permeability, measured as exudation of [125I]human serum albumin, in amounts exceeding 1 pmol, and was more than 1000 times as potent as histamine. The effect was independent of cell infiltration, as no accumulation of PMNs, measured as the amount of myeloperoxidase in the skin, occurred and as the response was unaltered in animals rendered neutropenic due to treatment with an antiserum against PMNs.

Steroid and anti-CD18 treatment have no effect on after-cataract formation following surgery in rabbits.

PURPOSE It is debated whether the inflammatory reaction that follows cataract surgery is important in the development of after-cataract. The aim of the present investigation was to study if reduction of the early inflammatory process had any effect on the development of after-cataract in the rabbit. METHODS Rabbits were divided into 3 groups: 1) no treatment (control), 2) steroids, and 3) IB4 (murine anti-CD18 mab). Aqueous humor (AqH) was aspirated from all rabbits just prior to, and 1 and 56 days after cataract surgery. On day 56 the weight of the after-cataracts was determined. The effect of the AqH on rabbit lens epithelial cell (LEC) proliferation in vitro was investigated. The AqH concentrations of protein, leukocytes, prostaglandin E(2) (PGE(2)), basic fibroblast growth factor (bFGF), and active transforming growth factor-beta (TGF-beta) were determined. RESULTS In AqH collected on day 1 postoperatively from the control group, the concentration of leukocytes, protein and PGE(2) were 152.1/microl, 20.6 mg/ml, and 441.5 pg/0.1 ml, respectively. The corresponding levels in the steroid group were significantly lower (55.8/microl, 12.1 mg/ml, and 140.2 pg/0.1 ml). In the IB4 group, the concentrations of leukocytes and protein were significantly lower, 17.9/microl and 11.1 mg/ml, respectively. However, the proliferative effect of AqH on rabbit LEC, the concentrations of bFGF and active TGF-beta on day 1, and the weight of after-cataract on day 56 were not reduced by the anti-inflammatory treatments. CONCLUSIONS This study indicates that reduction of the early inflammatory response after cataract surgery is not important in the development of after-cataract.

Inflammatory Reaction and Collagen Accumulation in an Experimental Model of Open Wounds in the Rat: A Comparison between Gauze and Debrisan® Treatment

Debrisan treatment was compared with the use of gauze in an experimental model of open wounds in the rat. The amounts of exudate absorbed by the gauze and by Debrisan were measured daily during the post-wounding period and the myeloperoxidase (MPO) activity in the exudate was determined. Blood flow, water content and accumulation of the collagen amino acid hydroxyproline in the granulation tissue were measured on days 3, 5, 7 and 10 post-wounding. The amount of exudate absorbed both by gauze and by Debrisan reached a peak on post-wounding days 4-5 and was greater in Debrisan-treated wounds. MPO activity in the Debrisan-absorbed exudate, on the other hand, was lower throughout the study period than in the exudate absorbed by gauze. Granulation tissue blood flow and water content reached maximum values on day 7 post-wounding, irrespective of treatment. Blood flows were 39% and 40% lower in the Debrisan-treated wounds on post-wound days 3 and 7, respectively, than in the gauze-treated wounds, whereas the water contents of the former wounds were 23% and 15% lower on days 3 and 10, respectively. The hydroxyproline content of the granulation tissue increased continuously from day 3 to day 10 and was similar in the two wounds. These results suggest that Debrisan is more effective than gauze for absorbing wound exudate, when applied on an openly secreting wound. In addition, the inflammatory reaction taking place in Debrisan-treated wounds seems to be less severe than in wounds treated with gauze.

The role of histamine and serotonin in the inflammatory reaction in an experimental model of open wounds in the rat.

The role of histamine and serotonin in the inflammatory reaction in the granulation tissue of open wounds in the rat was studied. The model involved plastic chambers attached to the edges of two open circular full-thickness skin wounds. Five days post-wounding, agonists or antagonists were applied in one of the two chambers, the adjacent wound serving as control. Thereafter blood flow and albumin extravasation were measured. Application of histamine (100 microM) caused an increase in granulation tissue blood flow by 36%, but left albumin extravasation unaffected. Treatment with mepyramine (H1 antagonist, 20 microM), cimetidine (H2 antagonist, 20 microM) or methysergide (serotonin antagonist, 20 microM) did not influence the level of either blood flow or albumin extravasation. It is suggested that endogeneous histamine and serotonin play a minor role in the inflammatory process in the granulation tissue of this model of healing wounds.

REGIONAL DIFFERENCES IN DERMAL INFLAMMATORY REACTIONS

Regional differences in dermal inflammatory reactions in the dorsum of rat trunk were studied in three commonly used inflammatory models, i.e., reverse passive Arthus reaction (RPAR), passive cutaneous anaphylaxis (PCA), and histamine-induced inflammatory (HI) reaction. The RPAR showed an increasing severity from cranial to caudal regions, as measured by water content in the skin lesions. The PCA reaction, as measured by Evans blue leakage was not influenced by regional differences. The HI reaction, as measured by water content and leakage of radioactively labeled human serum albumin ([125I]HSA), was significantly smaller in the central regions of the dorsum than in the most cranial and sacral regions. However, no regional differences were observed when the reaction was evaluated by protein-bound Evans blue leakage. A comparison of the three different methods to determine the HI reaction showed a correlation (r=0.70) between measurements of water content and [125I]HSA leakage. There was less correlation of these two methods with measurements of Evans blue skin lesion diameter (r=0.31 and 0.56, respectively). In conclusion, regional differences in inflammatory responses, and methodological differences to measure them, may influence the results of commonly used tests like RPAR, PCA and HI reactions. Such differences should be considered when quantitating dermal inflammatory reactions.