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Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of
G A Brito1, J L Falcão, S N Saraiva
1Departamento de Fisiologia e Farmacologia, Unidade de Pesquisa Clínica, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE, Brasil. gerly@fortalnet.com.br
Abstract:
In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 +/- 0.19 and Dex = 0.35 +/- 0.13 vs saline (S) = 2.85 +/- 0.59; fMLP: Ptx = 0.43 +/- 0.09 and Dex 0.01 +/- 0.01 vs S = 1.08 +/- 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 +/- 1.4 and Dex = 3.06 +/- 0.76 vs S = 15.94 +/- 3.97; fMLP: Ptx = 3.85 +/- 0.56 and Dex = 0.40 +/- 0.16 vs S = 7.15 +/- 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 microns), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 +/- 0.38 vs S = 4.20 +/- 1.01; fMLP: Dex = 0.25 +/- 0.11 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 +/- 2.25 vs S = 4.20 +/- 1.01; fMLP: Ptx = 4.66 +/- 1.24 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.
Insights
Dexamethasone (Dex) and pertussis toxin (Ptx) reduce neutrophil migration in rats. Dex inhibits neutrophil arrival, while Ptx prevents their movement into inflamed tissue, revealing distinct anti-inflammatory mechanisms.
Area of Science:
- Immunology
- Pharmacology
- Histopathology
Background:
- Leukocyte migration is crucial in inflammatory responses.
- Understanding the mechanisms of anti-inflammatory agents is vital for developing new therapies.
Purpose of the Study:
- To quantify the effects of dexamethasone (Dex) and pertussis toxin (Ptx) on leukocyte migration in rat mesentery.
- To elucidate the distinct mechanisms by which Dex and Ptx exert their anti-inflammatory actions.
Main Methods:
- Histopathological analysis of rat mesentery.
- Intravenous administration of Dex or Ptx prior to inflammatory stimuli (LPS or fMLP).
- Quantification of neutrophil diapedesis and infiltration.
Main Results:
- Both Dex and Ptx significantly reduced neutrophil diapedesis and infiltration.
- Dex decreased neutrophils within venules, suggesting pre-arrival inhibition.
- Ptx increased neutrophils within venules, indicating inhibition of transmigration from the lumen.
- Both agents acted preferentially in the early stages of neutrophil transmigration, not in response to haptotactic gradients.
Conclusions:
- The described histopathological method is effective for quantifying inflammatory and anti-inflammatory effects.
- Dex and Ptx exhibit different mechanisms of action in modulating leukocyte migration.
- Ptx appears to inhibit neutrophil locomotion from the vascular lumen, while Dex may act at an earlier stage.