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Abstract

Observing Leukocyte Trafficking in the Presence of Induced Inflammation Using the Pallid Bat Wing Model

Josie Flowers1 and Dr. Christopher M. Quick 2

Veterinary Physiology and Pharmacology1, Texas A&M University, College Station, TX, USA

Veterinary Physiology and Pharmacology2, College Station, Texas A&M University, TX, USA

Abstract

Objective -  We hypothesize that the number of leukocytes will increase in the area where the Incomplete Freund’s adjuvant was administered, and that leukocyte trafficking and movement will become visible after induction of a local inflammatory response in the region. 

Animals or Sample Population -  Pallid Bats were used in these experiments to observe in vivo effects of an induced inflammatory response on leukocyte trafficking in the bat wing.  Five bats were administered the Incomplete Freund’s Adjuvant and then monitored for any changes in leukocyte activity.  The experiment was performed once per bat.

Procedure -  To test our hypothesis, a 27½ Gauge hypodermic needle is flushed with Incomplete Freund’s Adjuvant and used to create a slight scratch of a length no more than 1 cm and depth of no more than 50 microns in the wing membrane over a main artery and vein pair (Figure 4).  The scratch was not made to draw blood.  A small amount, approximately 10 ul, of Incomplete Freund’s Adjuvant is then applied to the break created in the wing membrane.  The bat was then placed under the microscope for observation.

Results -   The minor break in the membrane in accordance with the adjuvant induced a visually measurable immune response that was viewed using IVM.  We were able to monitor leukocytes in the venous return (Figure 5) and lymphatic vessel in the area where the adjuvant was applied.  A significant increase in leukocyte activity resulted once the inflammatory process began.  Through trial and error we discovered that more than 5 hours was required in order for the response to occur. 

Conclusions and Clinical Relevance -  The local inflammatory response resulted in a significant increase in leukocytes attracted to the area where the adjuvant was applied to the wing.  Those experiments conducted which allowed less than six hours for the reaction to take place did not show any significant increases in leukocyte activity.  Leukocyte activity occurring in the interstitial space surrounding the area of concern is still being investigated. We intend to monitor leukocyte migration, using IVM, from the vasculature to a distant area adjacent to the vessels by creating a small scratch in the wing membrane and applying adjuvant to this region.  In addition we hope to monitor leukocyte movement into the lymphatic vessel from the interstitial space in order to locate where along the vessel leukocytes enter and to have a more thorough understanding of leukocyte trafficking.  With this knowledge we hope to make possible the ability to monitor in vivo effects of various agents and therapy drugs on leukocyte activity without the influence of anesthesia.     

Impact for Human Medicine – These types of studies prove relevant to the ongoing research involving the pathogenesis of a multitude of diseases and how the immune system is subsequently affected.