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R.R. Pfister, C.I. Sommers; Arginine–Threonine–Arginine (RTR) Tetramer Inhibits the Initial PMN Influx in the Alkali–Injured Eye . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4364.
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© ARVO (1962-2015); The Authors (2016-present)
Polymorphonuclear leukocytes (PMNs) play a central role in corneal ulceration. In the alkali injured cornea, we found an initial transient, yet substantial, infiltration of PMNs, peaking at about 12–24 hr and limited to the peripheral cornea; this subsided by about 72 hr. Chemoattractants released from the alkali–injured cornea, primarily proline–glycine–proline (PGP) identified by this lab previously, are the primary triggers for early neutrophil invasion. These tripeptides, released within the corneal stroma by direct hydrolysis of proteins, are believed to diffuse to the limbus where they are chemotactic to PMNs circulating in the perilimbal vascular arcades. Molecular Recognition Theory was implemented to develop a complementary peptide, arginine–threonine–arginine (RTR). Our previous in vivo studies showed that RTR tetramer, used as drops, resulted in a significantly reduced incidence of corneal ulceration and severity. The focus of this study was to determine, morphometrically, if RTR inhibits the earliest invasion of PMNs, post–injury.
Twenty–four (24) New Zealand Dutch strain albino rabbits of both sexes, weighing 2.0 to 2.5 kg, were used for this study. Rabbits were alkali–injured as described previously. Rabbits were divided into two groups; (1) received RTR Tetramer drops every hour for 8 hr, 24 hr or 48 hr (2) received Phosphate Buffered Saline (PBS) control drops every hour for 8 hr, 24 hr or 48 hr. At the end of 8 hours; 4 rabbits from each group were killed for histology, and at 24 hours and again at 48 hours. The alkali–injured eyes were removed and placed in 10% formalin. The fellow eye was used as a control.
There was no appreciable increase in neutrophils in the alkali injured cornea from 12 to 48 hours in the RTR treated group while the control group increased in number and advanced further into the central cornea. The quantitative assessment of neutrophils support our belief that the RTR tetramer binds the chemoattractant, thus reducing the number of PMNs recruited to the injury site. Although PGP peptides are the primary, initial chemoattractants released from alkali injured cornea, they are by no means the only chemoattractants released.
In vitro and in vivo data show that binding of the PGP molecules by RTR tetramer deprives the cornea of this initial neutrophilic chemoattractant, resulting in fewer invading PMNs. Reduction of corneal PMNs diminishes the release of cytokines involved in recruitment of the second wave of PMNs. It is this second, more powerful wave of PMNs that is responsible for the incidence and severity of corneal ulceration.
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