Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1995

Abstract

In an effort to elucidate the mechanism by which indomethacin (IN) attenuates the stimulatory effect of estradiol (E$\sb2$) on rabbit splenic red pulp macrophages (RPM), thirty-nine female New Zealand White rabbits were divided into 10 groups: ovariectomized (OVX), OVX/IN at 0.1 and 5.0 mg/kg body weight (bw)/day; sham OVX (SOVX), SOVX/IN at 0.1 and 5.0 mg/kg bw/day; OVX/25 mg E2, OVX/25 mg E$\sb2$/IN at 0.1 and 5.0 mg/kg bw/day; intact Control. Quantitative changes in RPM population in response to the treatments were measured using a 0 to 4 histologic grading scale. Estradiol treatment resulted in increased RPM grade when compared to the OVX non-E$\sb2$ groups. Indomethacin addition decreased mean RPM grade in the SOVX/IN 5.0 group when compared to its E$\sb2$ control group. Indomethacin administration had no significant effect on levels of PGE$\sb2$ in the spleen, blood or urine (p $>$.05). Hematocrits were reduced in both OVX and OVX/E$\sb2$ groups and this decrease was exacerbated by the high IN dose. The results from this study suggest that the effect of IN on E$\sb2$-induced RPM activation may be mediated through a non-prostaglandin pathway. The observed hematocrit changes are possibly the result of direct action of IN and E$\sb2$ on erythrocytes. To further investigate whether a direct interaction of IN and E$\sb2$ with rabbit erythrocytes may be responsible for the decreases in hematocrit observed in vivo, an in vitro study was conducted to determine the effect of these drugs on erythrocyte fragility characteristics. Two ml aliquots of treated New Zealand White rabbit whole blood were assayed as; Control, IN (9.6 $\mu$g/ml), E$\sb2$ (500 pg/ml) and IN plus E$\sb2$, for changes in erythrocyte fragility. Osmotic (OF) and mechanical (MF) fragility were evaluated under approximate physiological conditions by measurement of hemoglobin release at 545 nm. Blood samples at 39.5$\sp\circ$C were assayed immediately after drug addition (initial) and again 4 hours after incubation (final). Eight replicates of each experiment were run. Results of the OF assays showed a significant increase (p $<$.05) in mean 50% hemolysis point between IN (final) and IN plus E$\sb2$ (final) when compared to their mean initial values and to the mean final Control value. The OF hemolysis dispersion was increased by IN and IN plus E$\sb2$ treatment when final values were compared to initial values. The mean final values for MF increased with IN, E$\sb2$ and IN plus E$\sb2$ treatment versus the mean final Control value (p $<$.05). While the increase in MF from IN was greater than that from E$\sb2$, the MF from the combination (IN plus E$\sb2$) was not greater than from IN alone (p $>$.05). The IN-induced increases in both OF and MF indicate a difference in degrees of interaction with the erythrocyte from that of E$\sb2$, which only affected MF and whose effect was not additive or synergistic with that of IN. The in vitro experimental results demonstrate that the increased fragility produced by IN and E$\sb2$ on rabbit erythrocytes may account for the observed in vivo reduction in hematocrit. Increased erythrocyte fragility would also lead to their accelerated clearance from the circulation by splenic RPM and subsequent increases in activity of these macrophages. This elevation in splenic RPM population may also be enhanced by direct E$\sb2$ stimulation of macrophage proliferation.

Document Type

Dissertation - unrestricted

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