van der Hoek KH, Maddocks S, Woodhouse CM, van Rooijen N, Robertson SA, Norman RJ.
Intrabursal Injection of Clodronate Liposomes Causes Macrophage Depletion and Inhibits Ovulation in the Mouse Ovary.
Biology of Reproduction. 2000 Apr 1;62(4):1059 –1066.
[toggle_content title=”Abstract”]To investigate the role of the ovarian macrophage population in ovulation, we examined the effect of depleting this population using liposome-encapsulated clodronate. Clodronate liposomes, saline liposomes, or saline alone was injected under the ovarian bursa in gonadotropin-primed adult mice, either 84 h (Day −3) or 36 h (Day −1) before ovulation. Ovulation rates were determined by counting the number of oocytes released. The numbers of graafian follicles and corpora lutea were also counted immediately before and after ovulation. Macrophage distribution within the theca and stroma of preovulatory ovaries was examined by immunohistochemistry with specific monoclonal antibodies to the macrophage antigens macrosialin, major histocompatability complex class II (Ia), and F4/80. Injection of clodronate liposomes on Day −1 did not affect ovulation rates, whereas administration on Day −3 caused a significant reduction in ovulation rate (mean oocytes ovulated = 5.25 ± 0.6 from clodronate liposome-treated ovaries and 9.13 ± 0.9 from saline-treated ovaries, respectively, P < 0.05). The numbers of macrosialin-positive macrophages present in the theca at ovulation were reduced by treatment with clodronate liposomes on Day −1, and treatment on Day −3 reduced the numbers of Ia-positive and macrosialin-positive macrophages present in the theca. When the subsequent ovarian cycles were examined by vaginal smearing, the metestrous-2/diestrous stage was found to be extended in clodronate liposome-treated animals (7.5 ± 1.3 days vs. 3.4 ± 0.4 days for saline liposome-treated animals, P < 0.05). These results suggest that thecal macrophages may be involved in the regulation of follicular growth and rupture, as well as being important for the normal progression of the estrous cycle.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes Control Free Clodronate
5 mg/ml 23.5 mg/ml EPC/Chol 84/16 MLV Saline ND
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/61 mice, female, 8-11 w 10 µl intra-bursal/ovary F4/80+FA/11+MHC II (Ia)+ macrophages no N/A

1Paper states that C57B16 mice were used; we assume that this is an error.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Liposome prep method cited—
    van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods. 1994 Sep 14;174(1–2):83–93.
[/toggle_content] [toggle_content title=”Results”]
  1. Intrabursally-injected fluorescent liposomes were confirmed to enter the theca and stroma of the ovary, but not the interior sections of the ovary. Unfortunately, the cellular localization of these liposomes was not determined, however the fluorescence signal decrease to baseline by 6 h. Therefore, we assume that the liposomes were phagocytosed and metabolized, but the cellular distribution of the liposomes would verify that all macrophages were accessible by liposomes.
  2. Macrophage depletion was only about 30% at best and varied dramatically depending on macrophage phenotype and tissue location. If a cellular distribution of fluorescent liposomes had been determined, the reason behind the limited depletion may have been clear.
  3. Clodronate liposomes, but not control liposomes, reduced the ovulation rate by 50-60% when the clodronate liposomes were dosed 3 days, but not 1 day, prior to induction of ovulation.
  4. The authors cite the possibility that rapid repopulation of ovarian macrophages may be a reason for incomplete depletion of the population. If this were true, additional systemic dosing should deplete circulating monocytes from which the ovarian macrophages mature, thus limiting the influx of newly recruited monocytes/macrophages.