A suspension of clodronate-liposomes prepared according to Protocol 1 contains about 6 mg clodronate per 1 ml suspension. Splenic macrophages can be depleted by intravenous administration of about 0.1 ml of a suspension of clodronate-liposomes (prepared according to Pro toco/ 1) per 10 g body weight. Macrophages in the liver (Kupffcr cclls) arc more susceptible and can be completely depleted by intravenous administration of 0.02 ml of the suspension per 10 g body weight. See the relevant literature for detailed information on the doses of clodronate-liposomes required to deplete different macrophage (sub)populations in various organs (for references see next paragraph).
Liposomes are not able to cross vascular barriers such as capillary walls. For that reason it is important to choose an administration route allowing an unhindered access of liposomes to macrophages targeted for depletion. Four administration routes for clodronate-liposomes that are frequently used are shown in Figure 2. Given the open blood circulation system in spleen, liver, and bone marrow, intravenous administration of clodronate-liposomes allows the depletion of macrophages in these organs (Figure 2a) (16). Since the peritoneal cavity of mice and rats is drained by parathymic lymph nodes from where the lymph is ultimately carried into the circulation, intraperitoneal administration of clodronate-liposomes may be used for depletion of macrophages in peritoneal cavity, parathymic lymph nodes, liver, and spleen (Figure 2b) (17). Intratracheal instillation of the liposomes can be used for depletion of the alveolar macrophages in the lung (Figure 2c) (18). For obvious reasons, the interstitial macrophages in the lung can neither be depleted by intratracheal instillation nor by intravenous administration. Subcutaneous injection in the draining area of lymph nodes induces macrophage depletion in these lymph nodes. Macrophage depletion may also be induced in the next lymph node stations on the efferent path. However subcutaneously administered liposomes are usually given in a low dose, so that splenic and liver macrophages are not affected by the few liposomes that reach the circulation (Figure 2d) (19). In some organs, such as the testis, where collagen fibres are only loosely packed (20), or in knee joints where phagocytic synovial lining cells can be found (21), depletion can be achieved by direct injection in the organ.
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