2German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany
††S.A.G.'s current address is F. Hoffmann-La Roche Ltd, pRED, Pharma Research & Early Development, DTA CNS, Basel, Switzerland
Osmotic minipumps represent a convenient and established method for targeted delivery of agents into the brain of small rodents. Agents unable to cross the blood brain barrier can be directly infused into the brain parenchyma or lateral ventricle through implanted cannulas. The small volume of the minipump reservoir typically limits the infusion time to 4–6 weeks. Pump changes with reattachment of a new pump reservoir to the cannula might lead to brain tissue irritation or increased intracranial pressure associated with hydrocephalus. Here, we describe a pump reservoir exchange technique using a Y-shaped connection piece (Y-con) between the infusion cannula and the pump reservoir. This allows repeated replacement of a subcutaneously installed pump reservoir for brain delivery of agents in mice. Experimental evaluation of Y-con pump replacement revealed no signs of tissue irritation or hydrocephalus and allowed extended controlled delivery of infusion agents in the brain.
The invention of Alzet osmotic pumps in the 1970s allowed constant, reproducible, and long-term delivery of drugs, peptides, and antibodies to small laboratory rodents (1, 2). Subcutaneously implanted osmotic pumps reduce animal stress and diminish the risk of infections, compared with repeated acute injections. Subsequent advances in the development of targeted delivery using osmotic minipumps provided broader treatment implementations (3) and brain infusion devices that circumvent the blood brain barrier (4-6). In so-called Brain Infusion Kits, a subcutaneously placed osmotic minipump is connected with a plastic tube to an implanted stainless-steel cannula that delivers the agent to the brain parenchyma or the ventricle.
Osmotic minipumps represent an established technique for targeted delivery of agents into the brain of small animals. Thus, agents unable to cross the blood brain barrier can be directly infused in the brain parenchyma or into the ventricles through implanted cannulas. We describe a pump reservoir exchange technique allowing repeated replacements of a subcutaneously installed pump reservoir for brain delivery of agents in mice.
While the surgical replacement of subcutaneously placed osmotic pumps has been described (7), no established technique is available to exchange pump reservoirs connected to an intracerebral implanted cannula. This limitation shortens the duration of intracerebral infusions, especially in laboratory mice, which can only carry relatively small subcutaneous pump reservoirs due to their small body size. An extended infusion period would be of great advantage for various intracerebral applications in laboratory mice.
Direct replacement of the pump reservoir displaces the liquid volume in the tubing during reservoir reattachment and could result in increased agent release and tissue pressure at the injection site. Intracerebroventricular (i.c.v.) infusions have been reported to cause increased intracerebral pressure and associated brain deformation (8). To circumvent these putative adverse effects, we have developed a novel Y-shaped connection piece (Y-con) between the minipump and the cannula. The Y-con features a pressure release exit channel (Figure 1A) that allows excessive liquid to leave the device during a pump change, preventing increased tissue pressure.
To make the Y-con, a circular brass bar with a diameter of 2.5 mm was used (Hardware store HORNBACH-Baumarkt-AG, Tübingen, Germany). A longitudinal hole and a second connecting hole at a 45° angle from the first were drilled with a diameter of 0.78 mm at a distance of 0.2 mm from the end of the brass bar (Drill beads: Zecha GmbH, Konigsbach-Stein, Germany; Drill: Bergeon Swiss Tools, Le Locle, Switzerland). Inching of a 1.2 mm thick cross-sectional slice of the brass bar (0.2 mm distance from both sides of the holes) was performed with a turning machine at a precision of 3 µm (Bergeon Swiss Tools, Le Locle, Switzerland). An outer channel diameter of 0.8 mm was chosen to fit the tubing provided in the Alzet Brain Infusion Kits (Durect Corperation, Cupertino, CA). The channel, which is slightly larger in diameter than the hole, is applied to the hole with a certain amount of force to fix it to the relatively soft brass. The metal channels from the Brain infusion Kits were attached to the brass slice (about 0.5 mm deep), then fixed mechanically and with glue (UHU GmbH & Co. KG, Buehl/ Baden, Germany). This allows a constant flow rate and facilitates firm attachment to the tubing (Figure 1B). The 2 opposing 4 mm long channels connect to the tubing that reaches the pump reservoir and the brain infusion cannula. The shorter 1.4 mm long channel provides pressure release by enabling liquid displacement during pump reservoir replacement. The reduced length in combination with the 45° angle of this channel allows stable closure of the pressure release exit using dental cement (Figure 1C). This closure is achieved by subsequent sealing of the pressure release exit and filling of the space between the two channels by applying additional dental cement, ensuring free animal movement after the first pump reservoir replacement. Potential tissue irritation is minimized by the lack of sharp edges or notches and by installing the Y-con channels parallel to the skin during surgery. To avoid metal dissociation, oxidation, and copper-dependent Fenton reactions, the Y-con received a >5 µm thick gold coating (Puramet 202 finegold bath, Doduco GmbH, Pforzheim, Germany) (9, 10).
To evaluate the Y-con based osmotic pump change technique for brain delivery, we used male hemizygous CD11b- HSVTK (TK) mice (11) expressing a herpes simplex virus thymidine kinase under the monocytic CD11b promoter. Mice were kept under pathogen-free conditions and experiments were approved by the local authorities. Intracerebroventricular application of the thymidine-analog Ganciclovir (GCV) via Alzet osmotic minipumps to these mice (see also Supplementary Material), resulted in an almost complete microglia ablation in the neocortex within a two week timeframe, while PBS control application did not affect TK mice (12). Using this treatment paradigm, we showed that application of PBS into TK mice via minipumps for two weeks and subsequent Y-con dependent reservoir change to GCV resulted in microglia depletion highly comparable to the direct GCV application for two weeks via minipumps, without any deleterious side effects.
In the first group of mice, PBS was infused i.c.v. via minipumps for two weeks and the animals were subsequently sacrificed (PBS group). In a second group, GCV was infused i.c.v. via minipumps for two weeks and sacrificed (GCV group). A third group of mice received an i.c.v. PBS infusion for two weeks, followed by pump reservoir replacement and GCV infusion for another two weeks (PBS + GCV group) (Figure 2). Fourteen days after implantation of the osmotic minipump, a small incision was made in the skin to access the subcutaneous pump reservoir. The tubing was cut close to the pump reservoir, which was subsequently removed, and the new reservoir with an attached Y-con was placed in the subcutaneous pocket. The opposing channel of the Y-con was inserted into the tube connected to the CNS. After equilibration of liquid pressure, the pressure release exit was sealed with dental cement. (Further pump reservoir replacements can be achieved by removing the dental cement at the pressure release exit and resealing after pump exchange.) Finally, the cut was sutured, positioning the pressure release exit parallel to the skin to avoid tissue irritation.