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Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.
Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 µg of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.
The blood brain barrier (BBB) forms a semipermeable border separating the central nervous system (CNS) from the blood circulation. Reaching the CNS with therapeutics is however necessary in context of various diseases, like brain tumors, Alzheimer’s disease (AD) or Parkinson’s disease (PD) among others1. This becomes important in the development of new therapies, especially if the tested drug exhibits poor BBB permeability or its systemic exposure can lead to dangerous toxicity1,2. Some of the clinically used antibodies display both of these features. A solution to this problem would be to deliver the therapeutics directly behind the BBB.
Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes. This is achieved by surgically installing one or more catheters in the target area. During the drug application, a pressure gradient is formed at the opening of the catheter, which becomes the driving force of the infusate dispersion in the tissue3,4. It is thus the duration of infusion and not the diffusion coefficients that determine the perfusion range2,4,5. This provides uniform delivery of the infusate over a much larger brain volume compared to conventional, diffusion based intracerebral injection methods2,6. At the same time, this delivery modality has a lower risk of tissue damage2. Accordingly, CED can enable safe and efficacious administration of conventional chemotherapeutics for treatment of CNS tumors, as well as delivery of immunomodulatory agents or agonistic and antagonistic antibodies in a multitude of other CNS disorders2,7,8,9. CED is currently tested in therapies of Parkinson’s disease, Alzheimer’s disease, as well as high-grade glioma2,7,8,10,11.
Catheter design and the injection regimen are among the most important factors influencing the outcome of CED 10,12,13,14,15,16. Furthermore, it requires specific physicochemical properties of the infusate, including moderate size of the particles, an anionic charge, and low tissue affinity 10,17. Each of these parameters has to be potentially adjusted according to the histological features of the brain region to be targeted2,10,17.
Here we describe methodology for performing CED of an antibody solution into the caudate putamen (striatum) of mice. Furthermore, the protocol includes preparation of step catheters in a laboratory setup, testing them in vitro and performing the CED.
There are multiple catheter designs available in the literature, differing by the shape of the cannula, the materials used and the number of catheter openings12,15,18,19,20,21,22. We are using a step catheter made of a fused silica capillary protruding 1 mm from a blunt end metal needle. This catheter design can be easily manufactured in a research laboratory and reproducibly gives good CED results when tested in vitro with agarose blocks with physical parameters resembling brain parenchyma in vivo23.
Moreover, we implement a ramping regimen for delivering 5 µL of infusate in vivo. In such a protocol the injection rate is increased from 0.2 µL/min to a maximum of 0.8 µL/min, thus minimizing chances of infusate reflux along the catheter as well as risk of tissue damage16. Using this protocol, we have successfully administered mice with up to 20 µg of antibody in 5 µL of PBS over the course of 11 min 30 s.
The protocol can be readily adjusted for other infusion volumes or for injecting various other substances, e.g. chemotherapeutics, cytokines, viral particles or liposomes2,10,14,18,22. In case of using infusate with drastically different physicochemical properties compared to a phosphate buffered saline (PBS) or artificial cerebrospinal fluid (aCSF) solution of antibodies, additional validation steps are recommended. For catheter assembly, validation and CED, we describe all steps using a stereotactic robot with a drill and injection unit mounted onto a regular stereotactic frame. This procedure can also be performed with a manual stereotactic frame connected to programmable microinfusion pump that can drive the described glass microsyringes.
All methods described here have been approved by the Swiss Cantonal Veterinary Office under license number ZH246/15.
1. Preparation of the Step Catheters
2. Convection-enhanced Delivery of Antibody Solution into the Murine Brain
NOTE: Depending on local animal welfare regulations, various types of anesthetics, analgesics and antibiotics can be implemented for this procedure. This protocol describes the use of injection anesthesia. Inhalation anesthetics such as isoflurane can also be used by mounting a nose mask on the stereotactic frame. In addition, we recommend adding antibiotics to the drinking water for infection prophylaxis.
This protocol enables preparation of step catheters (Figure 1) for use in the CED procedure in a laboratory environment. In order to control the catheters for leakage, reflux along the needle tract and clogging, we recommend performing injections of a dye, e.g., trypan blue solution, into an agarose block. Figure 3 depicts a cloud of trypan blue forming after injection of 1 µL at 0.5 µL/minute using a CED catheter (Figure 3A
Convection-enhanced delivery, or pressure-mediated drug infusion into the brain, was first proposed in the early 19903. This approach promises perfusion of large brain volumes behind the blood brain barrier in a controlled manner2. However, so far, only a few clinical trials have been performed using this approach, partially because CED in a clinical setup has shown to be technically demanding24,25. Recent developme...
Johannes vom Berg is mentioned as an inventor on patent application (PCT/EP2012/070088) of the University of Zurich. Michal Beffinger, Linda Schellhammer and Johannes vom Berg are mentioned as inventors on a patent application (EP19166231) of the University of Zurich. The authors have no additional financial interests.
This work was supported by grants of the University of Zurich (FK-15-057), the Novartis Foundation for medical-biological Research (16C231) and Swiss Cancer Research (KFS-3852-02-2016, KFS-4146-02-2017) to Johannes vom Berg and BRIDGE Proof of Concept (20B1-1_177300) to Linda Schellhammer.
Name | Company | Catalog Number | Comments |
10 μL syringe | Hamilton | 7635-01 | |
27 G blunt end needle | Hamilton | 7762-01 | |
Agarose | Promega | V3121 | |
Atipamezol | Janssen | ||
Bone wax | Braun | 1029754 | |
Buprenorphine | Indivior Schweiz AG | ||
Carprofen | Pfizer AG | ||
Dental drill bits, steel, size ISO 009 | Hager & Meisinger | 1RF009 | |
Ethanol 100% | Reuss-Chemie AG | 179-VL03K-/1 | |
Fentanyl | Helvepharm AG | ||
FITC-Dextran, 2000 kDa | Sigma Aldrich | FD2000S | |
Flumazenil | Labatec Pharma AG | ||
Formaldehyde | Sigma Aldrich | F8775-500ML | |
High viscosity cyanoacrylate glue | Migros | ||
Iodine solution | Mundipharma | ||
Medetomidin | Orion Pharma AG | ||
Microforge | Narishige | MF-900 | |
Midazolam | Roche Pharma AG | ||
Ophthalmic ointment | Bausch + Lomb | Vitamin A Blache | |
PBS | ThermoFischer Scientific | 10010023 | |
Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647 | Jackson Immuno | ||
Scalpels | Braun | BB518 | |
Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm | Postnova | Z-FSS-100165 | |
Stereotactic frame for mice | Stoelting | 51615 | |
Stereotactic robot | Neurostar | Drill and Injection Robot | |
Succrose | Sigma Aldrich | S0389-500G | |
Topical tissue adhesive | Zoetis | GLUture | |
Trypan blue | ThermoFischer Scientific | 15250061 | |
Water | Bichsel | 1000004 |
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