Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Summary

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.

Abstract

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.

Introduction

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 toxicity^1,2. Some of the clinically used antibodies display both of these features. A solution to this prob....

Protocol

All methods described here have been approved by the Swiss Cantonal Veterinary Office under license number ZH246/15.

1. Preparation of the Step Catheters

1. Preparation of a fused silica tubing for the step of the catheter
   1. Cut the fused silica capillary with inner diameter of 0.1 mm and wall thickness of 0.0325 mm tubing to a length of 30 mm.
   2. Examine the tubing for cracks and heat polish the ends using a microforge to ensure the tubing openings have a smooth surface.
2. Fixation of the inner tube in a metal needle
   1. Mount a 27 G needle on a 10 &....

Results

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

Discussion

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 demanding^24,25. Recent developme.......

Materials

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