Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Summary

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Abstract

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

Introduction

The treatment of neurological and psychiatric disease is severely hindered by the presence of the blood-brain barrier (BBB) which prevents over 95% of all potential pharmaceutical agents from reaching the central nervous system^1-3. For example, Glial Derived Neurotrophic Factor (GDNF) has been shown to be effective in treating Parkinson's Disease when injected directly into the brain, however is ineffective when delivered systemically because it cannot penetrate the BBB^4-6.

Numerous approached have been developed to try to circumvent this problem. Improvement in systemic delivery of neurotheraputics has been demons....

Protocol

Prior to surgery make sure all procedures to be done are approved by IACUC and any additional ethical or legal authorities and use humane animal treatment practices. This includes using sterile surgery conditions, anesthetizing the mouse using IACUC approved method, lubricating mice eyes with vet ointment during surgery, and providing postsurgical care. Do not proceed with surgery if there is any question whether aspects of the procedure are approved. All procedures performed herein were approved by the Boston University Institutional Animal Care and Use Committee.

1. Preparation of Animals and Surgical Supplies

1. Autoclave all su....

Results

Obtaining a large enough nasal septum explant is crucial for the subsequent steps. This can be accomplished by drilling at the location on the donor mouse's skull shown in Figure 1a. Cutting along this path will produce an explant of sufficient the size as shown in Figure 1b. If the drilling depth is not deep enough, the graft will be truncated and it will be hard to obtain a large enough membrane to cover the brain surface. Drilling more laterally than the suggested path is not advised .......

Discussion

The most difficult step of the procedure described herein is the successful transfer of an adequately sized mucosal membrane onto the brain surface. This step is made significantly easier if the harvested nasal septum is large enough and cleaned well. If the ventral portion of the septum is truncated, a new graft should be obtained. The drilling angle should be perpendicular to the mouse head to ensure that the mucosal membrane is not damaged by the drill. If a wider than recommended drilling path is taken it will be muc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mice	Taconic	C57BL/6
Isoflurane	Piramal Healthcare
Student fine scissors	Fine Science Tools	91461-11
Pneumatic drill	MTI Dental	333-CB
Drill bit
Forceps	Fine Science Tools	91106-12
0.9% Sodium chloride injection USP	Abbott Laboratories	4925
Polystyrene Petri dish	Fisher	08-757-12	for temporarily storing graft
Bead sterilizer	Fine Science Tools	18000-45
Oxygen/Isoflurane System	SurgiVet	V720100
Temperature Control System	Physitemp	TCAT-2LV
Small animal stereotaxic instrument	KOPF	Model 940
Eye ointment
Electric shaver
Cotton-tipped applicators	Fisher	23-400-106
7.5% Providone iodine			Betadine surgical scrub
70% Ethanol
Surgical blade stainless	Feather	2976#10
Scalpel handle - #3	Fine Science Tools	10003-12
3% Hydogen peroxide			for cleaning the skull
Vetbond tissue adhesive	3M	1469SB
Needles	Becton, Dickinson and Company	305176	needle tip cut off and used as well
Syringes	Becton, Dickinson and Company	309597
Nitrile gloves	Denville Scientific Inc	G4162	for well closure and protection of graft
5-0 Nylon suture thread
Student Halsey needle holder	Fine Science Tools	91201-13
Cyanoacrylate adhesive			commecially available super glue
Dental cement kit, 1 lb, pink opaque	Stoelting	51458
Isobutane (2-methylbutane)	Aldrich	M32631	for dry ice bath
Dry ice