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 demonstrated by using drug conjugates containing antibodies selective for transport proteins located on the brain capillary endothelium; however this method has not been shown to be applicable for a broad range of pharmaceuticals^7,8. Additionally, osmotic opening of the BBB has been used clinically, however this method suffers from systemic drug dosing as opposed to a more direct delivery to the brain region of interest⁹. Substantial effort has been put into optimizing transnasal delivery in the hopes of directly targeting the brain^10-12. Although some success has been achieved, conclusive results have only been obtained for drugs that possess endogenous receptors, such as insulin^13,14. Furthermore the mechanism of transnasal delivery has been controversial with evidence suggesting indirect entry into the brain via olfactory neuron uptake or through the bloodstream¹¹. Direct, transcranial delivery using implantable catheters has been achieved, however this procedure is highly invasive and associated with numerous complications^15,16. To date, there is no general, minimally invasive method to deliver high molecular weight compounds into the brain.

Presented herein is a murine surgical procedure that creates a semipermeable interface with the brain. This is accomplished by engrafting a mucosal membrane explant¹⁷ over a surgical craniotomy defect in a mouse. Using this procedure it has been shown that soluble compounds up to 500 kDa can be delivered into the central nervous system (directly into brain parenchyma as well as into cerebrospinal fluid) in both a time and molecular weight dependent fashion¹⁸. This method of bypassing the BBB is a model for skull base defect repairs in humans which uses vascularized mucosal grafts to repair holes in the skull following transnasal endoscopic surgery^19,20.

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 surgical instrument that will be used during the surgery.
2. Make sure all techniques that are to be performed are approved by the animal regulatory agencies.

2. Harvesting of the Mucosal Graft

1. Chose a genetically identical mouse of similar age as the experimental mouse and euthanize it in an IACUC approved method (here: isoflurane asphyxiation followed by cervical dislocation).
2. Using surgical scissors, remove the skin around the nasal region of mouse head exposing the skull.
3. With a pneumatic drill, mark with three lines two of which laterally flank the nasal region and a third in line with the eyes which connects the two lines perpendicularly.
4. Drill down ventrally in order to separate the nasal septum from the surrounding tissue. A wider path will prevent damage to the mucosal membrane however it will also make it more difficult to isolate the membrane. A narrow cut closer to midline is recommended.
5. Use scissors to cut the septum free from any tissue adhered to it and store it in a sterile saline solution. At this time the graft can be cleaned up to remove any connected tissue. The ideal situation is to have undamaged mucosal membranes exposed on both sides of the cartilage septum. One graft can supply membrane for two mice provided the surface area of the membrane is sufficient to cover the craniotomy sites. It is recommended that the graft is used as quickly as possible and the researcher proceeds to step 3 as soon as the graft is isolated.

3. Surgical Implantation of Mucosal Graft

1. Using standard, aseptic murine surgical procedures, anesthetize and mount a mouse in the stereological frame. Use approximately 2% isoflurane in pure oxygen using a rodent anesthesia machine.
2. Immobilize the mouse in a stereotaxic apparatus with ear bars and a nose holder. Apply ophthalmic ointment to the eyes and scrub the scalp with betadine and 75% ethanol for three rounds. Using either scissors or a hair trimmer, remove the fur on the head. Expose the skull with a razor blade and level the head. Perform a craniotomy above the location of the brain that will be dosed. For example, when targeting the striatum cut a 1.25 mm diameter circular hole in the skull (centered at AP: 1.00 mm; ML: 0.88 mm) using a pneumatic drill. Wet the drilled area with sterile saline and use a razor blade to remove the skull.
3. Carefully remove the dura using the tip of a needle. Additionally, this can be accomplished by applying a minimal amount of tissue adhesive to the moist dural surface. Once this layer has hardened, lateral motion with a razor blade tip can be used to remove the membrane.
4. Place the mucosal membrane above the brain surface taking extreme care to keep the epithelial side facing away from the wound. This is best done by transferring the entire septum on to the surface of the skull adjacent to the craniotomy site with tweezers. Using the tip of a pair of surgical scissors, pull the membrane off of the cartilage and onto the skull and brain surface. Do not let the membrane dry out or touch it with any absorbent material. The graft should generously overlap all bony edges of the craniotomy site.
5. Cover the graft with a sterile piece of nitrile. This acts to prevent adhesion of the skin to the graft during healing. The nitrile needs to be large enough to cover the entire mucosal membrane. Trim excessive membrane if necessary. Avoid any motion of the nitrile once it has come into contact with the graft.
6. Close the skin with a running 5-0 sterile suture and let the mouse recover for 3-7 days before proceeding to the next step. Take care not to perturb the nitrile barrier or the mucosal graft during skin closure.

4. Administration of Dosing Solution

1. After securing the anesthetized mouse in the stereotaxic frame, cut the suture with scissors and remove excess skin around the skull.
2. Remove the nitrile barrier and clean the surface of the skull. Use sterile saline and cotton swabs to clean the area until the graft is visible. It may be necessary to cut the graft with a razor if has grown larger than the desired surface area.
3. If the experiment will be longer than a few days, it is wise to implant at least two skull screws to reinforce the head implant.
4. Place the well above the graft so that the edges are in contact with the skull. Apply cyanoacrylate adhesive at the junction between the well and the skull. Fill the well with sterile saline and check to make sure there are no leaks. Wells are made from cut syringe needles.
5. Apply bone cement on the skull to secure the well in place.
6. Remove the saline from the well with a pipette. Wash the well several times to verify that adhesive has not leaked in. Add the desired solution; in this case 50 μl of fluorescent dextran is used. It is expected that water soluble compounds of a similar polarity will behave the same as dextran. Delivery of hydrophobic compounds or suspensions has not been explored with this method.
7. Cap the top of the well by using a circular piece of nitrile secured to the top using cyanoacrylate adhesive. Make sure the adhesive does not come in contact with the well contents.

5. Analysis of Transmucosal Delivery

1. After the desired amount of time has passed, anesthetize the mouse and exchange the well contents with a solution of Evan's blue dye. This dye is used to verify that the graft was intact.
2. After 30 min, anesthetize the mouse heavily, remove the dye solution, and euthanize via decapitation.
3. Manually remove the implant and remove the brain using surgical scissors. Be careful to keep the graft in place.
4. Once removed, flash-freeze the brain in a solution of isobutane cooled in a dry ice bath.
5. Embed the brain in Optimal Cutting Temperature (OCT) solution and slice at 50 μm.
6. Place the desired slices directly on a microscope slide.
7. Image the slice using a florescence microscope as soon as the OCT solution has dried.

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 advi...

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

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