Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Summary

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

Abstract

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and autism. As evidence suggests that remyelination can effectively ameliorate the disease symptoms, there is an increasing focus on drug development to promote the myelin regeneration process. Thus, a region-selectable and result-reliable drug delivery technique is required to test the efficiency and specificity of these drugs in vivo. This protocol introduces the osmotic pump implant as a new drug delivery approach in the lysolecithin-induced demyelination mouse model. The osmotic pump is a small implantable device that can bypass the blood-brain barrier (BBB) and deliver drugs steadily and directly to specific areas of the mouse brain. It can also effectively improve the bioavailability of drugs such as peptides and proteins with a short half-life. Therefore, this method is of great value to the field of central nervous system myelin regeneration research.

Introduction

The osmotic pump is a small implantable solution-releasing device. It can be used for systemic delivery when implanted subcutaneously or in the abdominal cavity. The surface of the osmotic pump is a semi-permeable membrane, and its inner side is a permeable layer. The osmotic pump operates by using the osmotic pressure difference between the osmotic layer and the tissue environment where the pump is implanted. The high osmolality of the osmotic layer makes the water in the tissue flow into the osmotic layer through the semi-permeable membrane on the pump surface. The osmotic layer expands and compresses the flexible reservoir inside the pump, thereby displacing the so....

Protocol

All animal procedures were conducted under institutional guidelines and protocols approved by the animal welfare and ethics committee of the Third Military Medical University.

1. Establishment of the lysolecithin-induced demyelination mouse model

1. Prepare 1% lysolecithin (also called L-α-Lysophosphatidylcholine) solution with sterile PBS.
2. Sterilize scissors, forceps, curved hemostat, and other surgical instruments by autoclave sterilization. Sterilize the surgical area and lay down sterile sheets. All materials and reagents used for surgery should be prepared aseptically. It is important to keep the sur....

Results

To verify the effect of the osmotic pump in myelin regeneration research, a lysolecithin-induced demyelination model was created in P56 mice, followed by implantation of osmotic pumps containing UM206 (1 mg in 1.5 mL 0.9% saline), a peptide with a short half-life and poor BBB permeability that has been recently reported to promote remyelination¹⁰. 0.9% saline was used as the control. Fourteen days after the model establishment, mice were transcardially perfused with 4% formaldehyde to isolate the .......

Discussion

This protocol describes the osmotic pump as a novel drug delivery technique for myelin regeneration research, which can deliver drugs directly to the treatment site and allow consistent drug delivery for a prolonged period, creating a stable drug concentration in the micro-environment of the central nervous system in the whole experimental duration. Compared with other drug delivery methods, the osmotic pump is more conducive to maintaining drug concentration in the demyelination lesion¹³. For exa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia Air Pump	RWD	R510-29	E05818-006
Brain Infusion kit 3	ALZET	0008851	1-3 mm
Carprofen	Macklin	C830557-1g	5 mg/kg every 24 h
Erythromycin eye ointment	Along technology	YCKJ-RJ-024780	Cover the surface of the eyeballs during anesthesia
Erythromycin ointment	pythonbio	RG180
Gas Evacuation Apparatus	RWD	R546W	E05518-002
L-α-Lysophosphatidylcholine	Sigma	L0906	Dissolve at 1% with sterile PBS
Microliter Syringe	Hamilton	65460-05	Syringe Series:1700, 10 µL, 33 gauge
Micro-smotic pump model 1002	ALZET	0004317	0.25 µL per hour, 14 days
PBS (pH = 7.3)	ORIGENE	ZLI-9061
Pentobarbital sodium	Shanghai Civi	CAS NO: 57-33-0	150-200 mg/kg intraperitoneal injection for euthanasia
Small Animal Anesthesia Machine	RWD	R520IE	E05807-006 M
Stereotaxic Equipment	RWD	E06382
STERI 250 sterilizer	Keller	31101	Rapid sterilization of surgical instruments
Surgical sutures	Shanghai jinhuan	F504	5-0
Syringe needle (1 mL)	Shanghai KDL	6930197811018	26 gauge (0.45 mm x 16 mm)
Testing drug and solvent	Experiment dependent	N/A
ThermoStar Homeothermic Monitoring System	RWD	69026	Maintain body temperature during anesthesia
Vetbond Tissue adhesive	3M	1469SB	Secure the brain infusion cannula , Adhere the skin incision