This work was supported by grants from the National Nature Science Foundation of China (NSFC 32070964, 31871045) to J.N. and the Shenzhen Basic Research Foundation (JCYJ20210324121214039) to Y.S.

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
<ol>
	<li>Prepare 1% lysolecithin (also called L-&#945;-Lysophosphatidylcholine) solution with sterile PBS.</li>
	<li>Sterilize scissors, forceps, curved hemostat, and other surgical instruments by autoclave sterilization. Sterilize...

<ol>
	<li>Theeuwes, F., Yum, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+the+design+and+operation+of+generic+osmotic+pumps+for+the+delivery+of+semisolid+or+liquid+drug+formulations.">Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations.</a> Annals of Biomedical Engineering. 4 (4), 343-353 (1976).</li><li>Herrlich, S., Spieth, S., Messner, S., Zengerle, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+micropumps+for+drug+delivery.">Osmotic micropumps for drug delivery.</a> Advanced Drug Delivery Reviews. 64 (14), 1617-1627 (2012).</li><li>Wei, E., Loh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+dependence+of+opiate-like+peptides.">Physical dependence of opiate-like peptides.</a> Science. 193 (4259), 1262-1263 (1976).</li><li>Pettigrew, J. D., Kasamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+perfusion+of+noradrenaline+maintains+visual+cortical+plasticity.">Local perfusion of noradrenaline maintains visual cortical plasticity.</a> Nature. 271 (5647), 761-763 (1978).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+oligodendrocyte+precursor+cell+impairs+astrocytic+development+in+early+life+stress.">Reduced oligodendrocyte precursor cell impairs astrocytic development in early life stress.</a> Advanced Science (Weinheim). 8 (16), 2101181 (2021).</li><li>Tang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+behave+as+a+functional+niche+for+the+maturation+of+newborn+neurons+through+the+secretion+of+PTN.">Neural stem cells behave as a functional niche for the maturation of newborn neurons through the secretion of PTN.</a> Neuron. 101 (1), 32-44 (2019).</li><li>Watanabe, S., Komine, O., Endo, F., Wakasugi, K., Yamanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+administration+of+Cystatin+C+ameliorates+disease+in+SOD1-linked+amyotrophic+lateral+sclerosis+mice.">Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1-linked amyotrophic lateral sclerosis mice.</a> Journal of Neurochemistry. 145 (1), 80-89 (2018).</li><li>DeVos, S. L., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intraventricular+delivery+of+drugs+to+the+rodent+central+nervous+system.">Direct intraventricular delivery of drugs to the rodent central nervous system.</a> Journal of Visualized Experiments: JoVE. , e50326 (2013).</li><li>Tang, C., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+a+mini-osmotic+pump+plus+stereotactical+injection+of+retrovirus+to+study+newborn+neuron+development+in+adult+mouse+hippocampus.">Implantation of a mini-osmotic pump plus stereotactical injection of retrovirus to study newborn neuron development in adult mouse hippocampus.</a> STAR Protocols. 2 (1), 100374 (2021).</li><li>Niu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglial+ring+finger+protein+Rnf43+is+an+essential+injury-specific+regulator+of+oligodendrocyte+maturation.">Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.</a> Neuron. 109 (19), 3104-3118 (2021).</li><li>Breitschopf, H., Suchanek, G., Gould, R. M., Colman, D. R., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+probes:+sensitive+and+reliable+detection+method+applied+to+myelinating+rat+brain.">In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain.</a> Acta Neuropathologica. 84 (6), 581-587 (1992).</li><li>Cree, B. A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clemastine+rescues+myelination+defects+and+promotes+functional+recovery+in+hypoxic+brain+injury.">Clemastine rescues myelination defects and promotes functional recovery in hypoxic brain injury.</a> Brain. 141 (1), 85-98 (2018).</li><li>Eckenhoff, B., Yum, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osmotic+pump:+novel+research+tool+for+optimizing+drug+regimens.">The osmotic pump: novel research tool for optimizing drug regimens.</a> Biomaterials. 2 (2), 89-97 (1981).</li><li>Thoenen, H., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins:+from+enthusiastic+expectations+through+sobering+experiences+to+rational+therapeutic+approaches.">Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches.</a> Nature Neuroscience. 5, 1046-1050 (2002).</li><li>Hagg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+infusion+of+neurotrophic+factors.">Intracerebral infusion of neurotrophic factors.</a> Methods in Molecular Biology. 399, 167-180 (2007).</li><li>Bittner, B., Thelly, T., Isel, H., Mountfield, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+co-solvents+and+the+composition+of+experimental+formulations+on+the+pump+rate+of+the+ALZET+osmotic+pump.">The impact of co-solvents and the composition of experimental formulations on the pump rate of the ALZET osmotic pump.</a> International Journal of Pharmaceutics. 205 (1-2), 195-198 (2000).</li><li>Arnot, M. I., Bateson, A. N., Martin, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide/propylene+glycol+is+a+suitable+solvent+for+the+delivery+of+diazepam+from+osmotic+minipumps.">Dimethyl sulfoxide/propylene glycol is a suitable solvent for the delivery of diazepam from osmotic minipumps.</a> Journal of Pharmacological and Toxicological Methods. 36 (1), 29-31 (1996).</li><li>Gullapalli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+ALZET+osmotic+pump+compatible+solvent+compositions+to+solubilize+poorly+soluble+compounds+for+preclinical+studies.">Development of ALZET osmotic pump compatible solvent compositions to solubilize poorly soluble compounds for preclinical studies.</a> Drug Delivery. 19 (5), 239-246 (2012).</li><li>White, J. D., Schwartz, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+osmotic+minipumps+for+intracranial+delivery+of+amino+acids+and+peptides.">Using osmotic minipumps for intracranial delivery of amino acids and peptides.</a> Methods in Neurosciences. 21, 187-200 (1994).</li></ol>

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...

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 solution from the flexible reservoir at a certain rate for a long duration1. The pump has three different reservoir volumes, 100 &#181;L, 200 &#181;L, and 2 mL, with their delivery rates varying from 0.11 &#181;L/h to 10 &#181;L/h. Depending on the selected pump type, the device can operate from 1 day to 6 weeks2. In this protocol, a 100 &#181;L osmotic pump with a transfer rate of 0.25 &#181;L/h that can operate for 14 days is used.
Back in the 1970s, the osmotic pump had been used in neuroscience research3,4. For instance, Wei et al. adopted the osmotic pump approach to inject opioid peptides into the ventricle in a study of drug addiction3. After continuous improvement, the osmotic pump has now been used in the study of the controlled delivery of thousands of drugs, including peptides, growth factors, addictive drugs, hormones, steroids, antibodies, and so on. In addition, with special catheters (Brain Infusion Kits) attached, it can be used for targeted infusion to specific tissues or organs, including the spinal cord, brain, spleen, and liver5,6,7.
In the study of remyelination, many drugs have been shown to promote myelin regeneration in vitro, but most of them have not achieved significant effects in vivo, possibly due to the lack of an appropriate administration method. Traditional administration methods such as intraperitoneal injection, subcutaneous injection, and intragastric administration have limitations in the bioavailability of the drugs. In addition, some drugs have poor blood-brain barrier permeability, which undermines their access to the brain parenchyma. Together, these limitations call for a novel efficient delivery method. In combination with the brain infusion kits, osmotic pumps can bypass the blood-brain barrier and deliver drugs directly to the corpus callosum, which effectively improves the bioavailability of drugs, especially for some polypeptide and protein drugs with a short half-life. Therefore, the osmotic pump as a new drug delivery technique is of great value to the field of central nervous system myelin regeneration research. The application of this technique will be introduced in detail below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia Air Pump</td>
			<td>RWD</td>
			<td>R510-29</td>
			<td>E05818-006</td>
		</tr>
		<tr>
			<td>Brain Infusion kit 3</td>
			<td>ALZET</td>
			<td>0008851</td>
			<td>1-3 mm</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Macklin</td>
			<td>C830557-1g</td>
			<td>5 mg/kg every 24 h</td>
		</tr>
		<tr>
			<td>Erythromycin eye ointment</td>
			<td>Along technology</td>
			<td>YCKJ-RJ-024780</td>
			<td>Cover the surface of the eyeballs during anesthesia</td>
		</tr>
		<tr>
			<td>Erythromycin ointment</td>
			<td>pythonbio</td>
			<td>RG180</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Evacuation Apparatus</td>
			<td>RWD</td>
			<td>R546W</td>
			<td>E05518-002</td>
		</tr>
		<tr>
			<td>L-&#945;-Lysophosphatidylcholine</td>
			<td>Sigma</td>
			<td>L0906</td>
			<td>Dissolve at 1% with sterile PBS</td>
		</tr>
		<tr>
			<td>Microliter Syringe</td>
			<td>Hamilton</td>
			<td>65460-05</td>
			<td>Syringe Series:1700, 10 &#181;L, 33 gauge</td>
		</tr>
		<tr>
			<td>Micro-smotic pump model 1002</td>
			<td>ALZET</td>
			<td>0004317</td>
			<td>0.25 &#181;L per hour, 14 days</td>
		</tr>
		<tr>
			<td>PBS (pH = 7.3)</td>
			<td>ORIGENE</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Shanghai Civi</td>
			<td>CAS NO: 57-33-0</td>
			<td>150-200 mg/kg intraperitoneal injection for euthanasia</td>
		</tr>
		<tr>
			<td>Small Animal Anesthesia Machine</td>
			<td>RWD</td>
			<td>R520IE</td>
			<td>E05807-006 M</td>
		</tr>
		<tr>
			<td>Stereotaxic Equipment</td>
			<td>RWD</td>
			<td>E06382</td>
			<td></td>
		</tr>
		<tr>
			<td>STERI 250 sterilizer</td>
			<td>Keller</td>
			<td>31101</td>
			<td>Rapid sterilization of surgical instruments</td>
		</tr>
		<tr>
			<td>Surgical sutures</td>
			<td>Shanghai jinhuan</td>
			<td>F504</td>
			<td>5-0</td>
		</tr>
		<tr>
			<td>Syringe needle (1 mL)</td>
			<td>Shanghai KDL</td>
			<td>6930197811018</td>
			<td>26 gauge (0.45 mm x 16 mm)</td>
		</tr>
		<tr>
			<td>Testing drug and solvent</td>
			<td>Experiment dependent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoStar Homeothermic Monitoring System</td>
			<td>RWD</td>
			<td>69026</td>
			<td>Maintain body temperature during anesthesia</td>
		</tr>
		<tr>
			<td>Vetbond Tissue adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>Secure the brain infusion cannula , Adhere the skin incision</td>
		</tr>
	</tbody>
</table>

osmotic pump-based drug-delivery for in vivo remyelination research on the central nervous system

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.

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 ...

Watch this Scientific Journal Video about Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System at JoVE.com

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

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.

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

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...

Osmotic pump is of great value to the research of myelin regeneration, especially for those drugs with a short half-life, poor blood-brain barrier permeability, and of various peripheral side effects. Osmotic pump can bypass the blood-brain barrier and deliver drugs directly to the corpus callosum, which effectively improves the bioavailability of drugs, especially for some drugs with a short half-life. To begin, secure the head of an anesthetized mouse in the stereotaxic apparatus with a tooth bar and ear bars.Cover the animal body except for the surgical site. Using a scalpel, make a one centimeter long midsagittal incision of the skin from the base of the neck to in between the eyes to expose the skull. Using a cotton swab containing 30%hydrogen peroxide, gently wipe the surface of the skull to visualize the cranial structures.Adjust the height of the tooth bar and ear bars to place the lambda point and bregma point at the same height. Place the tip of a 10 microliter and 33 gauge syringe needle gently at the bregma point, and reset the X, Y, and Z coordinates to zero. Move the syringe to the injection site.Slowly drill a small burr hole through the skull at the injection site without penetrating the dura with a one microliter 26 gauge and 0.45 millimeter syringe needle. Slowly insert the microliter syringe needle into the brain tissue through the hole until a certain depth is reached. Inject 1.5 microliters of 1%lysolecithin at a speed of 0.5 microliters per minute.Wait for five minutes before pulling out the syringe, and stitch the skin with 5-0 surgical sutures. Place the mouse that has undergone surgery alone in a cage, and monitor the mouse daily after the operation. Attach three depth adjustment spacers to the needle of the brain infusion cannula with tissue adhesive to achieve an injection depth of 1.5 millimeters that is close to the callosum.Fill the osmotic pump by attaching the syringe needle with a pump package to a one milliliter syringe, and aspirate the drug. Hold the pump in an upright position. Insert the syringe into the opening at the top of the pump and slowly inject the drug without creating an air bubble.Slowly pull out the syringe as the liquid flows out of the opening. Using scissors or pliers, remove the white flange from the flow regulator. Insert the flow moderator into the pump.To determine whether there are bubbles in the osmotic pump, weigh the osmotic pump separately, before and after filling. Trim the catheter to the required length according to the size of the animal. Attach the catheter to the brain infusion cannula.Fill the catheter with drugs using the syringe without introducing air. Connect the catheter to the flow moderator so that it covers about four millimeters of the exposed flow moderator. Immerse the filled pumps in sterile 0.9%saline or PBS at 37 degrees Celsius for at least four to six hours.Prefer to extend overnight after implementation. Secure the mice on the stereotaxic apparatus again. Open the previously stitched surgical incision and expand the incision up to the shoulder blades.Separate the skin from the subcutaneous connective tissue at the scapula, using hemostatic pliers or tweezers to open a cavity, and place the osmotic pump into the cavity. With a cotton swab, gently wipe and expose the pin hole on the surface of the skull created when establishing the demyelinisation model. Insert the brain infusion cannula through the pinhole perpendicularly and secure it on the skull with tissue adhesive.Take off the removable tab above the brain infusion cannula with a pair of scissors. Stitch the incision or attach it with tissue adhesive. Place the animal in a cage alone after the surgery.After performing implementation DAPI staining revealed that the pinhole in the brain tissue lies just above the white matter, indicating successful implementation of the brain infusion cannula of the osmotic pump. The in situ hybridization experiment was performed with a mature oligodendrocyte marker MAG probe was used to label newly differentiated oligodendrocytes. The results showed that the UM206 treatment yielded more MAG positive cells in the demyelinated region than the control group.Transmission electron microscopy of the demyelinated region showed that the number of myelinated axons was increased in the UM206 treatment group compared to the control group, suggesting that UM206 induced a higher level of re-myelinisation. When preparing the osmotic pump, be careful not to introduce any bubble into the system.

osmotic-pump-based-drug-delivery-for-vivo-remyelination-research-on

Research

JoVE Journal

Neuroscience

4.2K Views.  Third Military Medical University. Osmotic pump is of great value to the research of myelin regeneration, especially for those drugs with a short half-life, poor blood-brain barrier permeability, and of various peripheral side effects. Osmotic pump can bypass the blood-brain barrier and deliver drugs directly to the corpus callosum, which effectively improves the bioavailability of drugs, especially for some drugs with a short half-life. To begin, secure the head of an anesthetized mouse in the stereotaxic apparatus with a too...