Name: osmotic pump-based drug-delivery for in vivo remyelination research on the central nervous system
Uploaded: 2021-12-17T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System at JoVE.com

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 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 surgical area sterile throughout the procedure.</li>
	<li>Anesthetize a postnatal day 56 (P56) C57BL6 mouse as follows.
	<ol>
		<li>Place the mouse in the isoflurane chamber of the small animal anesthesia machine. Adjust the O2 flow to 300-500 mL/min and isoflurane to 3%-4%. After sufficient anesthesia, when the mouse becomes immobile with a slow and stable breath, transfer the mouse to the stereotaxic apparatus with a heating pad.</li>
		<li>Switch the gas output from the chamber to the anesthesia mask and adjust isoflurane to 1% - 1.5% to maintain the mouse in the anesthesia state. Wait until the mouse is fully anesthetized, inject ketoprofen (3 &#8211; 5 mg/kg) intraperitoneally to relieve pain. Before the operation, pinch the toes of the mouse and check its reaction to confirm successful anesthesia8.</li>
		<li>When the mouse is anesthetized, it cannot regulate its body temperature. Therefore, monitor and regulate the body temperature of the mouse during surgery. To keep the mouse&#39;s eyeballs moist while under anesthesia, cover the surface of the eyeballs with erythromycin eye ointment.</li>
	</ol>
	</li>
	<li>Secure the mouse head in the stereotaxic apparatus with tooth bar and ear bars. (Figure 1A).</li>
	<li>Use a razor to remove hair from the top of the head. Sanitize the head skin with three cycles of betadine and 75% ethanol. For ethical concerns, cover the animal body except for the surgery site. Using a scalpel, make a 1 cm long mid-sagittal incision of the skin from the base of the neck to in between the eyes to expose the skull (Figure 1B).</li>
	<li>Gently wipe the surface of the skull with a sterile cotton swab containing 30% hydrogen peroxide to visualize the cranial sutures (Figure 1C). Adjust the height of the tooth bar and ear bars to place the lambda point and bregma point at the same height (i.e., with the same z-axis coordinates when the needle tip touches the points), so that the sagittal suture is horizontal.</li>
	<li>Gently place the tip of the microliter syringe needle (10 &#181;L, 33 G) at the bregma point and reset the x, y, and z coordinates to 0 (Figure 1D). Move the syringe to the injection site (x: 1.04; y: 1.0, i.e., 1.04 mm lateral to the midline and 1.0 mm posterior to the bregma point) according to the prompt of the digital readout (Figure 1E).</li>
	<li>Slowly drill a small burr hole through the skull at the injection site without penetrating the dura with a 1 mL syringe needle (26 G, 0.45 mm) (Figure 1F). Slowly insert the microliter syringe needle into the brain tissue through the hole until a certain depth is reached (z = -1.62 mm for most P56 mice) (Figure 1G). 
	NOTE: Empirically, the insertion depth of -1.62 mm allows the needle tip to reach the middle of the corpus callosum of most P56 mice so that the lysolecithin could be directly delivered into the corpus callosum to induce demyelination.</li>
	<li>Inject 1.5 &#181;L of 1% lysolecithin at a speed of 0.3 &#181;L/min. After the injection, wait for 5 min before slowly pulling out the microliter syringe to prevent the leakage of liquid along the injection needle path.</li>
	<li>Stitch the skin with 5-0 surgical sutures (Figure 1H).</li>
	<li>Place the mouse on a heating pad to avoid a drop in body temperature. Administer&#160;a subcutaneous injection of 5 mg/kg carprofen every 24 h to relieve the pain. Apply erythromycin ointment to the incision every day to ensure that the wound heals properly. Place the mouse that has undergone surgery in a cage alone and feed it with moist food until fully recovered. Monitor the mouse daily after the operation.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63343/63343fig01.jpg" /> 
Figure 1: Establishment of the lysolecithin-induced demyelination mouse model. (A) Secure the mouse in the stereotaxic apparatus. (B) Open a 1 cm mid-sagittal incision to expose the skull. (C) Visualize the cranial sutures. (D) Reset the x, y, and z coordinates to 0 on the Bregma point. (E) Move the syringe to the injection site. (F) Drill a hole in the skull at the injection site. (G) Insert the needle into brain tissue slowly and inject lysolecithin. (H) Stitch the skin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63343/63343fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of the osmotic pump
NOTE: Key components of the pump are shown in Figure 2A.
<ol>
	<li>Determine the depth of insertion of the brain infusion cannula into the brain. Ensure that the needle of the brain infusion cannula used is 3 mm long and each depth-adjustment spacer is 0.5 mm. To achieve an injection depth of 1.5 mm (close to the callosum), attach three depth-adjustment spacers to the needle of the brain infusion cannula with tissue adhesive (Figure 2B, C).</li>
	<li>To fill the osmotic pump, attach the syringe needle that comes with the pump package to a 1 mL syringe and aspirate the drug. Hold the pump upright, insert the syringe into the opening at the top of the pump, and slowly inject the drug, being careful not to create bubbles9 (see Figure 2D). When the liquid flows out of the opening, slowly pull out the syringe.</li>
	<li>Remove the white flange from the flow regulator with scissors or pliers being careful not to bend or crush the flow moderator. Then, insert the flow moderator into the pump (Figure 2E). To determine whether there are bubbles in the osmotic pump, weigh the osmotic pump separately before and after filling.</li>
	<li>Trim the catheter to a certain length according to the size of the animal (20-25 mm catheters for P56 mice that weigh about 25 g). Attach the catheter to the brain infusion cannula.</li>
	<li>Fill the catheter with drugs using the syringe without introducing air (Figure 2F).</li>
	<li>Connect the catheter to the flow moderator. After attachment, ensure that the catheter covers about 4 mm of the exposed flow moderator (Figure 2G).</li>
	<li>To ensure that the osmotic pump can work instantly after implantation, immerse the filled pumps in sterile 0.9% saline or PBS at 37 &#176;C for at least 4 to 6 h (preferably extend to overnight) to pre-wet the semi-permeable membrane on the pump surface with solutions that have the same osmotic pressure as the tissue environment (Figure 2H).</li>
	<li>All solutions loaded into the pumps should be sterile. ALZET pumps are supplied sterile, having been exposed to a sterilizing dose of 60Co. However, If exterior contamination occurs, the surface of the pump can be cleaned by wiping it with isopropyl alcohol (70% in water).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63343/63343fig02.jpg" /> 
Figure 2: Preparation of the osmotic pump. (A) Key components of the osmotic pump. (B,C) Attach depth-adjustment spacers to the needle of the brain infusion cannula. (D) Fill the osmotic pump using a 1 mL syringe. (E) Insert the flow moderator into the pump. (F) Fill the catheter using the syringe. (G) Connect the catheter to the flow moderator. (H) Immerse the filled pumps in sterile 0.9% saline or PBS at 37 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63343/63343fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Implantation of the osmotic pump
<ol>
	<li>Wait for 3 days after the establishment of the corpus callosum demyelination model. Turn on the small animal anesthesia system. Disinfect scissors, tweezers, and hemostatic pliers and soak them in 75% alcohol solution. Lay sterile sheets in the surgical area.</li>
	<li>Anesthetize and secure the mice on the stereotaxic apparatus again. Cover the surface of the eyeballs with an eye ointment to prevent dryness.</li>
	<li>Disinfect the original wound with 75% alcohol. Open the surgical incision that was previously stitched (Figure 3A) and expand the incision to the shoulder blades (Figure 3B).</li>
	<li>Separate the skin from the subcutaneous connective tissue with hemostatic pliers or tweezers at the scapula to open a cavity (Figure 3C). Place the osmotic pump into the cavity (Figure 3D,E).</li>
	<li>With a cotton swab, gently wipe and expose the pinhole on the surface of the skull created when establishing the demyelination model (see step 1.8). Insert the brain infusion cannula through this pinhole perpendicularly and secure it on the skull quickly with tissue adhesive (Figure 3F).</li>
	<li>Remove the removable tab above the brain infusion cannula with a pair of scissors (Figure 3G, H). Alternatively, remove the tab first before inserting the cannula to avoid shaking in this process.</li>
	<li>Stitch the incision or attach it with tissue adhesive (Figure 3I).</li>
	<li>After surgery, place the mouse on a heating pad to avoid a body temperature drop. Administer a subcutaneous injection of 5 mg/kg carprofen every 24 h to relieve the pain. Apply erythromycin ointment to the incision every day to ensure that the wound heals properly. Place the animal in a cage alone and feed with moist food until fully recovered. Monitor the mice every day and check whether the brain infusion cannula was firmly attached.</li>
	<li>Euthanize the mouse 11 days after the surgery by injecting 150-200 mg/kg Pentobarbital sodium intraperitoneally followed by perfusing transcardially with 4% formaldehyde.</li>
	<li>To verify that the solution is delivered normally, carefully remove the osmotic pump and measure the residual volume in the pump reservoir before brain dissection.
	<ol>
		<li>To measure the residual volume, remove the brain infusion cannula, attach a 1 mL syringe to the catheter, and then aspirate the remaining solution to determine its volume. Compare the actual residual volume to the theoretical residual volume (initial volume - mean pumping rate * infusion duration). 
		NOTE: Excessive residual volume indicates unsuccessful infusion, which might be due to catheter occlusion or pump malfunction.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63343/63343fig03.jpg" /> 
Figure 3: Implantation of the osmotic pump. (A) Open the surgical incision. (B) Expand the incision to the shoulder blades. (C) Separate the skin from subcutaneous connective tissue to make a cavity. (D,E) Place the osmotic pump into the cavity. (F) Insert the brain infusion cannula in the pinhole on the surface of the skull and firmly secure it on the skull. (G,H) Remove the removable tab from the cannula. (I) Stitch the incision. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63343/63343fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

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 example, for certain neurotrophic factors, systemic medication cannot achieve any effect because of the low concentration of the drug at the lesion site. But if the dosage is increased, the side effects will be more significant14. In such cases, administering to a specific site through an osmotic pump can reduce peripheral side effects effectively15. In addition, many myelin regeneration-related drugs have poor blood-brain barrier (BBB) permeability or display a short in vivo half-life due to susceptibility to proteolytic degradation. These problems could be well addressed by osmotic pumps.
However, the osmotic pump method is not without caveats and limitations. First, being an invasive drug delivery system, it inevitably causes brain tissue damage and neuroinflammation at the brain infusion cannula insertion site, which might obscure the drugs&#39; effect. Thus, a proper solvent-only control group must be set up. Second, some drugs require solvents like dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP) to dissolve, but these solvents are incompatible with the reservoir material and can cause a significant failure of the pumps. For example, high concentrations of dimethyl sulfoxide (DMSO) and PEG400 have been shown to adversely affect pump release and may not be suitable for use in osmotic pumps16,17,18. Third, drugs that are unstable at 37 &#176;C might not be suitable for long-term infusion using the osmotic pump. All these issues are worthy of attention if planning to apply the osmotic pump.
Several steps in this protocol require extra attention during the experiments. For the normal operation of the osmotic pumps, researchers must ensure that the osmotic pump is assembled correctly and that no bubble is introduced into the pump, which will otherwise greatly undermine the infusion efficiency. In addition, catheter occlusion or osmotic pump malfunction may cause infusion failure19, which could be determined by the measurement of the residual volume in the pump reservoir after the experiment. For the application of the osmotic pump in younger mice with smaller brain sizes, a trial experiment is recommended to ensure a suitable depth of insertion. Furthermore, the brain infusion cannula must be firmly secured on the skull to minimize its movement during infusion.
At present, many in vitro studies have found a variety of drugs that can promote myelin regeneration, but due to poor BBB permeability, short half-life, and other problems, these drugs are difficult to be successfully validated in vivo. Therefore, the osmotic pump is of great value to the field of central nervous system myelin regeneration research, especially relevant for those drugs with a short half-life, poor BBB permeability, and obvious peripheral side effects.

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><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-&amp;alpha;-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 &amp;micro;L, 33 gauge</td></tr><tr><td>Micro-smotic pump model 1002</td><td>ALZET</td><td>0004317</td><td>0.25 &amp;micro;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 brains for sectioning, followed by in situ hybridization and transmission electron microscopy to evaluate the remyelination level.
Staining of DAPI revealed the pinhole in the brain tissue just above the white matter, indicating successful implantation of the brain infusion cannula of the osmotic pump (Figure 4A). In the in-situ hybridization experiment, the mature oligodendrocyte marker MAG probe was used to label newly differentiated oligodendrocytes as shown in previous studies10,11,12. The results showed that the UM206 treatment yielded more MAG-positive cells in the demyelinated region than the control group (Figure 4B). Transmission electron microscopy of the demyelinated region also showed that the number of myelinated axons was increased in the UM206 treatment group compared to the control group (Figure 4C), suggesting that UM206 induced a higher level of remyelination. These results show that the osmotic pump can efficiently deliver drugs to the corpus callosum in the remyelination research.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63343/63343fig04.jpg" /> 
Figure 4: Representative results. (A) Representative image of DAPI-stained slice showing the pinhole in the brain tissue. Scale bar: 1,000 &#181;m. (B) Representative images showing in situ hybridization of MAG in the demyelinated region as shown by DAPI staining. UM206 treatment increased the number of MAG-labeled oligodendrocytes. Scale bar: 100 &#181;m. (C) Representative transmission electron microscopy images of the demyelinated region. UM206 treatment increased the number of myelinated axons. Scale bar: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63343/63343fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.7K Views.  Third Military Medical University. Osmotische pomp is van grote waarde voor het onderzoek naar myeline regeneratie, vooral voor die geneesmiddelen met een korte halfwaardetijd, slechte bloed-hersenbarrière permeabiliteit, en van verschillende perifere bijwerkingen. Osmotische pomp kan de bloed-hersenbarrière omzeilen en geneesmiddelen rechtstreeks aan het corpus callosum leveren, wat de biologische beschikbaarheid van geneesmiddelen effectief verbetert, vooral voor sommige geneesmiddelen met een korte halfwaardetijd. Om te b...

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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Cancer Research

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

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.

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.

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 ...

Medicine

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

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

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 &#181;g 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.

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.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

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

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