Name: osmotic pump-based drug-delivery for in vivo remyelination research on the central nervous system
Uploaded: 2021-12-17T14:00:00
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...

<ol>
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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.

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Research

JoVE Journal

Neuroscience

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 ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.