The authors work was supported by research grant ID-BOF 2006 of the University of Antwerp (granted to PPo and AVdL), by research grant G.0136.11 and G.0130.11 (granted to AVdL, ZB and PPo) and 1.5.021.09.N.00 (granted to PPo) of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium), by SBO research grant IWT-60838: BRAINSTIM of the Flemish Institute for Science and Technology (granted to ZB and AVDL), in part by a Methusalem research grant from the Flemish government (granted to ZB), in part by EC-FP6-NoE DiMI (LSHB-CT-2005-512146), EC-FP6-NoE EMIL (LSHC-CT-2004-503569), and by the Inter University Attraction Poles IUAP-NIMI-P6/38 (granted to AVDL). Nathalie De Vocht holds a PhD-studentship from the FWO-Vlaanderen. Peter Ponsaerts is a post-doctoral fellow of the FWO-Vlaanderen.

1. Cell Preparation
<ol>
	<li>Experiments should be initiated using ex vivo cultured stem cell populations genetically engineered to express the Luciferase and eGFP reporter proteins. Here we use Luciferase/eGFP-expressing murine bone marrow-derived stromal cells (BMSC-Luc/eGFP) as previously described by Bergwerf et al.2, 5.</li>
	<li>Two days before cell labeling, plate BMSC-Luc/eGFP cells at a density of 8 x 105 cells per T75 culture flask in 15 ml complete expansion medium (CEM) supplemented with 1 &mu;g/ml Puromycine.</li>
	<li>Allow cells to grow for 48 hr at 37 &deg;C and 5% CO2.</li>
	<li>Add 5 x 106 Glacial Blue micron-sized iron oxide particles (GB MPIO) per ml culture medium to the growing cell culture (total: 75 x 106 particles per 15 ml culture medium in a T75 culture flask).</li>
	<li>Incubate for 16 hr to allow particle uptake via endocytosis.</li>
	<li>Wash away remaining particles and allow cells to grow for an additional 24 hr to obtain cell confluency and homogeneous distribution of the GB MPIO.</li>
	<li>Validate particle uptake visually using fluorescence microscopy by counting the ratio of eGFP+ and GB MPIO+ cells to the total amount of eGFP+ cells.</li>
	<li>Wash GB MPIO-labeled BMSC-Luc/eGFP cells twice with 10 ml PBS and harvest cells following trypsin-EDTA treatment (5 ml per T75 culture flask for 5 minutes).</li>
	<li>Wash harvested cells and resuspend at final concentration of 133 x 106 cells/ml in PBS.</li>
	<li>Keep cells on ice until transplantation.</li>
</ol>
2. Cell Implantation in the CNS of Mice
<ol>
	<li>Anaesthetize mice by an intraperitoneal injection of a ketamine (80 mg/kg) + xylazine (16 mg/kg) mixture.</li>
	<li>Wait for 5 minutes to allow mice to fall asleep.</li>
	<li>Shave the mouse head to allow for sterile manipulations.</li>
	<li>Place the mouse in a stereotactic frame.</li>
	<li>Desinfect the skin and wet the eyes of the mice to prevent dehydration.</li>
	<li>Make a midline scalp incision to expose the skull, and drill a hole in the skull using a dental drill burr at the given coordinates: 2 mm posterior and 2 mm lateral to Bregma.</li>
	<li>Vortex the cell suspension briefly and aspirate the cell suspension in the syringe.</li>
	<li>Place the syringe needle at a depth of 2.5 mm under the dura and allow pressure equilibration for 1 minute.</li>
	<li>Inject a 3 &mu;l cell suspension (4 x 105 cells) at a depth of 2 mm under the dura and at a speed of 0.70 &mu;l/min using an automated micro-injection pump.</li>
	<li>Wait for an additional 5 minutes to prevent backflow of the injected cell suspension.</li>
	<li>Slowly retract the needle and disinfect the skin borders before suturing the skin.</li>
	<li>Inject 300 &mu;l 0.9% NaCl solution subcutaneously in order to prevent dehydration, and place the mouse under a heating lamp to recover from anaesthesia. Administer post-operative analgesic in accordance with institutional standards.</li>
</ol>
3. In vivo Bioluminescence
<ol>
	<li>Anaesthetize the mice by a mixture of 3% isoflurane and oxygen.</li>
	<li>Place the mice in the Photon Imager and reduce anaesthesia level to 1.5% isoflurane and oxygen.</li>
	<li>Inject 150 mg D-luciferin per kg body weight intravenously.</li>
	<li>Acquire image for 5 minutes using the Photo Vision software.</li>
	<li>Perform image processing using the M3vision software. Quantify the observed signal using fixed regions of interest.</li>
</ol>
4. In vivo Magnetic Resonance Imaging
<ol>
	<li>Anaesthetize the mice with 3% isoflurane in a mixture of O2:N2O (3:7).</li>
	<li>Place the mice in the restrainer of a horizontal 9.4T MR system and reduce anaesthesia level to 1% isoflurane in a mixture of O2:N2O (3:7).</li>
	<li>Wet the eyes of the mice to prevent dehydration, attach a rectal probe to monitor the body temperature and monitor the breathing rate by placing a sensor underneath the mouse belly.</li>
	<li>Maintain breathing rate at 110 &plusmn; 10 breaths per minute and keep body temperature constant within a narrow range of 37 &plusmn; 0.5 &deg;C.</li>
	<li>Place the surface RF coil on top of the mouse head and position the mouse in the middle of the magnet.</li>
	<li>Acquire a set of 10 coronal T2-weighted spin echo (SE) images to obtain specific anatomical information and T2*-weighted gradient echo (GE) images in order to study stem cell migration with an in-plane resolution of 70 &mu;m2. Set sequence parameters as follows: repetition time (TR): 500 ms, echo time (TE): 8 ms (GE sequence) and repetition time (TR): 4200 ms, echo time (TE): 12.16 ms (SE sequence); field of view (FOV): 18x18 mm2, matrix: 256x256, 1 mm slice thickness and 1 mm slice separation (Paravision 5.1 software).</li>
	<li>Perform data processing using Amira 4.0 software.</li>
</ol>
5. Post Mortem Histology
<ol>
	<li>Anaesthetize the mice very deeply by inhalation of an isofluorane (4%), oxygen (0.5 L/min) and nitrogen (1 L/min) mixture for 2 minutes. Sacrifice the mice by cervical dislocation.</li>
	<li>Remove the mouse brain from the skull and fixate the brain tissue in 4% paraformaldehyde in PBS for 2 hours.</li>
	<li>Dehydrate the brain tissue by placing the brain subsequently in different gradients of sucrose: 2 hr in 5% sucrose in PBS, 2 hr in 10% sucrose in PBS, overnight in 20% sucrose in PBS.</li>
	<li>Freeze the brain tissue using fluid nitrogen and store the tissue at -80 &deg;C until sectioning.</li>
	<li>Section the brain tissue in 10 &mu;m thick sections using a cryostat.</li>
	<li>Screen unstained cryosections for blue fluorescence from the GB MPIO particles and green fluorescence from the eGFP expressing cells using a fluorescence microscope.</li>
	<li>Screen unstained cryosections for green/red background fluorescence from inflammatory cells.</li>
	<li>Perform further immunohistochemical and immunofluorescent stainings to identify endogenous cell populations (e.g. microglia, astrocytes, T cells,...) interacting with cellular grafts.</li>
</ol>
6. Representative Results
We here visually presented an optimized sequence of events for successful multimodal imaging of Luciferase/eGFP-expressing (stem) cell populations in the CNS of mice. At first, the described GB MPIO labeling procedure results in highly efficient labeling of BMSC-Luc/eGFP cells, which can easily be validated using fluorescence microscopy (Figure 2A). Next, upon grafting of GB MPIO labeled BMSC-Luc/eGFP in the CNS of mice, survival of the cellular graft can be monitored by in vivo BLI based on luciferase activity (Figure 2B). Additionally, the exact localization of grafted cells can be monitored by in vivo MRI based on the iron content of the GB MPIO (Figure 2C). Finally, histological analysis allows validation of the results obtained by BLI and MRI. Stable survival was confirmed by a stable eGFP expression in time (Figure 2D). For this, colocalization of eGFP-expressing cells with blue fluorescent MPIO allows for exact determination of localization and survival of grafted cells. In addition, this dual fluorescent labeling of the stem cells reduces the possibility to observe false positive results based on background fluorescence created by inflammatory cells5.
<img alt="Figure 1." src="/files/ftp_upload/3906/3906fig1.jpg" /> 
Figure 1. Overview of the handling sequence
<ol class="lroman">
	<li>Cell labeling: BMSC-Luc/eGFP are labeled with 5 x 106 GB MPIO particles per ml culture medium. After 16 hr of incubation and a following rest period of 24 hr, cells are harvested for intracerebral implantation.</li>
	<li>Cell implantation: GB MPIO labeled BMSC-Luc/eGFP are grafted in mouse brain at the following coordinates: bregma -2 mm, 2 mm right from the midline and 2 mm under dura.</li>
	<li>In vivo BLI: Following intravenous administration of 150 mg D-luciferin/kg body weight, the generated light was acquired during 5 minutes using the Photon Imager.</li>
	<li>In vivo MRI: A set of 10 coronal T2-weighted spin echo (SE) images and T2*-weighted gradient echo (GE) images with an in-plane resolution of 70 &mu;m2 were acquired. Sequence parameters were as follows: repetition time (TR): 500 ms, echo time (TE): 8 ms (GE sequence) and repetition time (TR): 4200 ms, echo time (TE): 12.16 ms (SE sequence); field of view (FOV): 18x18 mm2, matrix: 256x256, 1 mm slice thickness and 1 mm slice separation.</li>
	<li>Post-mortem histology: 10&mu;m thick cryosection were screened for eGFP expressing and blue fluorescently labeled BMSC using a fluorescence microscope.</li>
</ol>
<img alt="Figure 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> 
Figure 2. Multimodal imaging of stem cell grafts
<ol class="ualpha">
	<li>Direct immunofluorescence microscopy of Glacial blue (GB) MPIO labeled BMSC-Luc/eGFP.</li>
	<li>In vivo BLI of mice injected with 4x105 GB MPIO labeled BMSC-Luc/eGFP cells at week 1 and week 2 after implantation. Statistical analysis of detected BLI signals at week 1 and week 2 post-implantation. Data are expressed as average number of photons per second per steradian per square centimeter (ph/s/sr/cm2) from a 5 minute time period +/- standard error calculated from a fixed region of interest in mice showing a clear BLI signal at the injection site.</li>
	<li>Coronal two-dimensional MRI of GB MPIO labeled BMSC-Luc/eGFP (left panel: (i) T2-weighted spin echo image and (ii) T2*-weighted gradient echo image) and unlabeled BMSC-Luc/eGFP (right panel: (iii) T2-weighted spin echo image and (vi) T2*-weighted gradient echo image) grafts in mouse brain.</li>
	<li>Histological analysis of GB MPIO labeled BMSC-Luc/eGFP implants at week 2 post-implantation. (i) Direct immunofluorescence analysis for localization of eGFP-expressing and GB MPIO labeled BMSC-Luc/eGFP grafts. (ii) High magnification detail of (i). (iii) Immunofluorescent staining for GFAP antigen, indicating the development of astrocytic scar tissue in the surrounding of GB MPIO labeled BMSC-Luc/eGFP. (vi) Immunofluorescent staining for Iba-1 antigen, indicating the invasion and surrounding of GB MPIO labeled BMSC-Luc/eGFP by microglia.</li>
</ol>

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The authors declare no conflict of interest.

In this report, we describe an optimized protocol for the combination of three complementary imaging modalities (BLI, MRI and histology) for detailed characterization of cellular implants in the CNS of immune competent mice. A combination of reporter gene labeling of cells, based on genetic modification with the reporter genes firefly Luciferase and eGFP, and a direct cell labeling with GB MPIO, leads to an accurate assessment of stem cell grafts in vivo.
For particle labeling of BMSC...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Lonza</td>
 <td>BE12-722F</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Gibco</td>
 <td>10270-106</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>Gibco</td>
 <td>1605-122</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Gibco</td>
 <td>15290-018</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>14190</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Puromycine</td>
 <td>Invivogen</td>
 <td>ant-pr-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>trypsin</td>
 <td>Gibco</td>
 <td>25300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GB MPIO</td>
 <td>Bangs Laboratories</td>
 <td>ME04F/7833</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-luciferin</td>
 <td>Promega</td>
 <td>E1601</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ketamine (Ketalar)</td>
 <td>Pfizer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xylazine (Rompun)</td>
 <td>Bayer Health care</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Isoflo </td>
 <td>05260-05</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.9% NaCl solution</td>
 <td>Baxter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Merck</td>
 <td>1.04005.1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>sucrose</td>
 <td>Applichem</td>
 <td>A1125</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-injection pump</td>
 <td>KD scientific</td>
 <td>KDS100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Photon imager</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>9.4T MR scanner</td>
 <td>Bruker Biospin</td>
 <td>Biospec 94/20 USR </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BX51 microscope </td>
 <td>Olympus</td>
 <td>BX51</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mycrom HM cryostat</td>
 <td>Prosan</td>
 <td>HM525</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>syringe</td>
 <td>Hamilton</td>
 <td>7635-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30 gauge needle</td>
 <td>Hamilton</td>
 <td>7762-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Photo Vision software</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>M3vision software</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paravision 5.1 software</td>
 <td>Bruker Biospin</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amira 4.0 software</td>
 <td>Visage Imaging</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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).

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

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

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, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K Views.  University of Antwerp. Questo articolo descrive una sequenza ottimizzata di eventi per l'imaging multimodale di innesti cellulari nel cervello di roditori utilizzando: (i) in bioluminescenza vivo e la risonanza magnetica, e (ii) post mortem l'analisi istologica. La combinazione di queste modalità di imaging su un singolo animale consente la valutazione del trapianto cellulare ad alta risoluzione, sensibilità e specificità.

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

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

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 ...

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.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...