This work was supported by the Ministry of Science and Technology Grants, Taiwan (MOST 104-2923-B-038-004 -MY2, MOST 107-2314-B-038-063, and MOST 107-2314-B-038-042) and Taipei Medical University (TMU 105-AE1-B03, TMU 106-5400-004-400, TMU 106-5310-001-400, DP2-107-21121-01-N-05 and DP2-108-21121-01-N-05-01).

All procedures involving animals in this protocol were approved by the Institutional Animal Care and Use Committee, with the approval of the Ethics Committee for animal use in Taipei Medical University (approval no. LAC-2018-0574; 15.03.2019).
1. Labeling of MSCs with SPIO Nanoparticles
<ol>
	<li>To label MSCs with SPIO, add 6 mL of labeling media (25 &#181;g/mL of SPIO in Dulbecco&#39;s modified Eagle medium [DMEM] with no fetal bovine serum [FBS]) to a T75 flask containing MSCs (80% confluency).</li>
	<li>Incubate the cells with the labeling media in a CO2 incubator (37 &#176;C, 5% CO2) without shaking. After 24 h, gently remove the labeling media using a sterile Pasteur pipette with a plastic tip attached to a vacuum. Wash the cells monolayer 2x with 6 mL of phosphate buffered saline (PBS) to remove any traces of uninternalized SPIO.
	<ol>
		<li>To determine whether the cells have been successfully labeled, check the labeled cells under a fluorescent microscope or confocal microscope if the SPIO are tagged with a fluorophore (i.e., like those used here; Figure 1B,C). Alternatively, perform Prussian blue staining for the cells (see steps 6.2&#8211;6.4 for the staining protocol).</li>
	</ol>
	</li>
	<li>Harvest adherent cells by treatment with 3 mL of trypsin and incubate at 37 &#176;C. After 5 min of incubation, add 7 mL of pre-warmed DMEM media with 10% FBS (v/v) to inactivate the trypsin. Collect the cell suspension using a pipette into a 15 mL conical tube.</li>
	<li>Centrifugate the cell suspension at 300 x g for 5 min. Discard the supernatant and resuspend the cell pellet in PBS. Count the viable cells using trypan blue dye and a hemocytometer. 
	NOTE: The cell pellet of the labeled cells will appear as a dark color due to iron loading (Figure 1D). This protocol is relevant for MSC labeling and MRI imaging. The procedure for MSC labeling6 has been previously optimized, and only the steps to prepare labeled MSCs to track in vivo are included here, since in vivo tracking is the focus. The protocol for culturing and labeling of other cell types should be optimized by the researcher.</li>
	<li>Adjust the cell concentration using PBS to 150,000 cells (or a number that results in a sufficient MRI signal) in 18 &#181;L of PBS (or the total volume that will be used in the intranasal delivery procedure). 
	NOTE: It was noticed that a cell concentration higher than 150,000 cells/18 &#181;L of PBS leads to cell aggregation, which may affect the efficiency of intranasal delivery. If a higher number of cells is needed for intranasal delivery, increase the total volume of cell suspension and increase the number of intranasal administrations, as intranasal administration is a non-invasive procedure, and multiple dosing is possible.</li>
</ol>
2. Controlled Cortical Impact (CCI) Injury
NOTE: In this protocol, male C57 BL/6 mice (7&#8211;8 weeks old) were kept in a 12/12 h light/dark cycle with ad libitum access to food and water.
<ol>
	<li>To prepare each mouse for CCI injury, administer the zolazepam (50 mg/kg) and xylazine (20 mg/kg) anesthetizing cocktail via intraperitoneal (i.p.) injection (1 mL/kg). Ensure that the depth of anesthesia is sufficient by a lack of toe-pinch response. Alternatively, place the mouse in a chamber supplied with 2%&#8211;4% isoflurane for 60 s. </li>
	<li>Shave the fur of the dorsal surface of the skull between the ears using an electronic hair clipper. Clean the shaved area several times using a sterile cotton swab soaked in iodine. Use a cotton swab soaked in 70% ethanol to clean off the iodine.</li>
	<li>Place the anesthetized mouse in the stereotactic frame and secure the mouse using ear bars and nose bars. Make a midsagittal incision (approximately 2.5 cm) in the shaved skin using sterile scissors to access the surface of the skull.</li>
	<li>Remove the tissue on the bone using a cotton pad to expose the skull. Clean the skull surface using a cotton swab soaked in a 3% H2O2 for 10 s, then clean it with a dry cotton pad. 
	NOTE: The skull sutures and both bregma and lambda can now be easily identified.</li>
	<li>Identify the coordinates of choice on the skull surface for the CCI injury and draw a circle (4 mm diameter) around the coordinates using a pencil or proper marker. 
	NOTE: In this protocol, the coordinates at anteeroposterior (AP) -2.0 mm and mediolateral (ML) +1.5 mm were used for CCI induction.</li>
	<li>Use a microdrill and round burr (0.5 mm diameter) to thin the skull at the marked circle. Avoid applying pressure while drilling, as drilling through the bone may cause damage to the brain parenchyma. Clean bone dust away using a clean and dry cotton swab.</li>
	<li>Gently remove the bone flap using sterile fine forceps to expose the dura mater while keeping it intact. Remove the mouse from the stereotactic frame that was used for pre-injury preparation and place it into the stereotactic frame of the CCI device.</li>
	<li>Stabilize the head of the mouse using the ear bars and nose bars. Make sure the head of the mouse is level in the rostral-caudal direction and adjust the nose bars, if needed.</li>
	<li>Follow the instructions on the control box to zero the impactor tip to the exposed cortical surface. Make sure that the impactor tip is aligned directly above the desired cortex coordinates to be impacted using the X and Y control wheels on the base of the impactor.</li>
	<li>Set the experiment parameters using the control box with a velocity of 5 m/s, dwell time of 250 ms, and injury depth of 1 mm to induce mild injury in the mouse.</li>
	<li>Induce injury by pressing the &#8216;&#8217;impact&#8217;&#8217; button on the control box. Swab any bleeding that occurs using a sterile cotton swab.</li>
	<li>Remove the mouse from the stereotactic frame and close the incision using silk surgical sutures. Do not use metal clips to close the surgical site, since the mouse will be subjected to a magnetic field for MRI.</li>
	<li>Apply topical antibiotics (bacitracin neomycin) to the surgical site to prevent infections. Keep the mouse on the heating pad and monitor it closely during the recovery phase.</li>
	<li>Administer ketoprofen (2.5 mg/kg, IP) daily for 3 days after surgery, unless the ketoprofen administration contradicts with the study goals.</li>
</ol>
3. Intranasal Delivery
<ol>
	<li>At 1 day post-CCI induction, administer the zolazepam (50 mg/kg) and xylazine (20 mg/kg) anesthetizing cocktail via i.p. injection. Ensure that the mouse is deeply anesthetized by lack of toe-pinch response.</li>
	<li>Prepare the mouse for intranasal delivery of MSCs by hyaluronidase treatment.
	<ol>
		<li>Grab the mouse&#8217;s scruff and turn on its back firmly while immobilizing the skull. Place the tip of a pipette that contains hyaluronidase in sterile PBS (4 U/&#181;L) near the nostril of the mouse at a 45&#176; angle.</li>
		<li>Administer 3 &#181;L of hyaluronidase suspension in each nostril. Keep the mouse immobilized and facing upward on a clean pad for 5 min. Repeat hyaluronidase treatment 4x (total of 100 U hyaluronidase suspension).</li>
	</ol>
	</li>
	<li>After hyaluronidase treatment, keep the treated mouse on a clean pad facing up for 30 min.</li>
	<li>To deliver MSCs into the brain, hold the mouse firmly, as described in step 3.2.1. Administer 3&#8201;&#181;L/nostril of MSC suspension with a 3&#8201;s interval. Keep holding the mouse in the same position for 30 s until the sample drops have completely disappeared. 
	NOTE: Avoid forming air bubbles during administration.</li>
	<li>Repeat the administration with a 2 min interval up to 3x. 
	NOTE: The total number of cells to be delivered is 150,000, such that 18&#8201;&#181;L of the cell suspension can be delivered at a 3 &#181;L dosage for each nostril, 3x each.</li>
	<li>Return the mouse to its cage and monitor it closely until it fully recovers from anesthesia.</li>
</ol>
4. In Vivo Magnetic Resonance Imaging
NOTE: Histological staining of brain tissue has previously been used to confirm the successful delivery of stem cells after intranasal administration. However, this method can only be used as an endpoint of a study, not longitudinally. Using MRI probes to label therapeutic stem cells will allow for longitudinal, non-invasive, in vivo tracking of the cells using MRI. Importantly, this protocol efficiently reduces the number of animals required. In this protocol, MRI scanning was performed at days 1, 7, and 14 post-delivery of MSCs.
<ol>
	<li>To prepare the mouse for MRI scanning, anesthetize the mouse with isoflurane (5% isoflurane in 1 L/min of O2&#160;for induction, 1.5%&#8211;2% isoflurane for maintenance). Perform a toe-pinch to ensure that the mouse reaches the required anesthesia level.</li>
	<li>Place the mouse on the imaging holder and secure its position using taps or any other proper method. Move the holder to the center of the MRI coil (7 T/40 cm magnet) and connect the monitoring connection.</li>
	<li>To acquire T2*-weighted scans using a spin-echo sequence, set the repetition time (TR) to 1500 ms and echo time (TE) to 2.8 ms.</li>
	<li>Use a 16 mm x 16 mm field of view (FOV), 128 x 128 acquisition matrix (MTX), and slice thickness of 0.75 x 0.8 mm2 with four signal averages and a 90&#176; flip angle (FA).</li>
	<li>Retract the mouse holder from the MRI coil center after completing the scans. Return the mouse to its cage and monitor it closely until it completely recovers from anesthesia.</li>
	<li>To track and quantify the labeled MSCs on the T2*-weighted images, use ITK-SNAP software (version 3.8.0)7.
	<ol>
		<li>Transfer the raw data of the MRI scans from the MRI machine&#8217;s computer to the computer used for analysis in a DICOM (digital imaging and communications in medicine) format.</li>
		<li>Run the ITK-SNAP software and load the MRI images by clicking on the File button. Then, click on Open Main Image in the menu. Press on the Open Image button in the display window, then locate and open the MRI images using the Browse button. 
		NOTE: The visualization of labeled cells in the MRI images will appear as hypointense areas. If the image contrast needs to be adjusted, select Tools | Image Contrast.</li>
		<li>Create segmentations of the hypointense areas and lesion or other brain parts by selecting Active Label in the segmentation labels section. Use different label colors for different segments (if the segmentation of more than one part is needed).</li>
		<li>Use the Polygon tool in the Main Toolbar to draw around the hypointense areas representing the SPIO-labeled MSCs. Select Accept, located below the MRI image. The segmented areas will appear as the same color of the active label assigned to that particular segment. Repeat this segmentation step for all MRI slices.</li>
		<li>Develop a 3D map of the segmented areas to represent the MSC distribution in the whole brain by selecting the Scalpel Tool at the bottom of the 3D Toolbar, located in the Segmentation Labels section at the bottom of TheITK-SNAP toolbox. Then, press Accept at the bottom of the created 3D map.</li>
		<li>To perform a quantitative analysis (volume and intensity mean) of the segmented hypointense areas representing labeled cells, press the Segmentation button in the top panel and select Volume and Statistics.</li>
	</ol>
	</li>
</ol>
5. Fixation of the Mouse Brain and Cryosectioning
<ol>
	<li>To fix the mouse brain, perform transcardiac perfusion with 4% paraformaldehyde (PFA) following the last MRI scan, as previously described8.
	<ol>
		<li>Decapitate the head and extract the brain8. Fix the brain with 4% PFA for at least 48 h at 4 &#176;C.</li>
		<li>Dehydrate the brain by immersing it into a 30% sucrose solution at 4 &#176;C until the brain sinks to the bottom of the solution.</li>
	</ol>
	</li>
	<li>Embed the brain in the optimal cutting temperature (OCT) solution and freeze at -20 &#176;C. Section the brain with a cryostat microtome into slices with 14 &#956;m thickness and mount them onto slides. Store the sections slides at -20 &#176;C until further use.</li>
</ol>
6. Prussian Blue Staining
NOTE: Prussian blue staining is commonly used to detect the iron content in SPIO-labeled cells. Here, Prussian blue staining is used to confirm that the hypointense signals in the MRI images correspond to the SPIO-labeled MSCs and not to artifacts. Prussian blue staining is one of the most sensitive histochemical methods used to detect iron in tissues and can be used to identify even a single granule of iron in the cells.
<ol>
	<li>Wash the slides of brain sections with distilled water for 5 min.</li>
	<li>Perform&#160;Prussian blue staining by immersing the slides for 30 min in the staining solution, which contains equal parts hydrochloric acid (10%) and potassium ferrocyanide (10%) prepared immediately before use.</li>
	<li>Wash 3x with distilled water, for 5 min each. Counterstain the sections with nuclear fast red for 5 min. Rinse the slides 2x with distilled water.</li>
	<li>Dehydrate the sections gradually by immersing the slides in 95% and 100% alcohol for 2 min each. Add coverslip with a resinous mounting medium.</li>
	<li>Use a light microscope to detect the stained cells in the brain sections. 
	NOTE: The iron in the labeled cells will appear as blue colored deposits.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Mesenchymal+Stem+Cells+Overexpressing+Fibroblast+Growth+Factor+21+Facilitates+Cognitive+Recovery+and+Enhances+Neurogenesis+in+a+Mouse+Model+of+Traumatic+Brain+Injury.">Transplantation of Mesenchymal Stem Cells Overexpressing Fibroblast Growth Factor 21 Facilitates Cognitive Recovery and Enhances Neurogenesis in a Mouse Model of Traumatic Brain Injury.</a> Journal of Neurotrauma. , (2019).</li><li>Danielyan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+cells+to+the+brain.">Intranasal delivery of cells to the brain.</a> European Journal of Cell Biology. 88 (6), 315-324 (2009).</li><li>Karussis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunological+effects+of+mesenchymal+stem+cell+transplantation+in+patients+with+multiple+sclerosis+and+amyotrophic+lateral+sclerosis.">Safety and immunological effects of mesenchymal stem cell transplantation in patients with multiple sclerosis and amyotrophic lateral sclerosis.</a> Archives in Neurology. 67 (10), 1187-1194 (2010).</li><li>Danielyan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+bone+marrow-derived+mesenchymal+stem+cells,+macrophages,+and+microglia+to+the+brain+in+mouse+models+of+Alzheimer's+and+Parkinson's+disease.">Intranasal delivery of bone marrow-derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease.</a> Cell Transplant. 23, 123-139 (2014).</li><li>Shahror, R. 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The authors have nothing to disclose.

The protocol described here represents general procedures for the SPIO labeling of MSCs and MRI tracking of SPIO-labeled MSCs post-intranasal delivery. The protocol allows the opportunity to study the migration and biodistribution of MSCs post-delivery in vivo in the brain, using a non-invasive method.
MSCs are attractive candidates for stem cell-based therapies for CNS disorders and injuries due to their ability to secrete trophic factors that 1) trigger neurorestorative processes and 2) provide neuroprotection, owing to their anti-inflammatory effects within the injury area9,10,11,12. Although long-term MRI tracking and detection of SPIO-labeled MSCs may be limited due to the dilution of intercellular SPIO with cell division, labeled cells can be detected for up to several weeks post-transplantation in the brains of animal models13.
Also described here is the labeling protocol of MSCs with SPIO nanoparticles coated with dextran without transfection agents. Other protocols have been used in the literature14,15,16. However, in all cases, these protocols should be adjusted for cell type, SPIO size, incubation time, and SPIO concentration. MSCs have been shown to have impaired chondrogenic differentiation potential but not adipogenic differentiation upon SPIO labeling17. Therefore, it is highly recommended that differentiation assays be performed prior to stem cell delivery to evaluate the influence of SPIO on the differentiation potency of stem cells. In a previous study, it was demonstrated that MSC labeling with the same SPIO type and concentration used in the here did not affect the osteogenic or adipogenic differentiation potency of MSCs6.
The intranasal route of therapeutic stem cell delivery for brain disorders and injuries is a promising approach for the clinical application of stem cells. However, the intrinsic and molecular mechanisms that dictate the behaviors of stem cells in the nasal cavity remain unclear. Although the intranasal route is widely explored for the delivery of small molecules, the size and biodistribution behavior of the therapeutic stem differ from small molecules. The current protocol demonstrates that MSCs tend to migrate toward the injury site after intranasal delivery.
Here, T2*-weighted images were used to track the SPIO-labeled MSCs. Other reports have used gradient echo imaging. However, susceptibility artefacts are often observed in gradient echo imaging due to intercellular SPIO. In the current protocol, the location of the hypointense areas representing the SPIO-labeled MSCs on T2*-weighted images was the same as the location of the SPIO in brain sections as detected by histological examination (Figure 3). This indicates the adequate sensitivity of T2*-weighted spin echo imaging for SPIO-labeled MSC tracking in the brain.
In summary, the described protocol is beneficial for in vivo stem cell tracking studies of brain injuries and disorders. The&#160;longitudinal tracking of stem cells&#160;in vivo has traditionally been performed by sacrificing animals at multiple timepoints. The current protocol provides a non-invasive and efficient approach for MSCs delivery and tracking, which represents a potential procedure for stem cell-based therapy for brain injuries and disorders in clinical settings.

Mesenchymal stem cells (MSC) are attractive candidates for stem cell-based therapies in treatments of central nervous system (CNS) disorders and injuries in humans. Moreover, MSCs have been used as a vehicle for the delivery of therapeutic proteins at injury sites1,2. In recent years, promising innovations have been developed to establish 1) novel routes of cell delivery and 2) cell tracking for stem cell-based therapies of CNS disorders. The intranasal delivery of stem cells into the brain depends on the ability of cells to bypass the cribriform plate and enter the olfactory bulb partially via a parenchymal route3. The combination of intranasal delivery and the labeling of MSCs with superparamagnetic iron oxide (SPIO) nanoparticles represents a promising approach for clinical applications of MSCs in treating CNS disorders, since SPIO nanoparticles are safe probes for magnetic resonance imaging (MRI) and allow non-invasive sensitive longitudinal tracking of MSCs post-delivery by MRI3,4,5. Furthermore, intranasal delivery is a safe and non-invasive route that allows repeated administration within a short period of time.
This article describes a highly sensitive and non-invasive technique for tracking MSCs in vivo post-intranasal delivery in a mouse model of traumatic brain injury (TBI), which employs SPIO-labeled cells and MRI. One important advantage of the SPIO labeling is the sensitive detection of SPIO in tissue by MRI, which makes it possible to track cells efficiently and non-invasively. The SPIO nanoparticles used here are commercially available and tagged with a fluorescein isothiocyanate (FITC) fluorophore, which allows for the detection of SPIO in tissue without immunostaining or additional processing. Furthermore, it is possible to perform longitudinal real-time tracking and investigate the biodistribution of the delivered MSCs.

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		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Cell culture supplies (Plastics)</td>
			<td style="width:197px;">ThermoFisher Scientific</td>
			<td style="width:179px;">Varies</td>
			<td style="width:171px;">Replaceable with any source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Disposable Microtome Blade</td>
			<td style="width:197px;">VWR</td>
			<td style="width:179px;">95057-832</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">D-MEM/F-12 (1X) with GlutaMAX</td>
			<td style="width:197px;">GIBCO</td>
			<td style="width:179px;">10565-018</td>
			<td style="width:171px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:181px;">Embedding medium for frozen tissue specimens (O. C. T.)</td>
			<td style="width:197px;">Sakura Finetek</td>
			<td style="width:179px;">4583</td>
			<td style="width:171px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:197px;">GIBCO</td>
			<td style="width:179px;">12662-029</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Fluorescence Wild Field Microscope</td>
			<td style="width:197px;">Olympus</td>
			<td style="width:179px;">Olympus BX43</td>
			<td style="width:171px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Forcept</td>
			<td style="width:197px;">Fine Science Tools</td>
			<td style="width:179px;">11293-00</td>
			<td style="width:171px;">Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Gentamicin (10 mg/mL)</td>
			<td style="width:197px;">GIBCO</td>
			<td style="width:179px;">15710-064</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Hair clipper</td>
			<td style="width:197px;">Pet Club</td>
			<td style="width:179px;">PC-400</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Head Trauma Contusion device</td>
			<td style="width:197px;">Precision Systems and Instrumentation</td>
			<td>Model TBI-0310</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Hyaluronidase from bovine testes</td>
			<td style="width:197px;">MilliporeSigma</td>
			<td style="width:179px;">H3506</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:181px;">ITK-SNAP Software</td>
			<td style="width:197px;">Penn Image Computing and Science Laboratory (PICSL) at the University of Pennsylvania, and the Scientific Computing and Imaging Institute (SCI) at University of Utah</td>
			<td>ITK-SNAP 3.8.0</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Ketamine (Ketavet)</td>
			<td style="width:197px;">Pfizer</td>
			<td style="width:179px;">778-551</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Mice</td>
			<td style="width:197px;">National Laboratory Animal Center, Taiwan</td>
			<td style="width:179px;">C57BL6</td>
			<td style="width:171px;">Wild type mice strain used in the study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:181px;">Microdrill</td>
			<td style="width:197px;">Nakanishi</td>
			<td style="width:179px;">NE50</td>
			<td style="width:171px;">Combine with Burrs for generating the bone window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Microtome</td>
			<td style="width:197px;">Leica</td>
			<td style="width:179px;">RM2265</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Mouse (C57BL/6) Mesenchymal Stem Cells</td>
			<td style="width:197px;">GIBCO</td>
			<td style="width:179px;">S1502-100</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">MRI scanner</td>
			<td style="width:197px;">Bruker Biospec</td>
			<td></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Phosphate Buffer Saline (PBS)</td>
			<td style="width:197px;">Corning Cellgro/ThermoFisher</td>
			<td>21-031-CV</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Povidone-iodine 7.5%</td>
			<td style="width:197px;">Purdue product L.P.</td>
			<td></td>
			<td style="width:171px;">Surgical scrub</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Prussian Blue Stain</td>
			<td style="width:197px;">Abcam</td>
			<td style="width:179px;">ab150674</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Scissor</td>
			<td style="width:197px;">Fine Science Tools</td>
			<td style="width:179px;">14084-08</td>
			<td style="width:171px;">Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Stereotaxic frame</td>
			<td style="width:197px;">Kopf Instruments</td>
			<td style="width:179px;">Model 900</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:181px;">Superparamagnetic iron oxide (SPIO) nanoparticles</td>
			<td style="width:197px;">BioPAL</td>
			<td style="width:179px;">Molday ION EverGreen, CL-50Q02-6A-51</td>
			<td style="width:171px;">stem cells labeling for in vivo tracking using MRI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Suture monofilament</td>
			<td style="width:197px;">Ethicon</td>
			<td style="width:179px;">G697</td>
			<td style="width:171px;">Suture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:181px;">Timer</td>
			<td style="width:197px;">Wisewind</td>
			<td style="width:179px;"></td>
			<td style="width:171px;">Replaceable with any source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">TrypLE</td>
			<td style="width:197px;">GIBCO</td>
			<td style="width:179px;">12604-013</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:181px;">Xylazine (Rompun)</td>
			<td style="width:197px;">Bayer</td>
			<td style="width:179px;">QN05 cm92</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

tracking superparamagnetic iron oxide-labeled mesenchymal stem cells using mri after intranasal delivery in a traumatic brain injury murine model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Twenty-four hours following intranasal delivery, the SPIO-labeled MSCs were detected as strong hypointense areas medial to the cortical injury on T2*-weighted images (Figure 2B), indicating the targeted migration of SPIO to the injury site. This migration remained visible up to 14 days post-delivery, as the hypointense signals were found to be visible without significant reduction for this time period (Figure 2B). The injured animals treated with PBS showed no hypointense areas, indicating that the observed hypointense areas correspond to the SPIO labeled MSCs and not to signal artifacts (Figure 2A) The biodistribution of the labeled MSCs that were observed&#160;in vivo&#160;with MRI was visualized using 3D reconstruction (Figure 2C,D). The migration of MSCs to the injured cortex was confirmed histologically by Prussian blue staining and FITC channel detection of the FITC-tagged SPIO in the labeled MSCs (Figure 3A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60450/60450fig1.jpg" /> 
Figure 1: Schematic flowchart of the protocol and in vitro confirmation of SPIO uptake by MSCs. (A) MSCs were incubated with SPIO for 24 h for labeling. Then, the labeled MSCs were delivered into a TBI mouse model via an intranasal (IN) route. MRI at different timepoints was performed to track the labeled MSCs. Confirmation of sufficient labeling of MSCs by SPIO was achieved by (B) fluorescence microscopy and (C) confocal microscopy using the FITC channel, since SPIO nanoparticles were tagged with FITC. (D) The cell pellet of the labeled MSCs appeared dark in color due to iron loading. FITC = fluorescein isothiocyanate; SPIO = superparamagnetic particles of iron oxide; MSCs =&#160;mesenchymal stem cells; MRI = magnetic resonance imaging; IN = intranasal; TBI = traumatic brain injury. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60450/60450fig2.jpg" /> 
Figure 2: Real-time MRI enables the detection and tracking of SPIO-labeled MSC migration toward injury sites in the brains of TBI-induced mice. (A) Mice were subjected to TBI, followed by treatment with PBS or SPIO-labelled MSCs, administered via an intranasal route 24 h after injury. Coronal sections of T2*-weighted images showed the labeled MSCs as a hypointense area (arrowhead) on the edge of the injury site (outlined area) at 1, 7, and 14 days post-delivery. The PBS-treated mice show no hypointense area. (B) Segmentation process of the injury site area (green) and labeled MSCs (red) based on coronal T2*-MRI images. (C) 3D reconstruction of the mouse brain treatment based on T2*-weighted images illustrating the biodistribution of SPIO-labeled MSCs in the brain 14 days post-delivery. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60450/60450fig3.jpg" /> 
Figure 3: Histological analysis confirms the presence of SPIO-labeled MSCs in the brains of the treated animals. Prussian blue staining of brain sections of a (A) mouse treated with PBS (control) and (C) mouse treated with SPIO-labeled MSCs. SPIO-positive cells were detected in MSC-treated mouse (boxed cells, blue), while the control mouse showed no positive cells at the injury site in the cortex at 14 days post-delivery, confirming MRI observations. Fluorescence microscopy analysis of the cortex of a (B) control mouse treated with PBS and (D) mouse treated with SPIO-labeled MSCs was conducted 14 days post-delivery. The analysis revealed the presence of FITC-tagged SPIO-positive cells (boxed cells, green) at the injured cortex in the MSC-treated mouse, but no FITC signals were observed in the cortex of the PBS-treated mouse. Scale bars = 50 &#181;m, unless stated otherwise. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model at JoVE.com

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

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7.5K Views.  Taipei Medical University and National Health Research Institutes. Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Video: Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...