Name: intra-arterial delivery of neural stem cells to the rat and mouse brain: application to cerebral ischemia
Uploaded: 2020-06-26T13:30:00
Description: Watch this Scientific Journal Video about Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia at JoVE.com

This research was supported by the following: AHA Award 14SDG20480186 for LC, Subject innovation team of Shanxi University of Chinese Medicine 2019-QN07 for BZ, and Kentucky Spinal Cord and Head Injury Research Trust grant 14-12A for KES and LC.

All the procedures on animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky, and appropriate care was taken to minimize stress or pain associated with surgery.
1. Preparation of injection catheter and surgical hooks
<ol>
	<li>Construct the injection catheter (Figure 1). Gather necessary materials including: MRE010, MRE025, and MRE050 tubing, 20 G, 26 G and 27 G injection needles (<strong class="xfi...

<ol>
	<li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+research+in+2017:+surgical+progress+and+stem-cell+advances.">Stroke research in 2017: surgical progress and stem-cell advances.</a> The Lancet. Neurology. 17, 2-3 (2018).</li><li>Bliss, T., Guzman, R., Daadi, M., Steinberg, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+transplantation+therapy+for+stroke.">Cell transplantation therapy for stroke.</a> Stroke. 38, 817-826 (2007).</li><li>Boese, A. C., Le, Q. E., Pham, D., Hamblin, M. H., Lee, J. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Stem+Cells+the+Next+Generation+of+Stroke+Therapeutics.">Are Stem Cells the Next Generation of Stroke Therapeutics.</a> Stroke. 49, 1056-1057 (2018).</li><li>Wechsler, L. R., Bates, D., Stroemer, P., Andrews-Zwilling, Y. S., Aizman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Therapy+for+Chronic+Stroke.">Cell Therapy for Chronic Stroke.</a> Stroke. 49, 1066-1074 (2018).</li><li>Muir, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trial+design+for+stem+cell+therapies+in+stroke:+What+have+we+learned.">Clinical trial design for stem cell therapies in stroke: What have we learned.</a> Neurochemistry International. 106, 108-113 (2017).</li><li>Guzman, R., Janowski, M., Walczak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-Arterial+Delivery+of+Cell+Therapies+for+Stroke.">Intra-Arterial Delivery of Cell Therapies for Stroke.</a> Stroke. 49, 1075-1082 (2018).</li><li>Misra, V., Lal, A., El Khoury, R., Chen, P. R., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+delivery+of+cell+therapies+for+stroke.">Intra-arterial delivery of cell therapies for stroke.</a> Stem Cells and Development. 21, 1007-1015 (2012).</li><li>Argibay, B., 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=Intraarterial+route+increases+the+risk+of+cerebral+lesions+after+mesenchymal+cell+administration+in+animal+model+of+ischemia.">Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia.</a> Scientific Reports. 7, 40758 (2017).</li><li>Kelly, S., 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=Transplanted+human+fetal+neural+stem+cells+survive,+migrate,+and+differentiate+in+ischemic+rat+cerebral+cortex.">Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 11839-11844 (2004).</li><li>Chen, L., Swartz, K. R., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+microport+technique+for+applications+in+cerebrovascular+research.">Vessel microport technique for applications in cerebrovascular research.</a> Journal of Neuroscience Research. 87, 1718-1727 (2009).</li><li>Fischer, U. M., 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=Pulmonary+passage+is+a+major+obstacle+for+intravenous+stem+cell+delivery:+the+pulmonary+first-pass+effect.">Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect.</a> Stem Cells and Development. 18, 683-692 (2009).</li><li>Misra, V., Ritchie, M. M., Stone, L. L., Low, W. C., Janardhan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+in+ischemic+stroke:+role+of+IV+and+intra-arterial+therapy.">Stem cell therapy in ischemic stroke: role of IV and intra-arterial therapy.</a> Neurology. 79, 207-212 (2012).</li><li>Muir, K. W., Sinden, J., Miljan, E., Dunn, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+delivery+of+stem+cells.">Intracranial delivery of stem cells.</a> Translational Stroke Research. 2, 266-271 (2011).</li><li>Boltze, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dark+Side+of+the+Force+-+Constraints+and+Complications+of+Cell+Therapies+for+Stroke.">The Dark Side of the Force - Constraints and Complications of Cell Therapies for Stroke.</a> Frontiers in Neurology. 6, 155 (2015).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+noncontact+speckle+contrast+diffuse+correlation+tomography+of+cerebral+blood+flow+in+rats.">Noninvasive noncontact speckle contrast diffuse correlation tomography of cerebral blood flow in rats.</a> Neuroimage. 198, 160-169 (2019).</li><li>Wong, J. K., 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=Attenuation+of+Cerebral+Ischemic+Injury+in+Smad1+Deficient+Mice.">Attenuation of Cerebral Ischemic Injury in Smad1 Deficient Mice.</a> PLoS One. 10, 0136967 (2015).</li><li>Zhang, B., 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=Deficiency+of+telomerase+activity+aggravates+the+blood-brain+barrier+disruption+and+neuroinflammatory+responses+in+a+model+of+experimental+stroke.">Deficiency of telomerase activity aggravates the blood-brain barrier disruption and neuroinflammatory responses in a model of experimental stroke.</a> Journal of Neuroscience Research. 88, 2859-2868 (2010).</li><li>Walker, T. L., Yasuda, T., Adams, D. J., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+doublecortin-expressing+population+in+the+developing+and+adult+brain+contains+multipotential+precursors+in+addition+to+neuronal-lineage+cells.">The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells.</a> The Journal of Neuroscience. 27, 3734-3742 (2007).</li><li>Progatzky, F., Dallman, M. J., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+seeing+to+believing:+labelling+strategies+for+in+vivo+cell-tracking+experiments.">From seeing to believing: labelling strategies for in vivo cell-tracking experiments.</a> Interface Focus. 3, 20130001 (2013).</li><li>Bertrand, L., Dygert, L., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Ischemic+Stroke+and+Ischemia-reperfusion+in+Mice+Using+the+Middle+Artery+Occlusion+Technique+and+Visualization+of+Infarct+Area.">Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area.</a> Journal of Visualized Experiments. , (2017).</li><li>Leda, A. R., Dygert, L., Bertrand, L., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Microsurgery+Infusion+Technique+for+Targeted+Substance+Delivery+into+the+CNS+via+the+Internal+Carotid+Artery.">Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery.</a> Journal of Visualized Experiments. , (2017).</li><li>Chua, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+injection+of+neural+stem+cells+using+a+microneedle+technique+does+not+cause+microembolic+strokes.">Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes.</a> Journal of Cerebral Blood Flow and Metabolism. 31, 1263-1271 (2011).</li><li>Potts, M. B., Silvestrini, M. T., Lim, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Devices+for+cell+transplantation+into+the+central+nervous+system:+Design+considerations+and+emerging+technologies.">Devices for cell transplantation into the central nervous system: Design considerations and emerging technologies.</a> Surgical Neurology International. 4, 22-30 (2013).</li><li>Duma, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+intracerebroventricular+(ICV)+injection+of+autologous,+non-engineered,+adipose-derived+stromal+vascular+fraction+(ADSVF)+for+neurodegenerative+disorders:+results+of+a+3-year+phase+1+study+of+113+injections+in+31+patients.">Human intracerebroventricular (ICV) injection of autologous, non-engineered, adipose-derived stromal vascular fraction (ADSVF) for neurodegenerative disorders: results of a 3-year phase 1 study of 113 injections in 31 patients.</a> Molecular Biology Reports. 46, 5257-5272 (2019).</li></ol>

Stem cell therapy for neurological diseases is still at an early exploratory stage. One major issue is there is no established method for sufficient delivery of SCs or NSCs into the brain.
Although exogenous SCs/NSCs can be detected in the brain following intravenous (IV), intraperitoneal (IP) or intraparenchymal/intracerebral injection, each delivery approach has drawbacks. The detectable population within the brain is estimated to be very low with peripheral injection (IV or IP), representin...

Stem cell (SC) therapy holds tremendous potential as a treatment for neurological diseases, including stroke, head trauma and dementia1,2,3,4,5,6. However, an efficient method to deliver exogenous SCs to the diseased brain remains problematic2,6,7,8,9,10,11,12,13. SCs delivered through peripheral delivery routes, including intravenous (IV) or intraperitoneal (IP) injection, are subject to first-pass filtering in the microcirculation, especially in the lung, liver, spleen and muscle8,9,13,14, increasing chances of accumulation of cells in non-target areas. The invasive intracerebral injection method results in localized brain tissue damage and a very restricted distribution of SCs near the injection site2,6,8,14,15,16. We have recently established a catheter-based intra-arterial injection method to deliver exogenous neural SCs (NSCs), which is described here applied in a rodent model of focal ischemic stroke. We induce transient (1 h) ischemia-reperfusion injury in one hemisphere using a silicone rubber coated filament to occlude the left middle cerebral artery (MCA) in the mouse or rat17,18,19. In this model we have reproducibly observed approximately 75-85% depression of cerebral blood flow (CBF) in ipsilateral hemisphere with Laser Doppler or Laser speckle imaging17,19, yielding consistent neurological deficits17,18,19.
For time-saving purposes, the video is set to play at twice the normal speed and routine surgical procedures such as skin preparation and wound closure with suture and the use and setup of the motorized syringe pump are not presented. The method of intra-arterial delivery of NSCs is demonstrated in the context of the middle cerebral artery occlusion (MCAO) model of experimental stroke in rodents. Therefore, we include the transient ischemic stroke procedure in order to later demonstrate how the second surgery, the intra-arterial injection, is performed using the previous surgical site on the same animal. The feasibility of intra-arterial NSC delivery in rodent stroke models is demonstrated by assessing the distribution and survival of exogenous NSCs. The efficacy of NSC therapy to attenuate brain pathology and neurological dysfunction will be reported separately.

<table>
	<tbody>
		<tr>
			<td>20 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305175</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>26 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305111</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>27 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305136</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>4-0 NFS-2 suture with needle</td>
			<td>Henry Schein Animal Health</td>
			<td>56905</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>6-0 nylon suture</td>
			<td>Teleflex/Braintree Scientific</td>
			<td>104-s</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7922</td>
			<td>cell detachment solution</td>
		</tr>
		<tr>
			<td>blade</td>
			<td>Bard-Parker</td>
			<td>10</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Buprenorphine-SR Lab</td>
			<td>ZooPharm</td>
			<td>Buprenorphine-SR Lab&#174;</td>
			<td>analgesia (0.6-1 mg/kg over 3 d)</td>
		</tr>
		<tr>
			<td>Calcium/magnisum free PBS</td>
			<td>VWR</td>
			<td>02-0119-0500</td>
			<td>NSC dissociation</td>
		</tr>
		<tr>
			<td>DCX antibody</td>
			<td>Millipore</td>
			<td>AB2253</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Invitrogen</td>
			<td>180063</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>50562-1</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MCAO filament for mouse</td>
			<td>Doccol</td>
			<td>702223PK5Re</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MCAO filament for rat</td>
			<td>Doccol</td>
			<td>503334PK5Re</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MRE010 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE010</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>MRE025 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE025</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>MRE050 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE050</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>Nu-Tears Ointment</td>
			<td>NuLife Pharmaceuticals</td>
			<td>Nu-Tears Ointment</td>
			<td>eye care during surgery</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps - SuperGrip Tips JF-5TC Angled</td>
			<td>Fine Science Tools</td>
			<td>00649-11</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps - SuperGrip Tips JF-5TC Straight</td>
			<td>Fine Science Tools</td>
			<td>00632-11</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Pacer Technology</td>
			<td>15187</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Tuj1 antibody</td>
			<td>Millipore</td>
			<td>MAb1637</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>two-component 5 minute epoxy</td>
			<td>Devcon</td>
			<td>20445</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>00400-03</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Zeiss microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td>microscopy</td>
		</tr>
	</tbody>
</table>

intra-arterial delivery of neural stem cells to the rat and mouse brain: application to cerebral ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

GFP-labeled NSCs were readily detected in the ischemic brain, mostly in the ipsilateral hemisphere, especially in the penumbra and along the injury rim (Figure 6). The examiner was single-blinded during imaging and analysis.
For example, at 1 d after injection, NSCs were detected within the mouse hippocampus. A subset of NSCs showed co-expression of the immature neuron marker DCX in the dentate gyrus even at this early time point (Figure 6A</s...

Watch this Scientific Journal Video about Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia at JoVE.com

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Stroke is the leading cause of mortality and disability worldwide. Although thrombolysis and surgical removal of cerebral vascular occlusion can be performed in select patients, there are still an urgent need for effective therapies against stroke. Here, we demonstrate a catheter-based, intra-arterial delivery of stem cells to the ischemic hemisphere as a potential regimen for stroke therapy.First, we introduce how to prepare the injection catheter and surgical hooks. We designed two types of injection catheters for intra-arterial injection. Design one is used for injection of a solution or suspension that does not require flushing of the tube.Design two allows injection of neural stem cells, followed by a flush of the dead volume to deliver the full volume. To build a set of injection catheters, you will need 26 gauge and 20 gauge needles, a 10 to 15 centimeter length of MRE 25 catheter and three centimeter length of MRE 50 and MRE 10 catheters. Cut off both needle tips and polish the metal end on sandpaper to reopen the tube and smooth the edges and then flush with distilled water to clean out the bore and wash off debris.The figure shows a closed-end of a 20 gauge needle after clipping off the needle tip, which is reopened with a smooth bore after polishing. Under a microscope, insert the 20 gauge needle into the end of the MRE 50 tubing. Bend the 26 gauge needle and then penetrate the tubing below the end of the 20 gauge needle tip.Secure the 26 gauge needle with super glue. Then embed the two needle hubs and the tubing and epoxy to build a solid block to enhance strength. You will also need three surgical hooks to hold the tissue in position for better exposure of the surgical sites.Cut a one and a half to two centimeter long needle shaft from a 27 gauge needle and polish both ends on sandpaper until dull. Use a small hemostatic clamp to bend the shaft into a hook at one end and a ring-shaped at the other end. Insert a 10 to 15 centimeter long MRE 25 catheter through the ring and secure with clear surgical tape.Make two more hooks using the same method. The hooks and catheter system should be soaked overnight in seventy percent ethanol for sterilization. All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.We use ENT embryonic mouse brain to culture GFP, positive neural stem cells. GFP positive embryos can be readily visualized in the FITC channel on a fluorescent microscope. While wild-type embryos show no fluorescence using the same exposure.The autofluorescence of wild-type embryos is visible only when the exposure time is increased 100 times. Embryonic cortex is collected and used to establish neural stem cell cultures following the manufacturer's protocol. These neural stem cells can form neurospheres and can be passage over 10 generations and they can differentiate into glia are neurons.Panel of embryonic stem cell markers such as nanog, SSEA1, Oct4 and Sox3 can be used to identify the stem cell properties while in neurospheres. Neurospheres were cultured in media, following the manufacturer's protocol and use between passages three and five for neural stem cell delivery. Ischemic stroke was induced in the left hemisphere for both mice and rats, using a filament based, middle cerebral artery occlusion, MCAO with some modifications depending on the species.In mice, the filament is inserted through the common carotid artery, CCA and advanced into the internal carotid artery, ICA. While in rats, the filament is introduced through the external carotid artery, ECA into the ICA because of the larger working space. Here we demonstrate the mouse surgery.After making a midline incision along the neck, separate the underlying tissues using blunt dissection. Use the three microsurgical hooks made from 27-gauge needle shafts to achieve a better visual field. The black square indicates the surgical area for the next step.The muscle layer above the left CCA is carefully open to expose the left CCA and vagus nerve. Follow the CCA until you reach and identify the bifurcation. Caution should be exercised not to stretch, displace or squeeze this CCA or vagus nerve.Carefully open the clear connective tissue, covering the CCA and vagus nerve. Free the space underneath this CCA with a tip of the forceps and isolate the CCA stem as much as possible. Thread a double-stranded six O braided nylon surgical suture underneath the CCA.Cut the middle point to leave to sutures underlying the CCA. Place another nylon suture under the CCA. Tie a slipknot on the upper suture, keeping it loose.Separate the two lower sutures. Tie a slip knot with the middle suture, keeping it loose for now. Tie a surgeon's knot with a lower suture and move it to the proximal end as far as possible.Trim down the suture ends. Further expose the CCA beyond the bifurcation. Push the upper loose slipknot to the bifurcation and tighten it to prevent backflow of blood.Lay the silicon rubber coated 70 nylon MCAO suture next to the CCA. Using micro scissors, cut a small incision on the CCA, next to the lower knot. Insert the tip of the MCAO suture into the CCA through this incision and advance it to the upper knot.Adjust the position of the middle knot and tighten it until it can hold the MCAO suture in position. Do not excessively tighten the knot. Release the upper knot, but make another loose slip knot at the same location.Advance the MCAO suture into the ICA. Confirm the MCAO suture passes the bifurcation into the ICA. The path of the suture should be relatively straight, entering deeper into the tissue as it advances.Check the silver marker on the suture end. Make sure it reaches the bifurcation. Check the direction of the MCAO suture again.Adjust upper and middle knots to ensure no blood leakage. Further advance the MCAO suture, tighten the two upper knots to secure the MCAO suture in place. Start recording time for one hour of ischemia.After one hour of ischemia, we anesthetize the animal and reopen the skin wound. Expose the surgical area. Locate upper and middle slipknots.Gently release the upper knot. Gently pull out the MCAO suture until the white silicone rubber part reaches the middle knot. Tighten the upper knot to hold the MCAO suture in place to avoid bleeding.Gently release the middle slipknot. Pull out the MCAO suture until its end passes the upper knot. Tighten the upper knot.Place the middle slipknot right above the incision on the CCA. Tighten the middle slipknot. Remove the upper knot.Trim suture ends of the middle knot. Close the skin wound and allow the animal to recover. Based on our experience, neural stem cells safely injected through this arterial route between one and three days after stroke.Again, there are minor variations between mouse and rat surgeries for neural stem cell injection. And here we demonstrate the mouse surgery. On day three post-stroke, anesthetize the animal, reopen the wound and re-expose the CCA.Lay a double-stranded surgical suture under the CCA and cut it at the midpoint to get two sutures. Place the two sutures properly. Make a slipknot with the upper suture.Place it right below the bifurcation. Tighten the upper slipknot. Make another slipknot with the middle suture.Cut a small incision right above the trimmed knot remaining from the stroke surgery. Trim the tip of the MRE 10 catheter with an angle of approximately 45 degrees. Use this sharp end to enter the incision on the CCA.Be careful to properly adjust the entry angle and the force used to insert the catheter. Otherwise, the catheter may not enter the CCA or may puncture it. Insert the catheter and advance until the tip reaches the upper knot.Tighten the middle slipknot to hold the catheter in place and ensure no blood linkage. Release the upper knot. Make a slip knot with the upper suture.Adjust the angle of the catheter within the CCA and the positions of both knots until a quick backflow of blood is visible, indicating successful communication between the catheter and CCA flow. Start the syringe pump. The blood in the catheter will be flushed back into the CCA.Terminate the injection after five minutes. Ligate the upper knot. Release the middle knot and withdraw the catheter.Ligate the middle knot. Trim all knots. Close the wound and allow the animal to recover.Following intra-arterial injection, GFP labeled neural stem cells are mostly found in the ipsilateral hemisphere. Here, we show the distribution in the hippocampal dentate gyrus at one day after injection. In the ipsilateral hemisphere, some of the GFP positive neural stem cells localized in the cortex and striatum, express double cort, a marker of immature neurons.These are representative images from sham and stroke animals with or without neural stem cell injection at seven days after injection. At 30 days after delivery, only minor auto fluorescence can be detected through FITC channel in the animals that receive vehicle injection. While in stem cells injected animals, GFP labeled neural stem cells can be found in the injured brain with expression of various cell markers like the gleo marker GFP or the neuronal marker tujuan.The white arrows in each image indicate neural stem cells expressing GFP or tujuan markers respectively. In conclusion, intra-arterial injection is a feasible method for delivery of neural stem cells for stroke therapy. Neural stem cells can survive and differentiate into different cell types within the injured brain.And finally, the intra-arterial injection method introduced here can also be used for delivery of solutions and suspensions.

Research

JoVE Journal

Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...