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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+therapy+for+subacute+and+chronic+ischemic+stroke.">Neural stem cell therapy for subacute and chronic ischemic stroke.</a> Stem Cell Research &amp; Therapy. 9, 154 (2018).</li><li>Kokaia, Z., Llorente, I. L., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customized+Brain+Cells+for+Stroke+Patients+Using+Pluripotent+Stem+Cells.">Customized Brain Cells for Stroke Patients Using Pluripotent Stem Cells.</a> Stroke. 49, 1091-1098 (2018).</li><li>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=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.

intra-arterial-delivery-neural-stem-cells-to-rat-mouse-brain

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 clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

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 are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo 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 chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

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. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

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 adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

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 pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

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 into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

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 characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver 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

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

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 multispectral diffuse reflectance imaging system.

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

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.