Name: Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus (Video) | JoVE Journal
Uploaded: 2011-07-10T22:00:00.000+00:00
Duration: 8 min 33 s
Description: Learn neural stem cell transplantation into the spinal cord of JHMV-infected mice for studying remyelination in demyelinating diseases. This method enables researchers to investigate cell replacement therapies and functional recovery mechanisms.

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5 minutes

1.	Preparation
<ol>
	<li>Prepare Xylazine-ketamine solution (Ketamine is a controlled substance. Detailed records should be kept and solutions stored in a safe, locked location).</li>
	<li>Clean and sterilize equipment.</li>
	<li>Prepare surgery area by wiping with aseptic agent and then covering with sterile paper toweling, and set up the micromanipulator.</li>
</ol>
2.	Preparation of Cells for Transplant
<ol>
<li>Cells should be resuspended to a concentration of 100,000 cells/É l and, although only 250,000 cells are transplanted per mouse, an
excess of cells is required for syringe loading purposes (prepare at
least 300,000 cells per mouse receiving cells).</li>
<li>Wash cells to be transplanted 3 times in HBSS in a 50 ml conical vial. Count the cells before the final spin.</li>
<li>After the final spin down, decant HBSS and leave vial in upside down position to prevent droplets from reaching the pellet.</li>
<li>Dry inside walls with a UV-irradiated, sterile Kimwipe. Do not allow the pellet to dry.</li>
<li>Holding the tube upright, slowly and very delicately resuspend the cells in half the final desired volume of HBSS.</li>
<li>Measure the total volume by pipetting the majority of the suspension into a pipette tip and then adjusting the dial on the pipetter until the entire suspension is in the pipette tip. This will tell you how much more HBSS is needed for the desired concentration.</li>
<li>Bring volume to the desired amount by adding HBSS.</li>
<li>Place back on ice. Check viability of cells if they must be on ice for longer than 2 hrs.</li>
</ol>
3.	Preparation of mice for surgery and transplantation
<ol>
	<li>Anesthetize the mouse by intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg) in ~100 μl doses or an equivalent anesthetic. The entire procedure from surgical prep to suturing will take 30-40 minutes.</li> 
	<li>(Optional: apply numbered, colored tape to the tail of each mouse to ensure identification)</li>
	<li>Shave the dorsal area of the mouse from the lower back to the neck, and extending 2 cm bilateral from the midline, with electric clippers. The hair should be cut as close as possible (it may be necessary to go over the area several times).</li>
	<li>To remove the remaining hair, apply a thin layer of hair removal cream (Nair) with a gauze tipped applicator.</li>
	<li>After 1-2 minutes, wipe Nair off with gauze wetted lightly with soapy water. The prepared area should be clean bare skin without any stray pieces of hair that could get into the wound during the subsequent surgical procedure. 
Sterilize the prepared area with iodide solution.</li>
</ol>
4.	Laminectomy
<ol>
	<li>Frequently change and/or sterilize gloves throughout the procedure. Position the mouse dorsal side up with head pointing to the left (if you are right handed). Drape animal to ensure sterility and that only the shaved area is exposed. Make a vertical incision (˜1.3cm) over the laminecomy site spanning from about thoracic vertebrae T8 to T12.</li>
	<li>With the graefe forceps held in the left hand, firmly secure the spinal column at T9 (Figure 1A) and lift the mouse up to exaggerate the spinal curvature.</li>
	<li>Use the scalpel to score the junction between T10 and T11, the space between the two spiny protrusions. Further expose the junction by carefully scrapping the muscle layer away to expose the bone (see Figure 1B,C).</li>
	<li>Use the scissors to further clear muscle away from the lamina and around the pedicle with small snips. This will open up a small space between the vertebrae. Slowly and delicately insert one blade of the scissors into this gap and snip the pedicle. Make sure the curvature of the scissors is always positioned laterally, away from the cord. Repeat on the other side. (see Figure 1D,E)</li>
	<li>Lift the lamina to expose the cord and carefully snip it off. Be sure not to leave any free or jagged bone fragments behind. (see Figure 1F)</li>
	<li>Prior to injection, clean any blood away with sterile cotton swabs.</li>
</ol>
5.	Injection of cells
<ol>
<li>Attach the needle with needle nut to the Hamilton syringe and clean them by flushing several times with water, then 70% ethanol, and finally HBSS. Insert plunger after each fill with water, 70% ethanol, or HBSS.</li>
<li>Prepare the Hamilton syringe for cell loading by removing the plunger and loosening the needle nut and pulling the needle away from the syringe to prevent backpressure. Ensure that careful handling of the needle and nut are done with sterile gloves.</li>
<li>Load 15μl of cells into pipette tip and press the tip tightly into the back end of the syringe to load the cells into the syringe.</li>
<li>Insert the plunger about 5mm and then tighten the needle nut.</li>
<li>Depress plunger until some of the cell suspension is seen exiting the needle.</li>
<li>Make sure there are no bubbles in the syringe and lay the syringe down in the horizontal position to prevent the cells from making a gradient by gravity.</li>
<li>Grab hold of the laminectomized mouse by the spinalis dorsi muscle connecting the spines of T8 and T9 (Figure 2B, C).</li> 
<li>Clamp the hemostat to the left (vertical) micromanipulator arm so that the mouse’s front paws are in the air and its rear paws lightly touching a platform of sterile paper towels as in Figures 2A and 2B.</li>
<li>Attach the syringe to the right micromanipulator arm (at a 70° angle) and slide the syringe to the lowest position possible before clamping.</li>
<li>Stabilize the mouse by pinning its tail against the paper towels and slowly lower the syringe (Figure 2B).</li>
<li>Lower the needle towards the cord and insert the needle 1mm into the opposite hemisphere through the dorsal midline (Figure 2C). The tip of the needle should be in the gray matter close to the central canal.</li>
<li>Slowly inject 2.5μl of cells. Inject at a rate of 1μl / 5 seconds. After injecting the cells, wait a 10 seconds and retract the needle a tenth of a turn at a time every 10 seconds until the needle is out of the cord. Pay attention to possible efflux of cell suspension.</li>
<li>Quickly retract the syringe and detach it from the micromanipulator arm. Lay the syringe down horizontally.</li>
<li>Release the mouse and transfer to suturing table.</li>
<li>Repeat steps 5.7-5.14 for each mouse until the syringe is emptied. Sterilize tools (in the sterilizer) and the needle (by wiping with ethanol) between animals. Discard cells and reload if clumping is visible.</li>
<li>Clean the syringe as in step 5.1 between loads.</li>
</ol>
6.	Sutures and post-op care
<ol>
<li>Suture the incision. Suture needle is inserted into the superficial fascia on both sides of the incision. The thread is strung through, pulling superficial fascia together (Figure 3A), thereby covering the exposed spinal cord at the site of removed lamina. Do not suture the cutaneous muscle attached to the skin or the skeletal muscle of the back. Pull the entire thread through, leaving approximately 1/2". Using the needle holder, three knots are formed and thread is trimmed as close to the knot as possible.</li>
<li>Close the incision by applying two to three staples (depending on size of incision) to the skin (Figure 3B). Carefully pull skin away from the mouse to avoid stapling the underlying muscle.</li> 
<li>Using a 26G3/8 needle inject 0.5ml Lactated Ringers sub-cutaneously in the lower back away from the incision.</li>
<li>Place mouse back in its cage. To ensure it is able to breathe
comfortably while under anesthesia, the mouse should be placed on its
side in the cage, avoiding contact between the surgery site and cage
bedding. Cages should be placed on heating pads. </li>
<li>Mice are monitored after anesthesia wears off to ensure bleeding subsides, sutures remain closed, and that mice return to pre-surgery mobility.</li>
<li>Treat mice one time with the analgesic buprenorphine (0.05-0.1 mg/kg) following surgery.</li>
<li>Ensure impaired mice have sufficient access to food and water: water bottles are fitted with extended 3.5 inch spouts and mice that are unable to walk are hand fed water and/or high-calorie dietary supplement (Nutri-Cal, Tomlyn).</li>
</ol>
7. Representative Results:
Desired results will be identifiable by the lack of efflux of the cell suspension during the injection and by the intact appearance of the spinal cord following the procedure. To this end, it is vital to have bright and direct lighting on the mouse’s spine during the laminectomy and during the injection. Optimal lighting is facilitated by fiber optic lighting (Figure 2A).
<img src="/files/ftp_upload/2834/2834fig1.jpg" alt="Rodent surgery process, spinal cord exposure, diagram illustrating experimental method and anatomy." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1- Laminectomy. (A) Once vertebra T9 is solidly held with the Graefe forceps, (B, C) score the spine with scalpel between T10 and T11 to facilitate entry of the micro scissors. (D) Carefully slide the micro scissors through the space between T10 and T11(arrows and inset, E) and cut the pedicles on each side (dashes, E) to free the dorsal lamina. (F) Flip the lamina up rostrally and cut it off.
<img src="/files/ftp_upload/2834/2834fig2.jpg" alt="Brain surgery technique on rodent; imagery of a stereotaxic setup and cortical exposure process." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Injection of NSCs. (A) The general setup of the micromanipulator with the hemostat holding the mouse attached to the left arm and the Hamilton syringe on the right arm at a 70 angle. (B) The hemostat hold the spinalis dorsi muscle connecting the spines of T8 and T9. (C) The needle is lowered through the midline and into the gray matter on the opposite hemisphere, proximal to the central canal.
<img src="/files/ftp_upload/2834/2834fig3.jpg" alt="Surgical technique diagram on rodent wound closure with sutures and staples for tissue repair." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Sutures and wound closure. (A) Sutures are applied to the superficial fascia on both sides of the incision. (B) The incision is closed with 2-3 staples as needed (one staple shown on a wound needing 3).

<ol><li>Bergmann, C. C., Lane, T. E., Stohlman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronavirus+infection+of+the+central+nervous+system%3A+host-virus+stand-off%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Coronavirus infection of the central nervous system: host-virus stand-off.</a> Nat Rev Microbiol. 4, 121-132 (2006).</li>
<li>Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+challenge+of+multiple+sclerosis%3A+how+do+we+cure+a+chronic+heterogeneous+disease%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease.</a> Ann Neurol. 65, 239-248 (2009).</li>
<li>Fleming, J. O., Trousdale, M. D., Bradbury, J., Stohlman, S. A., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+demyelination+induced+by+coronavirus+JHM+%28MHV-4%29%3A+molecular+identification+of+a+viral+determinant+of+paralytic+disease%5BTitle%5D)+AND+%22Microb+Pathog%22%5BJournal%5D)">Experimental demyelination induced by coronavirus JHM (MHV-4): molecular identification of a viral determinant of paralytic disease.</a> Microb Pathog. 3, 9-20 (1987).</li>
<li>Totoiu, M. O., Nistor, G. I., Lane, T. E., Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remyelination%2C+axonal+sparing%2C+and+locomotor+recovery+following+transplantation+of+glial-committed+progenitor+cells+into+the+MHV+model+of+multiple+sclerosis%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis.</a> Exp Neurol. 187, 254-265 (2004).</li>
<li>Carbajal, K. S., Schaumburg, C., Strieter, R., Kane, J., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migration+of+engrafted+neural+stem+cells+is+mediated+by+CXCL12+signaling+through+CXCR4+in+a+viral+model+of+multiple+sclerosis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis.</a> Proc Natl Acad Sci U S A. 107, 11068-11073 (2010).</li>
<li>Hardison, J. L., Nistor, G., Gonzalez, R., Keirstead, H. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+glial-committed+progenitor+cells+into+a+viral+model+of+multiple+sclerosis+induces+remyelination+in+the+absence+of+an+attenuated+inflammatory+response%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response.</a> Exp Neurol. 197, 420-429 (2006).</li>
<li>Blakemore, W. F., Crang, A. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBlakemore%2C+WF+%22Transplantation+of+glial+cells+into+areas+of+demyelination+in+the+adult+rat+spinal+cord%22">Transplantation of glial cells into areas of demyelination in the adult rat spinal cord</a>. , Oxford UP. Oxford. (1992).</li>
<li>Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+stem+cells+differentiate+into+oligodendrocytes+and+myelinate+in+culture+and+after+spinal+cord+transplantation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.</a> Proc Natl Acad Sci U S A. 97, 6126-6131 (2000).</li>
<li>Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+embryonic+stem+cell-derived+oligodendrocyte+progenitor+cell+transplants+remyelinate+and+restore+locomotion+after+spinal+cord+injury%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.</a> J Neurosci. 25, 4694-4705 (2005).</li>
<li>Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+patient+enters+trial+to+test+safety+of+stem+cells+in+spinal+injury%5BTitle%5D)+AND+%22BMJ%22%5BJournal%5D)">First patient enters trial to test safety of stem cells in spinal injury.</a> BMJ. 341, c5724-c5724 (2010).</li>
</ol>

No conflicts of interest declared.

A well-executed transplant will hinge primarily on the careful laminectomy and injection of the cells. The primary pitfall to avoid during a laminectomy is the damaging of the spinal cord. This can occur during the procedure itself or by damage caused by sharp bone fragments left behind following the procedure. To avoid these, ensure that the points of the curved micro scissors are always facing away from the cord and carefully examine the laminectomized spine to ensure that all bone fragments are cleared and that the remaining vertebral structure does not have overtly protruding or jagged edges. 
As previously mentioned, the detection of efflux will be possible if the light is shining brightly and directly onto the exposed spinal cord during the injection. Efflux is most likely to happen with 30 gauge needles (versus 33 gauge) and if the injection is done too rapidly. Though this protocol has given us good results, others have reported longer waiting periods (up to 5 minutes) before retracting the needle following injection 8,9. Also, smaller gauge needles are preferable, but we have observed that some cells are too easily lysed when passed through 33 gauge needles.
To maximize efficiency, a transplant team manning the four different stations (mouse prep, laminectomy, injection, and sutures) is desirable. Furthermore, the timing for each procedure should be optimized to minimize the time the cells are waiting on ice. For example, we transplant our mice in groups of four (the number of doses in each load of the syringe), the person injecting the cells starts loading the syringe after the third mouse has been laminectomized and the person preparing the mice should anesthetize the following group after the second mouse of the previous group has been laminectomized. In this manner, we can transplant cells (or control media) into 40 mice in about 3 hrs even though each mouse will take approximately 30-40 min. 
Cell replacement therapies for the treatment of some CNS disorders are currently in clinical trials 10. There is no substitute for in vivo models of NSC transplantation and our protocol for the engraftment of NSCs into the spinal cords of mice with viral induced demyelination facilitates the use of an important model of MS and can also be easily adapted to other models.

<table><tbody><tr><td>Ketaject</td><td>Phoenix Pharmaceuticals, Inc.</td><td>NDC 57319-542-02</td><td></td></tr><tr><td>Xylazine Hydrochloride</td><td>MP Biomedicals</td><td>158307</td><td></td></tr><tr><td>Nair</td><td>Church &amp;amp; Dwight Co.</td><td></td><td></td></tr><tr><td>10 &amp;mu;l Hamilton Syringe w/ Removable needle</td><td>Hamilton Co</td><td>7635-01</td><td></td></tr><tr><td>Hamilton Needles, 30G or 33 G, &amp;frac12; inch, 30&amp;#x00B0; bevel </td><td>Hamilton Co</td><td>7803-077803-05</td><td>Test the viability of your cells following passage through needles to identify the best gauge to use</td></tr><tr><td>Micro Scissors</td><td>World Precision Instruments, Inc.</td><td>555500S</td><td></td></tr><tr><td>Small Graefe Forceps</td><td>Fine Science Tools</td><td>11053-10</td><td></td></tr><tr><td>Stereotaxic</td><td>Kopf Instruments</td><td>Model 1772 Universal Holder</td><td></td></tr><tr><td>Stereotaxic</td><td>Kopf Instruments</td><td>Model 1773 Electrode Holder</td><td></td></tr><tr><td>Stereotaxic</td><td>Kopf Instruments</td><td>Model 902 small animal stereotaxic</td><td></td></tr><tr><td>Stereotaxic</td><td>Kopf Instruments</td><td>Model 960 left electrode carrier</td><td></td></tr><tr><td>Sutures</td><td>Ethicon Inc.</td><td>95057-064</td><td></td></tr><tr><td>Lactated Ringers</td><td>Hospira Inc.</td><td>NDC 0409-7953-03</td><td></td></tr><tr><td>Staples</td><td>Fine Science Tools</td><td>12032-07</td><td></td></tr><tr><td>Hemostat</td><td>Fine Science Tools</td><td>13010-12</td><td></td></tr><tr><td>Scalpels, sizes 10,11</td><td>Fisher Scientific</td><td>268878, 268879</td><td></td></tr><tr><td>Sutures</td><td>Ethicon Inc.</td><td>1676G</td><td>size 5-0, 3/8&quot; circle, 19mm needle, 45cm braided thread</td></tr><tr><td>Reflex 7 wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7mm Reflex wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td></td></tr><tr><td>Olsen-Hegar needle holder</td><td>Fine Science Tools</td><td>12502-12</td><td></td></tr></tbody></table>

surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

The overall goal of this procedure is to promote remyelination and clinical improvement in animals with established demyelination via transplantation of neural stem cells into the spinal cord of JHMV infected mice. This is accomplished by first preparing the animal. The second step of the procedure is to perform a laminectomy at thoracic vertebrae.10 cells are then injected into the exposed spinal cord. After a successful injection, the wound is closed. Ultimately, presence of mouse neural stem cells in the spinal cord.Parenchyma can be visualized by methods such as fluorescent microscopy, Neuro stem cell transplantation into the spinal cord of JHMB. Infected mice can help answer key questions into the field of cell replacement therapies for the treatment of demyelinating diseases, such as how the cells migrate, proliferate, and differentiate also how the cells affect the central nervous system. And if we can modify the cells or the spinal cord environment they're transplanted into to enhance their therapeutic benefits.Although this method can provide insight into cell replacement therapy in a viral model of demyelination, it can also be applied to other models of demyelination and injury, such as experimental autoimmune encephalomyelitis, EAE, and spinal cord injury. Before preparing cells for injection, prepare all necessary solutions. Next, clean and sterilize all equipment.Prepare the surgical area and set up the micro manipulator to prepare the cells for transplantation trypsin eyes, and resuspend them in a 50 milliliter conical vial. Wash the cells to be transplanted three times in HBSS. Count the cells before the final spin.After the final, spin down decant HBSS and leave the vial in an upside down position to prevent droplets from reaching the pellet. Dry the inside walls with a UV irradiated sterile Kim wipe. Do not allow the pellet to dry.Holding the tube upright slowly and very delicately. Re suspend the cells in half the final desired volume of HBSS. Ultimately, the cells should be at a concentration of 100, 000 cells per microliter.Measure the total volume by pipetting the suspension into a pipette tip, and then adjusting the dial on the pipetter until the entire suspension is in the pipette tip. This will tell you how much more HBSS is needed for the desired concentration. Bring up the cells to the desired volume.By adding HBSS. You may transfer the cells to an eph tube. Place them back on ice.Check the viability of the cells if they must be on ice for longer than two hours. To begin the surgical procedure, confirm the proper level of anesthesia through the toe pinch reflex. Use electric clippers to shave the dorsal area of the mouse from the lower back to the neck.Once the area is shaved, apply a thin layer of hair removal cream to remove any remaining hair. After one to two minutes, wipe the area clean with dry gauze and then with cause lightly soaked in soapy water. The surgical area should now be free of hair.Lastly, sterilize the prepared area with Betadine solution and apply ointment to the eyes. To avoid dryness, position the mouse dorsal side up. Make an incision over the laminectomy site spanning from about thoracic vertebrae.T eight to T 11. Exaggerate the curvature of the spine and locate T 10. It is the first vertebrae with a flat process and is generally two vertebrae rostral to the apex of the curvature of the spine.With the gray forceps, firmly secure the spinal column. At T nine, use a scalpel to score the space between the two spiny protrusions of T 10 and T 11. Next, scrape the muscle layer away to expose the bone underneath, making sure that the curvature of the scissors is pointing away from the cord.Slowly insert one blade of the scissors between T 10 and T 11. Slide the blade between the cord and the pedicle of T 10 and snip the pedicle Repeat on the other side. Lift the lamina to expose the cord underneath and carefully snip it off.Be sure not to leave any free or jagged bone fragments behind. Once all the bone fragments are cleared, wipe any blood away with sterile cotton swabs. First, prepare the Hamilton syringe by attaching the needle with the needle nut.Insert the plunger and flush several times with water. 70%ethanol and HBSS once cleaned. Remove the plunger and loosen the needle nut.Pull the needle away from the syringe to prevent back pressure. When loading the cells load 15 microliters of cells into the syringe by pressing the tip of the pipette tightly into the back of the syringe. Insert the plunger about five millimeters and then tighten the needle nut.Depress the plunger until some of the cell suspension is seen exiting the needle. Make sure there are no bubbles in the syringe, and lay the syringe down horizontally to prevent the cells from making a gradient by gravity. Returning back to the animal, use hemostats to grip the S spinous doci muscle.Connecting the spines of T eight and T nine. Clamp the hemostat to the left micro manipulator arm so that the mouse's front paws are in the air and its rear paws. Lightly touch a platform of sterile paper towels.Attach the syringe to the right micro manipulator arm at a 70 degree angle and slide the syringe to the lowest possible position before clamping. Stabilize the mouse by pinning its tail against the paper towels. Slowly lower the needle towards the cord.Insert the tip one millimeter into the opposite hemisphere through the dorsal midline. The tip of the needle should be in the gray mat or close to, but not in the central canal. Slowly inject 2.5 microliters of cells at a rate of one microliter per five seconds.After injecting the cells, wait 10 seconds and retract the needle. A 10th of a turn. Retract at this pace every 10 seconds until the needle is out of the cord.Injecting the cells too fast or removing the needle too quickly may cause flx of cell suspension. If this occurs, discard the mouse from the study. Once removed from the cord, quickly retract the needle and detach the syringe from the micro manipulator arm.Again, laying it down horizontally. Release the mouse and transfer it to a suturing table. Repeat this procedure for each mouse until the syringe is emptied.Discard the cells and reload if clumping is visible, flushing and cleaning the syringe between loads. After the injection, the animal is transferred to a separate location To be sutured. Pass the suture needle through the superficial fascia on both sides of the incision.Pull the suture through to bring the fascia together and to cover the exposed spinal cord at the site of the removed lamina. Use a needle holder in forceps to tie three knots and trim away as much threat as possible. One suture is usually sufficient while pulling away the skin from the mouse.To avoid stapling the underlying muscle, close the incision with two to three staples. Once the wound is closed, inject 0.5 milliliters of lactated ringer solution subcutaneously into the lower back, but away from the incision. Place the mouse on its side in its cage, making sure the surgery site does not contact the cage bedding.Monitor the animals until fully recovered. This image shows successfully transplanted GFP labeled mouse neural stem cells in a coronal section of the spinal cord. 21 days post-transplant.After watching this video, you should have a good idea of how to transplant neuro stem cells into the spinal cord of JHMV infected mice Once mastered. It is possible to perform this technique on 40 mice in about three hours.

surgical-transplantation-mouse-neural-stem-cells-into-spinal-cords

Research

JoVE Journal

Neuroscience

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Source: Weber, R. Z., et al. Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain. J. Vis. Exp. (2022)This video demonstrates the stereotaxic transplantation of genetically modified human neural progenitor cells (NPCs) into the mouse brain. Researchers inject NPCs expressing luciferase and fluorescent proteins, enabling in vivo bioluminescence imaging and post-mortem fluorescence microscopy to study cell survival, migration, and...

Transplantation of Human Neural Progenitor Cells in the Mouse Brain

Source: Furmanski, O., et al. Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells. J. Vis. Exp. (2019)In this video, human-induced pluripotent stem cell-derived neural cells are stereotaxically injected into the gray matter-white matter border of the deep cortex in a mouse model of traumatic brain injury. The transplanted cells integrate into the tissue, promoting functional recovery and circuit...

Stereotaxic Transplantation of Neural Cell Suspension into a Mouse with Traumatic Brain Injury

This video demonstrates the intranasal delivery of oncolytic virus loaded therapeutic neural stem cells into a brain-tumor bearing mouse model. The non-invasive intranasal administration of stem cells bypasses the blood brain barrier, facilitating their delivery to the tumor site in the brain for therapeutic applications.

Intranasal Delivery of Therapeutic Neural Stem Cells (NSCs) in Murine Model: A Procedure for Tumor-targeted Delivery of Oncolytic Virus Loaded NSCs into Brain Tumor Bearing Mouse Model

Source: Parajuli, B., et.al. Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route. J. Vis. Exp. (2022).This video demonstrates the trans-nasal transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) cells into a mouse brain. Hyaluronidase is applied to the nostrils of a microglial-depleted mouse to enhance permeability. iPSMG suspension is administered intranasally, and the mouse is...

Transnasal Transplantation of Induced Pluripotent Stem Cell-Derived Human Microglial Cells into the Mouse Brain

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Learn neural stem cell transplantation into the spinal cord of JHMV-infected mice for studying remyelination in demyelinating diseases. This method enables researchers to investigate cell replacement therapies and functional recovery mechanisms.

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus (Video) | JoVE Journal

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

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