Department of Neuroscience, Thomas Jefferson University Medical College
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Lepore, A. C. Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury. J. Vis. Exp. (55), e3069, doi:10.3791/3069 (2011).
Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.
1. Cell Preparation
As an example, we will describe the procedure for preparing glial progenitor cells 12 for transplantation because of our experience with this cell type. However, the specifics of the protocol, including medium and use of trypsin for example, will depend on the particular cell type being used for transplantation.
Optional: Add 5.0 mL/T-75 flask of 1.0 mg/ml soybean trypsin inhibitor in DMEM/F12.
Do not use 0.75 mL tubes, as the Hamilton syringe/needle cannot fit deeply into this tube. Multiple injections will likely be made during the surgery sessions, and one wants to avoid disturbing the same tube of cells many times. Try to prepare at least 50% more volume of cell suspension than is needed. Keep cells on ice throughout the surgery session. Transplant cells within 4-5 hours of preparing cell suspensions in order to assure greatest viability of cells post-transplantation.
2. Preparation Prior to Surgery
3. Surgery: Animal Preparation and Surgery
Troubleshooting Tips for Problems Associated with Various Steps of Protocol
Lack of/poor cell survival: This is likely not a technical issue associated with the injection, but is probably due to properties of the cell type being injected and/or to the immunesuppression regimen. These issues need to be empirically evaluated on a cell-type and animal model specific basis. The availability of a number of immune-deficient rat and mouse models are also available to circumvent problems with immunesuppression; however, these animals also present difficulties such as cost, the need for maintaining a colony, and additional caution necessary during surgery and housing.
Functional deficits observed following surgery: In our experience, we have not observed the occurrence of any functional deficits following this procedure when assessed by measures such as forelimb and hindlimb grip strength and phrenic nerve/diaphragm compounds muscle action potentials, even with 6 injections sites (of 2μL each) in the cervical spinal cord. Tissue destruction has also not been observed. Possible reasons for the occurrence of these problems include: not using the suggested needle gauge, injecting larger volumes and/or cell numbers, unintentional damage caused by improper laminectomy, lack of delicacy while cutting the dura (such as pinching the cord with the #5 forceps) or during the process of inserting the Hamilton needle into the spinal cord (replace a dull needle).
Cells not found in ventral gray matter: This could be due to a number of issues. If the injection misses the target medially or laterally, make sure to properly line up the injection system parallel with the axis of the spinal cord, and insert the needle just medial to the entry of the dorsal rootlets. If the injection misses the target dorsally or ventrally, make sure to zero your z-axis reading when the tip of the needle is just barely touching the dorsal surface of the spinal cord, use the suggested Hamilton needle with the appropriate bevel (a longer bevel can affect targeting), assure that the spinal cord does not compress when inserting the needle, take care in exactly measuring depth using the micromanipulator, and use animals within the suggested weight range. Adjust your technique accordingly if you are consistently injecting the cells in a location (which requires histological evaluation and subsequent technique modification). If the injections are randomly scattered at all of your injection sites, you will need to practice to improve consistency. If the cells tend to be located mostly at the dorsal aspect of the spinal cord along the needle track, wait longer before and after cell injection, and extend the actual injection over a longer period of time.
4. Representative Results:
Mouse-derived glial progenitor cells were transplanted (50,000 cells/site) into the ventral horn of spinal cord level C4 in an adult SOD1G93A rat. Mouse-derived transplanted cells can be distinguished from host rat tissue via detection with the mouse-specific antibody, M2. This image shows the survival of M2+ transplant-derived cells at 1 month post-transplantation (see Figure 12). The cells localized to the ventral gray matter, but the injection site medially missed the lateral ventral horn (the location of most phrenic motor neurons: denoted by dotted line). No cyst formation was seen when 50,000 cells were injected per site, and no behavioral impairment resulted from the injection procedure. However, injection of much higher numbers of cells (the actual number varies according to cell type, and should be systematically evaluated) results in damage at the injection site and along the needle track (see Figure 13: immunohistochemistry with the astrocyte marker, GFAP).
Figure 1. Initial incision of skin.On the lowest microscope magnification (we use 8 x magnification), use scalpel blade to make midline incision. Stretch skin laterally with other hand to make skin taut (which makes the skin easier to cut), and make incision (denoted by thick dotted line)from base of skull (at the level of the back of the ears) to shoulder blade (denoted by thin dotted line).
Figure 2. Exposure of surgical field.The surgical exposure should be square/rectangle-shaped. This shape can be achieved by pulling the surrounding muscle towards 4 corners using the 4 retractors. Tape string that is attached to retractors to surgical board in order to secure retractors and properly keep field open.
Figure 2-inset. Retractors for exposure of surgical field.The retractors are used to pull back muscle in order to create a surgical field with both clear visibility of the spinal cord and adequate space to perform surgery. Retractors can be made by shaping sturdy paperclips into the desired shape and size. Autoclave the retractors before for surgery. Tie string to retractor.
Figure 3. Vertebrae. Following removal of paraspinal muscle, thoroughly clean dorsal surface of vertebrae with rongeur. Individual lamina can be seen, as well as dorsal rootlets entering from lateral aspect of spinal column.
Figure 4. Laminectomy.Start laminectomy at spinal cord level C5. Secure spinal column by holding muscle overlying level C2 with rat toothed forceps. Grab entire lamina (see diagram: grab near midline) with rongeur. Position rongeur so that tool is completely perpendicular to axis of spinal column. Slowly crush lamina. Do not push down into spinal cord, as this will cause damage to spinal tissue. Crush and gently pull broken piece of bone upwards. Rongeur should crush piece so that one can easily pull away to remove. If piece of bone is still attached to rest of laminae, do not tug as this will cause hemorrhage and possible injury to spinal cord. Rongeur should be clean and sharp.
Figure 5. Exposure of spinal cord tissue following laminectomy.Extend laminectomy to expose all of C4-C6 spinal cord. Make 1 continuous opening in the bone over 3 spinal levels. Do not extend laminectomy too far laterally because this will cause hemorrhage. In order to target ventral horn, the injection site is relatively medial, so it is unnecessary to extend laminectomy to the complete lateral extent of the vertebral bone.
Figure 6. High magnification of dorsal surface of spinal cord.The prominent dorsal blood vessel can be seen running down the midline of the spinal cord. This blood vessel pattern is observed in most cases; however, some animals display a non-midline trajectory of the blood vessel. The dorsal rootlets can be seen at the lateral aspects of the spinal cord dorsal surface. Relative to the dura overlying the spinal cord, these nerves have a hazy appearance.
Figure 7. Injection setup.Line up syringe/needle parallel with axis of spinal column to properly target desired anatomical region. The needle is angled just enough (approximately 80-degrees relative to the surgical table) to not bump the surgical scope head, but as close to 90-degrees as possible (left panel).Lower injection tip towards the spinal cord dorsal surface using the microscope (right panel). Gently touch surface of spinal cord with tip of needle. Slightly depress cord with needle. Retract needle until spinal cord is back to normal flat state. Record this position as z = 0.0 using the ruler on the micromanipulator.
Figure 8. High magnification of spinal cord injection Aim needle just medial to the entry zone of the dorsal rootlets (denoted by dotted line).
Figure 9. Diagram of spinal cord: how to target anatomical region of interest. When attempting to target the ventral horn, incise dura parallel to axis of spinal column just medial to the entry zone of the dorsal rootlets. This will allow one to target the ventral horn. Lower needle to depth of 1.5 mm to target ventral horn in adult rats (the age and sex of the animal does not make much of difference on depth). Lower needle to depth of 0.75 mm to target ventral horn in adult mice. Of course, depth and lateral position depend on the specific region of interest.
Figure 10. Closure of surgical site.Suture closed three overlying muscle layers at one time with 4-0 suture. Suture muscles at 3 locations in rostral-caudal axis.
Figure 11. Stapling of skin.Staple skin closed with 9.0 mm wound clips. Tighten staples with needle holders to prevent the animal from pulling off staples prior to full wound healing. Space staples approximately 0.5 mm apart.
Figure 12. Transplantation of glial progenitors into cervical ventral horn. 50,000 mouse-derived glial progenitor cells were transplanted into the ventral horn of spinal cord level C4 in an SOD1G93A rat. M2+ mouse-derived transplanted cells survived at 1 month post-transplantation. The cells localized to the ventral gray matter, but the injection site medially missed the lateral ventral horn (denoted by dotted line).
Figure 13. Tissue injury following transplantation of high numbers of neural precursor cells. Injection of much higher numbers of cells (the actual number varies according to cell type, and should be systematically evaluated) results in damage at the injection site and along the needle track.
For studies involving SOD1G93A mice and rats, age- and sex-match the animals within a group, and distribute animals within the same litter to different groups. It is preferable to use all animals from the same sex for both ALS and SCI models because disease processes may differ between males and females; however, it may also be useful to have enough animals from both sexes to detect possible sex-specific effects, as this phenomenon has been reported with SOD1G93A rats 13 and SOD1G93A mice 14,15. Consider the age/point of disease (for ALS animals) or time post-injury (for SCI models) for transplantation of cells into animals. This strategy will likely differ depending on the specific cell mechanisms that are being addressed.
The protocol described here can be used for both rat and mouse cervical spinal cords 11, as well as for transplantation into other levels of the rat and mouse spinal cords such as thoracic and lumbar levels 14. In order to target specific anatomical structures (other than ventral horn) in these various spinal cord levels, it is advisable to first test the medial/lateral position and depth of injections using a dye or some other means to evaluate the accuracy of the coordinates.
Outcome measures include detailed analysis of cell transplant fate, biochemical, histological, and functional states of the spinal cord, and behavioral changes relevant to the location of transplantation. The specifics of behavior testing, histological analysis and other outcome measures analyzed following transplantation should be tailored to the experimental needs/model.
Having a surgical assistant to anesthetize, shave, "open" and "close" the animals is quite helpful for smoothly getting through the most animals as possible. In addition, a surgical assistant can help the surgeon to remain sterile throughout the procedure.
The optimal number and concentration of cells injected will vary depending on the size of the cells, the species of the recipient animal, and the disease condition. For example, compared to the intact spinal cord, increased numbers of cells can be delivered into a SCI site if a cavity has already formed 16. However, the specific parameters need to be systematically optimized prior to undertaking a large study.
Prior to conducting a large study with a specific NPC type, evaluate the long-term effectiveness of the immune suppression regimen. In addition, cell survival may vary for a given cell type depending on the disease model or the timing and location of transplantation.
No conflicts of interest declared.
I would like to thank: all members of the Lepore, Maragakis and Rothstein labs for helpful discussion; The Paralyzed Veterans of America and the Craig H. Neilsen Foundation for funding.
|HBSS||GIBCO, by Life Technologies||14170|
|0.05% Trypsin||GIBCO, by Life Technologies||25300|
|Soybean Trypsin Inhibitor (optional)||Sigma-Aldrich||T-6522|
|Acepromazine maleate (0.7 mg/kg)||Fermenta Animal Health|
|Ketamine (95 mg/kg)||Fort Dodge Animal Health|
|Xylazine (10 mg/kg)||Bayer AG|
|#11 Feather surgical blade||Electron Microscopy Sciences||72044-11|
|Cotton-tipped applicators (6 inch)||Fisher Scientific||23-400-101|
|Rat-toothed forceps||Fine Science Tools||Rat: 11023-15; Mouse: 11042-08|
|Medium-sized spring scissors||Fine Science Tools||15012-12|
|Mini spring scissors||Fine Science Tools||15000-10|
|Rongeur||Fine Science Tools||Rat: 16121-14; Mouse: 16221-14|
|Microknife||Fine Science Tools||10056-12|
|Needle holders||Fine Science Tools||12502-14|
|Staples: 9 mm||Autoclip||427631|
|Stapler: 9 mm (Reflex #203-1000)||World Precision Instruments, Inc.||5000344|
|Warm water pump (T/Pump)||Gaymar Industries||P/N 07999-000|
|Cyclosporin A: 250.0 mg/5.0 mL ampules||Novartis AG||NDC 0078-0109-01|
|Injector||World Precision Instruments, Inc.||UMP2|
|Micro 4 Microsyringe Pump Controller||World Precision Instruments, Inc.||UMC4|
|Micromanipulator||World Precision Instruments, Inc.||Kite-R|
|10.0 ÁL Hamilton syringe||Hamilton Co||80030|
|Hamilton needles: 33-gauge, 45° bevel, 1 inch||Hamilton Co||7803-05|
|Glass 20.0 ÁL microcapillary pipettes (optional)||Kimble Chase||71900-20|