Procedures and Methods



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Figure 6. Biological Safety Hood
 

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Figure 7. CT image

Clinical cases of IVDD present at different stages of the disease and so require customizing of therapy. Some pets present with mild behavioral abnormalities such as reluctance to jump, climb or descend stairs, or painful or stiff back or neck. Some pets have mild to moderate proprioception deficits. Other pets have ataxia, crossing of the limbs and reluctance or inability to stand while the most severe pets have no pain perception and are tetraparetic or quadriplegic. Almost all pets are candidates for stem cell therapy as a prevention or treatment with or without surgical decompression. However, the best decisions are made after complete neurologic examination and imaging. MRI imaging is the preferred modality because not only can compression of the spine be perceived but also the amount of water within the disc is an indication of disc health and can be seen on T2 weighted MRI images.

Figure 7 & Figure 9. CT images have proven adequate when MRI is not available, and CT scans outperform myelogram studies when evaluating IVDD .

Autologous Adipose Derived Stem Cells (ADCs) are obtained from the patient by harvesting the fat filled falciform ligament through a 2cm midline abdominal incision anterior to the umbilicus. The fat is minced, washed with PBS and mixed with collagenase type I then incubated, agitated, and centrifuged at 37°C to separate fat from the Stromal Vascular Fraction (SVF). The fat is transferred to a sterile container and processed in a biological safety hood. (See Figure 6 -top of page)

 

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Figure 8. C-Arm
 

The SVF contains mesenchymal stem cells, preadipocytes, endothelial progenitor cell, T cells, B cells, and mast cells, as well as adipose tissue macrophages. The SVF is filtered through a 100 µm pore filter then filtered again through a 60 µm pore filter. The cells are counted and a ratio of live cells to dead cells is used to calculate live cell doses.

 

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Figure 9. CT image
 

In a separate process, blood from the patient is harvested for the creation of Platelet Rich Plasma (PRP). The stem cells are mixed with PRP to dilute them and stimulate them to release cytokines. The PRP is activated with autologous thrombin and mixed with the SVF in the proper proportions for administration of 100 µl volume containing 1 x 107 live cells/ml.

 

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Individual luer-lok syringes are prepared containing 100 µL stem cell mixture for each disc to be injected. This process takes about 2 hours. The pet is prepared for general anesthesia and the area of skin over the site is surgically prepared. A C-Arm radiology unit is used to properly guide 24ga spinal needles into the disc space.

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Figure 11. Positioning is confirmed at opposing 90° angles. The stem cells are injected into the center of the nucleus pulposus and the needle is then removed.
 

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Figure 12

 

For the animals who have significant neurologic disease, hemilaminectomy surgery is recommended with stem cell therapy as an adjunct to the surgery and to treat other discs that may be affected. The surgical technique for hemilaminectomy is the standard removal of the bony wall lateral to the spinal cord. Disc material is removed with small atraumatic instruments. The spinal cord is then injected with stem cells. A syringe is prepared with pure platelet rich plasma enriched adipose derived stem cells containing 150 µL volume with 1 x 107 stem cells. These cells are injected using a 24GA spinal needle through the dura matter in three locations of 50µL each at the location of damage and above and below the area of damage. (See Figure 12)

In animals with spinal cord damage as indicated on physical examination or diagnostic imaging, but that are not receiving decompressive surgery, the stem cell and PRP mixture is injected sub-arachnoid much the same as if doing myelography using the cisterna magna or lower lumbar access areas. Total volume of stem cell mixture should be 150 µL per kg and contain 1 x 107 cells per ml.

Intravenous administration of stem cells is optional but may be recommended if cells are not to be cryopreserved or cultured. The average sized pet will yield between 40 and 80 million live cells. Each disc requires about 1 million cells and intrathecal administration may use an additional 20 million cells. This may leave an additional 20 million cells that would be wasted if not given intravenously. The SVF has significant anti-inflammatory properties and there is evidence that intravenous stem cells will seek damaged tissue to regenerate. It has been our experience that pets receiving additional intravenous stem cells seem to recover faster with less pain.

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Figure 13. When neurons damaged by IVDD are stimulated they will release cytokines when the nerve impulses reach the damaged area.

Physical therapy is an important part of the post-operative recovery plan. The day after the procedure, the pet should be receiving spinal proprioceptive inputs in the form of spatial alterations using foam or inflatable “peanuts” to roll the pet back and forth. Limb placement and massage also stimulate ascending neurons.

These cytokines influence the genetic neurogenesis of the stem cells enabling more accurate repair. Utilizing electrostimulation, massage, or passive range of motion exercises for paraparetic or quadraparetic patients enables spinal tracts to receive information destined for the brain . Mental stimulation using the pet’s name, treats and other encouragements creates impulses along the cerebrospinal tracts also liberating cellular chemical signals that communicate to the newly engrafted stem cells. The importance of these chemical messengers in the instruction of stem cell development cannot be overemphasized. Two to three times a day the pet’s spinal and centrally mediated reflexes need to be stimulated using different and alternating modalities. Strength training with water treadmill needs to be started as soon as possible, taking into consideration the need to protect a surgical wound from the water.