A Combinatorial Treatment for Spinal Cord Injury: A Research Proposal
Spinal cord injury (SCI) is a traumatic and complex condition that often results in chronic sensory and motor paralysis below the level of injury. There are approximately 17,000 new SCIs in the U.S. each year and on average 62% of these injuries have been incomplete . However, basic research has focused more attention on treating motor complete SCI for a number of reasons. Many reactionary cellular mechanisms alter the microenvironment following acute SCI inhibiting functional nerve regeneration. Mechanisms including robust inflammation and glutamatergic excitotoxicity cause massive cell death. Formation of an astroglial scar and myeline accumulation inhibit the growth and reconnectivity of axons .
There are many types of therapies aimed at various mechanisms although two recent therapies have been shown to be very promising. Bioscaffolds that bridge the cavity caused by SCI can be seeded with stem cells and neurotrophic factors to induce axonal growth, guidance and regeneration .
In addition, several recent studies have shown that applying electrical stimulation to the spinal cord can induce involuntarily and ultimately voluntary step-like movements in motor complete SCI patients. These same studies claim that residual effects such as the ability to activate lower extremity muscle groups are seen even after the stimulation is turned off .
This proposal will investigate the efficacy of a bioscaffold seeded with appropriate stem cells and neurotrophic factors implanted at the time of spinal fusion following incomplete SCI. We will then determine how a specific therapeutic regiment focused on noninvasive electrical stimulation can enhance the regenerative capacity of the spinal cord.
Aim 1: To engineer a bioscaffold seeded with stem cells and neurotrophic factors to induce axonal growth and guidance
Our goal is to design a neurospinal scaffold that will facilitate axonal growth and guidance. The scaffold will be optimized for biocompatibility, biodegradation, morphology and ultimately SCI specific nerve regeneration. The propriospinal interneuron network (PN) will be targeted as a possible regenerative substrate. Because the PN contains neurons that sprout laterally into grey matter as well as neurons that connect spinal cord segments together, it is a very promising target for complete and incomplete SCI regeneration .
Aim 2: To use noninvasive electrical stimulation to neuromodulate the lumbosacral spinal cord circuitry to induce step-like movements
Targeting the lumbosacral circuitry with electrical stimulation has been shown to induce involuntary step-like movements in both healthy individuals and individuals who have suffered motor complete SCIs . We believe that electrical stimulation may activate dormant pathways that project supraspinally to the lumbosacral central pattern generator (CPG). This will be the first human trial to test noninvasive electrical stimulation on patients with incomplete SCI.
While treatment of motor complete paralysis is important for providing proof of concept it is considered to be by far the most difficult type of SCI to treat clinically. On the other hand, incomplete SCIs hold far more potential for regeneration and functional recovery which is why we are proposing to treat this population with our multifaceted approach first.
If successful this strategy will be completely noninvasive, aside from the obvious spinal fusion following SCI, and will not be as intense as current SCI therapy paradigms. Furthermore, if patients reach our predicted goal it will enhance the health and quality of life while reducing the degree of disability for thousands of children and adults who have been paralyzed due to SCI.
Due to the novelty of both technologies there has never been a study designed to determine the synergistic effects of a surgically implanted bioscaffold and noninvasive electrical stimulation on SCI regeneration. Our approach is unique in that we are attempting to bridge the spinal cord lesion as well as stimulate motor circuits below the level of injury. We believe that treating SCI from the top-down and from the bottom-up holds more potential for regeneration than either method alone. With the success of recent electrical stimulation of complete SCI we believe there is reason to have very high expectations for patients with incomplete SCI.
Preliminary Data: Bioscaffolds
Scaffolds offer the advantage of concentrating appropriate factors at a specific site and enhancing neural regeneration through contact mediated axonal guidance . Scaffolds can also be seeded with stem cells, neurotrophic factors, nucleic acids material and other pharmacological drugs as needed. This method of delivery is aimed at improving efficiency of the drug or stem cell delivered and reducing the side effects of conventional drug delivery such as multiple injections . SCI leaves a very hostile environment that is not conducive to axonal growth or regeneration as stated above. Using a bioscaffold can help in the physical and chemical reorganization that is needed to promote proper growth and reconnection of spinal cord axons [13, 15]. Scaffolds have been used in every area of tissue engineering for a long time however, neurospinal scaffolds are the most complicated and least studied due to the intrinsic complexity of the CNS. Still, there have been many studies investigating how a neurospinal scaffold should be engineered with positive results .
The figures above describe the data that were collected from a number of behavioral tests administered to rats who were given a unilateral (incomplete) SCI. Groups were assigned as such, one with a bioscaffold seeded with neural stem cells (NSCs), one with only a bioscaffold, one with only NSCs and one control group. (A) Data from the BBB open-field walking test for the ipsilateral side of SCI shows that the bioscaffold+NSC group scored significantly higher than both the bioscaffold only and NSC only groups. (B) BBB open-field walking test for the contralateral forelimb showing very similar data. (C) Data from an inclined plane test where the upward orientation was not altered significantly but the downward orientation was. The bioscaffold+NSC group improved significantly from day 14 onward. (D) Represents the righting reflex results. The bioscaffold+NSC group shows a significantly higher percentage of righting compared to other groups. (E) Represents the percentage of animals with a normal pain response. (F) Represents the percentage of animals with a spastic response to the same painful stimuli. Figures were taken from , description was adapted from .
Preliminary Data: Non-invasive Electrical Stimulation
A recent study  have successfully induced step-like movements using an invasive epidural stimulation device in four complete SCI patients. The patients regained the ability to move their legs after several weeks, and in one case several months of training with the electrical stimulation. However, the epidural device had to be surgically implanted along the patients’ lumbar spinal vertebrae. In addition, leg movement was only seen when the epidural stimulation device was on, no residual effects were seen. Instead of an invasive stimulation a new form of noninvasive electrical stimulation is suggested instead of the epidural device. This non-invasive method is known as transcutaneous electrical nerve stimulation (TENS). TENS is not new in itself but applying this method of stimulation to the spinal cord is relatively new. TENS works by strategically placing electrodes onto the skin above the spinal cord, specifically at the lumbosacral junction in humans. In a more recent study  five motor complete SCI patients who were at least two years post injury were able to move their legs in a gravity neutral position while the TENS unit was activated. Surprisingly, after a number of training sessions each of the five patients showed some degree of movement without the TENS unit turned on. Because this study was done in motor complete SCI patients who were at least two years post injury we have every reason to believe that an acute, incomplete SCI patient will respond even better to the TENS stimulation
Lower extremity EMG activity during voluntary movement occurred only with epidural stimulation in four individuals with motor complete spinal cord injury.
Lower extremity EMG activity during voluntary movement occurred only with epidural stimulation in four individuals with motor complete spinal cord injury. EMG activity during attempts of ankle dorsiflexion (A) without stimulation and (B) with stimulation. Force was not collected for Patient B07. Electrode representation for each subject denotes the stimulation configuration used. Although stimulation was applied throughout the time shown in B, in all four subjects EMG bursts were synchronized with the intent to move. Grey boxes are cathodes and black boxes are anodes, white boxes are inactive electrodes. Stimulation frequency varied from 25 to 30 Hz.
Figure and Description were taken directly from .
Approach: Bioscaffold Design
Several conditions must be met in order to maximize reconnectivity and subsequent functional recovery. Biocompatibility is the most important characteristic to consider when designing a SCI model scaffold due to the risk of inflammation which could cause further neurological damage . Furthermore, biodegradability is crucial, especially in the SCI model where removal of a non-degradable scaffold would warrant secondary surgery which may result in unnecessary complications. After the scaffold has assisted in axonal growth it should degrade through endogenous enzymatic activity. Another quality to consider is that of mechanical strength. The SCI scaffold will not have to stretch or expand but it will need to be able to withstand a certain amount of pressure caused by inflammation. The morphology of a bioscaffold for SCI should also be highly porous and contain large pore sizes, allowing for cell attachment and axonal growth.
For the reasons stated above, a chitosan scaffold with collagen hydrogel as a filler is proposed. Chitosan is a natural, biodegradable polymer used extensively by tissue engineers. The scaffold will contain the extra cellular matrix proteins fibronectin and laminin which have been shown to improve cell adhesion and axonal guidance . Furthermore, evidence has shown that axons and axonal growth are extremely sensitive to the surface and architecture of scaffolds. Specifically, microchannels have proved successful in extending neurite growth with an optimal channel diameter of 20-30 nanometers . For this reason the chitosan scaffold will be a solid conduit instead of an injectable scaffold. The scaffold will also be seeded with appropriate cells and neurotrophic factors that can modulate regeneration.In most cases after SCI the axons of the injured neurons will retract proximally and distally from the site of injury leaving a gap or cavity in the spinal cord . Therefore, it will be beneficial to seed the bioscaffold with stem cells that will directly replace the cells that died. A popular stem cell line used for inducing spinal cord regeneration are neural stem cells (NSCs). NSCs are stem cells that are committed to becoming neural cells and are believed to facilitate spinal cord regeneration by differentiating into, and thus directly replacing, lost neurons and glial cells (Iwanami et al., 2005). Another advantage of NSCs is that they are believed to facilitate host axonal growth, that is, axons that survived the SCI, by secreting neurotrophic factors. For these reasons we are choosing NSCs to seed our chitosan bioscaffold. Several neurotrophic factors have been identified as playing important roles specific to the propriospinal interneuron network (PN) in axonal regeneration and plasticity. Brain-derived neurotrophic factor (BDNF) contributes to regeneration by promoting plasticity and increasing remyelination (Fouad et al., 2012). Specifically, BDNF administered to the cortex resulted in a greater number of propriospinal axon sprouting and a more complete preservation of corticospinal tract (CST) axons . Additionally, ciliary-derived neurotrophic factor (CDNF) prevents degeneration of axons after axotomy  and glial derived neurotrophic factor (GDNF) has been shown to promote plasticity of the PN directly . Other members of the neurotrophin family, including NT-3 and NT-4 are known for promoting axonal sprouting and growth . These five neurotrophic growth factors are among some the most extensively studied and are well described in spinal cord regenerative literature . Therefore, they will constitute the basis of the neurotrophic factors that will be seeded into the bioscaffold.
Approach: Noninvasive Electrical Stimulation Design
As soon as our recruited patients would be able to begin conventional physical therapy following a SCI we will instead start them on a therapeutic plan centered on noninvasive electrical stimulation. We will work with Thomas Jefferson University Hospital in Philadelphia, PA to recruit eight individuals who have recently suffered an incomplete SCI. We are attempting to recruit two groups of four, each group being comprised of similar injuries (ie. Level, type and severity of injury). We will begin our study by applying the TENS at the lumbosacral junction tonically for 45 minutes at a time or as long as the patient can stand the stimulation. We will have two sessions a day, five days a week for 90 days. The default stimulation parameters we will use will be adapted from Edgerton et al. from his 2015 study which are 8v at 15Hz. We will also record EMG data at three time points (d=30,d=60,d=90) from three groups of muscle synergies including hip, knee and ankle. The hip flexor, quadriceps and hamstring muscles. From the ankle we will record from the plantar flexor and plantar extensor. We will compare the data we collect from incomplete controls.
The bioscaffold approach may result in a number of clinical issues. It will be difficult to personalize the bioscaffold to each SCI until we have a much better understanding of the different types of SCI and how different types of bioscaffolds work in each environment. Also, the solid conduit bioscaffold has a higher risk of causing inflammation and further neurological damage in comparison to an injectable scaffold. However, the bioscaffold allows for much more effective targeting of stem cells and neurotrophic factors and thus a higher potential for successful regeneration. The balance between the regeneration and inflammation of the solid conduit and injectable scaffold needs to be investigated further. There are a few potential problems with the TENS therapy. Patients may not be able to tolerate an intensity of electrical stimulation that is sufficient to provide the response needed for regeneration. Also, some patients will undoubtedly be able to tolerate higher levels of stimulation which may make interpreting results complicated. Some patients may need longer and more frequent electrical stimulation which may render the noninvasive method pointless at which point the patient may want to
At the end of 90 days our goal is that at least 6 out of 8 of our patients will be able to take a few steps with the help of parallel bars, a walker or crutches. We expect that 6 out of 8 of our patients will be able to bear at least 75% of their body weight. We believe that being treated with a bioscaffold and training for 90 days with the TENS unit these SCI patients will improve significantly relative to controls. Further studies may want to address the relevancy of Lokomat training in addition to TENS therapy. Although at least one study determined that Lokomat training was no more beneficial than conventional physical therapy there may be reason to investigate the effect of the TENS with the Lokomat training. This may be a promising combination because TENS stimulation is thought to stimulate a central pattern generator and the Lokomat helps the patient to generate rhythmic walking motion.
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