Yvette S. Nout, DVM, MS, PhD, DACVIM, DACVECC
Acute traumatic damage to the spinal cord occurs in most species, but is most commonly seen in humans, rabbits, dogs, and horses. In dogs the most common causes of spinal cord injury (SCI) are intervertebral disk extrusion and traumatic penetrating or non-penetrating injuries (1). Explosive protrusion of an intervertebral disk can cause both concussion and compression of the spinal cord. It has been reported that 1-2% of all dogs admitted to veterinary hospitals have injuries to the spinal cord resulting from disc disease (2) but total incidence of SCI in hospitalized dogs has not been reported. In horses, trauma to the central nervous system is the most common cause of neurologic disease, accounting for 22% of neurologic disorders in one study (3). In this same study, 50% of horses with traumatic central nervous system disease were diagnosed with lesions in the cervical spinal cord. Spinal cord injury occurs predominantly in young horses and is most commonly the result of falls. In horses the spinal cord is well protected in the thoracic and lumbar vertebral column, leaving the cervical and sacral vertebral column the most susceptible to trauma. There are no reports on incidence of SCI in horses; however, since in general SCI is the result of high velocity / high impact forces, prognosis for life is guarded. In humans, it is estimated that the annual incidence of SCI, not including those who die at the scene of the accident, is approximately 40 cases per million population in the U.S., or approximately 11,000 new cases each year. The number of SCI patients in the U.S. who were alive in July 2005 was estimated to be approximately 250,000. Between 2000 and July 2005 vehicular crashes accounted for the majority (47.5%) of reported SCI cases, followed by falls (22.9%) and violence (13.8%) (The National SCI Statistical Center, 2005).
In order to study the consequences of spinal cord injury, a number of experimental animal models have been developed. Our laboratory uses a model in which rats are subjected to a weight-drop contusion injury. The main advantages of this model are the fact that the injuries that are created strongly resembles those that occur in human spinal cord injury, and the size of the rat is large enough to enable investigations of numerous body systems and anatomy following injury.
Use of Stem Cells in Spinal Cord Injury
Damage inflicted to the spinal cord occurs in 2 distinct phases. So-called "primary injury" refers specifically to the mechanical disruption of axons as a result of compression, penetration, laceration, shear, and/or distraction (4). Primary damage results in cell membrane disruption and rapid cell death which is followed by a second period of progression of tissue damage and cell death that can last several hours to days after initial insult (4,5). This is referred to as "secondary injury". Many well-characterized mechanisms of cell damage have been shown to play a role during this period of secondary damage, including 1) vascular compromise leading to reduced blood flow, loss of autoregulation, loss of microcirculation, vasospasm, thrombosis, and hemorrhage; 2) electrolyte shifts, permeablility changes, loss of cellular membrane integrity, edema, and loss of energy metabolism; 3) biochemical changes including neurotransmitter accumulation, arachidonic acid release, free radical and prostaglandin production, and lipid peroxidation; and 4) apoptosis (4,5). Secondary expansion of the lesion cavity that occurs through necrotic and apoptotic pathways is associated with the invasion of immune cells and activation of the resident microglial population. The period of ongoing secondary injury is considered to be the phase in which therapeutic measurements could be taken to halt the process of ongoing cell loss.
Endogenous repair has been shown to take place following a contusion lesion. Proliferation of ependymal cells that lie around the central canal has been shown to occur in fetal mammals, but also in adult rats (6,7). These cells appear to add tissue bridges referred to as trabeculae that begin to fill the lesion cavity (8,9). Glial cells and invading sensory axons can follow these cellular tracks. Over time, these trabeculae can support growth of axons from within the central nervous system, such as the corticospinal tract and fibers from the brainstem reticular formation (6). In distinct contrast to the limited distribution of neuronal stem cells, glial precursors are widely distributed in the developing and mature mammalian central nervous system. Furthermore glial progenitor cells that can give rise to both astrocytes and oligodendrocytes exist predominantly in the outer rim of the spinal cord (10). Proliferation of endogenous glia and/or glial precursor cells is thought to play an important role in restoring chronic function to the injured cord (6,11). It is likely that oligodendrocyte precursor cells rather than surviving mature oligodendrocytes contribute to the intrinsic remyelination after demyelinating insult. Oligodendrocytes, the cells that wrap axons in myelin and allow them to function, undergo apoptosis during the delayed phase of death after SCI and since each oligodendrocyte myelinates 10-20 different axons, the resulting loss of myelination and conductance through that segment has a large effect on the loss of function. Although remyelination occurs, this intrinsic repair is insufficient to overcome the insult. However, perhaps by increasing the number of these cells in the vicinity of the lesion, functionally significant repair could be obtained. Currently the use of various cell transplant procedures is being widely investigated in the field of SCI research. Rationales for using transplantation strategies are 1) the functional reconstruction of neuronal circuits; 2) the production of neurochemically active substances; and 3) remyelination of axons.
Several different kinds of cells have been used including Schwann cells, embryonic spinal cord, olfactory ensheathing cells, macrophages, choroid plexus ependymal cells, bone-derived mesenchymal stem cells, and neural stem cells. Among these cells, embryonic neural stem cells have been most enthusiastically studied. However, ethical problems make it impossible to use human fetal tissue as a practical and immediate source for therapeutic treatment. It has been shown that the optimal time window for transplanting cells into the lesion site is around day 9 after SCI (12). Although it is currently possible to make axons sprout from surviving but damaged neurons, it is not yet possible to make long axons grow and form appropriate reconnections. This ultimate goal may be achievable through ongoing studies and investigations into the potential of stem cell transplantation in neurologic diseases. At this time, it appears that optimizing the function of neuronal circuits that survive SCI is probably the most achievable strategy. Many current investigations attempt to do this through enhancement of glial cell function. Immature astrocytes promote axonal growth and perhaps promote survival after injury (13), whereas immature oligodendrocytes provide remyelination after injury (11). Furthermore, it has now become possible to isolate pluripotent cells from embryos and using treatments with growth factors, push these cells into lineage-specific states that produce either neuronal or glial-specific progenitor cells (14,15). For cell replacement in neurological conditions, experimental data to date suggests that lineage-committed immature neuronal or glial progenitor cells, rather than multipotential neural stem cells or mature neurons and glial cells, are most likely to result in predictable phenotypic differentiation and potential functional outcome. Key in these treatment strategies is the ability of cells to integrate into the neural circuitry in a functional manner and consequently contribute to the correction of functional deficits.
To improve integration and function of transplanted cells a wide variety of bio-molecular therapies are being investigated. For example neurotrophic factors, such as neurotrophins, have been found to promote a variety of neural responses such as survival and outgrowth of nerves and spinal cord nerve regeneration (16). Inducing up-regulation of neurotrophins, regeneration-associated genes, and antiapoptotic factors and / or blocking inhibitory biomolecules in the nervous system through use of genetically modified cells are methods used to complement cell transplantation strategies. Elevation of cyclic adenosine monophosphate (cAMP) concentrations has been shown to play an important role in regeneration of the adult central nervous system and in overcoming inhibitory aspects of the injured spinal cord milieu (17). Recently, elevation of cAMP through administration of the phosphodiesterase IV inhibitor rolipram in conjunction with transplantation of embryonic spinal tissue or Schwann cells resulted in improved functional outcome and promoted axonal regeneration after SCI (18).
Combination Glial Restricted Precursors and Elevation of CAMP
Our laboratory has previously shown that when glial restricted precursor (GRP) cells, derived from transgenic rats harboring the heat-stable human placental alkaline phosphatase (hPLAP) gene, are transplanted into the spinal cord immediately following SCI, these cells survive, differentiate, and alter the lesion environment through reducing astrocytic scarring and through reducing expression of inhibitory proteoglycans (19). Furthermore, GRP cell transplants, in combination with neuronal restricted precursors or enhanced expression of neurotrophins, have been shown to improve recovery of function after spinal cord lesions (20,21). In the following experiment we used a delayed combined transplant strategy that included transplantation of GRP cells and administration of cAMP. We asked whether combining cAMP with a GRP cell transplant would improve functional recovery, similar to that observed after combining cAMP with Schwann cell transplants (18). In addition to examining locomotor function, we studied recovery of sexual and bladder function. Autonomically mediated functions, such as urogenital tract function, following SCI and its subsequent complications are highly prevalent and clinically very important (22). Recently, we developed a method to assess recovery of micturition and erectile function in conscious freely moving rats by monitoring corpus spongiosum penis (CSP) pressure using telemetry (23,24).
Fifty-one adult, male Long-Evans hooded rats were used in the long-term survival part of this study. Groups: 1: age-matched control; 2: operated control; 3: GRP control; 4: cAMP control; 5: GRP cAMP. Twelve rats were used in the short-term survival part of this study. Groups: 1: uninjured control; 2: operated control; 3: cAMP control. All animal experiments were conducted after approval by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University and were performed in compliance with NIH guidelines and recommendations.
Our moderate contusion injury produced a very consistent lesion with the area of remaining tissue at the lesion center in the OP control group (12.4 ± 2.7%) in the same range as described previously for this level of injury (9.9 ± 4.8%). GRP cells and cAMP did alter the cellular composition of the lesion region. The presence of GRP cells resulted in a significant increase of tissue throughout the lesion region. The GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue in all animals that received a transplant. In animals that received exogenous cAMP, however, the area occupied by GRP cells at the lesion center and the volume of the GRP transplant throughout the lesion region were reduced. The histopathological alterations we found in the lesion region did not appear to affect serotonergic input to the DM and DL nuclei in the lumbosacral spinal cord, however, animals that were given exogenous cAMP showed greater reduction of amount of serotonin immediately distal to the lesion center compared to the control group.
As far as the author is aware, this is the only study that has quantified extent of transplant within the lesioned spinal cord. Certainly there are no other reports on the effect of cAMP on survival of transplanted cells within an injured spinal cord. Our study suggests that elevation of cAMP is not necessarily beneficial to survival of GRP cells. This is unlikely to be due to the altered immune response that is caused by cAMP elevation, since it has been well established that cAMP has predominantly anti-inflammatory effects. Macrophages failed to differentiate into activated macrophages when exogenous cAMP was added in an in vitro model (25) and phagocytotic function of macrophages is inhibited by cAMP (25,26). However, numerous reports have shown that cAMP is involved in regulation of cell survival, in particular of neoplastic and progenitor cells.
In conclusion, our study showed no beneficial effects on functional outcome measurements of transplanting GRP cells and administering cAMP after SCI. Although elevation of cAMP has been reported to have beneficial effects in neural regeneration and may have beneficial effects on lineage differentiation of GRP cells, from our study it appears that cAMP are not always reduced after SCI and that administering cAMP may reduce survival or proliferation of GRP cells following transplantation into the injured spinal cord. Continued research should be undertaken into effects of cAMP and effects of these combined treatment strategies for SCI. Transplant strategies appear promising; however it seems appropriate to use combinatorial strategies in which cell transplants are combined with other therapies such as treatments that induce differentiation of progenitor cells into populations with desired functional characteristics, treatments that affect regeneration, and/or treatments that alter the spinal cord milieu. It is important that investigators critically examine effects and interactions of all these treatments, not only on functional outcome measurements but also on histopathological features. Furthermore, effects of the complimentary treatments such as administered neurotrophins or cAMP administration may have different effects in normal adult cells vs. in progenitor cells such as used in current transplantation strategies.
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