Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells

Abstract

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.

Lumbar Intraspinal Injection of Neural Stem Cells in Patients with Amyotrophic Lateral Sclerosis: Results of a Phase I Trial in 12 Patients

“…More frequent measurements of individual patients are likely to identify ''noise'' that may not reflect true changes in disease progression. EIM, a newer and potentially more powerful biomarker of clinical progression, was performed at baseline for patients 1-6 and monthly for at least 3 months prior to surgery for patients [7][8][9][10][11][12]. The data are summarized in Figure 2, in which the major parameter, the 50 kHz phase averaged for all six muscles studied, for all 12 patients is plotted against time.…”

“…These cells, expanded in serum-free media more than many passages, have shown therapeutic benefit when injected into the ventral horn area of spinal cord in the G93A rat model of familial ALS [7] and in a rat model of ischemic spinal cord injury [8]. In these motor neuron disease paradigms, the majority of the transplanted HSSCs differentiated into neuronal populations expressing glutamatergic and GABAergic neurotransmitter markers [9].…”

“…Very few of the transplanted cells were shown to have characteristics of alpha motor neurons. Even so, injection of HSSCs rescued endogenous motor neurons of G93A rats, prolonged their survival, and induced partial motor recovery in rats with ischemic spinal cord injury [8]. These promising preclinical data led us to design a trial where spinal cord injection of HSSCs can be tested as a novel therapy for people with ALS.…”

Mesenchymal Stromal Cells and Neural Stem Cells Potential for Neural Repair in Spinal Cord Injury and Human Neurodegenerative Disorders

“…In addition, the loss of these specific inhibitory pools localized in the intermediate zone of the spinal grey matter, ultimately leads to an increase in the monosynaptic reflex and nearcomplete loss in spinal polysynaptic activity. A challenging study done in collaboration with anesthesiology research laboratory at University of California San Diego, has shown that NSCs derived from human fetal spinal cord grafted into a rat model of ischemic spastic paraplegia resulted into a progressive recovery of motor function with correlative improvement in motor evoked potentials (Cizkova et al 2007). Of note, transplanted NSCs became integrated into host neuronal circuits and displayed an extensive axo-dendritic outgrowth and active rostrocaudal/dorsoventral migration for about 8-12 weeks.…”

“…Patients who suffer from spinal cord trauma show limited functional recovery, which frequently leads to deficit of multiple sensory, motor and autonomic systems resulting to clinical signs of partial or complete paralysis with prominent spasticity and rigidity (Cizkova et al 2007). Because of the limited regenerative capacity of the adult CNS due to the inhibitory molecules, decrease of trophic factor support and scar tissue formation, the current functional treatments for SCI are not successful (Rowland et al 2008).…”