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

• Published in: Neuroscience Vol. 147; no. 2; pp. 546 - 560
• Main Authors: Cizkova, D, Kakinohana, O, Kucharova, K, Marsala, S, Johe, K, Hazel, T, Hefferan, M.P, Marsala, M
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 29.06.2007; Elsevier
• Subjects: Adult; Animals; Astrocytes - physiology; Biological and medical sciences; Cell Proliferation; cell replacement; Cell Survival - physiology; Cells, Cultured; Evoked Potentials, Motor - physiology; Female; Fundamental and applied biological sciences. Psychology; gamma-Aminobutyric Acid - metabolism; gamma-Aminobutyric Acid - physiology; Glutamate Decarboxylase - metabolism; Glycine Plasma Membrane Transport Proteins - metabolism; human neural stem cells; Humans; Immunohistochemistry; Interneurons - physiology; Isoenzymes - metabolism; Locomotion - physiology; Microscopy, Confocal; Muscle Rigidity - physiopathology; Muscle Rigidity - therapy; Muscle Spasticity - physiopathology; Muscle Spasticity - therapy; Neurology; Neurotransmitter Agents - metabolism; paraplegia; Paraplegia - therapy; Pregnancy; Rats; rigidity; spasticity; Spinal Cord - cytology; spinal cord ischemia; Spinal Cord Ischemia - pathology; Spinal Cord Ischemia - physiopathology; Spinal Cord Ischemia - therapy; Stem Cell Transplantation; Synaptophysin - metabolism; Synaptophysin - physiology; Tissue Fixation; Vertebrates: nervous system and sense organs; human neural stem cells; BBB; paraplegia; TX; normal goat serum; hNSE; hMOC; hSSC; rigidity; motor evoked potential; neuron specific enolase; spinal cord ischemia; mouse anti-neuron-specific enolase; human spinal stem cell; mouse anti-neural cell adhesion molecule; mouse anti-synaptophysin; Triton X-100; synaptophysin; cell replacement; NSE; Basso, Beattie and Bresnahan; SYN; hSYN; MEP; spasticity; NGS; Human; Spinal cord; Rat; Stem cell; Rodentia; Central nervous system; Cardiovascular disease; Vertebrata; Mammalia; Functional recovery; Ischemia; Animal
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2007.02.065

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 α-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 μl/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 α-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.