Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells.
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.
Cizkova
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D
Institute of Neurobiology, Centrum of Excellence, Slovak Academy of Science, Kosice, Soltesovej 4, Slovakia.
Kakinohana
O
O
Kucharova
K
K
Marsala
S
S
Johe
K
K
Hazel
T
T
Hefferan
M P
MP
Marsala
M
M
D000328:Adult; D000818:Animals; D001253:Astrocytes / Q000502:physiology; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019054:Evoked Potentials, Motor / Q000502:physiology; D005260:Female; D005968:Glutamate Decarboxylase / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007395:Interneurons / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D008124:Locomotion / Q000502:physiology; D018613:Microscopy, Confocal; D009127:Muscle Rigidity / Q000503:physiopathology / Q000628:therapy; D009128:Muscle Spasticity / Q000503:physiopathology / Q000628:therapy; D018377:Neurotransmitter Agents / Q000378:metabolism; D010264:Paraplegia / Q000628:therapy*; D011247:Pregnancy; D051381:Rats; D013116:Spinal Cord / Q000166:cytology*; D020760:Spinal Cord Ischemia / Q000473:pathology / Q000503:physiopathology / Q000628:therapy*; D033581:Stem Cell Transplantation; D016708:Synaptophysin / Q000378:metabolism / Q000502:physiology; D016707:Tissue Fixation; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000502:physiology
D050487:Glycine Plasma Membrane Transport Proteins; D007527:Isoenzymes; D018377:Neurotransmitter Agents; C493269:Slc6a5 protein, rat; D016708:Synaptophysin; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2