ABSTRACT
Axonal regeneration within the injured central nervous system (CNS) is hampered by multiple inhibitory molecules in the glial scar and the surrounding disrupted myelin. Many of these inhibitors stimulate, either directly or indirectly, the Rho intracellular signaling pathway, providing a strong rationale to target it following spinal cord injuries. In this study, we infused either control (PBS) or a ROCK inhibitor, Y27632 (2 mM or 20 mM, 12 microl/day for 14 days) into the intrathecal space of adult rats starting immediately after a cervical 4/5 dorsal column transection. Histological analysis revealed that high dose-treated animals displayed significantly more axon sprouts in the grey matter distal to injury compared to low dose-treated rats. Only the high dose regimen stimulated sprouting of the dorsal ascending axons along the walls of the lesion cavity. Footprint analysis revealed that the increased base of support normalized significantly faster in control and high dose-treated animals compared to low dose animals. Forepaw rotation angle, and the number of footslips on a horizontal ladder improved significantly more by 6 weeks in high dose animals compared to the other two groups. In a food pellet reaching test, high dose animals performed significantly better than low dose animals, which failed to recover. There was no evidence of mechanical allodynia in any treatment group; however, the slightly shortened heat withdrawal times normalized only with the high dose treatment. Collectively, our data support beneficial effects of high dose Y27632 treatment but indicate that low doses might be detrimental.
Subject(s)
Amides/administration & dosage , Axons/drug effects , Enzyme Inhibitors/administration & dosage , Pyridines/administration & dosage , Recovery of Function/drug effects , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/physiopathology , Actin Depolymerizing Factors/metabolism , Analysis of Variance , Animals , Axons/physiology , Behavior, Animal , Biotin/analogs & derivatives , Biotin/metabolism , Blotting, Western/methods , Cholera Toxin/metabolism , Dextrans/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Interactions , Immunohistochemistry/methods , Intracellular Signaling Peptides and Proteins , Male , Motor Activity/drug effects , Motor Activity/physiology , Myosin-Light-Chain Phosphatase/metabolism , Myosin-Light-Chain Phosphatase/pharmacology , Nerve Regeneration/drug effects , Pain Measurement/drug effects , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/pharmacology , Psychomotor Performance/drug effects , Random Allocation , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects , Rotarod Performance Test/methods , Spinal Cord Injuries/cerebrospinal fluid , rho-Associated KinasesABSTRACT
Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. We examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract (CST) dieback, and lesion size and to improve functional outcome after SCI. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending CST in segments (3-7 mm) both proximal and distal to the injury site. Surprisingly, the numbers of dying oligodendrocytes in the proximal and distal segments were comparable, suggesting more than the lack of axon-cell body contiguity played a role in their demise. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daily for 2 d. We report a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the AST after minocycline treatment, using immunostaining for active caspase-3 and Hoechst 33258 staining in combination with cell-specific markers. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials.
