The Intracellular Signaling Events Of Interleukin-1 Beta Induced Neuron Apoptosis After Spinal Cord Injury In Rats

Backgroud: Acute spinal cord injury (SCI) often results in complete loss of voluntary motor and sensory function below the site of injury. The functional decline following SCI is contributed to both direct mechanical injury and secondary pathophysiological mechanisms that are induced by the initial trauma. Extensive evidences indicate that secondary injury of the cord involving edema, degenerative and necrosis, as well as widespread apoptosis of neurons and oligodendroglia after the trauma contributes significantly to long-term neurological deficits.Interleukin-1 beta (IL-1β) is an important inflammatory cytokines. It induces apoptosis in neurons in vitro and in cultured human astrocytes and oligodendrocytes in vivo. IL-1β expression is upregulated rapidly at the lesion site after SCI. The rapid accumulation of IL-1β following SCI may initiate apoptosis in neurons and glial cells, so that results in motor and sense functional loss. The intracellular signaling events initiated by IL-1β that lead to apoptotic cell death in vivo, however, are not well understood. p38 mitogen-activated protein kinase (p38 MAPK) is a pivotal molecule of signal pathway initiated by IL-IRI. Apoptosis is involves in the caspase family of cysteine proteases, and caspase-3 is a potential mediator in apoptosis after CNS injury.OBJECTIVE: To further characterize the apoptotic cascade initiated by IL-1β after SCI, we examined the expression of IL-1β, p38 MAPK and caspase-3 after SCI, and further investigated whether p38 MAPK is involved in neuron apoptosis induced by IL-1β.METHODS: Adult rats were assigned randomly to either the SCI group, the sham-operatedcontrol group (n = 54 for each group) or the drug-treated group (including SB203580-ap38MAPK inhibitor; IL-IRa-IL-1 receptor antagonist; Vehicle, n = 18 for each group). Ratswere given contusion SCI at the T9-T10 vertebrae level with a weight-drop impactor (10 g weight dropped 25.0 mm). Drug treatments were given intrathecally immediately after the injury. Recombinant rat IL-1ra (10 μg) was given to antagonize IL-1 receptors and SB203580 (4 μg/10ul) was given to inhibit p38 MAPK. The animals in the sham-operated control group underwent a T9 laminectomy without injury and received intrathecal injections of only vehicle (10 ul saline).The expression levels of IL-ip, p38 MAPK and caspase-3 Bfter SCI were assessed with Western blots, immunohistochemistry staining and real time reverse transcription polymerase chain reactions (RT-PCR). Neuron apoptosis was assessed with the terminal deoxynucleotidyl transferase-mediated deoxyuredine triphosphate-biotin nick end labeling (TUNEL) method. The outcome of locomotor performance in rats was assessed using an open field locomotor scale (the Basso, Beattie and Bresnahan, BBB).Results: Increased levels of IL-ip and p38 MAPK were observed soon after injury, with a peak in expression levels within 6 h of injury. By 24 h after injury, caspase-3 expression was markedly increased in the injured spinal cord. TUNEL-positive cells were first observed in the lesioned area 6 h after SCI. The largest number of TUNEL-positive cells was observed at 24 h post-SCI. Intrathecal injection of the IL-IRa significantly reduced expression of p38 MAPK and caspase-3, and reduced the number of TUNEL-positive cells. Moreover, intrathecal injection of an inhibitor of p38 MAPK, SB203580, also significantly reduced the expression of caspase-3, and reduced the number of TUNEL-positive cells in the injured spinal cord. Moreover, the BBB rating scale indicated that thoracic spinal contusion rats had more severely affected hindlimbs than sham-operated animals. Comparing with saline group, animals receiving SB203580 or IL-IRa demonstrated significantly better locomotor function, P<0.01.Conclusions: In conclusion, our study provides evidence that IL-ip serves as an external apoptosis triggering signal in the spinal cord after injury. Our results further suggest that the apoptotic cell death induced by IL-ip is mediated, in part, by phosphorylation of p38 MAPK and subsequent activation of the apoptotic gene caspase-3. These findings suggest that inhibition of p38 MAPK or blocking 11-1 receptor after SCI (i.e. by SB203580) may prevent neuronal cell death after SCI, and improve neurological function.