Effect Of Calcineurin On The Axon After Traumatic Axonal Injury And Its Mechanism

Diffuse axonal injury(DAI) is a kind of injury with extensive axonal damage in brain white matter caused by trauma and one of the most severe traumatic brain injuries(TBI). With the development of imageology,the diagnosis of DAI has some progress.However, there are few effective ways for DAI treatment so far.DAI is the result of joint actions of direct or indirect mechanical force,secondary injury,self-repair of nervous system.As to its bio-mechanical mechanism,DAI might be the common character for all types of TBI and is also called traumatic axonal injury(TAI). Immediately as the injury takes place,axons would be disrupted or not,which depends on the power of mechanical force.Furthermore,some nondisruptive axons would finally be severed due to the participation of secondary injury within 4-24h after trauma.So the strategics to treat DAI is to prevent nondisruptive axon axotomy,promote self-repair and the regeneration of axon.Calcineurin(CaN) is a Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase,which is involved in the protein dephosphorylation reaction and regulated by a second messenger, Ca²⁺.It is highly localized in brain and mainly expressed in cytoplasma,neurites,dentrites and axons.Now some researches have found that CaN takes part in many neurocytes' regulation,such as immune inflammatory reaction,synapse plasticity,apoptosis of neurons, et al.The effect of CaN on the axon after TAI is paid more and more attention now.Some animal experiments have demonstrated the axons stained by APP,an index to reflect dysfunction of axonal anterograde transportation,decreased and the locomotion of animal improved by administrating the cyclosporine A(CsA) or FK-506,inhibitors of CaN,after TAI.These results suggest CaN get involved in the treatment after TAI.But,the effect and emechanism of CaN on axon after TAI is required to be further clarified.So we studied that in 3 ways:1) comparison of CaN activity among rats with different age with an aim of study the relationship between CaN and axonal outgrowth.2) 10nM CsA, brain derived neurotrophin factor(BDNF),tautomycin(TTM,specific inhibitor of PP-1) were given into cortex neurons of rats cultured for 3,7,10d respectively and the neurites length,CaN activity,and protein ofβⅢ-tubulin,^P-CREB,^P-GAP-43 was determined to clarify the effect of CaN on the axonal outgrowth and its mechanism.3) 10nM CsA and TTM were respectively given into cortex neurons after stretch which made TAI.CaN activity,and protein of APP,βⅢ-tubulin,^P-GAP-43 were determined at 1h,6h,24h,3d after injury to clarify the effect of CaN on the axonal self-repair or regeneration.The main results and conclusions are as follows:1.Enzymatic substrate coloration assay showed that with the development of rats' age,the activity of CaN is increased which is a probable clue to study the difference of self-repair between mature and immature neurons.2.It was found from enzymatic substrate coloration assay that the CaN activity was inhibited significantly by CsA except BDNF in cortical neurons which is suggested that CaN is the target of CsA.3.With contrast phase microscope digital photo system and SPOT software,it was found the length of neurites increased during 3-10d while CsA,BDNF,TTM promoted the increase significantly without notable difference between each other,which is suggested that CsA and TTM could have the similar effect on axonal outgrowth as BDNF and the effect of CsA could depend on the PP-1 signal way because pp-1 is known as a substrate of CaN.4.Western blot assay displayed that there was an increasing tendency forβⅢ-tubulin, ^P-GAP-43,^P-CREB during 3-10d after injury,but no statistical singnificance.CsA made all the proteins increase significantly while TTM only promoted notable increase ofβⅢ-tubulin and ^P-CREB.It is suggested that CaN could act on axonal outgrowth by ^P-GAP-43 directly and pp-1,CREB signal way indirectly.5.With stretching the neurons cultured for 7d with a special instrument in our lab,a TAI model is established.It is good to duplicate,easy to control,precise to make axonal injury.So it may simulate the TAI in vivo.6.With western blot and enzymatic substrate coloration assay,it was found that at 3d posttrauma APP protein attenuated significantly after CsA administration without TTM, which is suggested that CsA repairs the axonal injury via CaN,independence of PP-1 signal way,in the acute phase of TAI.7.Western blot assay displayed thatβⅢ-tubulin,^P-GAP-43 did not change significantly within 3d after TAI in control group but increased at 3d after CsA administration.The TTM treatment only increased theβⅢ-tubulin at 3d after TAI.These results indicate that self-repair could take place at 3d after TAI,andβⅢ-tubulin,^P-GAP-43 are the target of CaN.