80.3  The expression of mRNA for neurotrophins, and of their receptors is differentially upregulated after nerve injury  A strong role for BNDF, and its receptors Trk-B and p75 NTR, has been suggested based on their pattern of expression after nerve injury  NT-4/5, the other Trk-B and p75 NTR ligand, is also upregulated after nerve injury. However little is known about its role in axonal regeneration Purpose: To evaluate the role of endogenous NT-4/5 on peripheral axonal regeneration Axons of motoneurons are capable of nearly complete regeneration, but achieving favorable restoration of muscle function after injury of peripheral nerves remains a serious clinical problem. 1. Introduction  Mice containing the reporter yellow fluorescent protein (YFP) inserted under the control of the thy-1 promoter were used.  In these mice, YFP is expressed in a subset of axons in peripheral nerves and is easily visualized using fluorescent microscopy 2. The Model System This grafting technique was used not only because it allowed to study the effect of the environment on axonal regeneration but also because it provided the dark background necessary to study the regeneration of YFP labeled axons. Otherwise the determination of regenerating and degenerating axons is impossible because YFP fluorescence persists for up to two weeks in the cut distal stumps. Distance of the regenerating axons in recipients was measured at 1, 2, 4, 8, 14, and 28 days post surgery (1 recipient/time period) The common fibular nerve (CF) nerve on 6 YFP mice (recipients) was exposed bilaterally and cut using sharp scissors. A section of CP nerve aproximately 2-5 mm in length was removed from 3 wild type (NT-4/5 +/+ ) and 3 NT-4/5 -/- mice (donors) and connected to the proximal and distal stumps of the cut CF nerve of the recipients, using fibrin glue. 3. Time Series MG Sciatic Common Fibular Post. Tibial LG-S Sural Proximal Stump Distal Stump Common fibular nerve was cut bilaterally and the ends allowed to retract Graft from NT-4/5 -/-. Graft from NT-4/5 +/+ in contralateral cut CF 6. Electrical stimulation does not enhance axon regeneration in absence of NT-4/5 7. Axon regeneration is also inhibited when expression of NT-4/5 is reduced Proximal Stump Distal Stump Common fibular nerve was cut bilaterally and the ends allowed to retract Graft from NT-4/5 +/-. Graft from NT-4/5 +/+ in contralateral cut CF  Binding of NT-4/5 to p75 NTR might prevent the Nogo-induced activation of p75 NTR, and thus, activation of Rho  BDNF may exert a similar effect after binding to Trk-B or p75 NTR 9. Summary and Conclusion  Axon regeneration is inhibited in the absence of NT-4/5 or when the expression of this neurotrophin is reduced  The enhancement of axonal regeneration produced by electrical stimulation is prevented in the absence of NT- 4/5  We interpret these result to mean that NT-4/5 is required for normal peripheral axonal regeneration. Whether or not this requirement is a direct promotion of axon outgrowth or a decrease in p75-mediated inhibition is not yet known  Axon growth after injury is restricted by myelin-derived growth inhibitory signals  The inhibition requires the interaction of the nogo receptor (NgR) with p75 NTR 8. Hypothesized Signaling Mechanism NgR MAG Nogo GT1b OMgp p75 In absence of MAG or Nogo, Rho-GDI maintains Rho in the inactive state Active No Growth RhoGTP RhoGDI RhoGDP Inactive RhoGDI 1. MAG and Nogo, both present in myelin, bind to NgR. NgR, in turn, bind and activates p75 NTR 2.Activated p75 NTR sequester Rho-GDI away from Rho, allowing Rho to become activated NT-4/5 MAG Nogo GT1b OMgp X X BDNF NgR p75 Inactive Growth Inactive RhoGDI RhoGDP RhoGDI X RhoGDP Adapted from Huber et. al., 2003; and Kaplan & Miller, 2003 KS: p < 0.01 KS = Kolmogorov-Smirnov Two Sample Test Recipients and surgery same as Time Series This time, proximal and distal stumps of CF nerves of 4 recipients were connected with nerve grafts removed from 4 NT4/5 +/+ and from 4 NT4/5 +/- donors (n=4) Is normal expression of NT-4/5 required for normal axonal regeneration ES enhance axonal regeneration in NT4+/+ grafts (1 Week Data) KS: p < 0.01 KS = Kolmogorov-Smirnov Two Sample Test KS: p > Axon regeneration is inhibited in the absence of NT-4/5 Recipients and surgery same as in Time Series This time, proximal and distal stumps of CF nerves of 4 recipients were connected with CF nerve grafts removed from 4 NT-4/5 +/+ and from NT-4/5 -/- mice Distance of the regenerating axons in recipients was measured at 14 days post surgery (n=4) Quantitative Analysis 5. Can electrical stimulation enhance axon regeneration in the absence of NT-4/5?  Continuous electrical stimulation accelerates motor axonal regeneration after axotomy Recipients and surgery same as in Time Series. Nerve grafts from NT4/5 -/- donors only (n=2) Immediately after connecting the grafts to the proximal and distal stumps of the CF nerves, the sciatic nerve proximal to the cut was stimulated continuously via a cuff electrode. Stimulus intensity was 2 times above the threshold intensity needed to elicit muscle contraction in the innervated muscles. The left sciatic nerve was sham stimulated. 50mm Proximal Stump Distal Stump Graft from NT4/5 -/- in both cut CF Cuff electrode Electrical stimulation: 20Hz, 1 hr. Sciatic KS: p < 0.01 KS = Kolmogorov-Smirnov Two Sample Test NT-4/5 -/- Nerve Graft NT-4/5 +/+ Nerve Graft Prox Stump Dist. Stump Prox Stump Dist. Stump KS: p < 0.01 for NT-4/5 +/+ VS NT-4/5 -/- KS = Kolmogorov-Smirnov Two Sample Test NT-4/5 IS REQUIRED FOR NORMAL PERIPHERAL AXONAL REGENERATION D. I. Carrasco, K. L. Pierce, and A.W. English. Department of Cell Biology, Emory University, Atlanta GA, 30322