Conditioning Lesions Elicit Significantly Greater In Vitro Neurite Outgrowth from Adult DRG Neurons than cAMP
Adult DRG neurons cultivated on poly-L-lysine and isolated from animals 3 or 7 days after conditioning lesions exhibited a significant 2-fold increase in neurite length when compared to DRG neurons from naïve animals (). In contrast, infusion of the PDE-IV inhibitor mesopram for 3 or 7 days did not increase neurite length. Similarly, cultivation of adult DRGs in the presence of 2 mM db-cAMP did not significantly increase neurite extension on poly-L-lysine or myelin. (). Thus, conditioning lesions are more effective than cAMP in enhancing in vitro
outgrowth of adult
DRG neurites. In contrast to adult DRG neurons, cAMP significantly increased the mean neurite length of postnatal
DRGs and cerebellar granular neurons when cultivated on permissive or inhibitory substrates (Suppl. Fig. S1A–C
) (comparison to conditioning lesions was not made due to the young age of the subjects). Taken together, these experiments suggest that increased cAMP levels at the time of cell plating (db-cAMP incubation) or prior to cell plating (in vivo mesopram infusion) are insufficient to replicate conditioning effects on neurite growth of adult
Quantification of in vitro neurite growth and cAMP levels in adult DRG neurons after conditioning lesions and infusion of phosphodiesterase inhibitors
To determine whether infusion of the phosphodiesterase inhibitor mesopram results in cAMP increases comparable to conditioning lesions, we measured cAMP by ELISA in lumbar DRGs (L4–6). Conditioning lesions increase cAMP levels in DRGs approximately 2-fold as early as 1 day after sciatic nerve crush, consistent with previous reports (Qiu, et al., 2002
) (). Levels remain elevated 1 week later. Infusions of mesopram (or rolipram; Suppl. Fig S1D
) result in cAMP increases of the same magnitude for at least 3 days, but cAMP levels are no longer significantly elevated compared to intact animals by day 7 despite continuous infusion. Thus, while mesopram increases cAMP levels at 3 days to the same extent as conditioning lesions, neurite outgrowth capacity is significantly enhanced only after conditioning lesions. Microarray data and in vivo assays (below) further support this result.
Conditioning lesions in combination with NT-3 enhance axonal bridging beyond the lesion site
To further clarify the role of cAMP in mediating conditioning lesion effects in vivo, we compared axon growth-promoting effects of conditioning lesions to infusion of phosphodiesterase inhibitors and direct injections of a cell permeable analog of cAMP (db-cAMP), at various time points after spinal cord injury.
First we determined whether pre-conditioning lesions alone or in combination with Lenti-NT-3 delivery are effective in increasing the number and distance of axons regenerating beyond a central lesion. Animals received bilateral sciatic nerve crush lesions; one week later, the dorsal funiculus was transected at cervical level (C3) (Lu, et al., 2004
, Taylor, et al., 2006
) and animals received grafts of bone marrow stromal cells (BMSC) mixed with NT-3 protein into the lesion site to provide a cellular substrate for regenerating axons (). Lenti-NT-3 or -GFP as control was injected 2.5 mm rostral to the lesion as previously described (Alto, et al., 2009
, Taylor, et al., 2006
). Additional control animals received the same graft and virus injection but no conditioning lesion (Suppl. Fig. S2
A combination of pre-conditioning lesions and lentiviral NT-3 gene transfer results in significant axonal bridging across the lesion site
Tracing of ascending sensory axons with CTB showed significant axonal bridging across the graft into the rostral spinal cord only among animals that received Lenti-NT-3 (–). In animals that received no conditioning lesions (CL) or CL with cellular grafts and Lenti-GFP injections (but not Lenti-NT-3), rare axons reached the rostral host/graft interface but virtually no axons extended further in the rostral white matter of the spinal cord (). In animals with Lenti-NT-3 delivery without preconditioning lesions, a small number of axons extended across the rostral host/graft interface and for short distances beyond, as previously reported () (Taylor, et al., 2006
). This number was increased approximately 8-fold in animals that received pre-conditioning lesions in addition to Lenti-NT-3 (). Indeed, subjects that underwent conditioning lesions and injections of Lenti-NT-3 beyond the lesion exhibited significantly more axons compared to all other groups over distances up to 1600 µm beyond the graft/lesion site (; ANOVA p<0.001; Fischer’s posthoc p<0.01 comparing each group to conditioning lesion + Lenti-NT-3). Axons extended an average maximum distance of nearly 2.5 mm beyond the rostral host/graft interface, a distance six times greater than the distance of animals receiving only Lenti-NT-3 (Suppl. Fig. S3
). As growth distance was measured from the rostral host/graft interface, the total length of axon growth (measured from the caudal aspect of the lesion site) was in fact 3–3.5 mm. Axons crossing from the graft into the host spinal cord beyond the lesion were present at all dorsoventral levels within the dorsal funiculus, and extended preferentially in white matter rostral to the graft along regions of NT-3 expression (). Axons were frequently found to orient along GFAP-labeled processes and were occasionally associated with blood vessels. Anatomical sectioning of the medulla confirmed lesion completeness (data not shown).
Quantification of axons bridging across a C3 lesion site filled with bone marrow stromal cells in animals that received pre- or post-conditioning lesions, infusions of mesopram or db-cAMP injections
Ascending sensory axons extend beyond the graft/lesion site towards Lenti-NT-3-transduced cells in an animal that received pre-conditioning lesions and Lenti-NT-3 gene transfer
Thus, initiation of an axonal growth program by peripheral pre-conditioning lesions before spinal cord lesions allows axonal extension across a growth substrate into an otherwise inhospitable environment of degenerating white matter, but only if an additional NT-3 trophic stimulus is provided.
Conditioning lesions are effective when administered shortly after central lesions
Next we explored the temporal requirements for neuronal conditioning to allow axonal bridging beyond a spinal cord lesion. Previous studies have indicated that pre-conditioning lesions are most effective when applied 5–7 days before
central lesions. Analogous to the in vivo experiments described above, cell grafting and Lenti-NT-3 injections were made immediately following the C3 lesion, but conditioning lesions were placed at a delay of 1 or 7 days after
spinal cord lesions. Quantification of axonal growth beyond the lesion site indicated that conditioning lesions one day after central lesions were at least as effective as pre-conditioning lesions in promoting axonal bridging beyond the C3 lesion site (). A significant increase in the number of bridging axons was found up to 800 µm beyond the lesion site when compared to animals that received only Lenti-NT-3 and cell graft but no conditioning lesion (). In contrast, when conditioning lesions were delayed by one week after the central lesion, a substantial reduction in axonal bridging was observed. Despite the lack of a significant post-conditioning effect at this time point, axons extended for longer distances in animals with a one week conditioning delay compared to animals without conditioning lesions (Suppl. Fig. S3
; p<0.01) and axon numbers quantified at each distance beyond the lesion were slightly higher in animals with a 7 day “post-conditioning” lesion compared to animals without peripheral lesions.
Thus, signaling mechanisms underlying a conditioning lesion are still fully effective one day following a CNS lesion, but the effect is reduced when applied one week after spinal cord injury.
Conditioning lesions significantly enhance axonal regeneration compared to cAMP modulation
To determine whether direct cAMP augmentation exhibits equipotency compared to conditioning lesions in eliciting axonal regeneration, we examined central sensory axon regeneration after C3 spinal cord lesions. Both direct cAMP injections and infusions of the PDE-IV inhibitor mesopram (Dinter, et al., 2000
) were compared to conditioning lesions, in the same lesion/treatment paradigm described in the preceding paragraph. Subcutaneous infusions of mesopram were initiated 7 days prior to C3 dorsal funiculus lesions. Pumps were exchanged at the time of spinal cord lesions, cell grafting and viral NT-3 delivery, and infusions continued for one more week. Consistent with our in vitro data, quantification of axons beyond the lesion site 4 weeks after spinal cord lesions did not indicate significant increases in the number of axons beyond the lesion site/cellular graft when comparing animals that received a combination of mesopram infusion with Lenti-NT-3 injections to animals that received only Lenti-NT-3 injections (). Mesopram infusions alone (without rostral Lenti-NT-3) did not induce axonal growth beyond the rostral lesion border. Similar results were obtained with rolipram infusion (data not shown).
Next, we investigated whether a combination of Lenti-NT-3 with injections of a cell permeable analog of cAMP (db-cAMP) into L4–5 DRGs would enhance axonal bridging. As data reported above indicate that conditioning lesion one day after spinal cord lesions are equally effective as 7-day pre-conditioning lesions, db-cAMP or PBS (control) were injected directly into L4–5 DRGs 1 day after spinal cord lesions. Consistent with previous findings using db-cAMP injections 5 days before spinal cord lesions (Lu, et al., 2004
), a 2-fold increase in the number of bridging axons was detected comparing animals with db-cAMP injections and Lent-NT-3 to animals with PBS injections and Lenti-NT-3 (). This difference was significant up to 100 µm distal to the rostral lesion border. PBS injections in control animals also resulted in an increase in the number of bridging axons compared to Lenti-NT-3 alone (compare to ), likely a result of a partial conditioning effect from PBS injections into DRGs.
Taken together, cAMP injections with NT-3 delivery resulted in a modest number of axons (2-fold increase) bridging for short distances compared to conditioning lesions with NT-3 delivery (10-fold increase) indicating that effects of conditioning lesions on axonal regeneration exceed targeted cAMP modulation.
Genetic programs induced by conditioning lesions exceed cAMP effects
To investigate genetic mechanisms underlying conditioning lesion effects compared to cAMP, we examined changes in gene expression in lumbar DRGs (L4–6) by microarray analysis at 1, 3, 7 and 14 days after either conditioning lesions, mesopram or vehicle infusions, and after injection of db-cAMP or PBS into DRGs. Three animals/group were pooled for RNA isolation and for each microarray, and 3–4 microarrays were hybridized at each time point using a total of 210 animals and 70 microarrays ().
Analysis of changes in gene expression in DRGs that underwent peripheral nerve conditioning lesions demonstrated early and long-lasting changes in gene expression compared to intact DRGs, consistent with previous reports (Costigan, et al., 2002
, Kubo, et al., 2002
, Stam, et al., 2007
, Tanabe, et al., 2003
, Yang, et al., 2004
). Using a false discovery rate of 5% (pFDR<0.05), a significance level of p<0.05, and a fold-change in expression of at least 20%, a total of 4883 differentially expressed probe sets were detected (, Suppl. Table 2
). A large number of changes in gene expression occurred one day after conditioning lesions: 773 probe sets were upregulated and 497 downregulated. This number increased to 1721 upregulated probes and 1198 downregulated probe sets at 3 days, 1083 and 997 at 7 days, and 1867 and 1400 significantly changed probe sets at 14 days (). Several genes previously identified to exert an important role in peripheral nerve regeneration and to contribute to the priming effect of conditioning lesions were upregulated, including arginase-1, interleukin-6, ATF-3 and jun (see Suppl. Table 2
). Between 79% and 89% of significantly changed probes were altered by more than 20% (depending on the post-injury day of analysis), and 5–8% of probe sets changed by more than 2-fold over the 14 day period after conditioning lesions. 60% (881/1479) of probe sets that exhibited significant changes on day 1 were still significantly changed 14 days after peripheral lesions. Thus, rapid and sustained activation of genes occurs after peripheral conditioning lesions. The sustained activation pattern correlates with the in vivo observation that central projections of DRG neurons exhibit significantly increased growth when spinal cord injury is accompanied by a peripheral conditioning lesion.
Heat maps depicting fold changes in gene expression one day following conditioning lesions and infusions of the phosphodiesterase inhibitor mesopram
Changes in gene expression after conditioning lesions compared to naïve control animals
In contrast, infusion of the PDE IV inhibitor mesopram induced changes in a much smaller number of genes over time (544 probe sets using the same significance criterion of the preceding paragraph, and Suppl. Table 3
). The highest number of probe sets showing significant changes in expression compared to naïve controls occurred one day following the start of the infusion (524; pFDR<0.05): 275 genes were significantly upregulated and 249 probe sets showed reduced expression levels compared to intact animals. The number of differentially expressed probe sets rapidly declined at all subsequent time points, even before transient increases in cAMP levels returned toward baseline levels (see ). These changes in gene expression were due to mesopram infusion, and were not a result of the vehicle infusion or induction of anesthesia, because only 1 and 3 differentially expressed genes were identified in the latter groups, respectively (pFDR<0.05; Suppl. Table 4
Changes in gene expression after mesopram and control infusions compared to naïve control animals (pFDR<0.05)
Comparison of probe sets modulated by conditioning lesions versus mesopram (1 day after infusion) identified a limited but significant (p<0.0001) overlap in transcriptional responses (, Suppl. Table 5
). Of all probe sets significantly changed 1 day after mesopram infusion, 17% (88 probe sets) were also changed 1 day after conditioning lesions (at pFDR<0.05, more than 20% change). The 88 overlapping probe sets contained a large number of genes known to be regulated by cAMP such as cAMP responsive element modulator (crem) (Mioduszewska, et al., 2003
), CCAAT/enhancer binding protein beta (C/EBPbeta) and delta (C/EBPdelta) (Yukawa, et al., 1998
), c-fos and early growth response 1 (egr1, NGFI-A, zif268) (Vaccarino, et al., 1993
). In contrast, many classical regeneration associated genes such as GAP-43, jun and β-III-tubulin were increased by conditioning lesions but not mesopram infusion. Taken together, more than 90% of early
transcriptional responses induced by conditioning lesions were not induced by mesopram, despite equal increases in cAMP levels by ELISA. Virtually no persistent transcriptional modulation was achieved by mesopram infusion. Array data from animals that received cAMP injections into DRGs showed numerous changes compared to naïve control animals but similar changes were also observed after control PBS injections. Indeed, comparison of genes modulated by PBS and cAMP injections into DRGs did not indicate any significant differences using the same statistical criteria used for all other microarray studies (data not shown).
Network analysis using Ingenuity software confirmed that conditioning lesions influenced several broad networks of gene expression, including jun, p53 (Suppl. Fig. S4
) and Il-6/stat3 (not shown), whereas mesopram recruited far more narrow activation (). Indeed, mesopram infusions influenced only 6.9% of genes that changed after conditioning lesions at the same time point, reflecting the broader pattern of genomic alteration induced by conditioning lesions. Taken together, fundamental differences in transcriptional responses to conditioning lesions versus cAMP modifying compounds were detected in our analysis, likely contributing to differences observed in potency of neurite outgrowth in our in vitro and in vivo models.
Microarray data were confirmed for several genes by quantitative real time PCR (qRT-PCR), including Cebp/d, Egr1, Egr3, ATF3, Myc, Jun, Fos, Smad1, Sox11, Gadd45a, Gadd45g, ZFP367and NFIL3. In no case did results of PCR considerably differ from array findings. These data confirmed changes in gene expression identified by microarray methods in subjects with conditioning lesions, and further indicated that mesopram infusions resulted in more modest effects ().
qRT-PCR data confirming significant changes of selected genes in microarrays in DRGs of animals after mesopram infusions and conditioning lesions at day 1