Hypothesis / aims of study
Neurogenic detrusor overactivity (NDO) emergence following spinal cord injury (SCI) courses with abnormal lumbosacral extension of bladder afferents. Mechanisms regulating this aberrant sprouting are unresolved, but may involve changes at the lumbosacral cord in the expression of inhibitory cues that block axonal growth, including myelin-associated inhibitors (MAIs), such as Nogo-A, and chondroitin sulphate proteoglycans (CSPGs), such as Phosphacan. It is presently unclear whether bladder afferents recognize these repulsive cues and respond to them, regulating the expression of their receptors and adjusting growth responses, and how this process could be regulated. Here, we investigated changes in the expression of Nogo-A and Phosphacan and their specific receptors NgR1/Lingo1/p75 and/or TROY, NgR1/NgR3 and Rptps/Lar at the lumbosacral cord and dorsal root ganglia (DRG) neurons, respectively, following SCI. We also investigated whether these alterations were NGF-dependent.
Study design, materials and methods
Female Wistar rats (n=8/group) were left spinal intact (controls) or underwent a largely incomplete spinal cord transection (SCT) at T8/T9 segments and left to recover for 7 or 28 days. At each endpoint, lumbosacral cords (L5-S1) and associated ganglia (DRG) were collected. Lumbosacral cords were processed by Western Blotting to evaluate the expression of Nogo-A, Phosphacan and GAP43, a marker of axonal sprouting. Total RNA from lumbosacral DRG neurons was extracted, reverse-transcribed and analyzed by qPCR to evaluate the expression of Nogo-A receptor complex NgR1/Lingo1/p75 and/or TROY and Phosphacan receptor complexes NgR1/NgR3 and Rptpσ/Lar. L5-S1 DRG neurons from other control and SCT animals were collected and cultured for 22h in the presence of 0, 50 or 100 ng/mL of NGF.
Lumbosacral cord expression of Nogo-A and Phosphacan had a 3-fold increase at 7 days post-injury (dpi), compared to controls (Phosphacan: p≤0.05 vs. controls; Nogo-A: p≤0.001 vs. controls), returning to baseline at 28 dpi. A significant increase in GAP43 expression at the lumbosacral cord was also found following SCI.
In lumbosacral DRG, the expression of the Nogo-A receptor complex NgR1/Lingo1/TROY was time-dependently decreased, compared to controls (NgR1: p≤0.05 controls vs. 7 dpi, p≤0.001 controls vs. 28 dpi). Phosphacan receptor complexes NgR1/NgR3 and Rptps/Lar also showed time-dependent decreases in expression, compared to controls (NgR1: p≤0.05 controls vs. 7 dpi, p≤0.001 controls vs. 28 dpi; NgR3: p≤0.05 controls vs 7 and 28 dpi; Rptps and Lar: p≤0.05 controls vs. 28 dpi). To assess whether the alterations in RNA levels of these receptor complexes were dependent on NGF exposure, lumbosacral DRG were cultured in 0, 50 or 100 ng/ml of NGF. Preliminary results indicate that after 22h in culture, neurite length and branching increased in an NGF concentration-dependent manner, particularly in SCT groups. In contrast, RNA levels of receptor complexes decreased in an NGF concentration-dependent manner in all groups, in tandem with in vivo observations.
Interpretation of results
We observed alterations in the expression of known inhibitors of axonal growth, Nogo-A and Phosphacan, at the lumbosacral cord following thoracic SCI. Both proteins were overexpressed at the lumbosacral cord 7 dpi, even though axonal sprouting significantly increased at this site following thoracic SCT, as indicated by GAP43 levels. This process appears to be dependent on exposure to NGF, known to occur in vivo in peripheral tissues after spinal injury.
These results indicate that, despite the presence of a highly repulsive spinal environment, bladder sensory afferents can grow from the periphery into the lumbosacral spinal cord. This reflects a time-dependent down-regulation of the expression of receptors for those repulsive cues. This process appears to be NGF-dependent, suggesting that peripheral NGF control could be used to regulate abnormal axonal sprouting and, consequently, block NDO emergence.