Lymnaea epidermal growth factor promotes axonal regeneration in CNS organ culture

Abstract

Members of the epidermal growth factor (EGF) family are frequently implicated in the injury response of the mammalian nervous system. Although this implication is supported by extensive molecular evidence, it is not underpinned by conclusive functional data. Recently, we found that expression of an EGF homolog from the pond snail Lymnaea stagnalis (L-EGF) is upregulated after axotomy in the adult CNS, suggesting a role for this molecule in the injury response of the CNS. In the present study we asked whether L-EGF can promote axonal regeneration of three types of identified neurons in organ-cultured CNS. Treatment with purified L-EGF substantially enhanced axonal regeneration of all three types of neurons, an effect inhibited by submicromolar doses of PD153035, a specific EGF receptor (EGFR) tyrosine kinase inhibitor. In addition, PD153035 and K252a, a nonspecific kinase inhibitor, also reduced the degree of axonal regeneration that occurs without L-EGF supplementation, indicating that L-EGF or other EGFR ligands synthesized in the CNS participate in the regenerative response. An intriguing aspect of these results is that axonal regeneration of different, intrinsically L-EGF responsive and unresponsive neurons occurred in a coordinated manner. This observation suggests that indirect in addition to direct actions contribute to the beneficial effect of L-EGF. In conclusion, we provide functional evidence that an EGF homolog can promote axonal regeneration, substantiating existing molecular evidence implicating the EGF family in peripheral nerve regeneration and emphasizes the therapeutic potential of these molecules.

Fig. 1.

Experimental model and nerve injury procedure.A, Schematic representation of the dorsal aspect of theLymnaea CNS with the cerebral commissure cut and cerebral ganglia folded outward. LBu/RBu, Left and right buccal ganglia; LCe/RCe, left and right cerebral ganglia; LPe/RPe, left and right pedal ganglia;LPl/RPl, left and right pleural ganglia;LPa/RPa, left and right parietal ganglia;Vi, visceral ganglion; RPeD1, right pedal dorsal 1; RPA, right parietal A neurons;RPD1, right parietal dorsal 1; VD2/VD3, visceral dorsal 2 and 3 [nomenclature according to Benjamin and Winlow (1981)]. B, Microphotograph of aLymnaea CNS after retrograde staining of the RIP nerve (buccal ganglia not shown). Scale bar, 500 μm. In this case no nerves were crushed. Backfilling the RIP nerve labeled numerous neuronal somata. Particularly, RPeD1, VD2/VD3, and the RPA group motoneurons are clearly visible (also see enlarged inset of RPa ganglion). Scale bar, 100 μm.

Fig. 2.

Effect of proximal crush and organ culture on axonal projections into the RIP nerve. A, Microphotograph of a CNS (dorsal view, with the cerebral commissures cut) that was retrograde labeled immediately after isolation without crushing the RIP nerve. Scale bar, 500 μm. Numerous labeled neuronal somata are seen in several ganglia, including those of RPeD1, VD2/VD3, and several RPA neurons. B, Microphotograph of a CNS (dorsal view, with the cerebral commissures cut) in which the RIP nerve was backfilled with nickel-lysine immediately after the nerve was crushed. No neuronal somata are labeled in this preparation, indicating that axonal projections projecting into the RIP nerve were completely severed (note that the tracer is not transported across the crush). Scale bar, 500 μm.

Fig. 3.

Stability of RIP nerve axonal projections in organ culture. A, Frequency of preparations in which RPeD1 and VD2/VD3 were retrograde labeled by backfilling the RIP nerve either immediately (acute), after 2 d in culture (2 days), or after 7 d (7 days) in culture. Note that there was no difference in the frequency of labeled RPeD1 and VD2/VD3 somata between the three conditions. B, Frequency distribution of labeled RPA somata per CNS in preparations in which the RIP nerve was backfilled either immediately (acute; n = 20), after 2 d in culture (2 days), or after 7 d (7 days) in culture. Note that the data are distributed very similarly in all three cases, with a range of 5 to 15 and a median value of 10 labeled RPA somata per CNS (bin width = 2).

Fig. 4.

Regeneration of RIP axons in isolated CNS cultured in saline. A, Frequency of preparations in which RPeD1 and VD2/VD3 were retrograde labeled by backfilling the RIP nerve immediately after dissection of the CNS without nerve crush (no crush) or by backfilling the RIP nerve in crushed preparations after 2 d (crush, 2 days) or 7 d (crush, 7 days) in culture, respectively. Note that RPeD1 and VD2/VD3 are labeled in <40% of the preparations after 2 d in culture and that for both cell types this proportion did not significantly improve by keeping the preparations in culture for 7 d in culture. B, Frequency distribution of the number of retrograde-labeled RPA somata per CNS in preparations that were backfilled immediately after dissection without crushing the RIP nerve (no crush) and preparations in which the RIP nerve was crushed and backfilled after 2 d in culture (crush, 2 days) and 7 d (crush, 7 days) in culture, respectively.C, Average number of labeled RPA somata in the total 2 d data set (overall), a subset of the 2 d data in which neither RPeD1 nor VD2/VD3 was labeled (− −), and a subset of the 2 d data in which both RPeD1 and VD2/VD3 were labeled (+ +). ***p < 0.001.

Fig. 5.

Effect of L-EGF on regeneration of crushed RIP axons. A, B, Microphotographs of isolated CNSs (dorsal view, with the cerebral commissures cut) that were cultured for 2 d after receiving a crush to the RIP nerve in saline (A) or in saline plus 100 nmL-EGF (B). Scale bar, 500 μm. Comparison of both photographs illustrates that the number of labeled neuronal somata was dramatically enhanced in the presence of L-EGF. C, Frequency of preparations in which RPeD1 and VD2/VD3 were retrograde labeled after 2 d in the presence of 100 nm L-EGF (saline + L-EGF) and without (saline only). Treatment with L-EGF significantly enhanced the proportion of preparations that extended axons from RPeD1 and VD2/VD3 into the damaged RIP nerve. D, Frequency distribution of labeled RPA somata per CNS in preparations that were cultured for 2 d in the absence (saline only) and presence of 100 nm L-EGF (saline + L-EGF) (bin width = 2). Treatment with L-EGF significantly enhanced regeneration of RPA axons projecting into the RIP nerve.

Fig. 6.

Effect of the specific EGF tyrosine kinase receptor inhibitor PD153035 on L-EGF-enhanced axonal regeneration after nerve injury. A, Frequency of preparations in which RPeD1 and VD2/VD3 were retrograde labeled in injured preparations after 2 d in the presence of 100 nm L-EGF (L-EGF) or 100 nm L-EGF plus 100 nm PD153035 (L-EGF + PD153035). Significantly fewer preparations with labeled RPeD1 or VD2/VD3 somata were observed in the presence of PD153035. B, Frequency distribution of labeled RPA somata per CNS in injured preparations that were cultured for 2 d in saline plus 100 nm L-EGF (L-EGF) or 100 nm L-EGF plus 100 nm PD153035 (L-EGF + PD153035) (bin width = 2). Compared with controls, the number of labeled RPA somata was significantly lower in preparations treated with L-EGF + PD153035.

Fig. 7.

Effect of the specific EGF tyrosine kinase receptor inhibitor PD153035 on axonal regeneration after RIP nerve crush in saline without L-EGF supplementation. A, Frequency of preparations in which RPeD1 and VD2/VD3 were retrograde labeled by backfilling the RIP nerve after 2 d in culture in saline (saline only) or saline plus 100 nmPD153035 (saline + PD153035). Significantly fewer preparations with labeled RPeD1 or VD2/VD3 were observed in the presence of the kinase inhibitor. B, Frequency distribution of labeled RPA somata per CNS in preparations that were cultured for 2 d in saline plus 100 nm L-EGF (saline only) or saline plus 100 nm PD153035 (saline + PD153035) (bin width = 2). Compared with controls, treatment with PD153035 caused a significant reduction in number of labeled RPA somata.