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First published online February 1, 2008
Journal of Experimental Biology 211, 491-501 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013102
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RGD-dependent mechanisms in the endoneurial phagocyte response and axonal regeneration in the nervous system of the snail Lymnaea stagnalis

Petra M. Hermann1, Jennifer J. Nicol1, Andrew G. M. Bulloch2 and Willem C. Wildering1,2,*

1 Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada, T2N 1N4
2 Department of Physiology and Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1


Figure 1
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  		Fig. 1. Treatment with RGD-peptides modulates axonal regeneration in organ-cultured CNS. (Ai and Aii) Microphotographs of isolated CNS (dorsal view) that were cultured after receiving a crush to the right internal parietal (RIP) nerve (arrow) in ABS only (i) and ABS + 100 µmol l–1 GRGDS (ii). Comparison of the two photographs illustrates that the number of labelled right parietal A (RPA) somata was substantially reduced in the presence of GRGDS. (B) Average number of retrograde nickel-lysine labelled RPA somata in preparations that were cultured in ABS only, ABS + 100 µmol l–1 control peptide SDGRG and ABS + 100 µmol l–1 GRGDS. (C) Average number of retrograde nickel-lysine labelled RPA somata in preparations that were cultured in ABS only or in the presence of different concentrations of cGRGDSPA (10 nmol l–1 to 100 µmol l–1). Treatment with cGRGDSPA had a concentration-dependent biphasic effect on the regeneration of RPA neurons projecting into the RIP nerve. ***P<0.001.

 

Figure 2
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  		Fig. 2. Linear RGD-peptides reduce the percentage of spreading endoneurial phagocytes in vitro. (Ai and Aii) Photographs of non-spreading (i) and spreading (ii) endoneurial phagocytes cultured in ABS. (B) Percentage of endoneurial phagocytes showing a spreading response after culturing in the presence of uncoated monodispersed polystyrene carboxylated Fluoresbrite YG microspheres in ABS only, ABS + 100 µmol l–1 SDGRG and ABS + 100 µmol l–1 GRGDS. The addition of GRGDS significantly reduced the percentage of spreading cells. (C) Percentage of endoneurial phagocytes showing a spreading response after culturing in the presence of fibronectin-coated microspheres in ABS only, ABS + 100 µmol l–1 SDGRG and ABS + 100 µmol l–1 GRGDS. Again, the addition of GRGDS significantly reduced the percentage of spreading cells. Scale bar in A, 10 µm. ***P<0.001.

 

Figure 3
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  		Fig. 3. Linear RGD-peptides reduce the percentage of phagocytic active cells in vitro. (Ai) Photograph of an isolated endoneurial phagocyte cultured in the presence of monodispersed polystyrene carboxylated Fluoresbrite YG microspheres. (Aii) Photomicrograph showing the large number of fluorescent microspheres engulfed by the phagocyte shown in Ai. (Bi) Percentage of spreading cells that internalized uncoated microspheres when cultured in ABS only, ABS + 100 µmol l–1 SDGRG and ABS + 100 µmol l–1 GRGDS. (Bii) The distribution of the number of uncoated microspheres engulfed by spreading cells under the three culture conditions. Note that the addition of GRGDS had no effect on the percentage of phagocytic cells engulfing uncoated microspheres nor on the distribution of the number of engulfed uncoated microspheres. (Ci) Percentage of spreading cells that phagocytized fibronectin-coated microspheres when cultured in ABS only, ABS + 100 µmol l–1 SDGRG and ABS + 100 µmol l–1 GRGDS. The addition of GRGDS significantly reduced the percentage of phagocytic cells. (Cii) Distribution of the number of engulfed fibronectin-coated microspheres by spreading cells is shifted to the right (i.e. more microspheres are engulfed) when the cells were cultured in ABS or ABS + 100 µmol l–1 SDGRG. In contrast, treatment with GRGDS did not result in a similar increase in internalization of fibronectin-coated microspheres. Scale bar in A, 10 µm. ***P<0.001.

 

Figure 4
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  		Fig. 4. Circularized RGD-peptides modulate activation of endoneurial phagocytes in vitro. (A) Percentage of endoneurial phagocytes showing a spreading response after culturing in the presence of fibronectin-coated microspheres in ABS only, and in the presence of ABS + 10 nmol l–1 cGRGDSPA, ABS + 1 µmol l–1 cGRGDSPA or ABS + 100 µmol l–1 cGRGDSPA. (B) Percentage of spreading endoneurial phagocytes that engulfed fibronectin-coated microspheres when cultured in ABS only, and in the presence of ABS + 10 nmol l–1 cGRGDSPA, ABS + 1 µmol l–1 cGRGDSPA or ABS + 100 µmol l–1 cGRGDSPA. Treatment with cGRGDSPA had a significant concentration-dependent biphasic effect on both the percentage of spreading cells and the percentage of phagocytic active cells. (C) Distribution of the number of engulfed fibronectin-coated microspheres by spreading cells is shifted to the right, i.e. more microspheres are engulfed, when the cells were cultured in ABS only compared with the cells cultured in the presence of 1 µmol l–1 cGRGDSPA.

 

Figure 5
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  		Fig. 5. Reactive oxygen species production depends on the activation status of endoneurial phagocytes. (Ai and Aii, left panels) DIC images of a spreading endoneurial phagocyte (i) and non-spreading endoneurial phagocyte (ii). (Ai and Aii, right panels) Epifluorescence images showing CM-H2DCF fluorescence (i), or the absence thereof (ii), in the same cells as shown in the corresponding left panels. Note that the CM-H2DCF fluorescence is contained inside the activated endoneurial phagocyte. (B) Percentage of CM-H2DCF fluorescence-positive endoneurial phagocytes when cultured in ABS only, ABS + 1 µmol l–1 cGRGDSPA and ABS + 100 µmol l–1 cGRGDSPA. Note that independent of culture condition, the majority of the spreading cells are CM-H2DCF fluorescence positive while only a very small percentage (<3%) of the non-spreading cells display CM-H2DCF fluorescence. Image acquisition conditions were exactly the same for all cells. Scale bar in A, 10 µm.

 

Figure 6
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  		Fig. 6. Circularized RGD-peptides modulate oxidative burst in the injured nerve. (A–C, left panels) Phase contrast photomicrographs showing the injured RIP nerve cultured in ABS only, 1 µmol l–1 cGRGDSPA and 100 µmol l–1 cGRGDSPA, respectively. Arrows indicate crush zones. (A–C, right panels) Corresponding CM-H2DCF fluorescence signal of the nerves shown in the left panels. (D) Average CM-H2DCF fluorescence intensity of preparations cultured for 1 h in ABS only, ABS + 1 µmol l–1 cGRGDSPA or ABS + 100 µmol l–1 cGRGDSPA. (E) Average CM-H2DCF fluorescence intensity of preparations cultured for 48 h in ABS only, ABS + 1 µmol l–1 cGRGDSPA or ABS + 100 µmol l–1 cGRGDSPA. Note the attenuated CM-H2DCF fluorescence signal in the preparations cultured in the presence of 1 µmol l–1 cGRGDSPA. Image acquisition conditions were exactly the same for all preparations. Scale bar in A–C, 100 µm.

 

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© The Company of Biologists Ltd 2008