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Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

Vecino E, Heller JP, Veiga-Crespo P, Martin KR, Fawcett JW - PLoS ONE (2015)

Bottom Line: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions.We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites.Adult rat RGCs can survive and grow in the presence of different ECM tested.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Histology, University of the Basque Country, UPV/EHU, Leioa, Vizcaya, Spain; John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Purpose: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites?

Methods: Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against βIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, β1 or β3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed.

Results: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1β1 or α3β1 on L, α1β1 on CI and CIV, and α5β3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK).

Conclusions: Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

No MeSH data available.


Related in: MedlinePlus

Influence of the substrata and cell distribution.In all cases the statistical difference was (p<0.05). A: Influence of the substrata on cell survival. The influence of the substrate on the number of observed cells PL and L (•) show significant differences compared to the other substrates; CI ($) shows significant differences in the number of cells compared to the other substrates and CIV and F (#) have significant differences compared to the other substrates B: Distribution of RGC complexity (number of neurites per cell). B1: Low level of complexity. Significant differences were found for PL and L (•) against the rest of the substrates; CI ($) against the rest of substrates and CIV and F (#) with the rest of the substrates. B2: Medium level of complexity. Significant differences were found for each substrate compared to the other substrates. B3: High level of complexity. Significant differences were found for PL, L and CIV (•) against CI and F; CI ($) and F (#) was significantly different to all other substrates. C: Distribution of RGC neurites length between the different substrates. C1: Short length RGCs growing in PL and L (•) are significantly different to the other substrates; and the rest of the RGCs growing in CI ($), CIV (#), or F (##) each was different to the other substrates. C2 Medium length. CI ($) was significantly different to the other substrates, but there was no difference between them PL, L, CIV and F (•). C3 Long length neurites were more frequent when RGCs were plated on L (••) and this difference was significant compared to the other substrates. PL and F (•) were the second substrate in which long neurites were present; CI and CIV had significantly fewer long neurites.
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pone.0125250.g001: Influence of the substrata and cell distribution.In all cases the statistical difference was (p<0.05). A: Influence of the substrata on cell survival. The influence of the substrate on the number of observed cells PL and L (•) show significant differences compared to the other substrates; CI ($) shows significant differences in the number of cells compared to the other substrates and CIV and F (#) have significant differences compared to the other substrates B: Distribution of RGC complexity (number of neurites per cell). B1: Low level of complexity. Significant differences were found for PL and L (•) against the rest of the substrates; CI ($) against the rest of substrates and CIV and F (#) with the rest of the substrates. B2: Medium level of complexity. Significant differences were found for each substrate compared to the other substrates. B3: High level of complexity. Significant differences were found for PL, L and CIV (•) against CI and F; CI ($) and F (#) was significantly different to all other substrates. C: Distribution of RGC neurites length between the different substrates. C1: Short length RGCs growing in PL and L (•) are significantly different to the other substrates; and the rest of the RGCs growing in CI ($), CIV (#), or F (##) each was different to the other substrates. C2 Medium length. CI ($) was significantly different to the other substrates, but there was no difference between them PL, L, CIV and F (•). C3 Long length neurites were more frequent when RGCs were plated on L (••) and this difference was significant compared to the other substrates. PL and F (•) were the second substrate in which long neurites were present; CI and CIV had significantly fewer long neurites.

Mentions: The mean number of surviving attached cells after ten days on PL and L was higher than the number of cells surviving on the other 3 substrates (PL 474 ± 59, 24,9% and L 476 ± 57, 25,1%). The lowest number of RGCs was present on CI (227 ± 17, 11,9%), while CIV and F had similar numbers of cells (337 ± 29 and 350 ± 34, respectively). These differences were statistically significant (p<0.05). In conclusion, PL and L favoured the survival of RGCs (Fig 1).


Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

Vecino E, Heller JP, Veiga-Crespo P, Martin KR, Fawcett JW - PLoS ONE (2015)

Influence of the substrata and cell distribution.In all cases the statistical difference was (p<0.05). A: Influence of the substrata on cell survival. The influence of the substrate on the number of observed cells PL and L (•) show significant differences compared to the other substrates; CI ($) shows significant differences in the number of cells compared to the other substrates and CIV and F (#) have significant differences compared to the other substrates B: Distribution of RGC complexity (number of neurites per cell). B1: Low level of complexity. Significant differences were found for PL and L (•) against the rest of the substrates; CI ($) against the rest of substrates and CIV and F (#) with the rest of the substrates. B2: Medium level of complexity. Significant differences were found for each substrate compared to the other substrates. B3: High level of complexity. Significant differences were found for PL, L and CIV (•) against CI and F; CI ($) and F (#) was significantly different to all other substrates. C: Distribution of RGC neurites length between the different substrates. C1: Short length RGCs growing in PL and L (•) are significantly different to the other substrates; and the rest of the RGCs growing in CI ($), CIV (#), or F (##) each was different to the other substrates. C2 Medium length. CI ($) was significantly different to the other substrates, but there was no difference between them PL, L, CIV and F (•). C3 Long length neurites were more frequent when RGCs were plated on L (••) and this difference was significant compared to the other substrates. PL and F (•) were the second substrate in which long neurites were present; CI and CIV had significantly fewer long neurites.
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getmorefigures.php?uid=PMC4446304&req=5

pone.0125250.g001: Influence of the substrata and cell distribution.In all cases the statistical difference was (p<0.05). A: Influence of the substrata on cell survival. The influence of the substrate on the number of observed cells PL and L (•) show significant differences compared to the other substrates; CI ($) shows significant differences in the number of cells compared to the other substrates and CIV and F (#) have significant differences compared to the other substrates B: Distribution of RGC complexity (number of neurites per cell). B1: Low level of complexity. Significant differences were found for PL and L (•) against the rest of the substrates; CI ($) against the rest of substrates and CIV and F (#) with the rest of the substrates. B2: Medium level of complexity. Significant differences were found for each substrate compared to the other substrates. B3: High level of complexity. Significant differences were found for PL, L and CIV (•) against CI and F; CI ($) and F (#) was significantly different to all other substrates. C: Distribution of RGC neurites length between the different substrates. C1: Short length RGCs growing in PL and L (•) are significantly different to the other substrates; and the rest of the RGCs growing in CI ($), CIV (#), or F (##) each was different to the other substrates. C2 Medium length. CI ($) was significantly different to the other substrates, but there was no difference between them PL, L, CIV and F (•). C3 Long length neurites were more frequent when RGCs were plated on L (••) and this difference was significant compared to the other substrates. PL and F (•) were the second substrate in which long neurites were present; CI and CIV had significantly fewer long neurites.
Mentions: The mean number of surviving attached cells after ten days on PL and L was higher than the number of cells surviving on the other 3 substrates (PL 474 ± 59, 24,9% and L 476 ± 57, 25,1%). The lowest number of RGCs was present on CI (227 ± 17, 11,9%), while CIV and F had similar numbers of cells (337 ± 29 and 350 ± 34, respectively). These differences were statistically significant (p<0.05). In conclusion, PL and L favoured the survival of RGCs (Fig 1).

Bottom Line: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions.We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites.Adult rat RGCs can survive and grow in the presence of different ECM tested.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Histology, University of the Basque Country, UPV/EHU, Leioa, Vizcaya, Spain; John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Purpose: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites?

Methods: Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against βIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, β1 or β3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed.

Results: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1β1 or α3β1 on L, α1β1 on CI and CIV, and α5β3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK).

Conclusions: Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

No MeSH data available.


Related in: MedlinePlus