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Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats.

Zhu Q, Liu Z, Wang C, Nie L, He Y, Zhang Y, Liu X, Su G - Mol Med Rep (2015)

Bottom Line: The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05).The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups.In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of growth-associated protein-43 (GAP-43) on bone marrow mesenchymal stem cell (BMSC) differentiation in a rat model of traumatic optic neuropathy (TON). GAP‑43 and short hairpin (sh)RNA‑GAP‑43 were inserted into pGLV5 and pGLV3 lentiviral vectors, respectively. The stable control, GAP‑43‑overexpression and GAP‑43‑knockdown cell lines (GFP/BMSCs, GAP‑43/BMSCs and shGAP‑43/BMSCs, respectively) were established. The expression of GAP‑43, neuron‑specific enolase (NSE), nestin, neurofilament (NF), neuron‑specific nuclear‑binding protein (NeuN) and βIII‑tubulin were detected in the GAP‑43/BMSCs and shGAP‑43/BMSCs with retinal cell‑conditioned differentiation medium using semi‑quantitative polymerase chain reaction (PCR), western blotting and cell immunofluorescence. In addition, the BMSCs were observed under fluorescence microscopy. The Sprague‑Dawley rat models of TON were established and identified by retrograde labeling of retinal ganglion cells (RGCs) with fluoroGold (FG). The lentiviral‑mediated GAP‑43‑modified BMSCs were then transplanted into the rat model of TON. The expression of GAP‑43 was detected in the retinal tissues using qPCR and western blotting. The histopathology of the retinal tissues was observed using hematoxylin and eosin (H&E) staining. The GAP‑43/BMSCs exhibited positive expression of NSE, NF, nestin and βIII‑tubulin, and exhibited a neuronal phenotype. The shGAP‑43/BMSCs markedly inhibited expression of NeuN, NSE, NF, nestin and βIII‑tubulin induced by retinal cell‑conditioned differentiation medium. The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05). The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups. In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

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Intravitreally injected BMSCs overexpressing GAP-43 promote the nerve repair process in a rat model of TON. (A) Fluorogold staining revealed that the number of labeled RGCs was decreased in the TON model rats, compared with normal rats at day 7. (B) Number of labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction as the duration extended in TON model rats, compared with the normal rats. *P<0.05, vs. normal group. (C) mRNA levels of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSC control group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using quantitative polymerase chain reaction. (D) Protein expression of GAP-43 was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSCs group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using western blotting. The graph depicts the results of the densitometric quantification of the expression of GAP-43. 1, sham group; 2, GAP-43/BMSC group; 3, shGAP-43/BMSCE group; 4, PBS group; 5, GFP/BMSC group. (E) Hematoxylin and eosin staining revealed that pathological changes in the retina were markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups at day 3. Scale bar=50 µm. *P<0.01 GAP-43/BMSC, vs. GFP/BMSC group, #P<0.05 shGAP-43/BMSC, vs. GFP/BMSC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; shGAP-43, transduced with shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; RGCs, retinal ganglion cells; TON, traumatic optic neuropathy; PBS, phosphate-buffered saline.
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f4-mmr-12-04-5691: Intravitreally injected BMSCs overexpressing GAP-43 promote the nerve repair process in a rat model of TON. (A) Fluorogold staining revealed that the number of labeled RGCs was decreased in the TON model rats, compared with normal rats at day 7. (B) Number of labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction as the duration extended in TON model rats, compared with the normal rats. *P<0.05, vs. normal group. (C) mRNA levels of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSC control group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using quantitative polymerase chain reaction. (D) Protein expression of GAP-43 was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSCs group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using western blotting. The graph depicts the results of the densitometric quantification of the expression of GAP-43. 1, sham group; 2, GAP-43/BMSC group; 3, shGAP-43/BMSCE group; 4, PBS group; 5, GFP/BMSC group. (E) Hematoxylin and eosin staining revealed that pathological changes in the retina were markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups at day 3. Scale bar=50 µm. *P<0.01 GAP-43/BMSC, vs. GFP/BMSC group, #P<0.05 shGAP-43/BMSC, vs. GFP/BMSC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; shGAP-43, transduced with shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; RGCs, retinal ganglion cells; TON, traumatic optic neuropathy; PBS, phosphate-buffered saline.

Mentions: FG staining revealed that the labeled RGCs were significantly decreased in the TON model rats, compared with the normal rats (P<0.05; Fig. 4A). This suggested that the TON model had been successfully established. The labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction with longer periods of time (Fig. 4B), suggesting that the optimum period of nerve repair was limited to 3 days. At each time-point, the mRNA level of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group (P<0.01), compared with the GFP/BMSC group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSC group (P<0.05; Fig. 4C). Similar results were observed for GAP-43 protein (P<0.05; Fig. 4D). The retinal tissue was harvested at day 3 post-surgery (early injection) and subjected to H&E staining. As observed in the sections from the sham group (Fig. 4E), all layers were normally organized, the thickness of the retina was normal and no cell apoptosis was evident. In the PBS group, the outer nuclear layer of the retina became thinner, the inner nuclear layer of retina did not change and the ganglion cell layer became gradually thinner. In the GAP-43/BMSC group, all layers exhibited normal thicknesses and no significant decrease in ganglion cells was observed. The pathological changes in the GFP/BMSC group were similar to those in the GAP-43/BMSC group, with the exception that ganglion cells decreased, compared with the GAP-43/BMSC group. In the shGAP-43/BMSC group, all layers became thinner and ganglion cells exhibited marginal nuclear pyknosis, loss of nucleoli and cytoplasmic vacuolation. The degree of pathological changes was markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups (Fig. 4E).


Lentiviral-mediated growth-associated protein-43 modification of bone marrow mesenchymal stem cells improves traumatic optic neuropathy in rats.

Zhu Q, Liu Z, Wang C, Nie L, He Y, Zhang Y, Liu X, Su G - Mol Med Rep (2015)

Intravitreally injected BMSCs overexpressing GAP-43 promote the nerve repair process in a rat model of TON. (A) Fluorogold staining revealed that the number of labeled RGCs was decreased in the TON model rats, compared with normal rats at day 7. (B) Number of labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction as the duration extended in TON model rats, compared with the normal rats. *P<0.05, vs. normal group. (C) mRNA levels of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSC control group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using quantitative polymerase chain reaction. (D) Protein expression of GAP-43 was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSCs group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using western blotting. The graph depicts the results of the densitometric quantification of the expression of GAP-43. 1, sham group; 2, GAP-43/BMSC group; 3, shGAP-43/BMSCE group; 4, PBS group; 5, GFP/BMSC group. (E) Hematoxylin and eosin staining revealed that pathological changes in the retina were markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups at day 3. Scale bar=50 µm. *P<0.01 GAP-43/BMSC, vs. GFP/BMSC group, #P<0.05 shGAP-43/BMSC, vs. GFP/BMSC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; shGAP-43, transduced with shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; RGCs, retinal ganglion cells; TON, traumatic optic neuropathy; PBS, phosphate-buffered saline.
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Related In: Results  -  Collection

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Show All Figures
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f4-mmr-12-04-5691: Intravitreally injected BMSCs overexpressing GAP-43 promote the nerve repair process in a rat model of TON. (A) Fluorogold staining revealed that the number of labeled RGCs was decreased in the TON model rats, compared with normal rats at day 7. (B) Number of labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction as the duration extended in TON model rats, compared with the normal rats. *P<0.05, vs. normal group. (C) mRNA levels of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSC control group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using quantitative polymerase chain reaction. (D) Protein expression of GAP-43 was significantly increased in the GAP-43/BMSC group, compared with the GFP/BMSCs group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSCs group at days 3, 7, 14 and 28, determined using western blotting. The graph depicts the results of the densitometric quantification of the expression of GAP-43. 1, sham group; 2, GAP-43/BMSC group; 3, shGAP-43/BMSCE group; 4, PBS group; 5, GFP/BMSC group. (E) Hematoxylin and eosin staining revealed that pathological changes in the retina were markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups at day 3. Scale bar=50 µm. *P<0.01 GAP-43/BMSC, vs. GFP/BMSC group, #P<0.05 shGAP-43/BMSC, vs. GFP/BMSC group. GAP-43, growth-associated protein-43; shRNA, short hairpin RNA; NC, transduced with negative control scramble lentiviral vector; shGAP-43, transduced with shRNA-GAP-43-619; BMSCs, bone marrow mesenchymal stem cells; RGCs, retinal ganglion cells; TON, traumatic optic neuropathy; PBS, phosphate-buffered saline.
Mentions: FG staining revealed that the labeled RGCs were significantly decreased in the TON model rats, compared with the normal rats (P<0.05; Fig. 4A). This suggested that the TON model had been successfully established. The labeled RGCs began to reduce at day 3 and reached the lowest point at day 7, with no further reduction with longer periods of time (Fig. 4B), suggesting that the optimum period of nerve repair was limited to 3 days. At each time-point, the mRNA level of GAP-43 in the retinal tissues was significantly increased in the GAP-43/BMSC group (P<0.01), compared with the GFP/BMSC group, and decreased in the shGAP-43/BMSC group, compared with the GFP/BMSC group (P<0.05; Fig. 4C). Similar results were observed for GAP-43 protein (P<0.05; Fig. 4D). The retinal tissue was harvested at day 3 post-surgery (early injection) and subjected to H&E staining. As observed in the sections from the sham group (Fig. 4E), all layers were normally organized, the thickness of the retina was normal and no cell apoptosis was evident. In the PBS group, the outer nuclear layer of the retina became thinner, the inner nuclear layer of retina did not change and the ganglion cell layer became gradually thinner. In the GAP-43/BMSC group, all layers exhibited normal thicknesses and no significant decrease in ganglion cells was observed. The pathological changes in the GFP/BMSC group were similar to those in the GAP-43/BMSC group, with the exception that ganglion cells decreased, compared with the GAP-43/BMSC group. In the shGAP-43/BMSC group, all layers became thinner and ganglion cells exhibited marginal nuclear pyknosis, loss of nucleoli and cytoplasmic vacuolation. The degree of pathological changes was markedly improved in the GAP-43/BMSC group, compared with the GFP/BMSC and shGAP-43/BMSC groups (Fig. 4E).

Bottom Line: The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05).The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups.In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of growth-associated protein-43 (GAP-43) on bone marrow mesenchymal stem cell (BMSC) differentiation in a rat model of traumatic optic neuropathy (TON). GAP‑43 and short hairpin (sh)RNA‑GAP‑43 were inserted into pGLV5 and pGLV3 lentiviral vectors, respectively. The stable control, GAP‑43‑overexpression and GAP‑43‑knockdown cell lines (GFP/BMSCs, GAP‑43/BMSCs and shGAP‑43/BMSCs, respectively) were established. The expression of GAP‑43, neuron‑specific enolase (NSE), nestin, neurofilament (NF), neuron‑specific nuclear‑binding protein (NeuN) and βIII‑tubulin were detected in the GAP‑43/BMSCs and shGAP‑43/BMSCs with retinal cell‑conditioned differentiation medium using semi‑quantitative polymerase chain reaction (PCR), western blotting and cell immunofluorescence. In addition, the BMSCs were observed under fluorescence microscopy. The Sprague‑Dawley rat models of TON were established and identified by retrograde labeling of retinal ganglion cells (RGCs) with fluoroGold (FG). The lentiviral‑mediated GAP‑43‑modified BMSCs were then transplanted into the rat model of TON. The expression of GAP‑43 was detected in the retinal tissues using qPCR and western blotting. The histopathology of the retinal tissues was observed using hematoxylin and eosin (H&E) staining. The GAP‑43/BMSCs exhibited positive expression of NSE, NF, nestin and βIII‑tubulin, and exhibited a neuronal phenotype. The shGAP‑43/BMSCs markedly inhibited expression of NeuN, NSE, NF, nestin and βIII‑tubulin induced by retinal cell‑conditioned differentiation medium. The FG staining revealed that the number of labeled RGCs were significantly decreased in the TON model rats, compared with normal rats (P<0.05). The H&E staining revealed that the degree of pathological changes was improved in the GAP‑43/BMSC group, compared with the GFP/BMSC and shGAP‑43/BMSC groups. In conclusion, GAP‑43 promoted BMSC differentiation into neuron-like cells, and intravitreally injected GAP-43/BMSCs promoted the process of nerve repair in a rat model of TON.

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Related in: MedlinePlus