Histomorphological study of the spinal growth plates from the convex side and the concave side in adolescent idiopathic scoliosis

Asymmetrical growth of the vertebrae has been implicated as one possible etiologic factor in the pathogenesis of adolescent idiopathic scoliosis. The longitudinal vertebral growth derives from the endochondral ossification of the vertebral growth plate. In the present study, the growth plates from the convex and concave side of the vertebrae were characterized by the method of histology and immunohistochemistry to evaluate the growth activity, cell proliferation, and apoptosis. Normal zoned architectures were observed in the convex side of the growth plate and disorganized architectures in the concave side. The histological grades were significantly different between the convex and the concave side of the growth plate in the apex vertebrae (P < 0.05). The histological difference was also found significant statistically between end vertebrae and apex vertebrae in the concave side of vertebral growth plates (P < 0.05). The proliferative potential indexes and apoptosis indexes of chondrocytes in the proliferative and hypertrophic zone in the convex side were significantly higher than that in the concave side in the apex vertebral growth plate (P < 0.05). There was a significant difference of the proliferative potential index (proliferating cell nuclear antigen, PCNA index) between convex side and concave side at the upper end vertebra (P < 0.05). The difference of the proliferative potential index and apoptosis index were found significant statistically in the concave side of the vertebral growth plate between end vertebrae and apex vertebrae (P < 0.05). The same result was also found for the apoptosis index (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling assay, TUNEL index) in the convex side of vertebral growth plate between end vertebrae and apex vertebrae (P < 0.05). Some correlation were found between radiographic measurements and proliferation and apoptosis indexes. The difference in histological grades and cellular activity between the convex and concave side indicated that the bilateral growth plate of the vertebrae in AIS patients have different growth kinetics which may affect the curve progression

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma

The list of genes and primers.

<p>The list of genes and primers.</p

The survival rate and neurite outgrowth of primary RGCs co-cultured with transfected hNPs under various conditions.

<p>Dead/live (green/red fluorescence) cell analysis of primary RGCs co-cultured with hNP^TD (A) and hNP^IGF-TD (B) cells shows increased live cells in the latter group. ß-III tubulin staining of co-cultured cells indicates that neurites were rarely observed in RGCs co-cultured with hNP^TD cells (C) as compared with RGCs co-cultured with hNP^IGF-TD cells (D) (Cy3, orange-red fluorescence). (E—F) Quantification of survival rate and neurite length in RGCs co-cultured with hNP^IGF-TD or hNP^TD cells in presence and absence of IGF antagonists Data was analyzed with Mann-Whitney U test. RGC survival rate was significantly higher in hNP^IGF-TD co-cultures; IGF antagonists reduced both survival rate and neurite outgrowth of RGCs. The boxes in (E) and (F) represent the 0.25, median and 0.75 quantiles. On either side of the box, the whiskers extend to the minimum and maximum. Detailed index of each transfection is presented in the text. The red dash line in each box is the mean value. Abbreviations: NP, neuronal progenitor cells; TD, neuronal progenitor cells expressing TD (hNP^TD); IGF-1, neuronal progenitor cells expressing IGF-TD fusion protein (hNP^IGF-TD); Scale bar: 200 μm (A—B) and 50 μm (C—D).</p

RGC loss in the experimental glaucoma model.

<p>(A—E) Retina sections immmunostained for ß-III tubulin (red fluorescence) confirm microbead-induced glaucomatous RGC loss (SA, NP and TD) and RGC rescue in eyes transplanted with hNP^IGF-TD cells (IGF-TD). Brn3a antibody (red fluorescence) was used to verify the accuracy of RGC density stained with ß-III tubulin (green fluorescence) on retinal cross sections. (F—K) and retinal flatmounts using confocal microscopy (L—N). RGC population was slightly overestimated (1.7%) using ß-III tubulin staining compared to Brn3a staining, but the difference was not significant (Mann-Whitney U test, <i>P</i> > 0.05). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell; IGF-TD, transplanted hNP^IGF-TD cells after microbead injection; TD, transplanted hNP^TD cells after microbead injection. hNP, untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection. Scale bar: 50 μm in A—E.</p

Quantification of RGC density using ß-III tubulin (red fluorescence) in retinal flatmounts under different experimental conditions.

<p>(A—E) Confocal images show significant and robust RGC loss is visualized in the saline, hNPs and hNP^TD transplanted groups after microbead injections. RGC density and distribution in microbead-injected eyes transplanted with hNP^IGF-TD cells (E) were similar to the control group (A). (F) Quantification of RGC density in various experimental groups. Data are presented as the fitted geometric means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. <i>P</i> < 0.05 compared to NO group; #, <i>P</i> < 0.05 compared to IGF-TD group. Scale bar: 50 μm in A—E. Abbreviations: IGF-TD, transplanted hNP^IGF-TD cells after microbead injection; TD, transplanted hNP^TD cells after microbead injection. hNP, transplanted untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.</p

Detection of tdTomato (TD) and IGF1-TD fusion protein in human neuronal progenitor cells (hNP).

<p>(A) Expression of TD protein in transfected hNPs by their red fluorescence. (B) TD protein can also be detected by using immunohistochemistry (FITC, green). (C) Expression of IGF-TD fusion protein in hNPs by their red fluorescence. (D) Detection the IGF-1 moiety of the IGF-TD fusion protein in transfected cells by immunostaining (FITC, green). (E) Expression of IGF-1 component of the IGF-TD mRNA in hNP^IGF-TD cells by qRT-PCR; IGF-1 mRNA is undetectable in untransfected hNP and hNP^TD cells. (F) Western blot analysis of IGF-1 component of the IGF-TD protein in hNP^IGF-TD cell lysates confirms the expected molecular weight of approximately 60 kD. IGF-1 was not detected in untransfected hNP and hNP^TD cells. (G) IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein (red triangles). Data are presented as mean ± SE for the day 0 wells (B; blank wells). Symbols correspond to the fitted means ± the 95% confidence limits of the measurements conducted in conditioned medium from the difference hNPs (open symbols; empty vector, yellow symbols; TD-transfected cells, green symbols; IGF-TD—transfected cells). Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences <i>versus</i> all groups at the same time point. Abbreviations: hNP, neuronal progenitor cells; TD, tdTomato; IGF-TD, IGF-1-tdTomato. Scale bar in A—D: 50 μm.</p

Quantification of axons on semi-thin cross sections of optic nerve under different experimental conditions.

<p>(A) Cross section of axons from the intraorbital section of the optic nerve after saline injection (no glaucoma). Significant axon degeneration is observed in the saline, hNPs and hNP^TD transplanted groups after microbead injection (B—D). There is better preservation of axons in the hNP^IGF-TD transplanted group (E). (F) Quantification of axon cross-sections indicates that transplantation of hNP^IGF-TD cells show a trend to enhanced survival of axons in the glaucomatous environment (data are presented as the fitted means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the square of the data. Crosses indicate a trend (<i>P</i> < 0.1) compared to the NO and IGF groups). Scale bar: 5 μm in A—E. Abbreviations: IGF-TD, transplanted hNP^IGF-TD cells after microbead injection; TD, transplanted hNP^TD cells after microbead injection. hNP, transplanted (untransfected) hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.</p

Mouse treatment groups receiving different injections.

<p>hNP: human neuronal progenitor cells; hNP^TD_: cells expressing TD; hNP^IGF-TD: cells expressing IGF1-TD fusion prote</p><p>Mouse treatment groups receiving different injections.</p