Table 1.
In vivo treatment groups.
Figure 1.
NSPCs respond to dbcAMP in vitro.
A) Dose response curve of dbcAMP on neuronal differentiation of NSPCs after 7 days in culture. B) Differentiation profile of NSPCs after 7 days. NSPCs were treated with media containing 1 mM dbcAMP for 0, 1 or 7 days. Only sustained exposure to dbcAMP resulted in increased number of BetaIII-positive neurons. C–J) Representative images of NSPCs after 7 days in culture for markers of progenitors cells (nestin), neurons (BetaIII), oligodendrocytes (RIP), and astrocytes (GFAP). Scale bar represents 100 µm. K) Cell numbers at 1, 3, and 7 days in culture with or without 1 mM dbcAMP. L) Ki67 staining for proliferating cells after 3 days. M) Cell differentiation over time with or without 1 mM dbcAMP treatment. Data represented as mean ± standard (n = 3 to 9). Statistical differences denoted by *, p<0.05.
Figure 2.
Microsphere-loaded channels effectively release dbcAMP in vitro.
A) Cumulative release profiles of dbcAMP from free-floating microspheres and microsphere-loaded channels. The process of embedding microspheres into channel walls is likely responsible for early degradation of PLGA and faster drug release from channels. B) Schematic of the entubulation strategy. NSPCs are seeded on fibrin scaffold within a chitosan channel. Drug-loaded PLGA microspheres release the differentiation factor dibutyryl cyclic-AMP in a local and sustained manner, influencing NSPCs to preferentially differentiate into neurons. C) Viability of NSPCs in a three-dimensional fibrin scaffold. Simultaneous staining of CalceinAM (green) and Ethidium homodimer (red) for live and dead cells respectively show good cell viability of NSPCs in fibrin scaffolds at 1 week. Scale bar represents 100 µm. D–G) Immunostaining of NSPCs for DAPI-nuclear stain and betaIII-tubulin with various dbcAMP treatments. Scale bar represents 100 µm. H) Quantification of betaIII-tubulin immunostained NSPCs with various dbcAMP treatments. I,J) Quantitative RT-PCR data for (I) betaIII tubulin and (J) nestin mRNA expression with various dbcAMP treatments, normalized to housekeeping gene HPRT. Data represented as mean ± standard (n = 3 to 6). Statistical differences denoted by *, p<0.05.
Figure 3.
Channel implantation after spinal cord transection facilitates tissue bridging, NSPC survival, and behavioural improvement over time.
A) Photograph of the surgical implantation of fibrin-filled chitosan channels. B) Tissue bridges obtained from animals 2 weeks after implantation. C,D) Longitudinal section of tissue bridge demonstrating NSPC survival after 6 weeks in an animal receiving dbcAMP pre-treatment (dbcAMP, 4div). Boxed area in (C) is magnified in (D). E) NSPC survival after 2 and 6 weeks for various treatment groups. F) Assessment of functional recovery using the BBB locomotor scale. After 6 weeks, rats receiving transplants of dbcAMP-pre-treated NSPCs show a statistically significant increase in hindlimb function relative to untreated animals (*, p<0.05). Mean ± standard deviation shown for n = 4 to 6.
Figure 4.
Differentiation profiles of NSPCs are impacted by dbcAMP treatment.
A–L) Representative images of tissue samples demonstrating NSPC differentiation profile of (A–C) nestin-positive progenitor cells, (D–F) BetaIII-positive neurons, (G–H) CC1-positive oligodendrocytes, and (J–L) GFAP-positive astrocytes. Scale bar represents 50 µm. M) Quantification of NSPC differentiation profile for the various treatment groups. Mean ± standard deviation are plotted, n = 3 to 5; significant differences noted with an asterisks, p<0.05. N) Deconvoluted confocal image of betaIII-positive NSPC-derived neurons (arrows) 6 weeks post-transplantation. Scale bar represents 50 µm.
Figure 5.
The regenerated bridge tissue contains host axons, blood vessels, and fibroblasts.
A) Representative image of endogenous axonal regeneration into the tissue bridge based on betaIII tubulin staining. B) Evidence of association between betaIII-positive endogenous axons with surviving GFP-positive NSPCs at six weeks. Synaptophysin staining is observed at the interface (inset). C) RECA1 staining for endothelial cells show blood vessel formation throughout the tissue bridge at 2 weeks. D) Prolyl-4-hydroxylase (rPH) staining of bridge tissue indicates that the majority of cells are collagen producing fibroblasts.