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3D meshes of carbon nanotubes guide functional reconnection of segregated spinal explants

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Science Advances  15 Jul 2016:
Vol. 2, no. 7, e1600087
DOI: 10.1126/sciadv.1600087
  • Fig. 1 3D CNF scaffolds redirect neurite outgrowth between spinal organotypic slices.

    (A) SEM micrographs (left) of 3D CNF. At higher magnification (inset), the random skeleton of interconnected MWCNTs is shown. Confocal 3D reconstruction (right; reflection mode) of the same CNF scaffold. (B) Spinal slices cocultured in Control and in 3D CNF after 14 days of growth. Immunofluorescence is for neuron-specific microtubules (β-tubulin III; red), neurofilament H (SMI-32; green), and nuclei [4′,6-diamidino-2-phenylindole (DAPI); blue]. (C) Confocal micrographs showing β-tubulin III–positive neuronal projection appearance in Control (oriented; left) and in 3D CNF (random; right) and the corresponding plots (bottom) of fiber angle direction distribution. In the example, oriented field fibers are characterized by a mean directionality value of 37.6° and a dispersion value of 2.3°, whereas random field fibers show values of 22.3° and 25.1°, respectively (values evaluated from the Gaussian fitting; see Materials and Methods). (D) Top histograms summarize the mean values of fiber orientation dispersion in Control and 3D CNF (***P < 0.001); bottom histograms depict the percentage of visual fields in which fibers are aligned with a degree of dispersion less than 15°. In Controls, all samples (100%) contained oriented fibers with less than 15° of dispersion, whereas 7% of 3D CNF contained fibers with less than 15° of dispersion (***P < 0.001). Scale bars, 250 and 25 μm (inset) (A), 500 μm (B), and 100 μm (C).

  • Fig. 2 3D CNF favors neuronal process paths in the third dimension.

    (A) Detailed Z stack distribution of neuronal processes. Control neuronal processes are compared to 3D CNF ones by confocal microscopy. (B) Tilted confocal reconstruction of the intricate network of 3D CNF (white) and neuronal projections (β-tubulin III; red) (note the emergence of a 3D hybrid network knitted by the neuronal processes and the MWCNTs). (C) SEM micrograph of the hybrid network [same sample as in (B)]. (D) Volume rendering of the Z stacks of the neuronal processes in Control and 3D CNF samples. Neuronal processes under control conditions are relatively flat, whereas processes form a thicker layer when supported by 3D CNF. (E) Plots quantify the thicknesses of β-tubulin III–positive and SMI-32–positive processes (*P < 0.05, Student’s t test). (F) Spontaneous LFPs recorded in Control and 3D CNF. (G) Spontaneous LFP mean frequencies. Scale bars, 25 μm (horizontal) and 10 μm (vertical) (A), 40 μm (C), and 25 μm (D).

  • Fig. 3 3D CNF guides the functional reconnection of ventral outputs in segregated spinal organotypic slices.

    (A) Sketch of the experimental setting for double-slice ventral recordings. (B) LFP bursting induced by strychnine and bicuculline recorded simultaneously from left (L) and right (R) slices in Control and 3D CNF. Right insets are the corresponding cross-correlation plots. (C) Average CCF from Control and 3D CNF (***P < 0.001). (D) The fraction of spinal explants that were significantly correlated is significantly larger in 3D CNF (***P < 0.001, χ2 test). (E) Sketch of the experimental setting for dorsal stimulation. (F) Bursting LFP entrainment by dorsal electrical stimulation (dots) of left slices (arrow) in Control and 3D CNF slice pairs (note 3D CNF premotor output entrainment in both slices). (G) 3D CNF increases the fraction of cross-entrained explants (*P < 0.05, χ2 test). (H) Age in vitro is not correlated with CCF values (gray and black lines: regression lines for Control and 3D CNF conditions, r = 0.249 and −0.113, respectively; blue open and filled circles highlight significant correlated pairs).

  • Fig. 4 Tissue reaction to CNF scaffolds implanted into the adult rat visual cortex.

    (A) GFAP fluorescence intensity profile as a function of the distance from the edge of the implant in brain tissue. The average GFAP intensity at 4 weeks after implantation peaked at 10 μm from the implant edge on average and gradually decreased away from the implant edge. (B) GFAP-positive cells (green) are found surrounding the implant and within the material; boxed areas indicate high-magnification images shown in (C); inset: contralateral hemisphere used as a control. (C) High magnification of GFAP reactivity at the implant edge demonstrates the minimal and irregular cellular localization around the scaffold. (D) Iba1-positive area (as a fraction of total tissue area in the ROI) varies from 0.08 ± 0.05 to 0.17 ± 0.05 (roughly 8 to 17% of the tissue area) at 4 weeks after implantation, with an average of 0.13 ± 0.08. (E) Iba1-positive cells (red) are dispersed consistently throughout the tissue and within the material; boxed areas indicate high-magnification images shown in (F); inset: contralateral hemisphere used as a control. (F) High-magnification images of the Iba1 reactivity demonstrate no obvious border at the implant edge to indicate scar formation. (G) Top left: β-Tubulin III–positive cells (red; DAPI, blue) within the scaffold and surrounded by Iba1-positive microglia (green) (note the absence of colocalization). Top right: β-Tubulin III–positive cells (red; DAPI, blue) within the scaffold from another animal, suggestive of the fact that neuronal process infiltration may be consistent at 4 weeks after implantation. Bottom: NeuN-positive cells (green; DAPI, blue) within the scaffold and two different areas are shown. (H) NeuN-positive cells (green; DAPI, blue) within the contralateral hemisphere; inset: high magnification of NeuN-positive cells. Scale bars, 200 μm (B and E), 50 μm (C and F), 20 μm (G), and 100 and 10 μm (inset) (H).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1600087/DC1

    Supplementary Materials and Methods

    fig. S1. Illustration of the permutation test to assess the statistical significance of the synchrony between the bursting activities of two cocultured explants.

    fig. S2. Organotypic spinal slices cultured on 2D MWCNT substrates and on 3D-PDMS scaffolds.

    fig. S3. Extracellular voltage transients represent evoked or spontaneous synaptic, action potential–mediated activity.

    fig. S4. Directionality analysis of spinal neuronal process outgrowth.

    fig. S5. Immune reaction over time to CNF scaffolds implanted into the adult rat visual cortex as a pilot study.

    References (5661)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Illustration of the permutation test to assess the statistical significance of the synchrony between the bursting activities of two cocultured explants.
    • fig. S2. Organotypic spinal slices cultured on 2D MWCNT substrates and on 3D-PDMS scaffolds.
    • fig. S3. Extracellular voltage transients represent evoked or spontaneous synaptic, action potential–mediated activity.
    • fig. S4. Directionality analysis of spinal neuronal process outgrowth.
    • fig. S5. Immune reaction over time to CNF scaffolds implanted into the adult rat visual cortex as a pilot study.
    • References (56?61)

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