PMC

GFAP⁺ cells (red) 8 wks after injury. (a) Immunofluorescence of GFAP⁺ cells from bridge implants delivering lentivirus encoding SHH. Dashed line indicates border between bridge implant and host tissue. GFAP⁺ immunofluorescence at higher magnification from bridges delivering (a) FLuc, (b) SHH, (c) NT3, or (d) NT3 and SHH. Interface between bridge and contralateral tissue is visible in each image. Arrows indicate GFAP⁺ cells. Brightness and contrast were adjusted for clarity. (e) Quantification of GFAP⁺ cells in FLuc, NT3, SHH and NT3+SHH conditions (mean +/− SD).

Sox2⁺(red)/Hoescht⁺(blue) nuclei 8 wks after injury. (a) Immunofluorescence of Sox2⁺ cells from bridge implants delivering lentivirus encoding SHH. Dashed line indicates border between bridge implant and host tissue. Sox2⁺ immunofluorescence at higher magnification from bridges delivering (b) FLuc, (c) SHH, (d) NT3, or (e) NT3 and SHH. Arrows indicate Sox2⁺ nuclei. (f) Quantification of Sox2⁺ nuclei in FLuc, NT3, SHH and NT3+SHH conditions (mean +/− SD). (g) Quantification of sub-populations of Sox2⁺/Hoescht⁺ nuclei (co-expression of GFAP or Olig2) as a percentage of total Sox2⁺/Hoescht⁺ cells (mean +/− SD). (h) Quantification of sub-population densities of Sox2⁺/Hoescht⁺ nuclei (co-expression of GFAP or Olig2) (mean +/− SD).

Myelinated axons 8 wks after injury. (a) Immunofluorescence of myelinated (NFM⁺/MBP⁺: red/green, respectively) and total (NFM⁺: red) axons from bridge implants delivering lentivirus encoding SHH. Dashed line indicates border between bridge implant and host tissue. NFM+/MBP⁺ Immunofluorescence at higher magnification from bridges delivering (b) FLuc, (c) SHH, (d) NT3, or (e) NT3+SHH. White arrows show unmyelinated neurofilaments and yellow arrows show myelinated neurofilaments. Brightness and contrast were adjusted for clarity. (f) Quantification of total axon numbers in FLuc, NT3, SHH and NT3+SHH conditions (mean +/−SD). (g) Percentage of axons that were myelinated (mean +/− SD).

Olig2⁺(red)/Hoescht⁺(blue) nuclei 8 wks after injury. (a) Immunofluorescence of Olig2⁺ cells from bridge implants delivering lentivirus encoding SHH. Dashed line indicates border between bridge implant and host tissue. Olig2+ Immunofluorescence at higher magnification from bridges delivering (b) FLuc, (c) SHH, (d) NT3, or (d) NT3 and SHH. Arrows indicate Olig2⁺ nuclei. Brightness and contrast were adjusted for clarity. (e) Quantification of Olig2⁺ nuclei in FLuc, NT3, SHH and NT3+SHH conditions (mean +/−SD). (f) Quantification of sub-populations of Olig2⁺/Hoescht⁺ nuclei (co-expression of GFAP or Sox2) as a percentage of total Olig2⁺/Hoescht⁺ cells (mean +/− SD). (h) Quantification of sub-population densities of Olig2⁺/Hoescht⁺ nuclei (co-expression of GFAP or Sox2) (mean +/− SD).

Source of myelination. (a) Widefield view of immunofluorescence of Schwann cell- (NFM⁺/MBP⁺/P0⁺: blue/red/green, respectively) and oligodendrocyte- (NFM⁺/MBP⁺/P0⁻) derived myelin fibers from bridge implants delivering lentivirus encoding SHH. Dashed line indicates border between bridge implant and host tissue. Immunofluorescence at higher magnification from bridges delivering (b) FLuc, (c) SHH, (d) NT3, or (e) NT3+SHH. White arrows show fibers wrapped by Schwann cell-derived myelin and yellow arrows show fibers myelinated by oligodendrocytes. Brightness and contrast were adjusted for clarity. (f) Quantification of total axon numbers in FLuc, NT3, SHH and NT3 and SHH conditions (mean +/− SD). (g) Percentage of axons that were myelinated by oligodendrocytes (mean +/− SD).

(a) PLG bridge. (b) Schematic of bridge implantation into lateral hemisection injury. (c) Distribution of transgene expression along the spinal cord 4 weeks after injury (mean +/− SD). Note the bridge is implanted at T9/10. (d) Sustained expression of FLuc in vivo over 8 weeks with simultaneous delivery of GFP and FLuc lentiviral vectors from the bridge. Background signal remained less than 1×10³ photons/s during all measurements (e,f) Immunohistochemistry of spinal cord tissues extracted 8 weeks after injury with co-delivery of GFP and FLuc vectors (e) or bridge alone with no lentiviral delivery (f). Panels in (e) and (f) show staining for FLuc (upper left, red), GFP (upper right, green) and Hoescht (lower left, blue). are shown in the (green - GFP, upper right; red - FLuc, upper left; blue - Hoescht, lower right). Bottom right panels show overlaid images, where cell co-expressing GFP and FLuc appear yellow. Dashed lines denote the interface between bridge (left) implants and spinal cord tissue (right).