Инновационный потенциал и инновационная активность вузов УрФО

<p>Characteristics of the study patients.</p

The growing sciatic nerve intermingled with the dorsal root ganglion in 3D Gelfoam® histoculture.

<p>(A) A sciatic nerve was placed in Gelfoam® histoculture next to a dorsal root ganglion, both from a ND-GFP mouse. At day 38, many ND-GFP-expressing fibers were seen extending from both the sciatic nerve and co-cultured dorsal root ganglion. Bar: 500 µm. (B) Immunofluorescence staining of β-III tubulin (red) demonstrated that many β-III tubulin-positive fibers extended from both the sciatic nerve and the dorsal root ganglions. The fibers consisted of ND-GFP expressing cells. Bar: 500 µm. (C) Magnified image of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g007" target="_blank">Figure 7B</a> shows that many β-III tubulin-positive fibers extended widely and radially both from the sciatic nerve and the dorsal root ganglion. β-III tubulin-positive fibers from both nerves intermingled with each other. Bar: 500 µm.</p

Location and characteristics of ND-GFP-expressing cells in the sciatic nerve.

<p>(A) The first day of Gelfoam® histoculture of a sciatic nerve bundle removed from the ND-GFP transgenic mouse. The sciatic nerve contained ND-GFP-expressing cells which had long processes (white arrows). Bar: 100 µm. (B) High-magnification image of the sciatic nerve showed that ND-GFP expressing cells and their processes are located between Schwann cells. Bar: 10 µm. (C) Transverse sections of the sciatic nerve of the ND-GFP mouse. The ND-GFP-expressing cells did not express β-III tubulin and S100. Bar: 10 µm. (D) Transverse and longitudinal sections of the sciatic nerve of the ND-GFP mouse. The ND-GFP-expressing cells (white arrows) co-expressed p75^NTR. Bar: 10 µm.</p

Sphere formation and differentiation from the sciatic nerve in suspension culture.

<p>(A) The sciatic nerve was cultured in DMEM-F12 medium containing bFGF. The ND-GFP-expressing cells proliferated and formed spheres by day 32. Bar: 100 µm. (B) The spheres expressing ND-GFP co-expressed p75^NTR and CD34 but did not express β-III tubulin S100 and GFAP. Bar: 100 µm. (C) The ND-GFP-expressing spheres were switched to RPMI 1640 medium containing 10% FBS from DMEM-F12 containing B-27, N2 and bFGF and began to differentiate. At 7 days after switching into medium containing FBS, β-III tubulin-positive neuronal cells which expressed ND-GFP were observed. Bar: 10 µm. (D) At 14 days, the ND-GFP-expressing cells differentiated to GFAP-positive glial cells. Bar: 10 µm (E) At 7 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to K15-positive keratinocytes, some of which still expressed nestin. Bar: 10 µm. (F) At 30 days after culture in FBS medium, the ND-GFP-expressing cells differentiated to α-SMA-positive smooth muscle cells. Bar: 50 µm.</p

Intermingling of growing sciatic nerve in 3D Gelfoam® histoculture.

<p>(A) A sciatic nerve from a ND-GFP transgenic mouse was placed on Gelfoam® next to the sciatic nerve from an RFP transgenic mouse. At day 14, the sciatic nerve from the ND-GFP mouse was enriched with ND-GFP-expressing cells and intermingled with to the RFP-expressing sciatic nerve. Bar: 500 µm. (B) Magnified images of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g006" target="_blank">Figure 6A</a> show that the ND-GFP-expressing cells proliferated in fibers growing from the nerve extending toward the other sciatic nerve. At day 9, the thickest fibers appeared between both sciatic nerves. At day 14, the two nerves intermingling with each other. Bar: 100 µm. (C) Magnified images of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g006" target="_blank">Figure 6B</a> show that the fibers consisted of ND-GFP-expressing spindle cells (white arrow heads) and ND-GFP-negative spherical cells (white arrows). The spherical cells formed a line between both sciatic nerves and ND-GFP-expressing spindle cells extended among the lines. Bar: 10 µm. (D) A section of the intermingling two nerves. High-magnification images show that ND-GFP-expressing fibers growing from the sciatic nerve from the ND-GFP mouse invaded deeply into the RFP-expressing sciatic nerve. Bar: 100 µm.</p

The injured sciatic nerve expressed ND-GFP in vivo.

<p>(A) 1: The left sciatic nerve (green arrow heads) was exposed between the short and long adductor muscles. 2: ND-GFP expression was not observed. 3: The sciatic nerve was severed (black arrows: the nerve stumps). 4: The transected nerve was rejoined with a 9-0 suture (blue arrow head). (B) At day 0, left plantar (red arrows) was paralyzed. At day 35, the left plantar could show digit flection. (C) At day 35, the injured nerves (white arrow heads) connected (blue arrow head: point of suture). Strong nestin expression was observed in the injured sciatic nerve. Note that the intact nerve (white arrows) did not express ND-GFP. (D) The injured sciatic nerve (white dashed line) expressed ND-GFP. Nestin expression was stronger on the distal side compared to the proximal side of the sciatic nerve and spine (white arrow heads). White arrows: dorsal root ganglions.</p

Fibers growing from the sciatic nerve in 3D Gelfoam® histoculture expressed β-III tubulin and contained tips expressing phalloidin-positive F-actin.

<p>(A) A sciatic nerve from an ND-GFP mouse was cultured for 19 days on Gelfoam®. The nerve became enriched with ND-GFP-expressing cells and many fibers extended from the growing nerve. Bar: 500 µm. (B) Immunofluorescence stained images of β-III tubulin (red) demonstrated that many β-III tubulin-positive fibers extended from the nerve stump and extended widely and radially around the nerve. Merged images of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g005" target="_blank">Figure 5</a> demonstrated that β-III tubulin positive fibers consisted of ND-GFP-expressing cells. Bar: 500 µm. (C) Tips of the β-III tubulin (white)-positive fibers had phalloidin (red)-positive F-actin. The presence of F-actin indicates that tips are axons growth cones. White arrows: tips of the β-III tubulin-positive fiber. Bar: 10 µm.</p

ND-GFP-expressing cells are located in dorsal peripheral nerve roots but not in the spinal cord in 3D Gelfoam® histoculture.

<p>(A) A spinal cord with posterior roots removed from an ND-GFP transgenic mouse was put in Gelfoam® histoculture. On day 0, some ND-GFP-expressing cells were observed in the origin of posterior root (enclosed by white dashed lines). (B) Cross section of the spinal cord just after removal show that there were a few ND-GFP-expressing cells in the posterior root. White bar: 50 µm. (C) At day 7, posterior roots were enriched with ND-GFP-expressing cells. (D) Cross section of the spinal cord cultured for 7 days show that ND-GFP-expressing cells proliferated in the posterior root but not in the cortex of spinal cord. White bar: 50 µm.</p

ND-GFP-expressing cells proliferated in the sciatic nerve and formed fibers in 3D Gelfoam® histoculture.

<p>(A) Time-course imaging of ND-GFP-expressing cell trafficking in the histocultured sciatic nerve from an ND-GFP mouse. The nerve became enriched with ND-GFP-expressing cells by day 14. At day 6, the ND-GFP-expressing cells proliferated in extending fibers and migrated into the Gelfoam®. At day 14, many fibers consisting of ND-GFP-expressing cells extended widely and radially around the nerve. Bar: 500 µm. (B) Magnified images of the growing nerve. At day 6, the ND-GFP-expressing cells proliferated as fibers and migrated into the Gelfoam®. Bar: 100 µm. (C) High-magnification images of the area inside the boxes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g003" target="_blank">Figure 3B</a>. At day 9, the ND-GFP-expressing cells grew in fibers (white arrow heads). Some spherical cells proliferated in the growing fibers. They did not express nestin (white arrows). At day 14, the spindle cells expressed ND-GFP (white arrow head). In addition, there were spherical cells without nestin expression (white arrow). Bar: 10 µm.</p

Univariate analysis of event free survival.

**<p><i>P</i><0.01.</p