Statistics Summary Table.

<p>Posthoc pairwise comparisons and their corresponding p-values. Asterisks (*) denotes significance.</p

Hematoxylin and eosin staining of moderate (<b>Figure 4A</b>, with a–e at 2× magnification, a1–e1 at 4× magnification, and quantified in C) and severe (<b>Figure 4B</b>, with a–e at 2× magnification, a1–e1 at 4× magnification, quantified in D) TBI models.

<p>The cortical infarcts in the samples that received no reconstruction (Panel a, 2×; Panel a1, 4×) and immediate skull reconstruction with the bone flap only (Panel b, 2×; Panel b1, 4×) were smaller than the infarcts observed in the samples that received skull reconstruction with bone wax only (Panel c, 2×; Panel c1, 4×) and bone wax and bone flap (Panel d, 2×; Panel d1, 4×). The infarcts in the delayed reconstruction group (Panel e, 2×; Panel e1, 4×) were significantly reduced in both moderate and severe TBI models compared to the immediate reconstruction groups that received bone wax or bone wax and bone flap. Bars represent mean ± SEM. Asterisks * indicate p<0.05 vs. no reconstruction, bone flap, and delayed reconstruction.</p

Flowchart of experimental procedures.

<p>All animals received moderate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033646#pone-0033646-g001" target="_blank">Figure 1A</a>) or severe (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033646#pone-0033646-g001" target="_blank">Figure 1B</a>) TBI using the CCI model. After the CCI surgery, animals received either no reconstruction (Group A), immediate reconstruction with bone flap (Group B), bone wax (Group C), or bone wax and bone flap (Group D). The animals assigned to Group E in received delayed skull reconstruction on day 2. All animals were euthanized on day 4. Half of the animals (n = 4) from each group were assigned to TTC analysis and the other half (n = 4) were assigned to immunofluorescence assay.</p

TTC Immediate skull reconstruction with bone wax alone or in combination with bone flap exacerbates cortical damage in TBI.

<p>TTC analysis of moderate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033646#pone-0033646-g003" target="_blank">Figure 3A</a>, quantified in C) and severe (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033646#pone-0033646-g003" target="_blank">Figure 3B</a>, quantified in D) TBI revealed that immediate reconstruction with bone wax only or bone wax and bone flap significantly increased cortical damage compared to no reconstruction or reconstruction with only the bone flap. Of interest, delayed reconstruction at 2 days after TBI significantly reduced cortical damage compared to immediate reconstruction with bone wax only or bone wax and bone flap. Bars represent mean ± SEM. Asterisks (*) indicate p<0.05 vs. no reconstruction, immediate reconstruction with bone flap, and delayed reconstruction; # indicates p<0.05 vs. no reconstruction, bone flap, bone wax, and bone wax and flap; § indicates p<0.05 vs. bone wax.</p

Brain swelling accompanies TBI.

<p>Following CCI, obvious brain swelling was detected in animals subjected to no skull reconstruction (Panels a and a1). Reconstruction with bone flap only provided partial skull reconstruction, thereby allowing a tempered brain swelling (Panels b and b1). Skull reconstruction with bone wax only (Panels c and c1), bone wax and bone flap (Panels d and d1), or delayed reconstruction with bone wax (Panels e and e1) afforded normalized skull structure, but also prevented visualization of brain swelling. Arrows are indicative of brain swelling following TBI. Dotted circles show the tempered brain swelling that occurred in the animals that received reconstruction with bone flap only.</p

Aquaporin-4 immunofluorescence of moderate (<b>Figure 5A</b>

<p><b>with a–e at 2× magnification, a1–e1 at 4× magnification, and quantified in C) and severe (</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033646#pone-0033646-g005" target="_blank"><b>Figure 5B</b></a><b>with a–e at 2× magnification, a1–e1 at 4× magnification, and quantified in C) TBI brains.</b> Sparse aquaporin-4 staining was detected in the cortical tissues from TBI animals that received no reconstruction (Panel a, 40×; Panel a1, 60×). A slight, but significant increase in aquaporin-4 staining was seen in immediate skull reconstruction with bone flap (Panel b, 40×; Panel b1, 60×). Widespread aquaporin-4 upregulation was detected in the samples that received immediate skull reconstruction with bone wax only (Panel c, 40×; Panel c1, 60×) and bone wax and bone flap (Panel d, 40×; Panel d1, 60×), which was significantly elevated compared to no skull reconstruction. Aquaporin-4 density was reduced in the samples that received delayed reconstruction compared to immediate reconstruction with bone wax or bone wax and bone flap, but was significantly elevated compared to bone flap only or no reconstruction (Panel e, 40×; Panel e1, 60×). Bars represent mean ± SEM. Asterisks * indicate p<0.05 vs. all other treatment groups. Scale bars represent 50 µm.</p

Results of ELISA analysis for VEGF and GDNF.

<p>(A, B) In the lesioned striatum, VEGF was significantly increased by SCS at 1 week after 6-OHDA lesion (*p<0.05). At 2 weeks after 6-OHDA lesion, VEGF level in the lesioned striatum also appeared elevated, but did not reach statistical significance. (C, D) GDNF in the striatum of both sides was not significantly increased by SCS at 1 and 2 weeks after 6-OHDA lesion. (C-lesion: control group lesioned side Striatum; C-intact: control group intact side Striatum; S-lesion: 50 Hz SCS group lesioned side Striatum; S-intact: 50 Hz SCS group intact side Striatum, n = 10, respectively).</p

Time course and SCS electrode, and the brain region punched out for protein assay.

<p>(A) Scheme showing overall experimental design. (B) Scheme showing experimental design for protein assay. (C) Photograph showing SCS electrode used in this study (diameter: 2 mm; wire length: 60 mm). (D) Scheme showing a rat during stimulation. (E) Brain tissue (diameter: 3 mm showing gray circle), corresponding to the striatum, was punched out from both the lesioned and the intact side.</p

TH immunostaining in the substantia nigra pars compacta (SNc), and the ratio to the intact side.

<p>(A) TH immunostaining in the intact SNc. Severe loss of TH-positive neurons was seen in the lesioned side SNc of control group. Preservation of TH-positive neurons was seen at the lesioned side SNc of 50 Hz SCS group. Scale bar: 200 µm. (B) Significant preservation of TH-positive neurons in the lesioned-side SNc of 50 Hz SCS group, compared to those of control group (*p<0.05, n = 10, respectively).</p

The results of cylinder test and amphetamine-induced rotation test.

<p>(A) Rats receiving 2 Hz and 50 Hz SCS showed reduction of the contralateral bias at 2 weeks after 6-OHDA lesion, compared to that of rats in control group. (B) The number of amphetamine-induced rotations in all SCS groups decreased, compared to that of control group. There was a significant amelioration in 50 Hz SCS group, compared to control group (*p<0.05, n = 10, respectively).</p