Non-opioid neuroprotective mechanisms accompanying combination therapy of moderate hypothermia, MSCs and DADLE.

<p>Results revealed significant upregulation of GDNF, but not NGF or BDNF after combination therapy with or without DADLE (OGD-Treated) compared to OGD-Untreated and Control (Panel A). GDNF was also upregulated slightly in OGD-Untreated condition, likely due to an endogenous compensatory mechanism of the PRNCs. Similarly, OGD-Treated significantly reduced apoptosis compared to Control and OGD-Untreated. Apoptosis via ApoAlert assay is quantified (Panel B) and representative images shown (Panel C) (a-c are Hoechst-stained PRNCs, whereas d-f are corresponding apoptotic stained cells; a/d: Control; b/e: OGD-Treated; c/f: OGD-Untreated). There were no significant differences in cell cycle condition across the three conditions (Panel D). Symbols represent **p<0.0005 vs. Control GDNF; *p<0.0005 vs. Control GDNF; §§p<0.0001 vs. OGD-Treated GDNF (Panel A); **p<0.0001 vs. control; *p<0.005 vs. control and untreated (Panel B). Dashed lines in Panels A, B and D (pink, red, and black, respectively) indicate combined treatment of moderate hypothermia and MSCs without DADLE. Solid lines in Panels A, B and D (pink, red, and black, respectively) indicate treatment with antibody against GDNF and combined treatment of moderate hypothermia and MSCs without DADLE.</p

Experimental design.

<p>The diagram captures time course of temperature, introduction of stem cells, evaluation of cell viability and mitochondrial activity under different treatment conditions.</p

Measurement of cell viability and mitochondrial activity in PRNCs.

<p>Moderate hypothermia treatment indicated significant increase cell viability compared to normothermic and sever hypothermia treatments, and enhanced stem cells’ neuroprotection. Black- and white-columns indicate non-implementation and MSCs implementation after 2 hours reperfusion, respectively. Control shows a normoxic condition, atmosphere at 37°C, 95% O₂, and 5% CO₂. + Under same temperature condition, <i>P</i> value of comparison non-MSCs with MSCs implementation is less than 0.05. *<i>P</i><0.05: **<i>P</i><0.01: ***<i>P</i><0.005. Similarly, moderate hypothermia treatment indicated significant increase in mitochondrial activity compared to normothermic and severe hypothermia treatments, and enhanced stem cells’ neuroprotection. Black- and white-columns indicate non-implementation and MSCs implementation after 2 hours reperfusion, respectively. Cont shows a normoxic condition, atmosphere at 37°C, 95% O₂, and 5% CO₂. + Under same temperature condition, <i>P</i> value of comparison non-MSCs with MSCs implementation is less than 0.05. *<i>P</i><0.05: **<i>P</i><0.01: ***<i>P</i><0.005. a.u. = absorbance unit.</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

Statistics Summary Table.

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

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

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

DPC co-culture protects rat neural cells against OGD.

<p>Co-culture of rat primary neural cells with DPCs afforded neuroprotection against OGD dose-dependently in both Trypan blue and MTT activity assays of cell viability (a–f). The percentages correspond to the ratio of DPCs co-cultured with rat primary neural cells as follows: 1∶1 (100%), 1∶2 (50%), 1∶4 (25%), and 1∶0 (0%). DPCs co-cultured with rat primary neural cells under non-OGD condition did not alter cell viability in both assays (g–l). Asterisk* corresponds to statistically significant difference (p<0.05 vs. 0%). a,g: 0%; b,h: 25%; c,i: 50%; d,j: 100%; e,k: Trypan blue stain quantification; f,l: MTT assay quantification.</p