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

Prior to TBI surgery (A), the number of errors on trial 1 (T1), trial 4 (T4), and the 30 minute retention trial (T5) for NT (yellow) and AD (blue) mice during the final two days of RAWM testing.

<p>Asterisk indicates significant improvement from T1 for that group at p<0.05 or higher level of significance. For Post-TBI testing, the number of errors on T1, T4, T5 for the RAWM at two weeks (<b>B</b>) and six weeks (<b>C</b>) after TBI. NT-Sham (hatched yellow, n = 9), NT-TBI (solid yellow, n = 10), AD-Sham (hatched blue, n = 8), and AD-TBI (solid blue, n = 7) mice. Single asterisk indicates the AD-TBI group is significantly different from all other groups at p<0.02. Double asterisk indicates the AD-TBI group is significantly different from AD-Sham at p<0.01. The error bars represent the SEM.</p

TBI accelerated extracellular Aβ deposits in the hippocampus of AD-TBI mice.

<p>Statistical analysis revealed a significant upregulation of extracellular Aβ deposits in the AD mice that received TBI compared to the NT mice that received TBI and the AD and NT mice that received sham surgery (Panel A). MAP2 staining revealed a significant decrease of MAP2 positive cells in the hippocampus of AD mice that received TBI compared to the AD and NT mice that received sham surgery (Panel B). Immunofluorescence for the detection of extracellular Aβ deposits and MAP2 (Panel C). Scale bars = 50 µm. The insets correspond to representative high magnifications of MAP2 images. Scale bars  = 100 µm. The brain illustration shows the location of the brain slices chosen for histological analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.</p

Body and brain temperature measurements for APPsw+PS1 transgenic (Tg) mice recorded prior to the start of EMF treatment (control), as well as at 5 days and 12 days into EMF treatment.

<p>For both control and treatment time points, there were no differences between EMF-treated and control Tg mice for either body or brain temperatures. No significant differences in OFF vs. ON temperatures (via paired t-test) were evident in EMF-treated Tg mice.</p

Working memory in the radial arm water maze (RAWM) task pre-treatment, 1 month, and 1.5 months into EMF treatment for the naïve first trial (T1) and working memory trial (T5) of APPsw transgenic (Tg) and non-transgenic (NT) mice.

<p>(Fig. 2A) Pre-treatment RAWM testing revealed Tg mice to be impaired vs. NT mice during working memory Trial 5 on the last block of testing. *p<0.002 vs. NT; (Fig. 2B) and (Fig. 2C). At both 1 month and 1.5 months into EMF treatment, Tg mice continued to be impaired in working memory (T5) performance on the last block of testing, irrespective of whether they had been receiving EMF treatment or not. *p<0.01 or higher level of significance vs. NT.</p

Experimental design is shown.

<p>For cohort 1, animals were trained in the RAWM prior to TBI surgery and then tested for cognitive deficits in the RAWM at two and six weeks post-TBI. For cohort 2, there was no pre-TBI behavioral training or post-TBI behavioral testing. All the animals were euthanized at six weeks post-TBI and the brains were harvested for histological analysis.</p

Body and brain temperature measurements for non-transgenic (NT) and APPsw transgenic (Tg) mice recorded prior to the start of EMF treatment (control), and at 1 Day, 1 week, 3 weeks, and 6 weeks into EMF treatment.

<p>Control NT and Tg mice (no EMF exposure) maintained stable body and brain temperatures throughout the 6 week recording period. By contrast, both NT and Tg mice being treated with EMF experienced small, but significant increases in body temperature during ON periods by 1 week into treatment and time points thereafter. Although brain temperature of EMF-treated Tg mice remained stable during ON periods through the 6 week recording period, EMF-treated NT exhibited small (but significant) increases in brain temperature during ON periods at 3 and 6 weeks into EMF treatment. *p<0.05 or higher level of significance for OFF vs. ON recordings (via paired t-test) at that time point. ^#p<0.02 vs. NT control on that day.</p

Cognitive performance of non-transgenic (NT) and APPsw transgenic (Tg) mice in the Y-maze task of spontaneous alternation (Fig. 1A) and the circular platform task of spatial/reference memory (Fig. 1B).

<p>(Fig. 1A) Both NT and Tg mice given EMF treatment exhibited nearly significant increases in Y-maze percent alternation. For both genotypes combined, a significant increased in percent alternation was evident in EMF-treated mice. *p<0.05 vs. control. (Fig. 1B) EMF treatment did not improve the poor performance of NT and Tg mice in the circular platform task, although Tg mice were impaired even more than NT mice irrespective of treatment. **p<0.02 vs. NT.</p