Effects of isoflurane on anesthesia and SS-31 pretreatment on ROS generation and ATP content in the hippocampus of aging mice.

<p>Peptide SS-31 (5 mg/kg) or PBS (vehicle) was intraperitoneally administered to 15-months old mice with a volume of 0.4 mL/kg 30 min before gas inhalation. Anesthesia was induced by placing the mice in an anesthetizing chamber prefilled with 1.8% isoflurane plus 30% oxygen for 10 min then changed to 1.5% isoflurane for 2 h. For control experiments, 30% O₂ was delivered for 2 h at the same flow rate. Con: control group without any intervention; Con + SS-31: control mice treated with SS-31; Iso: mice treated with isoflurane; Iso + SS-31: mice treated with SS-31 and isoflurane. ROS levels (A) and ATP production (B) in the hippocampus were measured immediately after the samples were prepared (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138256#sec002" target="_blank">Materials and Methods</a>) in six mice from each group. Values are presented as mean ± SEM (n = 6). *<i>p</i> < 0.05 versus the control group; ^#<i>p</i> < 0.05 versus the isoflurane group.</p

Effects of SS-31 pretreatment on the NLRP3 inflammasome-mediated inflammatory cytokines in the hippocampus of aging mice.

<p>(A) Western blotting analyses of NLRP3 and cleaved caspase 1 levels. (B) ELISA assays of IL-1β and TNF-α levels. Values are presented as mean ± SEM (n = 6). *<i>p</i> < 0.05 versus the control group; ^#<i>p</i> < 0.05 versus the isoflurane group.</p

Effects of SS-31 pretreatment on the intrinsic mitochondrion-dependent apoptotic signaling pathway activity in the hippocampus of aging mice.

<p>(A) Swelling of mitochondria. (B) and (C) Protein levels of activated caspase 3, total Cyt C, and cytosolic Cyt C, revealed by western blotting analyses. Protein levels were normalized to VDAC for total protein and β-actin for the cytosolic fraction, respectively. Values are presented as mean ± SEM (n = 6). *<i>p</i> < 0.05 versus the control group; ^#<i>p</i> < 0.05 versus the isoflurane group.</p

Effects of SS-31 pretreatment on cells positive for NF-κB p65, Bax, and Bcl-2 in the mouse hippocampal CA1 region.

<p>Representative images of NF-κB p65, Bax, and Bcl-2 cells immunohistochemical (IHC) staining in the hippocampal CA1 region are shown. Cells with brownish-yellow cytoplasm are positive for NF-κB p65, Bax, and Bcl-2. Scale bar: 25 μm. Lower panel represents statistic data from the four experimental groups.</p

Activation of caspase 3 was attenuated by SS-31 pretreatment in mouse hippocampal CA1 and DG regions.

<p>Representative images of cleaved caspase 3 immunohistochemical (IHC) staining in the hippocampal CA1 and DG regions are shown. Cells with brownish-yellow cytoplasm are positive for cleaved caspase 3. Scale bar indicates 100 μm. Lower panel presents statistic data from the four experimental groups.</p

Effects of SS-31 pretreatment on IκBα, Bax, and Bcl-2 expression in the hippocampus of aging mice.

<p>Protein levels of IκBα, Bax, and Bcl-2 were revealed by western blotting analyses. Values are presented as mean ± SEM (n = 6). *<i>p</i> < 0.05 versus the control group; ^#<i>p</i> < 0.05 versus the isoflurane group.</p

Dexmedetomidine Acts via the JAK2/STAT3 Pathway to Attenuate Isoflurane-Induced Neurocognitive Deficits in Senile Mice

<div><p>Background</p><p>Previous studies showed that isoflurane-induced cognitive deficits could be alleviated by dexmedetomidine in young animal subjects. In the current study, we examine whether dexmedetomidine could also alleviate isoflurane-induced cognitive deficits in senile animals.</p><p>Methods</p><p>Senile male C57BL/6 mice (20 months) received dexmedetomidine (50 μg/kg, i.p.) or vehicle 30 minutes prior to isoflurane exposure (1.3% for 4 h). Cognitive function was assessed 19 days later using a 5-day testing regimen with Morris water maze. Some subjects also received pretreatment with α₂ adrenoreceptor antagonist atipamezole (250 μg/kg, i.p.), JAK2 inhibitor AG490 (15 mg/kg i.p.) or STAT3 inhibitor WP1066 (40 mg/kg i.p.) 30 minutes prior to dexmedetomidine.</p><p>Results</p><p>Isoflurane exposure increased and reduced the time spent in the quadrant containing the target platform in training sessions. The number of crossings over the original target quadrant was also decreased. Dexmedotomidine attenuated such effects. Effects of dexmedotomidine were reduced by pretreatment with atipamezole, AG490 and WP1066. Increased phosphorylation of JAK2 and STAT3 in the hippocampus induced by isoflurane was augmented by dexmedetomidine. Effects of dexmedetomidine on JAK2/STAT3 phosphorylation were attenuated by atipamezole, AG490 and WP1066. Isoflurane promoted neuronal apoptosis and increased the expression of cleaved caspase-3 and BAD, and reduced Bcl-2 expression. Attenuation of such effects by dexmedotomidine was partially blocked by atipamezole, AG490 and WP1066.</p><p>Conclusion</p><p>Dexmedetomidine could protect against isoflurane-induced spatial learning and memory impairment in senile mice by stimulating the JAK2/STAT3 signaling pathway. Such findings encourage the use of dexmedetomidine in geriatric patients receiving isoflurane anesthesia.</p></div

Analysis of arterial blood gases and blood glucose in senile mice immediately after isoflurane exposure.

<p>Analysis of arterial blood gases and blood glucose in senile mice immediately after isoflurane exposure.</p

Effects of dexmedetomidine on p-CREB levels and JAK2/STAT3 pathway activation in hippocampus.

<p>Representative Western blots (A) showing levels of JAK2, p-JAK2, STAT3, p-STAT3, CREB, and p-CREB in hippocampus at 19d after animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164763#pone.0164763.g002" target="_blank">Fig 2</a>. Densitometry was used to determine ratios of the levels of p-JAK2 to JAK2 (B), p-STAT3 to STAT3 (C), and p-CREB to CREB (D). Data are represented as mean ± SD (n = 8). *<i>P</i> < 0.05 vs. control group; ^#<i>P</i>< 0.05 vs. ISO group; ^&<i>P</i>< 0.05 vs. DEX group.</p

Effects of dexmedetomidine on isoflurane-induced changes in expression of apoptosis-related proteins.

<p>Representative Western blots (A) showing levels of cleaved caspase-3, BAD, Bcl-2 and BDNF in hippocampus at 19d after animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164763#pone.0164763.g002" target="_blank">Fig 2</a>. Levels of caspase-3 (B), BAD (C), Bcl-2 (D) and BDNF (E) were quantitated relative to that of β-actin. Data are represented as mean ± SD (n = 8). *<i>P</i> < 0.05 vs. control group; ^#<i>P</i>< 0.05 vs. ISO group; ^&<i>P</i>< 0.05 vs. DEX group.</p