Fig 1.
Effects of isoflurane on anesthesia and SS-31 pretreatment on ROS generation and ATP content in the hippocampus of aging mice.
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 Materials and Methods) in six mice from each group. Values are presented as mean ± SEM (n = 6). *p < 0.05 versus the control group; #p < 0.05 versus the isoflurane group.
Fig 2.
Effects of SS-31 pretreatment on the NLRP3 inflammasome-mediated inflammatory cytokines in the hippocampus of aging mice.
(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). *p < 0.05 versus the control group; #p < 0.05 versus the isoflurane group.
Fig 3.
Effects of SS-31 pretreatment on the intrinsic mitochondrion-dependent apoptotic signaling pathway activity in the hippocampus of aging mice.
(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). *p < 0.05 versus the control group; #p < 0.05 versus the isoflurane group.
Fig 4.
Activation of caspase 3 was attenuated by SS-31 pretreatment in mouse hippocampal CA1 and DG regions.
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.
Fig 5.
The number of TUNEL-positive cells was diminished by SS-31 pretreatment in mouse hippocampal CA1 regions.
Representative images of TUNEL in the hippocampal CA1 region are shown. Green color indicates TUNEL-positive cells; blue, NeuN. Scale bar: 25 μm. The low panel shows statistical numbers of TUNEL-positive cells.
Fig 6.
Effects of SS-31 pretreatment on cells positive for NF-κB p65, Bax, and Bcl-2 in the mouse hippocampal CA1 region.
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.
Fig 7.
Effects of SS-31 pretreatment on IκBα, Bax, and Bcl-2 expression in the hippocampus of aging mice.
Protein levels of IκBα, Bax, and Bcl-2 were revealed by western blotting analyses. Values are presented as mean ± SEM (n = 6). *p < 0.05 versus the control group; #p < 0.05 versus the isoflurane group.
Fig 8.
SS-31 pretreatment mitigated isoflurane-induced learning and memory impairments.
Panels represent total distance traveled (A), time spent in the center (B), freezing time to tone (C), and freezing time to context (D), respectively. Values are presented as mean ± SEM (n = 8). *p < 0.05 versus the control group; #p < 0.05 versus the isoflurane group. The procedures of the open field test and fear conditioning test are in Materials and Methods.