Study design and verification of reproducibility of induced labor model mice.

(A) Experimental design of induced labor model and pups. At gestational day 18.5 an osmotic pump was implanted subcutaneously in anesthetized mice. The male pups were analyzed at 24 h (P1) after birth. (B) Time until labor for each group. Time until labor of the oxytocin (OXT) group was significantly shorter than that of the phosphate-buffered saline (PBS) and Wild groups (P < 0.001, Tukey–Kramer method). There were no significant differences between the PBS and Wild groups. (C) Survival rate of OXT, PBS and Wild groups at P1. There were no significant differences between each group. (D) Body weight of OXT, PBS and Wild groups at P1. The body weight of the OXT group was significantly lower than that of the PBS and Wild groups (P < 0.001, Steel–Dwass test). There were no significant differences between the PBS and Wild groups.</p

Comparison of architecture of PL and IL of vmPFC between OXT and PBS groups at P40.

Nissl-stained sections of OXT (A) and PBS (B) groups. Scale bar: 100 μm. (TIF)</p

Ultrastructure of dying cells in forceps minor of corpus callosum and ventromedial prefrontal cortex of the male pups at 24 h after delivery.

(A–D) Electron micrographs of forceps minor (FMI) of the male pups at 24 h after delivery of the OXT (A–C) and PBS (D) groups. (A) In the OXT group, cells containing pyknotic nuclei and debris of dying cells were abundant (asterisks). (B) An enlarged image of the phagocytic cell (P) shown in A containing many pyknotic nuclei and debris of dying cells. (C) A dying cell with chromatin condensation observed in the OXT group. (D) An enlarged image of the phagocytic cell (P) containing pyknotic nuclei and debris of dying cells in the PBS group. (E–G) Electron micrographs of ventromedial prefrontal cortex (vmPFC) of the male pups at 24 h after delivery of the OXT group. Dying cells with pyknotic nuclei indicated by an arrow and an arrowhead in E were enlarged and shown in F and G, respectively. (F) A dying cell with pyknotic nuclei that was not phagocytosed (arrow). (G) A dying cell with pyknotic nuclei that was encircled by the cytoplasm of the phagocytic cell (arrow). Scale bars: 4 μm (A–E) and 2 μm (F, G).</p

Increased number of pyknotic nuclei and TUNEL-positive cells in forceps minor of corpus callosum and infralimbic and prelimbic subregions of ventromedial prefrontal cortex of OXT group.

(A–D) TUNEL- and Nissl-stained sections of OXT (A, C) and PBS (B, D) groups. (A, B) In forceps minor (FMI) of corpus callosum (CC) TUNEL-positive and pyknotic nuclei were more abundant in the OXT group than in the PBS group. TUNEL-positive nuclei were often clustered (arrowheads). The insets are low magnification images of the sections indicating the regions of interest. dcw: deep cerebral white matter, IL: infralimbic cortex, LV: lateral ventricle, ne: neuroepithelium, PL: prelimbic cortex, vmPFC: ventromedial prefrontal cortex. Scale bars: 50 μm and 500 μm (in inset). (C, D) In PL and IL of vmPFC TUNEL-positive and pyknotic nuclei were detected more abundantly in the OXT group than in the PBS group. Squared areas with TUNEL-positive and pyknotic nuclei were enlarged and shown in insets. In the OXT group most of the TUNEL-positive and pyknotic nuclei appeared independently (arrows) rather than as a cluster (bold arrow). Scale bars: 50 μm and 10 μm (in inset). (E) The number of TUNEL-positive and pyknotic nuclei per square millimeter in FMI of CC in the OXT group was significantly larger than that in the PBS group (377.12 ± 48.93 vs 107.04 ± 42.10 for OXT and PBS groups, respectively, P = 0.002, Student’s t-test). (F) The number of TUNEL-positive and pyknotic nuclei per square millimeter in PL and IL was significantly larger than that in the PBS group (87.48 ± 10.57 vs 31.95 ± 6.32 for OXT and PBS groups, respectively, P = 0.002, Student’s t-test).</p

Infiltration of Iba-1-positive microglial cells in forceps minor of corpus callosum and ventromedial prefrontal cortex of the male pups at 24 h after delivery in OXT group.

(A) In low power view, the infiltration of Iba-1-positive microglial cells seemed increased in the infralimbic and prelimbic cortex (IL and PL) of the OXT group when compared with that of the PBS group (arrows). FMI: forceps minor, LV: lateral ventricle, ne: neuroepithelium. (B) Immunohistochemistry for Iba-1 in IL of OXT and PBS groups. A squared area was enlarged and shown in the inset. Nissl stain was used for counterstaining. In IL, pyknotic nuclei (arrows) appeared more abundantly and infiltration of Iba-1-positive microglial cells in IL was more pronounced in the OXT group than in the PBS group. In the OXT group some Iba-1-positive microglial cells were engulfing pyknotic nuclei (an arrow in the inset). (C) Images in high power view of the FMI from the same section shown in A. Double labeling for Iba-1 (green), TUNEL staining (red). In FMI, there were more TUNEL-positive nuclei (red) in the OXT group than in the PBS group. In the OXT group many of the Iba-1-positive microglial cells were engulfing clustered TUNEL-positive nuclei (arrowheads). In the PBS group many of the TUNEL-positive cells appeared independently and some Iba-1 positive cells contained TUNEL-positive cells (arrowheads). Scale bars: 100 μm (A) and 50 μm (B, C) and 150 μm (the inset in B). (D) The number of Iba-1-positive microglia per square millimeter in IL and PL in the OXT group was significantly larger than that in the PBS group (228.69 ± 64.71 vs 65.72 ± 5.88 for OXT and PBS groups, respectively, P = 0.016, Student’s t-test). The number of Iba-1-positive microglia per square millimeter in FMI of CC was also significantly larger than that in the PBS group (452.22 ± 124.08 vs 214.34 ± 50.88 for OXT and PBS groups, respectively, P = 0.016, Student’s t-test).</p