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. 2022 Feb 28;10:810980. doi: 10.3389/fcell.2022.810980

FIGURE 6.

FIGURE 6

The qualitative and quantitative differences in perineuronal nets (PNN) and parvalbumin-positive interneurons (PV) between control and hypoxia-treated animals at the age of 3.5 months confirm a permanent structural change in the connectivity of the cingulate cortex. (a–b’,A–B’) High magnification of cortical layers III-Va of MCC (a,b), cortical layers III-Va of RSC (a’,b’), both stained by Wfa, and quantitative findings for the number of PNN in all cortical layers of MCC (A,B) and RSC (A’,B’). The PNN are more numerous in hypoxia-treated animals in both cingulate areas. The Wfa staining of PNN is denser and thicker around the soma and proximal portion of the dendrites after perinatal hypoxia (b,b’) compared to controls (a,a’). (A–B’) Two-way ANOVA revealed a significant influence of hypoxia on the number of PNN/mm2: (A) in the MCC (Hypoxia = F (1.12) = 72.46; p < 0.0001; Sex = F (1.12) = 3.641; p = 0.0806; Interaction = F (1.12) = 0.1863; p = 0.6736), and (A’) in the RSC area (Hypoxia = F (1.9) = 10.11; p = 0.0112; Sex = F (1.9) = 0.01477; p = 0.9059; Interaction = F (1.9) = 0.7814; p = 0.3997). The hypoxia-treated animals of both sexes had a significantly higher number of PNN in the MCC area (B), and the same, but insignificant, trend was observed in RSC area (B’). (c–d’) High magnification of layers III-Va of MCC (c,c’), cortical layers III-Va of RSC (d,d’) stained by parvalbumin, and quantitative findings for the PV in all cortical layers of MCC (C,D) and RSC (C’,D’). The interneurons soma is strongly parvalbumin - positively stained as well as the proximal portion of the dendrites in the animals subjected to perinatal hypoxia (d,d’) compared to controls (c,c’). Two-way ANOVA revealed a significant influence of hypoxia on the number of PV per mm2: (C) in the MCC (Hypoxia = F (1.12) = 8.770; p = 0.0119; Sex = F (1.12) = 0.01956; p = 0.8911; Interaction = F (1.12) = 0.1141; p = 0.7413), and (C’) in the RSC area (Hypoxia = F (1.12) = 17.84; p = 0.0012; Sex = F (1.12) = 0.2751; p = 0.6095; Interaction = F (1.12) = 0.05901; p = 0.8122). The hypoxia-treated animals of both sexes had a significantly higher number of PV interneurons in the RSC area (D’), while in the MCC area (D), the difference is only indicative. Data are shown as mean ± standard error of the mean (SEM) (error bars). (E) A representative PNN (Wfa-positive, green) is visible around the cell body of PV (Parv, red) as a thin coat. The PV of the control animal also has larger soma when compared with PV in hypoxia-treated animal. (F) Representative PNNs (Wfa-positive, green) in the brain section of hypoxia-treated animal, show thicker and denser ECM-coat around the cell soma, surrounding the proximal portion of the dendrites, and often the initial axon segment [(F); arrow]. The PV stained with parvalbumin (Parv, red) reveals the smaller neurons soma in the cingulate cortex of hypoxia-treated animal compared to control. The actual image magnification for (a–d’) is shown with the scale bar in (d’), presenting 50 µm, for (E,F) is shown with the scale bar in (F), presenting 25 µm.