2.2.2. 4-Hydroxy-2-Nonenal (4HNE, a Marker for Lipid Peroxidation) Immunoreactivity

In the vehicle-sham group, 4HNE immunoreactivity was weakly found in the intact CA1-3 pyramidal cells (Figure 5(Aa,Ca)). In the vehicle-IR group, 4HNE immunoreactivity in the pyramidal cells of CA1 and CA2/3 was significantly increased (296% and 305% of the vehicle-IR group, respectively) at one day after IR (Figure 5(Ab,B,Cb,D)). At two days after IR, 4HNE immunoreactivity in the CA1-3 pyramidal cells was decreased compared to that shown in the vehicle-IR group at one day after IR; at this time, many of the CA1-3 pyramidal cells died (Figure 5(Ac,B,Cc,D)). At five days after IR, 4HNE immunoreactivity was very low due to a massive loss of the CA1-3 pyramidal cells (Figure 5(Ad,B,C,D)). In the ATX-sham group, 4HNE immunoreactivity in the CA1-3 pyramidal cells was similar to the vehicle-sham group (Figure 5(Ae,B,Ce,D)). In the ATX-IR group, 4HNE immunoreactivity in CA1 and CA2/3 pyramidal cells was also increased (214% and 221% of ATX-sham group, respectively) at one day after IR, but the immunoreactivity was significantly lower than that in the vehicle-IR group (Figure 5(Af,B,Cf,D)). Thereafter, 4HNE immunoreactivity in the CA1-3 pyramidal cells was gradually and slightly decreased with time due to the attenuation of IR-induced neuronal loss (Figure 5(Ag,Ah,B,Cg,Ch,D)). This finding means that lipid peroxidation in the pyramidal cells of CA1-3 was significantly increased at one day after severe IR, and ATX could attenuate the lipid peroxidation induced by severe IR.

**Figure 5.** (**A**,**C**) Representative photographs of 4HNE immunohistochemistry in CA1 (**A**) and CA2/3 (**C**) of the vehicle-treated (left column) and ATX-treated (right column) groups in sham (**Aa**,**Ca**,**Ae**,**Ce**), and on day 1 (**Ab**,**Cb**,**Af**,**Cf**), day 2 (**Ac**,**Cc**,**Ag**,**Cg**), and day 5 (**Ad**,**Cd**,**Ah**,**Ch**) after IR. Black asterisks (in (**Ab**,**Cb**)) indicate that, in the vehicle-IR group, 4HNE immunoreactivity is significantly increased in CA1-3 pyramidal cells at one day after IR. However, in the ATX-IR group, 4HNE immunoreactivity in CA1-3 pyramidal cells (white asterisks in (**Af**,**Cf**)) at 1 day after IR is significantly low when compared with in the vehicle-IR group. Note that, at five days after IR, 4HNE immunoreactivity is very weak (arrows in Ad and Cd) in the vehicle-IR group due to death of pyramidal cells. Scale bar = 60 μm; (**B**,**D**) ROD of 4HNE immunoreactivity in CA1 (**B**) and CA2/3 (**D**). The bars indicate the means <sup>±</sup> SEM (*<sup>n</sup>* = 7, respectively; \* *<sup>p</sup>* < 0.05 vs. corresponding sham group, # *<sup>p</sup>* < 0.05 vs. vehicle-IR group).

#### *2.3. Increase of SOD1 and SOD2 Expressions by ATX*

#### 2.3.1. SOD1 Immunoreactivity

SOD1 immunoreactivity, in the vehicle-sham group, was shown in the pyramidal cells of CA1 and CA2/3 (Figure 6(Aa,Ca)). In the vehicle-IR group, SOD1 immunoreactivity in the CA1 and CA2/3 pyramidal cells was significantly decreased (69% and 71% of vehicle-sham group, respectively) on day 1 after IR (Figure 6(Ab,B,Cb,D)). On day 2 after IR, SOD1 immunoreactivity in the CA1-3 pyramidal cells was further decreased (Figure 6(Ac,B,Cc,D)), and, on day 5 after IR, SOD1 immunoreactivity was very low (56% and 21% of the vehicle-sham group, respectively) (Figure 6(Ad,B,Cd,D)) due to an extensive loss of CA1-3 pyramidal cells. In the ATX-sham group, SOD1 immunoreactivity in the CA1 and CA2/3 pyramidal cells was significantly higher (144% and 155% of the vehicle-sham group, respectively) than the vehicle-sham group (Figure 6(Ae,B,Ce,D)). In the ATX-IR group, SOD1 immunoreactivity in CA1 and CA2/3 pyramidal cells was also decreased with time, but each immunoreactivity was significantly high (on day 5 after IR, 378% and 382% of the vehicle-IR group, respectively) when compared with the ATX-IP group (Figure 6(Af–Ah,B,Cf–Ch,D)). This finding means that SOD1 in the CA1-3 pyramidal cells was significantly decreased with time after severe IR, and ATX could attenuate the decrease of SOD1 induced by severe IR.

**Figure 6.** (**A**,**C**) Representative photographs of SOD1 immunohistochemistry in CA1 (**A**) and CA2/3 (**C**) of the vehicle-treated (left column) and ATX-treated (right column) groups in sham (**Aa**,**Ca**,**Ae**,**Ce**), and on day 1 (**Ab**,**Cb**,**Af**,**Cf**), day 2 (**Ac**,**Cc**,**Ag**,**Cg**), and day 5 (**Ad**,**Cd**,**Ah**,**Ch**) after IR. In the vehicle-IR group, SOD1 immunoreactivity in CA1-3 pyramidal cells is decreased with time after IR, and very low (arrows in (**Ad**,**Cd**)) on day 5 after IR. In the ATX-sham group, SOD1 immunoreactivity in CA1-3 pyramidal cells is significantly higher (asterisk in (**Ae**,**Ce**)) than in the vehicle-sham group. In the ATX-IR group, SOD1 immunoreactivity in CA1-3 pyramidal cells is decreased with time, but the immunoreactivity is significantly high (asterisks in (**Af**–**Ah**,**Cf**–**Ch**)) as compared with the vehicle-IR group. Scale bar = 60 μm; (**B**,**D**) ROD of SOD1 immunoreactivity in CA1 (**B**) and CA2/3 (**D**). The bars indicate the means <sup>±</sup> SEM (*<sup>n</sup>* = 7, respectively; \* *<sup>p</sup>* < 0.05 vs. corresponding sham group, # *<sup>p</sup>* < 0.05 vs. vehicle-IR group).

## 2.3.2. SOD2 Immunoreactivity

SOD2 immunoreactivity, in the vehicle-sham group, was mainly shown in the CA1 and CA2/3 pyramidal cells (Figure 7(Aa,Ca)). In the vehicle-IR group, a significant decrease of SOD2 immunoreactivity in the CA1 and CA2/3 pyramidal cells (65% and 68% of the vehiclesham group, respectively) was observed at one day after IR (Figure 7(Ab,B,Cb,D)). At two days after IR, SOD2 immunoreactivity in the CA1-3 pyramidal cells was more decreased (Figure 7(Ac,B,Cc,D)), and, at five days after IR, SOD2 immunoreactivity in the CA1-3 pyramidal cells was very low (51% and 18% of the vehicle-sham group, respectively) due to an extensive loss of pyramidal cells (Figure 7(Ad,B,Cd,D)). In the ATX-sham group, SOD2 immunoreactivity in the CA1 and CA2/3 pyramidal cells was significantly higher (205% and 196% of the vehicle-sham group, respectively) when compared with the vehicle-sham group (Figure 7(Ae,B,Ce,D)). In the ATX-IR group, SOD2 immunoreactivity in the CA1-3 pyramidal cells was also decreased with time, but each immunoreactivity was significantly high when compared with the vehicle-IR group (Figure 7(Af–Ah,B,Cf–Ch,D)). This finding means that SOD2 in CA1-3 pyramidal cells was also significantly decreased with time after severe IR, and ATX could attenuate the decrease of SOD2 induced by severe IR.

**Figure 7.** (**A**,**C**) Representative photographs of SOD2 immunohistochemistry in CA1 (**A**) and CA2/3 (**C**) of the vehicle-treated (left column) and ATX-treated (right column) groups in sham (**Aa**,**Ca**,**Ae**,**Ce**), and on day 1 (**Ab**,**Cb**,**Af**,**Cf**), day 2 (**Ac**,**Cc**,**Ag**,**Cg**), and day 5 (**Ad**,**Cd**,**Ah**,**Ch**) after IR. SOD1 immunoreactivity, in the vehicle-IR group, is decreased in CA1-3 pyramidal cells with time after IR, and very low (arrows in (**Ad**,**Cd**)) at five days after IR. In the ATX-sham group, SOD1 immunoreactivity in CA1-3 pyramidal cells is significantly higher (asterisk in (**Ae**,**Ce**)) than the vehicle-sham group. SOD1 immunoreactivity in CA1-3 pyramidal cells of the ATX-IR group is decreased with time, but the immunoreactivity is significantly high (asterisks in (**Af**–**Ah**,**Cf**–**Ch**)) when compared with the vehicle-IR group. Scale bar = 60 μm; (**B**,**D**) ROD of SOD2 immunoreactivity in CA1 (**B**) and CA2/3 (**D**). The bars indicate the means ± SEM (*n* = 7, respectively; \* *p* < 0.05 vs. corresponding sham group, # *p* < 0.05 vs. vehicle-IR group).
