**2. Results**

#### *2.1. Neuroprotection by ATX*

#### 2.1.1. Findings by Nissl Staining

In the vehicle-sham group, intact cells stained with cresyl violet (CV), which have intact Nissl substance (aggregation of rough endoplasmic reticulum) in the cytoplasm, were distinctly observed in the stratum pyramidale (SP) in the hippocampal proper (CA1- CA3) (Figure 1(Ba)). In the vehicle-IR group, CV stainability at one day after IR was not significantly altered compared with the vehicle-sham group (Figure 1(Bb)). However, at two days after IR, CV stainability was decreased in the SP of CA1 (Figure 1(Bc)), and, at five days after IR, a significant loss of CV stainability was observed in the SP of CA1-3

(Figure 1(Bd)). In the ATX-sham group, CV stainability in the SP of CA1-3 was not different from that of the vehicle-sham group (Figure 1(Be)). In the ATX-IR group, a significant change in CV stainability was not observed until two days after IR (Figure 1(Bf,Bg)), and, at five days after IR, CV stainability was weak in the SP of CA1 alone (Figure 1(Bh)). This finding means that severe IR damages Nissl substance, and ATX could protect it from IR injury.

**Figure 1.** (**A**) The chemical structure of ATX and experimental procedure. ATX is dissolved in saline and orally administered once a day for three days before IR induction. The gerbils are sacrificed on day 1, 2 and 5 after IR; (**B**) Representative photographs of Nissl staining in the hippocampus (CA1-3) of the vehicle-treated (upper low) and ATX-treated (lower low) groups in sham (**a**,**e**), and on day 1 (**b**,**f**), day 2 (**c**,**g**), and day 5 (**d**,**h**) after IR. In the vehicle-IR group, CV stainability in the SP of CA1 (arrows in (**c**)) is pale at two days after IR, and, at five days after IR, very pale CV stainability is shown in the SP of CA1-3 (black asterisks in (**d**)). In the ATX-IR group, pale CV stainability is shown only in the SP of CA1 (white asterisk in (**h**)) at five days after IR. DG, dentate gyrus; SO, stratum oriens; SR, stratum radiatum. Scale bar = 400 μm.

2.1.2. Findings by Neuronal Nuclei (NeuN, a Marker for Mature Neurons) Immunohistochemistry

In the vehicle-sham group, cells in the SP (named pyramidal cells or neurons) of CA1-3 were shown to have clear NeuN immunoreactivity (Figure 2(Aa,Ca)). In the vehicle-IR group, NeuN-immunostained cells (NeuN-pyramidal cells) of CA1-3 at one day after IR were not altered in morphology and numbers (Figure 2(Ab,B,Cb,D)). However, NeuNpyramidal cells of CA1-3 were significantly decreased (67% of the vehicle-sham group) in numbers at two days after IR (Figure 2(Ac,B,Cc,D)), and, at five days after IR, a massive loss (27% of the vehicle-sham group) of NeuN-pyramidal cells of CA1-3 were observed, showing that the number in CA1 and CA2/3 was 9 and 35 cells/300 μm2, respectively

(Figure 2(Ad,B,Cd,D)). In the ATX-sham group, NeuN-pyramidal cells in CA1-3 were not different in numbers and morphology from the vehicle-sham group (Figure 2(Ae,B,Ce,D)). In the ATX-IR group, NeuN-pyramidal cells were not significantly changed until two days after IR (Figure 2(Ae,Af,B,Cf,Cg,D)), and, at five days after IR, NeuN-pyramidal cells of CA1-3 were decreased in numbers, but the number in CA1 and CA2/3 was higher (603% and 268% of the vehicle-IR group) than the vehicle-IR group, showing that the number in CA1 and CA2/3 was 51 and 92 cells/300 μm2, respectively (Figure 2(Ah,B,Ch,D)). This finding means that severe IR damages the NeuN protein located in the nucleus, and ATX could protect it from IR injury.

**Figure 2.** (**A**,**C**) Representative photographs of NeuN 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, a loss of NeuN-cells (black asterisks in (**Ac**,**Cc**)) in the SP of CA1-3 is observed on day 2 after IR. At five days after IR, NeuN-pyramidal cells of CA1-3 (arrows in (**Ad**,**Cd**)) are markedly decreased; however, in the ATX-IR group, many NeuN-pyramidal cells (white asterisks in (**Ah**,**Ch**)) are shown. Scale bar = 60 μm; (**B**,**D**) Mean numbers of NeuN-pyramidal cells 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).
