The senile plaque: Morphological differences in APP knock‐in mice brains by fixatives

Abstract The morphology of senile plaques depends on the APP knock‐in mice brain fixative. Solid forms of senile plaques were detected in APP knock‐in mice after formic acid treatment with Davidson's and Bouin's fluid fixative as the brain of AD patients. Aβ42 was deposited as cored plaques and Aβ38 accumulated around Aβ42.

were used U-MNIBA (Olympus, Tokyo, Japan) for Aβ 38 and U-MWIG (Olympus, Tokyo, Japan) for Aβ42 detection. In the case of the whole sectioning images of the APP NL-G-F/NL-G-F mouse brain, an Olympus APX100 (Tokyo, Japan) was used for analysis. A Filter set was used U-FBNA (Olympus, Tokyo, Japan).

RESULTS AND DISCUSSION
Senile plaque (SP), one of the pathological hallmarks of Alzheimer's disease (AD) patient brains, can reproduce in its pathological model mouse To understand the specific morphology of SP in AD model mice brains, it is necessary to find SP-associated molecules. Therefore, we focused on SP detection methods to assess the morphology of SP in these mice brains. The generated amyloid β (Aβ) in APP NL-G-F/NL-G-F has made SPs in the brain since the age of 2 months. This generated Aβ has an Arctic genetic mutation at the E22 amino acid position and is more prone to aggregate than wild-type Aβ sequencing (Nilsberth et al., 2001). In previous studies, SPs in APP NL-G-F/NL-G-F and another AD mouse model were detected with antibodies after formic acid treatment or with thioflavin-S (Emre et al., 2022;Reinert et al., 2016;Saito et al., 2014;Sasaguri et al., 2022). We followed this formic acid treatment to 10month-old APP NL-G-F/NL-G-F brain sections. The antibody against the N-terminus of Aβ, 82E1, showed diffuse-like SPs around cored plaques, in which brain tissue was subjected to 4% paraformaldehyde (PFA) ( Figure 1A). Thioflavin-S detected the dense morphology of SP without formic acid treatment (Emre et al., 2022;Saito et al., 2014), but it cannot distinguish Aβ species from those of SP. To investigate the reason for this diffuse-like SPs staining, 10-month-old APP NL-G-F/NL-G-F brain tissues were immediately subjected to Davidson's and Bouin's fluid fixative after saline perfusion. In these paraffin-embedded sections, the antibody 82E1 showed undiffused SP forms in the section after formic acid treatment ( Figure 1A). These undiffused SP morphologies were shown in the whole sections ( Figure S1) Next, SPs stained with both anti-Aβ38 and anti-Aβ42 antibodies in 4% PFA, Davidson's fluid, and Bouin's fluid fixation sections in the 10-month-old APP NL-G-F/NL-G-F brain tissues. In the PFA fixed sections, Aβ38 showed diffuse-like distribution as shown in 82E1 staining in Figure 1A, and Aβ42 accumulated in the core of SP ( Figure 1B). However, in Davidson's fluid and Bouin's fluid fixed sections, Aβ38 accumulated around Aβ42 without halation, and Aβ42 accumulated in the core of SP ( Figure 1B). To exclude the effect for the Arctic mutation, 12-month of age APP NL-F/NL-F mice were used which carries wild-type Aβ38 and Aβ42 sequencing. The left brain tissue was dipped in the 4% PFA fixative, right brain tissue was dipped in the Bouin's fixative from the same APP NL-F/NL-F after saline perfusion.
The overlapped Aβ38 and Aβ42 were detected in the SP from Bouin's fixative ( Figure 1C). However, Aβ38 could not detect from the PFA fixative. Only Aβ42 could be detected in the PFA section ( Figure 1C). These results mean that PFA could fix Aβ42, although, Aβ38 could not be sufficiently fixed against formic acid treatment in wild-type Aβ sequencing by PFA. To gain further insight into age dependency, 4-month-old APP NL-G-F/NL-G-F brains were stained after formic acid treatment. The number of SPs in the 4-month-old mice section was lower than that of those in the 10-month-old, but the staining patterns of Aβ42 were the same as those at 10 months of age in each fixative ( Figure S2). The diffuselike morphology of Aβ38 did not depend on the age of the mice ( Figure   S2) brains within a few months. Thus, the physiological characterization of SP would still be softer and more flexible in these mice brains. Even though the brain was fixed with 4% PFA, formic acid will dissolve the SP in mice brain sections. On the other hand, Davidson's and Bouin's fluid fixative may be able to fix SP more strongly than PFA. Therefore, appropriate fixation and immunohistochemical methods are required to investigate SP morphology. These methods have the potential to accurately locate SP-associated proteins in the brain of APP knock-in mice and other AD model mice. Furthermore, as with previous human brain studies (Kakuda et al., 2017;Kakuda et al., 2020), it can compare the SPs of each Aβ species.

ACKNOWLEDGMENT
We thank Prof. Nobuyuki Nukina for kindly supporting our study.

DATA AVAILABILITY STATEMENT
All data and materials are included in this article and supplementary information files.

ETHICS STATEMENT
The present study was approved by the animal ethics committees of Doshisha University (S.F.).