The degradation process of amyloid fibers by enzymes was evaluated over time via attenuation images using an ultrasonic microscope. The alterations in the AOS values due to changes in the expression levels of the proteins were statistically compared among specimens. The decrease in the attenuation values observed by SAM correlated with the reductions in the positively-stained areas observed by LM.
The ease of amyloid degradation differed depending on the type of amyloid, enzyme, or organ. The reduction in AOS in the amyloid portion was difficult to detect in cases where the AOS values were high in the background nonamyloid tissues, such as in the AL amyloidosis (Fig. 5). On the contrary, AOS decrease was readily seen in cases with low backgrounds, such as the brain amyloidosis (Figs. 4a and b). Protein degradation treatment is ideal when only the amyloid portions are affected without any background damage.
The method used for protease digestion on the FFPE sections has been reported previously for antigen retrieval treatment[13]. Formaldehyde forms methylene bridges between proteins, which can hinder epitope recognition by primary antibodies. Partial digestion of the proteins unmask the antigenic epitopes. Compared to antigen retrieval purposes, the present condition for amyloid degradation is more severe using higher dosage and longer incubation time. After digestion, the section retained its original structure for observation.
AOS observations using an ultrasonic microscope to evaluate protein degradation have several advantages (Table 1). First, it is easy to contrast with the tissue images. The attenuation image corresponds to the LM image stained with Congo red; hence, the location of the amyloid can be accurately grasped. Second, objective statistical comparison is possible owing to the numerical form of the attenuation value. Third, the same section can be used to observe the degradation process over time visually. Fourth, the enzymes applied to the sections can be selected, thereby making it possible to search for one that retains the background structure and specifically recognizes the target protein.
Table 1
Comparison of the two methods used to detect amyloid for breakdown.
| | SAM | LM |
| Principle | AOS decrease | Congo red stain |
| Staining | Unnecessary | Necessary |
| Number of sections | One section | Many sections |
| Thickness | 10 µm | Variable |
| Observation over time | Easy | Difficult |
| SAM:scanning acoustic microscopy, LM:light microscopy, AOS: attenuation of sound |
Alternatively, there are some disadvantages to using ultrasound microscopy. First, the material (or section), which is affected by the fixation and specimen preparation processes, may differ from the nature of the raw protein. Notably, the properties may change from those of the original protein when affected by cross-linking between proteins, organic solvents, acids, and alkalis. Unfixed frozen sections can be used for the evaluations, but it is not easy to obtain 10-µm-thick flat sections. Moreover, FFPE sections are readily available from stored paraffin blocks. Secondly, the observation area for a single scan is limited, with a maximum of 4.82 mm using our ultrasonic microscope. The third point is that the resolution depends on the transducer's performance. Although the resolution is directly proportional to the frequency, the available depth of observation becomes shorter due to the increase in the sound attenuation. A low-frequency transducer can be used to visualize thicker sections but cannot provide high-resolution images. Additionally, acquiring the skills for manual operation takes time. The distance between the transducer and the section should be adjusted to detect pulse waves from the glass slide and the surface of the section.
Amyloidoses comprise heterogeneous disorders characterized by the deposition of abnormally folded proteins in tissues[14, 15]. Amyloid deposits are formed from soluble proteins that undergo misfolding and aggregate into insoluble fibrils, leading to progressive organ damage.
The activation of enzymes that degrade amyloid fibrils can be used to treat amyloidosis. Several studies have reported the breakdown of amyloid by proteases, such as trypsin for lysozyme amyloid[16] and MMP-1 for AA amyloid[17]. Recently, the brain beta-amyloid has been gaining popularity among aged populations and causing social and economic problems; beta-amyloid was reportedly digested by neprilysin[18, 19] and insulin-degrading enzyme[20].
Various endopeptidases were used to digest amyloid in this study. It is important to note that the effectiveness of enzyme-based approaches may depend on the targeted amyloid fibrils and the conditions under which the enzyme is used. In addition, the safety using enzymes must be considered to avoid tissue damage. Endopeptidases have specific cleavage sites to cut amyloid proteins, and care must be taken to minimize the impact on surrounding proteins.
Neprilysin is a zinc metalloendopeptidase with relatively broad substrate specificity[21]. The enzyme is localized to the plasma membrane of cells, where it can function to degrade extracellular peptides. Structural studies show that neprilysin preferentially cleaves peptides on the amino side of the hydrophobic amino acids. Neprilysin has been implicated in the catabolism of the amyloid beta peptides in the brain[20].
Previous studies on the degradation of amyloid used purified amyloid fibrils instead of deposited tissues[16, 22, 23]. However, real-deposited tissues containing amyloid and surrounding extracellular materials were used in the current study to evaluate the specific digestion of amyloid and assess the damage to surrounding tissues.
This study has several limitations. First, amyloid degradation by enzymes was not compared between FFPE and fresh frozen sections. Fresh frozen tissues are more vulnerable to enzymatic digestion, causing more tissue damage. Second, more various samples with different types of amyloid and organs are necessary to investigate amyloid breakdown. Third, more precise observation using a higher frequency transducer of SAM and electron microscopy can show a higher resolution of the amyloid degradation process.
The definitive diagnosis and the identification of the specific type of amyloid depends on the biopsy sample. Biopsy samples are generally available for enzymatic degradation. We believe that the method described in the present study will aid in finding the proper treatment for removing amyloid deposits from the tissue.