Revisiting the relevance of Hirano bodies in neurodegenerative diseases

Hirano bodies (HBs) are eosinophilic pathological structures with two morphological phenotypes commonly found in the hippocampal CA1 region in Alzheimer's disease (AD). This study evaluated the prevalence and distribution of HBs in AD and other neurodegenerative diseases.

A single study of six cases of dementia with DLB reported five cases with moderate or severe AD pathology and HBs in the hippocampal CA1 region [18].
Despite this, the interplay between these pathological stages, the accumulation of abnormal proteins and the presence of HBs remains unexplored.
The three-dimensional structure of HBs and their morphological diversity has not been examined, and it has not been determined if the typical rod-shaped HBs are truly rod-shaped in three dimensions.
A 2012 report noted the predominance of relatively large round HBs, as opposed to the more common rod-shaped HBs, in CJD [12], indicating that HB morphology may be more diverse and potentially disease-specific.Nevertheless, subsequent investigations into the spectrum of HB morphology and whether morphological types of HB differ between neurodegenerative diseases are limited.Thus, our study delved into the pathological implications of neurodegenerative conditions (including AD, LBD, PSP and MSA), as well as the abnormal protein aggregates characteristic of these disorders, within hippocampal HBs using post-mortem brain specimens.Moreover, we aimed to investigate disease-specific variations in the morphological diversity of HBs by classifying them into distinct types.

Cases and tissue collection
We conducted a retrospective review of 256 neuropathological autopsies obtained from the University Health Network Neurodegenerative Brain Collection (UHN-NBC).All brain specimens were procured through post-mortem examinations, following the approval of the local ethics committee and adherence to appropriate consent protocols.

Key Points
• Hirano bodies can be categorised into three distinct morphological phenotypes.
• Each phenotype is associated with different background pathologies.
• Age does not seem to be a direct risk factor for the development of Hirano bodies.
• Pathological staging of Hirano bodies in the posterior hippocampus could offer a simplified assessment method.
• Hirano bodies may disappear before the loss of hippocampal pyramidal cells due to the progression of background pathology.
well as cases for which no sections of the posterior hippocampus were available.All 11 FTLD-TDP cases were Type A or Type A + B, with one Type C case excluded.Amygdala-only Lewy pathology was found in two cases, both of which were classified as AD due to high ADNC levels.

Pathological evaluation
Formalin-fixed, 4.5-μm paraffin-embedded tissue sections of the posterior hippocampus from all 193 cases were stained with haematoxylin, eosin and Luxol fast blue (HE/LFB).The hippocampus and its subregions were identified according to established anatomical criteria [38,39], with annotations performed by K. Y and reviewed by T. K and G.G.K. Subsequently, the number of HBs in the pyramidal cell layer of CA1 and the stratum lacunosum of CA1 (HBL) were manually counted.
Evaluation of HBs extended to the CA1, CA2, CA3, CA4, subiculum and stratum lacunosum within hippocampal subregions as well as in the entorhinal cortex.The number of pyramidal cells in the CA1 region was assessed as follows: pyramidal cells in the distal, central and proximal portions of CA1 were each randomly captured under a 100Â field of view, and the count of pyramidal cells with discernible nuclei was undertaken manually.The total number of pyramidal cells within the CA1 region was estimated by multiplying the average density from the three views by the overall CA1 area (Figure S1A).
For the assessment of vascular pathology, arterial wall thickening was evaluated at two locations in the frontal and basal ganglia regions using a 4-point scale (0-3).This evaluation followed the methodology outlined in the Vascular Cognitive Impairment Neuropathology Guidelines (VCING) system [40]; however, due to the availability of the frontal but not the occipital cortex in all cases, we focused on that region.Subsequently, the scores from both locations were summed to derive the vascular injury score (0-6).
The pan-actin antibody clone C4 (Millipore Sigma, 1:300000) was used as the primary antibody against HBs.Immunostained slides depicting posterior hippocampal p-tau, Aβ, α-synuclein and TDP-43 were digitally scanned at 40Â magnification using a TissueScope LE120 (Huron Digital Pathology, St. Jacobs, Ontario, Canada) and stored as comprehensive whole-slide images.Subsequently, these images underwent analysis via the Area Quantification module within the HALO software (Indica Labs, Albuquerque, New Mexico, USA) to determine the percentage of positive area of each immunostain within the CA1 region and the stratum lacunosum for all 193 cases (722 sections) (Figure S1B,C).

Three-dimensional image analysis
To analyse the three-dimensional structure of different morphological forms of HBs comprehensively, 20-μm-thick sections from the posterior hippocampus of each of the HB-positive cases were subjected to staining with a 200Â dilution of clone C4.The secondary antibody used was Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen/Ther-moFisher, 1:500), and sections were treated with Sudan Black B and sodium borohydride to suppress autofluorescence.Nuclei were counterstained with spectral DAPI (Akoya Bioscience).Utilising a Nikon confocal microscope (Eclipse Ti2, Nikon, Tokyo, Japan), $40 images were captured at 0.5-μm intervals at five locations for each section, generating Z Stack images.These images were subsequently subjected to denoising using NIS-Elements (version 5.30.04,Nikon, Tokyo, Japan) image analysis software.Following the denoising process, the images were reconstructed into three-dimensional representations using the same software.

Statistical analysis
The variation in characteristics among each case group was assessed using Kruskal-Wallis and c 2

Demographics and characterisation of examined cases
Table 1 summarises the characteristics of each case group, including mean age, sex distribution and the representative pathological stage.Significant variations were observed between groups for all variables except sex.Therefore, each group underwent a comparison to the control group in a post hoc analysis to evaluate these differences.
The AD and PSP groups had a significantly higher mean age of death compared with the control group.The number of CA1 pyramidal cells was significantly smaller in the AD, FTLD-TDP and DS groups, and there were four cases of severe hippocampal sclerosis with no pyramidal cells in the CA1 region (two cases of FTLD-TDP, one case of AD + LATE, and one case of AD + LATE).Vascular injury score was significantly higher in the MSA and PSP groups.We substituted the ABC score and ADNC level with values ranging from 0 to 3, subsequently comparing their means to those of the control group.The outcomes indicated significantly higher values in the AD and AD + LBD groups, as well as in the DS group.Additionally, only the B score was significantly elevated in the LBD and PSP groups.

Morphological diversity of Hirano bodies
We performed manual counts of HBs in the CA1 subregion, which displayed the highest prevalence among the evaluated subregions, and in the stratum lacunosum, which seemed to be independently affected.In addition to the CHBs originally described by Hirano et al. [1,2], and the balloon-shaped HBs (BHBs) identified as "bizarre" HBs by Martinez et al. [12], we identified novel, string-shaped HBs (SHBs) in the CA1 subregion, which we counted separately.Specifically, we defined these types of HBs as follows: CHBs appeared as convex lens-shaped HBs with varying diameter between the central and end components when viewed in a planar manner (Figure 1A,D,G), BHBs appeared circular or oval-shaped (Figure 1B,E,H), and SHBs had elongated string-like structures with a thin, uniform diameter along their length (Figure 1C,F,I).
The 3D structures of these HBs were confirmed via confocal microscopy, exhibiting the following characteristics: CHBs displayed elongated rugby ball or teardrop-like structures (Figure 1G), BHBs appeared as sphere-like shapes (Figure 1H), and SHBs exhibited elongated rod-like forms with smooth ends (Figure 1I).

Pathological stage of Hirano body in posterior hippocampus
Given our findings indicating that HBs in the CA2 subregion, subiculum and entorhinal cortex always coexist with HBs in the CA1 region, we established a hierarchical pathological staging system for HBs within the posterior hippocampus.This proposed staging scheme commenced with CA1, followed by CA2 as the most common sites and subsequent stages encompassed the subiculum and entorhinal cortex, with a progressive decrease in frequency.The order of prevalence continued with CA1 followed by CA2, then the subicular cortex and finally the entorhinal cortex.Consequently, cases exhibiting HBs solely in CA1 were categorised as Stage 1, while those with HBs   remained comparable to those obtained before implementing the control measures (Figure S2).

Correlations of Hirano bodies and the amount of protein inclusions
The correlation between each HB and each protein density was examined for each subgroup and the entire case series.The percentage of areas positive for phosphorylated tau staining (AT8) showed a positive correlation with all types of HBs when evaluated across the entire case series, particularly with CHBs in the AD group (ρ = 0.7075, p < 0.0001) and with SHBs in the PSP group (ρ = 0.6080, p < 0.0001).

Influence of background pathology, age and sex
To examine the association of background pathology and age with each HB, we first explored whether age correlates with each HB count and each pathological stage within the entire cohort.Our findings revealed that none of the HB types exhibited a significant correlation with age (Figures 4 and S3), whereas all pathological stages evaluated in this study showed a significant correlation with age (Figure S4).The mean number of HBs peaked in the 80s for CHB (21.8 ± 35.9) and in the 90s or older for the other types of HBs: BHB (4.7 ± 12.9), SHB (1.8 ± 2.8) and HBL (2.9 ± 7.0) (Figure S3).
Second, our cohort comprised cases with overlapping background diseases such as AD and LBD, and there was statistically significant variation in age and background pathology among each disease group classified.Also, the number of CHBs exhibited a significant positive correlation with the estimated number of pyramidal cells (ρ = À0.2079,p = 0.0042), suggesting a potential impact of cell loss on this association (Figure S1D-F).To account for these factors, we conducted an ANCOVA to adjust our findings (Table 2).The ANCOVA with MSA.However, age was not identified as an independent factor influencing the presence or absence of any type of HB.
In the majority of cases with HBs, CHBs were the predominant subtype.Notably, three cases exhibited dominance of BHBs, but these cases were complicated by multiple concurrent pathologies, making it challenging to ascertain which pathology primarily influenced this outcome (Table S1).

Changes in Hirano body counts with disease progression
To investigate the relationship between the number of HBs and disease progression, we analysed the correlation between HB counts and various pathological stages, including ADNC level [31], Lewy pathology consensus criteria [29], LATE-NC stage [28] and AGD Saito stage [27] (Figure S5).
T A B L E 2 Regression analysis (ANCOVA) for each type of Hirano body.The results revealed that ADNC level and Lewy pathology type exhibited significant positive correlations with the number of all types of HBs.Specifically, ADNC level showed a relatively strong correlation with the number of CHBs (ρ = 0.2784, p < 0.0001), while Lewy pathology type demonstrated a relatively strong correlation with the number of BHBs (ρ = 0.3607, p < 0.0001).The LATE-NC stage displayed a significant positive correlation solely with the number of BHBs (ρ = 0.2559, p = 0.0005), and the AGD stage exhibited a significant positive correlation exclusively with the number of SHBs (ρ = 0.1574, p = 0.0292).Additionally, HBL counts demonstrated a significant weak negative correlation solely with ADNC levels (ρ = À0.1678,p = 0.0197).These correlations remained consistent when each HB count was adjusted for pyramidal cell count, except for the correlation between AGD stage and SHB count (Figure S6).
Looking at the number of HBs in each stage separately, a linear increase in the number of HBs was observed with the progression of the disease in the AGD stage, while a peak was observed in the ADNC level and LATE stage at Intermediate and Stage 1, respectively.This change was particularly observed in the SHB counts of ADNC and LATE stages.The observed change was statistically significant for the ADNC level regarding SHB counts, but not for the decrease from Intermediate to High levels regarding CHBs and BHBs.The post hoc power of these changes was calculated statistically to be 50% for CHBs and 46% for BHBs, suggesting that a total of 229 and 270 cases with ADNC levels of Intermediate and High, respectively, would be required to achieve 80% power.

DISCUSSION
In this study, we conducted a cross-sectional analysis of 193 hippocampi across a range of neurodegenerative diseases to explore the relationship between the quantity and distribution of the different types of HBs and neuropathological variables.Our investigation revealed that HBs are not only present in AD but also in LBD, PSP, MSA, AGD and TDP-43 proteinopathy.Furthermore, we identified a previously undocumented and novel morphological variant of HBs, suggesting potential variations in their morphological appearance based on the underlying pathology.Additionally, we proposed a novel approach for staging HB pathology in the posterior hippocampus using HE-stained sections, offering a convenient means to assess the severity of HB-related changes.
We conducted an extensive analysis of the distribution and prevalence of HBs across seven distinct subregions within the posterior hippocampus.This scrutiny led to the identification of specific patterns, which in turn facilitated the creation of a pathological staging framework.Intriguingly, this staging system exhibited a strong correlation with the quantity of CHBs present in the CA1 region.Consequently, this staging approach provides a convenient means to assess HB severity through a straightforward semiquantitative assessment, obviating the need for direct HB counting within CA1.
While the three-dimensional structure of HBs has been elucidated through electron microscopy in previous studies [44,45], this current research marks an advance in our morphological understanding of HBs by introducing the first-ever macroscopic threedimensional visualisation.Our analysis has successfully visualised the three-dimensional morphology of each type of HB, uncovering distinct characteristics such as the rugby ball-like appearance of CHB, the spherical nature of BHB and the elongated rod-like structure of SHB.This three-dimensional insight helps to interpret their planar pathological findings.For example, it can be inferred that it is difficult to distinguish the cut surface shape of CHBs from that of SHBs, and as a result of evaluating SHBs, which were not delineated with a long diameter as were CHBs, the present study may have underestimated SHBs and overestimated CHBs.This could explain why no SHBdominant cases were detected in this study.In the future, it may be useful to distinguish between CHBs and SHBs in cross-sections of HBs by the length of their short diameters.Such three-dimensional insight has been obtained for several other neuropathological structures [46], and hopefully, more structures can be examined in this fashion.
While previous research has linked HBs to neurological disorders such as AD, Guam ALS-PDC, Pick's disease and CJD [1,[8][9][10][11][12], our study is the first to report and systematically evaluate HBs in association with LBD, PSP, MSA, AGD and TDP-43 proteinopathy.This observation implies that HBs might emerge within the context of various neurodegenerative pathologies, suggesting a common mechanism underlying their formation.
However, there might also be disease-specific aspects regarding the quantity, localisation and morphology of HBs.Notably, our results highlight a particularly strong influence of AD on the prevalence of HBs.Within the spectrum of AD-related pathologies, tau pathology appears to exert a more significant effect on HB formation compared with Aβ pathology.Although prior studies have indicated that Aβ peptides can induce actin polymerisation in hippocampal cells [47], the relationship between tau and HBs warrants further detailed investigation.
Additionally, our findings suggest that TDP-43 pathology might lead to the formation of larger HBs, akin to the BHBs described by Martinez et al. in CJD cases [12].On the other hand, conditions characterised by 4R tau pathologies like PSP and AGD appear to result in elongated HBs, resembling SHBs.In MSA, the presence of HBLs appears to be a distinct feature, providing novel insights into the distinct morphological patterns of HBs associated with different pathologies.
TDP-43, implicated as the major pathological correlate to BHB, serves as the major protein associated with hippocampal sclerosis [48,49] and preferentially affects CA1 pyramidal cells.Prominent cases of BHB have also been reported in CJD, a prion disease recognised for its association with severe neuronal loss that spares the hippocampus in the most frequent forms [12].AD is mainly associated with CHB and has been reported to be associated with atrophy and neuronal loss in CA1 [50][51][52], although it has not been identified as a cause of hippocampal sclerosis.PSP and AGD are associated with SHB and have not been reported to be associated with selective neuronal loss in CA1 but are more likely to affect the CA2 subregion [25].
These observations allow the hypothesis that neurodegenerative diseases highly affecting the CA1 subregion may result in the formation of BHBs, while those associated with less involvement may lead to the formation of SHBs.However, this hypothesis is not entirely supported by the relationship between BHBs and LBD.
In addition to the hippocampus, HBs have been reported in various brain regions.For example, Okamoto et al. observed HBs in three patients with hepatic encephalopathy, affecting regions such as the substantia nigra, dentate nucleus and frontal lobe [53].
Hepatic encephalopathy is a potentially reversible disease, and, intriguingly, HBs are observed in such a condition, suggesting that HBs are pathological findings secondary to various conditions.However, studies of HBs outside the hippocampus in pathologies other than these neurodegenerative diseases are very limited.Therefore, as an initial step, we conducted a comprehensive analysis of the posterior hippocampus in major neurodegenerative diseases.Additionally, there have been instances of HB-like actin polymerisation pathology detected at a microstructural level, which remains unobservable in standard HE-stained sections [1].This pathology has been documented in anterior horn cells and axons in motor neuron diseases, Purkinje cells in Crow-Fukase syndrome [54], nucleus basalis of Meynert in Parkinson's disease, myelinated axons in progressive multifocal leukoencephalopathy [16] and cerebral white matter axons in Nasu-Hakola disease [55,56].The extent to which these structures align with HBs as observed in HE sections, in terms of size and underlying mechanisms, remains uncertain.Nevertheless, these collective findings strongly suggest that the specific sites of HB occurrence may indeed be disease specific.As such, a more systematic investigation into this aspect is warranted.
Distinct patterns emerged among different neurodegenerative pathologies.In cases of AD and LATE-NC, HB counts tended to peak at a specific stage of disease advancement.Notably, AD displayed its peak at the intermediate stage, while LATE-NC exhibited its apex at Stage 1.In contrast, in Lewy pathology, a marked tendency for the number of HBs to increase with disease progression was observed, and this upward trend was also observed between the number of SHBs and the AGD Saito Stage.This may explain why AD + LBD cases exhibited higher HB counts than AD cases.
AD cases, characterised by more severe AD pathology, experienced a peak-out phenomenon in HB counts in a greater number of cases.
However, it was difficult to show statistical significance in the number of HBs between pathological stages, and the regression analysis could not cover the evaluation of disease duration because the overall number of cases was insufficient.This issue should be resolved in future studies.
Our study also indicated that HB may represent a transient pathological finding that manifests only during a specific period of disease progression.Considering the neuropathological context of AD and LATE-NC, both conditions are characterised by a propensity for hippocampal neuron depletion, primarily in the CA1 region [49][50][51][57][58][59].This cell loss may contribute to the observed phenomenon of transient increase of HB numbers in earlier stages.Our analysis indicated significant correlations between the counts of pyramidal cells and CHBs, as well as SHBs.Moreover, while not statistically significant, there was suggestive evidence of a positive correlation between BHB and SHB counts and pyramidal cell counts.Further regression analysis underscored the independent influence of pyramidal cell counts on the appearance of CHBs and BHBs, reinforcing the connection.Given that both AD and LATE-NC are notably linked with the loss of pyramidal cells in the CA1 region, it is logical that LATE-NC, presumed to be more strongly associated, reaches its zenith early on, specifically at Stage 1.However, an additional observation surfaced as the ratio of HBs to pyramidal cell counts exhibited a decline in tandem with the advancement of ADNC levels and LATE-NC stages.This intriguing trend suggests that HBs may diminish even before pyramidal cells undergo substantial loss.In summary, it appears that HBs are transient pathological features that present differently in various pathologies and tend to disappear before significant pyramidal cell loss occurs (Figure 5).
Contrary to the prevailing assumption of an age-related elevation in the number of HBs within CA1 [1,60], our present findings do not establish a distinct correlation between age and HB counts.However, each of the HBs tended to be more common in older cases, with the average number of HBs peaking in the 80s or older.This phenomenon can likely be attributed to the heightened incidence of various neurodegenerative disorders with advancing age.A multitude of neurodegenerative diseases exhibit an inclination to escalate in both severity and prevalence as age progresses [61].In our cohort, as illustrated in Figure S3, each pathological stage demonstrated a significant increase with age.As a result, the augmented prevalence of diverse neurodegenerative pathologies with age potentially accounts for the observed rise in HB numbers.
Cerebrovascular damage is indeed known to increase with age [62], and the vulnerability of the CA1 region to ischemic damage is recognised [63][64][65].Consequently, it could be assumed that HBs are also impacted by ischaemia.However, our results did not support this assumption.Nonetheless, conducting a comprehensive evaluation of ischaemia poses challenges due to the variety of assessment scales available.
Of particular interest, earlier studies have suggested a phenomenon known as a "peak-out" in HB counts within the stratum lacunosum with ageing, implying a unidirectional increase or decrease solely linked to age [1,60].However, our results challenge this notion.Our findings indicate that the presence of HBLs may be influenced by MSA and other underlying pathological conditions, with some reports even indicating an increase in chronic alcohol use disorder [66].These observations suggest the existence of specific background pathologies that lead to an elevation of HBLs.Moreover, our investigation These findings from these mouse and yeast models suggest that HBs are associated with exacerbated cognitive dysfunction, but their formation may be treatable.Our study has further demonstrated that HB occurrence may escalate not only in AD but also in other neurodegenerative diseases such as PSP, AGD and TDP-43 proteinopathy.Since cognitive decline is prevalent in these diseases as well [28,[72][73][74][75], and HBs may be associated with cognitive decline in many neurodegenerative diseases, common therapeutic approaches could be effective.However, evidence supporting these findings is currently limited, and further studies are warranted.
A limitation of our study is the scarcity of cases with pure background pathology in our cohort, and many cases presented multiple pathologies, complicating straightforward comparisons of each HB with background pathology.To address this challenge, we endeavoured to elucidate the association between each HB and background pathology as comprehensively as possible through regression analysis and other statistical methods.Additionally, the limited number of cases for CBD and CTE (five each) hindered a thorough evaluation.
Our study is largely an exploratory investigation, particularly for these pathologies with a small number of cases.
Our study introduced a new morphological category of hippocampal HBs and visually depicted its three-dimensional structure.
Furthermore, we proposed a pathological staging system to easily assess the extent of HB.The study also revealed an increased 001) and LBD (p < 0.05) groups compared with the control group.The mean number of SHB was as follows: control 0.0 ± 0.5, AD + LBD 1.6 ± 0.4, LBD 0.9 ± 0.6, MSA 0.0 ± 0.6, PSP 2.0 ± 0.4, CBD 0.4 ± 1.0, FTLD-TDP 0.1 ± 0.7, DS 0.0 ± 0.7 and CTE 0.0 ± 0.7.Significantly higher quantities were observed in AD + LBD ( p < 0.05) and PSP (p < 0.05) groups compared with the control group.The mean numbers of HBL were as follows: control 2.3 ± 5.2, AD 0.6 ± 2.0, AD F I G U R E 2 Pathological staging for Hirano body (HB).Proposed pathological stage of HB according to the distribution of HB in the posterior hippocampus.Stage 1 was defined when HBs were found only in CA1, Stage 2 when HBs were found in CA1 and CA2, Stage 3 when HBs were also found in the subiculum and Stage 4 when HBs extended to the entorhinal cortex (A,B).The correlation between this stage and the number of each type of HB in CA1 was examined using Spearman's correlation coefficient (C-E).All types of HB showed a statistically significant correlation, but it was especially strong in CHB (ρ = 0.7508, p < 0.0001).+LBD 0.9 ± 2.6, LBD 1.9 ± 3.4, MSA 8.4 ± 12.9, PSP 1.1 ± 3.7, CBD 0.6 ± 1.3, FTLD-TDP 0.0 ± 0.0, DS 0.4 ± 1.2 and CTE 0.0 ± 0.0, showing a trend towards higher quantities in MSA.Considering the potential impact of cell loss on the results, we endeavoured to control for the effect of pyramidal cell depletion by dividing each HB count by the estimated pyramidal cell count.Nevertheless, the results was not significant in the PD or MSA group.Phosphorylated TDP-43 did not show a strong correlation with any HB type but did exhibit a significant weak negative correlation with CHBs across cases (ρ = À0.1596,p = 0.0278).HBL showed a weak negative correlation with phosphorylated tau (ρ = À0.3381,p = 0.0108) and Aβ (ρ = À0.3980,p = 0.0024) in the AD group, and a stronger negative correlation with α-synuclein in MSA (ρ = À0.6462,p = 0.0125) (Figures 4 and

F I G U R E 4
Correlation of the number of Hirano bodies with each protein deposition and age.The correlation between the number of Hirano bodies (HBs) of each type in CA1, the number of HBs in the stratum lacunosum and the percentage of each protein deposition area and age were evaluated using Spearman's correlation coefficient, and the correlation coefficient (ρ) and significance are summarised.Each protein was also assessed separately for all cases and each background disease group.Age showed no significant correlation with any type of HBs.The correlation pattern between HBs and protein deposition differed depending on the type of HBs.Classic rod-shaped Hirano body (CHB) showed a significant positive correlation with phosphorylated tau (pTau), amyloid beta (Aβ), and α-synuclein, particularly with pTau (ρ = 0.5407, p < 0.0001).This correlation was notably strong in the Alzheimer's disease (AD) group (ρ = 0.7075, p < 0.0001).Balloon-shaped Hirano body (BHB) also demonstrated a significant positive correlation with pTau, Aβ and α-synuclein, with the correlation with α-synuclein being relatively robust (ρ = 0.3570, p < 0.0001).String-shaped Hirano body (SHB) exhibited a significant positive correlation with pTau and Aβ, with the strongest correlation observed with pTau in the progressive supranuclear palsy (PSP) group (ρ = 0.6080, p < 0.0001).HB in the stratum lacunosum (HBL) displayed a significant negative correlation with all proteins, especially with α-synuclein in MSA (ρ = À0.6462,p = 0.0125).These correlation plot figures are shown in FigureS3.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.results indicated that the presence of CHB was independently and significantly influenced by a high Braak NFT stage and the number of preserved pyramidal cells in the CA1 region.BHBs were independently and significantly affected by male sex, the number of pyramidal cells (NPC), LBD, and TDP-43 pathology.SHBs were independently and significantly affected by female sex, high Braak NFT stage, the presence of PSP, and AGD.Finally, HBL was significantly associated revealed a negative correlation between the percentage of α-synuclein-positive areas in the stratum lacunosum and the number of HBLs in MSA cases, suggesting a potential increase in HBLs during the early stages of mild MSA.This implies that HBLs may respond quickly to pathological changes caused by MSA and other factors and then decline early.Our understanding of the symptoms and potential treatments associated with HBs is progressively improving.The hippocampus, a prominent location for HBs, has long been recognised for its role in memory functions[67].Recent studies conducted in mouse models of HIRANO BODIES IN NEURODEGENERATIVE DISEASES HB have unveiled that alterations in synaptic responses within the hippocampus are responsible for impairments in spatial working memory[68,69].Furthermore, recent investigations using in vitro and yeast models have demonstrated that specific antimicrobial agents possess the capability to disrupt HB-like actin aggregates[70,71].
presence of HBs in the hippocampus across various neurodegenerative diseases, including AD, LBD, PSP, MSA, AGD and TDP-43 proteinopathy, and highlighted how its morphology and effects vary depending on the underlying pathology.This increased presence of HBs in the hippocampus across a variety of neurodegenerative diseases underscores the variations in its morphology and effects associated with different underlying pathologies.Although the pathological examination of HBs has received limited attention in recent years, their prevalence across multiple neurodegenerative diseases underscores the need for more comprehensive pathological elucidation, potentially opening doors to understanding disease pathogenesis.This study serves as an impetus to expedite further research into HBs and highlights that meticulous evaluation strategies of previously underappreciated histological alterations can reveal potential contributing pathogenic aspects.AUTHOR CONTRIBUTIONS Koji Yoshida: Study design, data collection and analysis, drafting the manuscript, tables, and figures.Shelley Forrest: Proofreading, antibody testing, contributed to the pathology evaluation, critically reviewed the manuscript.Shojiro Ichimata, Hidetomo Tanaka: contributed to the pathology evaluation, critically reviewed the manuscript.Tomoya Kon: Annotation check, contributed to confocal microscope evaluation, critically reviewed the manuscript.Maria Carmela Tartaglia, Charles H. Tator, Anthony E. Lang: Providing clinical information, critically reviewed the manuscript.Naoki Nishida: Supervision, critically reviewed the manuscript.Gabor G. Kovacs: Supervision, pathology evaluation, data analysis, critically reviewed the manuscript.F I G U R E 5 Hypothesis about the appearance and disappearance of Hirano bodies (HBs).Classic rod-shaped Hirano body (CHB) appears under the influence of Alzheimer's disease-associated tau (AD-tau), balloon-shaped Hirano body (BHB) appears under the influence of Lewy body disease (LBD) and TDP-43, and string-shaped Hirano body (SHB) appears under the influence of AD-tau, progressive supranuclear palsy (PSP), and argyrophilic grain disease (AGD), and disappears before the pyramidal cells disappear as the background pathology progresses.
Case summary.
elongated rod-like, and these movies are shown in Movies S1-S3.In addition, the three-dimensional location of each HB and pyramidal cell can be determined from the lipofuscin stained green with autofluorescence and the nuclei stained with DAPI.Scale bar: 50 μm.present Number of Hirano bodies in each disease group.The number of HBs in each group was compared with the control group using the Steel multiple comparison test.The number of classic rod-shaped Hirano bodies (CHBs) was significantly higher in Alzheimer's disease (AD), AD + Lewy body disease (LBD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) compared with controls (A); the number of balloon-shaped Hirano bodies (BHBs) was significantly higher in AD + LBD and LBD compared with controls (B); and the number of stringshaped Hirano bodies (SHBs) was significantly higher in AD plus LBD and PSP compared with controls (C).