Determination of the age-related changes in the rat cerebellar cortex by using histologic and histometric methods

doi:10.21203/rs.3.rs-6645348/v1

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Determination of the age-related changes in the rat cerebellar cortex by using histologic and histometric methods

https://doi.org/10.21203/rs.3.rs-6645348/v1

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This study was aimed to determine the effects of normal aging on the cerebellum by using histological and histometric techniques.

A total of 24 male Wistar albino rats were divided into three groups young (4–6 weeks), adult (20–22 weeks), and old (22–24 months). Cerebellar tissue samples were treated using histology techniques and embedded in paraffin. Serial sections of 5–6 µm thickness were stained with Hematoxylin-Eosin and Kluver-Barrera for general histological evaluations and histometric measurements. The PAS reactions were also carried out on the sections. Glial fibrillary acidic protein (GFAP) immunohistochemistry staining was performed to assess GFAP expression in astrocytes. The slides were evaluated using a light microscope. Molecular layer thickness was high in the adult group compared to the younger and older groups, whereas granular layer was significantly thicker in both the adult and elderly groups than in the young rat group (P < 0.05). The total cortical thickness exhibited statistically significant differences among all age groups. The thickest cortex was observed in the adult group (P < 0.05). PAS-positive aging pigment granules were observed in the cytoplasm of Purkinje cells in older rat groups.

Immunohistochemical evaluations indicated that the density of GFAP-immunoreactive (GFAP-IR) astrocytes in old rats were significantly increased compared to young and adult rats with distinct hypertrophy and strong GFAP immunoreactivity in astrocyte cell bodies.

It was established that, despite age-related variations exist, cerebellar folia height and width gradually increased from young to adult rat. In contrast, old rats have decreased cerebellar folia height and width than adults.

Cerebellum. GFAP. Immunohistochemistry. Aging

Aging, recognized as a risk factor for neurodegenerative diseases, has been defined as an irreversible process resulting in structural and functional decline across various organs and tissues (Jin et al. 2015). Numerous studies indicate that this process may arise from molecular events that do not seem to be managed by genetic programming, potentially leading to the accumulation of damaged cellular components (Mora 2013; Yeoman et al. 2012). The proportion of aged population have dramatically increased in worldwide. The World Health Organization (WHO) categorizes the age range of 65–75 as the youngest-old, 75–85 as the middle-old, and 85 and above as the oldest-old (WHO 2011). United Nations predictions indicate that the population 60 year-old and over will reach 3.1 billion by 2030, while the number of individuals 80 year-old and over is expected to rise from 70 million to 401 million between 2000 and 2050 (Chereshnev et al. 2020).

Neurodegenerative disorders, including Alzheimer's, Parkinson's, and dementia, are prevalent among the elderly population and significantly diminish quality of life. These conditions attracted increasing attention in recent years. (Li et al. 2018). The recent studies reveal that the aging process impacts many regions of the central nervous system (CNS) (Santos et al. 2024). Histopathological abnormalities specific for the hippocampus and cerebral cortex are evident in Alzheimer's disease (Demir et al. 2019), whereas motor capabilities are disturbed due to dopamine deficiency in the substantia nigra region in Parkinson's disease (Park et al. 2024; Stranahan and Mattson 2010). Likewise, aging is associated with both structural and functional alterations in the cerebellum, particularly in the cerebellar cortex. Research examining age-related alterations has documented histological changes, particularly within the cerebellar cortex, such as reduced cortical thickness, increased neuronal loss, and both hypertrophy and hyperplasia of astrocytes (Khaled et al. 2024; Zhang et al. 2006).

It is proposed that many problems, ranging from the onset of aging to neurodegenerative disorders, may result from abnormalities in communication between neurons and glial cells (De Keyser et al. 2008; Sofroniew 2009). It has been found that malfunction in glial cells, especially astrocytes, plays a role in age-related neuropathologies and participates in the initiation and progression of neurodegenerative disorders (Preininger and Kaufer 2022). Considering the involvement of astrocytes in these processes, it is important to discover markers to identify these cells (Jurga et al. 2021). Researches showed that glial fibrillary acidic protein (GFAP), an intermediate filament protein found in the cytoplasmic extensions of astrocytes, serves as a reliable and widely used marker for astrocytes (Potokar et al. 2020).

In studies on the effects of aging on astrocytes, it has been determined that GFAP expression increases (Clarke et al. 2018; Wu et al. 2005). The increase in GFAP expression is a common feature of reactive astrocytes, and these findings indicate that astrocytes show a reactive phenotype with age (Palmer and Ousman 2018). Astroglial proliferation, known as astrogliosis, is associated with some types of brain injury and other clinical disorders; also, aging is thought to increase the number of GFAP immunoreactive astrocytes (Mohamed and Sayed 2020; Yuan et al. 2024). In some studies, it has been determined that there is an increase in the size and extent of cell process in astrocytes without any numerical changes in some certain central nervous system disorders (Verkhratsky et al. 2023). Consequently, elucidating the histological alterations associated with aging in the cerebellar cortex and identifying GFAP expression are significant contributions to the current knowledge.

Neuroimaging and clinical studies indicate that cerebellar functions are executed by distinct compartments known as cerebellar lobules (foliums), the functions of which remain inadequately elucidated (Park et al. 2018). The main objective of the present study is to assess the effects of aging on each folia of cerebellum in rats by using histometric techniques and GFAP immunohistochemistry due to limited published articles on the effect of aging proccess on the each cerebellar folia. This study also aimed to obtain comprehensive reference data about the morphological and histological structures of normal folia for use in similar or more advanced studies to be conducted using neuroimage and histological techniques to determine cerebellar malformations.

Ethical approval

This research was conducted by the Department of Histology and Embryology at the Faculty of Veterinary Medicine, Selçuk University, with the approval of the Ethical Comittee of Selçuk University Chair of Experimental Medicine Research and Application Center (SUDAM) on 31.03.2022, under the reference number 2022/6. All procedures were conducted according to the European Guidelines for the Care and Use of Laboratory Animals (Directive 2010/63/EU) under.

Animals and experimental design

Twenty four healthy male Wistar albino rats were divided into three groups including eight animals of each one: 4-6 weeks (young), 20-22 weeks (adult), and 22-24 months (old). No experimental protocol was implemented on these animals. The rats were sacrificed by cervical dislocation under anesthesia with 50 mg/kg of ketamine and 10 mg/kg of xylazine injection via intraperitoneally.

Body weight and relative cerebellum weights

After the rats were weighed, the cerebella were removed and weighed. The relative cerebellum weights were calculated by using the formula as follow:

Relative Cerebellum Weight = Cerebellum Weight / Body Weight x 100. Body weights and relative cerebellum weights are given in Fig. 3.

Histological processes

Tissue samples were fixed in a 10% neutral-buffered formalin for 24 hours. After fixation, tissue samples were processed in routine histological methods and were embedded in paraffin (Basak et al. 2022). The serial midsagittal sections (5-6 μm thickness) were taken from paraffin blocks at 30 μm intervals. The sections were stained with Hematoxylin-Eosin and Kluver-Barrera staining. The PAS reaction was also performed on tissue sections (Culling et al. 1985).

Histometric measurements

Histometric measurements were performed on serial midsagittal sections of individual cerebellar vermal lobules. The thicknesses of the molecular layer, granular layer, and total cortex were measured at the apex of the cerebellar folium from a minimum of 20 distinct areas on 3 serial sections obtained from the midline of the cerebellar tissue of each specimen. The height and width of folia were measured in each of the 10 foliums on each of the serial sections, including both primary and secondary foliations. The height measurements of the primary foliations were measured as the distance between the apical points of the folium and the lowest region of the granular layer at the base of the folium, while the width measurements were determined as the maximum distance between the lateral edges of the folium. The total height of secondary foliations was calculated by adding the secondary height measurements on the primary/basal (main-root) height. At the same time, the widths were ascertained by measuring the distance between the lateral edges of each secondary foliation, like explained in measurement of the primary foliations (Fig. 1). The slides were analyzed using a Leica DM-2500 LED light microscope equipped with an MC170 HD camera attachment, and appropriate digital images were recorded for histometric measurements. All histometric evaluations were performed using the Leica IM50 measurement program (Leica, Germany).

Immunohistochemical staining

GFAP was stained immunohistochemically a sensitive peroxidase-labelled streptavidin-biotin detection system. For this purpose, the sections mounted on poly-L-lysine slides were dewaxed in xylene series and rehydrated. Then, the sections were washed, followed by boiling in a microwave oven (750W) in a citrate buffer (pH: 6) solution for antigen retrieval. Sections were immersed in a 3% hydrogen peroxide solution for 30 minutes to suppress endogenous peroxidase activity, followed by a 5-minute incubation in a blocking solution (ScyTek UHP 125, USA) to block nonspecific binding sites. The sections were subsequently treated with primary antibody (GeneTex GFAP antibody (1B4), dilution 1:500) for 1 hour at room temperature, followed by biotinylated goat polyvalent antibody (ScyTek UHP 125, USA) for 30 minutes and streptavidin-peroxidase (HRP, ScyTek UHP 125, USA) for an additional 30 minutes. DAB (3-3’-diaminobenzidine, ScyTek ACK 125) was used as the chromogen, whereas nuclear staining was performed with Mayer’s hematoxylin solution. Phosphate-buffered saline (PBS, 0.1 M, pH 7.4) was used for washing protocols.

Astrocytic density and image analysis of GFAP-IR astrocytes

All specimens were analyzed by using a Leica DM-2500 LED-type light microscope. A minimum of 10 randomly selected 1000 square micrometer (µm²) measurement areas from the stratum granulosum, substantia alba, and central white matter of cerebellar midvermis sections of each 10 foliums in cerebellar tissue were analyzed at X40 magnification. GFAP positive astrocytes were evaluated immunohistochemically by using the LAS image analysis program, and images of the appropriate regions were recorded (Fig. 2).

Statistical analysis

The SPSS 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp) statistical software was utilized for the statistical analysis of all data. The Kolmogorov-Smirnov test was employed to assess the normality of the data distribution (P>0.05). One-way analysis of Variance (ANOVA) accompanied by Duncan's post hoc test was employed for the comparison of three or more groups during data analysis. All data are presented as mean ± standard error (SE). The significance level of the tests was established at P<0.05.

Macroscopic results

No structural anomalies and malformations were observed in the cerebella.

Body weight and relative cerebellum weights

The average body weights of young rats were lower than those of adult and old rats (P<0.05). Conversely, it was shown that the relative cerebellum weights were significantly greater in young rats compared to adult and old rats (P<0.05, Fig. 3).

Histological structure of the cerebellum

All age groups, it was observed that ten folium and they were numbered from I to X rostro-caudal direction. The secondary foliations were observed in the folia III, VI, and IX. Each folium exhibited distinct layers of gray matter (cortex) and white matter (medulla). In the central part of the cerebellum, there was a white matter region, referred to as the central alba.

The histology of the cerebellum was similar in all animals. Light microscopic analysis demonstrated that the cerebellar cortex comprises three layers (Fig. 4). The molecular cell layer (stratum moleculare), situated in the outermost, consisted of diminutive neurons and glial cells. The granular cell layer (stratum granulosum), located next to the medulla (substantia alba), had tiny neurons distinguished by heterochromatic nuclei and small amount of cytoplasm. The ganglioner layer (stratum gangliosum) comprises Purkinje cells organized in a single row located between the molecular and granular layers (Fig. 4D, 4E, 4F).

In the cerebellar tissue sections from the elderly rat group, edema, and demyelination were observed, particularly in the white matter (Fig. 5A, 5B). In the sections of this group, senescence pigment granules exhibiting positive reactions in PAS staining were observed in Purkinje cells, predominantly situated in the upper part of the perikaryon, adjacent to the initial dendritic branching (Fig. 5C, 5D).

Histometric measurement results

Average thicknesses of the cortical layers

Histometric examinations of foliums revealed that the molecular layer thickness was increased in adults compared to the young and old rats, however, the granular layer thickness was lower in the young age group. The adult rats had the thickest cortex. The difference between the groups was statistically significant (Fig. 6, P<0.05).

Average folium heights

Analysis of the folium height in the adult rats revealed a consistent reduction throughout the aging process (Fig. 7). The highest folium I, IXA, IXB, IXC, and X were identified in the adult rats, compared to the old rats (P<0.05). The highest folium VIB was identified in the adult rats, and difference between young and adult animals was statistically significant (P<0.05). The highest folium VII and VIII were identified in the young age group (P<0.05) whereas the smallest foliums IXC and X were identified in the old rat group (P<0.05). Although the differences were not statistically significant (P>0.05), the highest folium II, IIIA, and IIIB were observed in adult rats, the highest folium V and VIA were identified in young rats, and the smallest folium IV was found in old rats.

Average folium widths

According to the results obtained from the average folium widths, the widest folium I, II, IIIB, VII, and VIII were observed in adults, and differences among the folium I, VII, and VIII were statistically significant (P<0.05); conversely, the widths of folium II and IIIB were similarly to both young and old rats (P>0.05). The largest folium IXB was observed in the old rats (P<0.05) while the narrowest folium VII, VIII, and X were identified in the old rats (P<0.05). However, folium X was similar in both young and adult rats (P>0.05). The narrowest folium IIIA and IXA were detected in young rats, whilst the widest foliums V and VIA were found in old rats. Although the widths of these foliums typically increase with age, it was observed that the maximum widths of foliums IV and IXC were recorded in young rats, whereas the width values in adult and elderly rat groups were comparable and diminished with age (Fig. 8).

Immunohistochemical results

Astrocytic soma and processes stained for GFAP were identified as dark brown and GFAP-immunoreactive (GFAP-IR) cells exhibited star-shaped. In contrast to the young and adult groups, astrocytes in the cerebellar tissue sections of the old rats exhibited hypertrophy, hyperplasia, and increased staining intensity. In old rats, the cytoplasmic extensions of astrocytes were more abundant and interwoven with each another (Fig. 9F, 9I, 9L). GFAP-immunoreactive astrocytes were identified in the granular layer and white matter of each folium in the cerebellar tissue, as well as in the central white matter regions situated along the mid-vermal line of the cerebellum (Fig. 9). GFAP-IR cellular extensions were also observed in the molecular layer (Fig. 9D, 9E, 9F).

The assessments revealed the greatest quantity of GFAP-IR astrocytes in the granular layer of the foliums in old rats (Fig. 9F, 9I). GFAP-IR astrocyte numbers in the granular layer of folium I, II, III, IV, V, VIII, IX, and X exhibited a significant increase in the old rat compared to the young and adult animals. Conversely, in folium VI and VII, the number of GFAP-IR astrocytes varied among all groups, showing significant increases in the order to the young, adult, and old groups, respectively (P<0.05, Fig. 10).

In white matter of cerebellar vermis, the highest number of GFAP-IR astrocytes was observed in old rats (Fig. 9I). The number of GFAP-IR astrocytes in folium I, II, and VII showed significant increases in all age groups. In folium III, IV, V, VI, VIII, and IX, the enhancement was significantly greater in the old rats compared to the young and adult (P<0.05). In folium X, the statistically significant change was observed solely between the young and old rat (P<0.05). The mean GFAP-IR astrocyte number in the stratum granulosum and white matter of the foliums are presented in Fig. 10 and Fig. 11.

The evaluation of total GFAP-IR astrocyte counts in the cerebellar folia (stratum granulosum + substansia alba) revealed significant differences in folia I, II, VI, VII, and X among all age groups, with the highest number observed in the old-rats. Additionally, significant increase density of GFAP-IR astrocyte was noted in the other folia (III, IV, V, VIII, and IX) in the old rat group compared to the young and adult groups (P<0.05, Fig. 12).

Analysis of GFAP-IR astrocyte number in the central alba region of the mid-vermal sections of the cerebellum revealed that the old rat group exhibited the highest values (Fig. 9L, P<0.05, Fig. 13).

Aging is characterized as a process resulting in the structural and functional decline of numerous essential organs and tissues (Jin ve ark. 2015). The etiology of sensory-motor control and functional problems in this process is multifaceted and linked to the central nervous system (Seidler et al. 2010). Nonetheless, the cerebellum, which receives inputs from various brain regions and responsible for motor motions and balance perception, is also influenced by this process (Bhandari et al. 2022). It has been established that with aging, there are numerous alterations in the cerebellar cortex such as neuronal loss and reduction in the number of synapses (Jäschke et al. 2023). A study investigating age-related alterations in the cerebellar cortex of young adult and elderly cats revealed a significant reduction in the molecular layer and overall cerebellar cortex thickness in elderly cats, accompanied by an increase in granular layer thickness (Zhang et al. 2006). A study on young adult (3-6 months) and old (22-26 months) rats revealed a reduction in the molecular layer thickness of the cerebellar cortex, an increase in granular layer thickness, and a decrease in total cortex thickness in old animals (Khaled et al. 2024).

A study examining the cerebrum and cerebellar tissues of young, old, and antioxidant-treated old rats at electron and light microscopic levels reported that reductions in the thickness of both the molecular and granular layers in the cerebellar cortex of old rats, attributed primarily to neuronal losses (Eşrefoğlu et al. 2010). A study examining the influence of the JAK-1/STAT-3 signaling pathway on the cerebellar cortex of aging rats demonstrated significant reductions in the overall thickness of the cerebellar cortex and the molecular layer, while the thickness of the granular layer increased with aging (Ahmed 2021). In the present study, a significant reduction was observed in molecular layer thickness and total cortex thickness in the cerebellar cortex of old rats compared to adult rats (P<0.05).

Degenerative granular formations that exhibit a positive reaction with periodic acid-Schiff (PAS), commonly referred to as "PAS positive granules," have been identified in the elderly brain (Manich et al., 2016). Research on old aged rats has indicated the existence of PAS-positive aging granules in the upper region of the perikaryon of Purkinje cells and other cerebellar cells. It is suggested that the potential variability of the distribution of pigment-containing cells in cerebellar folia might increase with not only some certain diseases but also with aging (Heinsen 1979, 1981). Firląg et al. (2013) conducted a study on alterations in the central nervous system with aging in various mammalian and primate species, and discovered that PAS-positive aging granules increased in Purkinje cells with age. A further study including humans and chimpanzees noted the occurrence of lipofuscin pigment, referred to as the aging pigment, in Purkinje cells, which has a positive reaction with PAS (Gilissen et al. 2016). In the present study it was found that the PAS-positive aging pigment granules in Purkinje cells of old rats are predominantly located in the upper half of the perikaryon, near the major dendritic branching.

Astrocytes, a type of glial cell in the CNS, have a crucial role in the maintenance of cerebellar function (Cerrato 2020; Khaled et al. 2024). GFAP is regarded as a crucial component of the astrocyte cytoskeleton (Yang and Wang 2015) and serves as a marker for astrogliosis (Hagemann et al. 2018). Astrogliosis is characterized by the abnormal growth of astrocytes, which play crucial functions in the repair of neural tissue (Kunchok et al. 2019). Many studies demonstrated a substantial increase of GFAP immunoreactive astrocytes in all CNS areas, indicative of the gliosis associated with aging and neurodegenerative alterations (Ahmed 2021; McKenney et al. 2016; Sabbatini et al. 1999). As male rats aged, their cerebellar tissues showed increased GFAP and caspase-3 immunoreactivity, and the expression levels of JAK1-STAT3-SOCS3 proteins changed with aging, according to research by Mohamed and Sayed (2020). There was an increase in SOCS3 levels and a decrease in JAK1 and STAT3 expression in the elderly rats when compared to the adult group. Sabbatini et al. (1999) conducted a study on the distribution of GFAP immunoreactive astrocytes in the granular layer and substantia alba of the cerebellum in young (3 months), adult (12 months), and old (24 months) rats. They observed significant increases in both the number and size of GFAP immunoreactive astrocytes in the granular layer of the cerebellar cortex in older rats, whereas the number of these astrocytes in the substantia alba diminished in adult and old rats when compared to the young rats. A study investigated the effects of aging and chronic ethanol consumption on astrocyte morphology and GFAP immunoreactivity in the cerebellum of both young and old rats, focusing on the molecular and granular layers of folia II, IV, VII, and X in the cerebellar vermis, as well as the central white matter in the mid-vermal sections of the cerebellum, using by a computer-aided analysis system (Rintala et al. 2001). This study demonstrated that chronic ethanol exposure did not influence GFAP-IR in the granular layer and central white matter; however, it indicated an age-related elevation in GFAP-IR in older male rats compared to younger male rats.

In this study, numerical alterations were assessed in astrocytes within the cerebellar tissue of young, adult, and old rat groups using by the GFAP immunohistochemistry. GFAP-IR astrocyte counts were performed in the granular layer and white matter of each cerebellar folium, as well as in the central white matter area. Upon reviewing the existing studies, it is evident that while numerous investigations have focused on age-related alterations in GFAP expression, there is the limited study that assessed each folium of the cerebellum individually. Our study aimed to elucidate the histological structure and regional variations of astrocytic density in the foliums of the cerebellum at different ages. It was found that the number of GFAP-IR astrocytes significantly increased in the granular layer and white matter of all foliums in old rats compared to young and adult rats (P<0.05). It was also established that astrocytes displayed hypertrophy and were stained more intensely by GFAP immunohistochemistry. Age-related increases in the total number of GFAP-IR astrocytes in the granular layer of the folium and the white matter, as well as in the number of GFAP-IR astrocytes in the central alba area, were statistically significant (P<0.05).

Cerebellar folia are regarded by certain researchers as distinct structural and functional units that share afferent and efferent pathways in the cerebellum (Maryenko and Stepanenko 2021). Iwaniuk et al. (2007) investigated folium size variations in the cerebellum tissue of 96 avian species, and they found a complex relationship characterized by one folium more height and the other more shorter depending on talent of bird species. They concluded that folium size is influenced by both phylogenetic history and avian behavior. The study (Iwaniuk et al. 2007) indicated that there was a clear evidence of a correlation between powerful legs and the enlargement of the anterior lobe, noted significant reductions in the size of foliums I-III. Investigators also observed enlargements of foliums VI and VII in species identified as proficient fliers, suggesting that this enlargement likely corresponds to the demands of enhanced visual processing in species exhibiting rapid and/or agile flight. A study on cerebellar tissue from individuals 20-88 year-old, whose deaths were not attributed to CNS pathology, delineated variations in morphometric parameters (height and width) and shape of cerebellar folia to enhance their classification (Maryenko and Stepanenko 2021). This study's (Maryenko and Stepanenko 2021) findings indicate that human cerebellar folia exhibit variability in size and shape, and future research may reveal that the dimensions and configurations of all normal cerebellar folia align with one of the variants identified in this study.

Research on the cerebellum primarily focuses on elucidating the neurodegenerative processes within this structure. Hernández-Pérez et al. (2023) performed a meta-analysis of 116 studies, revealing that a particular lobe in the cerebellum exhibits a natural resistance mechanism to neurodegeneration. This study indicated that the anterior region of the cerebellum is the most sensitive and typically the first to degenerate, while the middle and posterior regions exhibit moderate sensitivity. Additionally, folium X, which is the evolutionary earliest to develop in the cerebellum and is present even in the most primitive vertebrates, demonstrates notable resistance to neuronal damage (Maryenko and Stepanenko 2021). The findings of this study lead us to believe that the cerebellum contains numerous hidden knowledge in its various lobes and foliations. This study identified variations in height and width among cerebellar folia in rat related to age. It was observed that the average height and width progressively increased from the young to the adult as the animals developed, as expected, however, these measurements exhibited a decline in the old animals relative to the adult group. A substantial increase in average height was observed in the folium I of the adult rats within the anterior lobe of the cerebellum (lobules I-V). Folium I, II, and IIIB were found to be wider in the adult animals, while folium IIIA and V were wider in the old group, and folium IV was wider in the young group compared to the other groups (P<0.05). In the posterior lobe of the cerebellum (VI-IX) the adult animals exhibited the highest folia VIB, IXA, and IXB, whereas the highest folia VII, VIII, and IXC were found in the young group. In the posterior lobe of the cerebellum, it was found that the widest folia VII and VIII were present in adults; the widest folia VIA and IXB were observed in the old, while the widest folium IXC was noted in the young (P<0.05). The X^th folium in the flocculonodular lobe of the cerebellum exhibited notable decreases in average height and width in the old group. The data indicate that age-related alterations occur in the dimensions of the cerebellar folium.

Morphological alterations in the cerebellar folia have been documented in numerous neurodegenerative disorders, cerebellar dysplasia, and hypoplasia, serving as fundamental criteria in the classification of cerebellar malformations (Patel and Barkovich 2002). Neuroimaging studies aimed at identifying pathological alterations in the cerebellar cortex revealed findings including irregularities and disorientation in the folia, abnormal folial structures, reduced folial size, and the absence of folia (Chatur et al. 2019; Poretti et al. 2014). This study aims to determine age-related alterations in cortical thickness, and immunoreactive astrocyte density per unit area for each folium by using histological techniques. In addition, this study also aimed to determine the age-related changes that may occur in the height and width of cerebellar foliums in rats. It is thought that the results obtained from this study might be useful as referrences data for the studies investigated cerebellar pathology including age related alterations.

The frequency and diversity of age-related neurodegenerative diseases necessitate just timely evaluation and diagnosis of clinical manifestations. As known, early therapy is possible with an early diagnosis. In order to comprehend the effects of aging on the cerebellar cortex and foliations, as well as any neurodegenerative diseases that may be associated with aging, evaluations using techniques such as histopathological examination, neuroimaging, electron microscopy, immunohistochemistry, and molecular biology methods are of significance.

Common methods used to study cerebellar foliations include morphology, histology, and neuroanatomy. These methods are believed to be significant in providing a novel perspective on the development, structural characteristics, and functions of cerebellar foliations. A thorough investigation of alterations in cerebellar folia with aging and the pinpointing of specific folia exhibiting initial indications of aging may aid in developing techniques for early detection and intervention. It may be suggest that the decline in movement, and balance abilities due to aging may be linked to the decrease in the average width of foliums II and VIII (convert sensory inputs into motor responses) and folium VII (involved in postural balance, and coordination), especially in old rats. Furthermore, the average height and widht of folium X, known for its resistance to various neurodegenerative damages, considerably diminishes in the elderly, indicating that age must be included in experimental investigations on neurodegeneration. The considerable reduction in the average height of folium IX in the elderly was assessed as an important finding in our current investigation. It is deemed essential to consider other foliums, particularly folium IX, whose function remains partially understood, and whose height and width measurements vary numerically across age groups, despite the absence of statistically significant differences, in forthcoming structural or experimental investigations. One of the results of this study is that Purkinje cells in aged rats exhibit distinct senescence granules that respond positively to PAS. These granules are an aging pigment known as lipofuscin, which is present in numerous cells in different organs. This study also concludes that future research should analyze these granules on a folium basis. Another significant discovery of the study presented is that the elderly exhibit a substantial increase in GFAP-IR astrocytes. It is thought that this increase is similar in that the increase in fibrous connective tissue, which is developed to compensate the loss of parenchymal tissue in peripheral organs. Additionally, the aging process results in advanced neuronal degeneration in the central nervous system. It is also thought that this increase in GFAP-IR astrocytes may be a protective response that arises to mitigate the damage triggered by neuronal loss in the elderly and to maintain the continuity of intercellular communication, which diminishes as neurons are lost.

Author Contribution

Conceptualization, T.K and E.S.; Methodology, T.K and E.S.; Software, T.K and E.S.; Validation, E.S.; Formal Analysis, E.S.; Investigation, T.K and E.S.; Resources, T.K and E.S.;Writing—Original Draft, T.K and E.S.; Preparation, T.K and E.S.; Writing—Review & Editing, T.K and E.S.; Visualization,T.K and E.S.; Supervision, E.S.; Project Administration, E.S.; Funding Acquisition, E.S.All authors reviewed the manuscript

Acknowledgement

The authors express gratitude to the academic members of Selçuk University’s academic of Veterinary Sciences Department of Histology and Embryology: Prof. Dr. Yasemin Öznurlu, Prof. Dr. Tuğba Özaydın, and Asst. Prof. İlknur Ündağ. These individuals were unwavering in their support of the laboratory experiments throughout the research. Prof. Dr. Tamer Çağlayan, a faculty member of the Department of Animal Science and Animal Nutrition at Selçuk University’s Faculty of Veterinary Medicine, participated to the statistical analysis of the collected data.

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Determination of the age-related changes in the rat cerebellar cortex by using histologic and histometric methods

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Body weight and relative cerebellum weights

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Body weight and relative cerebellum weights

Histological structure of the cerebellum

Histometric measurement results

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