Role of medical ozone in attenuating age-related changes in the rat cerebellum

Role of medical ozone in attenuating age-related changes in the rat cerebellum Abstract Aging is an inevitable biological process characterized by motor in coordination and decline in the ability to learn new motor skills. The purpose of this study is to investigate, for the first time, the beneficial antiaging effects of medical ozone (O3) on the age-related structural damage of the rat cerebellum. We have examined the cerebellum of albino rats at the ages of 6, 20 and 22, and the effect of intraperitoneal medical O3 administration (0.7 g/kg) by histological, immunohistochemical and morphometric studies. Age-related changes in the cerebellum were in the form of a significant reduction in the number of Purkinje cells (PCs), which appeared shrunken with a darkly stained cytoplasm and vacuolated spaces in all layers. The decrease in Nissl granule content of the PCs was also observed. A significant reduction in Mab2, Ki67 immunoreactivity associated with significant increase in glial fibrillary acidic protein, Caspase-3 and iNos immunoreactivity were also detected. Medical O3 administration reversed all these histopathological and immunohistochemical changes. This protective effect was mediated by reducing oxidative stress, apoptosis, astrocyte activation and improving both neuritogenesis and neurogenesis. We can conclude from the results of the present study that medical O3 can prevent the retardation of age-related changes in rat cerebellum. medical ozone, age, cerebellum, histopathology, immunohistochemistry Introduction Aging is associated with disturbances in motor and cognitive functions, especially learning and memory. The decline in fine motor control, gait and balance affect the ability of older adult to perform activities of daily living and to maintain their independence [1]. Gait and balance disorders are of particular interest in older adults as they are a major source of injury and morbidity. Most of older adults who fall suffer injuries that reduce mobility and quality of life [2]. The cerebellum provides an excellent model for the study of age-related changes at the cellular level due to its architectural and cellular simplicity [3]. The cerebellum does not only play a fundamental role in the postural control, equilibrium and motor coordination [4] but also involved in cognitive functions such as learning and memory [5]. Aging is a deteriorative process of antioxidants, antioxidant enzymes and repair systems associated with dysregulation of physiological signals and tissue damage [6]. Both free antioxidants and anti-oxidative enzymes not only protect cells from oxidation and inflammation but they may be able to reverse the chronic oxidative stress, which is a characteristic to Parkinson’s disease (PD) and Alzheimer’s disease (AD) [7]. Ozone (O3) is a gaseous molecule consisting of three oxygen (O2) atoms. It is an unstable structure. Ozone therapy is a treatment that involves O3–O2 gas mixture [8]. O3 has been suggested as an immunomodulator, antioxidant enzyme activator and cellular metabolic activator. It is inexpensive and useful in the treatment of different diseases including gastroduodenal ulcers, peritonitis, infected wounds, chronic skin ulcers, initial gangrene, burns and colitis [9]. Recently, Ozone therapy was effective in improving the ischemic damage of rat testis [10]. Because, the effect of medical O3 on age-related changes of rat cerebellum has not been studied before, the aim of this present study is to evaluate the beneficial effect of Ozone therapy on the cerebellum of aged rats. Material and method Chemicals Ozone treatment An O3/O2 mixture of 0.7 g/kg in the form of gas was administered intraperitoneally three times weekly for 8 weeks to the rats in the O3 group in which the flow rate of O2 was kept at 1/32 and the O3 flow rate was kept constant at 80 mg/ml concentration, 95% O2 + 5% O3 gas mixture [11]. This O3/O2 mixture was obtained from a Longevity Ozone Generator (EXT120; Canada) from the O3 unit, faculty of medicine Menoufia University. A special table depending on the flow rate of O2 and O3 concentration in the generator was used to calculate the output concentration. The glial fibrillary acidic protein (GFAP) was obtained from Midco Trade Company (Farid Shawqy St., Elaguza, Giza, Egypt), Microtubule-associated protein-2 (Map2), Ki67, Caspase-3, inducible NO synthase (iNos) were obtained from Lab Vision Corporation (47790 Westinghouse Dr, Fremont, California 94539, USA). Animals The 30 male Wister albino rats (150–200 g) used in this study were procured from EL Helw animal house, Tanta, Egypt. The rats were adapted for a period of 2 weeks before the study. They were housed in well-ventilated cages. Rats were provided with standard diet and unlimited water supply and subjected to a natural photoperiod of ~12-h light/dark cycle throughout this study. The international guidelines for the care and use of laboratory animals were followed and the study was approved by the institutional ethical committee on the animal experiment of the faculty of medicine, Menoufia University. Rats were divided into two main groups: Group I (Control group): This group consisted of 15 rats kept without treatment and were subdivided equally into three subgroups: Ia, Ib and Ic: 6, 20 and 22 months old, respectively. Group II (Ozone-treated group): This group consisted of 15 rats, which received Ozone therapy at a dose of (0.7 g/kg) administrated intraperitoneally three times/week for 8 weeks [11] and were divided equally into three subgroups: IIa, IIb and IIc: 6, 20 and 22 months old, respectively. At the end of the study, all rats were anesthetized by diethyl ether inhalation. The skull was opened and the brain was removed. The cerebellum was excised and a midsagittal section of the cerebellar vermis was done. The cerebellum was immediately placed in 10% Bourin’s solution to harden the brain and also to prevent enzymatic and other postmortem changes [12]. Slices were fixed in 10% buffered formalin for 24 h and then processed to prepare paraffin blocks. Histopathological, immunohistochemical and morphometric analysis For histopathology, 5 μm sections from the cerebellum were prepared then dehydrated using ethanol and stained with hematoxylin and eosin (H&E) and toluidine blue and for immunohistochemical study, paraffin sections (5 μm) were deparaffinized, rehydrated in descending grades of ethanol, and after antigen retrieval with 10 m mol/l citrate acid solution (pH 6), specimens were preincubated with blocking solution (goat serum) for 5 min and were then incubated overnight at 4°C with the primary antibody (GFAP, Midco; Map2, Ki67, Caspase-3) as well as iNos, (Lab Vision, working dilution 1:500). Sections were incubated with secondary biotinylated antibody (goat anti-mouse IgG; Sigma Aldrich, St. Louis, MO) for 20 min. Streptavidin–peroxidase complex was then applied to sections for 10 min. The secondary antibody binding was visualized by incubating sections with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich, St. Louis, MO). Finally, slices were rinsed with phosphate-buffered saline, counterstained with hematoxylin and mounted. For quantitative assessment, five different H&E stained and immunostained sections (400×) from five different rats from each group were analyzed to count the number of Purkinje cells (PCs), color intensity of toluidine blue staining of the PCs, the area percentage of GFAP and Mab2, number of positive cells of iNos, Caspase-3 and Ki67 immunostaining using Image J software (1.74 v; National Institute of Health, Bethesda, MD). Statistical analysis The results were expressed as mean ± SEM. The results were computed statistically (SPSS for Windows, version 14.0; SPSS Inc., Chicago, IL). All the comparisons among groups were carried out using one-way analysis of variance (ANOVA) followed by post hoc test. A P-value <0.05 was considered statistically significant. Results There was no difference noticed between subgroup Ia and subgroup IIa (control aged 6 months and aged 6 months that received O3). The cerebellar cortex of control rats aged 6 months (subgroup Ia) in H&E sections was made up of molecular (M), Purkinje cell (P) and granular (G) layers. The molecular layer consisted mainly of fibers with a few glial cells [basket cells and stellate cells]. Purkinje cells (PCs) were arranged in one row and appeared pyriform in shape and contained rounded nuclei with prominent nucleoli. The granular layer showed small rounded cells, tightly packed. In animals aged 20 months, PCs significantly decreased in number compared to control subgroup Ia, many PCs were shrunken, lost their characteristic shape with missing nuclei (ghost-like appearance) and become surrounded by many vacuoles. Other PCs show darkly stained pyknotic nuclei. Some vacuolization appeared between the cells of the granular layer. At the age of 22 months, most of the PCs show ghost-like appearance, excessive vacuolization of the cytoplasm. Activated glial cells surrounding the focal area of necrosis depicting the typical histomorphology of neuronophagia was detected. Vacuolated neurons in the molecular layer and multiple areas of vacuolization appeared in the granular cell layer. The thickness of the molecular cell layer was decreased while the thickness of the granular cell layer was increased compared to control subgroup Ia. In the O3-treated group, many PCs appeared normal in shape and number. However, some vacuolization, apoptotic neurons and PCs with pyknotic nuclei were still detected (Fig. 1). Fig. 1. View largeDownload slide Representative H&E staining of rat cerebellum of different groups: the cerebellar cortex of a control subgroup Ia (A) showing Purkinje cells (white arrow) in the Purkinje cell layer (P) are arranged in one row of large pyriform body with vesicular nucleus and prominent nucleoli, a molecular layer (M) formed of few small stellate cells (thin black arrow) and basket cells (crossed arrow) whereas the granular layer (G) shows tightly packed small rounded cells. Subgroup Ib and Ic (B, C), many Purkinje cells show pyknotic nuclei (black arrowheads), some neurons were surrounded by halos(white arrowheads). Neuronophagia (black circle) and vacuolization (curved arrow) of the Purkinje cells were more evident at the age of 22 months. Many Purkinje cells in the O3-treated subgroups IIb and IIc (D, E) show nearly normal appearance (white arrows). However, few of the Purkinje cells in the treated group appear pyknotic (black arrowheads), few neurons surrounded by halos (white arrowheads) and few vacuolizations was still evident. The number of Purkinje cells (f) were dramatically decreased with age; ●● P < 0.001, compared with control subgroup Ia. This decrease was significantly increased in the O3-treated subgroups; ○○ P < 0.001, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Fig. 1. View largeDownload slide Representative H&E staining of rat cerebellum of different groups: the cerebellar cortex of a control subgroup Ia (A) showing Purkinje cells (white arrow) in the Purkinje cell layer (P) are arranged in one row of large pyriform body with vesicular nucleus and prominent nucleoli, a molecular layer (M) formed of few small stellate cells (thin black arrow) and basket cells (crossed arrow) whereas the granular layer (G) shows tightly packed small rounded cells. Subgroup Ib and Ic (B, C), many Purkinje cells show pyknotic nuclei (black arrowheads), some neurons were surrounded by halos(white arrowheads). Neuronophagia (black circle) and vacuolization (curved arrow) of the Purkinje cells were more evident at the age of 22 months. Many Purkinje cells in the O3-treated subgroups IIb and IIc (D, E) show nearly normal appearance (white arrows). However, few of the Purkinje cells in the treated group appear pyknotic (black arrowheads), few neurons surrounded by halos (white arrowheads) and few vacuolizations was still evident. The number of Purkinje cells (f) were dramatically decreased with age; ●● P < 0.001, compared with control subgroup Ia. This decrease was significantly increased in the O3-treated subgroups; ○○ P < 0.001, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. The cerebellum of the control subgroup Ia showed dark blue staining indicating the presence of dense Nissl’s granules in the cytoplasm of PCs. There was a significant decrease in color intensity of toluidine blue staining in subgroups, Ib and Ic compared to subgroup Ia (P < 0.001, Fig. 2). O3 treatment up-regulated the toluidine blue color intensity compared to subgroups, Ib and Ic. (P < 0.001, Fig. 2). Fig. 2. View largeDownload slide Representative toluidine blue staining of rat cerebellum of different groups: the Purkinje cells of control subgroup Ia (A) showed dark blue staining, Purkinje cells of subgroups IIb and IIc (B, C) revealed a significant decrease in the color intensity as compared to the control subgroup Ia. O3 treatment {subgroup IIb and IIc (D, E)} attenuated age-induced decrease in the toluidine blue color intensity. ●● P < 0.001, compared with control subgroup Ia, ○ P < 0.05, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Statistical analysis (F). Fig. 2. View largeDownload slide Representative toluidine blue staining of rat cerebellum of different groups: the Purkinje cells of control subgroup Ia (A) showed dark blue staining, Purkinje cells of subgroups IIb and IIc (B, C) revealed a significant decrease in the color intensity as compared to the control subgroup Ia. O3 treatment {subgroup IIb and IIc (D, E)} attenuated age-induced decrease in the toluidine blue color intensity. ●● P < 0.001, compared with control subgroup Ia, ○ P < 0.05, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Statistical analysis (F). The sections of the cerebellar cortex processed for GFAP immunoreactivity showed astrogliosis in the granular layer of control subgroup (Ib and Ic) (age of 20 and 22 months, respectively) compared to control subgroup (Ia) (P < 0.05, P < 0.001, respectively, Fig. 3); this increase in area % of GFAP immunostaining was dramatically decreased in O3-treated subgroups (IIb and IIc) (P > 0.05, Fig. 3). Fig. 3. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: GFAP (A–C) immunoreactivity were significantly increased in the granular layer of subgroups Ib and Ic when compared with subgroup Ia. O3-treated rats showed a non-significant decrease in these immunoreactivities (D and E) in both molecular and granular layers while Mab2 (F–H) immunoreactivity showed significant decrease in the molecular layer of control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Mab2 (I and J) expression which was significant in the granular layer of subgroup IIc. ● P < 0.05 and ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Fig. 3. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: GFAP (A–C) immunoreactivity were significantly increased in the granular layer of subgroups Ib and Ic when compared with subgroup Ia. O3-treated rats showed a non-significant decrease in these immunoreactivities (D and E) in both molecular and granular layers while Mab2 (F–H) immunoreactivity showed significant decrease in the molecular layer of control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Mab2 (I and J) expression which was significant in the granular layer of subgroup IIc. ● P < 0.05 and ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. In the control subgroups, Ib and Ic, the expression of Mab2, marker of neuritogenesis was significantly decreased in the molecular layer compared to subgroup Ia (P < 0.05, P < 0.001, respectively, Fig. 3); this decrease showed an increase in O3-treated subgroups, which were significant in subgroup IIc (P < 0.001, Fig. 3). A significantly elevated expression of iNos, marker of oxidative stress, was found at the age of 20 and 22 months in all cerebellar layers (P < 0.001, Figs. 4 and 5). This expression was down-regulated in O3-treated subgroups (IIb and IIc) compared to control subgroups (Ib and Ic) (P < 0.001, Figs. 4 and 5). Fig. 4. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: Caspase-3 (A–C) and iNOS (F–H) immunoreactivities were dramatically increased in control subgroups Ib and Ic. O3-treated rats showed a significant decrease in these immunoreactivity (D, E, I and J) while Ki67 (K–M) immunoreactivity showed significant decrease in control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Ki67 (N and O) expression. Scale bar 50 μM, ×400. Fig. 4. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: Caspase-3 (A–C) and iNOS (F–H) immunoreactivities were dramatically increased in control subgroups Ib and Ic. O3-treated rats showed a significant decrease in these immunoreactivity (D, E, I and J) while Ki67 (K–M) immunoreactivity showed significant decrease in control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Ki67 (N and O) expression. Scale bar 50 μM, ×400. Fig. 5. View largeDownload slide Graphs representing iNOS (A), Ki67 (B) and Caspase-3 (C) positive cell number and Ki67/Caspase-3 ratio (D). ●P < 0.05, ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Fig. 5. View largeDownload slide Graphs representing iNOS (A), Ki67 (B) and Caspase-3 (C) positive cell number and Ki67/Caspase-3 ratio (D). ●P < 0.05, ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Caspase-3, marker of apoptosis was found to be significantly elevated in the three cerebellar layers of control subgroup Ib and Ic (P < 0.001, Figs. 4 and 5). O3-treated subgroups (IIb and IIc) showed significant reduction in Caspase-3 expression (P < 0.001, Figs. 4 and 5). There was a significant decrease in Ki67 immunohistochemical staining of PC layer in control subgroups Ib and Ic compared to subgroup Ia. However, Ki67 expression in O3-treated subgroup IIb showed no significant difference compared to control subgroups (Ib and IIc) (P > 0.05, Figs. 4 and 5). The ratio of proliferation (Ki67) to apoptosis (Caspase-3) is an important parameter describing cell turnover. Aging (subgroup Ib and IC) was associated with significant reduction in this ratio in all of the cerebellar layers (P < 0.001, Fig. 5). While in O3-treated subgroups (IIb and IIc), this ration showed significant increase compared to control subgroups (Ib and Ic) (P < 0.001, Fig. 5). Discussion The relationship between morphological change and functional decline in the central nervous system (CNS) has been documented in various studies. Aging is associated with loss of cortical thickness and neurons which viewed as the main cause of the decline in brain function. The PC as the sole output neurons of the cerebellum plays an important role in learning and motor coordination. As concluded, the inevitable loss of PC resulted in various motor disorders including autism, ataxia and Huntington’s disease [13]. Our histological data revealed PC degeneration and cell loss with a significant decrease in number. Zhao et al. [14] stated that deficiency of ceramide biosynthesis gives rise to cerebellar PC neurodegeneration and lipofuscin accumulation. The correction of the histopathological picture by medical O3 treatment might be attributed to reducing the lipofuscin pigment [15]. This study revealed a significant decrease in Nissl granule content of the PCs with age, this is in agreement with Wu et al. [16] who explained it by the decreased in gene transcription of proteins caused by aging process. Medical O3 treatment in this study was able to up-regulate Nissl granule content as observed by Frosini et al. [17]. Our results revealed a significant increase in area percentage of GFAP of the granular layer with age and this explains the increase in the granular cell layer thickness in H&E sections. Gliosis may be a reaction to intrinsic modifications in the CNS and such activation lead to progressive degeneration by triggering neurotoxic pathways [18], oxidative stress states [19] and may be an attempt to promote neuroprotective action through the production of neurotrophic factors [20]. Rats treated with O3 in this study showed significant suppression of the astrocytic marker GFAP; similar results were reported by El-Sawalhi et al. [21] who observed that Ozone therapy significantly suppressed the astrocytic marker GFAP and improved the age-related changes in oxidative stress markers in the hippocampus. Oxidative stress plays a causal role in neurodegenerative diseases [7]. Aging is associated with increased nitric oxide (NO) synthesis, through up-regulation of iNos and promotes apoptosis which is characterized by Caspase-3 elevation [22]. Our results showed increased expression of iNos in all cerebellar layers and Caspase-3 in both Purkinje and granular layers in aged rats. This is consistent with previous studies, which concluded that aging was associated with increased expression of oxidative stress markers (iNOS and gp47phox) and apoptotic regulatory proteins (Bax and caspase-3) [23]. Antioxidants protect cells from inflammation and oxidation and may be able to reverse the chronic oxidative stress [24]. Our results revealed that medical O3 ameliorated age-induced oxidative stress and apoptosis detected by histological (H&E) and immunohistochemical (Caspase-3) evaluation. This is in agreement with Díaz-Soto et al. [25] who found that O3 biochemically reduced oxidative stress in patients during ethanol withdrawal. Ozone therapy activates antioxidant protection systems, which activate nuclear factor-erythroid 2-related factor 2 (Nrf2) that can protect against neurodegenerative diseases, such as AD and PD [24]. Loss of neurons was not the only mechanism by which age may affect the cerebellum; atrophy of neural spines and decrease in their branching and loss of their dendrites (faint brown MAP2 immunoreactions) may also interfere with the exchange of information despite the survival of the cell bodies. Our results are in accordance with Zhang et al. [26] who added that the loss of PCs arborizations could directly limit the amount of information input, minimize afferent efficacy, and affect the integration of information and signal transmission in the aged cerebellum, as a result leading to a reduction in cerebellar function. Our results confirmed by the results of previous studies, showed that the cell proliferation rate (Ki67 expression) in aged rats declined exponentially without an intermittent plateau in adult hippocampus in C57 mice [27]. The mechanism of reduced neuronal differentiation and neuritogenesis in aged rats remains poorly understood, but evidence suggests that aging cells have been shown to accumulate oxidative DNA damage from numerous insults that affect the proliferation [28]. The proliferation/apoptosis ratio was significantly decreased with age tangles. Similar results were observed by Ben Abdallah et al. [27] who reported that cell death in relation to cell proliferation was increased from the age of 1 month to the age of 10 months with intermittent plateau in adult hippocampus in C57 mice. Our study showed that medical O3 is able to reverse the age-related suppression of neurogenesis and neuritogenesis, which indicates that oxidative stress is likely to be involved in the suppression of neurogenesis and neuritogenesis in the cerebellum of aged rats [29]. Ozone therapy-related neuroprotection may involve improved cerebral oxygenation, reduced apoptotic cell death, increased neural regeneration and enhancement of antioxidant defenses in the ischemic rat brain [30]. These factors altogether promote the regulation of endogenous NO concentrations and the decrease of the oxidative damage [24]. Moreover, Shehata et al. [15] found that the therapeutic use of O3 normalized mitochondrial superoxide dismutase activity and increased complex I activity as compared to the untreated aged control. Conclusion Possibly, the reduction in the number of cerebellar cells either due to increase of the degeneration or reduction in neurogenesis and associated alterations in communication elements (Purkinje neuron and glia), is implicated in the alterations in the motor information processed by the cerebellum. To the best of our knowledge, this is the first study elucidating the role of Ozone therapy in attenuating age-related changes in rat cerebellum. Our results have shown that O3 protective effect was mediated via reducing oxidative stress, apoptosis, astrocyte activation and improving both neuritogenesis and neurogenesis. It goes without saying that, Ozone therapy appears to be a very effective cheap treatment with no side effects. It can markedly reduce the medical costs, especially in less developed countries. If combined with already improved standardized treatments, it is capable of improving the health of many patients. 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Role of medical ozone in attenuating age-related changes in the rat cerebellum

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Abstract

Abstract Aging is an inevitable biological process characterized by motor in coordination and decline in the ability to learn new motor skills. The purpose of this study is to investigate, for the first time, the beneficial antiaging effects of medical ozone (O3) on the age-related structural damage of the rat cerebellum. We have examined the cerebellum of albino rats at the ages of 6, 20 and 22, and the effect of intraperitoneal medical O3 administration (0.7 g/kg) by histological, immunohistochemical and morphometric studies. Age-related changes in the cerebellum were in the form of a significant reduction in the number of Purkinje cells (PCs), which appeared shrunken with a darkly stained cytoplasm and vacuolated spaces in all layers. The decrease in Nissl granule content of the PCs was also observed. A significant reduction in Mab2, Ki67 immunoreactivity associated with significant increase in glial fibrillary acidic protein, Caspase-3 and iNos immunoreactivity were also detected. Medical O3 administration reversed all these histopathological and immunohistochemical changes. This protective effect was mediated by reducing oxidative stress, apoptosis, astrocyte activation and improving both neuritogenesis and neurogenesis. We can conclude from the results of the present study that medical O3 can prevent the retardation of age-related changes in rat cerebellum. medical ozone, age, cerebellum, histopathology, immunohistochemistry Introduction Aging is associated with disturbances in motor and cognitive functions, especially learning and memory. The decline in fine motor control, gait and balance affect the ability of older adult to perform activities of daily living and to maintain their independence [1]. Gait and balance disorders are of particular interest in older adults as they are a major source of injury and morbidity. Most of older adults who fall suffer injuries that reduce mobility and quality of life [2]. The cerebellum provides an excellent model for the study of age-related changes at the cellular level due to its architectural and cellular simplicity [3]. The cerebellum does not only play a fundamental role in the postural control, equilibrium and motor coordination [4] but also involved in cognitive functions such as learning and memory [5]. Aging is a deteriorative process of antioxidants, antioxidant enzymes and repair systems associated with dysregulation of physiological signals and tissue damage [6]. Both free antioxidants and anti-oxidative enzymes not only protect cells from oxidation and inflammation but they may be able to reverse the chronic oxidative stress, which is a characteristic to Parkinson’s disease (PD) and Alzheimer’s disease (AD) [7]. Ozone (O3) is a gaseous molecule consisting of three oxygen (O2) atoms. It is an unstable structure. Ozone therapy is a treatment that involves O3–O2 gas mixture [8]. O3 has been suggested as an immunomodulator, antioxidant enzyme activator and cellular metabolic activator. It is inexpensive and useful in the treatment of different diseases including gastroduodenal ulcers, peritonitis, infected wounds, chronic skin ulcers, initial gangrene, burns and colitis [9]. Recently, Ozone therapy was effective in improving the ischemic damage of rat testis [10]. Because, the effect of medical O3 on age-related changes of rat cerebellum has not been studied before, the aim of this present study is to evaluate the beneficial effect of Ozone therapy on the cerebellum of aged rats. Material and method Chemicals Ozone treatment An O3/O2 mixture of 0.7 g/kg in the form of gas was administered intraperitoneally three times weekly for 8 weeks to the rats in the O3 group in which the flow rate of O2 was kept at 1/32 and the O3 flow rate was kept constant at 80 mg/ml concentration, 95% O2 + 5% O3 gas mixture [11]. This O3/O2 mixture was obtained from a Longevity Ozone Generator (EXT120; Canada) from the O3 unit, faculty of medicine Menoufia University. A special table depending on the flow rate of O2 and O3 concentration in the generator was used to calculate the output concentration. The glial fibrillary acidic protein (GFAP) was obtained from Midco Trade Company (Farid Shawqy St., Elaguza, Giza, Egypt), Microtubule-associated protein-2 (Map2), Ki67, Caspase-3, inducible NO synthase (iNos) were obtained from Lab Vision Corporation (47790 Westinghouse Dr, Fremont, California 94539, USA). Animals The 30 male Wister albino rats (150–200 g) used in this study were procured from EL Helw animal house, Tanta, Egypt. The rats were adapted for a period of 2 weeks before the study. They were housed in well-ventilated cages. Rats were provided with standard diet and unlimited water supply and subjected to a natural photoperiod of ~12-h light/dark cycle throughout this study. The international guidelines for the care and use of laboratory animals were followed and the study was approved by the institutional ethical committee on the animal experiment of the faculty of medicine, Menoufia University. Rats were divided into two main groups: Group I (Control group): This group consisted of 15 rats kept without treatment and were subdivided equally into three subgroups: Ia, Ib and Ic: 6, 20 and 22 months old, respectively. Group II (Ozone-treated group): This group consisted of 15 rats, which received Ozone therapy at a dose of (0.7 g/kg) administrated intraperitoneally three times/week for 8 weeks [11] and were divided equally into three subgroups: IIa, IIb and IIc: 6, 20 and 22 months old, respectively. At the end of the study, all rats were anesthetized by diethyl ether inhalation. The skull was opened and the brain was removed. The cerebellum was excised and a midsagittal section of the cerebellar vermis was done. The cerebellum was immediately placed in 10% Bourin’s solution to harden the brain and also to prevent enzymatic and other postmortem changes [12]. Slices were fixed in 10% buffered formalin for 24 h and then processed to prepare paraffin blocks. Histopathological, immunohistochemical and morphometric analysis For histopathology, 5 μm sections from the cerebellum were prepared then dehydrated using ethanol and stained with hematoxylin and eosin (H&E) and toluidine blue and for immunohistochemical study, paraffin sections (5 μm) were deparaffinized, rehydrated in descending grades of ethanol, and after antigen retrieval with 10 m mol/l citrate acid solution (pH 6), specimens were preincubated with blocking solution (goat serum) for 5 min and were then incubated overnight at 4°C with the primary antibody (GFAP, Midco; Map2, Ki67, Caspase-3) as well as iNos, (Lab Vision, working dilution 1:500). Sections were incubated with secondary biotinylated antibody (goat anti-mouse IgG; Sigma Aldrich, St. Louis, MO) for 20 min. Streptavidin–peroxidase complex was then applied to sections for 10 min. The secondary antibody binding was visualized by incubating sections with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich, St. Louis, MO). Finally, slices were rinsed with phosphate-buffered saline, counterstained with hematoxylin and mounted. For quantitative assessment, five different H&E stained and immunostained sections (400×) from five different rats from each group were analyzed to count the number of Purkinje cells (PCs), color intensity of toluidine blue staining of the PCs, the area percentage of GFAP and Mab2, number of positive cells of iNos, Caspase-3 and Ki67 immunostaining using Image J software (1.74 v; National Institute of Health, Bethesda, MD). Statistical analysis The results were expressed as mean ± SEM. The results were computed statistically (SPSS for Windows, version 14.0; SPSS Inc., Chicago, IL). All the comparisons among groups were carried out using one-way analysis of variance (ANOVA) followed by post hoc test. A P-value <0.05 was considered statistically significant. Results There was no difference noticed between subgroup Ia and subgroup IIa (control aged 6 months and aged 6 months that received O3). The cerebellar cortex of control rats aged 6 months (subgroup Ia) in H&E sections was made up of molecular (M), Purkinje cell (P) and granular (G) layers. The molecular layer consisted mainly of fibers with a few glial cells [basket cells and stellate cells]. Purkinje cells (PCs) were arranged in one row and appeared pyriform in shape and contained rounded nuclei with prominent nucleoli. The granular layer showed small rounded cells, tightly packed. In animals aged 20 months, PCs significantly decreased in number compared to control subgroup Ia, many PCs were shrunken, lost their characteristic shape with missing nuclei (ghost-like appearance) and become surrounded by many vacuoles. Other PCs show darkly stained pyknotic nuclei. Some vacuolization appeared between the cells of the granular layer. At the age of 22 months, most of the PCs show ghost-like appearance, excessive vacuolization of the cytoplasm. Activated glial cells surrounding the focal area of necrosis depicting the typical histomorphology of neuronophagia was detected. Vacuolated neurons in the molecular layer and multiple areas of vacuolization appeared in the granular cell layer. The thickness of the molecular cell layer was decreased while the thickness of the granular cell layer was increased compared to control subgroup Ia. In the O3-treated group, many PCs appeared normal in shape and number. However, some vacuolization, apoptotic neurons and PCs with pyknotic nuclei were still detected (Fig. 1). Fig. 1. View largeDownload slide Representative H&E staining of rat cerebellum of different groups: the cerebellar cortex of a control subgroup Ia (A) showing Purkinje cells (white arrow) in the Purkinje cell layer (P) are arranged in one row of large pyriform body with vesicular nucleus and prominent nucleoli, a molecular layer (M) formed of few small stellate cells (thin black arrow) and basket cells (crossed arrow) whereas the granular layer (G) shows tightly packed small rounded cells. Subgroup Ib and Ic (B, C), many Purkinje cells show pyknotic nuclei (black arrowheads), some neurons were surrounded by halos(white arrowheads). Neuronophagia (black circle) and vacuolization (curved arrow) of the Purkinje cells were more evident at the age of 22 months. Many Purkinje cells in the O3-treated subgroups IIb and IIc (D, E) show nearly normal appearance (white arrows). However, few of the Purkinje cells in the treated group appear pyknotic (black arrowheads), few neurons surrounded by halos (white arrowheads) and few vacuolizations was still evident. The number of Purkinje cells (f) were dramatically decreased with age; ●● P < 0.001, compared with control subgroup Ia. This decrease was significantly increased in the O3-treated subgroups; ○○ P < 0.001, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Fig. 1. View largeDownload slide Representative H&E staining of rat cerebellum of different groups: the cerebellar cortex of a control subgroup Ia (A) showing Purkinje cells (white arrow) in the Purkinje cell layer (P) are arranged in one row of large pyriform body with vesicular nucleus and prominent nucleoli, a molecular layer (M) formed of few small stellate cells (thin black arrow) and basket cells (crossed arrow) whereas the granular layer (G) shows tightly packed small rounded cells. Subgroup Ib and Ic (B, C), many Purkinje cells show pyknotic nuclei (black arrowheads), some neurons were surrounded by halos(white arrowheads). Neuronophagia (black circle) and vacuolization (curved arrow) of the Purkinje cells were more evident at the age of 22 months. Many Purkinje cells in the O3-treated subgroups IIb and IIc (D, E) show nearly normal appearance (white arrows). However, few of the Purkinje cells in the treated group appear pyknotic (black arrowheads), few neurons surrounded by halos (white arrowheads) and few vacuolizations was still evident. The number of Purkinje cells (f) were dramatically decreased with age; ●● P < 0.001, compared with control subgroup Ia. This decrease was significantly increased in the O3-treated subgroups; ○○ P < 0.001, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. The cerebellum of the control subgroup Ia showed dark blue staining indicating the presence of dense Nissl’s granules in the cytoplasm of PCs. There was a significant decrease in color intensity of toluidine blue staining in subgroups, Ib and Ic compared to subgroup Ia (P < 0.001, Fig. 2). O3 treatment up-regulated the toluidine blue color intensity compared to subgroups, Ib and Ic. (P < 0.001, Fig. 2). Fig. 2. View largeDownload slide Representative toluidine blue staining of rat cerebellum of different groups: the Purkinje cells of control subgroup Ia (A) showed dark blue staining, Purkinje cells of subgroups IIb and IIc (B, C) revealed a significant decrease in the color intensity as compared to the control subgroup Ia. O3 treatment {subgroup IIb and IIc (D, E)} attenuated age-induced decrease in the toluidine blue color intensity. ●● P < 0.001, compared with control subgroup Ia, ○ P < 0.05, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Statistical analysis (F). Fig. 2. View largeDownload slide Representative toluidine blue staining of rat cerebellum of different groups: the Purkinje cells of control subgroup Ia (A) showed dark blue staining, Purkinje cells of subgroups IIb and IIc (B, C) revealed a significant decrease in the color intensity as compared to the control subgroup Ia. O3 treatment {subgroup IIb and IIc (D, E)} attenuated age-induced decrease in the toluidine blue color intensity. ●● P < 0.001, compared with control subgroup Ia, ○ P < 0.05, compared with subgroups Ib and Ic. Scale bar 50 μM, ×400. Statistical analysis (F). The sections of the cerebellar cortex processed for GFAP immunoreactivity showed astrogliosis in the granular layer of control subgroup (Ib and Ic) (age of 20 and 22 months, respectively) compared to control subgroup (Ia) (P < 0.05, P < 0.001, respectively, Fig. 3); this increase in area % of GFAP immunostaining was dramatically decreased in O3-treated subgroups (IIb and IIc) (P > 0.05, Fig. 3). Fig. 3. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: GFAP (A–C) immunoreactivity were significantly increased in the granular layer of subgroups Ib and Ic when compared with subgroup Ia. O3-treated rats showed a non-significant decrease in these immunoreactivities (D and E) in both molecular and granular layers while Mab2 (F–H) immunoreactivity showed significant decrease in the molecular layer of control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Mab2 (I and J) expression which was significant in the granular layer of subgroup IIc. ● P < 0.05 and ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Fig. 3. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: GFAP (A–C) immunoreactivity were significantly increased in the granular layer of subgroups Ib and Ic when compared with subgroup Ia. O3-treated rats showed a non-significant decrease in these immunoreactivities (D and E) in both molecular and granular layers while Mab2 (F–H) immunoreactivity showed significant decrease in the molecular layer of control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Mab2 (I and J) expression which was significant in the granular layer of subgroup IIc. ● P < 0.05 and ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. In the control subgroups, Ib and Ic, the expression of Mab2, marker of neuritogenesis was significantly decreased in the molecular layer compared to subgroup Ia (P < 0.05, P < 0.001, respectively, Fig. 3); this decrease showed an increase in O3-treated subgroups, which were significant in subgroup IIc (P < 0.001, Fig. 3). A significantly elevated expression of iNos, marker of oxidative stress, was found at the age of 20 and 22 months in all cerebellar layers (P < 0.001, Figs. 4 and 5). This expression was down-regulated in O3-treated subgroups (IIb and IIc) compared to control subgroups (Ib and Ic) (P < 0.001, Figs. 4 and 5). Fig. 4. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: Caspase-3 (A–C) and iNOS (F–H) immunoreactivities were dramatically increased in control subgroups Ib and Ic. O3-treated rats showed a significant decrease in these immunoreactivity (D, E, I and J) while Ki67 (K–M) immunoreactivity showed significant decrease in control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Ki67 (N and O) expression. Scale bar 50 μM, ×400. Fig. 4. View largeDownload slide Representative immunostaining of rat cerebellum of different groups: Caspase-3 (A–C) and iNOS (F–H) immunoreactivities were dramatically increased in control subgroups Ib and Ic. O3-treated rats showed a significant decrease in these immunoreactivity (D, E, I and J) while Ki67 (K–M) immunoreactivity showed significant decrease in control subgroups Ib and Ic. O3 treatment attenuated age-induced decrease in Ki67 (N and O) expression. Scale bar 50 μM, ×400. Fig. 5. View largeDownload slide Graphs representing iNOS (A), Ki67 (B) and Caspase-3 (C) positive cell number and Ki67/Caspase-3 ratio (D). ●P < 0.05, ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Fig. 5. View largeDownload slide Graphs representing iNOS (A), Ki67 (B) and Caspase-3 (C) positive cell number and Ki67/Caspase-3 ratio (D). ●P < 0.05, ●● P < 0.001, compared with control subgroup Ia, ○○ P < 0.001, compared with subgroups Ib and Ic. Caspase-3, marker of apoptosis was found to be significantly elevated in the three cerebellar layers of control subgroup Ib and Ic (P < 0.001, Figs. 4 and 5). O3-treated subgroups (IIb and IIc) showed significant reduction in Caspase-3 expression (P < 0.001, Figs. 4 and 5). There was a significant decrease in Ki67 immunohistochemical staining of PC layer in control subgroups Ib and Ic compared to subgroup Ia. However, Ki67 expression in O3-treated subgroup IIb showed no significant difference compared to control subgroups (Ib and IIc) (P > 0.05, Figs. 4 and 5). The ratio of proliferation (Ki67) to apoptosis (Caspase-3) is an important parameter describing cell turnover. Aging (subgroup Ib and IC) was associated with significant reduction in this ratio in all of the cerebellar layers (P < 0.001, Fig. 5). While in O3-treated subgroups (IIb and IIc), this ration showed significant increase compared to control subgroups (Ib and Ic) (P < 0.001, Fig. 5). Discussion The relationship between morphological change and functional decline in the central nervous system (CNS) has been documented in various studies. Aging is associated with loss of cortical thickness and neurons which viewed as the main cause of the decline in brain function. The PC as the sole output neurons of the cerebellum plays an important role in learning and motor coordination. As concluded, the inevitable loss of PC resulted in various motor disorders including autism, ataxia and Huntington’s disease [13]. Our histological data revealed PC degeneration and cell loss with a significant decrease in number. Zhao et al. [14] stated that deficiency of ceramide biosynthesis gives rise to cerebellar PC neurodegeneration and lipofuscin accumulation. The correction of the histopathological picture by medical O3 treatment might be attributed to reducing the lipofuscin pigment [15]. This study revealed a significant decrease in Nissl granule content of the PCs with age, this is in agreement with Wu et al. [16] who explained it by the decreased in gene transcription of proteins caused by aging process. Medical O3 treatment in this study was able to up-regulate Nissl granule content as observed by Frosini et al. [17]. Our results revealed a significant increase in area percentage of GFAP of the granular layer with age and this explains the increase in the granular cell layer thickness in H&E sections. Gliosis may be a reaction to intrinsic modifications in the CNS and such activation lead to progressive degeneration by triggering neurotoxic pathways [18], oxidative stress states [19] and may be an attempt to promote neuroprotective action through the production of neurotrophic factors [20]. Rats treated with O3 in this study showed significant suppression of the astrocytic marker GFAP; similar results were reported by El-Sawalhi et al. [21] who observed that Ozone therapy significantly suppressed the astrocytic marker GFAP and improved the age-related changes in oxidative stress markers in the hippocampus. Oxidative stress plays a causal role in neurodegenerative diseases [7]. Aging is associated with increased nitric oxide (NO) synthesis, through up-regulation of iNos and promotes apoptosis which is characterized by Caspase-3 elevation [22]. Our results showed increased expression of iNos in all cerebellar layers and Caspase-3 in both Purkinje and granular layers in aged rats. This is consistent with previous studies, which concluded that aging was associated with increased expression of oxidative stress markers (iNOS and gp47phox) and apoptotic regulatory proteins (Bax and caspase-3) [23]. Antioxidants protect cells from inflammation and oxidation and may be able to reverse the chronic oxidative stress [24]. Our results revealed that medical O3 ameliorated age-induced oxidative stress and apoptosis detected by histological (H&E) and immunohistochemical (Caspase-3) evaluation. This is in agreement with Díaz-Soto et al. [25] who found that O3 biochemically reduced oxidative stress in patients during ethanol withdrawal. Ozone therapy activates antioxidant protection systems, which activate nuclear factor-erythroid 2-related factor 2 (Nrf2) that can protect against neurodegenerative diseases, such as AD and PD [24]. Loss of neurons was not the only mechanism by which age may affect the cerebellum; atrophy of neural spines and decrease in their branching and loss of their dendrites (faint brown MAP2 immunoreactions) may also interfere with the exchange of information despite the survival of the cell bodies. Our results are in accordance with Zhang et al. [26] who added that the loss of PCs arborizations could directly limit the amount of information input, minimize afferent efficacy, and affect the integration of information and signal transmission in the aged cerebellum, as a result leading to a reduction in cerebellar function. Our results confirmed by the results of previous studies, showed that the cell proliferation rate (Ki67 expression) in aged rats declined exponentially without an intermittent plateau in adult hippocampus in C57 mice [27]. The mechanism of reduced neuronal differentiation and neuritogenesis in aged rats remains poorly understood, but evidence suggests that aging cells have been shown to accumulate oxidative DNA damage from numerous insults that affect the proliferation [28]. The proliferation/apoptosis ratio was significantly decreased with age tangles. Similar results were observed by Ben Abdallah et al. [27] who reported that cell death in relation to cell proliferation was increased from the age of 1 month to the age of 10 months with intermittent plateau in adult hippocampus in C57 mice. Our study showed that medical O3 is able to reverse the age-related suppression of neurogenesis and neuritogenesis, which indicates that oxidative stress is likely to be involved in the suppression of neurogenesis and neuritogenesis in the cerebellum of aged rats [29]. Ozone therapy-related neuroprotection may involve improved cerebral oxygenation, reduced apoptotic cell death, increased neural regeneration and enhancement of antioxidant defenses in the ischemic rat brain [30]. These factors altogether promote the regulation of endogenous NO concentrations and the decrease of the oxidative damage [24]. Moreover, Shehata et al. [15] found that the therapeutic use of O3 normalized mitochondrial superoxide dismutase activity and increased complex I activity as compared to the untreated aged control. Conclusion Possibly, the reduction in the number of cerebellar cells either due to increase of the degeneration or reduction in neurogenesis and associated alterations in communication elements (Purkinje neuron and glia), is implicated in the alterations in the motor information processed by the cerebellum. To the best of our knowledge, this is the first study elucidating the role of Ozone therapy in attenuating age-related changes in rat cerebellum. Our results have shown that O3 protective effect was mediated via reducing oxidative stress, apoptosis, astrocyte activation and improving both neuritogenesis and neurogenesis. It goes without saying that, Ozone therapy appears to be a very effective cheap treatment with no side effects. It can markedly reduce the medical costs, especially in less developed countries. If combined with already improved standardized treatments, it is capable of improving the health of many patients. 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MicroscopyOxford University Press

Published: Apr 9, 2018

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