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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1079-1084

A comparative study on the effect of aging on the hippocampal CA1 area of male albino rat


Anatomy and Embryology Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission16-Mar-2016
Date of Acceptance06-Jun-2016
Date of Web Publication04-Apr-2018

Correspondence Address:
Rasha R Selima
Anatomy and Embryology Department, Faculty of Medicine, Menoufia University, Sabry Abo-allam Street, Shibin el-koum, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.229200

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  Abstract 


Objectives
This work aimed to study the effect of aging on the structure of the hippocampus in the adult and aged male albino rats.
Background
Dementia is one of the most important problems nowadays. Aging is associated with learning and memory impairments. Aging is the progressive accumulation of changes with time that are associated with or responsible for the ever-increasing susceptibility to disease and death, which accompanies advancing age. The hippocampal formation is one of the most common brain areas affected by aging in both humans and other mammalian species.
Materials and methods
Eighteen male albino rats were divided into three groups of six rats each: Group I included adult animals aged 6 months; group II included rats aged 20 months; and group III included rats aged 24 months. All animals were killed after 8 weeks. Hippocampus sections were prepared for light microscopic examination. Morphometric and statistical analysis were carried out.
Results
In comparison with the control group aged 8 months, both groups aged 22 and 26 months showed a significant decrease in the number of pyramidal cells of the hippocampus (P<0.001), and a significant increase in the astrocyte surface area in glial fibrillary acidic protein immunostaining (P<0.001).
Conclusion
Aging process involves degenerative changes in the hippocampus. Aging is more serious as it can produce Alzheimer's disease-like pathological changes. Alzheimer's is the most common form of dementia, a general term for memory loss and other intellectual abilities serious enough to interfere with daily life. Alzheimer's disease accounts for 60–80% of dementia cases.

Keywords: aging, Alzheimer's disease, hippocampus, rat


How to cite this article:
El-Safti FE, El-Kholoy WB, El-Mehi AE, Selima RR. A comparative study on the effect of aging on the hippocampal CA1 area of male albino rat. Menoufia Med J 2017;30:1079-84

How to cite this URL:
El-Safti FE, El-Kholoy WB, El-Mehi AE, Selima RR. A comparative study on the effect of aging on the hippocampal CA1 area of male albino rat. Menoufia Med J [serial online] 2017 [cited 2018 Sep 19];30:1079-84. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1079/229200




  Introduction Top


Human aging is associated with a wide range of physiological changes that not only make us more susceptible to death but limit our normal functions and render us more susceptible to a number of diseases such as Alzheimer's (AD) and Parkinson's diseases[1]. One of the mechanisms underlying the aging process is proposed to be the oxidative damage caused by free radicals[2]. Several researchers have reported that oxidative stress parameters increased in the brain with increasing age[3].

Progressive disorders affecting behavior, thinking, memory, and daily activities is known as dementia syndrome. The most common type of dementia is AD[4]. A person who has dementia is always developing agitation and other behavioral symptoms, making it much harder to care for him or her[5]. In many vertebrates, the hippocampus forms a major component of the brain. The consolidation of information from short-term memory to long-term memory is a role of the hippocampus[6]. Cornu Ammonis 1 (CA1) is the zone that is most sensitive to various insults[7]. The hippocampal formation is one of the most common brain areas affected by aging in both humans and other mammalian species[8]. Thus, the present study was performed to investigate the effect of aging on the structure of the hippocampus in male albino rats.


  Materials and Methods Top


Glial fibrillary acidic protein (GFAP) was obtained from Midco Trade Company (Giza, Egypt), and used for immunohistochemical staining for GFAP of the astrocytes.

Eighteen male albino rats of Sprague–Dawley strain, weighing between 150 and 200 g were obtained from Tanta, Egypt. They were fed standard diet. Ethical approval was obtained from the Faculty of Medicine, Menoufia University. The animals were divided into three main groups of six each:

  1. Group I included adult animals aged 6 months.
  2. Group II included adult animals aged 20 months.
  3. Group III included adult animals aged 24 months.


Methods

At the end of the experiment (after 8 weeks), all rats were anesthetized using diethyl ether inhalation and killed. The skull vault was removed by means of dissection, and the brain was immediately placed in 10% buffered formalin. After 10 min, when brain soft tissue was hardened to avoid soft tissue dissipation, the temporal lobe of the right cerebral hemisphere was separated and coronal slices of 5–7 mm-thickness were taken at the level of the hippocampus. The slices were fixed in 10% buffered formalin for 24 h and processed to prepare paraffin blocks. Sections of 5 μm were obtained and stained for histological study:

Histological stains

  1. H and E for routine histological examination[8].
  2. Toluidine blue for Nissl granule[8].
  3. Silver stain for the senile plaques and neurofibrillary tangles (NFTs)[8].


Immunohistochemical stains

GFAP was used for the astrocytes[9].

Morphometric study

  1. Six different H and E sections from six different rats were examined from each group for the number of the pyramidal cells[10].
  2. Six different GFAP sections for the percentage of astrocyte surface area (area stained brown) were examined[10].


The data were obtained using image analyzer software, Image J analyzer, version 1.43o8 (National Institutes of Health, New York, USA) and Digimizer version 4.3.5, MedCalc software (Acacialaan 22, 8400 Ostend, Belgium)[11]. This was conducted in the Anatomy and Embryology Department, Menoufia University.

Statistical analysis

Statistical analysis was performed for the morphometric results. The results were collected, tabulated, and statistically analyzed using Statistical Package for Social Sciences (SPSS) version 20 on IBM compatible computer (SPSS Inc., Chicago, Illinois, USA).

The results were expressed as mean (X)±SD. The Mann–Whitney test is a test of significance used for comparison between two groups[12]. A P value of less than 0.05 was considered significant.


  Results Top


Histological results

Light microscopic results of H and E-stained sections in adult balanced diet-fed rat revealed that the hippocampus was composed of four areas: CA1–CA4. Each area appeared in three layers: Polymorphic, pyramidal, and molecular. Capillaries and different glial cells were scattered inside the molecular and polymorphic layers. Microglia, oligodendroglia, and astrocytes constituted the glial cell types. The pyramidal nerve cells appeared as large triangular cells with large vesicular nuclei and prominent processes.

On comparing the hippocampus of adult rats with that of the rats aged 22 months, it could be noted that the aged rats showed a decrease in hippocampus thickness, slight disorganization of the pyramidal cell layer, and degeneration of pyramidal cells.

On comparing the rats aged 26 months with adult rats, the hippocampus of the aged rats showed multiple vacuolations, especially in the CA1 area. Moreover, there were many degenerated shrunk enpyramidal cells and degeneration in the molecular layer with loss of the nuclear details.

Comparison between the adult and aged groups showed that there was a highly significant decrease in the mean number of pyramidal cells (P< 0.001) in all aged groups than that in the adult group [Table 1] and [Histogram 1 [Additional file 1]].
Table 1: Mean number of the pyramidal cells/5 HPF/rat

Click here to view


Light microscopic results of toluidine blue-stained sections recorded the strongest pyramidal cell activity (Nissl's granules density) in adult rats aged 8 months in comparison with other experimental groups. The activity became decreased in rats aged 22 months and highly decreased in rats aged 26 month.

Light microscopic results of silver-stained sections showed the pyramidal cell that appeared triangular in shape in adult rats aged 8 months. Senile plaques and NFTs appeared in rats aged 22 months and their number increased more in rats aged 26 months.

Immunohistochemical results for GFAP-stained sections revealed that the CA1 area had a cytoplasmic brown reaction in few small dispersed nonbranched astrocytes in adult rats aged 8 months, in small branched astrocytes in rats aged 22 months, and in large branched astrocytes in rats aged 26 months. The reaction became very strong in the latter. Comparison between adult and aged groups showed that there was a significant increase in the surface area of astrocytes (P< 0.0001) in all aged groups than in adult rats.

Morphometric results

Mean number of pyramidal cells

There was a highly significant decrease in the number of pyramidal cells of the hippocampus in rats aged 22 and 26 months (P<0.001) in comparison with group I (adult rats aged 8 months) [Table 1] and [Histogram 1].

The percentage of the surface area of the astrocytes in glial fibrillary acidic protein

There was a highly significant increase in the surface area of the brown color of the astrocytes in the rats aged 22 and a greater increase in the rats aged 26 months (P<0.001) in comparison with group I (adult rats aged 8 months) [Table 2] and [Histogram 2 [Additional file 2]].
Table 2: The percentage of the surface area of the astrocytes in glial fibrillary acidic protein

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  Discussion Top


Most central nervous system (CNS) diseases, such as AD, are caused by the degeneration and eventual death of neurons, and the reduced ability of nerves to regenerate[13].

Villanueva et al.[14] added that many pieces of evidence suggest that the CNS is highly sensitive to oxidative stress, because of its high content of unsaturated phospholipids, its high metabolic rate, and low content of some antioxidant enzymes, such as catalase, with the hippocampus, substantia nigra, and striatum being the most sensitive structures.

Tao et al.[15] suggested that aging is associated with impairment in certain aspects of cognition, especially learning and memory. Moreover, several studies demonstrated that aging increased the risk for impaired cognitive function.

Crisóstomo et al.[16] stated that aging induces several structural and functional changes in the brain. An imbalance in changes caused by increased generation of free radicals and decreased functional efficiency of the antioxidant system has been suggested to be one of the primary factors that contribute to the aging process.

Rat was utilized in this study as it is one of the most suitable animals for studies of aging in mammals. This was supported by Du and Yan[17], who stated that its life span is short. Therefore, the relationship between oxidative stress and aging has been extensively examined in rats. In most of the studies, male sex is especially preferred because female sex is known to be less susceptible to oxidative stress owing to estrogen's protection by decreasing oxidative stress and increasing antioxidant defenses.

In the present study, the hippocampus was chosen as it might be the main affected region in aging. The structural changes in the hippocampus and CA1 regions may serve as the main underlying neurobiological mechanisms of hippocampal dysfunction and may, therefore, be highly relevant to memory impairment in aging. Furthermore, these characteristics of hippocampal subfield atrophy in aging might be very similar to those in AD. This was supported by Hashish and colleagues[18],[19], who stated that the CA1 area is the zone that is the most sensitive to various effects.

In the present study, examination of the hippocampus of rats aged 22 months showed a decrease in hippocampus thickness and disturbed arrangement of the pyramidal layer with a significant decrease in the number of pyramidal cells. Some pyramidal cells were degenerated. There was a decrease in the Nissl granule content in many of the pyramidal cells.

However, the hippocampus of rats aged 26 months showed an increase in the degenerated shrunken pyramidal cells with loss of the nuclear details. There was faint blue coloration of the cytoplasm of the pyramidal cells. NFTs and senile plaques appeared more frequently in this age compared with the previous age.

Similarly, Ramesh et al.[20] observed a reduction in the mean thickness of senile rat hippocampus and added that those who have lower hippocampal thickness are more liable to cognitive decline as well as a number of disorders, including AD.

In the present study, immunohistochemical staining for GFAP showed its normal distribution in the adult animals as a mild positive reaction in the immunoreactive astrocytes and glial fibers. However, in aged rats, an increase in the amount and intensity of GFAP immunoreactive astrocytes was detected as the glial fibers and were thickened with increase in their staining intensity. In agreement with these results, Ghoneim et al.[21] stated that the hippocampus of aged rats showed an increase in the number and size of astrocytes detected using GFAP immunohistochemical stain, which were observed in the neuropil.

Moreover, Dhikav and Anand[22] found numerous astrocytes and microglial cells in the dentate gyrus of the hippocampus of their AD model. Moreover, Okasha[23] added that astrocytes represent the most abundant glial cell type in the CNS. Once these cells undergo reactive activation, they produce cytokines and other molecules involved in inflammatory response, which are thought to significantly contribute to expand brain damage.

Moreover, Roy[24] reported that GFAP expression significantly increased, reaching a 1.5–3.2-fold increase at end-stage AD, and they explained that this gradual increase in GFAP transcription is parallel to the progression of AD.

In the present study, senile neuritic plaques and NFTs were observed in the neuropil as detected using silver stain in the hippocampus of aged rats. This is in agreement with the findings of Polydoro et al.[25], who observed that hippocampal atrophy was associated with deposition of tau protein, formation of NFTs, and accumulation of β-amyloid.

Moreover, Serrano-Pozo et al.[26] added that NFTs were explained as insoluble twisted fibers found inside brain cells in Alzheimer-induced rats. These tangles are primarily formed of a protein called tau, and Nelson et al.[27] described the classic plaque as spherical areas with an argentophilic amyloid-positive core of aggregated fibrils consisting of β-amyloid surrounded by a clear halo with dystrophic neuritis in Alzheimer-induced rats. Moreover, Padurariu et al.[28] added that NFT-like aggregates lead to the damage of the major efferent projection to the dentate gyrus, the perforant pathway. Damage of this neural system in patients is thought to underlie memory impairment in AD and to be the first stage of what has been considered to be an irreversible cascade of lesions leading ultimately to widespread damage and dementia.

Furthermore, Hashem et al.[29] stated that the presence of even a few tangles in a single field in the cortex suggests a significant cognitive decline and is compatible with the diagnosis of AD made on the basis of clinical findings. They added that the numbers of tangles increase as cognitive decline increases. Moreover, Mokhtar et al.[30] observed that the criteria for neuropathological diagnosis of AD take into account the NFTs and senile plaques [Figure 1],[Figure 2],[Figure 3],[Figure 4].
Figure 1 Photomicrographs of H&E-stained sections of the hippocampus: (a) A photomicrograph of a coronal section in the hippocampus proper at the CA1 area of an adult rat aged 8 months showing the major two parts of the hippocampal formation hippocampus proper and dentate gyrus (DG) with narrow hippocampal sulcus (HS) in between. The hippocampus proper is composed of four areas (CA–C1 to CA–C4) (×40). (b) Three layers of the hippocampus: the molecular (M), pyramidal (P), and polymorphic (PM) layers; the pyramidal layer is formed of multiple regular rows of the pyramidal cells (×100). (c) The pyramidal cells appeared triangular in shape with large vesicular nuclei and basophilic cytoplasm. Notice: oligodendroglia cells (black arrows), astrocytes (red arrows), microglia (arrow head), nerve fibers (F), and blood vessels (BV) (×400). (d) Typical pyramidal cells as large triangular cells with basophilic cytoplasm, large vesicular nuclei, and prominent nucleoli (N). Notice: oligodendroglia cells (black arrow) (×1000). (e) A photomicrograph of a rat aged 22 months showing more degenerated pyramidal cells in the pyramidal cell layer (red arrows) and others still normal (black arrows). Notice: vacuolated areas (V) (×400). (f) A photomicrograph of a rat aged 26 months showing disturbed arrangement with a large number of degenerated shrunken pyramidal cells (black arrows). Notice: vacuolations (V) (×400).

Click here to view
,
Figure 2: Photomicrographs of (toluidine blue ×1000) toluidine-stained sections of the hippocampus: (a) A photomicrograph of an adult rat aged 8 months showing dense Nissl granule content inside the cytoplasm of the pyramidal cells (black arrows). (b) A photomicrograph of a rat aged 22 months showing scanty Nissl granule content in most of the pyramidal cells, with faint blue color of the cytoplasm. (c) A photomicrograph of a rat aged 26 months showing more faint blue color and scanty Nissl granules density in the pyramidal cells.

Click here to view
,
Figure 3: Photomicrographs of (silver ×1000) silver-stained sections of the hippocampus: (a) An adult rat aged 8 months showing normal appearance of the pyramidal cells, which were triangular in shape with large vesicular nuclei (blue arrows) and normal dendrites. (b) A rat aged 22 months showing neurofibrillary tangles (blue arrows), senile neuritic plaques (red arrows), and vacuolated neuropilic areas (yellow arrows). (c) A rat aged 26 months showing scattered silver-positive neuritic plaques within the polymorphic layer (red arrows), more neurofibrillary tangles (blue arrow), and vacuolated neuropilic areas (arrows heads).

Click here to view
,
Figure 4: Photomicrographs of glial fibrillary acidic protein (GFAP)-stained sections (GFAP ×400) of the hippocampus: (a) An adult rat aged 8 months showing a few astrocytes with small size, short branches, and faint brown immunoreaction (black arrows). (b) A rat aged 22 months showing an apparent increase in the number and size of astrocytes with intense brown positive immunoreaction in the cytoplasm and the processes of these astrocytes. (c) A rat aged 26 months showing an increase in the number of astrocytes with intense brown positive immunoreaction for GFAP in the cytoplasm and the processes of these astrocytes. Notice: increased in size with long processes (black arrows).

Click here to view



  Conclusion Top


Aging process has degenerative changes in the hippocampus and it can produce AD-like pathological changes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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