الفهرس 
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Abstract
The present study focused on the PFC and the hippocampus which are parts of the limbic system. The PFC is the largest area in the human brain and is localized in the anterior part of the frontal lobe. It has many functions including memory, problem solving, and intellectuality. Hippocampus is associated with memory consolidation and decision making. AASs are synthetic products of the male sex hormone testosterone. AASs abuse is considered a dominant current form of substance abuse, and is a growing public health concern in the world.
The aim of the current work was to study the effects of AASs, in the form of ND, on the PFC and the hippocampus of male adult albino rats in 5 consecutive days/ week for 2 weeks, both histologically and statistically. It also aimed to evaluate the reversibility of these histological and statistical changes in the selected areas after ND withdrawal. The present study used light microscopy, immunohistochemical and histomorphometric methods.
Twenty-four adult male albino rats were used in the current study and were divided into 3 groups; according to the procedure and treatment type:
group I (Control group): This group included twelve rats which were further subdivided into two subgroups, six rats each:
-Subgroup IA (6 rats): received no treatment (negative control group).
-Subgroup IB (6 rats): received 1 ml/KgBW olive oil daily IM in 5 consecutive days/ week for 2 weeks (positive control group).
group II (AASs group): This group included 6 rats and received ND 5 mg/ kgBW/day IM for 2 weeks. To obtain this dose, the ampule was dissolved in 10 ml olive oil, then a dose of 5 mg/ kgBW/day was given to each rat of groups II and III for 2 weeks (5 consecutive days /week).
group III (Withdrawal group): This group included 6 rats and received ND in the same dose and by the same route as in group II, and then left for two weeks after stoppage of the drug.
At the end of the experiment, the rats were anaethesized using isoflorane inhalation followed by decapitation and removal of the skull cap. Fixation of the brain using bouin solution was done first in situ followed by its removal and transfer to a glass bottle containing bouin solution for 24 hours to ensure complete fixation. The prefrontal cortex and the hippocampus were obtained through parasagittal section. Specimens were processed for preparation of paraffin blocks then stained with H&E stain, Cresyl Violet stain and immunohistochemical stain for GFAP antibody.
In the control group, examination of H&E stained sections of the PFC revealed its six layers and the covering thin regular layer of pia matter with flattened spindle shaped cells. The molecular layer (I) contained scattered spindle shaped cells. Moreover, the outer granular layer (II) revealed loosely arranged granule cells together with pyramidal cells. The outer pyramidal layer (III) showed loosely packed pyramidal cells and scattered granule cells. Furthermore, the inner granular layer (IV) demonstrated densely packed granule cells together with pyramidal cells. However, the inner pyramidal layer (V) appeared with densely packed pyramidal cells together with scattered granule cells. The sections demonstrated granule cells which appeared small and rounded with vesicular nuclei. It also showed pyramidal cells with large open vesicular nuclei and prominent nucleoli together with apical dendrites. Additionally, the multiform layer (VI) revealed rounded cells with open vesicular nuclei present within the neuropil.
Sections of the hippocampus stained with H&E showed the two regions of hippocampal formation; the dentate gyrus (DG) and the hippocampus proper (Cornu Ammonis, with its four regions; CA1, CA2, CA3 and CA4). CA1 region was examined as a representative to the other parts of the hippocampus proper. The cells forming the CA1 region were arranged in three layers; the molecular layer, the pyramidal layer and the polymorphic layer. The pyramidal layer was formed of densely packed pyramidal cells with large rounded open vesicular nuclei and prominent nucleoli. The molecular layer and polymorphic layer appeared lying superficial and deep to the pyramidal layer, respectively. Cresyl Violet stained sections showed that the cytoplasm of the pyramidal cells in the PFC and CA1 region of the hippocampus were studded with Nissel’s granules. In immunohistochemically stained section, the present work showed minimal apparent immune-reaction in the cytoplasm and processes of astrocytes in both the PFC and CA1 region of the hippocampus.
Regarding ND group, signs of neuronal damage were observed in both examined brain regions (the PFC and CA1 region of the hippocampus). H&E stained sections of the middle four layers of the PFC showed darkly stained rounded granule cells. Moreover, the middle four layers of the PFC and the pyramidal layer of CA1 region of the hippocampus demonstrated also dark shrunken pyramidal cells with deeply stained nuclei. Some of these pyramidal cells were flame-like with pointed end. In addition, the current study reported pericellular spaces around some of these affected degenerated cells. The study also showed statistical significant decrease of the normal pyramidal cell number in the inner pyramidal layer of the PFC compared with the control group. However, the decrease in the number of the normal pyramidal cells in CA1 region of the hippocampus was statistically highly significant in the ND group compared with the control group. Congested blood vessels, hemorrhagic areas and perivascular spaces were also reported in the current study. The morphometric studies showed statistically highly significant decrease in the thickness of the middle four layers of the PFC and the pyramidal layer of CA1 region of the hippocampus in the ND group compared to the control one.
Regarding PFC sections, weak demarcations starting from the outer pyramidal layer till the inner pyramidal layer were observed. The pia mater covering the PFC revealed apparent irregular thickening and hypercellurality. Additionally, the molecular and the multiform layers of the PFC showed apparent hypercellularity. Furthermore, vacuolation of the neuropil in the multiform layer was seen.
Cresyl violet stained sections showed degenerated pyramidal cells with unapparent Nissel’s granules in both examined brain regions. Assessment of area percentages of GFAP in sections of the PFC and CA1 region of the hippocampus showed that there was statistically highly significant increase in the ND group compared to the control group. Sections of both mentioned brain regions revealed apparent extensive brownish immunostaining of the astrocytes with their ramifying processes.
Regarding the withdrawal group, examination of H&E stained sections of the middle four layers of the PFC (the outer granular, the outer pyramidal, the inner granular and the inner pyramidal layers ) revealed the presence of normal rounded granule cells together with abnormal dark degenerated ones. Moreover, these middle four layers of the PFC in addition to the pyramidal layer of CA1 region of the hippocampus showed the presence of normal pyramidal cells together with distorted deeply stained flame like cells with pointed end. Furthermore, this group showed absence of hemorrhagic areas together with apparent decrease in pericellular spaces. Regarding the statistical assessment in the current study, it revealed an increase in the number of surviving pyramidal cells in the withdrawal group compared with the ND group. This assessment was significant in the inner pyramidal layer of the PFC, while it was highly significant in CA1 region of the hippocampus. Furthermore, there was statistical significant decrease in the number of surviving pyramidal cells in both brain regions on comparing the withdrawal group to the control one. Assessment of thickness of the middle four layers of the PFC and the pyramidal layer of CA1 region of the hippocampus, showed statistically highly significant increase in the withdrawal group compared to the ND group. However, it revealed statistically significant decrease in the withdrawal group compared to the control group in these regions. Moreover, the pia mater covering the PFC revealed apparent decrease in thickening together with apparent decrease in hypercellurality which was also demonstrated in both the molecular (I) and the multiform (VI) layers. In addition, the multiform layer of the PFC showed apparent less vacuolations within the neuropil compared to the ND group.
Cresyl violet stained sections of the withdrawal group showed apparent increase in Nissel’s granules in the cytoplasm of some pyramidal cells together with the presence of dark cells with no apparent Nissel’s granules in the examined brain regions. Assessment of area percentages of GFAP immune-reactivity showed statistically significant decrease in sections of the PFC and highly significant decrease in CA1 region of the hippocampus in the withdrawal group compared to the ND group. However, it revealed statistically significant increase of the same parameter in the withdrawal group when compared to the control group in both examined regions.