STRUCTURAL CHANGES IN THE PREFRONTAL CORTEX OF THE …

European Journal of Molecular & Clinical Medicine

ISSN 2515-8260 Volume 7, Issue 11, 2020

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STRUCTURAL CHANGES IN THE PREFRONTAL CORTEX

OF THE ALBINO RAT IN EXPERIMENTALLY INDUCED

PARKINSON΄S DISEASE Dina AbdallaArida, Reham Ismail Taha, Shaima M. Almasry, Dalia M. Saleh, Adel A.

Bondok

Human Anatomy and Embryology Faculty of Medicine,Mansoura University, Mansoura,

35516, Egypt

Email: [email protected], [email protected],

[email protected],[email protected],[email protected]

Abstract: In Parkinson's disease models, Rotenone, a popular pesticide and mitochondrial complex I

inhibitor, causes dopaminergic neuron loss (PD). Nonetheless, the rotenone neurotoxicity

mechanisms are still poorly defined. In this study, we used rotenone to induce Parkinson's

disease and investigate its effect on medial prefrontal cortex (mPFC). Thirty rats were

divided into two groups (control and Rotenone (n=15) each).Rats in rotenone group

received a daily dose of2 mg/kg of rotenone dissolved in 1ml of sun flower oil by

subcutaneous injection for 5 weeks. Rotenone group showed bothmotor dysfunction as

evaluated byrotaroad (RRT)and open field tests (OFT)and non- motor changes

(depression) as evaluated by forced swimming test (FST). Histologically,the rotenone

group has shown layer I widening and layer II and layer IIII condensation with marked

apoptotic changes and neuropil showed decrease distribution and absence of nerve fibers

compared to control group in the mPFC.Immunohistochemically,rotenone groupshoweda

depletion of dopaminergic cells in the midbrain (MB)and dopaminergic nerve fibers

inthemPFC and aggregation of Lewy bodies (LB) in both tissue together these changes

were associatedwith significant increase in the optical density (OD) of caspase- 3 immune

expression and significant decrease of OD of GFAP immune expression in the rotenone

group compared with control group.Also, rotenone group demonstratedsignificant increase

in the lipid peroxidation and nitric oxide as well as significant reduction in the glutathione

level in the MB and mPFC comparedwith control group. In conclusion rotenone induced

PD model in rats caused behavioral, neurochemical, histological, and

immunohistochemical changes in the mPFC.

Keywords: Rotenone, Medial Prefrontal Cortex (mPFC), Parkinson's disease (PD).

INTRODUCTION:

Parkinson’s disease (PD) in the elderly population is an increasingly crippling

neurodegenerative disorder that affects about 6 million individuals worldwide (Chaudhuri et

al., 2006).The mean age of onset of symptoms is 60 to 65, but about 10 % of patients with

PD experience motor symptoms before 40 years of age (Paul, 2011). PD results from

dopamine depletion in the limbic pathways of substantianigra pars compacta (SNpc) and

ventral tegmental area (VTA), leading to motor and non-motor disease symptoms, including

cognitive and behavioral disorders (Sgambato-Faure et al., 2005; Aarsland et al., 2010;Li

et al.,2017).Anatomically, the prefrontal cortex (PFC) forms the frontal pole of the cerebral

hemisphere. It is divided into the medial prefrontal cortex (mPFC) and the orbitofrontal

cortex (oFC). The mPFC is subdivided into infralimbic cortex, prelimbic cortex, and dorsal

anterior cingulate cortex (Akkoc and Ogeturk, 2017). The mPFC is predominantly

innervated by MB VTA dopaminergic nerve fibers(Spencer et al., 2015) which is named

mailto:[email protected]







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VTA mPFC fibers. These fibers are projected mainly to layer IV (inner granule cell layer)(Li

et al., 2017).

Rotenone isan insecticide; it causes inhibition of the mitochondrial electron transport chain

and nigro-striatal degeneration. Chronic rotenone exposure can inducePD in humans (Kamel,

2013). Many studies have addressed the adverse effects of PD on the midbrain but, little is

known about the possible corresponding changes in the mPFC. After establishment of rat

model of PD, this study aimed to investigate the hypothesis that the development of PD has

an impact on the mPFC as it is innervated by dopaminergic nerve fibers from the MB VTA.

MATERIALS AND METHODS:

Experimental animals:

This research used thirty adult male albino rats weighing 200-250 grams. Male rats were used

because it had been shown that rotenone is more toxic to the females and that the mortality rate

is higher in the females than in males (Gupta,2014). Before the start of the experiment, the

rats were kept in metal cages with soft wood bedding chips and fed two weeks of standard rat

chow diet and water ad libitum for acclimatization and to insure normal growth and behavior.

The study was carried on at the research lab of faculty of medicine of Mansoura

University.The experiment was performed in compliance with the guidelines of the National

Institutes of Health (NIH) for the maintenance and use of animals from the science

laboratory; NIH Publication 1986 (86/609/EEC). The research has been accepted by the local

institutional research committee (MDP.18.11.14).

The rats weredivided into two groups:

1. Control group: (n=15) received a daily dose of 1 ml of sun flower oil for a period of five

weeks by subcutaneous injection.

2. Experimental group: (n=15) received a daily dose of 2 mg/kg of rotenone dissolved in

1ml of sun flower oil by subcutaneous injection for five weeks (Sharma&Nehru, 2013).

Rotenone was obtained in powder form from Sigma –Aldrich (Germany), while sunflower oil

obtained from El-Gomhorya Company for pharmaceuticals, Egypt. The behavioral locomotor

activity of the rats was assessed after the last injection to evaluate the success of the PD rat

model.

I- Evaluation of behavioral locomotor activity, three tests was evaluated in the animals

to assess the PD model and examine its effect on the MB and mPFC.

i- Rotarod Test (RRT): (You et al., 2019)

By testing the capacity of rats to stay on a rotating rod, the Rotarod test was used to assess

motor coordination. Three day training of the rotaroad test was performed before injection of

rotenone. The apparatus is attached to the variable-speed motor by a horizontal rough metal

rod with a diameter of 3 cm. This 70cm long rod was split by wooden partitions into four

parts.To discourage the animals from jumping from the revolving rod, the rod was positioned

at a height of 50cm. The rotation rate has been adjusted to permit the normal rats to remain

on it for 5 minutes. Five trials were given to each rat before the actual reading was taken.

With the rotation of the rod, each trial begins and finishes with the drop of the rat. The

rotational speed was steadily increased to 10 rpm over 1 minute till the rats fell off the

rotating rods. Latency until fall was detected. (A score was given to each rat as follow; score

1=fall from 1-5 sec, score 2= fall from 6-10 sec, score 3= fall from 11-20 sec, score 4= fall

from 21-30 sec, score 5= fall after 30 sec).



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ii-Spontaneous Activity in the Open Field (Shallie et al., 2017):

The open field apparatus, measuring 72 x 72 cm with walls measuring 36 cm, was made of

white plywood.There was a transparent one of the walls, so rats could be seen in the

apparatus. On the floor, blue lines were drawn with a marker and were visible through the

translucent floor. The lines break the floor into sixteen squares measuring 18 x 18 cm. In the

middle of the open field, a central square (18cm x 18cm) was drawn. Rats were put in one of

the open field's four corners and permitted the apparatus to be explored for five minutes. Rats

were returned to their home cages after the five-minute test and the open field was washed

with 70 % ethyl alcohol and allowed to dry among tests. The number of line crosses and the

frequency of rearing have been used as measures of locomotor activity. As measurements of

exploratory behavior and anxiety, the number of central square entrants and the length of

time spent in the central square were also used. Every rat was then given a score that was

calculated as the sum of line crosses and number of rears for total locomotor activity.

iii-Forced swimming test (FST):(Campos et al., 2005)

In a glass jar (40 x 25 cm) comprising 15 cm of water held at 24 ° ± 2 °C, the rats were

individually forced to swim. Total immobility duration was measured in seconds over a span

of 6 minutes. When it stopped struggling and stayed floating in the water with minimal

motion, the rat was considered to be immobile. Before being brought back to their home

cages, the rats were permitted to dry for 15 minutes in a heat enclosure (32˚C). For Two

weeks, the test was performed once a day.

Specimen Collection:

After the last injection, chloral hydrate (300 mg/kg, intraperitoneal) was used to anaesthetize

all rats. Then, half of the rats were sacrificed byintracardiac perfusion through the left

ventricle with 10%buffered neutral formalin. Midbrainwas dissected out and processed for

TH+ and α-synuclein immunohistochemistry and the mPFCwas dissected out and processed

for histological studies (cresyl and silver staining) and immunohistochemicalstudies (Anti

TH+, Anti α synuclein antibodies, Anti Caspase 3, and Anti GFAP antibodies

immunohistochemistry). The MB and prefrontal cortex of the other half of the rats were used

for neurochemical study.

II- Neurochemical Study (ELISA of MB and mPFC homogenate).

For processing of homogenates,thefresh samples of MBand prefrontal cortex wereweighted

and Transported to a glass homogenizer and homogenized following the addition of 10

phosphate buffer volumes in an ice bath (pH 7.4). At 3000rpm for 10 minutes at 4˚C, the

homogenate was centrifuged utilizing a refrigerated centrifuge.Levels of

malondialdehyde(MDA)(Leão et al., 2017), nitric oxide (Bhidwaria, &Ashwlayan,

2017)and reducedglutathione(Subramaniam and Ellis, 2013)for assessing ROS(oxidizing

activity in the MB and mPFC).

III-Histological and immunohistochemicalStudy:

Specimens of the MB and the prefrontal cortex were kept in 10% buffered neutral formalin

then processed for paraffin sections and stained with:

i- Nissl stain: for examining the histology of the mPFC(Pilati et al., 2008).

ii- Silver stains: fordetecting the nerve fibers of the mPFC (Pilati et al., 2008).

iii- Tyrosine Hydroxylase (TH) immunohistochemistery: for detecting of TH positive

cells in theMB and dopaminergic nerve fibers in mPFC respectively(Li et al., 2017). .



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iv- α-synceline immunohistochemistry: for detecting lewy bodies in the MB and the

mPFC (Acosta et al., 2015).

v- Caspase 3 immunohistocemistery: for assessing of apoptotic activity inside the

mPFC(Omara et al., 2014).

vi- GFAP immunohistochemistery: for evaluating astrocytes activaty inside the mPFC

(Liu et al., 2017).

Antibodies Company Dilution Code

TH + Abcam, Egypt, 1/200 -

α –synuclein Abcam, Egypt 1/100 -

ActivatedCaspase-3 Servicebio, China 1/500 GB11532

GFAP ABclonal, USA 1/100 A14673

Iv-Morphometric study:

Images were captured using Olympus CX41 light microscope and photographed with a

Nikon CP5000 digital camera. Image analysis was performed using ImageJ 1.51j8 program.

Images were captured with 40× and100× objective lenses. TH positive neurons were counted

manually by image J program (Huang et al., 2017).For brightness modification, ImageJ

v2.35 (NIH) has been used to identify the existence and degree of TH positivedopaminergic

nerve fibers, GFAP and caspase3 immunostaining in the DAB images.To analyze the

cytoplasmic staining, an ImageJ plug-in was operated by assigning a histogram profile to the

deconvoluted DAB image utilizing the H-DAB vector to generate three distinctly colored

images: green, brown, and blue. DAB image calibration (brown-colored) was performed by

measuring the average intensity of five non-overlapping stained tissue regions. For legitimate

contrasts utilizing this method, the uniformity of section thickness has been considered. To

prevent bias in the results, the empty areas were excluded from the measurements. We used

the following formula to convert the intensity numbers into Optical Density (OD): OD = log (𝑀𝑎𝑥𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦|𝑚𝑒𝑎𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦):

where the maximum intensity = 250; mean intensity = mean grey value (EL-Tarhounyet al.,

2014).

Statistical Analysis:

Data was tabulated, coded and analyzed utilizing version 25.0 of the computer software SPSS

(Statistical package for social science). For the statistical comparison among the two groups,

thesignificance groups of parametric data of behavioral changes, neurochemical changes and

immunohistochemical changes.P ≤ 0.05 was deemed statistically significant.

RESULTS:

I-Confirmation of Parkinson's Rat Model:

i- Behavioral tests:

-The rotaroad testrevealed a highly significant reduction in latency before the rotary rod

dropped by the rotenone rats (2.8±1.5)compared to control rats (4.6±1.1)(p<0.001).

-The open field test showed highly significant decrease in the total score measured in the

rotenone rats (11.2±6.7) compared to control rats (32.6±6.69) (p<0.001).

-The forced swimming test revealed ahighly significant increase in the duration of the

rotenone rat immobility (44.7±24.4) compared to control rats(11.8±3.3) (p<0.001) suggesting

that rotenone treated rats showing behavioral despair (depression). Histogram (1)



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ii-Neurochemical results:

Themid brain specimen showedhighly significant increased levels of MDA (2.87±1.5)and

NO (1.1±.22) and highly significant decreased levels of GSH (0.73±0.12)in the rotenone

group compared to control group), (71±.13), (1.34±.071), (1.33±0.1) p < 0.001), respectively

(Histogram 2, 3 and4)

iii-immunohistochemical results in the mid brain:

-TH immune stained sections showed highly significant decrease in number of dopaminergic

cells in rotenone treated rats (2.014±0.72)compared to control rats (14.514±1.199) (p<

0.001). Histogram (5), Fig (1)

- α synculein stained sections showed lewy bodies appeared as rounded brown stained vesicle

surround by perivascular space in rotenone treated rats however, absence of lewy bodies in

control rats.Fig(1).

II- Results of medial prefrontal cortex:

i-Neurochemical results:

The medial prefrontal cortexspecimenshowed highly significant increased levels of

MDA(2.05±.26) and NO (2.12±.25) and highly significant decreased level of GSH (.8±.071)

in rotenone treated rats compared to controlrats (1.36±.055), (.8863±.02669), (1.397±.038)

(p< 0.001).(Histogram 2.3.4)

ii-Histological results:

-Cresyl violet stainedsections:

Control rats showed normal distribution of mPFC layers which were arranged in six layers

(molecular, outer granular, pyramidal, inner granular, ganglionic and multiform,

respectively).Layer one is the molecular layer, layer two is the outer granular layer, layer

three is the pyramidal cell layer, layer four is the inner granular layer, layer five is the

ganglionic layer and layer six is the multiform layer. There are big rounded vesicular nuclei

and prominent nucleoli in the granule cells. Vesicular nuclei and long peripheral processes

are owned by the pyramidal cells. The glial cells, with dark nuclei, are small in size. There

are neuronal and glial cellular processes in the neuropil. (Fig 2 A & B)Treated rats with

Rotenone revealed a widening of layer one and a condensation of layer two and layer three.

There are shrunken pyknotic nuclei and broad pericellular space in several neurons that

appear odd, shrunken, darkly stained (Fig 2 C & D)

-Silver stainedsections:-

Control rats showed normal cells with long processes and neuropilscontaining neuronal and

glial cellular processes and less beaded blood vesselson the other hand, rotenone group

showed short processes of neuronal cells and presence of more beaded blood vessels (Fig 2 D

& E).

iii-Immunohistochemical results:

THimmune stainedsections of Control rats showed highly positive reaction in the nerve fibers

of mPFC but in the sections of the rotenone treated rats there was a mild reaction. In all

examined sections TH immune reaction was more obvious in lower three layers than upper

three layers. (Fig 3A,B,C&D)

By image analysis, rotenone treated group showed highly significant decrease (0.26±.014) in

theOD of TH immune stain compared to control group (0.52±0.11) (p < 0.001). Histogram

(5)



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-α- synuclein immune stainedsections showedpresenceof lewybodides in rotenone treated

rats moreover, there is no detection of lewy bodies in controlrats (Fig 3 E &F).

-Caspase-3immue stained sectionsofcontrol rats showed moderated positive reaction to

caspase 3 but in sections of rotenone treated rats there is highly positive reaction (Fig 4

E&F).

By image analysis,rotenone treated group showed highly significant increase in the OD of

caspase 3 immune stain(0.9±0.03) compared to control group(0.22±0.08)

(p<0.001).Histogram (6)

-GFAPimmune stained sections ofrotenone treated rats showed decreased positive reaction

of GFAP activated astrocyte compared to control group (Fig 4A,b,C&D).

By image analysis,rotenone treated group showed significant decrease (1.3±.01) in the OD of

GFAP immune stain compared to control group (1.4±.01) (p≤0.004).Histogram (6)

Histogram (1):Behavioral changes (RRT, OFT and FST).

RRT: Rotarod test, OFT: Open field test, FST:Forced swimming test, P is significant to

control, P < 0.001is highly significant P < 0.05 is significant.

Histogram (2): Tissue levels of MDA in the mid brain and the medial prefrontal cortex of

the studied group.P is significant to control, P < 0.001is highly significant P < 0.05 is

significant.



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Histogram (3): Tissue levels of NO in the mid brain and the medial prefrontal cortex of the

studied group.

P is significant to control, P < 0.001is highly significant P < 0.05 is significant.

Histogram (4): Tissue levels of GSH in the mid brain and the medial prefrontal cortex of the

studied group.


Histogram (5):Number of TH positive dopaminergic cells in the mid brain and the mean

optical density (OD)of dopaminergic nerve fibers of the studied group:




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Fig (1):Photomicrographs of the midbrain sections of adult rat: (A):Control rat shows

deeply stained positive TH+ dopaminergic neurons (arrows).(B):Rotenone treated group

shows decreased number of TH+ dopaminergic neurons (arrow). (C): Control rat illustrates

normal spindle shaped dopaminergic neuron (arrow) and absence of α-synuclein aggregated

protein. (D)Rotenone treated group shows positive α-synuclein aggregated protein (Lewy

body) (LB). (A,B: Th+ immunhistochemistryx 400; C,D: α-synuclein immunohistochemistry

x400).

Fig(2):Photomicrographs of sectionsof adult rat mPFCs: (A,B)Control rat illustrates the

organized layers of mPFC; the molecular layer (LI), the outer granular layer (LII), the

pyramidal cell layer (LIII), the inner granular layer (LIV), the ganglionic layer (LV) and the

multiform layer (LVI).There are large rounded vesicular nuclei and prominent nucleoli in the

granule cells(Dottedarrow). Pyramidal cells (black arrow)have vesicular nuclei and long

peripheral processes and nissl granule(g). Neuropil showing neuronal and ganglionic

processes (n). (C,D) Rotenone treated rat illustrates: widening of the layer I (LI) and

condensation of layer II (LII) and layer III (LIII).Several neurons containing shrunken

pyknotic nuclei and broad pericellular space are irregular, shrunken, darkly stained (black

arrow), neuropil (n) showing vacuols (v). (E) Control rat illustrates: pyramidal shaped cells

with long processes (black arrows) and neuropil (n) showing interconnecting nerve fibers. (F)



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Rotenone treated rat illustrates: degenerated nerve cells (dotted arrow) and depletion of

interconnected nerve fibers (black arrow) and increased beaded blood vessels (blue head

arrows).(A, C: CVx100; B, D: CVx400; E, F SV x400).

Fig (3):Photomicrographs of sections in in adult rat mPFC :(A,B)Control rat (A) upper three

layers illustrates: less localization of TH positive immunoreactive dopaminergic nerve

fibers,(B) lower three layers showing more localization of TH positive immunoreactive

dopaminergic nerve fibers of cerebral cortex(black arrows).(C&D) Rotenone treated rat

illustrates: decreased expression of TH positive immunoreactive dopaminergic nerve fiber in

upper and lower layers .(E)Control rat illustrates normal rounded granular cells(black arrow)

absence of α-synuclein aggregated protein (Lewy body) (LB).(F) Rotenone treated rat

illustrates: +ve α -synuclein aggregated protein (Lewy body) (LB). (A, B, C, D; TH x 400; E,

F;α-synuclein immunohistochemistryx400).

Fig(4): Photomicrographs of sections in in adult rat mPFC: (A, B)Control rat

illustrates:highly positive reaction of GFAP positive astrocytic cells reaction(black

arrow).(B) showing processes of astrocytic cells and connection in between (head

arrow).(C,D) Rotenone rat illustrates:Mild GFAP positive astrocytic cells reaction(black



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arrow). (D) showing abscnce of astrocytic processes and decreased iterconnection in between

(black arrows). (E ) Control rat illustrates: mild positive caspase 3 reaction (black arrows).

(F) Rotenone treated rat illustrates: highly positive caspase 3 reaction (black arrows). (A:

GFAP x400; B: GFAP x1000; E,F: Casp 3 immunohistochemistry x 400).

Histogram (6): The mean optical density (OD)of Caspase 3 and GFAP immunostaining of

the studied group:


DISCUSSION:

A rotenone-induced Parkinsonian model in albino rats displayed histological alternations in

mPFC neurons in the current study.The histological changes were associated with increased

OD of dopaminergic nerve fibers and CASP-3immunostaining and decreased OD of GFAP

immunostaining.

Here, we found that daily subcutenous administration of rotenone produces degenerative

lesion of dopaminergic neurons in SNpc of MB with aggregation of α-synuclein protein

(Lewy bodies),thus a PD model was established and this was associated with apoptotic and

dgenerative lesions of nerve cells and fibers with decreased activation of astrocytes in

mPFC.

In the present study there is highly significant decrease in locomotor activity that proved by

rotarod and open field test and this result was reported in previous recent studies, (Moreira et

al., 2011, Liu et al., 2015 and Wrangel et al., 2015).Owing to changes in locomotor activity

induced by central nervous system stimulants, behavioral changes and immobility can be

caused. As rotenone administration causes degeneration of dopaminergic neurons of SNpc of

MB and basal ganglia (striatum), it could lead to decreased dopamine secretion which is

responsible for inducing mobility(Tremblay et al., 2015).Moreover rotenone treated Rats

showed a significant increase in the floating time of forced swimming test that may indicate a

depressive behavior as reported by (Santiago et al., 2010 and McDowell &Chesselet, 2012).

The observed immobility in this test model signifies ‘behavioral despair which may be due to

affection of VTA-dopaminergic pathway to mPFC leading to depletion of dopamine and

affecting function of frontal cortex (personality center) that its affection causing

depression(Santiago et al., 2010 andBeaudoin-Gobert et al., 2015 ).

In the present study, Lipid peroxidation (MDA) and NOwere showing highly significant

increase and there was highly significant decrease in GSH in rotenone treated group in mPFc



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and MBin rotenone treated rats and that was agreed with (Subramaniam and Ellis, 2013,

Abdel-salam et al., 2014, González-Burgos et al.,2015, and Xiong et al., 2015).

By compromising the integrity of weak neurons, oxidative stress is thought to play a

significant role in neuronal degeneration and death of nigral cells in PD; mitochondrial

dysfunction, enhanced metabolism of dopamine which can generate excessive hydrogen

peroxide and other reactive oxygen species (ROS) and impaired antioxidant function are

potential causes of excess oxidative stress output (i.e. decreased levels of reduced

glutathione)(Jenner 2003) in the SNpc(Schapira et al., 1990) and other locations (Parker et

al., 1989 andKrige et al., 1992, Sherer et al. 2002; Moore et al. 2005).However, Swarnkar

et al., 2010showed no association among decreased GSH and increased MDA except in MB,

GSH was reduced in striatum without MDA change whereas MDA was increased in

hippocampus without GSH change and the frontal cortex stayed unaffected in terms of

oxidative stress caused by rotenone in the area (Mbiydzenyuy et al., 2018).

Histological finding of the present study revealed degeneration of cell bodies of nerve cells of

the mPFC that result was reported by Abdel-Salam, et al., (2014) and Abdel-Salam, et al.,

(2018). Silver staining methods in rodent models of acute and chronic neurodegeneration

may define damaged or degenerating neurons (Tenkova and Goldberg, 2007).Silver staining

revealed loss of nerve fibers and synapses between nervecells that was agreed with Schulz-

Schaeffer, 2010 and Sonia Angeline et al., 2012).The outcomes of the number of TH (+)

dopaminergic neurons in the MB revealed highly significant decrease in rotenone treated

group and this result was demonstrated by Galhom et al., (2017) and Liu et al., (2015)and

this is due to degeneration ofdopaminergic neurons due to inhibition of the mitochondrial

electron transport chain and nigro-striatal degeneration and decreased GSH generation as a

protective mechanism to dopaminergic cells. Due to its lipophilic nature, Rotenone promotes

the formation of alpha-synuclein fibrils and crosses both the blood-brain barrier and the cell

membrane (Allam and Schmidt, 2002).The findings of this experiment showed that TH+

nerve fibers were scattered in the control group across the cortical layers, mainly in layer IV

that is substantiated by a prior anatomical study (Liu et al., 2015) that there was a decline in

OD of TH+ nerve fibers compared to the rotenone group, suggesting a decline in TH+ nerve

fibers. This finding showed that VTA damage caused by rotenone injection could trigger

anterograde nerve damage to the projected fibers of the nerve. This result was supported by

earlier studies by Sweet et al. (2014), which found that damage to SNpc and VTA in a broad

range of brain regions was correlated with a reduction in nerve fibers.

Compared with the control group in the present research, the OD of caspase 3 activity

increased in the mPFC of the rotenone treated group and apoptotic cells were increased and

scattered throughout the cortical layers and that was agreed with (Norazit et al., 2010 and

Sonia Angeline et al., 2012).Other studies have shown that the activity of caspase-3 in the

PD brain (Mogi et al. 2000) and levels (Tatton 2000; Jiang et al. 2012) in the SN and

temporal cortex have increased. Hartmann et al. (2000) found, in comparison to these

findings, a substantial decline in caspase-3-positive pigmented neurons in PD patients' SNpc

relative to controls, which may be due to changes in the distribution of caspase 3 within the

dopaminergic neurons in SNpc, as it is increased in dorsal part than ventromedial

part.Accumulation of α-synuclein in dopaminergic neurons results in decreased

mitochondrial complex I activity and increased generation of reactive oxygen species, as well

as degradation of dopamine products undergoing autoxidation, leading to increased

generation of reactive oxygen species that may reflect a potential significant mechanism

contributing to dopaminergic neuronal death via apoptosis (Erekat,2018).



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The present study have showed that significant decrease in the OD of GFAP

immunohistochemistry of the mPFC in rotenone treated group compared with control group

and this result was detected by (Abdel-Salam et al., 2018),van den Berge et al., 2012 and

Tong et al., 2015) due to accumulation of α syncline in astrocytes causing degeneration of

astrocytes (Halliday and Stevens 2011)and decreased GFAP activity. However, otherstudies

were showed that astrocytes activityincreasedand increased GFAP expression in mid brain

(Abdel-Salam et al., 2018) and striatum (Ojha et al., 2016). Nonetheless, many findings

suggest that an inflammatory response in the Parkinsonian brain is ongoing (Orr et al.,

2005).Halliday and peers(Halliday and Stevens, 2011) speculate that α-synuclein

accumulation in astrocytes is the primary etiopathological factor in PD and other

synucleinopathies, could damage SN astrocytes and thus promote dopamine neuron

degeneration. Overexpression of α-synuclein in astrocyte culture induced glial cell

death(Stefanova et al., 2001).

CONCLUSION:

Rotenone induced a rat model of PD proofed by behavioral changes, neurochemical changes,

decreased number of TH+ dopaminergic neurons and presence of lewy bodies in mid brain

followed by studying of structural and neurochemical changes in mPFC which showed

increase in MDA and NO and decrease in GSH. Histopathological study showed

degeneration of neuronal cells bodies and disconnection of nerve fibers synapses. Caspese- 3

showed increased activity while GFAP showed decreased activity and this is the first study to

collecting data about changes in mPFC in amodel of PD induced by rotenone subcutaneous

injection.

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