Effects of Paracetamol and Monosodium Glutamate on ...medicaljournalofcairouniversity.net/Home/images/pdf/2014/march/73.pdf · that Cytochrome P450 is also expressed in the brain

Med. J. Cairo Univ., Vol. 82, No. 2, March: 289-302, 2014

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Effects of Paracetamol and Monosodium Glutamate on Cerebellar

Granule Cells of the Adult Male Albino Rats: A Histological and

Morphometric Study

MOHAMED H. MOHAMED, M.D., HAZEM A. MOHAMED, M.D. and ASHRAF H. ABD EL-HAKEM, M.D.

The Department of Anatomy & Human Embryology, Faculty of Medicine, Assiut University

Abstract

Background: Paracetamol (Acetaminophen, PAM) and

monosodium glutamate (MSG) are widely used nowadays.

PAM is used to treat mild pain and fever in most countries of

the world. While MSG is ubiquitous in nature and is present in all living organisms. MSG is a principal excitatory neu-rotransmitter mainly in the central nervous system, and is used as a food additive that is commonly marketed as a flavor

enhancer.

Aim of the Study: The main objective in this study was to determine the effect of chronic simultaneous administration

of PAM with MSG on the cerebellar granule cells (CGc) of

adult male albino rats. This would be monitored through quantitative and qualitative studies on a control group and

experimental groups.

Material and Methods: In the current study, a total number

of twenty three adult male albino rats were used. The animals

were divided into four groups: A control group (G1) and three

experimental groups. The experimental groups included: An

experimental group that was given PAM and MSG (G2), an

experimental group that was given MSG only (G3) and an

experimental group that was given PAM only (G4). A quali-tative and quantitative methods were used to evaluate the

results of the current study.

Results: In the current study, the histological examination by light and electron microscope of G2 and G3 revealed that,

beside of the presence of some normal CGc, some CGc were

lost and others appear pyknotic and dark. These pyknotic and dark CGc appeared shrunken with irregular outline and in-creased staining intensity, dilated perinuclear cisterna and cytoplasmic vacuolation. However, the changes were more pronounced in G2 than G3. Quantitative parameters confirmed

the previous mentioned morphological changes that were

observed by the light and electron microscope examination.

Cerebellar weight showed a highly significant decrease in G2, a significant decrease in G3 and none significant decrease

in G4 as compared with that of G1. For the CGc nuclear diameter (ND) and numerical density (NV), a highly significant

Correspondence to: Dr. Mohamed H. Mohamed, The Department of Anatomy & Human Embryology, Faculty of Medicine, Assiut University

decrease in G2, a significant decrease in G3 were noticed as compared with that of G1. On the other hand, none significant

change in the ND nor NV of G4 was noticed as compared to

that of G1. Regarding CGc axonal length (AL) of the control

and the experimental animals, there was a highly significant

decrease in G2, a significant decrease in G3 as compared with

that of G1. A non-significant change of AL was noticed in G4 as compared to that of G1.

Conclusions and Significance: The results of the present study found out a direct potentiating action of Paracetamol

for increasing the neurotoxicity of Monosodium Glutamate

on the cerebellar granule cells. Therefore, it is important that Paracetamol should not be used chronically with Monosodium Glutamate.

Key Words: Paracetamol – Monosodium glutamate and cer-ebellar granule cells.

Introduction

THE anatomy of the cerebellar cortex could be

described as a two layered network; the input and

output layers. The input layer-involves the granule cells-which processes the incoming mossy fiber signals and transmits them via the parallel fiber system to the output layer (mainly Purkinje neu-rons). They considered that cerebellar granule cells (CGc) are the masters of Purkinje cells and CGc

in turn is mastered by Golgi neurons [1] .

CGc account for more than half of all neurons in the CNS of vertebrates. Approximately 85% of

the cerebellar granule cells are generated postnatally

mostly during the first year in human. Thus the

human cerebellum has a much higher functional

plasticity during the first year of life than previously thought, and may respond very sensitively to in-ternal and external influences during this time [2] . Affection of CGc has important implications for several neuropsychiatric conditions in which cer-ebellar involvement has been demonstrated [3,4] .

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290 Effects of Paracetamol & Monosodium Glutamate

The CGc was reported to be one of the few

regions of the mammalian brain where neurogenesis

continue to occur throughout adulthood. The neu-rogenesis in the CGc was thought to play an im-portant role in memory [5] . Theoretical work has suggested that the abundance of CGc is advanta-geous for sparse coding during memory formation.

Findings indicate that a minority of functionally

intact CGc is sufficient for the maintenance of basic motor performance, whereas acquisition and stabilization of sophisticated memories require higher numbers of normal CGc [6,7] .

Paracetamol (acetaminophen; PAM) is consid-ered a non-steroidal anti-inflammatory drug, even

though in clinical practice and in animal models

it shows little anti-inflammatory activity [8] . How-ever, like NSAIDs, PAM is used to treat pain and

fever and it has become one of the most popular

“over-the-counter” non-narcotic analgesic agents.

For instance, this compound has been taken, at

least once, by more than 85% of children in the UK [9] . In the US, about 79% of the general pop-ulation regularly takes PAM, including more than 35% of pregnant women [10] .

The most frequently reported adverse effect

associated with PAM is hepatotoxicity, which

occurs after acute over dosage [11] and, less fre-quently, during long term treatment with doses at

the higher levels of the therapeutic range or in the

presence of precipitating factors like fasting, nu-tritional impairment or alcohol intake [12,13] . Be-sides hepatic toxicity, no PAM toxic actions have been described in the nervous system, although it is well known that PAM crosses the blood-brain barrier both in rodents and humans [14] .

PAM is mainly metabolised in the liver but, a small fraction is metabolized by cytochrome P-450 [15,16] forming a chemically reactive metabo-lite, n-acetyl-p-benzoquinone imine (NAPQI),

which reacts with glutathione (GSH) to form a non-toxic conjugate that will be excreted. Once GSH is exhausted, NAPQI binds to cellular pro-teins, including mitochondrial proteins, leading to

cellular death [17,18] . It has also been described that Cytochrome P450 is also expressed in the brain [19] , suggesting that PAM might be metabo-lised by neurons producing the toxic metabolite

that lead to neurotoxicity. Although there is a previous report indicating that PAM-mediated toxicity in neuroblastoma, information on direct

PAM neurotoxicity has not been described [20] .

Monosodium Glutamate (MSG) is a crystalline sodium salt of glutamic acid used in cooking pri-

marily to enhance the flavor of food or as a preser-vative. MSG is absorbed very quickly into the blood stream as compared to glutamic acid. MSG

is recognized as a standard of identity ingredient

in several commercial food preparations. It is

principally used in the preparation of canned and

dried soups; some meat, fish products, vegetables and fowl [21]

MSG has neurotoxic effect leading to degener-ative changes in neurons and astrocytes in cerebellar

cortex of albino rats [22] . They found that by his-tological examination of MSG treated group, de-generative changes as pyknotic Purkinje and gran-ule cells with areas of degeneration surrounded by

inflammatory cells in the granular cell layer

Martinez-Contreras et al., [23] observed that neonatal administration of a MSG induces reactivity of the astrocytes and microglia cells in the fronto-parietal axis cortex which is characterized by

hyperplasia and hypertrophy. Also previous studies

have shown that neonatal administration of a dosage

of MSG to the newborn rats caused toxic damage

of the glial cells of the adult brain of Wistar rat

[24] . According to Ekpo and Jimmy [25] who studied the adverse effect of MSG on the hypothalamic

lesion which corresponds to human experience on

severe headache of the users of monosodium salt. A study of experiment showed that whereas expo-sure to MSG caused mature but not young, brain cells to die, the action was dependent on a lack of

calcium [26] .

Aim of the study:

The principal aim is to determine the effect of chronic simultaneous administration of paracetamol

(acetaminophen) and monosodium glutamate on cerebellar granule cells of the adult male rats. This was monitored by quantitative and qualitative evaluations on control and experimental animals.

Material and Method

I- Scope, place and time of study:

Scope of study included histological and mor-phometric analysis and the study was conducted

between June July 2013 in Assiut University.

II- Study design:

The study was experimental design post-test

only one control group. The samples were kept for 1 week for adaptation before they randomly divided into 1 control and three experimental groups.

Mohamed H. Mohamed, et al. 291

III- Inclusion and exclusion criteria: For inclusion criteria, the rats were albino rats,

males, of age 3 months for all rats at the beginning of the study and weighted 200-250gm.

For exclusion criteria, the rats died before the

end of the experiment and showed violent activities were excluded.

IV- Animals: A total number of twenty three male albino rats

(average weight 200-250gm.) were randomly used

in this study. The animals were isolated in clean

properly ventilated cages in the Animal House of

Assiut University under normal conditions with

an appropriate temperature, normal light and dark

cycle and free access to food and water. Each cage

contained three rats. All animals of the four groups were at age of 3 months at beginning of the exper-iment. They were kept for 1 week adaptation before

intervention. They were exposed for a period of 6 weeks of intervention [27] .

V- Animal grouping: The animals were divided into four groups (one

control and three experimental):

Group1 or Control (G1): This group included five male rats. The rats of this group received

distilled water.

Experimental groups: These were as follows:

Group 2 (G2): This group includes six male rats that were received PAM and MSG. PAM was

given in distilled water via Lavage tube. MSG was given dissolved in distilled water.

Group 3 (G3): This group includes 6 male rats

that were received MSG distilled water.

Group 4 (G4): This group includes 6 male rats

that were received PAM in distilled water via

Lavage tube.

VI- Drug dosage: - The dose of PAM was given as 100mg/kg body

weight dissolved in 1 ml of warm distilled water. - The dose of MSG was given as 3mg/kg body

weight in 1ml of distilled water. The dose for both was administered daily for sex weeks. The

dose was calculated according to Barnes and Elthertington [28] and methodology according to Al-Agha [29] .

VII- Study variables:

Independent variables included the various doses of MSG and PAM. The dependent variables included the cerebellar vermal tissues.

VIII- Experimental analysis: Two methods of analysis were used in the

present study: A- Histological examination; in which the pu-

tative effects of the administration of PAM and

MSG on CGc were clarified through studying the

morphological changes in CGc and layering pattern of the cerebellar cortex. This was attained by

examination of the sections under light and electron

microscope though: - Heamatoxylin and eosin staining. - Toluidine blue staining for semithin sections. - Golgi impregnated sections. - Ultrathin sections by transmission electron micr-

oscope.

B- Quantitative analysis; in which the putative

effects of the administration of PAM and MSG on CGc were clarified through studying the numerical

differences in CGc between the control and exper-imental groups. This was attained by measuring the following parameters: - Cerebellar weight (CW). - Nuclear diameter of CGc (ND).

- Numerical density of CGc (NV).

- Axonal length of CGc (AL).

IX- Experimental methodology:

Each animal of the control and treated groups

was anaesthetized with ether, its heart was exposed,

and saline solution was perfused through the left

ventricle until the coming out fluid ,from the right

atrium after being opened, was blood-free. Then Bouin’s fixative was done for light microscopy, and with 4% cold gluteraldhyde (at 4ºC) in a buff-ered cacodylate solution pH 7.4 for electron mi-croscopy. The cranial cavity was opened; the brain

was carefully dissected out and left immersed in the fixative and undisturbed for one hour. Then,

the middle 1 /3 of the cerebellum or the ve rmal area was sectioned.

For light microscopy, paraffin sections (5-7µm) of tissue specimens were prepared and stained with

Harris haematoxylin and eosin according to Drury and Wallington [30] . For semithin sections (0.5-lµm) of the specimens fixed in 4% gluteraldhyde (at 4ºC)

were stained with toluidine blue Gupta [31] , and were examined with light microscope. Subsequently,

thin sections (0.05-0.08 µm) were obtained for the selected areas in semithin sections, contrasted with

uranyl acetate and lead citrate Reynolds [32] , and studied with the transmission electron microscope,

JEOL (J.E.M.- 100 CXI 1) and photographed at 80

K.V. in Assiut University Electron Microscope Unit.

Nv = N / a_____ D - + t


For Golgi stained sections, a modified Golgi-Kopsch technique for impregnation the neural tissue according to Riley [33] was used. The cere-bellum from each animal groups was cut into slices.

The slices were placed in 4:1 mixture of 5% potas-sium dichromate and concentrated formaldehyde

(40%) for 4 days. The slices were transferred to 3.6% potassium dichromate for 4 days. Then they were washed in 0.75 sliver nitrate and then placed

in the last solution for 4 days. The last two steps were repeated twice. The slices were dehydrated

then placed in xylene. Embedding process was

made in paraffin wax. Serial sections were made

at 40 µm. The sections were de-waxed by using

xylene. The sections were mounted in Canada

balsam.

For estimation of CGc diameter and numerical density, a number of non-overlapping diagrams

were made by Camera Lucida (Leitz Wetzlar, Ger-many) using a Leitz light research microscope. These diagrams were drawn for stereological pro-cedures. A digitizing set consisted of Digitizer KD 3040 B connected to IBM compatible personal computer, was used with a specially prepared

program to measure lengths. The major diameter (a), which is the widest diameter and narrowest diameter (b), which is the widest diameter perpen-dicular over (a). The diameter of equivalent circle

(D -) was calculated (D - = ab). Schwartz-Saltikov correction procedure was applied to obtain more

reliable estimates of the true mean nuclear diameter (D -).The numerical density (Nv) of the CGc per

unit volume of the cerebellar cortex was calculated

as follow: Where, N: The number of calculated

cells, A: The area in which the number of calculated cells were measured, D - : The corrected mean diameter of the nucleus and T: The tissue thickness.

For estimation of the length of CGc axons, Golgi stained preparations were used. With the aid of Camera Lucida (Leitz Wetzlar, Germany) which

was fitted to a research light microscope. A suitable

sized white sheet was fitted under the field of the Camera Lucida. Under the field of Camera Lucida, a complete granule neuron was projected on the paper. The projection was done by using a suitable uniball pencil (ub- 103). The process was repeated

for at least 10 granule neurons. Tracing of complete

neuron, necessitated changing the focus of the

microscope at different segments of the neuron to trace its whole parts. At the same setting of the microscope and Camera Lucida, the image of an

objective stage micrometer was projected on the

same white sheet and the final magnification was

calculated. The length of axons was measured from

the white sheet. Using the value of the final mag-nification the true length of the apical dendrites

was estimated.

X- Statistics:

The cerebellar weight and other parameters

were performed to each animal, then later were

pooled to estimate the mean±SD. The data were

analyzed using the computerized statistical package

'SPSS Version 17. Student t-test was used to show any statistically significant difference in absolute

neuronal count between the control and treatment

groups.

Results

G1 (Control group):

The haematoxylin and eosin (H&E) stained

sections of the cerebellar cortex of G 1 showed that

the cerebellar cortex consisted of three layers, an

outer molecular layer, a middle Purkinje cell layer,

and an inner granule cell layer. In the granule cell layer, cerebellar granule cells (CGc) were densely

packed with rounded or oval shaped nuclei (Plate 1, Fig. 1).

The semithin sections stained with toluidine blue (TB) showed that the granule cell layer was

having dense packing of the granule bodies with little intervening tissue. The CGc assumed oval shape. Their nuclei appeared pale, rounded or ovoid with single dense peripheral nucleoli and finely dispersed chromatin. Nuclei were surrounded

by thin rim of pale staining cytoplasm (Plate 2, Fig. 1).

In Golgi impregnated sections, the CGc bodies

were seen into a thick layer at the bottom of the

cerebellar cortex. A granule cell emitted only three

to four dendrites. The axons of CGc directed ver-tically to the upper (molecular) layer of the cortex, where they split into two, with each branch travel-ing horizontally to form a parallel fiber; the splitting

of the vertical branch into two horizontal branches

giving rise to a distinctive “T” shape (Plate 3, Fig. 1 -A, B).

By electron microscope, the granule cell layer

showed the characteristic closely packing of cell

bodies of CGc with little intervening tissue. The

CGc cell bodies appeared nearly oval in shape. Their nuclei were oval to round in shape with

uniformly dispersed chromatin and single electron

dense nucleoli. The cytoplasm formed a thin shell around the nucleus with mitochondria (Plate 4, Fig. 1).


G2 (PAM and MSG given group):

The H&E stained sections of the cerebellar

cortex of G2 showed an obvious loss of CGc. CGc.

cell bodies appeared widely separated, assumed irregular profiles with irregular outline and showed an increase staining intensity. Their nuclei were irregular in shape and deeply stained with no visible nucleoli (Plate 1, Fig. 2).

The TB stained sections of the cerebellar cortex

show many of the granule cells are shrunken. The

nuclei of these neurons appear darkly stained with hardly visible deeply stained nucleoli and surround-ed by thin rim of stained cytoplasm, which shows small vacuoles. Other granule cell bodies show

pale stained, clear, vacuolated cytoplasm and their

nuclei relatively normal in shape and staining

intensity (Plate 2, Fig. 2).

In Golgi impregnated sections, appearance of

damaged CGc was observed. CGc were character-ized by dendritic atrophy. Some CGc were appar-ently having distorted shape. CGc dendrites were mostly lost. Only few CGc dendrites were still seen with fine architecture. The axons of CGc were

still running vertically, but with very shorter course and could not be traced in to the molecular layer. The splitting of the vertical branch into two hori-zontal branches that gives rise to a distinctive “T”

shape was not seen in many CGc (Plate 3, Fig. 2).

By electron microscope, Many CGc showed

increased electron density, shrinkage and irregu-larity. Their cytoplasm showed distorted mitochon-dria which appeared swollen. Heterogenous lipo-fuscin pigment bodies were seen in their cytoplasm.

Organelle-free areas were observed in the cyto-plasm. Some degenerated CGc with irregularities of their outlines were noticed. Their nuclei appeared

homogenous with no distinct chromatin and their

scanty cytoplasm appeared with ill-defined or-ganelles (Plate 4, Fig. 2).

G3 (MSG given group): The H&E stained sections of the cerebellar

cortex of G3 group showed a similar histological

features of G2 but the changes that were observed

here were lesser. So, there was some loss of granular cells. The cell bodies of these neurons was having irregular with irregular outline. Their nuclei were

irregular in shape and deeply stained with no visible nucleoli. (Plate 1, Fig. 3).

The TB stained sections of the cerebellar cortex

show similarly a similar histological features of G2 but the changes that were observed here were lesser. Therefore, CGc was having different staining

intensity; many of them appeared darkly stained,

shrunken and irregular in outlines with ill-defined

nuclei. Other CGc cell bodies showed pale stained,

clear, vacuolated cytoplasm (Plate 2, Fig. 3).

In Golgi impregnated sections, the appearance

of CGc showed some histological features that

seen in G2. CGc dendrites were mostly lost. Only few CGc dendrites are still seen. The axons of

CGc were still seen running vertically to the mo-lecular layer of the cortex, but with short course

and thin caliber. Similarly the splitting of the vertical branch into two horizontal branches that

gives rise to a distinctive “T” shape was not seen

(Plate 3, Fig. 3).

By electron microscope, the granular cells of

the cerebellar cortex showed similar histological

features of G2 to a lesser degree. Some CGc showed

increased electron density, shrinkage and irregu-larity of outlines. Many similar ultrastructural

changes that were observed in G2, are also observed

in this group. However these changes was lesser than that of G2 (Plate 4, Fig. 3).

G4 (PAM given group):

The H&E stained sections of the cerebellar

cortex of G4 showed some histological features of G1 and G3. Therefore, the histological features of G3 were manifested by some loss of CGc. The cell

bodies of these neurons were having irregular

outline and showed increase staining intensity.

Their nuclei were irregular in shape and deeply stained with no visible nucleoli. On the other hand, the histological features of G 1 were manifested by

the presence of some CGc which assumed oval

shape. Their nuclei fairly appeared rounded or

ovoid (Plate 1, Fig. 4).

Also, by the TB staining, cerebellar cortex of G4 showed some histological features of G1 and G3. So, the histological features of G3 were man-ifested by the presence of some CGc which have cell bodies with irregular outline and increased staining intensity. Their nuclei are irregular in shape and deeply stained with no visible nucleoli.

On the other hand, the histological features and presentation resembling that of G 1 were manifested

by the presence of some granule cells which assume

polyhedral shape. Their nuclei fairly appear rounded

or ovoid (Plate 2, Fig. 4).

Similarly, in Golgi impregnated sections, some of CGc were similar to that seen in G1. However

the dendrites architecture and axons could not been

traced as that of G1. Similarly the axons of CGc directed vertically to the molecular layer of the

cortex, but the splitting of the vertical branch into

two horizontal branches gives rise to a distinctive


“T” shape could not be traced in some neurons (Plate 3, Fig. 4).

By electron microscope, the CGc show some histological features similar to that seen in G 1 and

G3. Therefore, the histological features of G1 were manifested by the presence of some CGc which

were having oval rounded nuclei with finely dis-persed chromatin with clumps of peripherally

dispersed chromatin and distinct cell membrane.

Other CGc showed shrinkage and increased electron

density (Plate 4, Fig. 4).

Quantitative analysis: Comparing the CW of the control and the ex-

perimental animals, Tables (1-4) and Bar charts 1, 2, 3 and 4 showed a highly significant statistical decrease in the CW of G2 (where the t=25.922 and the p-value of t test was <0.0 1) as compared with that of G1. There was a significant statistical

decrease in the CW of G3 (where the t=2.840 and the p-value of t test was = 0.019) as compared with that of G1. On the other hand, there was no signif-icant statistical decrease in the CW of G4 as com-pared with that of G1 (where the t=1.834 and the p-value of t-test was = 0. 1).

On comparing the control and the experimental

ND, there was a highly significant statistical de-crease in G2 (where the t=49.494 and the p-value of t-test was <0.01) as compared with that of G1.

Table (1): Mean±standard deviations of means of CW (in mg)

in the control and experimental groups.

Groups and number of animals

Parameter per each group (n)

There was a significant statistical decrease in the

ND of G3 (where the t=2.427 and the p-value of t-test was = 0.038) as compared with that of G1.

However, the decrease of ND in G4 did not show

any statistical significance as compared to that of

G1 (where the t=0.523 and the p-value of t-test was = 0.613).

On comparing the control and the experimental

NV, there was a highly significant statistical de-crease in G2 (where the t=65.322 and the p-value of t-test was <0.01) as compared with that of G1.

There was a significant statistical decrease in the

NV of G3 (where the t=2.694 and the p-value of t-test was = 0.025) as compared with that of G1.

However, the decrease of NV in G4 failed to show any statistical significance as compared to that of

G 1 (where the t=0.45 7 and the p-value of t-test was = 0.658).

Regarding AL of the control and the experi-mental animals, there was a highly significant statistical decrease being noticed in G2 (where the

t=14.062 and the p-value of t-test was <0.01) when compared with that of G1. There was statistical significant decrease in G3 (where the t=2.876 and the p-value of t-test was = 0.0 18) as compared

with that of G1. Whereas, no significant statistical difference was noticed in G4 as compared with

that of G1 (where the t=1.248 and the p-value of t-test was = 0.244).

Table (3): Mean±standard deviations of means of NV (/mm 3) in the control and experimental groups.

Groups and number of animals

Parameter per each group (n)

G1 n=5 G2 n=6 G3 n=6 G4 n=6 G1 n=5 G2 n=6 G3 n=6 G4 n=6

CW 351±2.5 315±1.7** 334±11.9 * 341±10.8 NV 2.61x103±

0.05 1.23x103±

0.01 ** 2.35x103

±0.19* 2.51x103

±0.44

CW : (Cerebellar weight). G3 : (Received MSG).

G1

: (Control group). G4 : (Received PAM). G2

: (Received PAM and MSG). * : Significant statistical difference, where p<0.05. ** : Highly significant statistical difference, where p<0.01.

Table (2): Mean±standard deviations of means of ND (in µm)


Groups and number of animals Parameter per each group (n)

NV

: (Numerical density) of CGc. G3 : (Received MSG). G1

: (Control group). G4: (Received PAM). G2

: (Received PAM and MSG). * : Significant statistical difference, where p<0.05. ** : Highly significant statistical difference, where p<0.01.

Table (4): Mean±standard deviations of means of AL (in µm)


Groups and number of animals Parameter per each group (n)

G1 n=5 G2 n=6 G3 n=6 G4 n=6 G1 n=5 G2 n=6 G3 n=6 G4 n=6

ND 5.43±0.11 3.01±0.01 ** 5.27±0.09* 5.37±0.21

ND : (Nuclear diameter) of CGc. G3 : (Received MSG).

G1


: (Received PAM and MSG). *

: Significant statistical difference, where p<0.05 and ** : Highly significant statistical difference, where p<0.01.

AL 144.8±1.7 123±2** 131.4±9.3 * 140.2±7.3

AL : (Axonal length) of CGc. G3 : (Received MSG).

G1


: (Received PAM and MSG).

*

: Significant statistical difference, where p<0.05. ** : Highly significant statistical difference, where p<0.01.


Plate (1):

Fig. (1): A section in the cerebellar cortex of G1 stained with H&E showing the three layers; M (molecular), P (Purkinje) and G (granular). Nuclei of CGc (N) and dense clumps of chromatin (arrows) (x1 000).

Fig. (2): A section in the cerebellar cortex of G2 stained with H&E showing; part of Pukinge layer (P), the granular layer (G) containing

widely separated CGc with areas of cell loss (white stars) and increased

staining intensity with invisible nucleoli (horizontal arrows) (x1000).

Fig. (3): A section in the cerebellar cortex of G3 stained with H&E showing; part of molecular layer (M), part of Pukinge layer (P) and the granular layer (G) containing CGc with shrunken irregular cellular outlines

(vertical arrows) and increased staining intensity with fairly visible nucleoli

(horizontal arrows) (x1 000).

Fig. (1): A section in the cerebellar cortex of G1 stained with toluidine

blue (TB) showing; the granular layer containing nuclei of CGc (N), thin

rim of cytoplasm (vertical arrows) and dense clumps of chromatin (hori-zontal arrows) (x1000).

Fig. (4): A section in the cerebellar cortex of G4 stained with H&E showing; similar presentations of Fig. (9) but with lesser changes; part of

molecular layer (M), part of Pukinge layer (P) and the granular layer (G)

containing CGc with shrunken irregular outline (vertical arrows) and

increased staining intensity with fairly visible nucleoli (horizontal arrows)

(x1000).

Fig. (2): A section in the cerebellar cortex of G2 stained with TB

showing; the granular layer containing CGc with increased staining

intensity with invisible nucleoli (white vertical arrows). Some CGc with

irregular shape (yellow vertical arrows) and others have pale stained

irregular shape (horizontal arrows). Few normal CGc (N) (x1000).

Plate (2):


showing; CGc with increased staining intensity with invisible nucleoli (white horizontal arrows). Some CGc with irregular shape (yellow vertical arrows) and vacuolated areas (V). Many normal CGc (N) (x1000).


showing; part of molecular layer (M), part of Pukinge layer (P) and the granular layer (G) containing CGc with irregular outline (yellow horizontal arrows) and increased staining intensity (horizontal white arrows) and

vacuolated areas (V) (x1000).

Fig. (4): A section in the cerebellar cortex of G4 impregnated with Golgi stain showing; more or less some fertures of normal neuron; CGc with three dendrites (vertical arrows). The

perikaryon is visible clearly (PK). The axon rises vertically to the molecular and can be traced up (horizontal arrows) (x1000).


Plate (3):

Fig. (1 -A): A section in the cerebellar cortex of G 1 impregnated

with Golgi stain showing; normal CGc with three dendrites (vertical arrows). The perikaryon is visible

(PK). The axon rises vertically to the molecular layer and can be traced up (horizontal arrows)

(x1000).

Fig. (1-B): Another section in the cerebellar cortex of G1 impregnated with Golgi stain showing; the axon

rises vertically to the molecular layer (horizontal arrows) and its splitting into tow horizontal branches forming the distinctive T shape or parallel

fibers (horizontal arrows) (x1000).

Fig. (2): A section in the cerebellar cortex of G2 impregnated

with Golgi stain showing; damaged CGc with dendritic atrophy (vertical arrows). The cell bodies are distorted (PK). The axons very shorter course

and could not be traced up (horizontal arrows) (x1000).

Fig. (3): A section in the cerebellar cortex of G3 impregnated with Golgi stain showing; damaged CGc with dendritic atrophy (vertical arrows). The cell bodies are distorted (PK). The axons very shorter course

and could not be traced up (horizontal arrows)

(x1000).

Plate (4):


Fig. (1): An electron micrograph of a section in cerebellar cortex of G1 showing; a part of the granule cell layer

in which the granule cells have pale oval rounded nuclei with finely dispersed chromatin (N), clumps of peripherally dispersed chromatin (horizontal white arrows), cytoplasmic membrane (vertical yellow arrows) and few mitochondria (M) (x5000).

Fig. (2): An electron micrograph of a section in cerebellar

cortex of G2 showing; CGc with increased electron density, shrunken and irregular (N) and distorted

mitochondria (M). A degenerated neuron can be seen

(horizontal arrow). Some lipofuscin pigments can

be seen (vertical yellow arrows) and vacuolated areas (V) (x5000).

Fig. (3): An electron micrograph of a section in cerebellar cortex of G3 showing similar presentations of fig.8

but with lesser changes; CGc with increased electron density, shrunken and irregular (horizontal arrows),

distorted mitochondria (M) and vacuolated areas

(V). Some normal CGc could be seen (N) (x5000).

CW

360

350

340

330

320

310

300

290 G1 G2 G3 G4

Bar Chart (1): The means of cerebellar weight (in mg) in the

control and experimental groups.

Fig. (4): An electron micrograph of a section in cerebellar

cortex of G3 showing more or less some histological

features of normal neurons; CGc with oval rounded

nuclei with finely dispersed chromatin (N), clumps of peripherally dispersed chromatin (horizontal white arrows) and distinct cell membrane (curved white

line) (x5000).

ND

6

5

4

3

2

1

0 G1 G2 G3 G4

Bar Chart (2): The means of CGc nuclear diameter (in µm)


G1 G2 G3 G4

3

2.5

2

1.5

1

0.5

0 G1 G2 G3 G4

150

145

140

135

130

125

120

115

110


NV

Bar Chart (3): The means of CGc numerical densities (/mm 3)


AL

Bar Chart (4): The means of CGc axonal length (in µm) in

the control and experimental groups.

Discussion

The choice of CGc may be attributed to the cellular pattern of cerebellum which suggested that the anatomy of the cerebellar cortex could be

described as a two layered network; the input and output layers. The input layer involves the granule

cells which processes the incoming mossy fiber

signals and transmits them via the parallel fiber system to the output layer (mainly Purkinje neurons). In both layers, activity is controlled by

inhibitory neurons. The Golgi cells is inhibitory for the input layer, and basket and stellate cells of

the output layer. It could be pointed out that

cerebellar granule and Golgi cells are considered

as one functional unit [1] . They considered that CGc are the masters of Purkinje cells.

CGc characteristically have a late maturation

so, these cells are more vulnerable to the exposure

of any stressful stimuli. Also the particular functional connectivity of CGc is another reason.

In this regard, CGc are excitatory neurons that use

glutamate neurotransmitter while, Purkinje cells are inhibitory neurons using gamma aminobutyric acid. So, any morphological changes, even minor, could reflect precisely the putative effects [34] .

The choice of the cerebellar vermis for study was performed according to Bedi and Warren [35] . The determination of crus I and II in the cerebellar vermis was done according to Larsell and Jansen

[36].Crus I and II were chosen since several studies

have reported that the most posterior lobules are known to develop before the more anterior ones

[37].According to Nguyen et al., [2] about 85% of the cerebellar granule cells are generated postnatally

mostly during the first year in human. So, the anterior crura (crus I and II) may respond very

sensitively to internal and external influences.

The use of Golgi impregnation in the present study was for clarifying the axonal and dendritic architecture of CGc qualitative changes that were

expected. Also, the use of Golgi impregnation technique was permitted to study the quantitative changes. A modified Golgi-Kopsch technique for

impregnation the neural tissue according to Riley [33] since it permits a rapid impressive technique.

Male albino rats were used to avoid the female hormonal effect. That was supported by previous

investigator who suggested that estrogen enhanced

cell proliferation during proestrus resulted in more

immature neurons in the cerebellum of females compared with males and present the possibility that these new cells exert an important influence

on CGc since the cerebellum is one of the few

brain structures currently known to have high rates

of neurogenesis in adult rats [38] .

In the present study, PAM was chosen since it

is one of the commonest used drug all over the

world as a mild pain reliever and antithermic [39] . MSG was chosen since it is one of the most abun-dant naturally occurring subjects. MSG is an amino acid readily utilized by glutamate receptors throughout the mammalian body. These glutamate

receptors are present in the central nervous system

as the major mediators of excitatory neuro-transmission. Neural injury associated with, stroke, epilepsy, and many neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s

diseases and amyotrophic lateral sclerosis may be mediated by excessive activation of the glutamate

receptors. Neurotoxicity associated with excitatory

amino acids encountered in food, such as mono-sodium glutamate, has also been linked to glutamate

receptors [40] .


Four parameters were used for studying the

quantitative analysis; the cerebellar weight CW),

the nuclear diameter (ND), the numerical densities (NV) and the axonal length (AL) of CGc. These

parameters represent a simple and sensitive indicator for studying the quantitative effects. On

the other hand the number of basal dendrites was

excluded from the quantitative parameters since, CGc has 3-4 dendrites which may be of not so

much statistical significance [41] .

Actually there is much controversies about the

effect of administration of PAM. It had previously

been described that PAM did not affect the total

number of mesencephalic neurons [42] . On the other hand, Bisaglia et al., [43] have shown that PAM in low dose (100 µM) protects hippocampal neurons. Moreover, PAM (100 µM) pre-treatment

also prevented menadione-induced neurotoxicity

(Tripathy and Grammas [44] , and PAM (1 00mg/kg) protected similarly oxidative neurotoxicity in vivo at four hours after its administration every hour for three hours [14,45] . However, when PAM was raised to 1mM, the protection was only apparent

soon after the application and was lost when higher

concentrations (2 & 9.2mM) were used. Moreover, it has been recently shown that intraperitoneal

administration of PAM (5-100mg/kg) seems to have protective effects on oxidative stress-induced brain toxicity by inhibiting free radical production [46,47] .

On the other hand, it was recently found that

PAM reduces creatine kinase activity (CK) in the

cerebellum and hippocampus but not in other brain

areas. The decrease in CK activity may affect the

mitochondrial status, which in turn may potentiate

the toxicity of other concurrent administered

subjects [48,49] .

The results of this study point to the fact that

administration of PAM increased the adverse effects

of independent variable (MSG) on the CGc. Fakunle et al., [27] studied the effect of chronic administration of acetoaminophen and ethanol on

the rat cerebellar Purkinje cells. They reported that

the PAM increases the toxicity of ethanol. They also reported a decrease in the number of total

Purkinje population suggesting that PAM increased

the adverse effects of the independent variable

(ethanol). In agreement of the results of the present

study, Hashem et al., [22] studied the effect of monosodium glutamate on the cerebellar cortex of male albino rats. They reported that MSG has neurotoxic effect leading to degenerative changes in neurons and astrocytes in cerebellar cortex of albino rats and attributed this for oxidative stress.

The morphological alterations that were observed in the present study, are in agreement

with the studies of Musa and Sunday [50] , who found an increase of oxidative DNA damage in neurons which is suggestive of a degenerative

process due to administration of MSG in specific

populations of neurons. They studied the pyramidal

cells of adult rats following oral administration of

MSG. They revealed histological findings such as

clumping and elongation of the nuclei material of pyramidal cells in the frontal lobe of the

experimental groups, with higher clumping and elongation observed in group administered with monosodium glutamate.

The morphological alterations in the experimental animals of G2 and G3 showed that some CGc were dark. However, the changes were

more pronounced in G2 than G3. These dark granule neurons appeared shrunken with irregular outline and increased staining intensity, dilated

perinuclear cisterna and cytoplasmic vacuolation.

This could be attributed to that PAM increasesd the adverse effects of MSG on the CGc. So, the

results of the present study is in accordance with

that of Huang et al., [51] who reported that stunted growth and delirious effects in brain were increased

with use of PAM.

The quantitative alterations for CW, ND, NV and AL that were observed in the present study

showed a highly significant decrease in G2, a significant decrease in G3 and none significant decrease in G4 as compared with that of G1. For

ND and NV, a highly significant decrease in G2, a significant decrease in G3 were noticed as

compared with that of G1. These quantitative alterations inforce the morphological alterations

seen in the experimental animals of G2 and G3 and indicating that PAM increasesd the adverse

effects of MSG on the CGc.

Analysis of this quantitative alterations revealed

that the highest recorded change was recorded in

G2. Furthermore, analysis of the results reveals that the highest recorded change was in NV parameter (the t=65.322). This was followed by that of ND (the t=49.494). This was followed by that of CW (the t=25.922). Finally, the smallest change was recorded in AL (the t= 14.062). In this regards Ahmed et al., [41] and Borst et al., [52] pointed out that, one of the mechanisms that if a

neuron is facing a deleterious factor, it starts to

lose the parts of the surface area (the axons and

dendritic field branches) of the neuron before

affection of the perikaryon.


There was noticeable reduction in the delirious

-associated changes in the G4 than with those of

G2 and G3 animals. This may be also attributed to that PAM is mainly hepatotoxic. In the present

study, PAM may potentiate the MSG neuronal

death through free radical production or via

inducing liver toxicity directly [53,54] . It has also been described that PAM causes a decrease in

glutathione levels might be related to an increase

in reactive oxygen species (ROS) production Lorenzo et al., [55] and Palade et al., [56] that can activate different death signalling pathways in

neuronal tissues [57] . In the present study, it seems

reasonable that PAM potentiate the toxicity of

MSG via PAM-mediated ROS high levels or via depletion of glutathione levels which will cause neuronal damage.

Another possible mechanism for PAM adverse effects on the CGc, is that mitochondria play a key

role in regulating the apoptotic mechanisms and

also in some forms of cell death by necrosis [58] . Calcium overload or free radical production induce the mitochondrial inner membrane that promotes

mitochondrial swelling, outer membrane rupture

and release of interamembrane proapoptotic proteins such as cytochrome C and apoptosis inducing factor to the cytoplasm [59,60] .

Conclusions and significance: The results of the present study found out a

direct potentiating action of Paracetamol for

increasing the neurotoxicity of Monosodium Glutamate on the cerebellar granule cells, and a

possible toxic effect of Paracetamol on cerebellar

granule cells in case of using higher doses or the

presence of other risk factors. Therefore, it is

important that Paracetamol is not used chronically

with Monosodium Glutamate. Also, it is highly recommended to proceed for further studies to

clarify the exact mechanism of the potentiating

action of it and the expected damage on the

cerebellar granule cells in case of using Paracetamol

with other risk factors or at higher doses.

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Effects of Paracetamol and Monosodium Glutamate on ...medicaljournalofcairouniversity.net/Home/images/pdf/2014/march/73.pdf · that Cytochrome P450 is also expressed in the brain

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