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Research Article
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Some Histological Observations and Microstructural Changes in the Nissl Substances in the Cerebellar Cortex of Adult Wistar Rats following Artesunate Administration |
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A.J. Ajibade,
P.B. Fakunle
and
P.D. Shallie
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ABSTRACT
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Several neurological disorders following abuse of artesunate used in chloroquine resistant malarial cases have been reported but little attention has been given the effects of this drug on histology of cerebellar cortex and Nissl substances. Twenty-four adult wistar rats of both sexes weighing between 150 and 230 g were randomly separated into four groups, N = 6. Rats in group D (control), received distilled water throughout exposure period while groups A, B and C (experimental groups) received 4 mg kg-1 b.wt. of artesunate orally on treatment day 1. Group A continued with (4 mg kg-1 b.wt.) for the next 3 days while group B and C received 2 mg kg-1 b.wt. of artesunate orally for the next 6 and 13 days, respectively. Groups A, B and C rats were sacrificed respectively on 5th, 8th and 15th day of treatment while Group D rats were sacrificed on the 15th day. Cerebellum of each rat was carefully dissected out and fixed in 10% formal saline for routine histological techniques. Histological findings showed normal cortical layers in control rats compared with degenerated and loss of purkinje cells, cellular hypertrophy with intercellular vacuolation appearing in the stroma of cerebellar cortex of treated rats. Nissl substances in cerebellar cortex of treated rats stained less intensely and appeared degenerated compared to more intensely stained and distinctly distributed Nissl substances in control rats. The observed vacuolation and neuronal loss in the cortical layers of the treated rats may adversely affect cerebellar functions while less intensely stained and degenerative changes in the Nissl substances in the cerebellar cortex of the treated rats may adversely affect protein synthesis in relation to neuronal functions.
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How
to cite this article:
A.J. Ajibade, P.B. Fakunle and P.D. Shallie, 2012. Some Histological Observations and Microstructural Changes in the Nissl Substances in the Cerebellar Cortex of Adult Wistar Rats following Artesunate Administration. Current Research in Neuroscience, 2: 1-10.
DOI: 10.3923/crn.2012.1.10
URL: https://scialert.net/abstract/?doi=crn.2012.1.10
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Received: February 23, 2011;
Accepted: July 25, 2011;
Published: July 10, 2012
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INTRODUCTION
Artesunate is an antimalarial drug commonly used as an alternative in chloroquine
resistant cases of plasmodum falciparum infection (Nwanjo
and Oze, 2007). Artemisinin which is the parent compound, is the antimalarial
principle of these compounds and is derived from the leaves of a plant called
sweet wormwood (Artemisia annua). Artemisinin was isolated by the Chinese
scientists from Artemisia annua leaves (Ngokere et
al., 2004). Artesunate is the most widely used member of artemisinin
derivatives in the treatment of chloroquine resistant malaria (Woodrow
et al., 2005). The effectiveness of artesunate has been attributed
to its rapid and extensive hydrolysis to Dihydroartemisinin (DHA) which is three
to five fold more active and more toxic, than the parent compound (Li
et al., 2002). Many physicians are now using new drugs like artemisinin
derivatives, due to very high rates of treatment failure with conventional treatment.
Although, number of clinical studies mainly conducted in Africa and Asia, have
identified a favorable tolerability profile for these drugs, however, many patients
have experienced adverse neurological effects (Price, 2000).
Artemisinin derivatives are potent antimalarial drugs, but concern has been
raised as to their neurotoxic potential (Li et al.,
2006). Several studies have shown that high doses of artesunate can produce
neurotoxicity such as selective damage to brain stem centers in mice and rats
(Nontprasert et al., 2002). All the adverse effects
of artesunate are still not well known, therefore, the potential for neurotoxicity
and other form of toxicity of artesunate is an issue that must be studied (Luo
et al., 2003). The safety profile of artesiminin derivatives has
been questioned because of their potential neurotoxic effects (Santiago
et al., 2008). Neurotoxic symptoms were observed in rats with 50
and 100 mg kg-1 from day 6 upward following artemether administration
(Oyemitan et al., 2007) Similarly, Obianime
and Aprioku (2011) had reported that artesunate and DHA may be toxic to
the liver, testis and hematopoietic cells resulting from increase in oxidative
stress via stimulation of protein kinase C activity.
Changes in serum glucose and triacylglycerol levels induced by the Co-administration
of Two Different Types of Antimalarial Drugs among Some Plasmodium falciparum
malarial patients in edo-delta region of Nigeria had been reported by Onyesom
and Agho (2011). It has been shown that artesunate and dihydroartemisinin
interacted more strongly with Fe (III) PPIX than artemisinin. Although, findings
have shown that hemin and endoperoxide lactone derived antimalarials slowly
react to give rise to several stereoisomers of artesunate while in contrast,
only heme (Fe2) was found to react with artemisinin based drugs in
previous studies (Mpiana et al., 2007). Artequin,
a combination of artesunate and mefloquine has been reported to be effective
against multidrug-resistant Plasmodium falciparum malaria in Nigeria
(Agomo et al., 2007). Neurotoxicity has been
detected in the various regions of the rat brain following monosodium glutamate
administration (Waggas, 2009). Adebayo
et al. (2009) also had reported brain damage in rats that were treated
with chemical neurotoxins. Long term exposure of rats to electromagnetic field
has been shown to be detrimental to the cerebellum (Ozra
et al., 2010). The neuropathological effect of Tributyltin (TRB)
on the neurons of the cerebral ganglia in snails has been reported by Essawy
et al. (2011). Aluminium acetate has similarly, been implicated as
a neuro-environmental factor responsible for neurodegenerative diseases. (Sushma
et al., 2006).
The cerebellum consists of three distinct layers and contains five major types
of neurons (Llinas et al., 2004) The molecular
layer is the outermost layer which contains few nerve cells. The purkinje cell
layer is the middle layer that contains a monolayer of purkinje cells sandwiched
between the molecular and granular layers. The granular layer is the inner layer
that consists of densely packed granule cells. Cerebellar injuries have been
reported to result from toxins, autoantibodies, structural lesion and inherited
cerebellar degeneration (Hain, 2009). In view of the
reported adverse neurological effects of artesunate on the central nervous system,
this study investigated the microstructural changes in the Nissl substances
and the histology of the cerebellar cortex in adult wistar rats following experimental
artesunate administration.
MATERIALS AND METHODS Twenty four wistar rats of both sexes weighting between 150 and 230 g were used for this study (2009). The rats were maintained under standard laboratory conditions. They were fed daily with rat chow purchased from Global Farms Ogbomosho and water was given to the rats ad libitum. The wistar rats were subjected to a period of three weeks of acclimatization before the treatment. The wistar rats were separated into four groups, each contained six rats. Group A, B and C (n =18) served as treatment groups while group D (n = 6) was the control. The rats in treatment groups A, B, C orally record 4 mg kg-1 b.wt. of artesunate base dissolved in distilled water through orogastric tube the first day. Wistar rats in the treatment group A continued with this dosage for the next three days while rats in group B and C received 2 mg kg-1 once daily for the next six and thirteen days, respectively. This was done to investigate some effects of acute, therapeutic and chronic treatment of artesunate on the cerebellum. The control group D received equal volume of distilled water as contained in the experimental doses.
Artesunate tablets were obtained from Salem pharmacy, Ibadan Oyo State, Nigeria.
One tablet which contains 50 mg of the active ingredient was dispersed in 50
mL of distilled water following the method of Mesembe et
al. (2004).
The treated rats in group A, B and C were sacrificed by cervical dislocation
on the 5th, 8th and 15th day of the study respectively, while the control group
D was also sacrificed on the 15th day of the study. Respective tissues specimens
were processed for routine histological procedures and sectioned at 6μ
and then stained for H and E and nissls substances as previously described by
Carleton (1967), respectively. Permanent digital microgrpahs
of the desired sections were obtained to record morphologic and microstructural
observations using a digital camera. The observations were made using a research
microscope.
Permanent digital microgrpahs of the desired sections were obtained to record
morphologic and microstructural observations using a digital camera. The observations
were made using a research microscope.
RESULTS Histological findings: Figure 1 shows the cerebellar cortex from control rats. The cerebellar cortex of the control rats showed a characteristic appearance of an outer molecular layer and an inner granular layer. The molecular layer contained distinct neurons. The middle cortical layer contained a monolayer of flash shaped purkinje cells sandwiched between the outer molecular and inner granular layers and the three cortical layers appeared normal in Fig. 1. Figure 2 shows the cerebellar cortex from artesunate-treated rats. The histological section of the cerebellar cortex of the artesunate-treated rats showed some cortical degenerative changes and vacuolations. The purkinje cell layer showed loss and degeneration of purkinje cells which appeared as vacuolations and smaller sized purkinje cells in the purkinje cell layer (Fig. 2). Figure 3 shows the section of cerebellar cortex from artesunate treated rats that received an initial 4 mg kg-1 b.wt. dose of artesunate followed by a 2 mg kg-1 b.wt. dose of artesunate for 6 days. The purkinje cell layer particularly showed increased degeneration and loss of purkinje cells. Loss of purkinje cells became more pronounced in Fig. 3 compared with Fig. 2.
Figure 4 shows the treated section of cerebellar cortex from
artesunate treated rat after receiving an initial 4 mg kg-1 b.wt.
dose of artesunate followed by a 2 mg kg-1 b.wt. dose of artesunte
for 13 days. There were vacuolations, distortion of cortical layers degeneration
and loss of neurons in this section.
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Fig. 1: |
(Group IV): Control section of the cerebellar cortex (Mag.
X400) |
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Fig. 2: |
(Group I): Treatment section of the cerebellar cortex that
received 4 mg kg-1 of artesunate for 4 days (Mag. X400) |
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Fig. 3: |
(Group II): Treatment section of the cerebellar cortex that
received 4 mg kg-1 1st day and thereafter 2 mg kg-1
for 6 days of artesunate (Mag. X400) |
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Fig. 4: |
(Group III): Treatment section of the cerebellar cortex that
received 4 mg kg-1 1st day and thereafter 2 mg kg-1
for 13 days of artesunate (Mag. X400) |
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Fig. 5: |
(Group D ): Control section of the cerebellar cortex of wistar
rats showing intensely stained Nissl substances in the cortical layers (Mag.
X400) |
Increased vacuolations which resulted from loss of cellular components, degeneration
and loss of neurons became more evident in this figure. Increased degeneration
and loss of purkinje cells became more pronounced in the purkinje cell layer
as shown in Fig. 4.
Histochemical findings on nissl substances: The cerebellar cortex of the control rats in group D revealed distinct and intensely stained Nissl substance in the cortical layers. The neurons in the cortical layers appeared normal, distinct and intensely stained (Fig. 5). The purkinje cells are distinct, deeply stained and consist of a monolayer sandwiched between the outer molecular layer and inner granular layer. The section of the cerebellar cortex from artesunate-treated rats (group A) showed some degenerative changes which made the neurons indistinct. The neurons in the cortical layers appeared to show some degenerative changes which them indistinct with a reduced staining intensity compared with the control section. Loss of neuronal cells and cellular components in the cortical layers are prominent as shown in Fig. 6.
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Fig. 6: |
(Group I): Treatment section of the cerebellar cortex that
received 4 mg kg-1 of artesunate for 4 days showing less intensely
stained and degenerated Nissl substances in the cortical layers Cresyl Violet
(Mag. X400) |
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Fig. 7: |
(Group B): Treatment section of the cerebellar cortex that
received 4 mg kg-1 1st day and thereafter 2 mg kg-1
for 6 days of artesunate showing less intensely stained and degenerated
Nissl substances in the cortical layers Cresyl Violet (Mag. X400) |
Section of the cerebellar cortex from group B treated rats also showed degenerative changes which made the neurons in the three cortical layers indistinct with reduced staining intensity compared with intensely stained and distinct neurons in the cortical layers of the control rats. The Nissl substances in the cortical layers as shown in Fig. 7 appeared degenerative with loss of staining intensity that made the neuronal cells indistinct. Some neurons particularly the purkinje cells in the middle layer appeared to have been lost in this section. Section of the cerebellar cortex from group C artesunate-treated rats also revealed some degenerative changes in the three cortical layers. The neurons in the three cortical cell layers of Fig. 8 appeared degenerative which made them indistinct with remarkable loss of staining intensity in the three cortical layers compared with the control section. Complete loss of purkinje cells and distortion of cortical layers became prominent in Fig. 8 as shown in this section.
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Fig. 8: |
(Group C): Treatment section of the cerebellar cortex that
received 4 mg kg-1 1st day and thereafter 2 mg kg-1
for 13 days of artesunate showing less intensely stained and degenerated
Nissl substances in the cortical layers Cresyl Violet (Mag. X400) |
DISCUSSION
Histological findings: The result of the haematoxylin and eosin stain
showed degenerative changes and loss of cellular components with reduced population
of purkinje cells in the treatment groups compared with the control section
of the cerebellar cortex of the adult wistar rats. Cells death has been reported
to result from neuronal degeneration (Waters et al.,
1994). Cell death may result from necrosis or apoptosis. Apoptosis and necrosis
differ morphologically and biochemically. Necrosis is a pathological change
which may result from toxic, thermal, traumatic and mechanical factors while
apoptosis is an organized form self destruction also known as programme cell
death that is mediated by intrinsic and active mechanisms (Wyllie
et al., 1980). Pathological or accidental cell death is regarded
as necrotic and could result from extrinsic insults to the cells such as osmotic,
thermal, toxic and traumatic effect (Faber et al.,
1981). Cell death in response to neurotoxin might trigger an apoptotic death
pathway within brain cells.
Artesunate crosses membrane and affects the cellular integrity of tissue. Artesunate
in this study, possibly might have acted as a neurotoxin to the cerebellar cortex
thereby affecting neuronal integrity and causing disruption in membrane permeability
and tissue homeostasis.. The degenerative effect of artesunate on the cortical
layers of the cerebellum observed in this study may be responsible for the cerebellar
degeneration. Clinical studies have reported adverse effects with artemisinin
derivatives when used alone or in combination with other antimalarials in therapeutic
doses. Adverse neurological effects after treatment include acute psychosis,
depressant syndrome, sleep disturbance (McIntosh and Olliaro,
2000) and post-cerebellar syndrome-ataxia and slurred speech (Sabchareon
et al., 1998). It is not impossible that artesunate had irreversible
neurotoxic effect on the cortical layer of the cerebellum which resulted in
distortion, loss of cellular component and purkinje cells in the cortical layers
of the treated cerebellar section. These findings agree with the earlier report
of Ajibade et al. (2006) on cerebellar cortex
of wistar rats following quinine administration. The degenerative changes observed
in this study may underlie the possible neurological symptoms such as ataxia,
tremor slurred speech and gait disturbance following artesunate administration
which has been reported Miller and Panosian (1997).
Histochemical findings on nissl substances: The sites in the cytoplasm
of basophilic ribosomes stain intensely with such basic dyes as methylene blue,
toludine blue and haematoxyhin. These basophilic regions have been described
as early as 19th century and were named according to the cells being studied.
They were called Nissl bodies in neurons. When appropriate stains are used Rough
Endoplasmic Reticulum (R.E.R) and free ribosomes appear under the light microscope
as basophilic granular areas called Nissl bodies. The purkinje cells appeared
distinct when nissl substances were stained which is consistent with the finding
of Gharravi et al. (2007). Neuronal degeneration
has been reported to cause a reduction in Nissl boches (Martin
et al., 1998). The observed neuronal degeneration in association
with loss of Nissl bodies with resultant reduced staining intensity of the Nissl
substances in the cerebellar cortex of the artesunate-treated rats in this study
is consistent with findings of Ajibade et al. (2009).
Injury to axons or neuronal exhaustion resulting from strong or prolong stimuli
causes a reduction in the number of Nissl bodies. This alteration which is called
chromatolysis which occurs simultaneously with nuclear migration to the periphery
of the perikaryon and consequently the RNA level is reduced (Louis
et al., 1986) Chemical and toxic substances affect the Nissl substances
thereby influencing their metabolic activity (Davis and Robertson,
1991). The findings from this study agreed with degeneration and vacuolar
changes which have been observed in many large brain stem neurons in mice that
were treated with trimethyl chloride. These neurons acquired a chromatolytic
character with eccentric nuclei and loss of Nissl substances which became progressive
in ultra structure (Change et al., 1983). Similarly,
research findings have shown that fatigue from over exertion; produced in the
brain cells similar to those changes produced by fear resulted in exhaustion
and consequently enormous reduction in Nissl substance. These changes were proportional
to the amount of exertion which is in agreement with observation on nissl substances
in this study. Loss of Nissl substance observed in this study is also supported
by the earlier findings of Classen et al. (1999)
reporting loss of Nissl substance in neurons and shrinkage of nucleus which
became prominent in the cerebellar roof, pontine and vestibular nuclei of dog
following intramuscular administration of artemether.
CONCLUSION This study concluded that chronic administration and high doses of artesunate administered to adult wistar rats resulted in loss of cellular components, distortion of the cortical layers and loss of Nissl substances in the cerebellar cortical layers. These pathological changes may impair cerebellar functions. ACKNOWLEDGMENT The authors wish to express their gratitude to the technical staff of the Anatomy Department Lautech, Ogbomoso, Nigeria, for their support in the execution of this present work.
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