Effect Of Bee Venom To Cell Death And Spatial Memory In Mice (Mus Musculus)
i
EFFECT OF BEE VENOM TO CELL DEATH AND
SPATIAL MEMORY IN MICE (Mus musculus)
RIAN OKTIANSYAH
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
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iii
STATEMENT ABOUT THESIS
I hereby declare that thesis entitled Effect of Bee Venom to Cell Death and
Spatial Memory in Mice (Mus musculus) is original result of my own research
supervised under advisory committee and has never been submitted in any form at
any institution before. All information from other authors cited here are mentioned
in the text and listed in the reference at the end part of the thesis.
Bogor, June 2016
Rian Oktiansyah
NIM G352140241
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SUMMARY
RIAN OKTIANSYAH. Effect of Bee Venom to Cell Death and Spatial Memory in
Mice (Mus musculus). Supervised by BERRY JULIANDI, KANTHI ARUM
WIDAYATI, and VETNIZAH JUNIANTITO.
Bee venom has been used as an alternative medicine as well as prevention
of various diseases. It contains complex compounds and some of its content act as
allergen and inflammation agents. It is expected to cause cell death in brain and to
affect spatial memory. The objective of this study was to determine dose of bee
venom that causes neuronal cell death in dentate gyrus, amygdala, and cerebral
cortex as well as analyze the alteration of mice behaviour, particularly spatial
memory. Fifteen male mice of Deutche Denken Yoken (DDY) were divided into
control and treatment groups. Bee venom was injected six times for two weeks
intraperitoneally with doses 1.88 mg/kg (P1), 3.76 mg/kg (P2), 5.6 mg/kg (P3), and
7.48 mg/kg (P4), respectively. Brain slices were made by paraffin method in 5 µm
coronal section and stained by haematoxylin eosin. The Y maze method was used
for behaviour assay. Parameters observed were the number of neuronal cell death
in hippocampus, amygdala, and cerebral cortex, and percentage of alteration mice
behaviour in Y maze. This study used complete randomized design method which
consists of five treatments and three repetitions. Data was analyzed using one-way
(ANOVA) in SPSS.
The results showed differences in the mean of neuronal cells death in dentate
gyrus, amygdala, and cerebral cortex. Mean of neuronal cells death varied in each
treatment compared to control. The highest mean of dead neuron in dentate gyrus,
amygdala, cortex upper layer (2-4), and cortex deep layer (5-6) was 1510 cell/mm2,
1754 cell/mm2, 1415 cell/mm2, and 1168 cell/mm2 while the lowest mean was 220
cell/mm2, 326 cell/mm2, 420 cell/mm2, and 365 cell/mm2, respectively. The highest
mean of alteration after 24 h bee venom injection was 74.57 % and the lowest was
61.07 %. The highest mean of alteration after 72 h bee venom injection was 76.03 %
and the lowest was 57.50 %. Analysis of variance (ANOVA) showed that effect of
bee venom was significantly different in the number of neuronal cell death in
dentate gyrus and amygdala, but no significant effect in cerebral cortex as well as
mice behaviour. The Duncan’s Multiple Range Test (DMRT) results showed that
P4 significantly different to neuronal cells death in dentate gyrus and amygdala
compared to other treatments. Based on the study, bee venom induced neurotoxic
effect because it caused neuronal cell death in dentate gyrus, amygdala, and cerebral
cortex and tend to affect spatial memory.
Keywords : bee venom, neuronal cell death, mice behaviour
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RINGKASAN
RIAN OKTIANSYAH. Pengaruh Racun Lebah terhadap Kematian Sel dan Memori
Spasial Mencit (Mus musculus). Dibimbing oleh BERRY JULIANDI, KANTHI
ARUM WIDAYATI, dan VETNIZAH JUNIANTITO.
Racun lebah digunakan sebagai obat alternatif untuk berbagai penyakit.
Racun lebah mengandung senyawa kompleks dan beberapa senyawa tersebut
berperan sebagai alergen dan agen inflamasi. Senyawa tersebut diduga
menyebabkan kematian sel di otak dan mempengaruhi memori spasial. Tujuan
penelitian ini adalah untuk menentukan dosis racun lebah yang menyebabkan
kematian sel saraf di hipokampus, amygdala, dan korteks dan menganalisis
perubahan perilaku mencit, terutama memori spasial. Mencit jantan galur Deutche
Denken Yoken (DDY) sebanyak lima belas ekor dibagi kedalam kelompok kontrol
dan kelompok perlakuan. Racun lebah diinjeksi enam kali selama dua minggu
secara intraperitoneal dengan dosis 1.88 mg/kg, 3.76 mg/kg, 5.6 mg/kg, dan 7.48
mg/kg. Sayatan histologi otak digunakan metode parafin dengan ketebalan 5 µm
secara koronal dan diwarnai dengan hematoksilin eosin. Metode Y maze digunakan
untuk pengujian perilaku. Parameter yang diamati adalah jumlah neuron yang mati
dan persentase perubahan perilaku mencit. Penelitian ini menggunakan metode
rancangan acak lengkap yang terdiri atas lima perlakuan dan tiga ulangan. Data
dianalisis menggunakan analisis one-way ANOVA di software SPSS statistics 20.
Hasil penelitian menunjukkan jumlah neuron yang mati di hipokampus
(dentate gyrus), amygdala, dan korteks dan perubahan perilaku mencit. Rata-rata
tertinggi jumlah neuron yang mati di dentate gyrus, amygdala, korteks lapisan atas
(2-4), korteks lapisan bawah (5-6) adalah 1510 sel/mm2, 1754 cell/mm2, 1415
cell/mm2, dan 1168 cell/mm2 sedangkan terendah adalah 220 sel/mm2, 326
cell/mm2, 420 cell/mm2, and 365 cell/mm2. Rata-rata tertinggi perubahan perilaku
mencit setelah 24 jam injeksi racun lebah adalah 74.57 % dan terendah 61.07 %.
Rata-rata tertinggi perubahan perilaku mencit setelah 72 jam injeksi racun lebah
adalah 76.03 % dan terendah 57.50 %. Analisis varians (ANOVA) menunjukkan
bahwa pengaruh racun lebah berbeda signifikan pada jumlah neuron yang mati di
hipokampus dan amygdala, namun tidak signifikan berpengaruh pada bagian
korteks dan perilaku mencit. Hasil uji Duncan menunjukkan bahwa P4 berbeda
signifikan terhadap jumlah neuron yang mati di hipokampus dan amygdala
dibandingkan perlakuan lain. Berdasarkan penelitian, racun lebah memiliki efek
neurotoksik karena menyebabkan kematian sel saraf di dentate gyrus, amygdala,
dan korteks, serta cenderung mempengaruhi memori spasial.
Kata kunci: racun lebah, kematian sel, perilaku mencit,
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© Copy Right by IPB, 2016
All rights reserved
It is prohibited to cite all or a part of this thesis without referring to and mentioning
the source. Citation is permitted for the purpose of education, research, scientific
paper, report, or critism writing only; and it does not defame the name and honour
of Bogor Agricultural University.
It is prohibited to republish and reproduce all or a part of this thesis without
permission from Bogor Agricultural University.
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EFFECT OF BEE VENOM TO CELL DEATH AND SPATIAL
MEMORY IN MICE (Mus musculus)
RIAN OKTIANSYAH
A Graduate Thesis
in partial fulfillment of Master Science degree in Animal Biosciences
Faculty of Mathematics and Natural Science
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
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Examiner: Prof drh Arief Boediono, PhD, PAVet
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PREFACE
I thank to Allah SWT for all His blessings so that this thesis successfully
completed. The title of this thesis is Effect of Bee Venom in Cell Death and Spatial
Memory in Mice (Mus musculus).
I want to send my gratitude to my supervisors Dr Berry Juliandi, MSi, Dr
Kanthi Arum Widayati, MSi, and drh Vetnizah Juniantito, SKH PhD for all
guidance and encouragement as well as invaluable academic advices for the whole
period of my study and research, and to my examiner, Prof drh Arief Boediono,
PhD PAVet for the generous support and great discussion. Thank to Lembaga
Pengelola Dana Pendidikan (LPDP) for the financial support. Special thank to my
parents and my family for your love and support, BSH 2014 for togetherness, and
to all people in Zoo Corner, for their supports for my study. Hopefully this paper
can be useful.
Bogor,
Rian Oktiansyah
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CONTENTS
LIST OF FIGURES
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INTRODUCTION
1
MATERIALS AND METHOD
Time and Place
Research Animal
Experimental Unit
The Treatments of Animals
Mice Brain Histology
Spatial Learning Test
Experimental Design and Data Analysis
2
2
2
2
2
2
2
3
RESULTS
Effect of Bee Venom to Neuronal Cells Death
Spatial Learning
3
3
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DISCUSSION
Effect of Bee Venom to Neuronal Cell Death
The Ability of Spatial Learning in Mice
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7
9
CONCLUSION
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REFERENCES
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BIOGRAPHY
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LIST OF FIGURES
1 Mean of neuronal cells death in hippocampus (dentate gyrus), amygdala,
and cortex
2 Representative image of hippocampus (dentate gyrus) in each treatment
stained by haematoxylin eosin
3 Representative image of amygdala in each treatment stained by
haematoxylin eosin
4 Representative image of cerebral cortex stained by haematoxylin eosin
5 Mean of behavioural mice alteration
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4
5
6
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1
INTRODUCTION
Bee venom is used as an alternative medicine as well as prevention of various
diseases, such as arthritis, rheumatism, pain, cancer, and neurodegenerative
diseases (Bellik 2015; Worlitzer et al. 2012; Yang et al. 2010; Kwon et al. 2001).
Bee venom is produced by two glands in bee worker sting. Bee venom increases
during the first week of adult bee worker life and reaches the maximum amount
when bee worker involves in nest defense and forage. However, Bee venom
decreases along with age (Krell 1996). Bee venom contains complex compounds,
including polypeptides, enzymes, lipids, and amino acids. Some of the content of
bee venom act as allergen, anti-inflammation, and inflammation agents (Lee and
Bae 2016). It is expected to cause cell death.
Cell death in multicellular organisms play an important role during
development. Cell death controls the number of cells and protects the organism by
removing all of damaged cells that caused by disease, aging, infections, genetic
mutations, and exposure of toxic substances. Cell death in nature is categorized into
two types, namely necrosis and apoptosis (Saikumar and Venkatachalam 2009).
Necrosis and apoptosis due to the external factors, such as toxic substances
and allergens is prominently found (Cudrici et al. 2006). Morphologically, necrosis
indicated by shrinkage, fragmentation, or fusing the nucleus of the cells. In contrast
to necrosis, apoptosis is characterized by the presence of the bleb on the plasma
membrane and separation of cytoplasm and organelles of cells (apoptotic body)
(Majno and Joris 1995). Necrosis and apoptosis can occur in tissue or organ,
including in the brain regions, such as amygdala, cerebral cortex, and hippocampus
that affect emotion, motoric response, and spatial memory, respectively.
Spatial memory is the capability to code, store, and retrieve information
relate to spatial space in environment. Spatial memory has important function for
human to learn a route, path, direction, or remember the location of objects.
(Kessels et al. 2000; Langley 2012; Banikowski 1999). There is a product alleged
to affect spatial memory but it is not reported yet scientifically, that is bee venom.
There are several studies showed that bee venom can also cause cell death.
Bee venom induced cell death in human lymphocytes (Gajski and GarajVrhovac, 2011). Bee venom contains phospholipase A that induced cell death by
inactivating p38 MAPK (Jeong et al. 2011). Associated with beevenom acts as an
allergen, there are few studies on allergen caused cell death, such as NFAT (Nuclear
Factor of Activated T Cell) which triggers the inflammation in the skin and it caused
cell death (Kwon et al. 2016). Mice were injected with 2.4-dinitrofluorobenzen
induces keratinocyte apoptosis due to the expression of FAS (Hedrych-Ozimina et
al. 2011). However, it has not been reported yet about bee venom that causes the
neuronal death affecting the behaviour of mice. The objective of this study was to
determine dose of bee venom that causes neuronal cell death and analyze alterations
in the behaviour of mice, particularly spatial memory.
2
MATERIALS AND METHOD
Time and Place
The study was conducted in Division of Animal Function and Behaviour,
Departement of Biology, Faculty of Matemathics and Natural Sciences and
Laboratory of Pathology, Faculty of Veterinary Medicine, Bogor Agricultural
University. The study was carried out on October 2015 until March 2016.
Research Animal
The study used three months old male mice Deutche Denken Yoken (DDY),
with weight ranging from 20-30 g. Mice were obtained from Non Ruminant
Laboratory and Prospective Animals, Faculty of Animal Sciences, Bogor
Agricultural University.
Experimental Unit
Fifteen mice used for this study were divided into control and treatments
groups. Each treatment group consisted of three mice. Mice were reared to different
cages, according to the group.
The Treatments of Animals
Mice were acclimatized for a week before treatments. Mice were maintained
in the animal maintenance unit at room temperature and with wood chips. Mice
were given drink and feed continuously throughout the maintenance (Clark et al.
2008). Bee venom was injected intraperitoneally after acclimatization. Bee venom
was injected for two weeks at days 1, 4, 7, 10, 13, and 16 with doses 1.88 mg/kg
(P1), 3.76 mg/kg (P2), 5.6 mg/kg (P3), and 7.48 mg/kg (P4) (Bogdanov 2015). Bee
venom was obtained from apitheraphy clinic.
Mice Brain Histology
Brain tissues isolation was done three days after the last injection of bee
venom using perfusion method (Matsuda 2007; Erwin et al. 2013). Morphological
examination on brain tissue was performed with sectioned tissue stained by
Haematoxylin-eosin (HE). Each section was observed by using light microscope.
Spatial Learning Test
Behaviour test was done after injection. The purpose of behaviour test was
to examine spatial learning using Y maze apparatus. The movement of mice was
score correct if the mice moved sequentially to different Y maze arm (such as ABC,
ACB, CBA, BCA) and was score incorrect if the mice moved to the same maze
(such as AAA, ABA, ACA, BCC) (Onaolapo et al. 2012). Behaviour test was done
between 1.30 to 4.30 pm (Juliandi et al. 2015).
3
Experimental Design and Data Analysis
This study used complete randomized design method which consists of five
treatments and three repetitions. Parameters observed were the percentage of mice
to explore correct Y maze arms and the number of neuronal cell death in dentate
gyrus, amygdala, and cerebral cortex. Data was analyzed using one-way analyses
of variance (ANOVA) in SPSS statistics software.
RESULTS
Effect of Bee Venom to Neuronal Cells Death
Based on observation, there were variation of mean neuronal cells death in
several brain regions (Figure 1). The variation showed that bee venom has influence
to neuronal cells death in hipocampus (dentate gyrus), amygdala, and cerebral
cortex (Figure 2, Figure 3, and Figure 4). The results showed differences in the
mean of neuronal cells death in dentate gyrus, amygdala, and cortex. The mean of
neuronal cells death varied in each treatment compared to control. The highest mean
of dead neuron in dentate gyrus, amygdala, cortex upper layer (2-4), and cortex
deep layer (5-6) was 1510 cell/mm2, 1754 cell/mm2, 1415 cell/mm2, and 1168
cell/mm2 while the lowest mean was 220 cell/mm2, 326 cell/mm2, 420 cell/mm2,
and 365 cell/mm2, respectively.
Figure 1 Mean of neuronal cells death in hippocampus (dentate gyrus), amygdala,
and cerebral cortex
4
Control
P1
P2
P3
P
P
P4
Figure 2
Representative image of brain sections containing dentate gyrus in
each treatment stained by haematoxylin eosin. Dead neurons are
shown by arrow (bars: 50 µm)
Brain histology in dentate gyrus stained by haematoxylin eosin showed
normal and dead neuron. Dead neuron were showed by dark colour cell and nucleus
was unseen (Figure 2). Analysis of variance (ANOVA) showed that effect of bee
venom was significantly different in the number of neuronal cell death in dentate
gyrus (F = 7.040, p < 0.01). It means that bee venom has a role in neuronal cell
death regulation in dentate gyrus. Thus, the Duncan’s Multiple Range Test (DMRT)
5
is required to examine the effect of treatments in neuronal cells death regulation in
dentate gyrus.
The DMRT results showed that P4 significantly different to other
treatments. Treatment with the highest dose showed the highest number of neuronal
cells death in dentate gyrus. Based on the study, doses of bee venom gave
significant effect on neuronal cells death. Furthermore, the effect of bee venom
against neuronal cells death significantly was showed by P4 (7.48 mg/kg).
Control
P1
P
Figure 3
P4
P2
P3
Representative image of brain sections containing amygdala in each
treatment stained by haematoxylin eosin (bars: 100 µm)
Amygdala was also examined to show effect off bee venom to neuronal cells
death (Figure 3). Results of ANOVA showed that effect of bee venom was
significantly different in the number of neuronal cell death in amygdala (F = 3.940,
p < 0.05). It means that bee venom has a role in neuronal cell death regulation in
amygdala. The DMRT results showed that P4 was significantly different to other
treatments. Treatment with the highest dose showed the highest number of neuronal
cells death in amygdala. Based on the study, doses of bee venom gave the
significant effect on neuronal cells death. Significant effect of bee venom to
neuronal cells death was showed by P4 (7.48 mg/kg).
6
upper layer
deep layer
Figure 4
Representative image of brain sections containing cerebral cortex
stained by haematoxylin eosin (bar: 50 µm)
Brain histology in cerebral cortex stained by haematoxylin eosin (HE)
showed neuron morphology in each layer (Figure 4). ANOVA showed that
neuronal cell death density in cerebral cortex upper layer and deep layer was not
significantly different (upper layer: F = 2.196, p > 0.05; deep layer: F = 2.267, p
>0.05). It means that bee venom has no effect to regulate neuronal cell death in
cerebral cortex.
Spatial Learning
The exploration of mice to the Y maze arms was observed in this study. The
results showed differences in the mean of alteration percentage in mice behaviour.
The alteration percentage in Y maze varied in each treatment as compared to
control. The highest mean of alteration after 24 h bee venom injection was 74.57 %
and the lowest one was 61.07 %. The highest mean of alteration after 72 h bee
venom injection was 76.03 % and the lowest one was 57.50 %. The variation of
mean showed that bee venom has influence in alteration of behaviour in Y maze.
The variation can be seen in Figure 5.
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Figure 5 Mean of correct alteration in Y maze arms
Results of ANOVA showed that effect of bee venom was not significantly
different to mice behaviour (after 24 hour: F = 0.856, p > 0.05; after 72 hour: F =
1.483, p > 0.05). It means that bee venom has no influence to mice behaviour. The
percentage of correct alteration in Y maze was congruent to the number of neuronal
cells death in the brain.
DISCUSSION
Effect of Bee Venom to Neuronal Cell Death
Based on this study, bee venom affected neuronal cell death in dentate gyrus.
DMRT test showed that the highest dose of bee venom was significantly different
to other treatments on neuronal cell death in dentate gyrus. Dose of 4 mg/kg body
weight of rats is the maximum dose of bee venom to anti-inflammation effect while
the higher dose shows neurotoxic effects (Bogdanov 2015). Figure 2 showed that
neuronal cells death found in subgranular zone (SGZ). According to study, we
proposed that dead neuron was neural progenitor cells (NPC) because it was found
in the innermost layer of SGZ. However, it needs special markers to confirm the
cell types.
We examined amygdala and cortex, other brain regions that relate to
hippocampus in regulation of memory. However, we found that bee venom-treated
mice influence neuronal cell death in amygdala but it was not significantly
difference in cerebral cortex. Although bee venom has no effect to cause neuronal
cell death in cerebral cortex, we could find that increasing in the number of neuronal
cells death in P4 compared to control (Figure 1). Cerebral cortex consists of six
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layers (Figure 4). Each layer has kind of neuronal type and different functions
(Feldmeyer 2012). Layers III and V of the cerebral cortex contains a large number
of pyramidal neurons with high levels of acetylcholinesterase activity (Mesulam
and Geula 1991; Viswanathan et al. 2006) while layer II and IV receive information
from hippocampus (Gomez-Isla 1996). Cell death in tissues or organs can upset the
function. The redundant of neuronal cells death in brain can cause variety of
neurodegenerative diseases, such as parkinson (Ruberg et al. 1997; Anglade et al.
1997), alzeimer (Shimohama 2000; Bamberger and Landreth 2002), huntington
(Dean 2008). Taken together, these result suggested that bee venom treatment
contribute to the poor performance in learning and memory test.
Increasing in the number of neuronal cells in dentate gyrus, amygdala, and
cerebral cortex in P4 suggested as an effect of inflammatory compounds of the bee
venom. Previous studies have demonstrated the role of melittin, phospholipase A2,
apamin, and adolapin in high doses contained in bee venom can cause allergies,
lysis of erythrocytes, myonecrosis, and neurotoxic effects (Ovcharov et al. 1976;
Ownby et al. 1997; Raghuraman & Chattopadhyay 2006; Ali 2012; Abdu &
Alahmari 2013; Elhakim et al. 2014; Bogdanov 2015; Eze et al. 2016; Lee & Bae
2016). Therefore, bee venom can affect the peripheral tissues.
The influence of bee venom in peripheral tissues is expected to produce
cytokines which sends signals to the brain, so that it can cause neuronal cells death
in hippocampus (dentate gyrus), amygdala, and cerebral cortex. Dantzer et al.
(2008), Dilger & Johnson (2008), and Maier et al. (2012) demonstrated that
cytokines from peripheral tissues transported into the brain through the endocrine
system and induce neuronal cell death. Palombella & Vilcek (1989) reported that
melittin and phospholipase A2 can activate cytokines, which is TNF. Although it
still needs further confirmation, administration of bee venom is also supposed to
have indirectly effect on neuronal cells death in the brain through cytokines.
Another suggestion about the mechanism of neuronal cell death because of
bee venom, is the ability of bee venom compounds in passing through the blood
brain barrier (BBB). BBB plays an important role in regulating the molecule that
can be in and out of the brain (Ransohoff & Engelhardt 2012; Takeshita &
Ransohoff 2012). Oller-Salvia et al. (2013) and Mourre et al. (1997) reported that
apamin, compound of bee venom, can pass through the blood brain barrier and
cause neuronal cell death. However, other compounds of bee venom have not been
reported to pass through the BBB. Therefore, apamin suggested neuronal cells death
in brain.
Linear effect on neuronal cell death in brain regions was not affected by the
increasing dose of bee venom (Figure 1). The higher doses of bee venom did not
indicate higher number of neuronal cells death. In this study, P2 showed the number
of neuronal cells death increased compared to control, P1, and P3. Anomaly result
in P2 did not affect the conclusion of this study. It is caused the efficiency of bee
venom utilization depicted by P1. P1 has the lowest of neuronal cell death in brain
regions compared to the other treatments. Lee et al. (2004) and Lee et al. (2011)
reported that low dose of bee venom has antiinflammation effect. It is predicted to
increase neuronal durability and proliferation. However, neuronal proliferation
needs further assesment to know amount of new born cell. These results provide
straightforward information about efficiency of administration to the clinic
apiteraphy as well as people who want to do the bee sting therapy.
9
The Ability of Spatial Learning in Mice
Spatial learning ability of mice is shown by the correct-arm alternation in
Y-maze. The higher percentage of correct-arm alternation showed that the better
spatial memory in mice.Y-maze was used to examine spatial learning underlying
mice activity to explore new environment. Wolfe (1969) reported that mice prefers
to investigate new object and environment. ANOVA test showed that number of
neuronal cell death was not significantly different to alteration mice behaviour.
Conrad and Roy (1993) reported that as much as 80% of neuronal cell death in
dentate gyrus do not affect spatial memory. Nonetheless, the results showed that
bee venom administration tend to affect the spatial learning ability of mice (Figure
5).
The tendency of these effects can be caused by neuronal cells death in the
dentate gyrus. Dentate gyrus plays a role in memory formation, distinctively related
to cognitive function and spatial memory (Silva et al. 1998; Eichenbaum et al.
1999). The results showed that treatment with the most of dead neuron had the
lowest correct-arm alternation in Y-maze (Figure 5). We suggested that NPC was
dead neuron in SGZ. Dead or damage NPC causes the mature neuron will be
reduced while the mature neuron that still remain will undergo programmed cell
death. It will affect the process of learning and spatial memory in mice. Lawyer et
al. (2006), Plessen et al. (2006), Thomann et al. (2012), and Juliandi et al. (2015)
reported that the abnormal morphology in the hippocampus associated with the
reduce neurogenesis which correlated with the cognitive deficits.
Cognitive deficits associated with bee venom-treated might not due to the
abnormal morphology in hippocampus (dentate gyrus), there is possibility
responses in emotion and motoric function that caused by abnormal in amygdala
and cerebral cortex. We have shown neuronal cell death in amygdala and cerebral
cortex (upper and deep layer) (Figure 1). Preston and Eichenbaum (2013)
demonstrated that hippocampus and cerebral cortex engages to form meaningful
contexts in which relate to memory retrieval, as well as the amygdala. Amygdala
and hippocampus act synergistically when emotion and memory came together
(Richter-Levin and Akirav 2000; Phelps 2004). Hippocampus, amygdala, and
cerebral cortex have a role in limbic systems, link to another brain region.
Abnormalities in those region will affect the tissues functionally. Deficiency in
amygdala and cerebral cortex contributes to cognitive deficit. However, other brain
regions contribute to the deficits. It still needs further investigation to complete
possibility.
CONCLUSION
Based on the study, bee venom had neurotoxic effect because it caused
neuronal cell death in dentate gyrus, amygdala, and cerebral cortex as well as
behaviour alteration in mice. The dose of bee venom that caused the highest
neuronal cells death in dentate gyrus, amygdala, and cerebral cortex was P4 (7.48
mg/kg). Bee venom tends to affect spatial memory.
10
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14
BIOGRAPHY
Author was born in Prabumulih in October 2nd, 1991. Author is the
youngest child of four from the parents, Ali Imron and Mulyati. In 2009 author
studied in Sriwijaya University, Faculty of Teacher Training and Education, major
of Biology education and graduated from Sriwijaya University in 2013. In August
2014, author entered college in the Graduate School, Major of Animal Biosciences,
Bogor Agricultural University at its own expense, and in the third semester author
got thesis scholarship from LPDP.
EFFECT OF BEE VENOM TO CELL DEATH AND
SPATIAL MEMORY IN MICE (Mus musculus)
RIAN OKTIANSYAH
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
ii
iii
STATEMENT ABOUT THESIS
I hereby declare that thesis entitled Effect of Bee Venom to Cell Death and
Spatial Memory in Mice (Mus musculus) is original result of my own research
supervised under advisory committee and has never been submitted in any form at
any institution before. All information from other authors cited here are mentioned
in the text and listed in the reference at the end part of the thesis.
Bogor, June 2016
Rian Oktiansyah
NIM G352140241
iv
SUMMARY
RIAN OKTIANSYAH. Effect of Bee Venom to Cell Death and Spatial Memory in
Mice (Mus musculus). Supervised by BERRY JULIANDI, KANTHI ARUM
WIDAYATI, and VETNIZAH JUNIANTITO.
Bee venom has been used as an alternative medicine as well as prevention
of various diseases. It contains complex compounds and some of its content act as
allergen and inflammation agents. It is expected to cause cell death in brain and to
affect spatial memory. The objective of this study was to determine dose of bee
venom that causes neuronal cell death in dentate gyrus, amygdala, and cerebral
cortex as well as analyze the alteration of mice behaviour, particularly spatial
memory. Fifteen male mice of Deutche Denken Yoken (DDY) were divided into
control and treatment groups. Bee venom was injected six times for two weeks
intraperitoneally with doses 1.88 mg/kg (P1), 3.76 mg/kg (P2), 5.6 mg/kg (P3), and
7.48 mg/kg (P4), respectively. Brain slices were made by paraffin method in 5 µm
coronal section and stained by haematoxylin eosin. The Y maze method was used
for behaviour assay. Parameters observed were the number of neuronal cell death
in hippocampus, amygdala, and cerebral cortex, and percentage of alteration mice
behaviour in Y maze. This study used complete randomized design method which
consists of five treatments and three repetitions. Data was analyzed using one-way
(ANOVA) in SPSS.
The results showed differences in the mean of neuronal cells death in dentate
gyrus, amygdala, and cerebral cortex. Mean of neuronal cells death varied in each
treatment compared to control. The highest mean of dead neuron in dentate gyrus,
amygdala, cortex upper layer (2-4), and cortex deep layer (5-6) was 1510 cell/mm2,
1754 cell/mm2, 1415 cell/mm2, and 1168 cell/mm2 while the lowest mean was 220
cell/mm2, 326 cell/mm2, 420 cell/mm2, and 365 cell/mm2, respectively. The highest
mean of alteration after 24 h bee venom injection was 74.57 % and the lowest was
61.07 %. The highest mean of alteration after 72 h bee venom injection was 76.03 %
and the lowest was 57.50 %. Analysis of variance (ANOVA) showed that effect of
bee venom was significantly different in the number of neuronal cell death in
dentate gyrus and amygdala, but no significant effect in cerebral cortex as well as
mice behaviour. The Duncan’s Multiple Range Test (DMRT) results showed that
P4 significantly different to neuronal cells death in dentate gyrus and amygdala
compared to other treatments. Based on the study, bee venom induced neurotoxic
effect because it caused neuronal cell death in dentate gyrus, amygdala, and cerebral
cortex and tend to affect spatial memory.
Keywords : bee venom, neuronal cell death, mice behaviour
v
RINGKASAN
RIAN OKTIANSYAH. Pengaruh Racun Lebah terhadap Kematian Sel dan Memori
Spasial Mencit (Mus musculus). Dibimbing oleh BERRY JULIANDI, KANTHI
ARUM WIDAYATI, dan VETNIZAH JUNIANTITO.
Racun lebah digunakan sebagai obat alternatif untuk berbagai penyakit.
Racun lebah mengandung senyawa kompleks dan beberapa senyawa tersebut
berperan sebagai alergen dan agen inflamasi. Senyawa tersebut diduga
menyebabkan kematian sel di otak dan mempengaruhi memori spasial. Tujuan
penelitian ini adalah untuk menentukan dosis racun lebah yang menyebabkan
kematian sel saraf di hipokampus, amygdala, dan korteks dan menganalisis
perubahan perilaku mencit, terutama memori spasial. Mencit jantan galur Deutche
Denken Yoken (DDY) sebanyak lima belas ekor dibagi kedalam kelompok kontrol
dan kelompok perlakuan. Racun lebah diinjeksi enam kali selama dua minggu
secara intraperitoneal dengan dosis 1.88 mg/kg, 3.76 mg/kg, 5.6 mg/kg, dan 7.48
mg/kg. Sayatan histologi otak digunakan metode parafin dengan ketebalan 5 µm
secara koronal dan diwarnai dengan hematoksilin eosin. Metode Y maze digunakan
untuk pengujian perilaku. Parameter yang diamati adalah jumlah neuron yang mati
dan persentase perubahan perilaku mencit. Penelitian ini menggunakan metode
rancangan acak lengkap yang terdiri atas lima perlakuan dan tiga ulangan. Data
dianalisis menggunakan analisis one-way ANOVA di software SPSS statistics 20.
Hasil penelitian menunjukkan jumlah neuron yang mati di hipokampus
(dentate gyrus), amygdala, dan korteks dan perubahan perilaku mencit. Rata-rata
tertinggi jumlah neuron yang mati di dentate gyrus, amygdala, korteks lapisan atas
(2-4), korteks lapisan bawah (5-6) adalah 1510 sel/mm2, 1754 cell/mm2, 1415
cell/mm2, dan 1168 cell/mm2 sedangkan terendah adalah 220 sel/mm2, 326
cell/mm2, 420 cell/mm2, and 365 cell/mm2. Rata-rata tertinggi perubahan perilaku
mencit setelah 24 jam injeksi racun lebah adalah 74.57 % dan terendah 61.07 %.
Rata-rata tertinggi perubahan perilaku mencit setelah 72 jam injeksi racun lebah
adalah 76.03 % dan terendah 57.50 %. Analisis varians (ANOVA) menunjukkan
bahwa pengaruh racun lebah berbeda signifikan pada jumlah neuron yang mati di
hipokampus dan amygdala, namun tidak signifikan berpengaruh pada bagian
korteks dan perilaku mencit. Hasil uji Duncan menunjukkan bahwa P4 berbeda
signifikan terhadap jumlah neuron yang mati di hipokampus dan amygdala
dibandingkan perlakuan lain. Berdasarkan penelitian, racun lebah memiliki efek
neurotoksik karena menyebabkan kematian sel saraf di dentate gyrus, amygdala,
dan korteks, serta cenderung mempengaruhi memori spasial.
Kata kunci: racun lebah, kematian sel, perilaku mencit,
vi
© Copy Right by IPB, 2016
All rights reserved
It is prohibited to cite all or a part of this thesis without referring to and mentioning
the source. Citation is permitted for the purpose of education, research, scientific
paper, report, or critism writing only; and it does not defame the name and honour
of Bogor Agricultural University.
It is prohibited to republish and reproduce all or a part of this thesis without
permission from Bogor Agricultural University.
i
EFFECT OF BEE VENOM TO CELL DEATH AND SPATIAL
MEMORY IN MICE (Mus musculus)
RIAN OKTIANSYAH
A Graduate Thesis
in partial fulfillment of Master Science degree in Animal Biosciences
Faculty of Mathematics and Natural Science
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
ii
Examiner: Prof drh Arief Boediono, PhD, PAVet
iv
PREFACE
I thank to Allah SWT for all His blessings so that this thesis successfully
completed. The title of this thesis is Effect of Bee Venom in Cell Death and Spatial
Memory in Mice (Mus musculus).
I want to send my gratitude to my supervisors Dr Berry Juliandi, MSi, Dr
Kanthi Arum Widayati, MSi, and drh Vetnizah Juniantito, SKH PhD for all
guidance and encouragement as well as invaluable academic advices for the whole
period of my study and research, and to my examiner, Prof drh Arief Boediono,
PhD PAVet for the generous support and great discussion. Thank to Lembaga
Pengelola Dana Pendidikan (LPDP) for the financial support. Special thank to my
parents and my family for your love and support, BSH 2014 for togetherness, and
to all people in Zoo Corner, for their supports for my study. Hopefully this paper
can be useful.
Bogor,
Rian Oktiansyah
v
CONTENTS
LIST OF FIGURES
vi
INTRODUCTION
1
MATERIALS AND METHOD
Time and Place
Research Animal
Experimental Unit
The Treatments of Animals
Mice Brain Histology
Spatial Learning Test
Experimental Design and Data Analysis
2
2
2
2
2
2
2
3
RESULTS
Effect of Bee Venom to Neuronal Cells Death
Spatial Learning
3
3
6
DISCUSSION
Effect of Bee Venom to Neuronal Cell Death
The Ability of Spatial Learning in Mice
7
7
9
CONCLUSION
9
REFERENCES
10
BIOGRAPHY
14
vi
LIST OF FIGURES
1 Mean of neuronal cells death in hippocampus (dentate gyrus), amygdala,
and cortex
2 Representative image of hippocampus (dentate gyrus) in each treatment
stained by haematoxylin eosin
3 Representative image of amygdala in each treatment stained by
haematoxylin eosin
4 Representative image of cerebral cortex stained by haematoxylin eosin
5 Mean of behavioural mice alteration
3
4
5
6
7
1
INTRODUCTION
Bee venom is used as an alternative medicine as well as prevention of various
diseases, such as arthritis, rheumatism, pain, cancer, and neurodegenerative
diseases (Bellik 2015; Worlitzer et al. 2012; Yang et al. 2010; Kwon et al. 2001).
Bee venom is produced by two glands in bee worker sting. Bee venom increases
during the first week of adult bee worker life and reaches the maximum amount
when bee worker involves in nest defense and forage. However, Bee venom
decreases along with age (Krell 1996). Bee venom contains complex compounds,
including polypeptides, enzymes, lipids, and amino acids. Some of the content of
bee venom act as allergen, anti-inflammation, and inflammation agents (Lee and
Bae 2016). It is expected to cause cell death.
Cell death in multicellular organisms play an important role during
development. Cell death controls the number of cells and protects the organism by
removing all of damaged cells that caused by disease, aging, infections, genetic
mutations, and exposure of toxic substances. Cell death in nature is categorized into
two types, namely necrosis and apoptosis (Saikumar and Venkatachalam 2009).
Necrosis and apoptosis due to the external factors, such as toxic substances
and allergens is prominently found (Cudrici et al. 2006). Morphologically, necrosis
indicated by shrinkage, fragmentation, or fusing the nucleus of the cells. In contrast
to necrosis, apoptosis is characterized by the presence of the bleb on the plasma
membrane and separation of cytoplasm and organelles of cells (apoptotic body)
(Majno and Joris 1995). Necrosis and apoptosis can occur in tissue or organ,
including in the brain regions, such as amygdala, cerebral cortex, and hippocampus
that affect emotion, motoric response, and spatial memory, respectively.
Spatial memory is the capability to code, store, and retrieve information
relate to spatial space in environment. Spatial memory has important function for
human to learn a route, path, direction, or remember the location of objects.
(Kessels et al. 2000; Langley 2012; Banikowski 1999). There is a product alleged
to affect spatial memory but it is not reported yet scientifically, that is bee venom.
There are several studies showed that bee venom can also cause cell death.
Bee venom induced cell death in human lymphocytes (Gajski and GarajVrhovac, 2011). Bee venom contains phospholipase A that induced cell death by
inactivating p38 MAPK (Jeong et al. 2011). Associated with beevenom acts as an
allergen, there are few studies on allergen caused cell death, such as NFAT (Nuclear
Factor of Activated T Cell) which triggers the inflammation in the skin and it caused
cell death (Kwon et al. 2016). Mice were injected with 2.4-dinitrofluorobenzen
induces keratinocyte apoptosis due to the expression of FAS (Hedrych-Ozimina et
al. 2011). However, it has not been reported yet about bee venom that causes the
neuronal death affecting the behaviour of mice. The objective of this study was to
determine dose of bee venom that causes neuronal cell death and analyze alterations
in the behaviour of mice, particularly spatial memory.
2
MATERIALS AND METHOD
Time and Place
The study was conducted in Division of Animal Function and Behaviour,
Departement of Biology, Faculty of Matemathics and Natural Sciences and
Laboratory of Pathology, Faculty of Veterinary Medicine, Bogor Agricultural
University. The study was carried out on October 2015 until March 2016.
Research Animal
The study used three months old male mice Deutche Denken Yoken (DDY),
with weight ranging from 20-30 g. Mice were obtained from Non Ruminant
Laboratory and Prospective Animals, Faculty of Animal Sciences, Bogor
Agricultural University.
Experimental Unit
Fifteen mice used for this study were divided into control and treatments
groups. Each treatment group consisted of three mice. Mice were reared to different
cages, according to the group.
The Treatments of Animals
Mice were acclimatized for a week before treatments. Mice were maintained
in the animal maintenance unit at room temperature and with wood chips. Mice
were given drink and feed continuously throughout the maintenance (Clark et al.
2008). Bee venom was injected intraperitoneally after acclimatization. Bee venom
was injected for two weeks at days 1, 4, 7, 10, 13, and 16 with doses 1.88 mg/kg
(P1), 3.76 mg/kg (P2), 5.6 mg/kg (P3), and 7.48 mg/kg (P4) (Bogdanov 2015). Bee
venom was obtained from apitheraphy clinic.
Mice Brain Histology
Brain tissues isolation was done three days after the last injection of bee
venom using perfusion method (Matsuda 2007; Erwin et al. 2013). Morphological
examination on brain tissue was performed with sectioned tissue stained by
Haematoxylin-eosin (HE). Each section was observed by using light microscope.
Spatial Learning Test
Behaviour test was done after injection. The purpose of behaviour test was
to examine spatial learning using Y maze apparatus. The movement of mice was
score correct if the mice moved sequentially to different Y maze arm (such as ABC,
ACB, CBA, BCA) and was score incorrect if the mice moved to the same maze
(such as AAA, ABA, ACA, BCC) (Onaolapo et al. 2012). Behaviour test was done
between 1.30 to 4.30 pm (Juliandi et al. 2015).
3
Experimental Design and Data Analysis
This study used complete randomized design method which consists of five
treatments and three repetitions. Parameters observed were the percentage of mice
to explore correct Y maze arms and the number of neuronal cell death in dentate
gyrus, amygdala, and cerebral cortex. Data was analyzed using one-way analyses
of variance (ANOVA) in SPSS statistics software.
RESULTS
Effect of Bee Venom to Neuronal Cells Death
Based on observation, there were variation of mean neuronal cells death in
several brain regions (Figure 1). The variation showed that bee venom has influence
to neuronal cells death in hipocampus (dentate gyrus), amygdala, and cerebral
cortex (Figure 2, Figure 3, and Figure 4). The results showed differences in the
mean of neuronal cells death in dentate gyrus, amygdala, and cortex. The mean of
neuronal cells death varied in each treatment compared to control. The highest mean
of dead neuron in dentate gyrus, amygdala, cortex upper layer (2-4), and cortex
deep layer (5-6) was 1510 cell/mm2, 1754 cell/mm2, 1415 cell/mm2, and 1168
cell/mm2 while the lowest mean was 220 cell/mm2, 326 cell/mm2, 420 cell/mm2,
and 365 cell/mm2, respectively.
Figure 1 Mean of neuronal cells death in hippocampus (dentate gyrus), amygdala,
and cerebral cortex
4
Control
P1
P2
P3
P
P
P4
Figure 2
Representative image of brain sections containing dentate gyrus in
each treatment stained by haematoxylin eosin. Dead neurons are
shown by arrow (bars: 50 µm)
Brain histology in dentate gyrus stained by haematoxylin eosin showed
normal and dead neuron. Dead neuron were showed by dark colour cell and nucleus
was unseen (Figure 2). Analysis of variance (ANOVA) showed that effect of bee
venom was significantly different in the number of neuronal cell death in dentate
gyrus (F = 7.040, p < 0.01). It means that bee venom has a role in neuronal cell
death regulation in dentate gyrus. Thus, the Duncan’s Multiple Range Test (DMRT)
5
is required to examine the effect of treatments in neuronal cells death regulation in
dentate gyrus.
The DMRT results showed that P4 significantly different to other
treatments. Treatment with the highest dose showed the highest number of neuronal
cells death in dentate gyrus. Based on the study, doses of bee venom gave
significant effect on neuronal cells death. Furthermore, the effect of bee venom
against neuronal cells death significantly was showed by P4 (7.48 mg/kg).
Control
P1
P
Figure 3
P4
P2
P3
Representative image of brain sections containing amygdala in each
treatment stained by haematoxylin eosin (bars: 100 µm)
Amygdala was also examined to show effect off bee venom to neuronal cells
death (Figure 3). Results of ANOVA showed that effect of bee venom was
significantly different in the number of neuronal cell death in amygdala (F = 3.940,
p < 0.05). It means that bee venom has a role in neuronal cell death regulation in
amygdala. The DMRT results showed that P4 was significantly different to other
treatments. Treatment with the highest dose showed the highest number of neuronal
cells death in amygdala. Based on the study, doses of bee venom gave the
significant effect on neuronal cells death. Significant effect of bee venom to
neuronal cells death was showed by P4 (7.48 mg/kg).
6
upper layer
deep layer
Figure 4
Representative image of brain sections containing cerebral cortex
stained by haematoxylin eosin (bar: 50 µm)
Brain histology in cerebral cortex stained by haematoxylin eosin (HE)
showed neuron morphology in each layer (Figure 4). ANOVA showed that
neuronal cell death density in cerebral cortex upper layer and deep layer was not
significantly different (upper layer: F = 2.196, p > 0.05; deep layer: F = 2.267, p
>0.05). It means that bee venom has no effect to regulate neuronal cell death in
cerebral cortex.
Spatial Learning
The exploration of mice to the Y maze arms was observed in this study. The
results showed differences in the mean of alteration percentage in mice behaviour.
The alteration percentage in Y maze varied in each treatment as compared to
control. The highest mean of alteration after 24 h bee venom injection was 74.57 %
and the lowest one was 61.07 %. The highest mean of alteration after 72 h bee
venom injection was 76.03 % and the lowest one was 57.50 %. The variation of
mean showed that bee venom has influence in alteration of behaviour in Y maze.
The variation can be seen in Figure 5.
7
Figure 5 Mean of correct alteration in Y maze arms
Results of ANOVA showed that effect of bee venom was not significantly
different to mice behaviour (after 24 hour: F = 0.856, p > 0.05; after 72 hour: F =
1.483, p > 0.05). It means that bee venom has no influence to mice behaviour. The
percentage of correct alteration in Y maze was congruent to the number of neuronal
cells death in the brain.
DISCUSSION
Effect of Bee Venom to Neuronal Cell Death
Based on this study, bee venom affected neuronal cell death in dentate gyrus.
DMRT test showed that the highest dose of bee venom was significantly different
to other treatments on neuronal cell death in dentate gyrus. Dose of 4 mg/kg body
weight of rats is the maximum dose of bee venom to anti-inflammation effect while
the higher dose shows neurotoxic effects (Bogdanov 2015). Figure 2 showed that
neuronal cells death found in subgranular zone (SGZ). According to study, we
proposed that dead neuron was neural progenitor cells (NPC) because it was found
in the innermost layer of SGZ. However, it needs special markers to confirm the
cell types.
We examined amygdala and cortex, other brain regions that relate to
hippocampus in regulation of memory. However, we found that bee venom-treated
mice influence neuronal cell death in amygdala but it was not significantly
difference in cerebral cortex. Although bee venom has no effect to cause neuronal
cell death in cerebral cortex, we could find that increasing in the number of neuronal
cells death in P4 compared to control (Figure 1). Cerebral cortex consists of six
8
layers (Figure 4). Each layer has kind of neuronal type and different functions
(Feldmeyer 2012). Layers III and V of the cerebral cortex contains a large number
of pyramidal neurons with high levels of acetylcholinesterase activity (Mesulam
and Geula 1991; Viswanathan et al. 2006) while layer II and IV receive information
from hippocampus (Gomez-Isla 1996). Cell death in tissues or organs can upset the
function. The redundant of neuronal cells death in brain can cause variety of
neurodegenerative diseases, such as parkinson (Ruberg et al. 1997; Anglade et al.
1997), alzeimer (Shimohama 2000; Bamberger and Landreth 2002), huntington
(Dean 2008). Taken together, these result suggested that bee venom treatment
contribute to the poor performance in learning and memory test.
Increasing in the number of neuronal cells in dentate gyrus, amygdala, and
cerebral cortex in P4 suggested as an effect of inflammatory compounds of the bee
venom. Previous studies have demonstrated the role of melittin, phospholipase A2,
apamin, and adolapin in high doses contained in bee venom can cause allergies,
lysis of erythrocytes, myonecrosis, and neurotoxic effects (Ovcharov et al. 1976;
Ownby et al. 1997; Raghuraman & Chattopadhyay 2006; Ali 2012; Abdu &
Alahmari 2013; Elhakim et al. 2014; Bogdanov 2015; Eze et al. 2016; Lee & Bae
2016). Therefore, bee venom can affect the peripheral tissues.
The influence of bee venom in peripheral tissues is expected to produce
cytokines which sends signals to the brain, so that it can cause neuronal cells death
in hippocampus (dentate gyrus), amygdala, and cerebral cortex. Dantzer et al.
(2008), Dilger & Johnson (2008), and Maier et al. (2012) demonstrated that
cytokines from peripheral tissues transported into the brain through the endocrine
system and induce neuronal cell death. Palombella & Vilcek (1989) reported that
melittin and phospholipase A2 can activate cytokines, which is TNF. Although it
still needs further confirmation, administration of bee venom is also supposed to
have indirectly effect on neuronal cells death in the brain through cytokines.
Another suggestion about the mechanism of neuronal cell death because of
bee venom, is the ability of bee venom compounds in passing through the blood
brain barrier (BBB). BBB plays an important role in regulating the molecule that
can be in and out of the brain (Ransohoff & Engelhardt 2012; Takeshita &
Ransohoff 2012). Oller-Salvia et al. (2013) and Mourre et al. (1997) reported that
apamin, compound of bee venom, can pass through the blood brain barrier and
cause neuronal cell death. However, other compounds of bee venom have not been
reported to pass through the BBB. Therefore, apamin suggested neuronal cells death
in brain.
Linear effect on neuronal cell death in brain regions was not affected by the
increasing dose of bee venom (Figure 1). The higher doses of bee venom did not
indicate higher number of neuronal cells death. In this study, P2 showed the number
of neuronal cells death increased compared to control, P1, and P3. Anomaly result
in P2 did not affect the conclusion of this study. It is caused the efficiency of bee
venom utilization depicted by P1. P1 has the lowest of neuronal cell death in brain
regions compared to the other treatments. Lee et al. (2004) and Lee et al. (2011)
reported that low dose of bee venom has antiinflammation effect. It is predicted to
increase neuronal durability and proliferation. However, neuronal proliferation
needs further assesment to know amount of new born cell. These results provide
straightforward information about efficiency of administration to the clinic
apiteraphy as well as people who want to do the bee sting therapy.
9
The Ability of Spatial Learning in Mice
Spatial learning ability of mice is shown by the correct-arm alternation in
Y-maze. The higher percentage of correct-arm alternation showed that the better
spatial memory in mice.Y-maze was used to examine spatial learning underlying
mice activity to explore new environment. Wolfe (1969) reported that mice prefers
to investigate new object and environment. ANOVA test showed that number of
neuronal cell death was not significantly different to alteration mice behaviour.
Conrad and Roy (1993) reported that as much as 80% of neuronal cell death in
dentate gyrus do not affect spatial memory. Nonetheless, the results showed that
bee venom administration tend to affect the spatial learning ability of mice (Figure
5).
The tendency of these effects can be caused by neuronal cells death in the
dentate gyrus. Dentate gyrus plays a role in memory formation, distinctively related
to cognitive function and spatial memory (Silva et al. 1998; Eichenbaum et al.
1999). The results showed that treatment with the most of dead neuron had the
lowest correct-arm alternation in Y-maze (Figure 5). We suggested that NPC was
dead neuron in SGZ. Dead or damage NPC causes the mature neuron will be
reduced while the mature neuron that still remain will undergo programmed cell
death. It will affect the process of learning and spatial memory in mice. Lawyer et
al. (2006), Plessen et al. (2006), Thomann et al. (2012), and Juliandi et al. (2015)
reported that the abnormal morphology in the hippocampus associated with the
reduce neurogenesis which correlated with the cognitive deficits.
Cognitive deficits associated with bee venom-treated might not due to the
abnormal morphology in hippocampus (dentate gyrus), there is possibility
responses in emotion and motoric function that caused by abnormal in amygdala
and cerebral cortex. We have shown neuronal cell death in amygdala and cerebral
cortex (upper and deep layer) (Figure 1). Preston and Eichenbaum (2013)
demonstrated that hippocampus and cerebral cortex engages to form meaningful
contexts in which relate to memory retrieval, as well as the amygdala. Amygdala
and hippocampus act synergistically when emotion and memory came together
(Richter-Levin and Akirav 2000; Phelps 2004). Hippocampus, amygdala, and
cerebral cortex have a role in limbic systems, link to another brain region.
Abnormalities in those region will affect the tissues functionally. Deficiency in
amygdala and cerebral cortex contributes to cognitive deficit. However, other brain
regions contribute to the deficits. It still needs further investigation to complete
possibility.
CONCLUSION
Based on the study, bee venom had neurotoxic effect because it caused
neuronal cell death in dentate gyrus, amygdala, and cerebral cortex as well as
behaviour alteration in mice. The dose of bee venom that caused the highest
neuronal cells death in dentate gyrus, amygdala, and cerebral cortex was P4 (7.48
mg/kg). Bee venom tends to affect spatial memory.
10
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BIOGRAPHY
Author was born in Prabumulih in October 2nd, 1991. Author is the
youngest child of four from the parents, Ali Imron and Mulyati. In 2009 author
studied in Sriwijaya University, Faculty of Teacher Training and Education, major
of Biology education and graduated from Sriwijaya University in 2013. In August
2014, author entered college in the Graduate School, Major of Animal Biosciences,
Bogor Agricultural University at its own expense, and in the third semester author
got thesis scholarship from LPDP.