Sample final draft
-
Upload
jasper-obico -
Category
Documents
-
view
663 -
download
4
Transcript of Sample final draft
ON THE POSSIBLE INVOLVEMENT OF AN OPIOIDERGIC MECHANISM IN THE ANTINOCICEPTIVE EFFECT OF ASPARTAME IN MICE (Mus musculus)
Maria Luisa J. AgravanteFaye B. Garciano
Submitted to theDepartment of Biology
College of Arts and SciencesUniversity of the Philippines Manila
Padre Faura, Manila
In partial fulfillment of the requirementsFor the degree of
Bachelor of Science BiologyMarch 2008
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
ii
Department Of BiologyCollege of Arts and Sciences
University of the Philippines – ManilaPadre Faura, Manila
Announcement ofUndergraduate Thesis Presentation
MARIA LUISA JOSE AGRAVANTEFAYE BONDOC GARCIANO
Entitled
ON THE POSSIBLE INVOLVEMENT OF AN OPIOIDERGIC MECHANISM IN THE ANTINOCICEPTIVE EFFECT OF ASPARTAME IN MICE (Mus musculus)
For the degree ofBachelor of Science in Biology
2:00 PM, 10 March 2008Room 11C, Rizal Hall
THESIS ADVISER THESIS CO-ADVISERMaria Ofelia M. Cuevas, M.S. Miriam P. de Vera, M.S.Associate Professor Assistant ProfessorDepartment of Biology Department of BiologyUP Manila UP Manila
THESIS READER THESIS READERElisa L. Co, Ph.D. Kimberly S. Beltran, M.S.Associate Professor InstructorDepartment of Biology Department of BiologyUP Manila UP Manila
Endorsed by: Authorized by:
Maria Ofelia M. Cuevas, M.S. Arnold V. Hallare, Ph.D.Chair ChairThesis Committee Department of BiologyCAS, UP Manila
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
iii
Department of BiologyCollege of Arts and Sciences
University of the Philippines – ManilaPadre Faura, Manila
ENDORSEMENT
The thesis attached hereto, entitled “On the Possible Involvement of an Opioidergic Mechanism in the Antinociceptive Effect of Aspartame in Mice” prepared and submitted by Maria Luisa Jose Agravante and Faye Bondoc Garciano, in partial fulfilment of the requirements for the degree of Bachelor of Science in Biology was successfully defended on March 10, 2008.
MARIA OFELIA M. CUEVAS, M.S. MIRIAM P. DE VERA, M.S. Thesis Adviser Thesis Co-Adviser
ELISA L. CO, Ph.D. KIMBERLY S. BELTRAN, M.S. Thesis Reader Thesis Reader
This undergraduate thesis is hereby officially accepted as partial fulfilment of the requirements for the degree of Bachelor of Science in Biology.
ARNOLD V. HALLARE, Ph.D. REYNALDO H. IMPERIAL, Ph.D.Chair DeanDepartment of Biology College of Arts and Sciences
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
iv
BIOGRAPHICAL DATA
I. Personal Information
Name: Maria Luisa Jose AgravanteNickname: RiaBirthday: March 20, 1986Birthplace: Manila, PhilippinesAddress: 25 St. Joseph Street, Paradise Village
Project 8, Quezon CityMobile number: 09189918319Email address: [email protected]: Julian Federico Agravante
Luz Mary Lou Jose Agravante
II. Educational Background
Primary: Colegio San Agustin (1993-2000)Dasmariñas Village, Makati City, Philippines
Secondary: Colegio San Agustin (2000-2004)Dasmariñas Village, Makati City, Philippines
Collegiate: University of the Philippines – Manila (2004-2008)Padre Faura Street, Ermita, Manila
III. Organizations
Member, Biological Sciences SocietyMember, Biology Majors’ AssociationMember, Quod Erat DemonstrandumMember, UP Forensic Society
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
v
BIOGRAPHICAL DATA
I. Personal Information
Name: Faye Bondoc GarcianoNickname: FayeBirthday: March 19, 1988Birthplace: Quezon City, PhilippinesAddress: 30 Libra Street Camella Homes II-B
Bacoor, CaviteMobile number: 09172414017Email address: [email protected]: Filomeno Rebano Garciano, Jr.
Corcini Bondoc Garciano
II. Educational Background
Primary: Southville International School (1994-1998, 1999-2000)B.F. Homes International, Las Piñas City
Philippine School Doha (1998-1999)Doha, Qatar
Secondary: Statefields School, Inc. (2000-2004)National Road, Molino III, Bacoor, Cavite
Collegiate: University of the Philippines Manila (2004-2008)Padre Faura Street, Ermita, Manila
III. Organizations
Vice President, Biology Majors’ AssociationMember, Biological Sciences SocietyMember, Quod Erat Demonstrandum
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
vi
ACKNOWLEDGEMENT
(Sgd.) (Sgd.) Maria Luisa J. Agravante Faye B. Garciano
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
vii
TABLE OF CONTENTS
TITLE PAGE ............................................................................................................. i
ANNOUNCEMENT .................................................................................................. ii
ENDORSEMENT ...................................................................................................... iii
BIOGRAPHICAL DATA .......................................................................................... iv
ACKNOWLEDGEMENT ......................................................................................... vi
TABLE OF CONTENTS ........................................................................................... vii
LIST OF TABLES..................................................................................................... viii
LIST OF FIGURES ................................................................................................... ix
LIST OF APPENDICES........................................................................................... . x
ABSTRACT ............................................................................................................... xi
INTRODUCTION..................................................................................................... 1
REVIEW OF RELATED LITERATURE ................................................................ 5
MATERIALS AND METHODS............................................................................. 9
RESULTS................................................................................................................. 13
DISCUSSION .......................................................................................................... 15
CONCLUSION........................................................................................................ 19
RECOMMENDATIONS......................................................................................... 20
LITERATURE CITED ............................................................................................. 21
APPENDICES........................................................................................................... 27
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
viii
List of Tables
PAGE
Table 1. Mean weights of mice negative control groups and aspartame-treatedgroups.................................................................................................................. 25
Table 2. Mean amount of aspartame solution consumed by mice...................... 25
Table 3. Mean index of analgesia (±S.E.M) of mice negative control groups and aspartame-treated groups…………………..…............................................ 25
Table 4. Mean differences of mice negative control groups and aspartame-treated groups………………….…………..………………………………….... 26
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
ix
List of Figures
PAGE
Figure 1. Mean index of analgesia of mice negative control groups andaspartame-treated groups…………………………………………………………. 28
Figure 2. Mean index of analgesia of mice negative control groups and aspartame-treated groups comparing the effect of aspartame after one and 14 days of administration. ………………………………………………………... 28
Figure 3. Mean index of analgesia of mice aspartame-treated groups comparing the effect of naloxone after one and 14 days of administration of treatment. ………………………………………………………………………. 29
Figure 4. Mean index of analgesia of mice negative control groups comparing the effect of naloxone after one and 14 days of administration of treatment. ………………………………………………………………………. 29
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
x
List of Appendices
PAGE
Appendix A1. Structure of Aspartame (N-L-aspartyl-L-phenylalanine-1-methyl ester)……………………………………………………………………...... 31
Appendix A2. Structure of Naloxone molecule…………………………………… 31
Appendix B. Table of analysis of variance for within subject effects…………….. 32
Appendix C. Mean values of index of analgesia (± S.E.M.) of mice treatmentgroups (for all trial runs)……………………………………………………………33
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
xi
ABSTRACT
To test whether aspartame-induced antinociception involves opioidergic mechanisms, male IRC mice received either distilled water or aspartame solution (0.16% w/v) and then subcutaneously injected with either saline or naloxone, an opioid receptor antagonist. Aspartame solution and distilled water were given for one day or 14 days. Latency responses, measured via the hot plate and the tail flick test methods, were normalized into an index of analgesia. Within-subject interaction effects were significant for the variables of aspartame treatment, behavioral test applied, duration of administration and presence of naloxone. The mean index of analgesia of aspartame-treated mice was higher by 93% compared to that of the negative controls following a long-term (14-day) duration of administration. In addition, the presence of naloxone was observed to inhibit aspartame-induced antinociception during the said period. On the other hand, following short-term (24-hour) duration of administration, the mean index of analgesia of water-treated mice given with naloxone was higher by 135% compared to those of the negative controls. The findings suggest that opioidergic mechanisms and both the spinal and supraspinal structures may be involved in aspartame-induced antinociception which could be influenced by the duration of aspartame treatment. However, the results do not negate the possible transient antinociceptive effect of naloxone in the absence of aspartame.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
1
INTRODUCTION
Background of the study
In recent years, much research has been conducted on the pain-relieving properties of
various substances. Sweet palatable substances such as sucrose, fructose, glucose, and saccharin,
have been reported to produce antinociception, a decrease in the response of an animal to
injurious stimuli. It is claimed that the mechanism of the analgesic effects produced by the
mentioned sweet substances may be attributed to their sweet taste, which triggers the release of
endogenous opiates in the body (Pepino and Mennella, 2005).
Aspartame is a non-caloric artificial sweetener that is widely used in a variety of food and
beverages. Discovered accidentally by American drug researcher James Schlatter in 1965, it goes
by the chemical name N-L-aspartyl-L-phenylalanine-1-methyl ester and is available
commercially under such brand names as NutraSweet® and Equal®. It is primarily composed of
two amino acids, aspartic acid and phenylalanine, wherein a liter of aspartame-sweetened soft
drink is said to contain about 400 mg of the former (Yellowlees, 1983; Romanowski, 2002). In
its pure form, aspartame appears as a white, odorless, crystalline powder and is said to be unique
for it provides sweetness without the caloric content that other sweeteners possess (Romanowski,
2002).
Since aspartame was made available commercially, much controversy has surrounded its
consumption. Articles and researches claim that the two amino acids in aspartame, phenylalanine
and aspartic acid, can damage the brain and cause other neurotoxic effects. However, these two
components of aspartame will only have adverse effects when taken by individuals suffering
from phenylketonuria and at very high doses (100mg/kg) (Stegink, 1980; Stegink, 1987). With
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
2
this, aspartame has been approved by the United States Food and Drug Administration as a
tabletop sweetener and is still widely used as of today (Romanowski, 2002).
Despite the abovementioned concerns, it has been suggested that aspartame exhibited
analgesic properties (Sharma, et al., 2005). Abdollahi and his collaborators suggested that the
inactivation of N-methyl-D-aspartate (NMDA) receptors by aspartame causes the analgesic
effect of the said compound (Abdollahi, et al., 2003). However, very little research has tested the
involvement of the endogenous opioid system in aspartame-induced antinociception. If naloxone,
an opioid receptor antagonist inhibits the antinociceptive effect of aspartame, then aspartame
induces a decrease in pain response by interacting with the endogenous opioid system.
Statement of the problem
Does the administration of aspartame in mice induce spinally-mediated antinociception
involving opioidergic mechanism?
Research objectives
This study aims to investigate whether the administration of aspartame induces opioid
receptor-mediated antinociception in mice based on behavioral response tests. It specifically
intends (1) to test whether the oral ingestion of aspartame ad libitum would result in the increase
in latency responses of male IRC mice; (2) to measure and compare the latency responses of
mice subjected to tests involving spinal and supraspinal structures; (3) to assess whether a short-
term and a long-term period of aspartame administration would increase pain tolerance in mice;
and (4) to measure and compare the latency responses, normalized to an index of analgesia, of
mice administered with an opioid receptor antagonist and a negative control.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
3
Significance of the study
Concerns have been addressed with regards to anesthetic agents such as the drug
meperidine, which are used in the treatment of pain, particularly post-operative pain. It is said
that the use of such anesthetic agents pose the possibility of incurring respiratory problems like
respiratory depression and apnea. This concept is of primary concern both in children and adults
(Ren, et. al., 1997). Thus, other treatments to pain such as sweet-taste analgesia are being looked
upon as alternatives. Moreover, aspartame is a promising alternative for sucrose in producing
sweet-taste analgesia particularly to patients who are at risk of obesity since aspartame is a non-
caloric sweetener (Romanowski, 2002). Aspartame analgesia may also be an alternative to
sucrose analgesia for patients with diabetes since aspartame does not affect blood sugar level
(Lean and Hankey, 2004). Moreover, the data that will be gathered in this experiment may shed
light on the modulatory effect of aspartame in neural paths and neurotransmitters involved in
pain nociception.
Scope and Limitations
The experimental design was based on a randomized complete block design involving
four factors: aspartame treatment, duration of administration of treatment, type of analgesic test,
and the presence or absence of an antagonist which is naloxone. Antinociceptive effects via
induction of pain tolerance in mice were evaluated through behavioral tests involving spinal and
supraspinal mechanisms without invasive clinical procedures. The said behavioral nociceptive
tests only used thermal noxious stimuli. In addition, this study tested the short-term and long-
term effects of aspartame administration within a two-week period per trial run. However, a
dose-dependent antinociceptive effect of aspartame was not conducted due to constraints in the
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
4
supply of opioid antagonist. The experiment utilized repeated measures. Moreover, this study
examined the involvement of opioidergic mechanism in aspartame-induced antinociception.
However, the type of opioid receptor that aspartame activates was not specified.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
5
REVIEW OF RELATED LITERATURE
Aspartame or N-L-aspartyl-L-phenylalanine-1-methyl ester (Appendix A1) is primarily
synthesized from phenylalanine and aspartic acid or aspartate. These two amino acids have a
sweet isomer and a bitter isomer. It is then a must for the appropriate isomers of phenylalanine
and aspartic acid to be used in synthesizing aspartame for it to be able to bind to the sweet
receptors of the tongue. This idea then accounts for the sweet taste of aspartame (Ophardt, 2003;
Romanowski, 2002).
Several researches have shown that sweet solutions, such as saccharin, sucrose, and
aspartame induce analgesia or antinociception. Analgesia is the absence of the sense of pain
without loss of consciousness. Antinociception, on the other hand, is defined as a decrease in
response of the sensory systems of the body to harmful or painful stimuli. Saccharin, the oldest
known artificial sweetener that goes under the brand name Sweet ‘N Low® by Cumberland
Packing Corporation in Brooklyn, New York, USA, has been shown to increase pain tolerance of
rats subjected to the hot plate test, which is a behavioral nociceptive test (Segato, et al., 1997).
Several studies regarding the analgesic effects of sucrose have also been conducted. Segato and
his team (1997) showed that orally-administered sucrose produced an increase in pain tolerance
in male albino Wilstar rats subjected to the tail flick test. Moreover, it was found that oral
administration of sucrose provided an increase in tolerance to persistent pain and hyperalgesia in
infant rats (Ren, et al., 1997).
Studies on the analgesic properties of sucrose have also been conducted in humans.
Johnston and others (2002) showed that routine intake of sucrose increased pain tolerance in
infants less than 31 weeks of age. The said infants were able to tolerate pain caused by invasive
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
6
and non-invasive but uncomfortable medical procedures such as injections and endotracheal tube
suctioning, respectively. Similar findings were shown by Ramenghi and his collaborators (1999),
wherein human infants of about 32 to 36 weeks showed an increase in pain tolerance to heel
pricks when administered with sucrose.
Aspartame has also been found to have analgesic properties. According to Abdollahi and
his team (2001), aspartame is able to control pain-related behavior through a nitric oxide/cyclic
guanosine monophosphate (cGMP)/glutamate release cascade by inactivating NMDA (N-
methyl-D-aspartate) receptors. NMDA receptors are ionotropic receptors for glutamate whose
activation results in the opening of an ion channel that is nonselective to cations. In addition,
another study by Abdollahi and others (2003) suggested that since aspartate, one of the major
components of aspartame is an excitatory amino acid (EA) like glutamate, it can interact with
NMDA receptors and modulate pain sensation.
Pepino and Mennella (2005) suggested that the analgesic effect of the previously
mentioned sweetening agents is said to involve afferent signals from the sense organ (Pepino and
Mennella, 2005). Ramenghi and his collaborators (1999) provided evidence wherein the direct
gastric loading of sucrose yielded no analgesic effects to human infants as compared to orally
administered sucrose wherein analgesia was observed. Also, the release of endogenous opiates is
said to be triggered by the taste of sweet substances since naloxone, a competitive opioid
receptor antagonist was said to be able to inhibit the analgesic effects of sucrose in rats (Segato,
et al., 1997). Naloxone acts by binding to the opioid receptors with a greater affinity than the
agonists for a specific binding site. It blocks the receptor so that the agonists cannot bind and
activate it (Sauro and Greenberg, 2005). However, it is a non-specific opioid antagonist. This
means that it can bind to any of the three types of opioid receptor, namely μ, δ, and κ. The
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
7
studies that were mentioned used the cold pressor, heel prick, and tail flick tests to assess the
antinociceptive properties of sweetening agents (Pepino and Mennella, 2005).
Typical behavioral tests that can be used to measure antinociception include the warm
water immersion tail flick, hot plate, acetic acid-induced writhing, and formalin tests. The warm
water immersion tail flick and hot plate tests are used to measure, via thermal stimulus of
cutaneous and short origin, the antinociceptive effect of different substances involving spinal and
supraspinal mechanisms respectively (South and Smith, 1998). The acetic-acid-induced writhing
and formalin tests on the other hand, use chemical stimuli (Chapman and Loeser, 1989).
The acetic acid-induced writhing test is a reflexive model wherein acetic acid is injected
into the animal and writhing or dorsoflexion of the back, stretching of hind limbs, and abdominal
contraction is considered as the animal’s pain response. In this model, the stimulus is applied for
a longer period of time (approximately 60 minutes). However, ethical issues are raised with
regards to this antinociceptive test since the pain stimulus (acetic acid) is applied for a longer
period of time and renders the animal unable to escape from the pain induced (Chapman and
Loeser, 1989).
One counterpart of the abovementioned analgesic test is the formalin test. It is similar to
the type and duration of stimulus application in the acetic acid-induced writhing test. Like the
hot plate test, it is an organized behavioral measure of nociception. The procedure involves a
dilute solution of formalin which is injected subcutaneously into one of the hind paws of the
animal. To measure the response in mice, the time spent licking or biting the injected part of the
foot is recorded (Chapman and Loeser, 1989).
The tail flick test is a reflexive measure of nociception wherein the reflex removal of the
tail is considered as a response (Chapman and Loeser, 1989). The hot plate test, on the other
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
8
hand, is an organized behavioral measure of nociception, wherein behavior toward a painful
stimulus is considered. This technique is also said to be the complement of the tail flick test,
since the hot plate test uses a thermal noxious stimulus as does the tail flick test. However, this
method involves supraspinal structures instead of spinal (South and Smith, 1998).
Since the first two tests mentioned, which use chemical stimuli pose ethical concerns, the
tail flick and hot plate tests were chosen for this present study to test whether the short-term and
long-term administration of aspartame would result in the decrease in the latency responses of
male IRC mice. Lastly, since no previous report has suggested the involvement of endogenous
opioid mechanisms in aspartame-induced antinociception, this present study will look into the
inhibitory effect of naloxone on the antinociceptive effects of aspartame, as an increase in
latency response of mice will indicate the involvement of endogenous opioid receptors.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
9
MATERIALS AND METHODS
Experimental Animals
Forty-eight male IRC mice with a mean weight of 18 (± 0.4) grams each, purchased from
the Bureau of Animal Industries (BAI), Diliman, Quezon City, served as the test subjects in this
study.
Each mouse was weighed before acclimatization, and once a week after the habituation
period. The mice were housed two to a cage with a 12:12 hour light/dark cycle, with food and
water available ad libitum. In the subsequent experiment proper, the treatment solutions were
placed in feeding bottles and replaced each week. The amount of treatment solution consumed
by the treatment animals was estimated per day by weighing each feeding bottle at the start of
the day. To account the consumption of each mouse, the amount of treatment solution consumed
was divided by two since the mice were distributed two to a cage with one feeding bottle per
cage.
Chemicals
A one-kilogram pack of aspartame (99% purity) was purchased from Baler Industrial
Corporation in Quezon City. Normal saline solution (0.9% NSS) from a local medical supplies
store in Bambang, Manila and naloxone solution (0.4 mg/mL) from San Juan De Dios Hospital
in Pasay City were also purchased for the experiment.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
10
Experiment Proper
Three batches of test animals were used in this study, wherein a batch pertains to a single
run of the experiment. A period of roughly one to two months was observed between each run.
1. Treatment Groups
A single experimental run involved 16 mice randomly distributed into four groups of four
mice each. These four groups were designated as follows: (1) WS, which received distilled water
ad libitum, and was subcutaneously injected with saline, (2) WN, which received distilled water
ad libitum, and was subcutaneously injected with naloxone, (3) AS, which received aspartame
solution (0.16% w/v) ad libitum and was given saline, (4) AN, which received aspartame
solution (0.16% w/v) ad libitum and was given naloxone, where W is water, A is aspartame, S is
saline, and N is naloxone. Each group was then subdivided into two wherein the latency response
of the first subgroup was measured via hot plate, while that of the second subgroup was
measured via tail flick methods.
All mice were acclimatized for one week under standard laboratory conditions prior to
experimentation. The mice were then subjected to a habituation period for three days. This was
done in order for the mice to be accustomed to the researcher and to either the hot plate or the tail
flick apparatus. The mice in the first subgroup were habituated by placing each mouse on top of
the hot plate apparatus for thirty seconds. On the other hand, the mice in the second subgroup
were habituated by placing each mouse inside the restraining plastic cylinder and immersing the
tip of its tail in water for seven seconds.
On the day after the habituation period, baseline latencies were measured for each mouse.
Distilled water in feeding bottles was placed in the cages of the WN and WS groups, while
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
11
aspartame dissolved in drinking water (aspartame solution) in feeding bottles was placed in the
cages of the AN and AS groups.
Each mouse was then subjected to two latency measurements: the first conducted after
one day of distilled water or aspartame solution consumption, and the second conducted after 14
days. Latency responses were measured by either the hot plate test or the tail flick method.
2. Hot Plate Test
The hot plate test was performed as conducted by South and Smith (1998). The first
subgroups of mice (eight per trial run) were subjected to this test. Ten minutes prior to hot plate
latency (HPL) measurement, the WN and AN groups were subcutaneously injected with
naloxone solution (1mg/kg) while the WS and AS groups were subcutaneously injected with
saline. Each mouse was then placed on the hot plate apparatus heated to a temperature of 40 ±
0.5 ºC, which was surrounded by a transparent enclosure. The timer was stopped automatically
and the mouse was removed once the first behavioral sign of nociception (i.e. licking a hind paw,
vocalization, escape response) was observed and the duration of latent response (in seconds) of
which was recorded. When no sign of nociception is observed within 30 seconds, the mouse was
removed from the hot plate apparatus to prevent paw tissue damage. The period of 30 seconds
was then considered to be its HPL. Each HPL measurement was normalized by an index of
analgesia (IA) using the formula:
where IA is the Index of Analgesia, HPLtest is the hot plate latency after treatment, HPLcontrol is the
average of three hot plate latencies before treatment. The value 30 refers to the maximum time
(in seconds) of exposure to the thermal stimulus.
3. Tail Flick Method
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
12
The tail flick method was performed as conducted by Tabarelli and his collaborators
(2003). The second subgroups of mice (eight per trial run) were subjected to this method. Ten
minutes before tail flick latency (TFL) measurement, the WN and AN groups were
subcutaneously injected with naloxone while the WS and AS groups were subcutaneously
injected with saline. Then, each mouse was placed inside a plastic tube and the last three
centimeters of its tail was submerged into a hot water bath with a temperature of 52 ± 0.5 ºC.
The timer was stopped and the tail of the mouse was removed from the water bath once its tail
flicked in response to the thermal stimulus, the duration of latent response (in seconds) of which
was recorded. When the mouse failed to flick its tail within seven seconds, the tail was removed
from the water bath to prevent skin damage and the period of seven seconds was considered to
be its TFL. Each TFL measurement was normalized by an index of analgesia (IA) using the
formula:
where IA is the Index of Analgesia, TFLtest is the tail flick latency after treatment, TFLcontrol is the
average of three tail flick latencies before treatment. The value 7 refers to the maximum time (in
seconds) of exposure to the thermal stimulus.
Statistical analysis
All values were expressed as mean ± standard error of mean (S.E.M.). The data gathered
were subjected to repeated measures analysis of variance (ANOVA). The level of statistical
significance was set at p ≤ 0.5 level. The software Statistical Package for the Social Sciences
(SPSS) version 15.0 for Windows Operating System was the computation package utilized in the
study.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
13
RESULTS
Measured Weights and Consumption Rates
During the course of each experimental run, the weight of each experimental mouse was
measured. On the average, each mouse showed a 23% increase in weight from the first day to the
fifteenth day of the experiment proper, and a 51% increase in weight from the first day to the last
day of the experiment proper (Table 1). In addition, throughout the course of the experiment
proper, the AS group consumed an average of 14.85 ml aspartame solution while the AN group
consumed an average of 11.92 of the said solution (Table 2).
Interaction Effect
The hot plate and the tail flick tests were used to assess the antinociceptive effect of
aspartame whether it involves supraspinal or spinal structures. The Analysis of Variance for
within subject effects showed that the interaction of duration, treatment (with or without
aspartame), behavioral test, and naloxone administration was not significant. The interaction
effect of variables, namely duration, treatment, naloxone, and experimental run was also not
significant (Appendix B).
In the subsequent sections, the effect of the particular independent variables involved has
incorporated the effect of the type of behavioural test employed.
Effect of Treatment and Duration of Administration
Aspartame treated groups administered with saline and negative control groups were
compared to test whether aspartame induces antinociception in mice. Following a 24-hour (short-
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
14
term) administration of treatment, results showed that the mean index of analgesia of the
aspartame treated groups AS did not differ significantly with that of the negative control groups
WS. On the other hand, for the 14-day (long-term) administration the reverse effect was
observed wherein the mean index of analgesia of the aspartame treated group S was higher by
95% compared to the negative control group WS14 (Table 3, Figures 1 and 2).
In order to test whether the antinociceptive effect of aspartame depends on the duration of
administration, the effect of aspartame after 24-hour administration of treatment was compared
to its effect after 14-day administration. Unlike in the 24-hour administration of treatment,
aspartame-induced antinociception was observed after 14 days of treatment since the mean index
of analgesia of the AS group was higher than that of the WS group after 14 days of
administration of treatment, while the mean index of analgesia of the AS group did not differ
significantly from that of the WS group after one day of administration of treatment (Table 3,
Figure 1 and 2).
Effect of Naloxone
Naloxone, an opioid receptor antagonist was used to test whether the antinociceptive
effect of aspartame involves opioidergic mechanism. If naloxone inhibits the antinociceptive
effect of aspartame, then aspartame-induced antinociception involves opioidergic mechanism.
Results show that following a 14-day administration of aspartame, the mean index of analgesia
of the AS group was higher by 93% compared to the AN group, the mean difference of which
lies within a 95% confidence interval of 0.046 to 0.704 (Table 4, Figures 1 and 3). However,
when treatment was administered for only one day, the mean latency response of the WN group
was higher by 135% compared to the WS group (Table 3, Figures 1 and 4).
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
15
DISCUSSION
Spinal and supraspinal structures may be involved in pain response or nociception
(Chapman and Loeser, 1989). The mechanism of aspartame-induced antinociception may be
assessed through the use of two different types of behavioral nociceptive tests. In the tail flick
test, the pain response is said to be processed at the spinal level whereas in the hot plate test,
nociception is mediated by supraspinal structures. Since the interaction effect of duration,
treatment (with or without aspartame), behavioral test, and naloxone administration was not
significant, the antinociceptive effect of aspartame involves both supraspinal and spinal
structures. Moreover, since the interaction effect of duration, treatment, naloxone, and
experimental run was not significant, the effect of all treatment combinations is consistent in all
three runs. Thus, it can be inferred that the test subjects are homogenous and extraneous
variables are minimal.
Regardless of the type of behavioral nociceptive test applied, the 14-day administration
of 0.16% (w/v) aspartame solution produced an observable antinociception in mice implying that
spinal and supraspinal structures are involved in aspartame-induced antinociception as was
mentioned in the previous paragraph. In accordance with the data, there are findings that show
that sweetening agents such as aspartame boost morphine-induced antinociception (Abdollahi, et
al., 2003). However, no antinociceptive effect of aspartame was detected after a short period of
24-hour administration only. Since the 24-hour and 14-day treatment administration were used
to compare the short and long-term effects of aspartame on antinociception, this result signifies
that the antinociceptive effect of aspartame is dependent on the duration of administration, which
was also observed for sucrose (Segato, et al., 1997).
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
16
The compound naloxone, an opioid receptor antagonist, was observed to inhibit the
antinociceptive effect of aspartame since the mean index of analgesia of the AS14 group is
higher compared to the AN14 group. These findings suggest that following the long-term
administration of aspartame, the antinociceptive effect of the said substance may involve
opioidergic mechanisms. These results are supported by the report of Segato and his
collaborators (1997) who revealed that antinociception induced by sweetening agents may
involve endogenous opioids.
The taste buds of the tongue are the primary sense organs which perceive gustatory
sensation. The facial nerve (cranial nerve VII) innervates the anterior two-thirds of the tongue,
while the glossopharyngeal nerve (cranial nerve IX) innervates the posterior third. The vagus
nerve (cranial nerve X) on the other hand, innervates the other taste buds. Taste information such
as sweet taste is then carried from the three mentioned cranial nerves to the nucleus of the
solitary tract which is part of the brain stem. From the said structure, the information is relayed
to certain regions of the brain, including the thalamus, cerebral cortex, amygdala, and
hypothalamus (Weiner, et al., 2003).
The hypothalamus contains the endogenous opioid β-endorphin (Shigeru, et al., 2003).
Nikfar and his peers (1997) reported that the antinociceptive effect of sweet substances such as
saccharin and sucrose was accompanied by an increase in the levels of β-endorphins in the
hypothalamus. Moreover, Taddio et al. (2003) stated that the sweet taste of sucrose triggered the
release of the said endogenous opioid. After being released, endogenous opioids bind to their
receptors. When β-endorphins bind to opioid receptors located in presynaptic terminals, a
decrease in calcium ion influx would result. The action potential brought about by pain stimulus
would then be less generated, thereby producing antinociception. On the other hand, when β-
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
17
endorphins bind to opioid receptors found in the postsynaptic terminals, there will be a resultant
increase in potassium ion efflux. This phenomenon then leads to postsynaptic hyperpolarization,
thus yielding a decrease in action potential generation brought about by pain stimuli.
Antinociception will thus occur (Golan, 2007). Therefore, when β-endorphin binds to opioid
receptors, modulation of pain is achieved (Kruger, 2001). Furthermore, the receptors of β-
endorphin is said to be located both in the brain and spinal cord, thereby suggesting the
involvement of spinal and supraspinal structures (Law, et al., 1979; Ferrara and Li, 1980).
Antinociception was also observed in water-treated, naloxone-administered groups for
the 24-hour administration of treatment since the mean index of analgesia of the WN1 group is
higher than that of the WS1 group. This suggests that naloxone administered at 1 mg/kg
subcutaneously may produce an analgesic effect in mice in the absence of aspartame. Atamer-
Simsek and his team (2000) reported that naloxone administered intraperitoneally at 2 mg/kg
was able to produce an antinociceptive effect by itself. In addition, Tsuruoka and his team (1998)
and Walker and her collaborators (1994) observed that antinociception induced by naloxone
administered intraperitoneally at 5 µg/kg and 5 mg/kg may involve serotonergic mechanisms.
Yohimbine, pirenperone, and ritanserin, which are antagonists of serotonergic receptors, were
able to reverse naloxone-induced antinociception. However, methiothepin, a 5-HT1 serotonergic
receptor antagonist, and MDL 72222, a 5-HT3 serotonergic receptor antagonist, did not inhibit
the antinociceptive effect of naloxone. These results suggest that the binding of naloxone to
opioid receptors induce antinociception involving 5-HT2 receptors (Walker, et al., 1994).
The hypothesis is that, naloxone, in acting on opiate receptors, may release serotonin (5-
hydroxytryptamine or 5-HT), which is involved in the regulation and processing of nociception
(Diaz-Reval, et al., 2002). The periaqueductal grey area (PAG) is the key part in the descending
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
18
inhibitory pathways, which regulate antinociception, in the central nervous system. Information
from different brain regions is received by the PAG, which is considered as the entry in the
modulation of nociception, particulary in the dorsal horn. The nucleus raphe magnus (NRM) and
some fibers in the spinal cord which has synaptic connections with the dorsal horn interneurons,
are primarily stimulated by PAG. The chemical 5-HT is the main neurotransmitter at these
synapses. Transmission in nociceptive pathways is then inhibited when the pathway from the
NRM to the substantia gelatinosa of the dorsal horn is activated due to the increase in the 5-HT
levels (Duman, et al., 2004).
Antinociception induced by aspartame, involving opioidergic mechanism, could be a
potential therapy for acute pain. It can be an alternative for sucrose in providing sweet-taste
analgesia to obese patients who require a low calorie diet. However, since aspartame contains
phenylalanine, it cannot be used on patients with phenylketonuria wherein elevation of blood
phenylalanine can be dangerous. Further studies are still needed in order to discover the actual
therapeutic place of aspartame in pain therapy for humans.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
19
CONCLUSION
Aspartame-induced antinociception via supraspinal and spinal structures involves
opioidergic mechanisms. In addition, the effect of aspartame was consistent in all three trial runs
in the present investigation. As far as the present findings are concerned, aspartame would
exhibit an antinociceptive effect following a relatively long period of administration of
treatment.
Furthermore, for a brief duration wherein treatment was administered for one day,
naloxone in the absence of aspartame administration can induce temporary antinociception in
mice. Since the observed antinociceptive effect of naloxone is very transient, it is not an ideal
alternative for pain medication. Reduction in pain response produced by aspartame could be
used as potential therapy for acute pain, which may be an alternative for sucrose in providing
sweet-taste analgesia to obese patients who require a low calorie diet.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
20
RECOMMENDATIONS
The possible areas for further investigation involving aspartame antinociception are the
following: first, the treatment (aspartame and control vehicle) should be administered with the
use of a gavage in order to ensure an even administration of treatment. Second, a positive control
group such as mefenamic acid may be included in the experiment for comparison with
aspartame. Third, the sample size should be increased to guarantee statistical precision of
experimental design. Fourth, the antinociceptive effect of aspartame should also be tested in a
chronic pain model wherein nociception is persistent such as carrageenan-induced inflammation
to assess whether aspartame can be used for pain relief in arthritis and other related health
problems. Lastly, other opioid receptor antagonists such as naltrindole and nor-binaltorphimine
should be used to identify the specific opioid receptor involved in aspartame-induced
antinociception, whereas proglumide and theophylline should be used in order to uncover other
possible mechanisms by which aspartame induce antinociception.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
21
LITERATURE CITED
Abdollahi, M., S. Nikfar and N. Abdoli. 2001. Potentiation by nitric oxide synthase inhibitor and calcium channel blocker of aspartame-induced antinociception in the mouse formalin test. Fund. Clin. Pharmacol. 15: 117-23.
Abdollahi, M., J. Sarrafzadeh, S. Nikfar and F. Roshanzamir. 2003. Mechanism of aspartame-induced antinociception in mice. Indian J. Pharmacol. 35: 37-41.
Atamer-Simsek, S., H. Olmez-Salvarli, O. Guc and L. Eroglu. 2000. Antinociceptive effect of amikacin and its interaction with morphine and naloxone. Pharmacol. Res. 41: 355-360.
Chapman, C.R. and J.D. Loeser. 1989. Issues in pain measurement. Raven Press. New York.
Diaz-Reval, M.I., R. Ventura-Martinez, M. Deciga-Campos, J.A. Terron, F. Cabre and F.J. Lopez-Munoz. 2002. Involvement of serotonin mechanisms in the antinociceptive effect of S(+)-ketoprofen. Drug Dev. Res. 57: 187-192.
Duman, E.N., M. Kesim, M. Kadioglu, E. Yaris , N.I. Kalyoncu and N. Erciyes. 2004. Possible involvement of opioidergic and serotonergic mechanisms in antinociceptive effect of paroxetine in acute pain. J. Pharmacol. Sci. 94: 161-165.
Ferrara, P. and C.H. Li. 1980. B-endorphin: characteristics in binding sites of rabbit spinal cord. Proc Natl Acad Sci. 77: 5746-5748.
Golan, D.E. 2007. Principles of pharmacology: The pathophysiologic basis of drug therapy. Lippincott Williams and Wilkins. Maryland.
Horne, J., H.T. Lawless, W. Speirs and D. Sposat. 2002. Bitter taste of saccharin and acesulfame-K. Chem. Senses 27: 31-38.
Johnston, C.C., et al. 2002. Routine sucrose analgesia during the first week of life in neonates younter than 31 weeks’ postconceptional age. Pediatrics 110: 523-528.
Kruger, L. 2001. Methods in pain research. CRC Press. Florida.
Law, P., H.H. Lo and C.H. Li. 1979. Properties and localization of β-endorphin receptor in rat brain. Proc. Natl. Acad. Sci. 76: 5455-5459.
Lean, M.E.J. and C.R. Hankey. 2004. Aspartame and its effects on health. Br. Med. J. 329: 755-756.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
22
Nakashima, K. and Y. Ninomiya. 1998. Increase in inositol 1,4,5-triphosphate levels of the fungiform papilla in response to saccharin and bitter substances in mice. Cell Physiol. Biochem. 8: 224-230.
Nikfar, S., M. Abdollahi, F. Etemad and M. Sharifzadeh. 1997. Effects of sweetening agents on morphine-induced antinociception in mice by formalin test. Gen. Pharmacol. 29: 583-586.
Ong, B. and R. Segstro. 1994. Respiratory depression associated with meperidine spinal anaesthesia. Can. J. Anaesth. 41: 725-727.
Pepino, M.Y. and J.A. Mennella. 2005. Sucrose-induced analgesia is related to sweet preferences in children but not adults. J. Pain. 119: 210-218.
Porth, C. Pathophysiology: Concepts of altered health states. 7th ed. Lippincott Williams and Wilkins. Maryland.
Ramenghi, L.A., D.J. Evans and M.I. Levene. 1999. “Sucrose analgesia”: Absorptive mechanism or taste perception? Arch. Dis. Child Fetal Neonatal Ed. 80: 146-147.
Ren, K., E.M. Blass, Q-q. Zhou and R. Dubner 1997. Suckling and sucrose ingestion suppress persistent hyperalgesia and spinal Fos expression after forepaw inflammation in infant rats. Proc. Natl. Acad. Sci. 94: 1471-1475.
Sauro, M.D. and R.P. Greenberg. 2005. Endogenous opiates and the placebo effect: A meta-analytic review. J. Psychosom. Res. 58: 115-120.
Segato, F.N., C. Castro-Souza, E.N. Segato, S. Morato and N.C. Coimbra. 1997. Sucrose ingestion causes opioid analgesia. Braz. J. Med. Biol. Res. 30: 981-984.
Sharma, S., N.K. Jain and S.K. Kulkarni. 2005. Possible analgesic and anti-inflammatory interactions of aspartame with opioids and NSAIDs. Indian J. Exp. Biol. 43: 498-502.
Shigeru, A., A. Kazuhito, H. Hiroyuki, H. Tadashi and S. Michio. 2003. Enhancement of beta-endorphin levels in rat hypothalamus by exercise. Jpn. J. Phys. Fitness Sports Med. 52: 159-166.
South, S.M. and M.T. Smith. 1998. Apparent insensitivity of the hotplate latency test for detection of antinociception following intraperitoneal, intravenous or intracerebroventricular M6G administration to rats. J. Pharmacol. Exp. Ther. 286: 1326-1332.
Stegink, L.D., L.J. Filer, G.L. Baker and J.E. Mcdonnell. 1980. Effect of an abuse dose of aspartame upon plasma erythrocyte levels of amino acids in phenylketonuric heterozygous and normal adults. Nutrition 110: 2216-2224.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
23
Stegink, L.D., L.J. Filer, E.F. Bell, and E.E. Ziegler. 1987. Plasma amino acid concentrations in normal adults administered aspartame in capsules or solution: lack of bioequivalence. Metabolism 36: 507-512.
Tabarelli, Z., D.B. Berlese, P.D. Sauzem, C.F. Mello and M.A. Rubin. 2003. Antinociceptive effects of Cremophor EL orally administered to mice. Braz. J. Med. Biol. Res. 36: 119-123.
Taddio, A., V. Shah, P. Sha and J. Katz. 2003. Β-endorphin concentration after administration of sucrose in preterm infants. Arch. Pediatr. Adolesc. Med. 157: 1071-1074.
Tsuruoka, M., Y. Hiruma, K. Matsutani and Y. Matsui. 1998. Effects of yohimbine on naloxone-induced antinociception in a rat model of inflammatory hyperalgesia. Eur. J. Pharmacol. 348: 161-165
Wall, P.D. and R. Melzack. 1994. Textbook of pain. 3rd ed. Longman Group. New York.
Walker, M.J., C.X. Poulos and A.D. Le. 1994. Effects of acute selective 5-HT1, 5-HT2, 5-HT3
receptor and α2 adrenoreceptor blockade on naloxone-induced antinociception. Psychopharmacology. 113: 527-533.
Weiner, I.B., D.K. Freedheim, J.A. Shinka, and W.F. Velicer. 2003. Handbook of psychology. John Wiley and Sons. Singapore.
Yellowlees H. 1983. Aspartame. Br. Med. J. 287: 912-913.
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
24
TABLES
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
25
Table 1. Mean weights of (±S.E.M.) mice negative control groups and aspartame-treated groups.
Treatment Groups Initial Weight Weight at Day 15 Weight at Day 28
AS 18.84167 ± 0.90942 24.34167 ± 0.95374 27.85000 ± 1.06867
WS 18.16667 ± 0.75321 22.67500 ± 1.08321 27.39091 ± 1.08321
AN 17.71667 ± 0.81861 22.95000 ± 0.62468 26.69167 ± 0.67974
WN 17.52500 ± 0.96767 23.21667 ± 0.86013 26.98333 ± 0.068753W, water; A, aspartame; S, saline, N, naloxone.
Table 2. Mean amount of aspartame solution consumed by mice treatment groups.
Treatment Groups Amount of Aspartame solution consumed (ml)
AS 14.847045 ± 2.18144
AN 11.92449 ± 1.06454A, aspartame; S, saline, N, naloxone.
Table 3. Mean index of analgesia (±S.E.M.) of mice negative control groups and aspartame-treated groups.
Duration of Administration of Treatment
Water AspartameSaline Naloxone Saline Naloxone
1 day -0.46845 ± 0.34076
0.16445 ± 0.16055
-0.08608 ± 0.06012
-0.12643 ± 0.24182
14 days -0.64620 ± 0.38005
-0.19701 ± 0.22795
-0.02777 ± 0.11212
-0.40277 ± 0.27306
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
26
Table 4. Mean differences of mice negative control groups and aspartame-treated groups.
95% Confidence IntervalTreatment Groups Mean Difference Lower Bound Upper boundAS (1-d) versus WS (1-d) 0.38237 -0.00321 0.76795AS (14-d) versus WS (14-d) 0.61843§ 0.17688 1.05998AS (1-d) versus AN (1-d) 0.04035 -0.23732 0.31801AS (14-d) versus AN (14-d) 0.37499§ 0.04606 0.70393WS (1-d) versus WN (1-d) 0.63290§ 0.21315 1.05265WS (14-d) versus WN (14-d) 0.44919 -0.04465 0.94303W, water; A, aspartame; S, saline, N, naloxone; 1-d, one day; 14-d, 14 days.§Mean difference is significant at α = 0.05
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
27
FIGURES
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
28
Figure 1. Mean index of analgesia of mice negative control groups and aspartame-treated groups. W, water; A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *T = differences in treatment effect are significant; *A = differences in antagonist effect are significant.
Figure 2. Mean index of analgesia of mice negative control groups and aspartame-treated groups comparing the effect of aspartame after one and 14 days of administration. W, water;
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
29
A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *T = differences in treatment effect are significant.
Figure 3. Mean index of analgesia of mice aspartame-treated groups comparing the effect of naloxone after one and 14 days of administration of treatment. A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *A = differences in antagonist effect are significant.
Figure 4. Mean index of analgesia of mice negative control groups comparing the effect of naloxone after one and 14 days of administration of treatment. W, water; S, saline, N,
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
30
naloxone. Range bars represent S.E.M. for 12 mice in each group. *A = differences in antagonist effect are significant.
APPENDICES
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
31
Appendix A1. Structure of Aspartame (N-L-aspartyl-L-phenylalanine-1-methyl ester)
Appendix A2. Structure of Naloxone molecule
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
32
Appendix B. Table of analysis of variance for within subject effects.
Source
Type III Sum of Squares df
Mean Square F P
Duration 1.072 1 1.072 18.879 .000
Duration * Treatment .104 1 .104 1.824 .192
Duration * Test .974 1 .974 17.153 .001
Duration * Naloxone .039 1 .039 .696 .414
Duration * Run .032 2 .016 .278 .760
Duration * Treatment * Test .004 1 .004 .066 .800
Duration * Treatment * Naloxone .250 1 .250 4.400 .049
Duration * Test * Naloxone .062 1 .062 1.098 .307
Duration * Treatment * Test * Naloxone .233 1 .233 4.103 .056
Duration * Treatment * Run .365 2 .182 3.212 .062
Duration * Test * Run .115 2 .058 1.016 .380
Duration * Treatment * Test * Run .271 2 .136 2.387 .118
Duration * Naloxone * Run .264 2 .132 2.325 .124
Duration * Treatment * Naloxone * Run .075 2 .037 .660 .528
Duration * Test * Naloxone * Run .544 2 .272 4.787 .020
Duration * Treatment * Test * Naloxone * Run
.140 1 .140 2.464 .132
Error(Duration) 1.135 20 .057
Antinociceptive Effect of AspartameAgravante and Garciano, 2008
33
Appendix C. Mean values of index of analgesia (± S.E.M.) of mice treatment groups(for all trial runs)
RunType of
Behavioral Test
1-Day 14-DayWater Aspartame Water Aspartame
Naloxone Saline Saline Naloxone Naloxone Saline Saline Naloxone
First
Hot plate 1.00±0.00
-1.62±0.10
0.07±0.20
0.58±0.42
0.16±0.01
-2.68±.
0.40±0.15
-0.46±0.17
Tail flick 0.04±0.10
-0.01±0.00
0.06±0.08
0.25±0.24
-0.01±0.08
-0.22±0.14
0.06±0.05
0.69±0.31
Second
Hot plate . -3.15±.
-0.32±.
-1.66±0.74
. -3.51±.
-1.04±.
-1.95±1.00
Tail flick 0.00±0.08
-0.03±0.05
0.06±0.01
0.06±0.02
0.02±0.05
-0.13±0.03
0.00±0.05
-0.13±0.05
Third
Hot plate -0.22±0.68
0.70±0.28
-0.23±0.13
0.11±0.00
-1.12±1.31
0.20±0.08
-0.02±0.05
-0.39±0.25
Tail flick 0.00±0.02
-0.03±0.03
-0.15±0.24
-0.10±0.04
-0.04±0.01
0.07±0.04
-0.18±0.14
-0.07±0.08