|
 
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| ORIGINAL ARTICLE |
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| Year : 2019 | Volume
: 3
| Issue : 4 | Page : 153-160 |
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To investigate the role of Withania somnifera in a mouse model of posttraumatic stress disorder
Ravjot Kaur1, Amteshwar Singh Jaggi2, Anjana Bali1
1 Department of Pharmacology, Akal College of Pharmacy and Technical Education, Mastuana Sahib, Sangrur, Punjab, India
2 Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India
| Date of Submission | 29-Jul-2019 |
| Date of Acceptance | 21-Nov-2019 |
| Date of Web Publication | 23-Dec-2019 |
Correspondence Address:
Dr. Anjana Bali
Department of Pharmacology, Akal College of Pharmacy and Technical Education, Mastuana Sahib, Sangrur - 148 001, Punjab
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/hm.hm_14_19
The present study was conducted to explore the role of Withania somnifera in posttraumatic stress disorder (PTSD) in mice. Swiss albino mice were subjected to a 2-day electric foot-shock stress of 5 min, which included 15 alternating inescapable foot shocks of 0.8 mA intensity with 10-s duration and 10-s intershock interval on the electrified grid floor. It was followed by 3 week re-exposures (on day 3, 7, and 14) in the same context (as situational reminders) for 5 min without delivering any foot-shocks. Trauma and situational reminders results a significant development of behavioral deficits and reduced serum corticosterone levels, as assessed on the 21st day. A significant development of freezing behavior was also observed in response to situational reminders on 3rd, 7th and 14th days. Repeated administration (for 21 days) of W. somnifera (50 mg/kg and 100 mg/kg) considerably restored the behavioral changes and normalized the corticosterone levels. W. somnifera led to significant reduction in the freezing behavior in response to situational reminders suggesting the inhibition of formation of aversive fear memory. It may be concluded that W. somnifera may be beneficial in preventing the PTSD symptoms in response to a traumatic event. Keywords: Behavior, foot-shock trauma, memory, posttraumatic stress disorder, Withania somnifera
How to cite this article:
Kaur R, Jaggi AS, Bali A. To investigate the role of Withania somnifera in a mouse model of posttraumatic stress disorder. Heart Mind 2019;3:153-60 |
| Introduction | |  |
Posttraumatic stress disorder (PTSD) is a psychiatric illness, precipitated by exposure to traumatic events such as natural disasters, domestic violence, wars, sexual abuse or other traumatic events. It is characterized by the development of re-experiencing, recurring flashbacks as well as nightmares, avoidance and hyperarousal symptoms in response to a traumatic event.[1],[2] It is the most serious condition which significantly affects and reduces the quality of life in the patients.[3] Several treatments have been proposed to prevent and treat PTSD patients. Selective serotonin reuptake inhibitor, sertraline and paroxetine, and serotonin-norepinephrine reuptake inhibitor agents such as venlafaxine have been approved for the treatment of PTSD symptoms.[4] Despite their efficacy against the PTSD-induced traumatic symptoms, their use is limited due to number of adverse effects, which emphasize the need for more research on therapeutic targets in this area.[5]
A number of studies have been conducted to investigate the Withania somnifera Dunal, family-Solanaceae, also known as Indian Ginseng, for its medicinal actions.[6],[7],[8] In Ayurveda, Unani and Siddha system of medicine, this plant has gained much interest and commonly used in more than 100 formulations. The decoction of the roots of Withanioa somnifera is used for colds and chills as well as to increase the tone of uterus after miscarriage or birth.[9] Furthermorte, W. somnifera possesses the anti-inflammatory[10] hepatoprotective,[11] antibacterial,[12] anticancer,[13],[14] and diuresis-inducing[15] properties. In addition, W. somnifera also possesses anxiolytic,[16] neuroprotective,[17] and cognition-promoting[18] properties. It has been shown to significantly reduce the stress-induced ulceration,[19] attenuated stress-induced inhibition of male sexual behavior[20] and abolished the adverse effects of stress.[21] Moreover, the selected plant is highly effective as an anti-stress agent[22] and as an anti-aging medicinal herb.[23] A study by Gupta and Rana found that W. somnifera significantly improves depression and anxiety-associated behavior caused by social isolation in rats.[24] A recent study has discovered the W. somnifera induced neuroprotection in bisphenol induced-cognitive dysfunctioning in mice.[25] Further, a clinical study has also shown the effectiveness and safety of Ashwagandha root extract in enhancing both immediate and general memory, attention, and information processing speed in people with mild cognitive impairment.[20] Considering antistress role of ashwagandha, this study was designed to delineate the neuroprotective potential of the hydroalcoholic extract of W. somnifera roots in attenuating contextual fear memory in electric foot shock stress-induced PTSD mice.
| Materials and Methods | | |
To execute and achieve the aim and objectives of this study, the following materials and methods were employed.
Experimental animals
Swiss albino mice (25–30 g) were procured from Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar. Animals were housed as six animals in a group per cage at temperature 23°C ± 1°C in the animal house of department. The animals were exposed to natural light and dark cycles of 12:12 h and were fed on the standard feed and water. The experimental protocol was approved by the Institutional Animal Ethics Committee (Reg. No. 1407/PO/Re/S/11CPCSEA) and care of the experiment on animal was carried out as per the guidelines of CPCSEA, Ministry of Environmental and Forests, Government of India.
Drugs and chemicals
Sertraline was obtained from Neuro Vision, Sirmour (HP) and was dissolved in physiological saline.
Preparation of plant extract
The plant material, i.e., the roots of W. somnifera were collected, dried and finely powdered. The powder was extracted three times in methanol-water mixture at ration 3:1 by stirring at room temperature for 1 h and every time. Afterwards, the extract was filtered out and 50°C under reduced pressure, the solvent was completely removed. The extract yield was 20% (w/w) in terms of dried starting material.[22] The dried hydroalcoholic extract was suspended in 0.5% carboxymethyl cellulose and it was administered to animals in two doses i.e., 50 and 100 mg/kg p.o. The dose volume was adjusted to 10 ml/kg for each mouse.
Acclimatization
The mice were acclimatized for 5 min on the behavioral test apparatus for 3 days before the start of actual protocol. The acclimatization to test apparatus before actual experimentation significantly avoids the confusing effects due to the novelty and thus, reduces the variation in the experiment data.[26.27]
Induction of electric foot shock trauma stress
On day 0 and 1, the mice were subjected to a 2-day electric foot-shock stress by introduction of the animal in a plexiglass chamber (26 cm × 21 cm × 26 cm), with a electrified stainless steel rods grid floor (0.3 cm diameter, 0.1 cm apart). After 5 min, the mice were delivered 15 inescapable electric foot shocks for 5 min with 0.8 mA intensity, 10-s and 10-s inter-shock interval delivered. It was followed by 3 week, i.e., day 3rd, day 7th and day 14th re-exposures to the same context (as situational reminders) for 5 min without any foot-shocks.[28]
Behavioral assessment of posttraumatic stress disorder
On 21st day, at the end of protocol, different behavioral tests including the actophotometer, hole board, open field, and social interaction tests were performed in animals with a time gap of 5 min between the successive behavioral tests. Every test apparatus was cleaned with alcohol and water after every test. The behaviors in the behavioral test apparatus were recorded and analyzed. The freezing behavior in response to weekly situational reminder was assessed on 3rd, 7th, and 14th day following stress exposure.
Freezing behavioral test
Freezing behavior has been defined as the lack of body movement and has been considered as the key feature of PTSD. The freezing response upon re-exposure to the similar stress context is an effective measure of aversive fear memory.[29] In this study, the animals were exposed to the situational reminders by placing in the same circular tank for 5 min and the duration of freezing i.e., total time (seconds) spent in freezing was noted.[30],[31]
Actophotometer test
The actophotometer activity has been considered as an index of alertness. The digital actophotometer was made up of square metallic chamber with arena (30 cm × 30 cm) with six in-built photosensors and digital counters. The animal's movement interrupts the beam of light falling on the photocell and then a count is digitally recorded. The number of counts in actophotometer is directly related to the movement of animals. Accordingly, the numbers of counts were used to indicate the locomotor activity of subjected animal. All the animals were kept in the actophotometer for 10 min and their locomotor activity was assessed as count per 10 min.[31],[32],[33]
Hole board test
The test is conducted to measure the exploratory behavior of animal. The number of head dippings and rearings in the familiarized environment represent the explorative behavior. The low number of head dippings reflects the anxious state, and the number of rearing represents the exploration in the novel surroundings. The mouse was kept in the center of the apparatus (hole-board) and was allowed to explore it for 10 min.[31],[32],[33]
Open field test
The open field test is used to assess the emotionality of animals. The open field test was carried out in a large wooden box, divided into 25 equal squares (10 cm × 10 cm each) and left uncovered at the top.[34] The walls were painted with black color and the floor was painted with white color. Mice were placed in a center of the test apparatus on the surface in a dim light room. Each animals was exposed to the test for 10 min during which its behavior including the number of lines crossing and time spent in the peripheral as well as central areas were recorded.[35]
Social interaction test
The social interaction test was used to assess stress-associated behavioral changes during which the mouse behavior with its social partner was observed for a specified period. During the experimental sessions, an experimental mouse (test mouse) spends time with an unfamiliar mouse (test partner) with free choice. Rodents are actively pro-social and demonstrate apparent altruistic behavior to fellow animals.[36] During a test of 10 min, each mouse (test) was allowed to interact with a marked partner mouse (partner), which was socially housed and not subjected to any kind of stressor. The behavior of test mice with test partner including close proximity, facing and sniffing the partner, physical contact and climbing over each other was considered as a social behavior. And the remaining time period was considered as a nonsocial behavior which included turning away from each other, keeping distance with the partner, freezing, self-grooming and sitting alone.[37]
| Biochemical Parameters | | |
Serum corticosterone estimation
The blood samples were taken by a tail vein nick procedure, by gently placing animal in a restrainer. Blood sample collected with needle was inserted perpendicular to the vessel in a quick punctuate type motion and free flowing blood drops were collected in the tube. Thereafter, the serum was cautiously isolated by centrifugation and further assayed for corticosterone levels using corticosterone enzyme immunoassay kit (K014-HI, Arbor assay).
Experimental protocol
Total six groups with five mice per group were employed in the present study.
Group I: Normal control
In normal control group, the animals were placed in the foot shock chamber on day 1 and no stress was applied. Thereafter, mice were exposed to weekly situational reminders on 3rd, 7th and 14th day by placing in a foot shock apparatus again without any stress and freezing behavior was analyzed on these days. Thereafter, all behavioral and biochemical tests were performed on 21st day.
Group II: Posttraumatic stress disorder
Mice were exposed to foot shock trauma stress by placing in an apparatus for 30 s on day 1. The mice were subjected to situational reminders on 3rd, 7th and 14th day by placing a foot shock apparatus without subjecting to stress and freezing behavior was analyzed on these days. Thereafter, various behavioral and biochemical parameters were performed on 21st day.
Groups III and IV: Hydroalcoholic extract of Withania somnifera (50 and 100 mg/kg)
The hydroalcohlic extract of aswahgandha (50 and 100 mg/kg p.o.) was administered for 21 days to foot shock-subjected animals. Thereafter, animals were subjected to behavioral and biochemical tests and procedures as described earlier.
Group V: Ashwagandha per se group
The hydroalcohlic extract of aswahgandha (100 mg/kg) was administered orally daily for 21 days in normal mice and the behavioral and biochemical parameters were performed as described earlier in group II.
Group VI: Sertraline (15 mg/kg i.p.) in posttraumatic stress disorder subjected animals
Sertraline was administered for 21 days in electric foot shock subjected animals. The behavioral and biochemical parameters were performed as described earlier.
Statistical analysis
The results were expressed as mean ± standard deviation. Except freezing behavior, all the data was statistically analyzed by using One-way ANOVA followed by Tukey's multiple range test. The freezing behavior data were analyzed using repeated measure Two-way ANOVA followed by Bonferroni test. The value of P < 0.05 was considered to be statistically significant.
| Results | | |
Effect of foot shock stress and weekly situational reminders on behavioral parameters
Animals exposed to foot shock stress followed by weekly situational reminders led to significant behavioral alterations on 21st day, including decreased locomotor activity in actophotometer [Figure 1], decreased number of head dips [Figure 2] and decreased rearing in the hole board [Figures 3], decreased total motor activity, i.e., number of rearings and line crossings in the open field [Figure 4] and [Figure 5], decreased social behavior in the social interaction test [Figure 6] and [Figure 7]. There was a significant increase in freezing time in response to situational reminders assessed on 3rd, 7th and 14th days as compared to the normal control, indicates the development of fear to the context associated with traumatic events [Figure 8]. | Figure 1: Effect of Withania somnifera on locomotor activity in electric foot shock-subjected mice in terms of counts in 10-min time interval in the actophotometer test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05,αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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 | Figure 2: Effect of Withania somnifera on frequency of head dips in electric foot shock – subjected mice in terms of counts in 10-min time interval in the hole board test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05,αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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 | Figure 4: Effect of Withania somnifera on exploratory behavior in electric foot shock stress subjected mice in terms of counts in 10-min time interval in the open-field test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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 | Figure 5: Effect of Withania somnifera on motor activity in electric foot shock stress-subjected mice in terms of counts in 10-min time interval in the open-field test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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{Figure 6} | Figure 7: Effect of Withania somnifera on avoidance time in electric foot shock stress-subjected mice in terms of counts in 10-min time interval in social interaction test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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 | Figure 8: Effect of Withania somnifera on freezing behavior time in electric foot shock– subjected mice to situational reminder. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) perse in normal group
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Effect of Withania somnifera extract and sertraline on other behavioral activities in posttraumatic stress disorder subjected mice
A daily administration of W. somnifera extract (50 and 100 mg/kg) for 21 days significantly attenuated foot shock trauma and situational reminders-induced behavioral deficits assessed on 21st day including increase in locomotor activity [Figures 1], number of head dips and rearing [Figure 2] and [Figure 3], number of line crossings and frequency of rearing [Figure 4] and [Figure 5]; improvement in social and attenuation of non-social behavior [Figure 6] and [Figure 7]. Ashwagandha (50 and 100 mg/kg) also significantly attenuated freezing time in trauma subjected mice in response to situational reminders on 3rd, 7th and 14th days in dose-dependent manner [Figure 8]. A daily administration of sertraline (15 mg/kg) also restored trauma-induced behavioral deficits and freezing response. | Figure 2: Effect of Withania somnifera on frequency of head dips in electric foot shock – subjected mice in terms of counts in 10-min time interval in the hole board test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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 | Figure 3: Effect of Withania somnifera on frequency of rearing in electric foot shock – subjected mice in terms of counts in 10-min time interval in the hole board test. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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Effect of Withania somnifera root extract and sertraline on the corticosterone levels in posttraumatic stress disorder subjected animals
In PTSD-subjected mice, a significant decrease in the serum corticosterone levels on the 21st day was observed as compared to normal control mice. Ashwagandha (50 and100 mg/kg) treatment for 21 days significantly attenuated PTSD-induced decrease in the corticosterone levels in a dose-dependent manner. The corticosterone restoration in response to ashwagandha (100 mg/kg) treatment was more significant as compared to ashwagandha (50 mg/kg) [Figure 9]. Moreover, daily administration of sertraline (15 mg/kg) also restored trauma-induced decrease in corticosterone levels. Administration of ashwagandha (100 mg/kg) did not modulate serum corticosterone levels in normal nonstressed mice. | Figure 9: Effect of Withania somnifera on serum corticosterone level in electric foot shock stress-subjected mice in corticosterone estimation. Data were statistically analyzed by using one-way ANOVA followed by Tukey's multiple range test, values are expressed as mean ± standard deviation within n = 5 in each group, P < 0.05, αP < 0.05 versus control;bP < 0.05 versus PTSD. PTSD: Posttraumatic stress disorder; sertraline (15 mg/kg) in PTSD; Withania somnifera (50 mg/kg) in PTSD; Withania somnifera (100 mg/kg) in PTSD; Withania somnifera (100 mg/kg) per se in normal group
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| Discussion | | |
The present study was conducted to investigate the effectiveness of the hydroalcoholic root extract of an Indian drug Ashwagandha in posttraumatic stress disorder in mice. To accomplish this objective, the study employed electric foot shock stress model followed by 3 weekly situational reminders, which significantly induce the symptoms of PTSD.[38] The different behavioral test including freezing behavior, hole board test, actophotometer, open field and social interactions test were conducted to assess the development of PTSD. Further to assess trauma-induced alteration in HPA axis, the serum corticosterone levels were measured. Corticosterone is considered as the main biomarker of stress and depressive disorders, and its levels are significantly depressed during PTSD.[27],[33]
In the present study, acute (a single) exposure of electric foot shock stress followed by three situational reminders (on 3rd, 7th and 14th day) produced significant alterations in terms of decreased locomotor activity, frequency of rearing, frequency of line crossing, frequency of head dips, social behavior and reduction in corticosterone levels assessed on 21st day following stress exposure. Moreover, situational reminders on 3rd, 7th, and 14th day led to significant development of freezing behavior in trauma subjected mice, suggesting the development of aversive memory of traumatic context. It is well documented that freezing behavior in response to situational reminder is an index of development of aversive fear memory.[29],[39] Electric foot shock stress is a commonly used stressor in rats and mice to induce anxious behavior,[40] stress,[41] and depression.[42] This model reproduces core symptoms which mimics the symptoms of PTSD, including avoidance and anxiety state in animals,[38] hyperarousal, aggression and re-experiencing flashbacks,[43] as well as sleep alteration.[43] It produces long-lasting behavioral effects that persist up to 3 or 4 weeks and may even get more intensified over time.[40] It has also been reported that situational reminders exposure is related to that traumatic event which reliably induces hyperarousal, physiological disturbance, anxious behavior, and increased fear response, all mimics the symptoms of PTSD.[31]
Ashwagandha is commonly recognized as Indian Winter cherry. Most of the Rasayana herbs act as anti-stress agents.[44] The roots of Ashwagandha are regarded as aphrodisiac, tonic, diuretic, diuretic, anthelmintic, narcotic and stimulant in nature. A large number of studies have demonstrated that ashwagandha improves the functioning of the brain and nervous system and consequently enhances the memory.[45] The beneficial role of Ashwangandha in stress and associated disorders has been attributed to its GABA-mimetic activity.[46] An interesting study demonstrated that repeated oral administration of withanoside IV attenuated the amyloid peptide Aβ induced axonal, dendritic and synaptic losses and memory deficits in mice.[45] The study proposed that withanoside IV present in root extract of W. somnifera may be metabolized into sominone, which considerably induce neurites and synapses revival. It also has been shown to enhance axonal and dendritic outgrowth as well as synaptogenesis.[13] Various studies shows the anti-stress effect of ashwagandha at different dose level. The alcoholic extract of ashwagandha (100 mg/kg) is shown to reduce stress-associated deleterious effects.[47] Furthermore, it is also shown to suppress foot shock stress-triggered alterations in body weight, basal core temperature, and marble burying behavior of mice.[48] A single dose of W. somnifera extract is found to possess anxiolytics and antidepressant-like activities in marble burying test.[49]
In the present study, administration of hydroalcoholic extract of W. somnifera (50 and 100 mg/kg) significantly reduced PTSD-associated behavioral and biochemical changes assessed on 21st day. It led to increase in the locomotor activity, frequency of rearing, frequency of line crossing, frequency of head dips, social behavior and increase in corticosterone levels. Furthermore, it also led to significant reduction in freezing behavior in trauma-subjected mice on exposure to situational reminders on 3rd, 7th and 14th days following stress exposure. However, per se administration of plant extract in normal mice did not show any behaviorial and biochemical changes. Administration of sertraline, a standard drug employed for PTSD, also modulated the behavioral and biochemical alterations assessed on 21st day. Sertraline is a Food and Drug Administration approved drug for the management of PTSD and is commonly employed as a standard drug in number of preclinical studies.[31],[50] We also found that (100 mg/kg) root extract W. somnifera was more effective as compared to low dose (50 mg/kg) of W. somnifera. Although the protective role of Ashwagandha in stress related anxiety has been identified.[51] yet it is the first study to depict the beneficial effects of W. somnifera in PTSD-induced behavioral and biochemical alterations. Based on the results, it may be proposed that W. somnifera triggers the activation of the HPA axis to normalize the depressed levels of serum corticosterone, which may possibly contribute in normalizing the behavioral alterations observed in PTSD. Nevertheless, future studies are required to elucidate the involvement of precise signaling pathway in W. somnifera-induced normalization of PTSD symptoms.
| Conclusion | | |
W. somnifera may be beneficial in preventing the PTSD symptoms following exposure to a traumatic event.
Acknowledgement
The authors are grateful to Akal college of Pharmacy and technical education, Mastuana Sahib, Sangrur (Punjab), India, for supporting this study and providing technical facilities for the work.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 2], [Figure 7], [Figure 8], [Figure 9]
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