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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 10  |  Issue : 1  |  Page : 1-8

Glial fibrillary acid protein expression and behavioral changes in hippocampus following prenatal co-administration of ethyl acetate leaf fraction of Tamarindus Indica and aluminum chloride in wistar rats


1 Department of Human Anatomy, Faculty of Biomedical Sciences, Kampala International University, Kampala; Department of Human Anatomy, College of Medicine and Health Science, Ahmadu Bello University, Zaria, Nigeria
2 Department of Human Anatomy, College of Medicine and Health Science, Ahmadu Bello University, Zaria, Nigeria
3 Department of Human Anatomy, Federal University Dutse, Jigawa, Nigeria

Date of Submission10-Aug-2021
Date of Decision28-Aug-2021
Date of Acceptance30-Aug-2021
Date of Web Publication01-Jul-2022

Correspondence Address:
Dr. Ibe Michael Usman
Department of Human Anatomy, Faculty of Biomedical Sciences, Kampala International University, Kampala
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_34_21

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  Abstract 


Background: The endowment of various plant parts with important phytochemicals needed in the management of human health breeds lots of hope. The present study investigated glial fibrillary acid protein (GFAP) expression and hippocampal behavioral changes following prenatal co-administration of ethyl acetate leaf fraction of Tamarindus indica (EATI) and aluminum chloride (AlCl3) in male Wistar rat pups. Methodology: Twenty pregnant Wistar rats were divided into five groups (n = 4). Group 1 received distilled water, while the treatment Groups 2 to5 received 200 mg/kg of AlCl3, followed by 400 and 800 mg/kg EATI in Groups 3 and 4, respectively, and 300 mg/kg Vitamin E in Group 5. All administrations lasted for 14 days from prenatal day 7 till parturition. The male pups (n = 6) were curled for Morris water maze (MWM) from postnatal day (PoND) 16–20, then sacrificed humanely on PoND 21. The brain tissues were harvested for oxidative stress studies (OSS) and the demonstration of GFAP antibody. Result: The result of the MWM showed significantly high mean latency to locate the platform in Groups 2 and 5 when compared to the control group (P < 0.05). The OSS revealed significantly higher superoxide dismutase concentration in Groups 4 and 5 compared to that observed in Group 2. The GFAP studies revealed significantly higher immunoreactivity scores in Group 2 when compared to every other group (P < 0.05) in the CA1 hippocampal region. Conclusion: EATI was associated with some protective potential during prenatal aluminum chloride exposure in Wistar rats.

Keywords: Aluminum toxicity, glial fibrillary acid protein, hippocampus, memory and learning, Tamarindus indica


How to cite this article:
Usman IM, Adebisi SS, Musa SA, Iliya IA. Glial fibrillary acid protein expression and behavioral changes in hippocampus following prenatal co-administration of ethyl acetate leaf fraction of Tamarindus Indica and aluminum chloride in wistar rats. Niger J Exp Clin Biosci 2022;10:1-8

How to cite this URL:
Usman IM, Adebisi SS, Musa SA, Iliya IA. Glial fibrillary acid protein expression and behavioral changes in hippocampus following prenatal co-administration of ethyl acetate leaf fraction of Tamarindus Indica and aluminum chloride in wistar rats. Niger J Exp Clin Biosci [serial online] 2022 [cited 2022 Aug 9];10:1-8. Available from: https://www.njecbonline.org/text.asp?2022/10/1/1/349560




  Introduction Top


The hippocampus is known for its cardinal role in learning and memory.[1],[2] In addition to its role in learning and memory, the hippocampus is also involved in spatial navigation, emotional behaviors, and regulation of hypothalamic function; the mentioned role of the hippocampus places the hippocampus at the helm of human survival.[3],[4] The role of the hippocampus in spatial navigation ensures the adequate acquisition of information about the environment, with coordinated cognitive strategies, and synchronous optimal behavior.[5] Despite the vital role associated with the hippocampus in mammalian survival, the hippocampus is not immune to the impact of environmental developmental neurotoxicants, including aluminum.[6],[7],[8] Just like other parts of the nervous system, the hippocampus contains glial cells including astrocytes.[9] Astrocytes provide necessary nutrients and facilitate the formation and stabilization of synapsis, thereby helping in the actualization of the various important functions the hippocampus performs.[9]

Aluminum has got a long history of beneficial uses among the human race: in medicine as an adjuvant in vaccines,[10] in domestic use as a component of kitchen utensils, and aluminum foil wrapping.[11],[12] Aluminum is used in water treatments and as food additives, fillers, and colors.[10] The major route of human exposure to aluminum for the general population is via oral ingestion.[13],[14],[15] The reported use of aluminum in various spheres of human existence for decades is based on the assumption that aluminum is safe.[10] However, recent studies have suggested the neurotoxic consequence of aluminum exposure.[10] Aluminum exposure unleashes structural and functional consequences on both neuronal and glial components of the central nervous system including the hippocampus.[16] The morphologic impact of aluminum on the neuronal and glial system of the hippocampus is linked to some behavioral deviations such as impaired memory and learning animal models.[6],[17],[18] Aluminum exposure has also been linked with developmental toxicity,[19] with reported functional and structural deficits extending into adulthood.[20] Aluminum exerts its damaging effects on the hippocampus by possible induction of oxidative stress, with the cellular consequence.[21] Given the detrimental consequence of aluminum exposure on the hippocampus, especially during its developmental phase, it is therefore important to look out for possible protective and therapeutic remedies.

The use of different plant parts is gaining lots of attention in alternative and complementary medicine, with emphasis on their rich phytochemical content.[22],[23] Tamarindus indica belongs to the monotypic genus Tamarind, commonly called “tsamiya” in Hausa based on its sour taste.[24] Phytochemical investigation on Tamarindus indica revealed the presence of important bioactive components, such as phenolic compounds, malic acid, glycosides, tartaric acid, pectin, arabinose, mucilage, galactose, xylose, glucose, and uronic acid.[25] An independent preliminary phytochemical study we conducted on ethyl acetate leaf fraction of Tamarindus indica (EATI) suggested the presence of flavonoids and carbohydrates (unpublished observation). The result from GCMS screening of EATI revealed the presence of oleic acid, n-Hexadecanoic acid, Phenol, 3,5-bis (1,1-dimethyl ethyl), and cis-9-Hexadecenal as its major chemical component (unpublished observation). The different phytochemicals present in the EATI suggest possible antioxidant and metal chelation potential. Some studies have reported Tamarindus indica pulp and seed to possess anti-inflammatory, antifungal, and antibacterial properties.[24],[26],[27],[28] Muhammad et al[29]investigated the neuroprotective effect of Tamarindus indica during aluminium chloride exposure. They reported a decrease in cerebral cortex levels of pro-inflammatory cytokines and lipid peroxidation products.[29] Tamarindus indica also protected against aluminum chloride induced memory impairment.[29] EATI was also linked with improved differential expression of different brain trace metal, suggesting a possible beneficial improvement in behavior (unpublished observation). The use of EATI in the present study is justified by the fact that most therapeutic intervention in aluminum and other heavy metal exposure relies on the metal chelating and antioxidant property of most drugs and other complementary remedies, coupled with the fact that there is no published report on possible changes in glial fibrillary acid protein (GFAP) expression and associated behavioral consequence following prenatal coadministration of EATI and aluminum chloride in Wistar rat. Therefore, the present study aimed to investigate GFAP expression and hippocampal behavioral changes following prenatal coadministration of EATI and aluminum chloride in Wistar rats.


  Methodology Top


Materials

Materilas used were twenty pregnant Wistar rats, light microscope, absolute ethanol, test tubes, Gemser reagent, aluminum chloride, Vitamin E, Tween 80, plant material (Tamarindus indica leaf), automatic vacuum tissue processor (model: KD-TS6B), citrate buffer, hydrogen peroxide, horse serum, mouse monoclonal anti-GFAP, biotinylated secondary antibody, avidin–biotin complex, diaminobenzidine, hematoxylin, water bath, distilled water, pestle, mortar, weighing balance, dissecting set, and Moris water maze setup.

Chemical and drug purchase/preparation

Aluminum chloride (CAS Number: 7446-70-0) was purchased from Zayo-Sigma Chemical Ltd., Jos Nigeria. Aluminum chloride stock solution was prepared by dissolving 1 g of aluminum chloride in 10 ml of distilled water. Emzo Vitamin E capsules (1000 mg) were purchased from a reputable drug store. The obtained capsules were used to prepare a stock solution by dissolution in Tween 80, ensuring 120 mg of Vitamin E is contained in 0.4 ml of the suspension, then kept away from direct light to avoid photodegradation.

Plant material acquisition and extraction/fractionation

Fresh leaves of Tamarindus indica were collected in the month of November from the Botanical Garden of Ahmadu Bello University, Zaria. The fresh leaves were taxonomically verified in Herbarium unit of the Department of Botany, Faculty of Life Science, Ahmadu Bello University, Zaria, and assigned a verification number 2417. The fresh leaves were shade dried for a week, then grounded using an electric blender. The fine leaf powder was then extracted by maceration, followed by subsequent fractionation as outlined by Ajiboye et al.[30] The stock solution of EATI was prepared by dissolution in Tween 80 since it was not soluble in water.

Experimental animals

Twenty adult nonpregnant females and 10 male adult Wistar rats were obtained and acclimatized for 2 weeks, after which a vaginal smear was taken from all the female rats and examined under a light microscope for the staging of their estrous cycle. The female rats in the proestrus phase were caged overnight with the mature male in the ratio of 2:1 (females: male); the presence of vaginal plugs the following morning indicated mating and assumed to be day zero of pregnancy.[31],[32],[33] Pregnant rats received humane care following the National Institute of Health guidelines for the care and use of laboratory animals until parturition, with their pups receiving the same.[34],[35] A minimal number of both dams and their pups were used for the present studies. The dams and their pups were allowed free access to feed and water before and during the experiment.

Dosage determination

A dosage of 200 mg/kg bw was adopted for AlCl3 based on previous studies using Wistar rat model.[36],[37] The adopted dosages for EATI were 400 and 800 mg/kg bw (low and high dose, respectively) based on the LD 50 (>5000 mg/kg bw) of EATI. A dosage of 300 mg/kg bw was adopted for Vitamin E based on previous studies using the Wistar rat model.[24],[26]

Experimental design and sample size

Twenty pregnant Wistar rats were grouped into five groups containing four rats on the 7th day of gestation. The sample size was determined using the resource equation,[38] where the minimum number of animals needed per group for the studies = (10/5) +1 = 3, and the maximum number of animals needed per group for the studies = (20/5) +1 = 5.

The choice of four rats per group in the present studies is therefore within the required limit for studies with five groups and quantitative variable output.

Group 1: 2 ml/kg bw of distilled water.

Group 2: 200 mg/kg bw of AlCl3.

Group 3: 200 mg/kg bw of AlCl3 and 400 mg/kg bwEATI

Group 4: 200 mg/kg bw of AlCl3 and 800 mg/kg bwEATI

Group 5: 200 mg/kg bw of AlCl3 and 300 mg/kg bw of Vitamin E.

Administrations were done orally by gastric intubation for 14 days from prenatal days 7–21.

Behavioral study

Male pups (n = 6) were curled and exposed to the Morris water maze (MWM) task on postnatal day (PoND) 16–20, considering possible sex differences in behavioral responses. MWM task is a well-established test for the assessment of memory and learning, a cardinal function of the hippocampus,[5] and was conducted in the present studies as outlined by Bazrgar et al.[39] The MWM used in the present study was a circular pool (140 cm in diameter, 60 cm high) filled with water (30 cm depth) at 24°C ± 2°C. The pool was divided into four quadrants of equal size. An invisible escape platform (10 cm diameter) was placed in the middle of one of the quadrants (2 cm below the water surface) equidistant from the sidewall and middle of the pool. The time taken for the pup to locate the platform was recorded. Four different starting were located. A trial began by placing the rat into the water facing the wall of the pool at one of the starting points. A pup that fails to locate the platform within 60 s was guided to the platform by the experimenter. Once on the platform, the pup was allowed to rest there for 30 s and then returned to their appropriate holder for intertrial rest. At the end of the trials, the pups were towel-dried and returned to their home cage. The trials on the first 4 days (morning and evening) served as the training session, while the trial on the last day served as the test session. The test session on the last day was aimed at assessing retention of the spatial training.

Animal sacrifice

On the PoND 21, the pups were weighed using a digital weighing balance and sacrificed humanely after anesthetizing them with thiopental sodium since this is ethically acceptable in experimental animals.[40],[41] The pups were intraperitoneally injected 5 mg/kg of thiopental sodium, well within the range (3–7 mg/kg) needed for the induction of anesthesia in experimental animal using thiopental sodium.[40],[42] The skull was dissected to expose the whole brain which was weighed before fixation in 10% neutral buffered formalin.

Oxidative stress studies

One gram of the harvested brain tissue was weighed and homogenized in 10 ml of phosphate buffer (pH of 7.2), then centrifuged for 10 min at 3000 rpm. The supernatant was collected for the determination of mean levels of superoxide dismutase (SOD) and malondialdehyde (MDA) (n = 4). Determination of SOD level was carried out as outlined by Fridovich,[43] with absorbance measured at a wavelength of 480 nm every 30 s up to 150 s, for subsequent calculations. Determination of MDA concentration was done as outlined by Zeb and Ullah,[44] with absorbance measured at 535 nm.

Tissue processing

The fixed brain tissues were trimmed and processed with the aid of an automated vacuum tissue processor (model: KD-TS6B)). The processed tissue was cut using rotary microtome at a thickness of 6 μ, then stained for glial reactivity.

Glial fibrillary acidic protein slide preparation

Staining for glial reactivity was evaluated using GFAP (DAKO, USA; CAT number: Z0334). Sections were treated with 0.01 M citrate buffer (pH 6.0) for 10 min to unmask the antigen. The sections were then incubated in 0.3% H2O2 for 30 min to get reed of tissue endogenous peroxidase activity followed by blocking with 5% horse serum for at least one to 2 h. The sections were then incubated with the primary antibody (1:500 mouse monoclonal anti-GFAP) for 18–20 h at a temperature of 4°C. The sections were washed, then incubated with biotinylated secondary antibodies (Vectastain ABC kit, 1: 200; CAT number: PK-4000) and then with avidin–biotin complex. The sections were finally developed with 0.05% diaminobenzidine, followed by counterstaining with hematoxylin before mounting.[45]

Immunoreactivity scoring

Klein's semiqualitative immune-reactive scoring approach was adopted as outlined by Fedchenko and Reifenrath[46] and Archibong et al.[47] In brief, the scoring was established by scoring each sample twice by two independent examiners. : The percentage labeling was scored thus; 0 for absence of astrocyte labeling, 1 for less than 30% astrocyte labeling, 2 for 30–60% astrocyte labeling, and 3 for greater than 60% astrocyte labeling. The intensity of the immunostaining was scored thus; 0 for no staining, 1 for weak staining, 2 for mild staining, and 3 for strong staining. Multiplication of both scores allowed the final quotation with a range between 0 and 9.

Statistical analysis

The obtained data were entered into excel, then imported to GraphPad Prism 8.3 (GraphPad Prism 8.3, San Diego, California, USA) for onward analysis. For the establishment of a significant difference, the data were analyzed using a one-way analysis of variance. Differences were considered significant at P < 0.05, where applicable a post hoc test was applied.


  Results Top


We observed a mix up in the data obtained from the pilot study and the main work, therefore, the result section, with the exception of the photomicrographs should be replaced with the following;

Behavioral studies

Morris water maze task was used to assess memory and learning. On the fourth day of the test, a significantly high mean time to locate the platform was observed in the 200 mg/kg of AlCl3 + 300 mg/kg of Vitamin E treated group when compared to the distilled water treated group (p<0.05). On the fifth day of the task, a significantly high mean latency to locate the platform was observed in the 200 mg/kg of AlCl3 and 200 mg/kg of AlCl3 + 300 mg/kg of Vitamin E treated group when compared to the distilled water treated group (P<0.05) [Figure 1].
Figure 1: Mean latencies during Morris water maze test following administration of EATI during prenatal AlCl3 exposure from PoND 16 to 20. a represent significance difference (P< 0.05) compared to the distilled water administered group. (n=6)

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Oxidative stress studies

The result of the OSS showed lower mean MDA concentrations in the 200 mg/kg AlCl3 + 400 mg/kg EATI and 200 mg/kg AlCl3 + 800 mg/kg EATI treated group when compared to the 200 mg/kg AlCl3 [Figure 2]a. A significantly lower mean concentration of SOD was observed in the 200 mg/kg AlCl3 and 200 mg/kg AlCl3 + 400 mg/kg EATI treated group when compared to the distilled water treated group (P < 0.05). On the other hand, significantly higher mean SOD levels were observed in 200 mg/kg AlCl3 + 400 mg/kg EATI and 200 mg/kg AlCl3 + 300 mg/kg Vit E treated groups when compared to 200 mg/kg AlCl3 treated group (P < 0.05) as shown in [Figure 2]b.
Figure 2: Oxidative stress study result on PoND 21 following administration of EATI during prenatal AlCl3 exposure (n = 4). MDA: Malondialdehyde SOD: Superoxide dismutase. Values are presented as mean ± standard error of the mean, a and b represents significance difference (P < 0.05) compared to the distilled water and the 200 mg/kg AlCl3 treated group respectively

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Immunohistochemical studies

The CA1 hippocampal area from rats in the different treatment groups on PoND 21 revealed significantly (P < 0.05) high immunoreactivity score in the groups treated with 200 mg/kg AlCl3, 200 mg/kg AlCl3 + 400 mg/kg EATI, 200 mg/kg AlCl3 + 800 mg/kg EATI, and 200 mg/kg AlCl3 + 300 mg/kg of Vitamin E [[Figure 5]a, [Figure 6]a, [Figure 7]a, and [Figure 8]a, respectively] when compared the control group (distilled water treated group) [Figure 3]a and [Figure 4]a. On the other hand, the CA3 hippocampal area revealed a higher immunoreactivity score in the groups treated with 200 mg/kg AlCl3, 200 mg/kg AlCl3 + 400 mg/kg EATI, and 200 mg/kg AlCl3 + 300 mg/kg of Vitamin E [[Figure 3]b, [Figure 5]b, [Figure 6]b, and [Figure 8]b, respectively] when compared to the control group (distilled water treated group) [Figure 4]b. Lower immunoreactivity score was observed in the group treated with 200 mg/kg AlCl3 + 800 mg/kg EATI [Figure 7]b.
Figure 3: Immunohistochemical score of the CA1 (3A) and CA3 (3B) hippocampal area of pups on PoND 21. Values are presented as mean ± SEM, a represent significance difference (P< 0.05) compared to the distilled water administered group

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Figure 4: Image from Wistar rat hippocampal area on postgestation day 21 from the control group. CA1 hippocampal area (a), CA3 hippocampal area (b), immune-positive cells (GFAP, ×400)

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Figure 5: Image from Wistar rat CA1 hippocampal area on postgestation day 21 exposed to 200 mg/kg bw of aluminum chloride. CA1 hippocampal area (a), CA3 hippocampal area (b), immune-positive cells (GFAP, ×400)

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Figure 6: Image from Wistar rat CA1 hippocampal area on postgestation day 21 exposed to 200 mg/kg bw of aluminum chloride and 400 mg/kg bw EATI. CA1 hippocampal area (a), CA3 hippocampal area (b), immune-positive cells (GFAP, ×400)

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Figure 7: Image from Wistar rat CA1 hippocampal area on postgestation day 21 exposed to 200 mg/kg bw of aluminum chloride and 800 mg/kg bw EATI, CA1 hippocampal area (a), CA3 hippocampal area (b), immune-positive cells (GFAP, ×400)

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Figure 8: Photomicrographs from Wistar rat CA1 hippocampal area on postgestation day 21 exposed to 200 mg/kg bw of aluminum chloride and 300 mg/kg bw of Vitamin E. CA1 hippocampal area (a), CA3 hippocampal area (b), immune-positive cells (GFAP, ×400)

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  Discussion Top


The availability of a variety of plant materials with important phytochemicals necessary for the maintenance and improvement of human health forms the core of alternative and complementary medicine.[22],[23] The result from the MWM task and the OSS revealed that prenatal aluminum chloride exposure interfered with memory and learning, and oxidative stress parameter in the pups; however, treatment with 400 and 800 mg/kg bw of EATI was associated with improved memory and learning, and fortified front against oxidative stress. Treatment with aluminum chloride was linked with lower GFAP expression in the CA1 and CA 3 areas of the hippocampus, with a dose-dependent improvement following treatment with EATI.

The observed interference with memory and learning, oxidative stress parameter, and GFAP expression may be linked with the reported possibility of persistence in the effect of prenatal aluminum.[20] The observed poor performance in acquisition and retention of memory in our studyis in line with the finding of Justin Thenmozhi et al.[48] who reportedly associated aluminum exposure with decreased spatial memory and accuracy in both acquisition and probe trials. Concurrent aluminum exposure and stress during pregnancy in rats are linked with longer avoidance in the passive avoidance learning task among their offspring.[48] The observed improvement in the spatial memory and learning and oxidative defense system in the present study may be linked with the possible metal chelating capability and antioxidant potential of EATI. Our observation is in line with the finding of Muhammad et al,[29] who reported that the administration of Tamarindus indica was associated with a decrease in cerebral cortex levels of proinflammatory cytokines and lipid oxidation products following aluminum chloride. Muhammad et al,[29] also reported that Tamarindus indica protected against aluminum chloride induced memory impairment.

Studies have linked neuronal damage with altered GFAP expression,[49] with behavioral consequences. Therefore, GFAP has now emerged as a classical marker for astrocytes, a structural protein synthesized in astrocytes made up of intermediate filaments. The observed increase in GFAP expression in the CA1 and CA3 hippocampal area of Wistar rat on PoND 21 revealed that prenatal aluminum chloride exposure adversely affected astrocyte immunoreactivity. This is not surprising as there are previous reports on the possibility of developmental neurotoxicants, including aluminum affecting astrocytes.[50],[51] The observed aluminum chlorideinduced adverse effect on astrocytes in both the CA1 and CA3 hippocampal area in the present study is in contrast with the findings of Inohana et al.[52] who reported decrease GFAP immunoreactivity in the hippocampus on post gestation 21 following prenatal aluminum chloride exposure, and concluded that prenatal exposure to aluminum chloride has an irreversible impact on postnatal hippocampal neurogenesis involving multiple functions in mice. More evidence suggests alterations in astrocyte functionality playing important role in the pathogenesis of diverse prenatally acquired dysfunction in various parts of the brain,[53] with some behavioral consequences manifesting later in life. The observed improved GFAP expression may not be unconnected to the behavioral improvement following treatment with EATI. These may be hinged on the rich phytochemical components of EATI, such as flavonoid and nHexadecanoic acid, with known antioxidant[54],[55] and metal chelation capability.[56] Flavonoids present in EATI are known to improve both structural and functional integrity of astrocytes with consequent efficient energy delivery, ion buffering, neurotransmitter clearance, and modulation of CO2 concentration by astrocytes.[57]


  Conclusion Top


The present study therefore, concluded that the administration of EATI was of therapeutic value during prenatal aluminum chloride exposure in Wistar rats, based on the recorded improvement in oxidative stress biomarker, spatial memory and learning, and GFAP reactivity.

Acknowledgments

The researchers acknowledge the support of the Human Anatomy Department Ahmadu Bello University, Zaria, for providing suitable working conditions for the present research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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