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Tabakwot et al
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IDOSR JOURNAL OF EXPERIMENTAL SCIENCES 8(1) 48-59, 2022. Evaluation of the Effects of Galinsoga parviflora Aqueous Leaf Extract on Motor Coordination (muscular strength and balance) on Adult Male Mice after Mercury Chloride Exposure.
John Ayuba Tabakwot, Okesina Akeem Ayodeji and Usman Ibe M. DepartmentofHumanAnatomy,KampalaInternationalUniversity,Uganda.ABSTRACT
The cerebellum forms a major part of the brain and its function is to control all muscular movements and coordination. Injury or damage to the cells of the cerebellum may lead to conditionslike;ataxia, Joubertsyndrome,and manyothers.Theseinjuries may bearesult of traumatic brain injuries or toxic agents such as mercury, tin and aluminum. For example,mercury exposure has been reported to be part of our daily lives, therefore it is important to understand the pathological implication that might follow these exposures andalsowiththehelpofalocalherb; Galinsogaparviflora (GP)tofindacheapandreadily available management solution to mercury toxicity in the cerebellum. This study was designed to evaluate the effects of Galinsoga parviflora aqueous leaf extract on motor coordination (muscular strength and balance) on adult male mice after mercury chloride exposure.Twenty-five adult male mice of an average weight of 25g were randomly divided into 5 groups of 5 rats each (n=5). The animals in Groups 1-5 received oral administration ofdistilledwater,2.3mg/kgbodyweightofHgCl2,2.3mg/kgHgCl2followedbyGPextract (800mg/kg), GP extract (800mg/kg) followed by 2.3 mg/kg HgCl2 and GP extract (800mg/kg)followedby2.3mg/kg HgCl2 concurrently respectively. Thesetreatments were followedbybehavioraltests.Theanimalsexhibitedabnormalneuromuscularfunction and uncoordinated balance and posture. In conclusion, the study revealed that mercury exposureinthecerebellumofmicecausedlossofneuromuscularfunction,lossofbalance and coordination. However, the introduction of Galinsoga parviflora in the cerebellum actedasatherapeuticagentagainstmercurytoxicityinthecerebellumofadultmalemice.
Keywords: Galinsogaparviflora, motorcoordination,muscularstrengthandbalance
INTRODUCTION
There are three functional areas of the cerebellum the cerebrocerebellum, spinocerebellum and vestibulocerebellum [1]. The spinocerebellar lobes play a role in the regulation of muscle tone, vestibulocerebellum lobes play a role in the maintenance of posture and balance and cerebrocerebellum plays a role in the coordination of voluntary motor activity [1]. Any possible damage to any parts of the cerebellum can affects the above mention parts which can lead to conditions such as ataxia, nystagmus, dysdiadochokinesia,anddysmetria[2]
Behavioral changes constitute a significant hallmark associated with nervous system disturbance in mercury exposure [3]. A review by [4] on mercury
involvement in neuronal damage and neurodegenerative diseases, suggested that mercury is dangerous to human health and in most cases associated with behavior deficit. Teixeira et al., [5] studiedtheeffectsofchronicintoxication with inorganic mercury on memory and motor control in rats; then found that long-term exposure to mercury chloride in the rat can cause motor function damage, anxiety-related disorder, and memory impairment. A study by [5] on the effect of inorganic mercury exposure on the motor cortex, indicated that chronic exposure to inorganic mercury decreases balance and motor coordination, an effect likely linked to cellular and molecular damages in some
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areas of the cerebellum. The enormity of the behavioral consequence of mercury exposure raises the need for possible therapeutic and ameliorative intervention [5]. According to [6] who reported that mice with cortical impact lesions often exhibitcontralateralslippingonthebeam and might be due to cortical impact lesion. A study by [7] on grip strength is potentially an early indicator of agerelated decline in mice, indicated that there was decreased in the strength of male mice 12 months of age compared to 8-month-oldmice,andcontinuedarobust declineto20monthsandthen28months of age when the study was terminated. The decline was not related to lean muscle mass, but extensive age-related carpal and digital exostosis could help explain the decreased grip strength times withincreasingage.Areportby[8]onthe use of a grip strength meter to assess the limbstrengthofMDX mice,indicated that the measure the disease progression of neuromuscular disorders can measure by the time the animal used to finish the tasks.
Ethnomedicinal use of plants has gradually moved to the fore in the struggle to curtail the impact of most environmental neurotoxicants including those associated with mercury exposure [9]. However, there is a paucity of literature on the effect of GP on the cerebelluminmercury,thereforetheneed for the present studies. Most ethnopharmacological interventions targeting possible protection/amelioration against mercuryinducedbehavioralchangesarehingedon the abundance of very important phytochemical composition of different plant materials [9]. A recent review by [10] on phytochemical and pharmacological properties of GP, suggested that GP contains aromatic esters, flavonoids, and phenolic acid
Study Location
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derivatives. Several flavonoids and polyphenolic compounds have been reported to possess antioxidant properties both in in-vivo and in-vitro studies [9]. A study by [11] on the differential effects of alkaloids on memory in rodents, suggested that alkaloids have the potential to improved learning and memory function. MoralesGarcía et al., [12] on the alkaloids of Banisteriopsiscaapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro. An alkaloid which is one of the phytochemicals of GP has been established that its modulation of antidepressant effects. Possible behavioral improvement following administration of GP during mercury exposuremaybelinkedwiththepresence ofheavy metalchelatorsandantioxidants (flavonoid) as one of its very important components [13,14,15,16,17,18]. A review by [2] emphasized the importance of various phytochemicals in the management of diverse behavioral alterations.Theirreviewalsolaiddiscreet emphasis on the neuroprotective potentialsofvariousphytochemicalssuch as fatty acids, phenols, alkaloids, flavonoids, saponins, and terpenes, therefore buttressing our choice of GP in the management of mercury exposure to ourWistarratmodel[2,19,20,21].
Aim of the Study
The aim of this study was to evaluate the effects of Galinsoga parviflora aqueous leaf extract on motor coordination (muscular strength and balance) on adult male mice after mercury chloride exposure.
Research Question
What are the effects of Galinsoga parviflora aqueous leaf extract on motor coordination (muscular strength and balance)onadultmalemiceaftermercury chlorideexposure?
METHODOLOGY
The study was conducted in the animal house of the Kampala International University, Institute of Biomedical Research (IBR) and Histology Laboratory of Kampala International University,
Western Campus (KIU-WC); which is situated in Western Uganda in Ishaka Municipality, Bushenyi District 350KM fromKampala,Uganda.
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Ethical Consideration
Ethical approval (KIU-2021-14) was obtained from the Kampala International University Research Ethics Committee before conducting the research and register in Uganda National Council for Science and Technology. The experimental study was carried out at the Institute of Biomedical Research Laboratory (IBRL) Kampala International University-Western Campus (KIU-WC) and the laboratory animal experiments were conductedfromtheKampalaInternational University Animal House. The 3Rs (Reduction,Replacement,andRefinement) were considered in determining the number of mice to be used in this study as well as the handling of animals (EuropeanMedicineAgency,2016).Atotal of 25 mice were used in this study. The study will use an animal model system and not an in vitro system to determine the effect of Galinsoga parviflora on the cerebellum after mercury chloride is introduced then the expression of antioxidant biomarkers in cells is determined. The report by ‘resource equation’ was considered when chosen the number of animals. The animals were grouped into 5 groups with five animals pergroupandwithgroups,threeandfour receiving the same doses but in reverse methodandalsoasuitabledosewasused according to literature to avoid using too many animals so that to be able to achieve the reduction principle. The researcher obtains training on how to handle laboratory animals before the study and work under the supervision of hissupervisors.Allproceduresandworks on animals were conducted following a guide for the care and use of laboratory animals (IACUC, 2010). The animals were treated in a humane way to prevent them from any discomfort like excessive pain and stress. The animals were housed five per group and there was fed on a daily basic morning and evening and also their beddingswerechangedweeklysothatthe animals were comfortable to avoid the fightandsoallowtorespondtotreatment inaventilatedenvironment.Attheendof 42 days, all animals were sacrificed humanely by subjecting them to light
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ether anesthesia followed by cervical dislocation. At the end of the study, all sacrificedanimalswereincinerated.
Collection and Preparation of Plant Extract
Galinsoga parviflora was obtained from the local garden in Ishaka- Bushenyi District, Uganda. A small sample of the leaves was taken to Mbarara University of Science and Technology, Faculty of Science Herbarium Unit, Mbarara, for botanical identification and deposition of voucher number. Extraction of the plant was carried out in the Biochemistry Departmental laboratory, Kampala InternationalUniversity,WesternCampus, Ishaka, Uganda. The plant’s leaves were driedatroomtemperature;groundedwith a grinding machineand followed by extraction. Sixteen grams (16g) of the powdered leaves were dissolved in 300ml of distilled water and left to stand for three days, shaken frequently until complete extraction of plant materials. Then the extracts were filtered through a filter into a funnel and the extract was kept in an oven at 400 C for three days to solidified[13].
Purchase of Chemical Mercuric chloride (May and Bakers, England) was purchased from a chemical storeinIshakatown,WesternUganda.
Animals
The animals were procured from the animal facility of Kampala International University Western Campus (KIU-WC) twenty-five (25) adult male mice were used for this study weighing 20-35g. All mice were housed in a well-ventilated plastic cage; the animals were left to acclimatize for two weeks with free accesstofoodandwater.
Study Sample Size
‘The resource equation’ approach was used to arrive at adequate sample size. According to the ‘resource equation’ method a value “E or DF” is measured. DF = degree of freedom of analysis of variance(ANOVA).
DF can be measured by the following formula: DF=N – k n=DF/k+1
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Where: N = total subjects number; k = numberofgroups;n=numberofsubjects in a group. Minimum DF range = 10 and maximumDFrange=20[14]. Thesamplesizeforthe studywillbe:N= 25malemice;K=5groups
DF = 25 – 5 = 25, n = DF/k+1 = 20/5+1 = 5(fivemicewillbeplacedpergroup)
Sampling Technique
The mice were sampled via simple random sampling procedures. A sequence
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of numbers was randomly used to direct how the animals will place in groups (table 1). After the randomization, the micewere placed in five groups comprising of 5 mice each labeled mercury and gallant soldier dose (MG), gallantsoldierandmercury(GM),mercury and gallant soldier dose concurrently(MGC), positive control (mercury dose) (PC) and negative control (NC)(table2).
Table 1: Randomization assignment sequence Table 2: Group placement after
Experimental Design
Dosage and Administration
Mercury chloride:2.3mg/kg of Mercury Chloride was administered. The stock solution was prepared daily to prevent the risk of degradation [15]. The dilution factorwas50mgin100ml.
Galinsoga parviflora 800mg/kg of GP was administered. The stock solution was prepared daily to prevent the risk of degradation [16]. The dilution factor was 100mg in 1ml. Mercury chloride and GP was administered orally for three weeks respectively
Reconstitution of Galinsoga parviflora Extract mercury Chloride for Animal Administration
AsuitabledoseofGPwasadministeredto the animals. The right amount of the extract which was guided by the OECD’s (organization of economic corporation
and developments) guidelineswas reconstituted and the calculated doses shall be administered according to each animal's body weight daily (between 9am and 10am). The mercury chloride on the other hand was constituted for administration on the mice according to the appropriate dose which was 800mg/kgasdescribeby[15].Thedosage of GP was prepared by dissolving in an appropriate volume of distilled water not exceeding 20 ml/kg [17]. The mercury chloride was guided by the OECD’S guideline and the powder be dissolved in distilled water daily. The mixture was kept in the refrigerator at 60C pending daily administration to the animals accordingtotheirbodyweight.
Dosage and preparation of stock solution will be calculated using the formula:
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Groupings
Animalswererandomlyassignedtofive(5)groupsoffive(5)animalsineachgroupi.e.N=5 pergroup.
Table 3: Animals Grouping
GROUP DOSAGE
Group1 Receivedistilledwateronly
Group2
Receivemercurychlorideonly(2.3mg/kg)
Group3 Receive mercury chloride (2.3mg/kg) followed byGalinsoga parviflora(800mg/kg)
Group4
Group5
Receive Galinsoga parviflora (800mg/kg) followed by mercury chloride(2.3mg/kg)
Receive mercury chloride (2.3mg/kg) and Galinsoga parviflora (800mg/kg)concurrently
Motor Coordination
Beam walking test: This test was conducted to assess motor coordination and balance. 1m long narrow wooden beam (1-2cm wide) suspended from its ends at an elevation of 30cm to the darkroom. The animal was gently be placed in the center of the Beam, facing one of the ends. The rat was allowed to walk to the end of the darkroom; the procedure was repeated three times, the animals were trained for a week once a day before administration and the test wasconductedthreetimesaweekandthe timetoreachtheendwasrecorded[18]
Forearm grip test
The neuromuscular/skeletal muscle functions were determined by the maximal peak force developed by the mice. The mice were placed on the
apparatus a 120cm by 50cm wire forearm grip apparatus. The wire was placed horizontally and the mice were placed in the center to balance the time it takes to reach the other end or falls will be recorded. The training was done for a week once a day before administration and the test was conducted three times a week[18]
Statistical Analysis
All the data of behavior and the biochemical study was statistically evaluated with SPSS 25 using ANOVA to compare the mean. Where necessary, Tukey post hoc test was used to find where the significant difference lies across the groups. All the results were expressedas mean±SDorSEMvalue was significantatp≤0.05.
RESULTS
Behavioral Studies of Adult Male Mice During Administration of Galinsoga Parviflora (Cav) Aqueous Leaf Extract on the Cerebellum After Mercury Chloride Exposure Beam Walking Test of adult male mice
Group 3 revealed there was a significant increase in the time taken for the mice to finished the apparatus especially at the beginning of thetest time, while during the end of the test time there was a significant reduction in time when comparedto group 1(P < 0.05).Although,whencomparedtogroup2 there was an increase in time during test time (Table 4). Group 4 revealed an increase in time taken for the mice to finished the apparatus during the beginning of test time and a significant reduction in time at the end of test timewhencomparedtogroup1(P<0.05), while when compared with group 2 there
was a reduction time in test time (Table 4). Group 5,revealed a reduction in time when compared with groups 1 and 2 duringtesttime(Table4).
Forearm
Grip Test of adult male mice
Group 3 revealed there was a significant decrease in timein both reaching and fallingtimewhencomparedtogroup1(P< 0.05). While when compared to group 2 there was a decrease in time in both reaching and falling time(Table 5).In group 4, both the falling and reaching time revealed that there was a significant decreaseintimewhencomparedtogroup 1(P < 0.05). When compared with group 2 revealed a reduction in both falling and reachingtime(Table5).Thefallingtimein
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group 5 revealed asignificant decreasein time (P < 0.05). Although, reaching time revealed an increase in time in week 1 then reduced from week 2 to 3 when compared with group 1. When compared with group 2 it revealed a reduction in falling time. While reaching time revealed an increase in groups 1 and 2 then reduced in week 3 (Table 5).
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Table 4:Beamwalkingtestofadultmalemiceduringadministrationof Galinsogaparviflora(cav) aqueousleafextractonthecerebellum aftermercurychlorideexposure
Groups TN 1(sec) TN 2 (sec) TN3 (sec) TN 4 (sec) TN 5(sec) TS 1 (sec) TS 2 (sec) TS 3 (sec) TS 4 (sec) Group 1 15.67±1.87 10.92±0.47 12.67±1.35 20.10±2.34 11.10±1.93 15.80±1.71 10.81±0.98 15.80±1.71 10.81±0.98
Group 2 13.81±1.88 11.79±1.30 11.43±1.62 20.75±2.60 16.02±1.29 19.80±3.25 15.68±3.40
Group3 17.50±1.96 12.70±1.84 11.50±1.19 21.77±3.69 20.42±2.48a 26.72±5.73 25.23±5.06a 10.30±2.39 20.99±4.43a Group 4 17.18±3.59 12.10±2.26 10.41±1.12 17.46±3.53 12.28±1.86 14.27±4.29 11.87±1.75 23.41±1.22a 9.87±1.22
Group 5 12.29±1.36 12.67±1.34 15.63±2.36 19.50±3.26 13.90±1.67 14.04±3.12 8.74±1.73
Fvalue Pvalue 0.969 0.446 0.225 O.921 1.596 0.214 0.265 0.897 3.809 0.018 1.907 0.149 4.507 0.009 11.175 0.002 8.681 0.005
N=5, TN=Training time, TS=Test time. The value was considered significantly different at p<0.05. b: indicates significance when test groupiscomparedagainstGroup2,a:indicatessignificancewhentestgroupiscomparedagainstGroup1.
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Table 5: Forearm Grip test of adult male mice during the administration of Galinsoga parviflora(cav) aqueous leaf extract on the cerebellumaftermercurychlorideexposure
Group s WBA W1 W2 W3 W4 W5
RT1(sec) FT1(sec ) RT2(se c) FT2(sec ) RT3(se c) FT3(sec ) RT4(se c) FT4(sec ) RT5(se c) FT5(sec ) RT6(se c) FT6(sec )
GP 1 24.2±8.0 3 109.8±3 7.33 8.0±3.5 1 131.2±3 2.96 61.8±10 .28 180.0±0 .00 29.4±10 .53 174.0±6 .00 42.0±6. 63 180.0±0 .00 42.0±9. 57 180.0±0 .00
GP 2 45.0±10. 82 180.0±0 .00 33.0±17 .07 164.0±1 6.0 20.4±5. 96 124.2±2 6.91 12.8±3. 07 84.0±26 .88
GP 3 24.2±16. 59 102.6±3 4.10 21.6±6. 97 96.6±31 .98 36.8±29 .15 79.0±36 .68 5.0±2.5 3a 43.4±2 1.12a 19.6±9. 79 39.2±1 4.04a 5.2±2.3 3a 102.0±3 6.43
GP 4 23.4±10. 70 116.2±3 9.07 20.6±5. 33 91.4±36 .86 16.6±7. 27 85.2±29 .93 12.8±4. 61 75.6±31 .17 9.4±1.0 3a 36.2±1 5.51a 15.4±8. 69 38.4±1 4.73a
GP 5 20.2±5.5 2 89.2±37 .78 44.0±32 .97 73.0±31 .29 21.8±8. 19 80.4±19 .99 8.4±3.6 1 59.6±2 5.76a
N=5, WBA=Before Administration, W: Weak, RT=Reaching Time, FT=Falling Time. The value was considered significantly different at p<0.05. b: indicates significance when test group is compared against Group 2, a: indicates significance when test group is compared againstGroup1
DISCUSSION
This study was centered on assessment effects of Galinsoga parviflora (cav) aqueous leaf extract on the cerebellum during mercury exposure in adult male mice, using neurobehavioral to observe the specific effect of this chemical on the brain using the mice as a model. The specific objective of the study was neurobehavioral studies using forearm grip and beam walk apparatus to assess locomotor function and motor coordination
The morphometric study indicated that groups treated with mercury chloride had adecreasein weight whencompared with the control (group1) might be due to the presence of damage of the cells, will those who have been treated with GP show an increase in weight might be due to the presence of phytochemicals present in the plant such as alkaloid, carbohydrate, flavonoid, polyphenol and glycoside. While organ body ratio did not showanysignificantdifference
The forearm gripapparatus is used to measureneuromuscular/skeletal muscle functions [18,21,22,23,24] The forearm grip test showed a significant decrease (p˂0.05) in treatment groups (3,4 and 5) compared to the control(group 1) in both falling and reaching a time of theforearm griptestandalsoadecreaseintimewhen compared to group 2.Thisresult suggests that mercury chloride may have the potential to decrease neuromuscular/skeletal muscle functions.Teixeira et al., [19] studied the effects of chronic intoxication with inorganic mercury on memory and motor control in rats; then found that long-term exposure to mercury chloride in the rat can cause motor function damage, anxiety-related disorder, and memory impairment. A report by [20,25,26,27] on the use of a grip strength meter to assess the limb strength of mice, indicated that the measure the disease progression of neuromuscular disorders can measure by
Tabakwot et althe time the animal used to finish the tasks. This means that a decrease in the neuromuscularfunctionofthecerebellum maybearesultofdamageofthePurkinje cellsasreportedby[5;20,28,29,30,31] Fine motor coordination and balance can be assessed by the beam walking test. Thisisusefulfordetectingsubtledeficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod [18]. In this study, the beam walk test was used to assess fine motor coordination and parameters such as the time it will take the mice to reach the darkroom.Results showed a significant increase in time in groups treatedwith mercury chloride when compare to a group treated with GP, which suggests poorfinemotorcoordinationintreatment groups given mercury chloride [32,33,34,35,36] The treatment groups had increase time of the group given mercury chloride on the beam walk when compared to the control, this suggests poorbalancewhichmaybeduetocortical impact lesion. This is in line with an earlier study reported by [21; 6] who reported that mice with cortical impact lesions often exhibit contralateral slipping on the beam. The poor fine motor coordination and poor balance observed in the beam walk test suggests motordeficits that may havebeencaused by CNS lesions. The motor deficits observed in the neurobehavioural studies may bedue to the pyknosis,degenerating myelinobservedinthehistomorphological studies of the cerebellum. The poor fine motor coordination and poor balance observed in the beam walk test suggests motordeficits that may havebeencaused by CNS lesions as aresult of mercury exposure The motor deficits observed in the neurobehavioural studies may be due to the pyknosis, degenerating myelin earlier observed in the histomorphological studies of the cerebellum.
CONCLUSION
In conclusion, the study revealed that mercury exposure in the cerebellum of mice caused loss of neuromuscular
function, loss of balance and coordination. However, the introduction of Galinsoga parviflora in the cerebellum
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acted as a therapeutic agent against mercury toxicity in the cerebellum of adultmalemice.
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