Introduction
Stress and western diets are one of the main causative factors for neurodegenerative diseases and dementia.1, 2 The no of people suffering with dementia in the world have more than doubled from 1990 to 2016.3 Alzhemiers disease is the major cause of dementia accounting to 60 to 80 percent of total cases.4 Studies have shown that stress and diets rich in saturated fat have led to cognitive decline and dementia.5, 6
Ginkgobiloba is a Chinese medicinal plant, which has been used in treating neurological disorders and for memory impairment.7 It has antioxidant, anti-apoptopic, and free radical scavenging and neuroregeneration properties.8 The benefit of Ginkgobiloba is attributed to its two active chemical components, flavonoids and terpenoids.9 Apart from that Ginkgobiloba has been proved to augment cholinergic system and increase the synaptic plasticity in central nervous system.10, 11
Ascorbic acid is an antioxidant found in citrus fruits and vegetables and strawberries.12 Studies have shown that Vitamin C enhanced the expression of genes involved in neurotransmission,13 neurogenesis,14 and maturation of embryonic stem cells of neurons.15 Recent studies proved that the free radical scavenging properties of ascorbic acid enhanced learning and memory in rats.16
Very few studies have addressed the changes in dendritic morphology of brain with exposure to stress along with intake of high saturated diet and the neuroprotective role of both Ginkgobiloba and Ascorbic acid against the toxicity caused on dendrites by stress and high saturated diet. With this back ground the present study was undertaken to explore the dendritic changes in various regions of brain.
Materials and Methods
Animal care and maintenance
Adult in-bred male Wistar rats weighing about 120-150g were obtained from the Central Animal House, Mamata Medical College, Khammam. Prior approval of institutional animal ethical committee was obtained for the study (IAEC/DP-05/C16 2012-2013). All the experimental procedures were carried out according to the guidelines prescribed by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India.
Animals were housed in polypropylene cages with paddy husk as the bedding material. Cages were maintained at standard temperature 25 ± 2°C, humidity (50-55%) under 12-hour light and dark cycle. Rats were fed ad libitum with a balanced diet containing 21.96% crude oil, 3.10% crude fibre, 7.37% ash, 1.38% sand silica.
Experimental design
Adult male Wistar rats were divided five groups with six animals in each group. The total duration of the study for 3 months.
Group I: Control animals received ad libitum diet and water (NC)
Group II: Fed with diet rich in high fat diet for three months and induced stress for 21 days (69th -90th day) (H+S).
Group III: Treated with Ginkgobiloba 100mg/kg.b.w for 15 days (75th- 90th day) (H+S+G).
Group IV: Treated with Ascorbic acid 100mg/kg.b.wfor 15 days (75th- 90th day) (H+S+C).
Group V: Treated with Ginkgobiloba 100mg/kg.b.w for 15 days and Ascorbic acid 100mg/kg.b.wt for 15 days (75th- 90th day) (H+S+G+C 100mg/kg).
Administration of high fat diet
Hyperlipidemia was produced by feeding with cholesterol-rich high-fat diet (H) for 3 months. Deoxycholic acid (5g) will be mixed thoroughly with 700g of powdered rat chow diet. Simultaneously cholesterol (5g) will be dissolved in 300g warm coconut oil. This oil solution of cholesterol will be added slowly into the powdered mixture to obtain a soft homogenous cake. This cholesterol-rich HFD will be molded into pellets of about 3g each and will be used to feed the animals.17
Induction of stress
Male wistar rats were placed in a wire mesh restrainer for 21 days, daily 6 hours. The restrainer was made up of a wooden base to which stainless steel wire mesh was hinged. A pad lock and latch were used to secure the rat. The dimensions were 8 cm (Length) x 4cm (Breadth) x 4 cm (Height). This type of wire mesh restrainer can only restrict the animal movement without any uneasiness, pain or suffocation.18
Administration of Ginkgobiloba
100 mg of Ginkgobiloba extract was dissolved in 1% gum acacia solution. This solution was administered to the animals at 100 mg/kg body weight with the help of oral gavage needle attached to a syringe for 15 days.19
Administration of ascorbic acid
200mg of Ascorbic acid was dissolved in the 20 ml of ordinary tap water, administered to the animals at the dose of 100mg/kg body weight with oral feeding needle.19
Golgi staining procedure
After 90 days of completion of experimental period, rats were deeply anesthetized with ether and sacrificed by cervical dislocation. Brains were removed quickly and placed in a petri dish containing freshly prepared Golgi- cox fixative. The hippocampus was dissected from both hemispheres of the brain. Tissue was processed for Golgi staining.18
Dendritic quantification
The dendritic quantification of hippocampal CA1 neurons was done by using the camera lucida technique.8 -10 well stained pyramidal neurons of CA1 region in hippocampus were selected from each rat and traced using camera lucida device (Dutta scientific works, Bangalore, India). Neurons that were darkly-stained throughout their arborization and with minimal overlap were selected. Neurons with truncated dendritic branches within a 100 μm radius from the cell body were excluded.
Quantification of dendritic branching points and dendritic intersections
The concentric circle method of Sholl was used for dendritic quantification. Concentric circles with radial distance of 20 μm were drawn on a transparent sheet and used for dendritic quantification. This sheet was placed on the cameralucida-traced neuron in order that centre of the cell body of the neuron coincided with the center of the concentric circles.
The number of branch points between two successive concentric circles i.e., within each successive 20 μm radial spheres were counted. The dendritic intersection was defined as the point where a dendrite touches or intersects the concentric circle. Both branch points and intersections were counted to a maximum radial distance of 100 μm from the center of the soma.18
Figure 1
Diagram showing a hippocampal CA3 neuron and the scheme of dendritic quantification. A transparent sheet with concentric circles of 20µ increment in radius (20µ-100µ) was placed on the camera lucida traced neuron so that the center of concentric circle coincides with the center of the cell body. Dendritic intersections at each concentric circle and dendritic branching points in each concentric zone werecounted. A- Apical dendrites, B- Basal dendrites, DBP- Dendritic branching points, DI- Dendritic intersections, S- Soma, CC- Concentric circles and 1 to 5- Concentric zones
Results
Apical dendritic intersections of CA3 neurons: Figure 2, Figure 6
Rats in H+S group showed significantly less number of apical intersections compared to NC 40, 60, 80 and 100 (p<0.001). Rats treated with only Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed significant increase in number of apical dendritic intersections compared to H+S. (H+S versus H+S+G): 60, 80 (p<0.01) and 100(p<0.05); (H+S versus H+S+C): 40, 80, 100 (p<0.01) and 60 (p<0.001). Rats treated with both Ginkgobiloba and Ascorbic acid showed higher significance in increase in number of apical dendritic intersections compared to H+S group.(H+S versus H+S+G+C):40, 60, 80 and 100(p<0.001).
Figure 2
Apical dendritic intersections of CA3 neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: ***p<0.001; H+S versus. H+S+ G:!p<0.05, !!p<0.01; H+S versus H+S+ C: &&& p<0.001,&&p<0.01; H+S versus H+S+G+C @@@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
Basal dendritic intersections of CA3 neurons Figure 3, Figure 6
Rats in H+S group showed significantly less number of dendritic intersections compared to NC 40, 60, 80 (p<0.001)and 100(p<0.01). Rats treated with only Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed significant increase in number of dendritic intersections (H+S versus H+S+G): 60, 80 (p<0.05); (H+S versus H+S+C): 60,100 (p<0.05) and 80 (p<0.001). Rats treated with both Ginkgobiloba and Ascorbic acid showed higher significance in increase in number of dendritic intersections compared to H+S group.(H+S versus H+S+G+C):40, 80, (p<0.001), 60 (p<0.01and 100(p<0.05).
Figure 3
Basal dendritic intersections of CA3 neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001, ** p<0.01; H+S versus. H+S+ G:!p<0.05; H+S versus H+S+ C: &&p<0.01, & p<0.05; H+S versus H+S+G+C @@@ p<0.001, @@ p<0.01@ p<0.05 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
Apical dendritic branching points of CA3 neurons Figure 4, Figure 6
A significantly less number of apical dendritic branching points were observed in H+S group compared to NC. 20-40, 80-100 (p<0.001) and 60-80 (p<0.01). A significant increase in number of apical dendritic branching points zones was observed in the rats treated with both Ginkgobiloba 100 mg/kg and Ascorbic acid 100mg/kg compared to H+S groups. H+S versus H+S+G+C):20-40, 60-80, 80-100 (p<0.01).
Figure 4
Apical dendritic branching points of CA3 neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001, ** p<0.01; H+S versus H+S+G+C @@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, Ascorbic acid 100 mg/kg body wt
Basal dendritic branching points of CA3 neurons Figure 5, Figure 6
A significantly less number of basal dendritic branching points were observed in the H+S group of rats compared to NC group. 20-40, 40-60, 60-80 and 80-100 (p<0.001). A significant increase in number of basal dendritic branching points was observed in the rats treated with only Ginkgobiloba 100 mg/kg (H+S versus H+S+G) 20-40, 60-80 (p<0.01) and 80-100 (p<0.001) and Ascorbic acid 100mg/kg compared to H+S group. (H+S versus H+S+C):20-40, 40-60, 60-80, 80-100 (p<0.01).
Rats treated with combination of both Ginkgobiloba and Ascorbic acid had higher significant increase in dendritic branching points when compard to H+S group. (H+S versus H+S+G+C):20-40, 40-60, 60-80, 80-100 (p<0.001).
Figure 5
Basal dendritic branching points of CA3 neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001, ** p<0.01; H+S versus H+S+G+C @@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt
Figure 6
Camera lucida tracings of CA3 neurons of Hippocampus showing the dendritic arborization in different groups (Golgi Cox staining). NC: normal control; H+S: High fat diet + Stress; G: Ginkgobiloba 100mg/kg; C: Ascorbic acid 100 mg/kg Note (i) increase in dendritic arborization in H+G & H+C compared to H+S group (ii) Significant increase in dendritic arborization in H+G+C compared to H+S group
Apical dendritic intrsections of Basolateral Amygdaloid neurons: Figure 7, Figure 9
Rats in H+S group showed significantly less number of apical dendritic intersections compared to NC 20, 40, 60, 80 (p<0.001) and 100(p<0.01). Rats treated with only Ginkgo biloba 100mg/kg showed significant increase in number of apical intersections compared to H+S. (H+S versus H+S+G): 20, 40, 60, (p<0.05), and 80(p<0.01). Rats treated with Ascorbic acid 100mg/kg also showed significant increase in number of apical intersections compared to H+S group. (H+S versus H+S+C): 20, 60, (p<0.05), 40, and 80(p<0.01). Rats treated with both Ginkgobiloba and Ascorbic acid showed higher significance in increase in number of intersections compared to H+S group. (H+S Vs H+S+G+C):20. 40, 80 (p<0.001) 60 (p<0.01) and (p<0.05) 100.
Figure 7
Apical dendritic intersections of Basolateral Amygdaloid neurons; Mean± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001, **p<0.01; H+S versus. H+S+ G:!p<0.05, !!p<0.01; H+S versus H+S+ C: & p<0.05, &&p<0.01; H+S versus H+S+G+C @ p<0.05, @@ p<0.01, @@@p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat dietplus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100mg/kg body wt
Basal dendritic points of Basolateral Amygdaloid neurons: Figure 8, Figure 9
H+S group of rats showed significantly less number of basal dendritic branching points at 20-40, 40-60, 80-100 (p<0.01) and 60-80 (p<0.001) zones compared to NC group. Rats that were administered only Ascorbic acid 100mg/kg showed significant increase in number of basal dendritic branching points compared to H+S group (H+S versus H+S+C): 80-100 (p<0.05). Rats treated with combination of Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg also showed significant increase in dendritic branching points. (H+S versus H+S+G+C): 20-40, 40-60, 80-100 (p<0.05).
Figure 8
Basal dendritic branching points of Basolateral Amygdaloid neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001, ** p<0.01; H+S versus H+S+C & p<0.05; H+S versus H+S+G+C @ p<0.05. (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt
Figure 9
Camera lucida tracings of Basolateral Amygdaloid neurons showing the dendritic arborization in different groups (Golgi Cox staining). NC: normal control; H+S: High fat diet + Stress; G: Ginkgobiloba 100mg/kg; C: Ascorbic acid 100 mg/kg Note (i) increase in dendritic arborization in H+G & H+C compared to H+S group (ii) Significant increase in dendritic arborization in H+G+C compared to H+S group
Apical dendritic intersections of Motor cortex neurons: Figure 10, Figure 14
Rats in H+S group showed significantly less number of apical intersections compared to NC 40, 60, 80 and 100 (p<0.001). Rats treated with only Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed significant increase in number of dendritic intersections compared to H+S. (H+S versus H+S+G): 40, 60, 80 (p<0.05) and 100(p<0.01); (H+S versus H+S+C): 40, (p<0.05) 60, 80 and 100 (p<0.01). Rats treated with combination of Ginkgobiloba and Ascorbic acid showed higher significance in increase in number of apical dendritic intersections compared to H+S group.(H+S versus H+S+G+C):40, (p<0.01) 60, 80 and 100 (p<0.001).
Figure 10
Apical dendritic intersections of Motor Cortex neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: *** p<0.001; H+S versus. H+S+ G:!p<0.05, !!p<0.01; H+S versus H+S+ C: && p<0.001,&p<0.05; H+S versus H+S+G+C @@ p<0.01 @@@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
Basal dendritic intersections of Motor cortex neurons: Figure 11, Figure 14
Rats in H+S group showed significantly less number of basal dendritic intersections compared to NC20, 80 (p<0.01)40, 60 and 100 (p<0.001). Rats treated with only Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed significant increase in number of dendritic intersections compared to H+S. (H+S versus H+S+G): 40, and 60 (p<0.05); (H+S versus H+S+C): 40 and 60 (p<0.01). Rats treated with combination of Ginkgobiloba and Ascorbic acid showed higher significance in increase in number of apical dendritic intersections compared to H+S group.(H+S versus H+S+G+C):40, 100 (p<0.01) and 60 (p<0.001).
Figure 11
Basal dendritic intersections of Motor Cortex neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: ** p<0.01 *** p<0.001; H+S versus. H+S+ G:!p<0.05; H+S versus H+S+ C: && p<0.001; H+S versus H+S+G+C @@ p<0.01 @@@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
Apical dendritic branching points of Motor Cortex neurons: Figure 12, Figure 14
A significantly less number of apical dendritic branching points were observed in H+S group compared to NC.20-40, 60-80, (p<0.001) and 80-100 (p<0.01). A significant increase in number of apical dendritic branching points zones was observed in the rats treated with only Ginkgobiloba 100 mg/kg and Ascorbic acid 100mg/kg compared to H+S groups. (H+S versus H+S+G): 60-80, 80-100 (p<0.05); (H+S versus H+S+C): 20-40, 60-80, (p<0.01) and 80-100 (p<0.05). Rats treated with combination of Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed higher significance in no of dendritic branching points.(H+S versus H+S+G+C):20-40, 60-80 and 80-100 (p<0.01).
Figure 12
Apical dendritic branching points of Motor Cortex neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: ** p<0.01 *** p<0.001; H+S versus. H+S+ G:!p<0.05; H+S versus H+S+ C: & p<0.05, && p<0.001; H+S versus H+S+G+C @@ p<0.01 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
Basal dendritic branching points of Motor Cortex neurons: Figure 13, Figure 14
A significantly less number of apical dendritic branching points were observed in H+S group compared to NC.20-40, 60-80, (p<0.001) and 80-100 (p<0.01). A significant increase in number of apical dendritic branching points zones was observed in the rats treated with only Ginkgobiloba 100 mg/kg and Ascorbic acid 100mg/kg compared to H+S groups. (H+S versus H+S+G): 20-40, 40-60 and 60-80 (p<0.05); (H+S versus H+S+C): 20-40, 60-80, (p<0.01) and 40-60 (p<0.05). Rats treated with combination of Ginkgobiloba 100mg/kg and Ascorbic acid 100mg/kg showed higher significance in no of dendritic branching points.(H+S versus H+S+G+C):20-40, 60-80 (p<0.001), 40-60 and 80-100 (p<0.01).
Figure 13
Basal dendritic branching points of Motor Cortex neurons; Mean ± SD is shown, n = 6 in each group. NC versus H+S: ** p<0.01 *** p<0.001; H+S versus. H+S+ G:!p<0.05; H+S versus H+S+ C: & p<0.05,&& p<0.001; H+S versus H+S+G+C @@ p<0.01, @@@ p<0.001 (One way ANOVA, Bonferroni’s test). NC: Control, H+S: High fat diet plus Stress, G: Ginkgobiloba 100 mg/kg body wt, C: Ascorbic acid 100 mg/kg body wt.
.
Figure 14
Camera lucida tracings of Motor Cortex neurons showing the dendritic arborization in different groups (Golgi Cox staining). NC: normal control; H+S: High fat diet + Stress; G: Ginkgobiloba 100mg/kg; C: Ascorbic acid 100 mg/kg Note (i) increase in dendritic arborization in H+G & H+C compared to H+S group (ii) Significant increase in dendritic arborization in H+G+C compared to H+S group
Discussion
Our study explored the neuroprotective role of Ginkgobiloba and Ascorbic acid against the high fat diet and stress induced neurotoxicity. The rats treated with Ginkgobiloba 100mg and Ascorbic acid 100mg showed significant increase in dendritic length and branching points but when given in combination there was higher significance in increase in dendritic length. The beneficial effects of Ginkgobiloba can be attributed to it pleitrophic effects.20
Ginkgobiloba have ameliorated the impairment of learning and memory abilities of rats induced by repeated high sustained +Gz (Positive acceleration) exposure.21 There is a clear line of evidence that Ginkgobiloba have improved the memory and learning deficits in rats induced by various stimuli such as chronic stress,22 fluoride,23 amyloid,24 aluminum,25 scopolamine,26 ischemia24 and aging.25
The cognitive enhancing properties of Ginkgobiloba can be ascribed to its potential effect on cholinergic system and neurotransmitters in brain.26 Few studies have shown that it has improved the microcirculation in brain by modulating the nitric oxide levels27 and by antagonizing the overproduction of endothelin-1 in the blood vessels.28 The beneficial action is mainly due to its antioxidant properties of the flavonoids present in it, for instance rats treated with Ginkgobiloba for fourteen days was found to enhance the antioxidant levels in hippocampus against sodium nitrite induced neurotoxicity.29 In another experiment it has reversed the mitochondrial dysfunction of hippocampi and platelets in middle aged ovariectomized rats.30
While the neuroprotective role of Gingkobiloba on dendrites of brain have been documented in very few studies for instance, aged rats treated with treated with Ginkgobiloba for eight weeks have shown increase in dendritic length and segments in CA1and CA3 regions of hippocampi.31 Barkats et al,32 reported that treatment with EGb 761 (50 mg/kg/day) for 7 months led to a significant increase in the projection field of intra- and infrapyramidal mossy fibers in the CA3 region of hippocampus. Dae Young Yoo33 et al, observed a significant increase in tertiary dendrites in hippocampus along with neuroblast differentiation in Dentate Gyrus after administration of Ginkgobiloba 40 mg/kg and 100mg/kg for 28 days. However Tchantchou et al34 did not find any significant change in the dendritic pattern in Alzheimer’s mice model when treated with Ginkgobiloba for six months. This discrepancy in the result may be related to the transgenic mouse model used in the experiment.
The neuroprotective effect of Ascorbic acid is due to its free radical scavenging and antioxidant properties.35 Many studies have revealed that Ascorbic acid have reduced the neurodegeneration caused by ischemia and exitotoxicity.36, 37, 38 Vitamin C protected the lead induced apoptosis in hippocampus39 and also attenuated the toxic effects of ethanol on neuroglial cells in brain.40, 41
In guinea pigs, Ascorbic acid deficiency have reduced the hippocampal volume and decreased the neuronal migration in dentate gyrus38, 42, 43, 44 Furthermore invitro studies have shown that on Ascorbic acid recycling have regulated the growth in neurite of brain 43 and enhanced the neural stem cells in the Midbrain by enhancing the generation of Midbrain type dopamine (mDA). However in another experiment treatment with Ascorbic acid did not alter hippocampal neuronal morphology or synaptic plasticity in CA1 region of young guinea pigs. 44 The difference in the result can be attributed to the area of hippocampus selected. In that experiment the author have only observed the changes of the dendritic morphology in CA1 region only.
Conclusion
In conclusion we observed that there was a higher significance in increase in dendritic brancing points and intersections in the brain in groups which were treated with both Ginkgobiloba and Ascorbic acid. Our findings are consistent with the findings of previous research. However the precise mechanisms responsible for this efficacy of Ginkgobiloba and Ascorbic acid have to be investigated in detail. Findings of our study provide a neuroanatomical basis and support therapeutic potential of Ginkgobiloba and Ascorbic acid in neurodegenerative diseases.
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