INTRODUCTION:

Radiation generates reactive oxygen species which induces toxicity to brain tissues. Brain being rich in lipid content, high oxygen demand and poor antioxidant system is the primary target for radiation-induced damage¹. Increased level of ROS produces oxidative stress which leads to damage glial cells and neurons². Literature survey shows, the radiation induced oxidative stress in the brain tissue initiating cell death signaling leading to programmed cell death and apoptosis^3-4. Oxidative stress in the cerebral cortex is a prime cause leading to various neurodegenerative disorders. This might be due the active role of cerebral cortex in various higher functions of the brain^5-6.

Endogenous and exogenous antioxidants are actively involved in inhibiting the formation of ROS⁷. Most exogenous antioxidants are derived from dietary constituents such as polyphenols, lipoic acid and carotenoids⁸. The radioprotective nature of plants has been studied by various researchers^9-11. In this regard, Cynodon dactylon (CD) (Dhub, Family: Poaceae), which has been used since ancient time has been shown to possess curative effects on spatial learning and motor coordination¹¹. Several in-vitro and in-vivo studies have validated the beneficial actions of CD^12-15. Neuroprotective effect of CD extract has also been documented in aluminum induced toxicity animal model¹⁶.Although various studies have reported the pharmacological actions of CD, very little is known about its action on radiation-induced neurotoxicity targeting specific brain tissues. The present study was focused to acquire more insight into interactions of CD with radiation-induced oxidative stress by taking into consideration the antioxidant level, inflammatory markers, neurogenesis along with the behavioral and cognitive parameters.

MATERIAL AND METHODS:

Ethics: All the procedures for the animal experiments were reviewed and approved by the Institutional Animal Ethical Committee, Kasturba Medical College, Mangalore (F. No. 25/439/2009; approved date: March 31, 2016). All the animal studies were conducted and maintained according to the guidelines proposed by the Committee for Control and Supervision of Experimentation on Animals, Government of India.

Experimental Animals:

Adult Swiss albino mice (25±2g) were procured from Central Animal House, Kasturba Medical College, Mangalore (Reg.No.213/CPCSEA). The experimental mice were placed in polypropylene cages with paddy husk beddings. All the animals were maintained at 25± 2°C temperature and 50±5% humidity with 12 h of dark-light cycles. Mice were provided with ad libtum access to laboratory food (commercial mice pellets from VRK nutritional solutions, India) and water.

Radiation procedure:

Swiss albino mice were placed in well-ventilated perspex box restrainers. Six mice at a time were exposed to whole body radiation of γ-rays (11 Gy/min). All the radiation procedures were carried in Centre for Application of Radioisotopes and Radiation Technology (CARRT), Mangalore University, Mangalagangotri, Karnataka, India.

Radiation dose selection:

In this experiment, it was found that at 4 Gy of radiation dose the mortality was 0%, whereas at 6Gy it increased to 33.3% and at 8 -14Gy it was 100%. Animals exposed to 8Gy of died in 25 days after irradiation, while mice exposed to 10Gy and above died within 10d with severe radiation sickness such as diarrhea, loss of appetite and loss of body hairs. The LD_50/30 was found to be 6.9Gy as explained in our previously published research¹⁰.

Details of Plant collection, extract preparation:

Entire plant of Cynodon dactylon with the roots was collected from the campus of Kasturba Medical College, Mangalore, Manipal Academy of Higher Education. The authentication of the plant was done by taxonomist .Cynodon dactylon was washed thoroughly in tap water and dried in room temperature for 15days. The dried plant was powdered and hydro alcoholic extract was prepared. Grinded plant powder (100g) and 50% of methanol in water (Total volume of 500 ml) was refluxed at 50-60˚C in a soxhlet apparatus for 72 hrs. The liquid extract was cooled and concentration was done by using rotary vacuum flash evaporator. The extract was kept in sterile bottle in cold storage, until further use.

Acute drug toxicity of hydroalcoholic extract of Cynodon dactylon:

Acute toxicity studies of hydroalcoholic extract of C. dactylon was done, and it was nontoxic up to 5g/kg body weight. It did not display any symptoms of behavioral changes and mortality up to 14days observation period¹⁰. Therefore, 1/20^th and 1/5^th of this dose, i.e., 0.25 and 1g/kg body weight, were used as low dose and high dose, respectively, in the subsequent study.

Experimental design:

Mice were randomly divided in to 6 groups each of 12 mice (n= 72 mice; n; number of mice) as follows:

Group-1 (Normal Group), administered with double distilled water

Group-2 (Radiation Control Group), exposed to sublethal dose (5Gy) of gamma rays

Group-3 (Low dose Group): administered a low dose (0.25g/kg b.w) of Cynodon dactylon extract for seven days;

Group-4 (High dose Group), administered a high dose (1g/kg b.w) of Cynodon dactylon extract for seven consecutive days

Group-5 (Low dose of plant extract+ radiation Group) received a low dose (0.25/kg b. w)of Cynodon dactylon extract for seven consecutive days and followed by irradiating sublethal dose (5Gy) of gamma rays

Group-6 (High dose of plant extract+ radiation Group), The mice of this group were administered a high dose (1g/kg b. w) of Cynodon dactylon extract for seven consecutive days and followed by irradiating sublethal dose (5Gy) of gamma rays.

After the 24 hours of irradiation mice of all groups were subjected to behavioral studies.

Open felid test:

In this experiment along with exploratory behaviour, locomotors activity and anxiety level of mice was evaluated. This test has been widely used to determine anxious performance by evaluating time spent in central and peripheral squares. Thus this test was carried out to assess the effect of Cynodon dactylon extract on irradiated Swiss albino mice by following method of Michael C and Michael L¹⁸. The open field apparatus consists of a rectangular wooden box (72x72x36cm); the floor place marked into 16 squares (18x18 cm). In this setup, mouse was placed in one corner of the box and allowed to explore the full arena for five minutes. The section of total exploratory time spent in peripheral area (close to wall) and in the central area will be measured for individual mice. Each mouse will have 3 sessions with 30 minute interval. In addition to this rearing (elevated hind limb & pelvis with elevation of fore limb), defecation and grooming (use of head, tongue and fore limb for the process of cleaning various part of the body) behavior were also quantified.

Object recognition test:

This test was used to study behavioral assay to analyze various aspects of learning and memory of rodents following procedure explained by Bevins et al¹⁹. Characteristically, it is carried out in two sessions in the open field box in specific time interval. In first session (familiarization session), the animal is free to explore two similar objects, and during the second session (test session), one of the objects is replaced by a novel, new object. Because mice have an inherent preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. In this experiment first the individual mice were exposed to the empty box for five minutes. Next after 15 minutes period gap a mice were subjected to apparatus where two objects A and B. The exploration time on each object was shown (as seconds) to indicate the exploring activity of mice. On the final phase mice were allowed to explore the open field in the presence of two objects: the familiar object A and a novel object C. Discrimination index calculated for each mouse

Discrimination index= [{Time spent on exploring Novel object/total exploration time} – {Time spent on exploring familiar object/total exploration time}].

After the completion behavioral and cognitive studies mice were scarified, whole brain was excised, quickly washed with chilled saline and removed blood and other debris. The cerebral cortex was separated out from the whole brain. The tissue homogenates (10%) were prepared in chilled phosphate buffer saline (pH 7.4) and centrifuged to collect the supernatant. Cytokines (TNF-α, IL-6) and Brain-Derived Neurotrophic Factor level were estimated by using commercially available kit following manufactures protocol. Total reduced glutathione was estimated in the cortex as described by Moron et al¹⁷. Radiation-induced oxidative stress in cortex of mice was analyzed by lipid peroxidation¹⁸, Catalase¹⁹, Superoxide dismutase²⁰, total protein content²¹ and nucleic acid content^25-26

Statistical analysis:

All the results were represented as Mean±SEM. Data was evaluated by one-way analysis of variance (ANOVA) following post hoc Tukey test using IBM SPSS statistics 20, P< 0.05was considered as significant.

RESULTS:

Effect on Cynodon dactylon on radiation-induced behavioral changes and cognitive impairments:

Irradiated Swiss albino mice (Group-2) spent significantly less time in the central arena (p<0.001) and spent more time in peripheral field compared to normal control mice (Group-1). Mice administered with CDE in low (Group-5) and high (Group-6) doses, displayed significant increase, (P=0.038), (P=0.016) prior to radiation increased time explored in center square and decreased time explored in peripheral squares compared to radiation control group (Group-2) Fig. 1

Fig.1: Time spent in central and peripheral arena by mice of different groups in the open field paradigm.

Each bar represents Mean ±SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; for comparison Group-2 and Group-6, #P<0.05.

Irradiated Swiss albino mice showed a significant reduction (p<0.001) in the rearing score compared to normal control and it was significantly more, (P=0.015), (P=0.017) in mice administered with CDE in low (Group-5) and high (Group-6) doses prior to radiation. Grooming scores were significantly (p<0.001) more in irradiated group (Group-2) compared to normal control group (Group-1) and it was significantly less (P=0.027), (P=0.021) in mice administered with CDE in low (Group-5) and high (Group-6) doses prior to radiation when compared to radiation control Table 1.

Table 1: Number of rearing, grooming, and defecation of mice in different groups in the open field exploration test.

	Rearing	Grooming	Defecation
G-1	17.63±0.44	2027±0.11	9.22±0.24
G-2	8.77±0.31***	6.50.31***	18.77±0.38***
G-3	17.880.43	2.19±0.23	10.36±0.33
G-4	18.33±0.61	2.061±0.22	9.94±0.19
G-5	10.69±0.17^$	5.55±0.25^$	17.22±0.21^$
G-6	11.19±0.37^##	5.52±0.13^#	17.16±0.19^##

Each value represents the number of times ± SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; for comparison Group-2 and Group-6, #P<0.05, ##P<0.01.

In Novel object recognition test mouse irradiated with a sub-lethal dose of 5Gy showed significant (P<0.001) low value of Discrimination Index (DI) for novel object compared to normal control mice. Radiation control mice spent more time in familiar objects and were more inactive throughout the sessions. Contradictory to this normal control mice were spending more time on novel object. Groups which pretreated with CDE in low (Group-5) and high (Group-6) doses, displayed significant increase (P=0.013), (P=0.003) in DI on familiar object compared to the radiation control group (Group-2) Fig.2.

Fig. 2 Discrimination index of the novel object on a familiar object by mice of different groups in Object recognition test.

Each bar represents Mean ± SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; comparison Group-2 and Group-6, ##P<0.01.

Effect on Cynodon dactylon on radiation-induced inflammation:

Gamma-irradiation of mice group (Group-2) at 5Gy dose exhibited a significant elevation (P<0.001) in inflammation as measured by TNF-α and IL-6, respectively. In contrast, a significant decrease in the inflammatory markers TNF-α was observed in CDE-treated in low (Group-5) and high (Group-6) doses, (P=0.038), (P=0.020), and IL-6was observed in CDE-treated in low (Group-5) and high (Group-6) doses, (P=0.050), (P=0.009) then gamma-irradiated mice group as compared irradiated mice group (Group-2) without pretreatment with CDE Fig.3 and Fig.4.

Fig .3 Changes in the level of IL-6 in the cortex of mice of different groups.

Each bar represents Mean ± SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; for comparison Group-2 and Group-6, #P<0.05.

Fig.4: Changes in the level of TNF- α in the cortex of mice of different groups.

Each bar represents Mean ± SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; for comparison Group-2 and Group-6, #P<0.05.

Effect on Cynodon dactylon on radiation-induced growth factor:

Gamma radiation exposure induced significant decline (P < 0.01) in the level of BDNF against normal control group (Group-1). C. dactylon extract treatment in low and high dose prior to radiation exposure displayed significant increased (P = 0.045), (P = 0.013) BDNF level in cortex compared to radiation control group (Group-2) Fig. 5.

Fig.5: Changes in the level of BDNF in the cortex of mice of different groups.

Each bar represents Mean ± SEM, n = 12. For comparison Group-1 and Group-2, ***P<0.001; for comparison Group-2 and Group-5, $P<0.05; for comparison Group-2 and Group-6, #P<0.05.

Effect on Cynodon dactylon on radiation induced biochemical changes:

Gamma radiation produced significant reduction (P< 0.001) in level of GSH, SOD, catalase, and significant enhance P < 0.001 in MDA, nitric oxide level in radiation control group (Group-2) against normal control group (Group-1). Pre treatment of CDE in low and high dose prior to radiation exposure showed significant increase in GSH (P = 0.039), (P =0.001); SOD (P= 0.023), (P =0.017); catalase (P =0.027), (P=0.015); and significant increase in MDA (P = 0.021), (P=0.007); nitric oxide level(P=0.030), (P=0.007). A significant decline in total protein, DNA and RNA content was observed in gamma irradiated radiation control group (Group-2) against normal control group (Group-1). Pre treatment of CDE in low and high dose prior to radiation exposure showed significant increase in protein (P = 0.038), (P =0.007) RNA (P=0.035), (P = 0.019) and DNA (P=0.036), (P =0.020); the results of various biochemical estimations are mentioned in Table.2.

Table 2: Biochemical changes in the cortex of mice of different groups.

Groups	Gsh (µ mole / g tissue	Sod (value /ml/mg protein)	CAT (µ / g of tissue)	MDA (µ mole/g tissue	No (µmole/g tissue)	Protein content (g/ml of tissue)	DNA content (ng/ml of tissue	RNA content ng/ml/of tissue
G-1	0.69±0.09	1.4±23	31.16±1.86	27.87±4	43.08±8.2	0.84±0.025	731.97±34	336.37±26.8
G-2	0.26±0.05^***	0.42±0.43^***	17.06±2.82^***	60.57±14.1^***	71.33±16.6^***	0046±0.023^***	332.15±36^***	148.81±13.9^***
G-3	0.74±0.08	1.13±0.18	31±1.17	27.2±1.9	44.41±7.22	0.85±0.022	741.38±30	343.19±26.5
G-4	0.74±0.06	1.14±0.38	31.59±1.68	26.53±3	41.66±6.24	0.84±0.023	744.85±34	344.61±.25.5
G-5	0.37±0.1^$	0.88±0.46^$	19.3±0.6^$	50.38±9^$	66±2.92^$	0.49±0.017	370.98±10^$	174.44±10.6^$
G-6	0.47±0.11^###	0.9±0.34^#	19.45±1.39^#	49.19±6.3^##	64.08±9.8^##	0.50±0.28^##	368.39±10^#	187.47±13.25^#

Each value represents Mean ±SD, n = 12. For comparison Group-1 and Group-2, ^*P<0.05,^**P<0.01***P<0.001; for comparison Group-2 and Group-5, ^$P<0.05^$P<0.01^$P<0.001; for comparison Group-2 and Group-6, ^#P<0.05, ^##P<0.01,^###P<0.001.

DISCUSSION:

External beam radiotherapy has been associated with neurological damage progressively detrimental over time leading to neural and cognitive deficiencies. cognitive impairment is a major concern in the recent years, due to increased incidence of cancer. Lack of research is available on radiation-induced oxidative stress on cerebral cortex. As the population of long-term survivors of radiotherapy continues to grow, understanding the irradiation effect on cerebral cortex is of prime importance with regard to neurological health concern. The use of radio protectors represents a distinguishable policy to improve the therapeutic index in radiotherapy. The development of radio protective agents has been the subject of intense research; however no ideal, safe synthetic radioprotectors are available till date. The side effects associated with the synthetic radioprotectors necessitated the search for alternative drugs, which could be less toxic and highly effective at optimum dose levels. This has focused attention of the researchers towards the plants and natural products focusing on a new drug discovery which would be safer than the available synthetic drug. One of the main limitations associated with the treatment of the brain tissues is the selective permeability of the radioprotective agent²⁴. The present study explored the CD protective effects against radiation-induced damage targeting cerebral cortex due to its active role in higher functions of the brain. No noticeable signs of toxicity were observed in our study in the CDE-treated mice.

Performance in an open field atmosphere needs a reaction from the rodents by a composite of innate and learned behavioral appearance²⁵. The locomotive action is usually analyzed as a behavioral paradigm because it indicates the total stage of arousal of the animal²⁶. In our study whole-body gamma irradiation of dose 5Gy induced a significant reduction in locomotive action and exploratory behavior of mice in comparison with normal control. Radiation control mice exhibited immobile posture in most of the test duration along with increased score of grooming decreased score of rearing. The observed effect can be well correlated with anxiety. Radiation induced damage to the intestine increased the defecation scoring in radiation control mice. Mice pretreated with CDE prior to exposure of 5Gy showed more active in novel environment by exploring more time in central squares rather than spending more time in peripheral squares. Anxiety was decreased so that it displayed low score of grooming and high score of rearing. The defecation score was also significantly less compared to radiation control which might be indicating the protective role of CDE against intestinal damage. Our study reports are in accordance with the study on hydroalcoholic extract of Vitis vinifera aluminum chloride-induced toxicity rats²⁷.

Object recognition test is based on the rodent’s nature to interest toward and explores novel objects that never been exposed earlier. Naturally, rodents have a tendency towards novelty due to their innate preference for newer things than familiar things. This nature of rodents utilized for checking their memory-related studies²⁸. Radiation-induced memory impairment was observed in gamma irradiated mice depicting the negative discrimination index towards novel objects compared to familiar objects. On the contrary, mice pretreated with CDE in both doses showed positive discrimination index on novel objects than familiar objects when compared to radiation control mice. Mice pretreated with CDE had attained memory of the first exposure by showing a strong bias for the new object. Similarly positive results were observed in scopolamine-induced dementia and oxidative stress²⁹.

Inflammation in the central peripheral and peripheral nervous system is distinguished by the up-regulation of cytokines and their receptors present in brain^30.The cytokines are substances released by peripheral microglia, immune cells, astrocytes and neurons in the nervous system. Augmented levels of inflammatory indicators tumor necrosis factor-alpha and interleukin-6 was observed in the brain cortex of irradiated mice indicating a of high level of inflammation. Brain tissue exposed to radiation induces free radicals creating oxidative stress subsequently stimulating cytokines IL-6 and TNF-α up-regulating the pro-inflammatory pathways. Il-6 and TNF-alpha are also having capacity to inhibit neurogenesis. Mice pretreated by CDE in both doses showed decline in level of pro-inflammatory markers TNF-α and IL-6 in cortex. Studies on ginger extract derivative from Zingiber officinale and Alpina galanga showed inhibition of translation of numerous genes implicated in the inflammatory reaction³¹.

Brain-derived neurotrophic factor (BDNF) is related to the neurotrophin family and very much studied relating to its positive effect on survival enhancement and synaptic regulation in the central nervous system. Studies showed that decreased BDNF and T TrkB-associated signaling are involved in development of neurodegenerative disorders³². BDNF has the capacity to promote the recovery process, protection against oxidative stress, restrain apoptosis in the neurons. Exposure to ionizing radiation leads to decline in BDNF levels³³.In our study whole-body exposure of mice to gamma radiation significantly declined BDNF levels in mice cortex.In the present study, was by γ-ray exposure and reversed in some degree CDE treatment prior to radiation. This suggests that CDE may have neuroprotective effects such as promoting neurogenesis by withstanding BDNF level on radiation-induced oxidative stress. Similar results were observed where the Kaempferia parviflora administered group significantly increased BDNF levels in the rat hippocampus compared to the valproic acid-treated group³⁰.

The present study showed reduction in level of reduced glutathione, catalase, superoxide dismutase, protein content deoxyribonucleic acid and ribonucleic acid contents in radiation control mice. In addition to this significant increase in of TBARS and NO in the brain cortex of gamma-irradiated mice indicates the high level of oxidative stress and free radicals generation. Pretreatment of CDE prior to exposure of gamma radiation showed significant augment in reduced glutathione, catalase, superoxide dismutase, protein content deoxyribonucleic acid and ribonucleic acid and a significant decrease in of TBARS and NO level in cortex of mice. Ionizing radiation-induced oxidative stress and neurotoxicity increased hydroxyl radicals by increased nitric oxide, lipid peroxides, and defective endogenous antioxidant systems.

In the present study, the pretreatment of mice with CDE has been assigned as a neuroprotective intermediary from ionizing radiation-induced oxidative stress altering cognitive dysfunction. This study evidenced the improvement. The justification of the observed protective role is significantly based on the observed results on potent biomarkers. The protective role of the CDE extract might be attributed to the different functional constituents of antioxidants flavonoids, polyphenols, apigenin, C-glycosides, beta-sitosterol, luteolin, citronellol, phytol, truxillic acid, docosanoic acid, linolenic acid, docosanoic acid, syringol, hexadecanoic acid, eicosanoic acid³⁴. Our previous studies also documented the presence of tannins, steroids, flavonoids, saponins, alkaloids and saponins. Bioactive components such as orientein, gallic acid, morin, rutin were also confirmed in our previous reports on CDE by HPLC analysis¹⁰. The various in-vitro study models of CDE also showed a dose-dependent increase in free radical scavenging^35-36. The polyphenols might have been counteracted deleterious effects of ionizing radiation by promoting DNA repair system and protect mRNAs of antioxidant enzymes such as glutathione, catalase, and superoxide dismutase³⁷. In our previous study pretreatment of CDE for seven days showed improvement in spatial learning, memory and motor coordination. In addition to this it the antioxidant enzymes were increased and oxidative stress markers were declined in cerebellum of mice¹⁰. The observed study reports are in consistent with the previous study findings^38-39.

The phytoconstituents of Cynodon dactylon ameliorates the deleterious effects of gamma irradiation and could be used as a neuroprotector or radioprotective agent. So Cynodon dactylon must be studied in extensive range to detect its activity as neuroprotective drug for different pathological conditions. Drastic development of technology has caused the exposure to radiation inevitable. In conclusion, CDE has exerted a significant neuroprotective effect aligned with ionizing radiation-induced oxidative stress and neuronal damage on cerebral cortex by scavenging the free radicals and by promoting the antioxidants. Further in depth studies are needed for the evaluation and isolation of active compounds facilitating the development of ideal neuroprotectors formulations in the near future.

ACKNOWLEDGEMENT:

We thank the Board of Research in Nuclear Science (BRNS), Mumbai, of Atomic Energy, Government of India, for providing the funding (Grant No. 34 (1)/14/38/2014-BRNS/1932/27/11/2014) and providing the financial support. We are also thankful to the Manipal Academy of Higher Education and Mangalore University for providing all the facilities needed for this research work

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 29.05.2020 Modified on 30.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2569-2575.

DOI: 10.52711/0974-360X.2021.00452