Effect of Loranthus longiflorus ethanolic Extract on Neuronal Damage Induced by Electromagnetic Radiation in Wistar Rats

The use of mobile phone is increasing with the generation of electromagnetic field (EMF). Several reports are available about the detrimental effects of microwaves generated by EMF (Ma et al., 2017).
       
EMF at low frequencies also leads to pathological changes including increased permeability of blood brain barrier (Eberhardt et al., 2008), decreased neuronal function, increased activity of the alpha, beta and gamma frequency bands in nearly every brain region in electroencephalograph (Roggeveen et al., 2015), altered regional cerebral blood circulation, anti-oxidant and oxidant imbalance, neurotransmitter imbalance and genomic response (Cichon et al., 2017).
       
The radiation from mobile phones can alter the ornithine decarboxylase activity (Nowotarski et al., 2018) and the autophagic genes and autophagic regulatory proteins were also significantly changed in the hippocampus which plays a key role in eliminating the intracellular damage and unwanted proteins (Kim et al., 2018). Experimental studies have shown that EMR from mobile phones can affect the brain in many ways (Yakubu et al., 2019).
       
Herbal extracts have antioxidant effect and are known to have minimal side effects and better protection from deleterious agents (Cui et al., 2019). Loranthus longiflorus belongs to the family of Loranthaceae, is a perennial climbing woody parasite, which has been used in indigenous system of medicine as a potential medicinal agent such as astringent, aphrodisiac, narcotic and useful in treating asthma, pulmonary tuberculosis, vesical and renal calculi. The plant has been shown to possess neuroprotective and anti oxidant effects against oxidative stress in NG108 15 cells (Daniel et al., 2011). It was also reported to inhibit AChE activity, ROS production and Ca2+ accumulation which aid memory enhancing and neuroprotective effect (Weon et al., 2014).The aim of this study was to find the effect of LLE on rats exposed to electromagnetic radiation in comparison with melatonin.

Plant material and preparation
 
The plant was procured from Tirunelveli District, Tamil Naduand India’ and was authenticated by Dr. V. Chelladurai, Taxonomist and Research Consultant, St. Xavier Institute of Research, Palayamkottai, Tirunelveli District. The shade dried powdered leaves of LLE plant were extracted with 80% (w/v) ethyl alcohol using Soxhlet apparatus (Chandrakasan et al., 2017). The extracts were concentrated, dried under vacuum and stored in air tight bottles.
 
Animals
 
Wistar rats weighing 200-300 g were obtained from the Centre for Laboratory and Animal Research, Saveetha Institute of Medical and Technical Sciences (CLAR, SIMATS). The rats were housed in polypropylene cages under natural light/dark cycle at 25°C+2°C with free access to food and water. The rats were acclimatized to laboratory conditions for 5 days prior to the commencement of the experiment. The animal experiments were carried out in the Centre for Laboratory Animal Research, Saveetha Institute of Medical and Technical Sciences in 2019-20, after obtaining approval from the Institutional Animal Ethics Committee (SU/CLAR/RD/ 025/2017).
 
Experimental design
 
Twenty-four animals were randomly segregated into four groups (I-IV) with each group containing six rats. Group I served as control that received only pelleted feed and water, the remaining groups received EMR exposure from mobile phone of 900MHz GSM (Global System of Mobile communication) at a specific absorption rate (SAR) of 2W. The mobile phone was kept in conversation mode for 2 hours per day for 28 days following the method of (Huang et al., 2017). Animals were free to move in the cage. The phone was kept on the roof within the cage and a wire mesh was tied to prevent the animals from contact with the phone. Meanwhile, group III animals received LLE 500 mg/kg bw p.o/day for 28 days and group IV animals received Melatonin once daily for 28 consecutive days using oral gavage. The optimum dose of LLE extract was fixed based on dose fixation study. After 10, 20 and 28 days of exposure, control and experimental group animals were tested for behavioural assays.
Group I- Control treated with vehicle (1ml).
Group II- EMR (2 h/day).
Group III- EMR + LLE (500 mg/day/ kg bwp.o).
Group IV- EMR + Melatonin (10 mg/day/ kg bwp.o).
 
Behavioural studies
 
Behavioural studies on rats were performed using Y Maze to evaluate memory and learning functions. Animals were placed in the centre of the maze and were given free access to the three arms. If the rat chooses a different arm than the one it earlier visited the choice is known as alteration, this is the correct response and if the previous arm is choosing it is taken as an error. An alteration was defined as entry into all three arms consecutively, for instance animal makes following arm entries; CBA, BAC, CAB, BCA, ABC, ACB and the sequence of arm entries was manually recorded. The equipment was cleaned with 5% alcohol and permitted to dry among sessions. The alteration percentage was then calculated as the ratio of actual to possible alterations. Number of triads = (total entries-2). Triad (set of three letters) containing all three letters is scored as alteration.                 
   
Measurement of neurotransmitter levels
 
On day 29, rats were anesthetized using isoflurane and the blood was collected from retro-orbital venous plexus in ethylene diamine tetraacetic acid tubes. The serum was separated by 3000 rpm for 10 minutes and was subjected to determine the levels of neurotransmitters γ-aminobutyric acid (GABA), dopamine and acetylcholine (ACh).
 
Tyrosine hydroxylase protein expression by Immunohistochemistry
 
Immunolocalization of Tyrosine hydroxylase [TH] protein in cells was carried out by ‘indirect peroxidase’ method. Tyrosine hydroxylase protein expression was viewed according to the method of (Nedoboy et al., 2016). A HRP/DAB detection IHC kit was used conferring to the manufacturer’s protocol. The immunohistochemistry images were quantified by Scion image software. TH immune reactivity was used as a marker of neurons in hippocampal sections and was detected with a monoclonal antibody.
 
Histological examination
 
Hippocampus were dissected out carefully from the brain tissue and fixed in 10% formalin. Later, the tissue were processed through a series of alcohol chambers containing low to high concentration of ethyl alcohol to remove excess water and formalin from the tissue. Before paraffinizing, the tissues were placed in xylene to remove alcohol. Paraffin blocks were made and 5 micron size sections were made carefully. These sections were then stained with haematoxylin and eosin. Analysis of neuron cell bodies in the pyramidal cell layer of CA-1 region of hippocampus was performed. The slides were then air dried and viewed under a light microscope and photomicrographs were taken at magnification of 40x and 10x.
 
Statistical analysis
 
The results were expressed as mean ± S.E.M. The obtained data were analysed by one way ANOVA with Bonferroni multiple comparison test and the level of statistical significance adopted was P<0.05. Sigma Plot 13 systat software was used.

Effect of LLE on behavioural effects
 
The EMR exposed group of rats exhibited a significant deficit in spontaneous alternation in contrast to control rats. On 28th day the animals were placed into a Y-maze, EMR exposed rat appeared to exhibit significantly more reducedspontaneous alternation relative to treatment group. Treatment with LLE and melatonin exhibited a significant increase in spontaneous alteration on day 28. Using the procedures outlined in this method, a significant restoration of spontaneous alternation was observed after treatment with the extract (Fig 1).
 

 
Effect of LLE on GABA, Ach and DOPA levels
 
Rats exposed to electromagnetic radiation showed decrease in the concentration of GABA and DOPA levels in serum. Treatment with LLE for 28 days has increased the levels in serum compared with the control group. Significant increase in the level of acetylcholine was observed in rats exposed to electromagnetic radiation. It was found that treatment with LLE extract could improve the serum neurotransmitters levels to their normal status. The results of the present study showed similar effect to that of melatonin against EMR reaction on neurotransmitters (Fig 2A and 2B).
 

 
Effect of LLE on pyramidal neurons in CA 1 region of hippocampus
 
The hippocampal tissue of control group shows normal histology of CA1 region with distinct parts of hippocampus (Fig 3A) and pyramidal layer neuronal cell bodies of CA-1 (Cornu Ammonis-1). The EMR exposed group showed a decrease in hippocampal pyramidal layer thickness and significant neuronal loss of hippocampal CA1 region (Fig 3B). The hippocampal region of the melatonin treated group showed normal number of pyramidal neurons extending into stratum radiatum of hippocampus (Fig 3C). LLE treated group showed increased number of surviving neurons in the CA1 region (Fig 3D).
 

 
Effect of LLE on tyrosine hydroxylase in hippocampus
 
Immunostaining intensity of tyrosine hydroxylase (arrow mark indicates dark brown colour positive expression) was increased in the region of hippocampus tissues of normal control rats (Fig 4A). The positive cells were less in number in EMR exposed rats (Fig 4B). However, melatonin treatment also significantly up regulated tyrosine hydroxylase expression in the CA1 of the hippocampus (Fig 4C). LLE treatment after EMR exposure protected the neurons and significantly increased tyrosine hydroxylase expression in the CA1 and DG regions of the hippocampus (Fig 4D). The quantification of relative expression of THY positive cells was depicted in the Fig 5. The expression of tyrosine hydroxylase was significantly reduced in EMR exposed group as compared to normal control whereas melatonin and LLE extract treatment showed similar THY expression, that significantly increased as compared to EMR exposed rats.
 

 

       
Nowadays, people are constantly exposed to electromagnetic radiation emitted from electronic devices particularly the mobile phones. Electromagnetic waves can break chemical bonds and harm the living tissues. Extensive researches have been done to explore the impact of electromagnetic radiation on human health. It has been reported that, radiation from electromagnetic fields (EMF) of generators augmented intracellular concentration of reactive oxygen species, activated apoptotic signalling and induced marked cell death (Parasuraman et al., 2018). At sufficiently high flux levels, different bands of electromagnetic radiation are found to cause deleterious health effects in people. It is important to assess the physiological action of long-term exposure to the EMR from smart phones (Lin et al., 2016). To find a remedy for this frequent exposure to EMR, the leaf extract of LLE has been considered for the study with melatonin as standard for comparison.
       
In this study, spontaneous alternation test with a Y maze apparatus was used to assess learning and memory and it showed that treatment with LLE increased spontaneous alterations in rats exposed to electromagnetic radiation. It also exhibited decreased exploratory behaviour in EMR exposed rats and treatment with LLE or melatonin showed improved behaviour. These changes have been shown to be occurred after EMR exposure might be due to changesin the ability of the radiation to change protein activity either inside or outside the cell (Turro, 1991; Kula et al., 1999; Levitt, 2008). The biological effects of electromagnetic fields may lead to the alternation in biological molecules structure (Adey, 1993). The protective effect of LLE against EMF induced damage in brain has been established in a study conducted by (Nagar et al., 2013). Thus, the present study also depicted concomitant results with the previous reports that give an evidence for memory enhancing property of LLE.
       
The levels of dopamine in control rats were lower than those of rats exposed to EMF. Increase in the dopamine levels were observed in rats treated with EMF (Reiter et al., 2016). Highest level of dopamine was observed in rats exposed to EMF for 28 days. Increases in dopamine concentration in rats exposed to EMF could account for stress-related activity of radiofrequency pulse exposure on dopaminergic cultures. Another mechanism to control neuron function is calcium homeostasis, proposed to be a mediator of cell response to radiofrequency exposure. It was also reported that EMR-induced alterations in the rat involved N-methyl- D-aspartate (NMDA) receptor in the brain (Kim et al., 2019).
       
Rats exposed to electromagnetic radiation showed remarkable increase in GABA and Ach neurotransmitter levels in serum. These alterations can be attributed to the increased rate O₂- and H₂O₂ formation. Decot et al., 2017 reported an increase in D-1 dopamine receptors and activity in the striatum of the rat after magnetic field exposure. These increases in dopamine concentration in rats exposed to EMF can be recognized to the stress-related activity of radiofrequency pulse exposure on dopaminergic cultures. Electromagnetic radiation also increased acetylcholine-esterase activity with decreased Ach concentration in serum (Hassanshahi et al., 2017). The present findings of the study substantiate that administration of LLE to rats controlled the harmful effect of EMR. This beneficial effect of LLE may be due to the suppression of calcium release from endoplasmic reticulum through binding to neuronal synaptic vesicle protein 2A receptor (Omura et al., 2015). Calciumaccumulation with respect to repetitive stimulation leads to glutamate over flow. Therefore, the binding of the synaptic vesicle protein receptor restored the ability of neurons to decrease the excessive glutamate overflow (Lenz et al., 2016). The present results further indicate that rats treated with LLE exhibited higher GABA levels as compared to control rats which may be due to a synergy of LLE associated with beneficial pharmacodynamics action (Azab et al., 2017).
       
In this study, the pyramidal neurons of CA1 region of hippocampus showed shortening of dendrites, loss of spine synapses and suppression of the neurogenesis in EMR exposed animals. This could be the cause for impairment of spatial memory shown in experimental animals. Whereas, administration of LLE protected the animals from radiation induced neuro-degeneration and decreased abnormal behavioural performance which was evident from the results of histology of hippocampus region.
       
EMR is capable of generating reactive oxygen species (ROS) comprising superoxide, hydrogen peroxide and hydroxyl radical leading to narcotic and oxidative damages (Houston et al., 2018). There are numerous cellular defences, which regulate the level of reactive oxygen species (ROS) and protect against the ill-effects of free radicals. The primary defence consist of two groups, the antioxidant compounds and the antioxidant scavenging enzymes. The secondary defence include lipolytic and proteolytic enzymes. Both groups of enzymes act as secondary lines of defence by “cleaning up” lipid or proteins that are damaged, altered or being turned over (Gamila et al., 2016).The antioxidant effect of LLE is mainly due to phenolic components, such as flavonoids, phenolic acid and phenolic diterpenes because of their redox properties, which could play a significant role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides. A positive correlation was evident between antioxidative and neuroprotective action due to its phenolic content. The result of the present study concludes that the changes in the neurotransmitters caused by EMR were restored by LLE. It also protected the neuronal cells in hippocampal tissue from the neuronal dysfunction, degeneration and cell death caused by EMR.

The present study reveals that Loranthus longiflorus ethanolic extract might be used as a potent antioxidant and neuroprotectant in treating neurodegenerative disorders caused by electromagnetic radiation in wistar rats implicating its protective potentials due to its neuroprotective capacity in comparison with melatonin. Further studies are desirable to provide greater insight in identifying and isolating the bioactive compound responsible for the antioxidant and neuroprotectant activity.

The author expresses their sincere gratitude to Dr. Ethirajan Sukumar, Dean Research and SIMATS for his help in plant extraction work and critically reviewing the manuscript.