A Study of Anxiety on Experimental Epileptic Rat Models using Dark Light Box

The word neurodegeneration refers to defects in neuronal structure and consequently its function. The main characteristics of these disorders are relentless progression and cognitive declination. Epilepsy is one of the neurodegenerative disorders, around 50 million people in the world are affected with. Though it is one of the major health problems in the present society, there are several gaps in understanding the consequences related to neurological disorders. As research works related to neurodegeneration is very much limited in India we have planned one as an initiative. We segregated 8 animal groups, each with 6 animals for this work. The animal groups are LC, CO, AC15, AC25, AC35, BA10, BA15 and BA20. This study was conducted on 10th day after the lesion by considering the day of lesion as day ‘1’ and the next day as day 2nd. All the animals were recovered completely within these 10 days and were put in the dark light box to analyse the anxiety level of the animals, so as to analyse the effect of the drug employed. This particular study clearly supported the efficacy of the drug as the drug group animals were less anxious or even behaved normal. Both the crude extract and the selected active principle have proved their efficacy by the study. Original Research Article Praba et al.; JPRI, 33(51B): 90-96, 2021; Article no.JPRI.77140 91


INTRODUCTION
Anxiety and stress became the normal part of the life now a days, that leads to loss of concentration, tension and irritability if persist for a long time. Epilepsy is one of the major neurodegenerative disorder related with anxiety that affects people of any gender, race and nation [1]. It is the fourth most common neurological disorder related with seizure, coexisting health conditions, abnormal behavior and sudden unexpected death. About 50 million people around the world are affected by epilepsy and temporal lobe epilepsy (TLE) is the most common form of focal epilepsy [2]. Focal epilepsy may be of simple type, may not affect the memory or behavior and complex type that affect the memory and behavior of the person (MedlinePlus) [3]. Drug-resistant TLE is even serious and associated with high risk for psychosocial impairment, cognitive decline and mortality.
The hippocampus was considered to be the generator of TLE. This view was due to the frequent observation of the histopathology of hippocampus of TLE patients. Surgical removal of the sclerotic hippocampus improved this epileptic condition, also the experimental neurophysiologists who work on normal hippocampi identified CA3 as the site of origin of discharge in a variety of models of experimental hippocampal epilepsy [4]. Because of strong interconnections, seizures beginning in either the medial or lateral temporal areas often spread to involve both areas and also to neighboring areas on the same side of the brain as well as the temporal lobe on the opposite side of the brain [5].
Anxiety and epilepsy have some underlying neurochemical features that involve GABA and serotonin in particular. GABAergic drugs such as valproate, phenobarbital and benzodiazepine have both seizure-and anxiety-reducing properties [6]. Reduced serotonin receptor binding has been shown both in patients with panic anxiety and in patients with epilepsy [7].
Scientists [8], studied the effects of individual housing as compared to conditions maintaining social contact on stress markers and epilepsy and concluded isolated pilo animals were very aggressive, social isolation constitutes a major stressful situation that can lead to a depressionlike profile.
As a medicinal plant Acorus calamus and its oil were employed in a number of neuronal disorders in olden days. It has a number of active principles and beta asarone is one of the main active principle.
Present days Memocare Plus is a noble herbal formulation that has blended these time-honored memory-enhancing herbs, which can enhance memory and reduce stress and anxiety. These herbs delay brain aging and stimulate regeneration of neurons [9].
In order to confirm the action of Acorus calamus in neurodegeneration related anxiety condition we formulated this study, collected the data and analyzed it. The results were amazing.

Objective
Anxiety is one of the symptoms of epilepsy [10] and for most of the epileptic patients the major point of anxiety is the seizure that arise at any place, any time without warning that even worsen the condition and so for animals too. So epilepsy and anxiety goes hand in hand.
As the animals, except control group, undergone neurodegenerative surgery that selectively damages the hippocampus, they experience epilepsy. This will give a state of alert and fear or anxiety that will make it difficult to handle the animals after surgery.
It is understandable, if the hippocampal damage is more that will be reflected as epilepsy and will worsen the mental status the animal as anxiety. So by analyzing the level of anxiety we can indirectly prove the level of neurodegeneration. The objective of this study is, To analyse the anxiety level of the animals using Dark Light box and it is an indirect measure to neurodegeneration.
To compare the anxiety level lesion control animals and drug treated animals with the control animals.
To study the efficacy of the drug employed based on the anxiety comparison study.
The above is the animal groups used for the study. AC 15, 25 and 35 groups were given crude extract of 15, 25 and 35mg's of Acorus calamus per kg body weight. Whereas groups BA 10, 15 and 20 were given 10, 15 and 20 mgs of Beta asarone, the principle component of Acorus calamus in IP, per kg body weight before and after 10 days of lesion with Kainic acid.
Drug dosages were decided based on the LD50 of the herb. The effective dosage was taken as mid value. One dosage above and one below was taken for comparison.
Authenticated by National Institute of Herbal Sciences, West Tambaram, Chennai.

Preparation of Ethanolic Extract of Acorus Calamus (AC)
The ethanolic extract is been prepared by soxhletion method [11]. It was proved that the ethanolic extract expressed more antioxidant activity than other extracts of Acorus calamus.
1 kg acorus calamus was been powered coarsely. 100 gram of powder was taken in a thimble and placed in a condenser. 100 -200 ml of ethanol was added to the powder and soxhletion was carried out for 10-12 hrs continuously.
After 12 hrs the solvent was collected in a turbo vial.
The solvent was then pre-concentrated using the turbovap.
Finally the pre-concentrated solution was collected and kept aside.
Similarly the procedure was repeated with 100 grams of powder and the extract was collected. Likewise total 10 samples were prepared and all the solutions were mixed together.
Totally 30-35 grams of Ethanolic extract is been collected from 1 kg of Acorus calamus powder.

Apparatus
• This test takes advantage of the natural conflict of a rodent between the exploration of a novel environment and the aversive properties of a large, brightly lit open field. • The test apparatus was a rectangular Plexiglas box was divided by a partition into two environments. One compartment was dark, and the other compartment was brightly illuminated.
• The compartments were connected by an opening located at the floor level in the center of the partition. The more time a mouse spends in the light compartment and the more transitions it makes the less it was considered anxious.
• This test was used to assess anxiety. The basic measure was the animal's preference for dark, enclosed places over bright, exposed places.

Procedure
• Each rat was taken from its home cage and placed into the dark chamber facing the end wall (parallel to the partition • Activities and time in the light-dark box was video-recorded for 5 min. • At the end of every light-dark box test, the number of fecal boli and urine puddles were recorded. • Duration in each chamber and the number of light-dark transitions were also recorded. • A single transition is counted when all the four paws entered a chamber.

Score
The more a mouse spends in the light compartment and the more transitions it makes the less it was considered anxious.

Dark and Light Field Test for Anxiety [12]
This test is used to access anxiety. The basic measure of this test is the animal's preference for dark, enclosed places over bright exposed places. The more a mouse spends time in light compartment the less it is considered anxious. Researchers [13] studied the anxiety level of adolescent and adult male rats by using dark light box and concluded this as a good technology in analyzing the anxiety of animals.

Scholars [14], analysed the anxiety level of different traits of rats by using Elevated Plus
Maize and categorized them based on anxiety.

Time in Dark Field (Fig-2)
This parameter shows the level of anxiety of the animals. The more the animal spend in the dark field the more the animal feels anxious that indirectly shows the ineffective nature of the drugs.
The animal group LC was spending more time in the dark field in comparison with the CO group. The animal groups AC 14 and BA 10 were spending significantly more time in dark field in comparison with CO group and equal with LC group. The drug groups AC 25, AC 35 (df=3,20. F=56) and BA 15 spend significantly more time in comparison with the CO group and less with the LC group and concluded as poor neuroprotective. The drug groups BA 20 (df=3,20. F=51) spends significantly low time in the dark field with LC group, that shows the drug dosage was effective in preventing epilepsy. Field (Fig-3) The more time the animal spends in the light field shows the low anxiety level of the animal the high neuroprotection of the drug and dosage.

Time in Light
LC group of animals spend most of the time in dark box and so were not ready to spend their time in light field this shows high anxiety in them. The drug groups AC 15 and BC 10 also spend more time in the dark field than light field. The drug groups BC 15 (df=3,20. F=47), AC 25 and AC 35 (df=3,20. F=32) spend significantly more times in light field in comparison with LC group and less significant with the CO group shows poor protection. The animals belongs to BA 20 spend more time in light field in comparison with LC group and was equivalent with the CO animals shows good neuroprotection.
The efficacy of the drugs employed was also analysed histologically, that clearly demarcated the level of lesion between drug employed and drug unemployed groups even with the unstained sections. With the lesion control group histology section the lesion was very much large and with the drug group the lesion was too small with some newly formed cells inside.

Observations Viewed on Unstained Sections Taken of the 2 nd day of Lesion
As the herbal drug employed here was given 10 days prior to the animals, to the day of lesion surgery, they protected the nerve cells from the deleterious effects of the exitotoxin employed at the time of lesion surgery, that was reflected in the epilectic status of the animal and so in anxiety.

CONCLUSION
Acoruscalamus is commonly known as sweet flag in India. Acorus calamus is a drug of choice for epilepsy, promotes intellect in children, memory and used to boost up the activities of brain in the form of brain tonic. Scholars [15] proved in his study that different fractions of Acorus calamus are effectivein preventing stress development neuroinflammation.
In this present study we also proved that Acorus calamus and its principle content, the Beta asarone has effective role in preventing epilepsy and neurodegeneration related anxiety in experimental epileptic rats.
It is an known fact that herbal drugs generally do not have side effects and this drug very much active in preventing neurodegeneration even used for some neurological disorders in patti vaithyam (older form of treatment given by the elders of family).
As neurodegenerarion is one of the commonest condition, that affects mostly all the elderly people and affect their lifestyle, we can employ this drug as a supplement along with tea or with other food items to protect their neurons so as to give a quality life in old age. Also this work can be expanded in future for better results.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Animal Ethic committee approval has been taken to carry out this study.