Preparation of Aluminum Chloride, Cannabidiol, Donepezil and D-galactose solutions

Upon completing the nine weeks of induction and treatment, Dr Amir prepared for the series of tests that will be conducted on the rats. The objective was to record and analyze the response of each rat treated with Cannabidiol (CBD) and Donepezil as compared to the negative control and baseline groups.

One of Dr Amir’s task during the study was to prepare the neurotoxin and treatment solutions in the lab. MASA was given the privilege to document his work and would like to begin this article by sharing the process of preparing the solutions. Dr Amir prepares the solutions based on the average weight of each rat (350 grams per rat) in the clean environment of the lab in University Putra Malaysia (UPM). As shown in the video above, he carefully scoops the required weight of the powdered Cannabidiol (CBD) isolate to be diluted in the emulsifier Polysorbate 80. Once mixed, each container was stirred until the powder completely dissolved. The required solutions were prepared in advance according to the timeline of the study to ensure that the supply was always available for daily administration to the rats.

Method of Aluminum Chloride, Water, Cannabidiol, Donepezil and D-galactose Administration by Injection

In the morning when Dr Amir arrived at the research site, each rat was checked and injected with the neurotoxin and treatment solutions accordingly as a continuous routine throughout the study. Once every lab rat have been injected as shown in the video above, they were placed back in their cages and are ready to participate in a series of cognitive function tests designed to highlight the Alzheimer’s disease symptoms. Alongside the baseline and negative group, each treated rat will need to take turns on the test rigs. Each test rig was placed on a table or on the floor parallel to the camera mounted on the ceiling of the research area. The camera captures and records the motor movement of each rat while they move around in the test environment. For this study, a custom designed research software was used to plot the the data into informative charts and graphs for Dr Amir to analyze.

Rat Head Colored with Artline Permanent Marker Pen

At the start of each test, each lab rat was taken out of their cage and the top of their heads were colored a dark color (blue, red or black) with a permanent marker pen. This was because the background color of room and rig setup was white, and the dark color stands out and is easily tracked by the camera and software when the lights are turned off for each session. Dr Amir has selected four separate cognitive function tests successfully used in international animal model studies to procure strong data that can be analyzed individually to form a conclusion. From the four, MASA has highlighted two setups below to explain how the motor movement of the rats help determine if it is affected with Alzheimer’s disease. The first experiment setup is called the Open-field and Novel Object Recognition Test and the second is called the T-maze Test.

Open-field and Novel Object Recognition Test

The open-field test (OFT) examines the behavior of the rats in the closed environment and how it responds to objects placed within the field. If a rat was found spending more time staying close to the walls, it is showing signs of fear or anxiety. However, this behavior usually disappears once the rat explores and familiarizes itself with the environment. To prepare each rat for the actual experiment, the rats were first placed in the environment and trained for the required study measurement time daily for up to four sessions. With the rats more relaxed, objects will be introduced (Novel Object Recognition Test) in the environment to analyze the time taken by each rat to explore objects and their memory of the object location. When ready, the actual experiment was repeated for three sessions on each rat. Based on the recorded data, Dr Amir can evaluate if the rat shows any sign of anxiety or impaired cognitive function.

T-maze Test recorded on software

The T-maze test is designed to study the memory of the rat when it navigates to the right or left of the T-maze. To do so, each rat was placed in the maze several times. On the first run if it chooses to go left, the rat will be confined to the left section (by blocking its path to other parts of the T-maze using a cardboard wall) for thirty seconds. Then the rat is lifted out and placed back in for the second run. On the second run, the rat with good memory will choose to go to the right section instead as it has explored and remembers the left section from the first run. From this experiment, Dr Amir found that the rats treated with CBD showed good memory when compared to the Donepezil and negative groups. A promising finding that was further confirmed by the Water Maze Test which was conducted as the final cognitive function experiment before the rats were killed from euthanasia. Samples of the brain, liver and other parts of the rat’s body will be used in other clinical studies to understand the biological affects of CBD on the organs, tissue and cells. After the first batch of 30 rats were euthanized, Dr Amir then began preparing the second batch of 30 rats that will undergo the same process to complete his study. Upon publishing this article, Dr Amir has completed tests on all 60 rats used for the study.

Dr Amir reviewing recorded data

When cannabis was re-introduced as a medicine (or health supplement) in countries like the United States of America, Thailand, Uruguay, Canada, Germany and Australia since the early 2000’s, more and more clinical trials and research have been completed to better understand the different strains of cannabis, the different active compounds contained within each strain, the medical applications of each compound for different diseases and the efficacy of cannabis as a treatment or preventive medicine. The overwhelming evidence and data collected by each country and the Expert Committee on Drug Dependence (ECDD) within the World Health Organization (WHO) led the United Nation Commission on Narcotic Drugs to vote for the removal of cannabis and cannabis resin from Schedule IV of the Single Convention on Narcotic Drugs 1961.

With Malaysia’s existing Dangerous Drugs Act 1952 and the Poisons Act 1952 ranking cannabis in Schedule I, the only clinical trials that could be conducted are on animals using extracted compounds from cannabis that are manufactured by a pharmaceutical company. This is the same if human trials were to be done in Malaysia. The National Pharmaceutical Regulatory Agency (NPRA) recognizes cannabis as a Schedule I drug and therefore treats it in that manner. Through more animal model clinical trials like that done by Dr Amir, more insight on cannabis can be shared locally. Local data like this must be presented to our rulers and leaders so that cannabis can be placed to the lower Schedule III of the Poisons Act 1952 which will allow for the cannabis extract and resin to be used as medicine governed by the Poisons (Psychotropic Substances) Regulations 1989.

This is an important factor as any one of the many cannabis plant strains contain an average of 400 active compounds that supports each other when consumed (also known as the entourage effect) to ensure the healthy homeostasis of the body. Patients using pharmaceutical medicine made using isolate compounds extracted from the cannabis plant like Epidolex (CBD isolate) and Sativex (THC isolate) reported adverse side-effects that led to the lack of interest in the products. In Dr Amir’s study, the CBD treated rats showed better cognitive function and a more relaxed behavior as compared to the Donepezil and other groups. This is a clear indication that CBD could essentially be an effective neuroprotectant for those in risk of developing Alzheimer’s disease. Dr Amir is now completing his thesis and will publish his findings in the selected journal soon.

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