A presentation by Avinash Kanakam outlining research on the abillity of endocannabinoids to produce neuroprotective and neuroregenerative effects on primary cortical neurocytes. The research is set with the goal to prove the above as well as move forward with research surrounding the endocannabinoid system and using it in the treatment of neurodegenerative disorders.

1. The Neuroprotective and
Neuroregenerative Effects of the
Endocannabinoid System on
Primary Cortical Neurocytes
By Avinash Kanakam

2. Statement of Purpose
The purpose of this experiment is to examine
the interactions that occur between the
chemicals and receptors of the
Endocannabinoid System and primary cortical
neurocytes.

3. Background
● The Endocannabinoid system consists of two receptors, the CB1 receptor and the CB2 receptor
○ The CB1 receptor is associated with the euphoric feeling that is often experienced by users of marijuana and
other things that include THC
○ The CB2 receptors is associated with the anti-inflammatory effects that commercial CBD tablets seem to
provide.
○ The Endocannabinoid system has it’s own versions of THC and CBD, with Arachidonylethylamine (AEA, or
anandamide) being similar to THC and 2- Arachidonlyglycerol (2-AG) being similar to CBD
● Neurocytes have been proven to carry CB1 receptors, and THC seems to have some neurogenerative effect. But
recently neurocytes were found to also contain CB2 receptors
○ CB2 receptors do not cause euphoric effects when activated, thus if stimulating neurogeneiss or
neuroprotection was possible via activating CB2, it would achieve results without inducing a high.

4. Background (Cont.)
● Endocannabinoids work via retrograde transmission, meaning they travel from the neuron to the
presynaptic terminal
○ Most often used by the body to stop the over-stimulation of a neuron by a certain stimulus
that are causing calcium channels to continuously produce neurotransmitters that pass
from the presynaptic terminal to the terminal
● Neurodegenerative insults are what cause neurodegenerative disorders, and are usually in the
form of exposure to toxins, alcohol, or trauma.
● One way to measure the neuroprotectIve actions taken by a neuron in certain situations is to
measure the amount of Brain-Derived Neurotrophic Factor (BDNF) which is responsible for the
creation of neurites that extend from the neuron

6. Hypothesis
If multiple wells containing primary cortical neurocytes are exposed to a neurodegenerative insult as
well as varying concentrations of CBD, then the wells that contain the highest amount of CBD will
produce the highest amount of BDNF and formazan crystals.

7. Materials
● Primary Cortical Neurocytes, 1 x
10^6 cells (Human)
● Complete Neurobasal™ Plus Medium
(47 mL)
● 50-mL conical culture tube (2)
● Water Bath
● Laminar Flow Hood
● CO2 Incubator
● Microplate reader
● 70% Ethanol (4 ml)
● CBD oil (.6 ml)
● Proto-D-Lysine (4.8 mL)
● Sterilized 24 well plates (2)
● Micropipette
● Inverted Microscope
● Distilled water (510 mL)
● Penicillin-Streptomycin (.5 mL)
● 15-mL conical tubes (4)
● MTT assay (with all reagents) (24
assays)
● Sterile PBS (1 mL)
● Hydrochloric Acid (.2 M, .5 mL)
● BDNF ELISA kit (with all reagents) (24
assays)
● Orbital shaker

8. Procedure
1. Culturing Neurocytes: Primary Cortical Neurocytes were cultured starting from the 24 well plates rather than
in a cell culture flask. The neurocytes required poly-d-lysine and the NeuroBasal Medium in order to grow properly
and without major incident. To make sure of an even distribution of neurocytes across every well, all the media
containing the neurocytes was placed in the same container; from the container, an equal amount of media was
taken every time, and was resuspended in order to ensure even distribution. The plates were incubated in a CO2
incubator set at 37 degrees celsius, and their general health was viewed using an inverted microscope.
2. Making CBD-ethanol solutions: 4 ml of 70% ethanol were initially received and then divided among 4
containers. From these 4 containers, varying amount of CBD oil had been added. In the first was .1 ml, in the
second was .2 ml, and the third had .3 ml of CBD oil. The 4th container had received no CBD. This mixture was
then shaken in an orbital shaker. The main reason the CBD amount had varied with ethanol, rather than ethanol
varying with CBD, is because it is important to give each well an amount of ethanol that is similar to another,
because ethanol has an effect on neurodegeneration. From here, .2 mL of each solution was added to a
corresponding row in each well plate.

9. Procedure (cont.)
3. MTT Assay: an MTT reagent was prepared using 5 mg of MTT component A and 1 ml of sterile PBS. 40
microliters of this reagent was added to each well, and then the well plate was incubated for approximately 4
hours. Before this time, a HCL-SDS solution was prepared using 10 ml .01 M HCL and 1 g of SDS. The .01 M HCL
solution was prepared using .5 ml of .2 M HCL and 9.5 mL of distilled water. 400 microliters of the HCL-SDS
solution was added to each of the wells, and then the well plates were incubated for another 4 hours. Following
this, the absorbance of each plate was read at 590 nm using a microplate reader.
4. BDNF ELISA Assay: Using a BDNF ELISA kit which had come with all associated reagents and chemicals
needed, the amount of Brain-Derived Neurotrophic Factor (BDNF) was measured in each of the wells. The only
thing to mention that differs from conventional ELISA kits is that the amount of reagents needed for all wells
used was multiplied by 4, due to the fact that this experiment had used a 24-well plate as opposed to a
conventional 96 well-plate for ELISA kits. Following the additions of all reagents and incubation times,
absorbance of the well plate was read at 450 nm using a microplate reader.

10. Results

14. Conclusions
This experiment had used varying concentrations of CBD carried in ethanol to protect and generate neurons in cell culture. Using
an MTT assay and BDNF ELISA kit, the experiment was accurately able to determine the magnitude of effect that the cannabinoids had on
neuronal cell cultures. The results showed that the cannabinoids did have a noticeable effect on the neurocytes, specifically how
neuroprotection greatly increased with the presence of CBD, and cell viability had also slightly increased with the presence of CBD. The
results support my hypothesis, which accurately predicted both the possible positive effect of cannabinoids and their magnitude. The
cannabinoids had a correlation with the amount of cells in the cell culture as well as the amount of BDNF present. This proves that the CBD
present in CBD oil does definitely have at least some effect on the neuroprotection and neuroregeneration of neurocytes.
The procedure seemed to be very effective for accurately determining the effect of the endocannabinoids, as the results seem to
match what background research would seem to suggest about this experiment. The control in this experiment were the wells that had
contained no CBD treatment. The constants were the amount of MTT assay and BDNF ELISA kit associated reagents added to each of the
wells, the temperature at which the two cell culture plates were incubated, the distribution of cell culture among the wells, and the amount
of Poly-D-Lysine that was given to each of the wells in the cell culture plates. Both of the assay results were put onto a scatter plot, and
then put through a regression analysis. The scatterplot for the MTT assay results resembled that of an exponential function, so the
logarithm of the data values were taken, and a near-linear equation was received, which had a very accurate R-squared value and p-value.
The BDNF results went through a transformed response to increase the regression of the final equation, where the R-squared value was also
accurate and the p-value still holds significance.

15. Future Applications
The applications of this experiment
are primarily understood in the context of
the prevention and treatment of
neurodegenerative diseases. As such, the
discovery that neuroprotection and
neuroregeneration is possible with
compounds such as cannabidiol, this
opens the door to treatments that use
marijuana-like compounds, but don’t have
a psychotropic effect. Thus, CBD
treatments would also be more accepted
in the field because of their lack of a
psychotropic effect.

16. Limitations
The limitations of this experiment were more
prominent in the delivery of the treatment. Since the
experiment was limited to CBD oil, and not a purified form
of CBD itself, it’s normal bioavailability is drastically
reduced, meaning the treatment isn’t completely absorbed
by the cell culture. Also, the only way to effectively
transport the CBD into the cell cultures were to create a
CBD-ethanol solution. However, ethanol is toxic to cells,
and though the experiment made sure to introduce the
same amount of ethanol to each well in order to ensure
that each well had a susceptibility to alcohol poisoning, the
usage of ethanol as a carrier solution is still a limitation.

17. Error Analysis
Systematic errors include inaccurate measurements
of the amount of liquids that were pipetted at any point of
time to any specific well. In any of the assays, if more or
less of any of the chemicals involved were poured
differently in any of the wells, it would present a major
error for data collection. Random errors include
temperature misreadings or temperature not being
properly achieved by the machines that were used for
incubation, especially in the BDNF ELISA assay, which
required multiple incubations/shakings at certain
temperatures during steps

18. Future Research
This research has the potential to be used in
neurogenesis, the formation of neurocytes via neural
stem cells. In such an examination as that, neural stem
cells would need to be studied and how they respond to
CBD-derived compounds. Though the reason CBD was
used over THC-like compounds in this experiment was
to avoid a psychotropic effect, but research should also
be done into inhibiting the psychotropic effects of THC
so that it may be used for it’s other helpful health
benefits. It is also important to enhance the delivery of
CBD oil to the body and it’s bioavailability. CBD oil is
poorly accepted all over the body, not just the brain, so
it would prove helpful if new methods were made to
easily deliver CBD oil to the body.

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