kiara vann neuroscience 391

Kiara VannNeuroscience 391Research Paper

Introduction:

This semester in Neuroscience 391, I worked on a collaborative project with Dr. Manju

Bhat and Dr. Jeffrey Overholt. The purpose of my project was to examine the mechanism of pain

in relation to Transient Receptor Potential channels and the effects of propofol and capsaicin on

TRP channels. Pain is defined as an unpleasant sensation resulting from localized discomfort.

Pain is caused by the interaction of painful stimuli that activates peripheral terminals of

nociceptors (sensory neurons that are activated by noxious chemical, mechanical or thermal

stimuli) that sends action potentials along the afferent axons to the spinal cord. The nociceptor

activates spinal neurons by transmitting across the spinal cord and up fiber tracts firing in the

medulla, midbrain and thalamic neurons reaches the somatosensory cortex. Transient Receptor

Potential cation channel subfamily V member 1(TRPV1) is activated by noxious stimuli such as

capsaicin, the active compound found in chili peppers and other spicy foods. Recent studies have

shown that nociceptors are sensitive to capsaicin. Nociceptor terminals eventually desensitize

capsaicin, the desensitization property makes it desirable as a treatment for chronic pain.

Propofol is one of the widely used general anesthetics that upon injection into the skin create an

intense burning sensation. A recent study claims that Transient Receptor Potential cation

channel subfamily A member 1(TRPA1) is mediating the activation of peripheral nerve endings

by general anesthetics. TRPA1 is also involved in the development of increased pain sensitivity

after inflammation. The purpose of the project was to compare the effects of capsaicin and

propofol on TRP channels in transfected HEK 293 cells. Techniques that were used are

transfection of HEK 293 cells with cTRPV1+ GFP and imaging to examine changes in

transfected HEK 293 cells and non-transfected HEK 293 cells.


Materials and Methods:

Passing of HEK(human embryonic kidney) cells:

Human embryonic kidney cells are standard cell lines that grow rapidly and can be easily

transfected. The process of passing cells begins with HEK wild type cells being cultured in a ten

milliliter cell culture dish with serum free medium for approximately a week. After a week, the

medium was aspirated and was replaced with five milliliters of Versene and it sat on for five

minutes. The purpose of the Versene was to dislodge the cells that adhere to the bottom of the

dish. A five milliliter pipette was used to pipette the mixture of Versene and cells up and down

into a fifteen milliliter conical tube. The cells required centrifugation to separate the supernatant

(Versene) and pellets (cells) from each other. The cells were centrifuged at 710 rpm for seven

minutes meanwhile the preparations of two dishes were necessary for the next passing and for

transfection. A ten milliliter cell culture dish containing nine milliliter of serum free medium

and a six well dish with one milliliter of medium was added into four of the wells for later usage.

Once the seven minutes of centrifugation was completed, the supernatant was aspirated and

seven milliliters of serum free medium was added to a fifteen milliliter conical tube containing

the cells that were pipetted up and down to resuspend the pellet. The resupsended pellet in the

medium was added to the nine milliliters of serum free medium in the ten milliliter cell culture

dish and the remaining pellet was added to four of the six wells in the six well dishes. The dishes

were placed in the incubator and were allowed to incubate for a period of 48 hours.

Transfecting of HEK(human embryonic kidney) with cTRPV1 + GFP:


Transfection is the process by which exogenous DNA (cTRPV1+GFP plasmid) in

solution was introduced into HEK 293 cells. After the incubation period of 48 hours, the cells

were ready for transfection. The beginning process of transfection is creating the plasmid

mixture.cTRPV1 is the plasmid that was used for transfection in this experiment. It is 3µg/1µl so

approximately 3µl was used. The 3µl of DNA was added to 100µl of serum free medium and

gently tapped and incubated at room temperature for fifteen minutes. Four polystyrene tubes

were used in the experiment, two polystyrene tubes contained the cTRPV1 and two polystyrene

tubes did not have cTRPV1 and they were used as a control. During the incubation, the

Lipofectamine cocktail was prepared by adding 85µl of Lipofecatmine (reagent responsible for

transfection) and 425µl of serum free medium in another polystyrene tube. The Lipofectamine

cocktail was gently tapped and incubated at room temperature for fifteen minutes. While the

Lipofectamine cocktail was incubating, two coverslips were placed into four of the six wells in

the six well dish. The cells were washed three times by aspirating existing serum free medium

and replacing it with one milliliter of fresh serum free medium and in the final wash of the cells,

800µl of serum free medium was added and placed back into the incubator.100µl of the

Lipofectamine cocktail mixture was added to the cTRPV1 and control mix to create a total

volume of 200µl. The 200µl mix was added dropwise to four of the six well dish and was

swirled uniformly and placed back into the incubator. The transfection mix remained on for four

hours and after four hours the transfection mix was aspirated and was replaced with a new

medium. The cells were fully expressed in two days.

Imaging of Transfected HEK 293 cells with cTRPV1+GFP:


After two days, cells were examined underneath a fluorescence microscope to check for

expression. The excess serum free medium was removed by gently dabbing the edges of the

coverslips. Vectorshield was used to mount two coverslips to each of the coverslides and nail

polish was added around the edges to make sure it remained mounted. The coverslides were

placed in -20ºC overnight and were removed the following day for imaging. The filters of

interest were FITC for detection of GFP and DAPI for detection of the normal cells.

Results:

Figure 1: HEK 293 cells that were used as a control.


Figure 2: HEK 293 cells that were used as a control, transfected HEK 293 cells with

cTRPV1+GFP underneath 20X magnification and 100X magnification.

Figure 3: Another set of transfected HEK 293 cells with cTRPV1+GFP underneath

20X,100X ,differential interference contrast ( DIC) magnification and a composite of DAPI,GFP

and FITC.


Figure 4: Another set of transfected HEK 293 cells with cTRPV1+GFP underneath 100X.

Discussion:


Figure 1 consisted of two panels that are HEK 293 cells that were used as a control.

Figure 2 consisted of six panels that were HEK 293 cells that were used as a control, transfected

HEK 293 cells with cTRPV1+GFP underneath 20X magnification and 100X magnification.

Figure 3 is consisted of five panels of another set of transfected HEK 293 cells with

cTRPV1+GFP underneath 20X,100X ,differential interference contrast ( DIC) magnification and

a composite of DAPI,GFP and FITC. Figure 4 consisted of three panels of another set of

transfected HEK 293 cells with cTRPV1+GFP underneath 100X. The images are from the

fluorescence microscope underneath the DAPI filter and the FITC filter that is the same

wavelength as GFP which made the transfected cells detectable.

Conclusion:

The results suggested that transfection was successfully because there was GFP (green

fluorescence protein) expression indicating that the HEK 293 cells had the cTRPV1 plasmid and

the controls did not have any GFP expression. The purpose of the control was to have HEK cells

without expression and to make sure there was not any cross-contamination and as an indicator

of the success of the transfection. In future experiments, whole-cell patch clamping will be used

to record differences between transfected HEK 293 cells with the application of capsaicin and

propofol.

kiara vann neuroscience 391

Documents