The Effects of Over-The-Counter Medication on Bleeding Time in Rats (Rattus Norvegicus)

7/30/2019 The Effects of Over-The-Counter Medication on Bleeding Time in Rats (Rattus Norvegicus)

1/16

The Effects of over-the-counter Medication on Bleeding

time in Rats (Rattus norvegicus)

Cory Lundeen

A Research Thesis

A Research Thesis Submitted in Partial Fulfillment of the

Requirements for the B.S. in Biology, Bethel University,

December, 2012

Page 1 of 16


2/16

Abstract

An increase in intraoperative bleeding time has been associated with the use of certain over-

the-counter medications. Excessive bleeding can pose a serious health risk to patients undergoing

surgical procedures. The risks include an increased risk of infection, difficulty with visualization in the

operative field, and the potential need for blood transfusions. The purpose of this experiment was

to determine the effects of certain over-the-counter medications on bleeding time in rats.

Each separate group of rats was treated with a specific over-the-counter medication for three

consecutive days. The bleeding time was measured prior to treatment and then was measured in

each rat, each day, until the average bleeding time normalized to the control bleeding time.

The over-the-counter medications fish oil, garlic oil, and flaxseed oil all showed a statically

significant, two-fold increase in bleeding time in rats. These results suggest that doctors may need to

advise patients as to the potential bleeding hazards of these over-the-counter medications taken

prior to surgical procedures.

Page 2 of 16


3/16

Introduction

In todays modern society, the availability and use of over-the-counter drugs (OTCs) is

extensive. The production and marketing of numerous OTC products has had a significant

economic impact on our society. The marketing styles often lead consumers to believe that since

these products are available over-the-counter they are safe to utilize. Oftentimes, consumers do

not consider OTC products to be medications and thus frequently, do not mention these products

to physicians prior to surgery. OTC products range from multi-vitamins to garlic oil to fish oil to

weight loss pills to aspirin. The availability of such drugs has allowed for significant improvements in

the quality of life for many people. However, many people do not understand the potential adverse

effects these drugs may have when it comes to intraoperative bleeding at the time of surgery. One

of the main concerns with OTCs and surgery is the potential effect on bleeding time. Bleeding time

is described as the time required for a standardsize wound to stop bleeding (Anderson 2008).

One of the dangers associated with drugs that increase bleeding time is the potential

complications during surgical procedures. Any increase in bleeding, at the time of surgery, can cause

not only complication of visualization for the surgeon during surgery, but can also cause post-

surgical complications. These include an increase in r isk for infection, the need for a blood

transfusion, and the potential complications which are associated with the infusion of blood

products. If one is just dealing with lifes normal scrapes and bruises, a slight rise in ones bleeding

time is not much of a problem. If these drugs are taken prior to extensive surgery, however, there

could be harmful, or even fatal consequences.

Drugs, like low dose aspirin, are known to increase bleeding time (Sahud and Cohen 1971). This

knowledge allows doctors to inform patients that they need to stop taking such drugs before

surgery. This helps prevent unnecessary bleeding in surgery, which could lead to complications.

Page 3 of 16


4/16

Unfortunately, some OTC drugs are overlooked when preparing a patient for surgery. Drugs like

fish oil, flax seed oil, and garlic oil, which are very commonly utilized in todays society, are often not

discussed during pre-op appointments. There is data suggesting that something as simple as garlic

supplements, which are used to help in the control of cholesterol, could increase bleeding time

(Chan et al. 2007). Despite the suspicion that many of these supplements affect intraoperative

bleeding, there is not much data available on the duration of the drugs effect on bleeding time.

The question my research seeks to address is how long these drugs have an effect clotting

time. If a patient is told to discontinue a certain drug 7 days before surgery, yet the drug has an

effect on bleeding time which lasts for 14 days, there may be unexpected bleeding during certain

surgeries. This bleeding may lead to severe complications both during and after surgery. In my

research, I tested the following drugs: fish oil, flax seed oil, and garlic oil. The test subjects for this

experiment were rats.

It is believed that one of the main etiologies for an increased bleeding time caused by some

herbal OTC medications, is the presence of omega-3 fatty acids in oil based supplements. Omega-3

fatty acids act in the fibrinolytic pathway. A fibrin clot is the main way in which bleeding from small

vessels is controlled by the body. Fibrin is a fibrous material that can be produced in a net like

structure over a wound, and stop the loss of blood from the wound. Omega-3 fatty acids are

believed to enter the complex fibrinolytic pathway and cause an increase in tissue plasminogen

activator and a decrease in plasminogen activator inhibitor. Both of these effects cause an increase

in fibrinolysis, and therefore an increase in the bleeding time of the wound. (Stone 1996).

My null hypothesis is that the various drugs tested will have no effect on the bleeding time of

rats. My first alternative hypothesis, for the various drugs, is that they will increase the bleeding time

of rats. My second alternative hypothesis, is that the same drugs will decrease the bleeding

Page 4 of 16


5/16

time.The importance of this research is to allow for a better understanding of the effects of certain

over-the-counter drugs on bleeding time. This data is crucial for both doctors and patients to

consider prior to surgery. The more informed both doctors and patients are about the potential

adverse affects of OTC products, the better patient care can be. Starting with an animal model to

obtain baseline effects of particular OTCs will help to tailor the future design of human studies to

address the specific effects on human bleeding time.

Page 5 of 16


6/16

Methods

In this experiment, the effects that fish oil, flaxseed oil, and garlic oil have on bleeding time were

tested. Sprague dawley rats (Rattus norvegicus) were utilized as the test subjects for this

experiment. This animal model was chosen due to rats relatively large size, which allowed for

multiple blood samples yet minimizing the potential effect of reduction of platelets. Rats were also

easily trained to be relatively calm. This was needed to avoid unnecessary stress and activity during

testing which would affect blood pressure. The training was done by handling the rats at a very

young age, and acclimating them to lab test procedures. For each different OTC medication used, a

total of five rats were utilized. The various OTC medications were administered via oral gavage

(Hoggatt, et al. 2010).

Before administration of the OTC medications, the bleeding time was measured for each

animal to serve as their baseline (control) bleeding time. The control tests, and eventually the

experimental tests, were accomplished using a modified Ivy bleeding time test. This test consisted of

creating an incision on the skin of the tail of a test subject and then recording how long it took for

the incision to stop bleeding. Each animal was placed in a restraint to minimize their motion, which

can affect the bleeding time. The rats tails were also raised up on a cutting board to prevent any

bend in the tail. The incisions were made using the corner of a razor blade. To keep a consistent

depth of the incision, tape was wrapped around the edge of the razor blade. This exposed only a

small portion of the razor blade to prevent it from cutting too deep into the tail. The incision site

was cleansed with alcohol, prior to each cut, to avoid infection. Each new incision site was on a

fresh location on the tail of the animal. The most distal site of the tail was utilized first and then the

cuts were moved successively cephalic to avoid issues of decreasing vascular blood flow, due to

prior incisions. Once the incision was made, a stop watch was started immediately. As the incision

Page 6 of 16


7/16

bled, blotting paper was used to draw the excess blood away from the site of the incision at five

second intervals. The time elapsed when no more blood was drawn unto the blotting paper was

documented as the bleeding time for that evaluation. (Central Texas College Staff)

In order to establish an average baseline control bleeding time, for each animal, each rats

bleeding time was be obtained each day, for three days. Each separate OTC medication was then

administered to a group of five rats, which were randomly assigned to one of the three specific

OTC medications as well as a control group (given just water via oral gavage). A total of 20 rats

were uitulized.

Using a 1cc syringe with a curved, 3 inch, 18 guage gavage needle, an average human dose of

each medication was injected into the rats stomach. The use of a human does of the OTCs may

seem excessive, however this does was chosen to account for the rapid metabolism of rats, in

comparison to the metabolism of a human. This dosing was administered once per day, and

continued for three days to obtain a stabilized blood level of the OTC medication in each animal.

Once the various OTC medications were administered for three days, testing of the bleeding time

resumed. Each animal was tested using the bleeding time technique (as described above) every day

until the bleeding time returned to its control level for at least two days.

Once the bleeding time data was collected, the ANOVA and the Tukey-Kramer post hoc tests

were used to determine if the selected OTC medications had led to a significant change in the

bleeding time and, if so, how many days were needed for the Ivy bleeding time to return to normal

once the medication had been discontinued.

Page 7 of 16


8/16

Results and Figures

Figure 1: The Overall Average Effects of Various OTC Medications On the Bleeding Time of Rats

The data is presented as average bleeding times ( SD). Significance is determined if the p

value was


9/16

Page 9 of 16


10/16

Figure 1 shows that all of the OTC medications caused a nearly 2-3 fold increase in the average

bleeding times of the rats after three days of treatment. Day one was the control bleeding time

data collected before any medication was administered. After the first days data was collected, the

administration of the medications was star ted. After three days of treatment, the bleeding time for

day three was collected at the end of the day. Day three was the only day that all of the OTC

medications showed a significant increase in the bleeding time, when compared to the control

group. A long lasting effect can be seen in the gar lic oil and flax seed oil treatment groups.

Figure 2: The Effects of Fish Oil on the Bleeding Time in Rats

Figure 2 shows the effect of fish oil on the bleeding time in rats, as compared to the control

group. The only significant increase in bleeding time was on day 3 of the treatment when,

compared to the control on day 3. Within one day of discontinuation of the fish oil the bleeding

time returned to the control level.

Page 10 of 16


11/16

Figure 3: The Effect of Garlic Oil on Bleeding Time in Rats

Figure 3 shows the effect of garlic oil compared to that of the control group. Days 3 and 4

demonstrate a significant increase in bleeding time, compared to the control group on days 3 and

4.

Figure 4: The Effect of Flax Seed on Bleeding Time in Rats

Page 11 of 16


12/16

Figure 4 shows the effect of flax seed oil compared to that of the control group. Only day 3

demonstrated a significant increase in bleeding time, compared to that of the control group on day

3.

Page 12 of 16


13/16

Discussion

This experiment was conducted to investigate the effects of certain over-the-counter

medications on the bleeding time in rats. The main alternative hypothesis was that the OTCs tested

(fish oil, flaxseed oil, and garlic oil), would cause an increase in the bleeding time. The Turkey-Kramer

test was used to determine whether or not each average bleeding time obtained in the test groups

was significant compared to the control group. Each of the OTCs studied, showed a slightly

different effect on bleeding, but the overall trend was that each OTC increased the bleeding time

of the rats tested.

In the group of rats receiving fish oil, there was a significant increase in the bleeding time

compared to the control group (see figure 2). This significant increase in the bleeding time was not

long lasting. It was statistically significant for only one day after discontinuation of the fish oil. The

result shows that although fish oil does cause an increase in bleeding time, it is not a long lasting

effect.

The next OTC medication tested was garlic oil. In this test group, the bleeding time increased,

just as it did in the fish oil group. The effect, however, was significant for two days, after

discontinuation of the garlic oil, instead of just one day (figure 3). On day five, the average bleeding

time was still higher than the control group, yet it was not statistically significant. This was due to the

finding that each individual bleeding time recorded for each rat in the garlic oil test group had a

much greater range on day five. This shows that each rat processed the garlic oil differently from

one another. Overall, garlic oil was shown to have a greater effect and longer lasting increase on

bleeding time.

The final test group was given flax seed oil as their OTC medication. Out of all the medications

tested, flax seed showed the largest, average increase in the bleeding time of the rats. However it

Page 13 of 16


14/16

only caused a significant increase for one day after discontinuation of the flax seed oil (figure 4). On

day four, the average bleeding time of the flax seed group was the same as day three. Even though

the average was the same for each day, the range of individual bleeding times, on day four, was very

large. It is likely that this increased range of bleeding times was caused by a varied individual

response, of the rats, to the flax seed oil. This is potentially caused by how the rats metabolized the

flax seed oil. Also, on day five the bleeding time rapidly decreased to the same bleeding time as

that of the control group. Flax seed showed a significant, but slightly unpredictable effect on

bleeding time.

All of the above test data confirms that the OTC medications tested have significant effects on

bleeding times in rats. The data was consistent with the proposed hypothesis that the OTCs would

increase bleeding time in rats. The manner in which both the control and test group data was

collected was controlled, as best as possible, for both technique and outside variables (stress,

normal diet, incision techniques, etc.). Trying to control as many outside variables as possible helps

increase the validity of the data collected.

Even with the care that was taken to keep the sampling as consistent as possible, there was no

way to completely remove all outside interference with the data. The main source of potential

error would likely come from inconsistent depths of cuts on the rats tails. In future research it

would be helpful to devise a set up that used the razor blade at the exact same depth for each cut

(likely a triggered, incision device). Future testing would need to include larger test and control

groups. Also, testing on humans would be an ideal next step. This would determine if the effects

that the OTC medications have on rats carry over to humans.

Page 14 of 16


15/16

Conclusion

Over all, the main conclusion that can be drawn from this experiment is that the OTC

medications tested do have significant effects on the bleeding time in rats. We can speculate that

these effects will carry over, to some extent, to humans. These results show that when it comes to

situations that have a high risk of bleeding (e.g. surgery) the OTC medication that a patient is taking

can have potentially dangerous effects. It is important for people to better understand all the

effects of the medications they take; both the good and bad. It is also important for surgeons to

have data on the effects of both prescription medications as wells as OTC medications, when it

comes to consulting patients prior to surgical procedures. With further research, both with animal

and human models, this important information will become available and help to minimize risks to

surgical patients.

Page 15 of 16


16/16

Literature Cited

Anderson DM, et al. 2008. Dorlands medical dictionary for heath consumers. Saunders.

[Electronic App]Central Texas College Staff. Bleeding time procedures [Homepage of Central Texas College],

[Online]. Available: http://www.ctcd.edu/mlt/mbyrd/plab1023/documents/6bBleedingtime.pdf [2011, 09/25].Chan K, Yin M, & Chao W. 2007. Effect of diallyl trisulfide-rich garlic oil on blood coagulation

and plasma activity of anticoagulation factors in rats. Food and Chemical Toxicology 45(3): 502-507.Hoggatt, Amber F., Jonathan Hoggat, Meghan Honerlaw, Louis M. Pelus. 2010. A spoon full of sugar

helps the medicine go down: a novel technique to improve oral gavage in mice. JAALAS 49(3): 329-334,Sahud MA, and Cohen RJ. 1971. Aspirin-induced prolongation of the ivy bleeding time. Its

diagnostic usefulness. California medicine 115(4): 10-13.Stone NJ. 1996. Fish consumption, fish oils, and coronary heart disease. AHA Circulation 94:

2337-2340

Page 16 of 16
http://www.ctcd.edu/mlt/mbyrd/plab1023/http://www.ctcd.edu/mlt/mbyrd/plab1023/

Documents

The Effects of Over-The-Counter Medication on Bleeding Time in Rats (Rattus Norvegicus)