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 Fulllment of the Requirements for the B.S. in Biology, Bethel University, December, 2012

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Abstract
An increase in intraoperative bleeding time has been associated with the use of certain overthe-counter medications. Excessive bleeding can pose a serious health risk to patients undergoing surgical procedures. The risks include an increased risk of infection, difculty with visualization in the operative eld, 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 specic 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 sh oil, garlic oil, and axseed oil all showed a statically signicant, 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.

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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 signicant 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 sh oil to weight loss pills to aspirin. The availability of such drugs has allowed for signicant 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 postsurgical complications. These include an increase in risk 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.

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Unfortunately, some OTC drugs are overlooked when preparing a patient for surgery. Drugs like sh oil, ax 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: sh oil, ax 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 brinolytic pathway. A brin clot is the main way in which bleeding from small vessels is controlled by the body. Fibrin is a brous 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 brinolytic pathway and cause an increase in tissue plasminogen activator and a decrease in plasminogen activator inhibitor. Both of these effects cause an increase in brinolysis, 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 rst 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

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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 specic effects on human bleeding time.

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Methods
In this experiment, the effects that sh oil, axseed 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 ve 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 modied 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 rst and then the cuts were moved successively cephalic to avoid issues of decreasing vascular blood ow, due to prior incisions. Once the incision was made, a stop watch was started immediately. As the incision

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bled, blotting paper was used to draw the excess blood away from the site of the incision at ve 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 ve rats, which were randomly assigned to one of the three specic 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 signicant 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.

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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). Signicance is determined if the p value was <0.05 when compared to the control group for the same day. (Figure one starts on the next page due to the graphs large size.)

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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 rst days data was collected, the administration of the medications was started. 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 signicant increase in the bleeding time, when compared to the control group. A long lasting effect can be seen in the garlic oil and ax seed oil treatment groups. Figure 2: The Effects of Fish Oil on the Bleeding Time in Rats

Figure 2 shows the effect of sh oil on the bleeding time in rats, as compared to the control group. The only signicant 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 sh oil the bleeding time returned to the control level.

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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 signicant 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

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Figure 4 shows the effect of ax seed oil compared to that of the control group. Only day 3 demonstrated a signicant increase in bleeding time, compared to that of the control group on day 3.

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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 (sh oil, axseed 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 signicant 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 sh oil, there was a signicant increase in the bleeding time compared to the control group (see gure 2). This signicant increase in the bleeding time was not long lasting. It was statistically signicant for only one day after discontinuation of the sh oil. The result shows that although sh 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 sh oil group. The effect, however, was signicant for two days, after discontinuation of the garlic oil, instead of just one day (gure 3). On day ve, the average bleeding time was still higher than the control group, yet it was not statistically signicant. This was due to the nding that each individual bleeding time recorded for each rat in the garlic oil test group had a much greater range on day ve. 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 nal test group was given ax seed oil as their OTC medication. Out of all the medications tested, ax seed showed the largest, average increase in the bleeding time of the rats. However it

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only caused a signicant increase for one day after discontinuation of the ax seed oil (gure 4). On day four, the average bleeding time of the ax 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 ax seed oil. This is potentially caused by how the rats metabolized the ax seed oil. Also, on day ve the bleeding time rapidly decreased to the same bleeding time as that of the control group. Flax seed showed a signicant, but slightly unpredictable effect on bleeding time. All of the above test data conrms that the OTC medications tested have signicant 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.

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Conclusion
Over all, the main conclusion that can be drawn from this experiment is that the OTC medications tested do have signicant 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.

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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 trisulde-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, sh oils, and coronary heart disease. AHA Circulation 94: 2337-2340

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