Journal Pre-proof

In vivo evaluation of effect of pre-operative ibuprofen on pro-inflammatory mediators

in irreversible pulpitis cases

Vicky Nguyen, DDS, MSD, Yen-Wei Chen, DDS, MSD, James D. Johnson, DDS, MS,
Avina Paranjpe, BDS, MS, MSD, PhD

PII: S0099-2399(20)30399-X
DOI: https://doi.org/10.1016/j.joen.2020.06.009
Reference: JOEN 4577

To appear in: Journal of Endodontics

Received Date: 13 March 2020

Revised Date: 25 May 2020
Accepted Date: 9 June 2020

Please cite this article as: Nguyen V, Chen Y-W, Johnson JD, Paranjpe A, In vivo evaluation of effect
of pre-operative ibuprofen on pro-inflammatory mediators in irreversible pulpitis cases, Journal of
Endodontics (2020), doi: https://doi.org/10.1016/j.joen.2020.06.009.

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Copyright © 2020 Published by Elsevier Inc. on behalf of American Association of Endodontists.

 In vivo evaluation of effect of pre-operative ibuprofen on pro-inflammatory mediators
in
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6 irreversible pulpitis cases.
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11 Vicky Nguyen, DDS, MSD*, Yen-Wei Chen DDS, MSD#, James D. Johnson,
DDS, MS*, Avina
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13 Paranjpe BDS, MS, MSD, PhD*.
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17 *Department of Endodontics, University of Washington, Seattle, Washington.
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18 Department of Restorative Dentistry, University of Washington, Seattle,
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Washington.
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22 Running title: Effects of ibuprofen in irreversible pulpitis.
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26 To whom correspondence and reprint requests should be addressed:
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Dr. Avina Paranjpe
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31 University of Washington, School of Dentistry,
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33 Department of Endodontics,
35 34 1959 NE Pacific Street,
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D-669 Health Science Center, Box 357448
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38 Seattle, WA 98195.
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40 Email: avina@uw.edu
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45 Acknowledgments: The authors deny any conflicts of interest related to this study.
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Abstract.

Introduction: Pre-operative administration of NSAIDs have been suggested as a method for

increasing the efficacy of local anesthetics and decreasing post-operative pain in symptomatic
irreversible pulpitis cases. However, the effects of ibuprofen at the molecular level are still
unknown. Hence, the purpose of this study is to compare the levels of pro-inflammatory
mediators in the dental pulp of teeth with irreversible pulpitis in patients who medicated with
pre-operative ibuprofen versus those who did not.
Methods: Thirty-four patients undergoing conventional endodontic therapy for teeth with
irreversible pulpitis were selected and randomly assigned into either the ibuprofen or non-
ibuprofen group. Four patients undergoing endodontic therapy for prosthodontic reasons served
as controls. Patients in the ibuprofen group were instructed to take 600mg of ibuprofen one-hour
prior to treatment, while patients in the non-ibuprofen and control groups were asked to avoid
analgesics prior to treatment. Blood samples obtained from the pulp upon access were tested for
levels of PGE2, TNF-α, IL-6, IL-1β and IFN-γ using high sensitivity ELISAs. Post-operative
pain levels were recorded for the groups. Data for the cytokine levels and post-operative pain
were analyzed using statistical analysis.

Results: There was a significant decrease in the levels of PGE2, TNF-α, IL-6, and IFN-γ in the
ibuprofen group as compared to the non-ibuprofen group. No significant differences were noted
in the post-operative pain levels between these groups.

Conclusions: The data demonstrated that the pre-operative ibuprofen significantly decreased the
levels of most pro-inflammatory cytokines in the dental pulp, which could possibly help with
anesthesia in irreversible cases.

Key words: inflammation; cytokines; ibuprofen; pain; irreversible pulpitis

1
Introduction.

Pulpitis is a common sequel of bacterial infections of the tooth. Studies have

demonstrated irreversible changes when caries reaches within 0.5mm of the pulp (1). These
irreversible changes are mediated by inflammation, which is mediated by the immune system in
response to this bacterial irritation. The inflammatory process includes recruitment of
inflammatory cells from the circulation and their subsequent transmigration. Adhesion
molecules, such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion
molecule-1 (VCAM-1), are expressed on dental pulp tissue in response to several inflammatory
stimuli and contribute to the vascular recruitment of inflammatory cells and transmigration (2).
The change in the pulp from the normal state to the inflamed state has also been attributed to a
barrage of impulses from certain peripheral nerves and continuous discharges from the
unmyelinated C fibers that are present in the dental pulp (3). Once the pulp has reached this stage
of inflammation and is diagnosed with irreversible pulpitis, non-surgical endodontic therapy is
the treatment of choice.

Previous research has demonstrated that achieving anesthesia in irreversible pulpitis

cases, especially with mandibular molars, is a significantly greater challenge than in teeth with
normal healthy pulps (4). This has been attributed to the fact that when the pulp undergoes
irreversible changes and there is an increase in inflammation and inflammatory cytokines like
IL-1β (interleukin-1β), IL-2 (interleukin-2), IL-6(interleukin-6), IL-8(interleukin-8), TNF-α
(tumor necrosis factor-α)and IFN-γ (interferon gamma) and an increase in prostaglandin (PG)
secretion (5-7). This inflammation causes an increase in the tetrodotoxin-resistant (TTXr) class
of sodium channels that have been shown to be resistant to the action of local anesthetics and are
sensitized by prostaglandins (8). Prostaglandins not only alter the kinetics of these sodium
channels, resulting in increased depolarization, but increase the expression of the channels
themselves (9). Another explanation for failure is that nerves arising from inflamed tissue have
altered resting potentials and decreased excitability thresholds (10). Wallace et al. demonstrated
that local anesthetic agents were not sufficient to prevent impulse transmission due to these
lowered excitability thresholds(11). Hence, the use of preoperative oral administration of non-
steroidal anti-inflammatory (NSAIDs) drugs like ibuprofen has been suggested as a strategy to
decrease inflammation and improve anesthetic success (12, 13). It has been shown that NSAIDs
exert their therapeutic effect by inhibiting the enzyme COX2 (cyclo-oxygenase 2), which is

2
highly expressed in the human dental pulp with a diagnosis of irreversible pulpitis, thereby
inhibiting the synthesis of PGs and decreasing the levels of pro-inflammatory cytokines(14).
This inhibition of PGs is hypothesized to increase the efficacy of local anesthetics (12).

Previous studies have also proposed the use of other drugs besides ibuprofen prior to
initiating treatment to help achieve better pulpal anesthesia. These include dexamethasone,
indomethacin and ketorolac among others (13, 15). However, NSAIDs have gained popularity in
the past decade and have been given pre-operatively in irreversible pulpitis cases. Numerous
studies have been conducted that support this treatment modality. Multiple randomized control
trails have been conducted that have evaluated the advantages of using pre-operative ibuprofen
on anesthetic success (16, 17). Lapidus et al. recently published a review that stated there is
moderate evidence to support the use of 600mg ibuprofen an hour before the administration of
local anesthetic to provide additional analgesia to the patient (18). However, there are other
studies that report that the use of pre-operative ibuprofen does not affect anesthetic success or
post-operative pain (12). It is important to understand the results from these studies were based
on clinical assessments of either pain on accessing the teeth or their response to vitality tests
prior to the procedure and not to the levels of inflammation in the dental pulp. Evidence at a
molecular level either supporting or disproving a decrease in inflammation in the dental pulp still
lacking. Some previous studies have demonstrated increased cytokine expression of IL-2, IL-8,
IL-10 (interleukin 10), TNF-α and IFN-γ in the inflamed pulp (6, 19, 20). However, the effects of
NSAIDs, namely ibuprofen, on these key inflammatory mediators and cytokine expression in
irreversible pulpitis have not been elucidated or well-studied. It is important to understand this,
as it would help in formulating better treatment modalities for patients with irreversible pulpitis.

This research aims to identify the cytokine levels in the pulp, which could possibly help
in better understanding pulpal inflammation. Furthermore, delineating the mechanism of
ibuprofen on key inflammatory mediators at this local site may offer a therapeutic potential for
the use of ibuprofen in management of teeth with irreversible pulpitis along with helping
endodontists to make better treatment decisions thereby improving methods of pain management
and patient experience.

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Materials and Methods.

Patient Selection.
The study protocol was approved by the Institutional Review Board (IRB) at the University of
Washington. Specific inclusion and exclusion criteria were used for recruitment of patients.
Inclusion criteria:
i) Male and female patients between the ages of 21-65.
ii) ASA class 1 or 2.
iii) Patients who needed a root canal treatment in the Graduate Endodontic Clinic on either an
upper or lower premolar or molar tooth.
iv) Teeth with an endodontic pulpal diagnosis of normal or symptomatic irreversible pulpitis.
v) Teeth with intact crowns, carious lesions, any existing partial or full coverage restorations.
vi) Patients who had not taken any analgesics the day of the root canal procedure unless they
belonged to the Ibuprofen group.
Exclusion criteria:
i) Any teeth with a diagnosis of pulpal necrosis, previously initiated, and previously treated.
ii) Teeth other than premolars and molars (incisors, canines).
iii) Teeth with any resorptions, anatomy anomalies or perforations.
iv) Patients who could not tolerate or were allergic to ibuprofen.
v) Patients taking pain medication for other systemic conditions.

Recruitment.
Thirty- eight patients were recruited for the study. Clinical and radiographic examination, which
included the sensibility testing with cold and EPT (electric pulp test), followed by three
periapical radiographs and one bitewing radiograph, indicated the pulpal and periapical status of
these teeth. Four patients that had pulpal diagnoses of normal/ vital pulp, requiring root canal
treatment solely for restorative purposes were allocated to the control group. The remaining
thirty-four patients with a pulpal diagnosis of symptomatic irreversible pulpitis were randomly
allocated to either the ibuprofen or the non-ibuprofen group. Randomization was completed
using a computerized randomization software (http://www.randomization.com). Patients
assigned to the ibuprofen group were dispensed three 200mg gel capsules of ibuprofen (600mg
in total) and were instructed to take the medication one hour prior to the initial treatment

4
appointment. The patients assigned to the non-ibuprofen group were instructed not to take any
NSAIDs or any other pain medication the day of the procedure.

Anesthesia.
Consents for treatment and inclusion in this study were obtained from the patients. Root canal
procedures were initiated, samples obtained, and treatment was completed in the Graduate
Endodontic Clinic by trained Endodontic residents. The number of cartridges, type of anesthetic
technique performed (regional block, infiltration, PDL injection, intra-osseous injection), and the
type of anesthetic used were recorded for each patient. The number of cartridges, type of
anesthetic, and anesthetic technique was not controlled for in this study.

Sample Collection.
The sample collection protocol mirrored that of a previous study (6). After anesthesia was
achieved and verified with Endo Ice (Coltene/ Whaledent Inc., Cuyahoga Falls, OH), the
patient’s tooth was isolated with a rubber dam, caries were excavated, and the pulp was exposed
with an excavator and/or round bur. Blood from the exposed surface of the pulp was collected
with two sterile cotton pellets (Henry Schein Inc., Melville, NY). The pellets were held at the
exposure site for about 60 seconds to allow absorption of the blood from the pulpal tissue. The
pellets were then placed in 1.0mL saline in heparin-coated tubes (VWR™, Radner, PA). Samples
were place on ice and stored/ refrigerated immediately at -20°C until they were ready to be
tested.

Sample Preparation.
Once the required number of samples was collected, the samples were prepared for analysis via
enzyme-linked immunosorbent assay (ELISA). After thawing the samples on ice, they were
centrifuged at 12,000g for 10 minutes at 4°C. The cotton pellets were removed after which the
samples were tested for cytokine levels using high sensitivity (HS) ELISA (Enzyme Linked
Immunosorbent Assay) kits.

Estimation of Cytokine Levels.

The levels of PGE-2 (prostaglandin E2), IL-1β, IL-6, TNF-α and IFN-γ were determined using

5
commercially available HS ELISA kits (Life Technologies Corp., Carlsbad, CA and Enzo Life
Sciences, Farmingdale, NY). The HS ELISAs has the following range for the respective
cytokines:

IL-6 has a sensitivity of 0.03 pg/mL.

TNF- alpha has a sensitivity of 0.13 pg/mL.
IFN- gamma has a sensitivity of 0.06 pg/mL.
IL-1beta has a sensitivity of 0.05 pg/mL.
PGE-2 can detect as little as 13.4 pg/ml.

These cytokines were selected based on previous studies that have reported an increase in
cytokines levels in irreversible pulpitis (6, 21). PGE-2 levels have also been shown to be
elevated in inflammation and ibuprofen has been shown to decrease these levels, which was why
it was included in our analysis (7). All samples were tested in triplicates. Optical density levels
were obtained using the accuSkan microplate reader (Fisher Scientific, Pittsburg, PA) and the
protein concentrations were then calculated based on the standards provided by the manufacturer
in the ELISA kits. A standard curve was generated, and the cytokine concentrations were
calculated based on this standard curve.

Determination of the total protein concentration.

The Bio-Rad protein assay (Bio-Rad, Hercules, CA) was used to determine total protein
concentration of the blood samples. Dye reagent was prepared by diluting one-part dye reagent
concentrate with four parts de-ionized distilled water. The solution was then filtered through
Whatman # 1 filter paper to remove particles. Ten microlitres of each diluted sample solution
was added into separate wells of microtiter plates. Two hundred microlitres of diluted dye
reagent was added to each well and mixed thoroughly. Samples were incubated at room
temperature for 30 minutes. Color intensity / absorbance was measured at 595 nm. Bovine serum
albumin (BSA) was used as control. Five dilutions of BSA were prepared with range between
0.05 mg/mL and 0.5 mg/mL to draw the standard curve. All samples were assayed in triplicate.
The final concentrations of all cytokine in each sample were calculated based on the total protein
concentrations of the samples.

6
Post –operative pain levels.
Patients from all groups (control, ibuprofen and non-ibuprofen) were asked to record whether
they had pain from 0-4 hours (yes/ no) after their anesthesia wore off and the numerical rating
scale (NRS) for pain that ranged from 0-10. Pain level was assigned as follow: 0: no pain ; 1–3:
mild pain; 4–6: moderate pain; 7-10: severe pain. (22, 23). The patients were contacted the day
after their treatment was initiated and the information was documented.

Data Analysis
SigmaPlot 11.0 (Systat Software, Inc. San Jose, CA) was used. A power analysis was completed
and the required sample size was calculated to be 12 samples for the experimental groups. This
gave the study at least 90% power to detect a difference between groups. Thirty-eight subjects
were recruited for our study. Median concentration values of each mediator for each group were
calculated. A Kruskal Wallis test for non-parametric comparisons were performed to evaluate the
obtained cytokine and PGE-2 concentration data. Fisher’s exact test was used to evaluate post-
operative pain data. Differences with p values (*, **) < 0.05 were considered significant.

7
Results.

Pro-inflammatory cytokine detection.

Sixteen patients were recruited to the ibuprofen group and eighteen patients were in the non-
ibuprofen group. The levels of pro-inflammatory cytokines were analyzed using HS ELISA kits
and their median levels were calculated. The levels of TNF-α were significantly lower in the
ibuprofen groups as compared to the non- ibuprofen groups and the control groups (Figure 1).
The levels of IFN-γ were statistically different between the ibuprofen and the non-ibuprofen
groups (Figure 2) and similar results were seen with the levels of IL-6 (Figure 3). IL-1β showed
no significant differences between the groups tested (Figure 4). In most cases, ibuprofen
decreased the levels of the proinflammatory cytokines to those of the control levels. In some
instances, like the IL-6 the median values for the ibuprofen group were lower than the control
group, which relates to the efficacy of ibuprofen lowering the concentrations of IL-6 to below
baseline levels.

Prostaglandin E2 (PGE-2) levels decreased in the presence of ibuprofen.

The levels of PGE2 were analyzed and the ibuprofen group showed lower levels of PGE2 as
compared to the non-ibuprofen group and the control groups (Figure 5).

Levels of post-operative pain.

Patients were contacted the following day after the initiation of endodontic treatment. They were
asked to use the previously explained NRS to rate and record their level of post-operative pain on
a scale of 0-10 for 0-4 hours after the effects of anesthesia wore off. Table 1 provides the mean
pain score provided for the non-ibuprofen and ibuprofen groups as well as the proportion of
patients in each group reporting post-operative pain (p=0.766). No statistically significant
differences were noted between the groups suggesting that ibuprofen did not affect the levels of
post-operative pain.

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Discussion.

Inflammation is associated with immune response of cells to bacteria. In the dental pulp, the
progression of inflammation could lead to symptoms of irreversible pulpitis. Previous studies
have demonstrated an increase in pro-inflammatory cytokines in caries exposed pulps (6). These
teeth with irreversible pulpitis have high levels of inflammation which could be the reason they
are harder to anesthetize and in many instances this can lead to anesthesia failures(24). Other
studies have demonstrated that more than 50% of patients with irreversible pulpitis were also
diagnosed with mechanical allodynia (25). Reducing this inflammation in the pulp could be a
key factor in contributing to anesthetic success. This would translate to the possible use of anti-
inflammatory drugs. Some previous studies have suggested that the use of ibuprofen pre-
operatively did not increase anesthetic success, however, some other studies have suggested
otherwise (12, 13, 18, 26-28). These studies mainly focused on the clinical signs and symptoms
during root canal procedures. However, no studies to date have demonstrated the effects of
ibuprofen at the molecular level in the dental pulp. Hence, this study analyzed the levels of pro-
inflammatory cytokines in the inflamed pulp in the presence or absence of ibuprofen.

Molars and premolars were selected for this study and anterior teeth were excluded as it
was not possible to obtain an adequate sample for analysis from the anterior teeth.
The gel capsule formulation of ibuprofen was selected for this study because a previous study
had shown that a higher plasma concentration could be achieved in a shorter period in
comparison to the tablet formulation of ibuprofen (29).

The levels of various cytokines were analyzed along with PGE-2. These five cytokines were
selected for the study because they are key cytokines involved in inflammation as explained
below.

PGE-2 has been previously shown to decrease pulpal nociceptor sensitization and induce
the stimulation of TTX-resistant sodium channels, which display resistance to lidocaine, which is
the anesthetic of choice during root canal procedures (8, 19). PGE-2, in turn, has been shown to
modulate the production various cytokines like IL-6 and TNF-α (30).
Interferon-gamma (IFN-γ), a pro-inflammatory cytokine was analyzed in this study. It is a
potent cytokine with strong inflammatory properties. Previous research has demonstrated higher

9
levels of IFN- γ in irreversible pulpitis compared to normal pulps (6). IFN- γ activates other
immune cells like macrophages that could potentiate the inflammatory process. IFN- γ is a potent
checkpoint switch for odontoblastic differentiation and can inhibit differentiation through the
JAK-STAT pathway (31). Hence, prolonged inflammation and higher levels of IFN-γ secretion
to impaired mineralization and differentiation, which in turn could possibly affect reparative
dentin formation (32).
TNF- α, another potent pro-inflammatory cytokine is well documented in inflamed pulps
(6). Bacterial lipopolysaccharide (LPS, endotoxin) are the main stimulants triggering TNF-α
production mainly by the macrophages and T cells and to a certain extent by other immune
cells like NK cells, B cells, mast cells etc., which are all present in inflamed pulps. Vascular
endothelial cells then respond to this TNF- α by undergoing a number of pro-inflammatory
changes, which increase leukocyte adhesion, transendothelial migration and vascular leak and
promote the inflammatory response (33).
IL-6 acts as a major mediator of the host response following tissue injury and infection as
well as inflammation. Higher levels of IL-6 have been demonstrated in pulps with irreversible
pulpitis (6). IL-6 is known to increase the levels of acute-phase proteins, C-reactive protein,
serum amyloid A and fibrinogen (34). Similar to TNF- α, IL-6 causes up-regulation of adhesion
molecules and induces angiogenesis leading to increase in vascular permeability and
inflammatory edema.
IL-1 is another pro-inflammatory cytokine that acts locally and promotes the recruitment of
inflammatory cells by upregulating the expression of cell adhesion molecules and other
proinflammatory cytokines, such as IL-6, IL-8, and TNF- α (35). Systemically, IL-1 induces the
acute phase response and induces the upregulation of IL-6 in this process. IL-1β along with IL-6
and TNF- α upregulate the production of agents, such as platelet activating factors,
prostaglandins, and nitric oxide, which are responsible for vasodialation and subsequent
inflammation (36) . Locally, IL-1 has a wide range of target cells and exerts multiple biological
effects, including synthesis of prostaglandin E 2 (PGE2), increased cell proliferation, and
synthesis of collagen and glycosaminoglycans (35). Other research has implicated the role of IL-
1β in the progression of pulpal and periapical disease (37). PGE-2, in turn, has been shown to
modulate the production various cytokines like IL-6 and TNF- α (30).

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 The data demonstrated a statistically significant decrease in the levels of PGE-2, IL-6 TNF-
α, and IFN-γ in patients taking 600mg of ibuprofen pre-operatively. These differences correlated
well to the mechanisms of activation and secretion of these cytokines. The potential mechanism
that may account for this decrease in the levels of these cytokines could possibly be attributed to
the decrease in the levels of PGE-2. Ibuprofen is a well-known NSAID that acts by blocking the
COX pathway, thereby blocking the production of PGE-2 and consequent vasodilation at the site
of inflammation. As mentioned previously PGE-2 modulates the various pro-inflammatory
cytokines. Hence, blocking PGE-2 in turn blocks the release of these pro-inflammatory cytokines
by the immune cells. This possibly decreases inflammation in the pulp and would decrease
vasodilation, decrease migration of inflammatory cells and inflammation, maintain the tissue pH,
decrease the phenomenon of ion trapping at a local anesthetic site and thus help to retain local
anesthetic at the target site for longer and increase the anesthetic efficacy. This strategy would be
particularly helpful in patients with a pulpal diagnosis of symptomatic irreversible pulpitis.

There were differences in the levels of IL-1β; however, these were not statistically
significant. This could be due to a variety of factors including patient variability and the levels
assayed by the ELISA. Another important finding from this study were the cytokine levels of
the control and the ibuprofen groups. As can been seen in figures 1- 5, a the median
concentrations of cytokines in the ibuprofen group very closely approximate those of their
respective control groups or in some cases (TNF- α and PGE-2) were lower than the control
groups suggesting ibuprofen decreases the levels of inflammation to at or below baseline levels.
Hence, ibuprofen has the potential to decrease inflammatory cytokine levels to baseline levels,
mirroring those of a normal, uninflamed pulp.

PGE2, IL-6, TNF-α, and IFN- γ are potent inflammatory mediators, and inflammation is
attributed to tissue acidosis, vasodilation, upregulation of tetrodotoxin- resistant (TTX-r) sodium
channels, and central sensitization that all play key roles in making pain management and
attaining local anesthesia in irreversible pulpitis cases so difficult. The results from this study
suggest that ibuprofen when taken pre-operatively, is able to diffuse into the pulp despite the
narrow diameter of the vasculature and help in decreasing these cytokines (38). This in turn
could possibly address some of the factors that can affect local anesthetic success. However, a
study related to anesthetic success and the use of pre-operative ibuprofen would be needed to
address this issue.

11
 The amount of local anesthetic and the type of injection used in each case were noted but this
study did not specifically control these parameters since it mainly focused on the levels of pro-
inflammatory cytokines in the pulp in the presence and absence of ibuprofen. No co-relations
were seen between the amount of anesthetic used and the levels of cytokines analyzed. Post-
operative pain was analyzed. As mentioned previously, patients were contacted the next day
following initiation of endodontic treatment and asked to self-report their level of post-operative
NRS for pain from 0-10 for 0-4 hours after the effects of anesthesia wore off. The NRS is
probably a more useful tool for pain assessment in research than the visual rating scale (VRS) or
visual analogue scale (VAS), because it is simpler and provides a descriptive numerical value to
the patient and is helpful in statistical analysis (23). Table 1 demonstrates that there were no
significant differences between the ibuprofen and non- ibuprofen groups. This data is in contrast
to some previous studies but supports another study, which suggested that pre-operative
ibuprofen may not be significant in reducing post-operative pain (23, 39).

In summary, the data in this study demonstrates the effectiveness of 600mg of ibuprofen on
pro-inflammatory cytokines in the dental pulp when taken one hour pre-operatively in cases of
teeth with irreversible pulpitis. The levels of PGE-2, IL-6, TNF-α, and IFN- γ were significantly
decreased in the in the ibuprofen group. This is the first paper until date to demonstrate the
effects of ibuprofen at the molecular level in the dental pulp. However, the clinician should
always consider other factors that could affect treatment of symptomatic irreversible pulpitis
cases.

12
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Figure Legends.

Figure 1. The levels of TNF-alpha were analyzed for the control, ibuprofen and non-ibuprofen
groups. Statistically significant differences were observed between the ibuprofen and non-
ibuprofen groups and the control and ibuprofen groups (*, ** p< 0.05)

Figure 2. The levels of IFN- gamma were analyzed for the control, ibuprofen and non-ibuprofen
groups. The differences in the levels of IFN- gamma were statistically significant between the
ibuprofen and non-ibuprofen groups (* p <0.05).

Figure 3. The levels of IL-6 were analyzed for the control, ibuprofen and non-ibuprofen groups.
The differences in the levels of IL-6 were statistically significant between the ibuprofen and non-
ibuprofen groups (* p <0.05).

Figure 4. The levels of IL-1 beta were analyzed for the control, ibuprofen and non-ibuprofen
groups. There were no significant differences seen between any of the groups.

Figure 5. The levels of PGE-2 were analyzed for the control, ibuprofen and non-ibuprofen
groups. The differences in the levels of PGE-2 were statistically significant between the
ibuprofen and non-ibuprofen groups (* p <0.05).

18
Table 1. Average post-operative pain scale ratings and the proportion of patients reporting post-
operative pain in each group.

PAIN NON- IBUPROFEN p-value

IBUPROFEN
AVERAGE PAIN
SCORE (0-10 2.072 1.857 0.70
scale)
PROPORTION
REPORTING 0.22 0.214 1.00
POST-OP PAIN

19