Aust Endod J 2019

ORIGINAL RESEARCH

Influence of different irrigants on substance P and IL-8

expression for single visit root canal treatment in acute
irreversible pulpitis
J.Evangelin, BDS, MDS1; I. Anand Sherwood, BDS, MDS, PhD1 ; Paul V. Abbott, BDSc, MDS, FRACDS(Endo)2 ;
Ramesh Uthandakalaipandian, MSc, Ph.D3; and Vijay Velu, MSc3
1 Department of Conservative Dentistry and Endodontics, CSI College of Dental Sciences, Madurai, Tamil Nadu, India
2 UWA Dental School, The University of Western Australia, Perth, Australia
3 Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India

Keywords
Dexamethasone, IL-8, ketorolac tromethamine,
post-operative pain, root canal irrigants,
substance P.
Correspondence
Dr I. Anand Sherwood, Department of
Conservative Dentistry and Endodontics, CSI
College of Dental Sciences and Research,
Anand Dental Clinic, No 1 Meenakshi Towers, P
T Rajan Road, Bibikulam, Madurai – 625002,
Tamil Nadu, India. Email:
anand.sherwood@gmail.com
doi: 10.1111/aej.12340
(Accepted for publication 2 March 2019.)
Abstract
The aim of this study was to assess the influence of ketorolac tromethamine
and dexamethasone on substance P and IL-8 expression when used as a root
canal irrigant for single visit root canal treatment for acute irreversible pulpitis.
A total of 42 patients with pain due to acute irreversible pulpitis in carious pre-
molar and molar teeth were included in this study. The four irrigation groups
were as follows: saline (n = 11), 3% sodium hypochlorite (n = 11), ketorolac
tromethamine (n = 10) and dexamethasone (n = 10). Blood samples S1 and
S2 were collected upon access opening and after canal preparation, respec-
tively. Quantification of substance P and IL-8 were done using ELISA test.
Post-operative pain was assessed by questioning the patients. The difference
between S1 and S2 sample values for the four different irrigant groups was not
significant. The sodium hypochlorite group had a higher mean expression of
substance P and IL-8 values. Dexamethasone irrigation was more effective in
controlling post-operative pain.

Of the many inflammatory mediators, IL-8 has been

Introduction
Providing pain relief for patients is one of the main
goals of root canal treatment. However, at times root
canal treatment can itself be the cause of post-operative
discomfort. This has been supported by findings from
various studies reporting on the incidence of post-
operative pain following root canal treatment (1–3).
Increased occurrence of post-operative pain following
root canal treatment in teeth presenting with pre-
operative symptoms and a diagnosis of acute irre-
versible pulpitis has been established in the literature
(1–5). The major cause of this pain is thought to be
because of the release of inflammatory mediators that
activate sensitive nociceptors surrounding the tooth
(4,6). The resultant stimulation of both central and
peripheral mechanisms is described as hyperalgesia
which is defined as an increase in the perceived magni-
tude of a painful stimulus (4,7).
extensively studied as a potential marker for irreversible
pulpitis (8,9). Increased expression of IL-8 is correlated
with increased polymorphonuclear neutrophils (PMNs)
within the pulp because IL-8 induces neutrophil
chemotaxis and release of degradation enzymes during
degranulation (9). The role played by neuropeptides
such as substance P has gained much attention in
recent times (10). Neuropeptides such as substance P
exert profound effects on blood flow, inflammatory
processes and immune responses such that they have
significant roles in inflammatory conditions such as
irreversible pulpitis (11–13). This peripheral mechanism
of stimulation should be able to be controlled by the
administration of an anti-inflammatory agent (4). Ster-
oidal anti-inflammatory agents work by inhibiting
phospholipase-A2 and non-steroidal anti-inflammatory
drugs act by blocking cyclooxygenase enzymes leading
to reduction in prostaglandins and leukotriene (14).

© 2019 Australian Society of Endodontology Inc 1

Irrigants for acute irreversible pulpitis
Ketorolac tromethamine, a potent NSAID available in
both oral and injectable forms, is over 400 times more
potent as a selective inhibitor of COX-1 over COX-2 than
many other drugs (15). An earlier study by the authors
revealed that when ketorolac tromethamine was used as
a root canal irrigant in teeth with irreversible pulpitis, it
was able to control the expression of substance P in the
periapical tissue (submitted for publication).
Dexamethasone is an adrenocorticosteriod that has
much greater anti-inflammatory action than other ster-
oids (16). The effectiveness of corticosteroids in reducing
periapical inflammation and the incidence of pain follow-
ing root canal preparation has been reported in the litera-
ture (17).
The aim of this study was to compare and assess the
influence of ketorolac tromethamine and dexamethasone
on substance P and IL-8 expression in the pulp and peri-
apical tissues when used as a root canal irrigant for single
visit root canal treatment in teeth with acute irreversible
pulpitis. Secondary objectives were to analyse the inci-
dence of intra-operative pain during the root canal treat-
ment and the amount of post-operative pain following
root canal treatment with the different irrigating solu-
tions.
Materials and methods
After obtaining approval from the Ethics Committee of
the institution, 42 patients consented to participate in the
study. A sample size of 41 patients was calculated to be
sufficient to detect clinical and biochemical data differ-
ence (alpha error of 0.05, power of 95% and effect size
0.7) (G power 3.1.9.2. software, Germany). The patients
(and where appropriate, their parent or guardian) were
informed about the nature of the treatment and the
study, and they were asked to sign an informed consent
form.
Patients referred to the Department of Conservative
Dentistry and Endodontics with pain due to acute irre-
versible pulpitis from carious premolar and molar teeth
requiring root canal treatment during the period April
2018 to June 2018 were evaluated as possible candidates
for this study. Subjects older than 15 years with no sys-
temic diseases and without having taken any medications
for pain in the previous 10 days prior to treatment were
included in the study. All patients reported moderate to
severe pain of either continuous or spontaneous nature,
nocturnal incidence, radiating or throbbing pain. All
teeth included in the study responded to cold pulp sensi-
bility tests (Endo-Frost, Coltene-Whaledent, Switzerland)
with an exaggerated response or pain with/without lin-
gering. In addition, profuse bleeding of the pulp was evi-
dent upon gaining access to the pulp chamber. A further
2 © 2019 Australian Society of Endodontology Inc
J. Evangelin et al.
criterion for inclusion in the study was a lack of any peri-
apical bone changes on the pre-operative periapical
radiographs.
Subjects were allotted to one of four treatment groups
according to the irrigant used during the root canal treat-
ment. Allocation was done by block randomization with
regard to pre-operative pain intensity (moderate or sev-
ere). The four groups were as follows: saline (n = 11),
3% sodium hypochlorite (n = 11), ketorolac trometha-
mine (n = 10) and dexamethasone (n = 10).
Experimental procedure and sample collection
The levels of pain for each patient pre-operatively and at
24 h post-operatively were recorded using a 10-point
visual analogue scale (VAS). The participants indicated
the intensity of their pain by choosing a number using
the following values: levels 0–3, mild pain, levels 4–7,
moderate pain, and levels 8–10 and severe pain.
Local anaesthesia (2.5 mL of 2% lignocaine with 1:
80 000 adrenaline–Lignox, Warren Pharmaceuticals,
India) was given prior to the root canal treatment pro-
cedure. Each tooth was isolated with rubber dam dur-
ing treatment. Pulp blood samples (S1) were collected
using a sterile paper point as soon as access was
gained to the pulp chamber. The collected sample was
placed in an Eppendorf tube and stored at 40°C for
further analysis.
Working length estimation was performed using a
Root ZX Mini Apex Locator (J Morita, Japan) and RaCe
files (FKG Dentaire, Switzerland) were used for root
canal preparation according to the manufacturer’s
instructions with an Endomate DT motor (NSK inc.,
Japan). Canal lubrication and smear layer management
were done with EDTA (10%) and carbamide peroxide
(15%) (Endoprep RC, Anabond Stedman Pharmaceuti-
cals, India). Depending on the participant’s allotment to
the irrigation groups, saline (NS 500 mL, Sodium chlo-
ride 0.9%, Fresenius Kabi, India Pvt. Ltd), 3% sodium
hypochlorite (Septodont Healthcare India Pvt. Ltd),
ketorolac tromethamine (Ketorolac tromethamine Inj.
IP 1 mL E.G. Pharmaceuticals, Solan (H.P), India) or
dexamethasone sodium phosphate (Dexalab Inj 2 mL,
Laborate Pharmaceuticals, Sahib (H.P), India) were used
as irrigants during the root canal preparation proce-
dures. In all cases, 2 mL of saline with 2% Povidone-
iodine (Puradine, Leeford Healthcare ltd, Mumbai,
India) was used as the initial irrigant and this was fol-
lowed by the sodium hypochlorite, ketorolac trometha-
mine or dexamethasone, according to the group
allocation, as mid-rinses using 1 mL for each canal.
Then, a final irrigation of each canal was performed
with 2 mL of saline with 2% Povidone-iodine solution.
J. Evangelin et al.
Initial irrigation of the root canal was done after a
glyde path establishment of apical size of up to 20 or
25 size K-file (Mani, Co., Japan). Mid-rinse and final
irrigation were employed after the usage of rotary
instrumentation. A total of up to 5 mL of irrigation
solution was used for each canal during the treatment.
Irrigation was performed with a standard syringe and a
24-gauge bevelled needle (Dispovan, India). The needle
was inserted as far apically into the canal as possible
but without any binding within the canal. Gentle force
was used on the syringe to deliver the irrigant, and the
needle was moved up and down inside the canal to
assist with irrigant flow and to ensure no binding of
the needle to the canal wall. If any patients required
additional local anaesthetic (either by block or infiltra-
tion injection) or if they experienced pain during root
canal treatment then this was recorded as intra-opera-
tive pain.
After completion of the root canal preparation, api-
cal patency was checked using a size 10 K-file (Mani
Co., Tokyo, Japan). A periapical blood sample (S2)
was then taken with a sterile paper point that was
placed 1–2 mm beyond the apical foramen. The paper
point (DiaDent Group, Seoul, Korea) was left in place
for 30 s, and then the collected sample was placed in
an Eppendorf tube and stored at 40°C for further
analysis. The root canal fillings were completed using
gutta percha points (DiaDentGroup, Seoul, Korea) with
zinc oxide eugenol-based cement (Prime Dental Prod-
ucts, Thane, India). Root canal filling was done using
a greater taper single gutta percha point (DiaDent
Group, Seoul, Korea) with zinc oxide eugenol-based
cement (Prime Dental Products, Thane, India). After
completing the procedure, the occlusion was checked
and relieved where necessary. A post-operative radio-
graph was taken to ensure the canals were filled to
the working length and there was no extrusion of fill-
ing material into the periapical tissues. Analgesics were
prescribed but the patients were advised to only take
them in the event of significant pain. The post-opera-
tive pain levels and the need for analgesics were
recorded after 24 and 48 h by telephoning each
patient to question them. If analgesics were required,
the patients were questioned about which medication
they used, the dosage, how often they had taken them
and whether they were effective.
Biochemical analysis
The paper points were fractionated and diluted by using
600 lL (pH 7.4) phosphate-buffered saline. Then the
samples were vortexed and centrifuged at 10 000 rpm
for 5 min. Quantification of substance P and IL-8 was
© 2019 Australian Society of Endodontology Inc
Irrigants for acute irreversible pulpitis
completed according to the manufacturer’s instructions
using an enzyme-linked immunosorbent assay (ELISA)
kit (Substance P ELISA kit Batch Number 0525422, Cay-
man Chemical, USA and Human IL-8 ELISA kit Batch
Number 1804010, Shanghai, China). The lower limits of
detection for substance P and IL-8 ELISA kits were 3.9
and 1.07 pg mL 1, respectively. The absorbency of each
sample was read at 420–450 nm wavelengths in a micro-
plate reader (SpectroMaxPlus 384, USA). A standard
curve was created using the standard concentrations of
substance P and IL-8. The concentrations of substance P
and IL-8 for each sample were calculated using the stan-
dard curve.
Statistical analysis
Statistical analysis was performed using IBM SPSS soft-
ware version 23 (IBM Corp., USA). Categorical values
of the pre-operative pain intensity, intra-operative pain
occurrence, analgesics requirement and post-operative
pain intensity were compared using the Chi-square
test. Pearson’s correlation coefficients were calculated
between the pain scores and substance P values. Nor-
mality of pre-operative pain, post-operative pain
scores, substance P and IL-8 data were checked by the
Shapiro–Wilk test. The data were skewed and deviated
from normal distribution; therefore, the comparison of
these values for the different irrigation groups was
done by non-parametric Wilcoxon signed-rank and
Kruskal–Wallis tests. The level of significance was set
at 5%.
Results
Forty-two patients (20 males and 22 females aged
between 15 to 65 years) participated in the study. The
mean pre-operative pain score was 7.09 ( 1.33). Four-
teen patients (33.3%) required analgesics post-opera-
tively for pain control and 28 patients (66.7%) did not
require analgesics (Table 1). Post-operative pain occur-
rence was 40.5% (17 patients). There was a significant
difference (P < 0.05) between the pre-operative and
24 h post-operative pain scores (Wilcoxon signed-rank
tests) (Fig. 1) with 24 h post-operative pain being signif-
icantly less. A significant difference (P < 0.05) between
the 24 and 48 h post-operative pain scores (Wilcoxon
signed-rank tests) was observed for all patients (Fig. 2)
with the mean 48 h pain score being zero. Female
patients had significantly higher (P < 0.05) 24 h post-
operative pain scores (mean score 1.86) and incidence of
post-operative pain (59.1%) (Chi-squared test) (Fig. 1).
No significant association was observed between gender
and the S1, S2 substance P, and IL-8 expression values.
3
Irrigants for acute irreversible pulpitis J. Evangelin et al.

Table 1 Distribution of the pain scores, substance P and IL-8 values (pg mL 1) and use of analgesics for different irrigation groups

Post-op Post-op
Pre-op Pain Pain
Post-operative
pain S1 Value S2 Value S1 Value S2 Value Scale Scale
Analgesics usage
scale substance P substance P IL-8 IL-8 24 hrs 48 hrs
Mean Mean Mean Mean Analgesics
[(Wilcoxon [Wilcoxon [Wilcoxon [Wilcoxon Analgesics not
Signed- Signed- Signed- Signed- required required
Rank Test Rank Test Rank Test Rank Test Number
Asymp. Asymp. Asymp. Asymp. Number of of
Mean Sig. (2-tailed)] Sig. (2-tailed)] Sig. (2-tailed)] Sig. (2-tailed)] Mean Mean patients patients

Irrigation Saline 6.73 83.45 [0.248] 43.68 [0.248] 16.27 [0.206] 11.27 [0.206] 0.91 0.00 3 8
Group Sodium 7.18 125.39 [0.500] 67.97 [0.500] 16.90 [0.462] 15.44 [0.462] 2.00 0.45 6 5
Hypochlorite
Ketorolac 6.90 54.69 [0.203] 56.64 [0.203] 13.10 [0.672] 11.00 [0.672] 1.40 0.00 3 7
Dexamethasone 7.60 64.06 [0.672] 60.55 [0.672] 14.40 [0.721] 12.40 [0.721] 0.90 0.00 2 8

Mean difference of substance P and IL-8 values (pg mL 1) between S1 and S2 samples for the different irrigation groups analysed using the Wilcoxon
signed-rank test.

It was noted that males had higher values of substance P

and IL-8 except for the S2 IL-8 values.
Lower molar teeth (12 of 17 patients) had significantly
(P < 0.05) higher pre-operative pain scores (7.94) com-
pared to the other teeth (Chi-squared test). Lower molar
teeth also had an increased incidence (seven patients) of
post-operative pain compared to the other teeth. In all
four irrigant groups, there were significant differences
(P < 0.05) between the pre-operative pain scores and the
24 h post-operative pain scores (Wilcoxon signed-rank
tests) (Table 1). A significant difference (P < 0.05)
between the 24 and 48 h post-operative pain scores was
observed only with the sodium hypochlorite group. The
mean post-operative 24 h (mean score 2) and 48 h pain
scores (mean score 0.45) were highest for the sodium
hypochlorite group compared to the other groups
(Table 1). Only one male patient in the study (from the Figure 1 Bar graph representation of pre-operative and post-operative
sodium hypochlorite irrigation group) reported pain at 24 h pain scores among the males and females (Z value -5.662, Asymp-
48 h post-operatively. Among the four irrigant groups, totic Significance (2-sided)–0.000) (mean pain score value in box).
dexamethasone had the least number of patients (two
patients) requiring analgesics for post-operative pain and significantly (P < 0.5) increased mean S1 substance P
also the lowest post-operative pain incidence (Table 1). value (112.41 pg mL 1) when patients experienced
The sodium hypochlorite group had the highest number intra-operative pain (Table 3).
of patients (six patients) requiring post-operative anal- Pre-operative and post-operative pain values did not
gesics and seven patients reported post-operative pain show significant associations with substance P and IL-8
(Table 1). values (Kruskal–Wallis test) (Table 4 and 5). The mean
Females (11 patients) had a higher incidence of intra- IL-8 S2 value was highest for patients with moderate
operative pain in this study. A significantly (P < 0.05) intensity 24 h post-operative pain and it was least for
higher number of lower molar teeth (13 of total 17 patients with no pain (Table 5). With substance P, the
patients) had intra-operative pain compared to the other highest values were observed with patients having no
tooth types (Chi-square test) (Table 2). The mean S1 val- 24 h post-operative symptoms (Table 5). The difference
ues of substance P (105.92 pg mL 1) and IL-8 between S1 and S2 sample values for substance P and IL-
(17.52 pg mL 1) were higher for lower molar teeth than 8 between the four different irrigant groups was not sig-
for the other tooth types (Fig. 3). There was a nificant (Wilcoxon signed-rank tests) (Table 1). The

4 © 2019 Australian Society of Endodontology Inc

J. Evangelin et al.
sodium hypochlorite group had a higher mean expres-
sion of substance P and IL-8 values in the S2 samples
than the other irrigant groups. Lower expressions of sub-
stance P and IL-8 S2 sample values were observed with
the saline and ketorolac tromethamine irrigation groups.
In patients requiring analgesics for post-operative pain
control, there was an increased expression of IL-8 values
in the S2 samples compared to patients who did not
require analgesics (Fig. 4).
Discussion
The main aim of the study was to assess the influence of
ketorolac tromethamine and dexamethasone on sub-
stance P and IL-8 expression in the pulp and periapical
tissues when used as root canal irrigants for teeth with
acute irreversible pulpitis. Only patients who had not
taken pre-operative analgesics prior to treatment were
included in the study. This was done to eliminate the
possible bias of analgesics on substance P and IL-8 expres-
sion and post-operative pain. In all irrigation groups,
Figure 2 Bar graph representation of post-operative 24 and 48 h pain
scores (Z value -3.655, Asymptotic Significance (2-sided)–0.000) (mean
pain score value in box).
Table 2 Comparison of intra-operative pain incidence with gender and tooth type analysed using the Chi-squared test. Percentage of incidence is
given in parenthesis
Gender Tooth Quadrant
Upper
Male Female premolar
Intra-operative Yes 7 (38.9%) 11 (61.1%) 1 (5.6%)
pain No 13 (54.2%) 11 (45.8%) 5 (20.8%)
© 2019 Australian Society of Endodontology Inc
Irrigants for acute irreversible pulpitis
saline with 2% povidone-iodine was used as the initial
and final irrigant. The povidone-iodine was added to the
saline in order to provide some anti-microbial effect in
the saline and dexamethasone irrigant groups as a pre-
cautionary measure.
The results reveal that ketorolac tromethamine was
able to better control the expression of substance P and
IL-8 compared to dexamethasone. Seventeen patients
(40.5%) reported post-operative pain. This observation is
higher than compared to a previous systematic review
(2). However, in contrast to that review study, the mean
pre-operative pain score in the current study was much
higher. The conclusions from another study for single
visit root canal treatment done in teeth with pulp status
of normal, irreversible pulpitis or pulp necrosis showed
that 46% of patients experienced post-operative pain but
there was no mention of the pre-operative pain intensity
(5). This data are in agreement with the present study
(5). The presence of pre-operative pain has been shown
to be a significant indicator of post-operative pain occur-
rence with one study reporting 57% post-operative pain
occurrence in teeth which were pre-operatively symp-
tomatic (3). Comparison of post-operative pain incidence
following root canal treatment from the literature is often
difficult as each study has used different parameters to
evaluate the pain and pulp status. Post-operative anal-
gesics were required for fourteen patients (33.3%) in this
study. All of the patients who required analgesics only
took one dose 24 h post-operatively, and this was able to
control the pain. The use of these analgesics in this study
was effective in controlling the post-operative pain, as
the mean 48 h post-operative pain score was reduced sig-
nificantly to zero. This conclusion is in concurrence with
an earlier observation (4).
Females had significantly higher 24 h post-operative
pain scores and incidence. Increased incidence of post-
operative pain in females has been previously reported in
the literature (4,18). This was in spite of findings of no
significant difference between substance P and IL-8
expressions between the two genders in the current
study. A possible explanation for the increased incidence
Lower
molar [Pearson
Lower Upper Chi-Square Asymptotic
premolar molar significance (2-sided)] Total
2 (11.1%) 2 (11.1%) 13 (72.2%) [0.002] 18 (42. 9%)
2 (8.3%) 13 (54.2%) 4 (16.7%) 24 (57.1%)
5
Irrigants for acute irreversible pulpitis J. Evangelin et al.

of post-operative pain in females is that women tend to
seek and accept treatment more willingly, as the presence
of symptoms is more readily perceived as indicators of
disease. Another possible explanation in females is the
differences in pelvic and reproductive organs leading to
Figure 3 Bar graph representation of mean substance P (S1) and IL-8
(S1) pg mL 1 values from different tooth types (mean substance P and
IL-8 pg mL 1 values in box).
possible local and distant hyperalgesia and fluctuating
hormone levels associated with menstrual cycles, oral
contraceptives or hormonal therapy (5,18,19). Compara-
ble to these observations a significantly higher number of
females (10 patients, 45.5%) reported with mild pain
post-operatively compared to males (two patients, 10%).
Lower molar teeth in this study had significantly
increased pre-operative pain intensity and a higher occur-
rence of post-operative pain. This is in accordance with
previous findings of increased pain intensity in mandibu-
lar molars. The reasons attributed for this are an increased
number of canals or foramina in molar teeth and the
mandible having a thicker cortical plate (3,5,19). The cur-
rent results show that the mean pre-operative substance P
and IL-8 (S1) values were higher for lower molar teeth
compared to other teeth, and this could explain the signif-
icantly higher incidence of pre-operative pain intensity in
these teeth. No further evidence could be found in the lit-
erature for this increased expression of these molecular
mediators in lower molar teeth. However, this could be
also because of the study design where the type of tooth
included was not controlled, and more lower molar teeth
with severe pain intensity were included.
The sodium hypochlorite irrigation group had higher
mean post-operative pain scores at 24 and 48 h com-
pared to the other irrigation groups. It was also observed

Table 3 Mean S1 values of substance P (pg mL 1) and IL-8 (pg mL 1) expression of patients with intra-operative pain analysed using the Kruskal–
Wallis test

Report
S1 Value substance 95% Confidence interval 95% Confidence interval
P (Kruskal–Wallis Test Lower bound S1 Value IL-8 (Kruskal–Wallis Test Lower bound
Intra-operative pain Asymp. Sig.) Upper bound Asymp. Sig.) Upper bound

Yes Mean 112.4133 (0.026) 16.3333 (0.406)

N 18 0.024 18 0.411
Std. Deviation 122.39167 0.030 9.31791 0.431
No Mean 58.0848 (0.026) 14.3043 (0.406)
N 23 23
Std. Deviation 104.59231 10.21334

Table 4 Mean pre-operative pain score comparison with substance P (S1) (pg mL 1) and IL-8 (S1) value (pg mL 1)

Report

S1 value substance P 95% Confidence interval 95% Confidence interval

(Kruskal–Wallis Test Lower bound S1 value IL-8 (Kruskal–Wallis Lower bound
Pre-operative pain value Asymp. Sig.) Upper bound Test Asymp. Sig.) Upper bound

Moderate pain Mean 82.0314 (0.162) 15.1364 (0.968)

N 22 0.159 22 0.975
Std. Deviation 141.37736 0.173 9.82851 0.980
Severe pain Mean 81.8263 (0.162) 15.2632 (0.968)
N 19 19
Std. Deviation 76.51801 9.95458

6 © 2019 Australian Society of Endodontology Inc

J. Evangelin et al. Irrigants for acute irreversible pulpitis

Table 5 Mean post-operative 24 h pain score comparison with substance P (S2) and IL-8 (S2) value (pg mL 1)

Report

S2 value substance P (Kruskal– 95% Confidence interval 95% Confidence interval

Wallis Test Lower bound S2 value IL-8 (Kruskal–Wallis Test Lower bound
Post-operative Pain 24 h Asymp. Sig.) Upper bound Asymp. Sig.) Upper bound

No pain Mean 63.6400 (0.743) 11.4783 (0.578)

N 24 0.746 23 0.583
Std. Deviation 61.64954 0.763 9.51969 0.603
Mild pain Mean 48.1767 (0.743) 12.5833 (0.578)
N 12 12
Std. Deviation 50.69512 11.54799
Moderate pain Mean 45.3140 (0.743) 16.4000 (0.578)
N 5 5
Std. Deviation 49.53294 10.23719

pain occurrence was highest during the first 48 h post-

Figure 4 Bar graph representing the comparison of mean substance P
and IL-8 (S2) (pg mL 1) values with post-operative analgesics require-
ment analysed using the Kruskal–Wallis test (mean values substance P
and IL-8 values in box).
that the sodium hypochlorite group had higher mean
substance P and IL-8 values (S2). Elevated levels of sub-
stance P and IL-8 in symptomatic irreversible pulpitis
have been mentioned in previous reports and have been
associated with the perpetuation and exacerbation of the
acute inflammatory process (9). In concurrence with
these findings, the results of this study have shown this
elevated expression was associated with higher post-
operative pain scores for the sodium hypochlorite irriga-
tion group. In a study by Farzeneh et al. 2018, using two
different concentrations of sodium hypochlorite as irrig-
ants for single visit root canal treatment in mandibular
molar teeth with irreversible pulpitis and no history of
spontaneous pain, it was shown that the post-operative
operatively with both concentrations of sodium
hypochlorite (20). However, it was also reported that
increased concentration of sodium hypochlorite had sig-
nificantly less post-operative pain occurrence (20).
Unlike that study, that the current study included only
patients with pre-treatment pain of moderate to severe
intensity, and the sodium hypochlorite irrigation was not
able to control the post-operative pain occurrence as
effectively as the other irrigation solutions. An earlier
study by the authors has revealed that sodium hypochlo-
rite did not have as much decreased expression of the
neuropeptide substance P compared to other irrigants
when used for single visit root canal treatment for teeth
with irreversible pulpitis (21). Therefore, the role and use
of sodium hypochlorite in the control of post-operative
pain following single visit root canal in teeth with acute
and chronic irreversible pulpitis needs to be reviewed.
Lower levels of expression of both inflammatory media-
tors were found for the saline and ketorolac tromethamine
irrigation groups. Ketorolac tromethamine with anti-
inflammatory properties has been previously reported by
the authors to be able to better control the expression of
substance P when used as root canal irrigant (21). Also, an
investigation by Rogers et al. has shown that the use of
ketorolac tromethamine as an intra-canal medicament
was able to provide significant pain relief (22). The saline
group appeared to be able to control the expression of
inflammatory mediators which was surprising since it was
not expected with an inert material. Future studies could
examine the role of saline in control of inflammatory
mediators and post-operative pain incidence.
Dexamethasone irrigation achieved the lowest post-
operative pain scores and the least requirement for anal-
gesics. This was despite the findings of not having the least
expression of substance P and IL-8 values in the S2

© 2019 Australian Society of Endodontology Inc 7

Irrigants for acute irreversible pulpitis
samples for this group. This may be explained by the
physiologic response to inflammation being a result of
complex interactions between multiple inflammatory
mediators (‘inflammatory soup’) and neuromediators
rather than the presence or amount of any one particular
mediator (23). The use of corticosteroids orally and as
intra-canal medicaments for the management of post-
operative pain following root canal treatment in teeth
with inflamed and necrotic pulps has been reported in the
literature (24,25). Criticism of corticosteroids being used
as intra-canal medicaments have been based on their pos-
sible systemic effects, but this was refuted with evidence
pointing to negligible quantities being detected in the
peri-radicular tissues and hence systemically (26). Dex-
amethasone as a root canal irrigant has earlier been
reported to have no significant improvement in post-
operative pain control (27). However, the sample size of
that study was too small to have any definitive conclu-
sion.
The intra-operative pain incidence of 42.9% in this
study is in agreement with previous investigations on pain
associated with root canal treatment of teeth with irre-
versible pulpitis (2,5). Previous observations of females
having increased susceptibility to intra-operative pain
have also been reinforced by the results of this study (5).
An increased risk of females with irreversible pulpitis hav-
ing intra-operative pain has been associated with signifi-
cantly higher levels of mechanical allodynia in addition to
the mentioned reasons discussed above (5). Lower molar
teeth had significantly increased incidence of intra-opera-
tive pain in this study, and this is in agreement with previ-
ously published reports of difficulty in attaining sufficient
anaesthesia in lower posterior teeth with irreversible pul-
pitis (5). Various theories and reasons have been attribu-
ted to the difficulty in attaining anaesthesia in these
patients (5,28,29). Patients with intra-operative pain in
this study had significantly increased expression of sub-
stance P. This higher expression of this neuropeptide is in
accordance with investigations from inflamed painful
pulps (30). The role of substance P in teeth with intra-
operative pain and strategies involved to control the
expression of these molecules needs further exploration.
The S1 values of substance P and IL-8 did not show sig-
nificant association with pre-operative pain intensity.
This may be because only patients with moderate and
severe pain intensity were included in this study. It was
also observed that patients with increased post-operative
pain intensity and those who also required analgesics for
post-operative pain had higher expression of IL-8 (S2 val-
ues). The role of higher levels of IL-8 in pulpitis has been
extensively reviewed and this molecule has been associ-
ated with PMNs (7). Pulpitis is clearly a PMN-driven
inflammatory process by extrapolation of the elevated
8 © 2019 Australian Society of Endodontology Inc
J. Evangelin et al.
levels of IL-8 which indicate increased neutrophils pres-
ence and could be considered a marker of tissue break-
down (9). In agreement with these findings, the results of
the current study reveal that elevated levels of IL-8 in the
periapical blood samples (S2) was associated with post-
operative pain. An earlier study by the authors did not
find an association with post-operative pain and periapical
substance P expression (21). Even in this study, it was
seen that substance P did not have association with post-
operative pain occurrence or the requirement for anal-
gesics. Substance P and IL-8 expression not having signifi-
cant association with pre-and post-operative pain levels
may be explained by the physiologic response to inflam-
mation being a result of complex interactions between
multiple inflammatory mediators (‘inflammatory soup’)
and neuromediators rather than the presence or amount
of any one particular mediator (23). Another factor may
be the very similar pre- and post-operative pain intensities
as can be observed in Figure 1.
Conclusions
Post-operative pain incidence following root canal treat-
ment in teeth with irreversible pulpitis is high. This pain
occurrence was effectively controlled by analgesics
within 48 h. Dexamethasone irrigation was more effec-
tive in controlling post-operative symptoms in acute irre-
versible pulpitis than the other irrigants tested. Females
were more susceptible to post-operative pain. IL-8
expression was elevated in patients with increased post-
operative pain intensity and those who required anal-
gesics for pain control. Substance P levels were signifi-
cantly increased in patients with intra-operative pain.
Lower molar teeth had higher incidences of pre-operative
pain intensity, intra-operative pain and post-operative
pain occurrence. These teeth also had higher levels of
substance P and IL-8 expression.
Author declarations
All the authors confirm their participation and significant
contribution in this study.
Conflicts of interest
The authors deny any conflicts of interest.
References
1. Sathorn C, Parashos P, Messer H. The prevalence of post-
operative pain and flare-up in single- and multiple-visit
endodontic treatment: a systematic review. Int Endod J
2008; 41: 91–9.
J. Evangelin et al.
2. Pak JG, White SN. Pain prevalence and severity before,
during, and after root canal treatment: a systematic
review. J Endod 2011; 37: 429–38.
3. Arias A, de la Macorra JC, Hidalgo JJ, Azabal M. Predictive
models of pain following root canal treatment: a prospec-
tive clinical study. Int Endod J 2013; 46: 784–93.
4. Menhinick KA, Gutmann JL, Regan JD, Taylor SE,
Buschang PH. The efficacy of pain control following non-
surgical root canal treatment using ibuprofen or a combi-
nation of ibuprofen and acetaminophen in a randomized,
double-blind, placebo-controlled study. Int Endod J 2004;
37: 531–41.
5. Segura-EgeaJ J, Cisneros-Cabello R, Llamas-Carreras JM,
Velasco-Ortega E. Pain associated with root canal treat-
ment. Int Endod J 2009; 42: 614–20.
6. Johnsen DC, Harshbarger J, Rymer HD. Quantitative
assessment of neural development in human premolars.
Anat Rec 1983; 205: 421–9.
7. Malmberg AB, Yaksh TL. Hyperalgesia mediated by spinal
glutamate or substance P receptor blocked by spinal
cyclooxygenase inhibition. Science 1992; 257: 1276–9.
8. Elsalhy M, Azizieh F, Raghupathy R. Cytokines as diag-
nostic markers of pulpal inflammation. Int Endod J 2013;
46: 573–80.
9. Zanini M, Meyer E, Simon S. Pulp inflammation diagnosis
from clinical to inflammatory mediators: a systematic
review. J Endod 2017; 43: 1033–51.
10. Fouad AF. Molecular mediators of pulpal inflammation.
In: Hargreaves KM, Goodis HE, Tay FR, eds. Seltzer and
Bender’s Dental Pulp. 2nd ed. Illinois, USA: Quintessence
publication; 2012. pp. 241–76.
11. Awawdeh L, Lundy FT, Shaw C, Lamey PJ, Linden GJ,
Kennedy JG. Quantitative analysis of substance P, neu-
rokinin A and calcitonin gene-related peptide in pulp tis-
sue from painful and healthy human teeth. Int Endod J
2002;35:30–6.
12. Shin SJ, Lee W, Lee JI et al. Matrix metalloproteinase-8
and substance P levels in gingival crevicular fluid during
endodontic treatment of painful, non-vital teeth. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2011; 4: 548–54.
13. Sacerdote P, Levrini L. Peripheral mechanisms of dental
pain: the role of substance P. Mediators Inflamm 2012;
2012: 1–6.
14. Seltzer S, Naidorf IJ. Flare-ups in endodontics: II. Thera-
peutic measures. J Endod 2004; 30: 482–8.
15. Harsh EV, Desjardins PJ, Trummel CL, Cooper SA. Non-
opoid analgesics, non-steroidal anti-inflammatory drugs
and anti-rheumatic and antigout drugs. In: Yagiela JA,
Dowd FJ, Johnson B, Mariotti A, Neidle EA, eds. Pharma-
cology and Therapeutics for Dentistry. 6th ed. United
States: Elsevier; 2014. pp. 324–58.
16. Goodman-Gilman A, Goodman LS, Rail TW, Murad F.
Goodman and Gilman’s: the pharmacological basis of
© 2019 Australian Society of Endodontology Inc
Irrigants for acute irreversible pulpitis
therapeutics. 7th ed. New York: Macmillan; 1985. pp.
1478–9.
17. Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium
hydroxide, antibiotics and biocides as antimicrobial medica-
ments in endodontics. Aust Dent J 2007; 52: S64–82.
18. Polycarpou N, Ng YL, Canavan D, Moles DR, Gulabivala K.
Prevalence of persistent pain after endodontic treatment
and factors affecting its occurrence in cases with complete
radiographic healing. Int Endod J 2005; 38: 169–78.
19. Ng YL, Glennon JP, Setchell DJ, Gulabivala K. Prevalence
of and factors affecting post-obturation pain in patients
undergoing root canal treatment. Int Endod J 2004; 37:
381–91.
20. Farzaneh S, Parirokh M, Nakhaee N, Abbott PV. Effect of
two different concentrations of sodium hypochlorite on
postoperative pain following single-visit root canal treat-
ment: a triple-blind randomized clinical trial. Int Endod J
2018; 51(Suppl 1): e2–11.
21. Bamini L, Sherwood A, Abbott PV, Uthandakalaipandian
R, Velu V. Influence of anti-inflammatory irrigants on
substance P expression for single visit root canal treatment
of teeth with irreversible pulpitis. Aust Endod J 2018; 44:
14–25 (submitted for publication).
22. Rogers MJ, Johnson BR, Remeikis NA, BeGole EA. Com-
parison of effect of intracanal use of ketorolac trometha-
mine and dexamethasone with oral Ibuprofen on post
treatment endodontic pain. J Endod 1999; 25: 381–4.
23. Diogenes A, Henry MA. Pain pathways and mechanisms
of the pulpo-dentin complex. In: Hargreaves KM, Goodis
HE, Tay FR, eds. Seltzer and Bender’s Dental Pulp. 2nd ed.
Hamilton, USA: Quintessence; 2012. pp. 159–84.
24. Glassman G, Krasner P, Morse DR, Rankow H, Lang J,
Furst ML. A prospective randomized double-blind trial on
efficacy of dexamethasone for endodontic interappoint-
ment pain in teeth with asymptomatic inflamed pulps.
Oral Surg Oral Med Oral Pathol 1989; 67: 96–100.
25. Ehrmann EH, Messer HH, Adams GG. The relationship of
intracanal medicaments to postoperative pain in endodon-
tics. Int Endod J 2003; 36: 868–75.
26. Abbott PV. Systemic release of corticosteroids following
intra-dental use. Int Endod J 1992; 25: 189–91.
27. Tarabishy K. Postoperative pain after the use of a dexam-
ethasone rinse as an irrigant prior to obturation. (Master’s
Thesis). 2013; Milwaukee, US. Marquette University.
28. Nusstein JM, Reader A, Drum M. Local Anesthesia strate-
gies for the patient with a ‘Hot’ tooth. Dent Clin North Am
2010; 54: 237–47.
29. Hargreaves KM, Keiser K. Local anesthetic failure in
endodontics. Mechanisms and management. Endod
Topics 2002; 1: 26–39.
30. Caviedes-Bucheli J, Mu~ noz HR, Azuero-Holguın MM,
Ulate E. Neuropeptides in dental pulp: the silent protago-
nists. J Endod 2008; 34: 773–88.
9