a pilot study to assess the levels of interleukin-6

A PILOT STUDY TO ASSESS THE LEVE LS OF INTERLEUKIN-6 FOLLOWING ADMINISTRATION OF DICLOFENAC, KETOROLAC AND TRAMADOL AFTER SURGICAL REMOVAL OF LOWER THIRD MOLARS

Dissertation Submitted In Partial Fulfillment For the Degree of Master of Dental Surgery (Oral and Maxillofacial Surgery )

Faculty of Dental Sciences Sri Ramachandra University Porur, Chennai-600116.

NOVEMBER 2012

DR.PUNEET SINGH

CERTIFICATE

This is to certify that the dissertation entitled “A pilot study to assess the levels of Interleukin-6 following administration of Diclofenac, Ketorolac and Tramadol after surgical removal of Lower Third Molars ” herewith submitted by Dr. Puneet Singh in partial fulfillment for the degree

of

Master

of

Dental

Surgery

in

Oral

and

Maxillofacial Surgery is a bonafide research work carried out by him under my supervision and guidance.

Dr. C. RAVINDRAN, MDS Professor and Head, Guide

Department of Oral and Maxillofacial Surgery Faculty of Dental Sciences, Sri Ramachandra University, Porur, Chennai – 600 116

ACKNOWLEDGEMENTS I consider it a privilege to express my most sincere and heartfelt gratitude to my p rofessor, head of the department, and my guide Prof. Dr. C. Ravindran, for his most

valuable

guidance,

constant

motivation

and

encouragement and excellent teaching, which has made this study possible. He has been a gentle driving force, always present to offer a helping hand, patiently bearing our shortcomings and striving to make us better individuals professionally

and

personally.

Without

his

constant

encouragement and confidence, this study would not have been possible.

I would like to express my gratitude to our Dean, Prof. Dr. D. Kandaswamy for permitting me to use the scientific literature and facilities available in the college.

I

would

like

to

extend

my

gratitude

towards

Prof. Dr. R. Suresh, Professor and Head, Department of Periodontics for permission and guidance to use the facilities available in the department.

I

would

like

to

sincerely

thank

my

co-guide

Dr. G.V.V. Giri for his relentless support, supervision, suggestions, and effort in bringing out my dissertation. He has been a constant driving force, pushing me to do my best. His attention to detail has been a source of inspiration to bring out the best in me.

I would like to thank Prof Dr. S.P. Thyagarajan, Pro

Chancellor

Prof

Dr.

(Research),

Chamundeeswari,

SRU

Principal,

and

College

of

Pharmacy, SRU for allowing me in using the Laboratory services at Central Research Facility for conducting the study.

I would also extend my gratitude towards Dr. M. K. Sangeetha, Senior Research Fellow, Herbal & Indian Medicine Research Laboratory for helping me execute the experimental procedure.

I wholeheartedly thank Dr.P.Ravishankar, Assistant Professor, Statistics, Department of Community M edicine for helping me with the statistical analysis needed for my study.

With a deep sense of gratitude, I would like t o express my

sincere

thanks

to

Prof.

Dr.

S.

Ramkumar

and

Prof. Dr. N. Nandakumar for their invaluable help, guidance,

encouragement

and

continuous

support

throughout my study. Without their innovative ideas, technical expertise, motivation and enco uragement, this study would not have been possible.

I

wholeheartedly

thank

Dr.

Emmanuel

Azaria,

Dr. Pinky Ganga, Dr. Deepak Pandiyan, Dr. Santhosh K., and Dr. Deepak C. for their suggestions and support.

I

would

also

like

to

thank

Dr.

Shanthi,

and

Dr. D. Swarnakantha for their support.

My thanks also go to my co-pgs, juniors and interns for all their help. Also my thanks goes to all the sisters and other non skilled assistants in my department.

I also have to thank Mr. Babu, Mr.Ramaraju.R and Mr.Thiyagu of Netway Prints for helping me with the printing work.

On a personal note, my heartfelt gratitude to my parents and siblings for their never ending support.

Above all, I thank God, the Almighty for everything.

CONTENTS

S. NO

PAGE. NO

1.

1. INTRODUCTION

1

2.

2. AIM AND OBJECTIVES

5

3.

3. REVIEW OF LITERATURE

7

4.

4. MATERIALS AND METHODS

88

5.

5. RESULTS

134

6.

6. DISCUSSION

159

7.

7. CONCLUSION

177

8.

8. BIBLIOGRAPHY

180

LIST OF TABLES

Table

Page Title

No.

No.

1

Overall sex distribution

135

2

Different types of Impactions

136

3

Sex distribution within the 3 groups

137

4

Type of Impactions in 3 groups

139

5

Mean values of IL-6 in 3 groups at different

144

intervals 6

ANOVA analysis of the mean values

145

7

Multiple Comparisons between 3 groups

146

8,9

Paired sample statistics for Diclofenac

147

10,11

Paired sample statistics for Ketorolac

148

12,13

Paired sample statistics for Tramadol

149

LIST OF GRAPHS

Graph

Page Title

No.

No.

1

Sample size calculation

107

2

Overall sex distribution

135

3

Types of Impaction

136

4

Sex distribution within the 3 groups

138

5

Type of Impactions in 3 groups

140

6

Pre-op values of IL-6 in 3 drug groups

150

7

24 hrs post op values of IL-6 in 3 groups

151

8

72 hrs post op values of IL-6 in 3 groups

152

9

1 week post op values of IL-6 in 3 groups

153

10

Values of IL-6 at 4 intervals in 3 groups

154

11

Values of IL-6 at 4 intervals in 3 groups

155

12

Individual variations in IL-6 at 4 intervals

156

in 3 groups 13

Individual variations in IL-6 at 4 intervals

157

in 3 groups 14

Variations in IL-6 at 4 time intervals in 3 groups

158

LIST OF CHARTS Chart

Title

Page

No. 1

No. Values of Interleukin-6 at different time

141

intervals in the 3 drug groups

LIST OF FIGURES

Figure

Title

Page

No.

No.

1

Acute Phase Response of An Organism

161

2

Role of IL-6

163

3

Sites

of

Action

of

Non-Opioids

in

170

regulation of IL-6 4

Cascade of signaling events in Chondrocytes stimulated with PGE 2

171

LIST OF ABBREVIATIONS AA

Arachidonic Acid

APR

Acute Phase Reactants

AP-1

Activator protein 1

BALB/c

Bagg Albino (Inbred strain of mice)

COX

Cyclooxygenase enzyme

cAMP

Cyclic Adenosine Monophosphate

CFA

Complete Freunds Adjuvant

CRP

C-reactive protein

DGLA

Dihomo-Gamma Linolenic Acid

DNA

Deoxyribonucleic Acid

EPG

Eicosapentanoic Acid

ELISA

Enzyme Linked Immunosorbant Assay

FMLP

N-Formyl-L-Methionile-L-Leucyl-LPhenylalanine

GM-CSF

Granulocyte Macrophage Colony Stimulating Factor

HLA

Human Leukocyte Antigens

iPGE 2

Immunoreactive Prostaglandin E 2

ISS

Injury Severity Score

IL-1β

Interleukin-1 beta

IL-1

Interleukin-1

IFNγ

Interferon Gamma

IL-6

Interleukin-6

IL-6Ra

Interleukin-6 receptor antagonist

JNK

Jun NH2-terminal kinase

KC

Kupffer Cell

LPS

Lipo Poly Saccharide

mRNA

Messenger Ribonucleic Acid

MRE

Multiple Response Element

MIP-2

Macrophage Inflammatory Protein-2

NF-IL6

Nuclear Factor for Interleukin-6

NF-κB

Nuclear factor kappa B

NE

Nor Adrenaline

NSAID

Non Steroidal Anti Inflammatory Drug

PKA

Protein Kinase A

PI3K

Phosphatidyl Linositol 3 Kinase

PGHS

Prostaglandin Endoperoxide Synthase

PGs

Prostaglandins

PGE 2

Prostaglandin E 2

PG/ML

Pico gram per millilitre

RA

Rheumatoid Arthritis

rIL-6

Recombinant Interleukin-6

sIL-6R

Soluble Interleukin-6 Receptor

STAT

Signal transducers and activators of Transcription

SOCS3

Suppressor of cytokine signalling 3

SLE

Systemic Lupus Erythmatosis

TNFα

Tumor Necrosis Factor Alpha

TXB 2

Thromboxane B 2

TNF-R

Tumor Necrosis Factor Receptor

VAS

Visual Analogue Scale

The mandibular third molar is the most common tooth to become impacted. Several studies have been carried out in developed countries, on impacted third molars and lots of investments are spent annually, on the management of impacted third molars. In fact, it is regarded as the most commonly

performed

oral

surgical

procedure.

The

frequency of this occurrence is illustrated by one study which revealed 65.6 % of males with an average age 19 years had embedded third molars equally divided between the four quadrants of the dental arches.

Teeth that fail to attain a functional position may be pathological and should be considered for removal. The indications

for

removal

include,

pain,

pericoronitis,

periodontal disease, caries, orthodontic therapy and various pathologies.

The

surgical

removal

of

impacted

third

molars

involves, trauma to soft and bony tissue and can result in considerable pain, swelling, and trismus. This postoperative sequel can cause distress to the patient and affect the patient’s quality of life after surgery.

1

There are a plethora of techniques available for the removal

of

mandibular

third

molars.

Oral

surgical

procedures can vary in difficulty and in the degree of trauma caused to the surrounding tissue. As the Oral and Maxillofacial surgeon performs a more invasive or difficult procedure, there will be an increased amount of trauma to the surgical site as well as surrounding tissues. The greater the amount of tissue injury the greater is the amount of inflammation in the perisurgical region.

Interleukin 6 is a protein that in humans encoded by the IL-6 gene. IL-6 is an interleukin that is secreted by many cells in response to trauma, especially burns or other tissue damage leading to inflammation.

IL-6 is both a pro inflammatory and anti-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate the immune response.

IL-6 is one of the most important mediators of fever and of the acute phase response. The concentration of IL -6 may change depending on the anti-inflammatory used during the post-operative period.

One of the major challenges after third molar removal is pain and swelling of surrounding tissues and resulting morbidity. Pain not only signals tissue injury, but also acts 2

as

an

impediment

to

dental

procedures

and

delays

resumption of normal activities after the procedure.

Various methods for achieving control of the post surgical morbidity are the use of steroids (IV and oral), use of

NSAIDs,

preemptive

analgesia,

synthetic

opioids,

combined use of steroids and analgesics etc.

Most Oral and Maxillofacial surgeons largely rely on non – steroidal anti – inflammatory drugs (NSAIDs) for pain management in ambulatory patients. Although NSAIDs are remarkably effective in management of pain and inflammation, their use is limited by several adverse effects including gastrointestinal bleeding and ulceration, impaired renal function and inhibition of platelet aggregation.

Numerous endogenous mediators are involved in nociception and in the inflammatory res ponse after the surgical procedures. Among these are pro inflammatory prostaglandins such as Prostaglandin E 2 and Prostaglandin I 2 . Cyclooxygenase (COX) constitutes the rate limiting step in the synthesis of these prostaglandins. It is commonly believed that NSAIDs exert their therapeutic effects by inhibiting the enzyme COX, which inhibits the synthesis of prostaglandins.

3

Carriches et al 1 0 concluded after a study that: a) After the extraction of lower third molars there was a significant increase in IL-6 levels in both groups which was evident 24 hours after surgery and remained high after 7 days. b) Patients treated with NSAIDs showed a higher amount of IL-6

during

the

first

day

as

compared

to

methylprednisolone c) In contrast to the above results , patients trea ted with methylprednisolone showed the high levels after 7 days as compared to diclofenac group By using three different drugs and collecting blood samples for varying Interleukin 6 levels, the drug which acts to inhibit IL-6 most at a molecular level can be found out. It would help the oral surgeons to better understand the effect of anti-inflammatory drugs on the immunological pathways leading to post-operative sequelae after third molar surgeries. Only after gaining such understanding it is possible to select drugs which maximize

its analgesic and anti-

inflammatory effect while minimizing the post-operative morbidity. IL-6 is also an early marker of tissue damage. Research

has

also

immunomodulatory

shown effects.

that In

Tramadol

addition

to

also

as

NSA IDs,

corticosteroids, opioids also can down regulate or suppress the immune system. 4

AIM The aim of this study is to evaluate the changes in serum Interleukin-6 levels following surgical removal of third molars under local anesthesia after administration of two NSAIDs Diclofenac and Ketorolac and opioid Tramadol post operatively.

5

OBJECTIVES  To find out the variation in Interleukin -6 following surgical

removal

of

lower

third

molars

after

administration of Ketorolac, Diclofenac and Tramadol administered orally.  To observe the variation in IL-6 levels after third molar surgery over a period of 7 days.  To find out a better protocol for administering a more effective NSAIDs and to henceforth reduce postoperative morbidity by increased analgesia and antiinflammatory activity.

6

James S. et al, 1983 3 0 non-steroidal anti-inflammatory drugs have been thought to act by inhibiting the production of prostaglandins. Until recently, PGs have been considered to be mainly pro-inflammatory compounds responsible for pain, increased blood flow and edema. PGE 2 has important immunoregulatory properties. Thus NSAIDs in vivo act as general

immunostimulants,

increasing

cellular

immune

responses in vitro and in vivo.

Prostaglandin synthesis inhibitors are now currently used as anti-inflammatory agents in many immunologic diseases such as rheumatoid arthritis (RA) and SLE. Although their anti-inflammatory activity is obvious, it is less clear what their effect is on the basic disease process. Data suggested that if endogenous PGE 2 tonically inhibits a suppressor cell for its immune globulin and auto antibody formation. This has relevance to the in vivo situation then PG synthesis inhibitors would be expected to improve the basic disease process.

Goodwin

J.S.

et

immunoregulatory immunomodulating

al,

1984 2 2

effects

of

actions

recent

studies

prostaglandin of

of

the

and

the

nonsteroidal

anti -

inflammatory drugs reveal that earlier reports of inhib ition 7

of

prostaglandin

synthesis

by

nonsteroidal

anti -

inflammatory drugs cannot alone fully explain the anti inflammatory properties of these agents. Data defining the complex inter-relationship of prostaglandin, the immune response, and nonsteroidal anti-inflammatory drugs were reviewed, and possible mechanisms of anti -inflammatory action of these agents were considered by the authors.

The effects of nonsteroidal anti -inflammatory drugs on cellular immune function and humoral immune response suggest that anti-inflammatory activity may be related to altered suppressor cell function, inhibition of monocyte collagenase release, or inhibition of neutrophil migration and activation.

Norihiro

Nishimoto

interleukin-6

(IL-6)

et and

al,

1989 4 8

acute

Serum

phase

levels

proteins

of

were

measured in patients who underwent surgical operation. Elevation of IL-6 preceded that of acute phase proteins, indicating that the measurement of serum IL -6 may be helpful for the early detection of an inflammatory state. Henrik Linder et al, 1990 2 6 in the present study the authors examined the effect of anti -inflammatory agents’ dexamethasone, diclofenac and indomethacin on parameters like

Interleukin-6

and

polymorphonuclear

8

leukocytes

(PMNL) and on the bacterial clearance from ki dneys of infected urinary tracts of C3H/HeJ mice.

They found out that dexamethasone reduced IL -6 secretion, the PMNL response and the bacterial clearance. Diclofenac abolished the urinary IL -6 response but reduced the PMNL response and bacterial clearanc e only at the highest concentrations. Indomethacin drastically decreased bacterial clearance without the corresponding effect on IL -6 production or PMNL response.

These results demonstrated that the inhibition of inflammation impairs bacterial clearance from the kidneys. But this however was not a direct function of inhibited IL -6 production or PMNL recruitment. Peter C. Heinrich et al, 1990 5 1 the acute phase response (APR) is the answer of the organism to disturbances of its homeostasis due to infection , tissue injury, neoplastic growth or immunological disorders. The APR is thought to be beneficial to the injured organism with the aim of restoring

the

disturbed

physiological

homeostasis.

It

consists of local reaction at the site of injury characterized by a number of responses such as

aggregation of platelets ,

clot formation, dilation and leakage of blood vessels , accumulation and activation of granulocytes , mononuclear cells, which in turn release cytokines.

9

In addition activated fibroblasts and endot helial cells are able to produce cytokines. These mediators act on specific receptors on different target cells leading to a systemic reaction characterized by fever, leukocytosis, increase

in

erythrocyte

sedimentation

rate,

increase d

secretion of ACTH and glucocorticoids, activation of complement and clotting cascades, decrease in serum level of iron, zinc, a negative nitrogen balance and by dramatic changes in the concentration of some plasma proteins. These proteins are called acute phase proteins.

The APRs patterns vary from species to species. C reactive protein and serum amyloid -A show highest increase during an acute phase response in man.

These changes in plasma levels of APPs are preceded by corresponding alternations in mRNA concentrations. In addition increases in the rates of secretion of some acute phase proteins have been observed.

In 1951 it was shown by Miller et al that liver is the major organ for synthesis of APPs. Ritchie and Fuller described and partially characterized a monocyte deri ved polypeptide involved in regulation of acute phase protein synthesis, which they called hepatocyte stimulating factor.

10

The molecular mass of hepatocyte stimulating factor from

human

monocytes

was

estimated

by

gel

chromatography to be 23-30 kDa.

The first evidence of IL-6 as an inducer of the acute phase response in the human system came from work with human cell lines. Increases in protein synthesis of β fibrinogen,

α 1 -acid

glycoprotein,

α 1 -antitrypsin

and

complement factor B are observed upon stimulation of HepG2 cells with recombinant human IL -6.

IL-6 stimulated a full spectrum of acute phase protein synthesis in man, IL-1 as well as TNFα had only a moderate effect on the positive acute phase proteins. Both inhibited the synthesis of β-fibrinogen, albumin and transferrin and failed to induce serum amyloid A and C -reactive protein. This data strongly suggests that IL-6 plays a key role in human acute phase protein synthesis. Pullicino E.A. et al, 1990 5 2 the authors investigated the possible role of Interleukin 6 (IL-6) and tumour necrosis factor (TNF) in the regulation of the acute phase response to injury. Thirteen subjects undergoing elective surgery or suffering from multiple accidental injuries were studied for this purpose. The temporal changes in the circulating concentrations of these cytokines were related to the circulating acute phase protein concentrations (C -reactive

11

protein and alpha 1 antichymotrypsin ), white cell count, blood loss and duration of surgery.

Circulating

acute

phase

protein

concentrations

increased in all subjects during a thirty hour period following injury but none of the subjects showed a detectable increase in circulating concentrations of TNF. Peak circulating concentrations of IL -6 were detected between 4-6 hours after surgery/trauma but these showed a poor correlation with blood loss, fever, white cell count or duration of surgery. The peak concentrations of IL -6 typically occurred before the rise in circulating acute phase protein concentration.

This data does not suggest a role for circulating TNF in the regulation of the acute phase response to injury. In contrast, it shows that IL-6 appears to be variably involved in the acute phase response but its precise role in mediating fever, leucocytosis and synthesis of acute phase proteins is as yet uncertain. A.M. Cruickshank et al, 1991 1 the authors aimed to investigate the role of IL-6 as a mediator of the acute phase response (APR) in man. They assessed the effect of under nutrition on serum/plasma IL-6 levels and it’s value as a prognostic indicator by measuring IL-6 in timed venous

12

samples taken from surgical patients and from protein deficient and control rats post -turpentine injections.

They found out that serum IL-6 raised in all surgical patients within 2-4 hours of incision. Peak levels differed among the groups and corrected significantly with duration of surgery. CRP did not rise in some patients and discriminated poorly among the groups. Patients who developed complications had higher IL -6 values at 24 hours post-incision that those making uneventful recoveries. Protein depleted rats had a significantly slower IL -6 response than controls.

They concluded that serum IL-6 is a sensitive, early marker of tissue damage which might help predict the development of surgical complications and also how under nutrition affects the IL-6 response in rats and may do so in human subjects. F. Di Padova et al, 1991 2 0 surgical intervention is a form of programmed trauma. Through the study of surgical patients,

changes

in

early

endogenous

mediators

of

inflammation, immune response and tissue repair can be investigated.

Here the authors have investigated the changes in serum levels of IL-1 inhibitors, IL-1β, IL-6, tumor necrosis

13

factor – alpha and cortisol in patients undergoing elective surgery. C-reactive protein was measured as a marker of the acute – phase response. Rises in serum levels of IL -1 inhibitors, IL-6 and cortisol were detected as early as 1 hour after the intervention. Peak levels were reached between 2 and 5 hours. Serum levels of IL -1 and TNF did not change after the intervention. CRP levels peaked on day 2. The communication system sustained by endogenous mediators was activated after surgery as shown by selective changes in IL-1 inhibitors, IL-6 and cortisol.

These mediators have different kinetics in serum and IL-6 is not the only early mediator detected. Some IL -1 inhibitors might be involved in immunological depres sion observed after major surgery, in the regulation of the inflammatory response or in tissue repair. IL -6 and cortisol seem to act synergistically to activate the acute phase response. Yoshio Oka et al, 1992 70 the authors examined postoperative serial changes in the levels of serum interleukin 6, serum acute phase reactants and plasma neutrophil elastase in patients with various cancers and reviewed these changes in patients who did and did not, show post operative complications. Serum IL-6 level was found to be elevated after surgery, peaking on the first post -operative

14

day. Elevation of serum APRs and Plasma NE levels also followed.

There was significant correlation between the serum IL-6 level and those of APRs and NE. There was a significant difference in the serum IL-6 level in patients with and without complications. The relationship between the serum IL-6 > 400 pg/ml and the incidence of post operative complications was also marked. These results suggested that circulating IL-6 is a clinically useful marker for the earliest detection and prediction of post -operative complications. R. J. Baigrie et al, 1992 5 5 the authors studied the systemic cytokine response to major surgical trauma in 20 patients undergoing elective aortic surgery and five pati ents after inguinal hernia repair. Tumour necrosis factor alpha and interferon gamma were not detected in these patients. An early and short-lived interleukin 1 beta (IL-1β) response to major surgery was detected only by intensive sampling in the perioperative period. The IL-1β peak preceded a more marked interleukin 6 response that peaked 4–48 h after surgery. IL-6 levels had fallen sharply by 48–72 h in all patients who had an uneventful postoperative course.

The IL-6 peaks were significantly lower after hernia surgery than after major aortic operations. IL -1β was not

15

detected in any samples. Three patients undergoing aortic surgery

developed

unexpected

major

postoperative

complications. IL-6 levels in this group were significantly higher than those of the other patients undergoing aortic surgery within 6–8 h of skin incision, and remained elevated for longer.

These rises in plasma IL-6 levels preceded the clinical onset of major complications by 12 –48 h. The systemic IL1β and IL-6 response to surgical trauma increased with the severity of the surgical insult. An early, exaggerated IL -6 response was associated with the subsequent clinical development of major complications. Brocks D.R. et al, 1993 7 Ketorolac is a new chiral nonsteroidal anti-inflammatory drug (NSAID) which is marketed for analgesia as the racemate. The drug is administered as the water soluble tromethamine salt and is available in tablets or as an intramuscular injection. The absorption of ketorolac is rapid, Cmax being attained between 20 to 60 min. Its oral bioavailability is estimated to range from 80 to 100%. The drug is extensively bound (> 99%) to plasma proteins and has a volume of distribution (0.1 to 0.3 L/kg) comparable with those of other NSAIDs.

Only small concentrations of ketorolac are detectable in umbilical vein blood after administration to women in

16

labour. The elimination half-life is between 4 and 6 hours and is moderate in comparison with other NSAIDs. The area under the plasma concentration -time curve of ketorolac is proportional to the dose after intramuscular administration of therapeutic doses to young healthy volunteers. Ketorolac is extensively metabolised through glucuronidation and oxidation. Little if any drug is eliminated unchanged. Most of the dose of ketorolac is recovered in the urine as conjugated drug.

Although ketorolac is excreted into the breast milk, the amount of drug transferred comp rises only a small fraction of the maternal exposure. Little stereoselectivity was present in the pharmacokinetics of ketorolac in a healthy volunteer receiving single intravenous or oral doses. The elderly exhibit reduced clearance of the drug. Renal insufficiency appears to cause an accumulation of ketorolac in plasma, although hepatic disease may not affect the pharmacokinetics. Roland Claude R. et al , 1993 5 7 the Kupffer cell (KC) is the resident hepatic macrophage, whose functions include local intrahepatic immune responses implicated in tolerance induction, participation in the septic state, and regulation of hepatic regeneration. The ability of the KC to participate in these biologically diverse functions is thought to be due to its release of pleiotropic cytokines, such as interleukin 6

17

(IL-6), which can act locally in a paracrine fashion or as hormones at distant sites. Many of the KC′s secretory responses are carefully regulated in an autocoid fashion by the eicosanoid prostaglandin E 2 (PGE 2 ). The degree of regulation depends on the particular cytokine and local environmental factors.

The authors have described a method for isolating KCs by their adherence to plastic and for testing their IL -6 and PGE 2 secretory responses to lipopolysaccharide. I n comparing

the

responses

of

KCs

from

normal

and

regenerating rat livers, the authors described an in vitro KC secretory pattern of eicosanoid inhibition of IL -6, whereas both responses to LPS are augmented in the KC during hepatic regeneration.

The authors concluded such an enhancement was found to be consistent with the shared putative supportive roles of IL-6 and PGE 2 in liver regeneration. Kiyoshi Sakamoto et al, 1994 3 8 the author investigated the effect of surgical trauma and other factors on post -operative elevation of serum IL-6 and examined changes in IL-6 concentrations after major thoracoabdominal surgeries. The serum IL-6 levels reached the maximum concentration on the first post-operative day in all 38 patients with peak ranging from 1400.8 pg/ml to 29.3 pg/ml among the six

18

groups who underwent surgery at different sites. The IL -6 peak was significantly correlated with the surgical trauma as defined by the operation length and the volume of blood loss during the surgery.

The peak concentration of serum IL-6 in patients undergoing esophagectomy was significantly higher than those

undergoing

concentration

pancreaticoduodenectomy.

observed

in

a

patient

who

Peak

IL -6

underwent

esophagectomy was 100 fold greater in fluid drained from thorax than in the peripheral blood. IL-6 mRNA could not be detected in leucocytes from thoracic and abdominal exudates at 6, 24, and 48 hours after surgery.

In contrast, IL-6 mRNA could not be detected in leucocytes from peripheral blood. Similar findings were also observed for IL-8. However IL-1β and TNF-α were detected only once after surgery in the drainage fluid.

These results indicate that IL-6 and IL-8 are induced and secreted in the field and subsequently enter the peripheral blood to induce cytokinemia. The operation length, volume of blood loss and thoracotomy are factors influencing the concentrations of cytokines in the blood. Kumar G.S. et al, 1994 4 1 cytokines, released by T cells, participate in inflammation and produce tissue injury.

19

Excess production of cytokines such as interleukins (ILs) and tumor necrosis factor (TNF) are believed to be involved in the pathobiology of conditions such as septicemia and septic shock, collagen vascular diseases, gl omerulonephritis etc. On the other hand, prostaglandins (PGs) are known to modulate

inflammation,

immune

response,

and

T -cell

response to antigens. But relatively little information is available on the effects of PGs and PG precursors on the release of cytokines.

Here the authors present data which suggests that PGs including thromboxane B2 (TXB2) and their precursors such as dihomo-gamma linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) can inhibit T cell proliferation and influence their ability to secrete IL-2, IL-4, IL-6 and TNF in vitro. These results may have relevance

to

inflammatory

the

use

conditions

of

PG-precursors

including

in

collagen

various vascular

diseases. Ueo H et al, 1994 6 3 it has been shown that IL-6 plays a central role in the acute phase of inflammation after surgical injury. The serum concentration of IL -6 increases during an operation. The authors have tried to determine the possibility of production of IL-6 at the operative wound site and its regulation by humoral factors in surgical patients. The IL-6 secretions of biopsied skin obtained from an

20

operative wound before and after the operation were quantified by using organ culture techniques.

The authors found out that when skin explants obtained from the uninjured skin were cultivated and the amounts of IL-6 secreted into the culture medium were measured, IL-6 secretion increased exponentially during culture, which indicated that the stress of the skin incision induced IL-6 production. The skin specimens obtained from the

operative

wounds

post-operatively

secreted

a

significantly larger amount of IL -6 than those obtained from

uninjured

skin

either

pre-operatively

or

post-

operatively, implying that skin at the site of the operative wound had been more sensitized to produce IL -6 because of the surgical injury. The IL-6 secretion by skin explants was significantly enhanced either by tumor necrosis factor or interleukin-1, while it was inhibited by corticosteroids.

They concluded that interleukin -6 production at the site of the operative wound is partly responsible for the elevation of the serum IL-6 level during the operation. Organ cultures of the skin explants may provide a fe asible system for research on the cytokine networks in surgical patients.

21

Ulrich Dendorfer et al, 1994 6 4 the goal of their study was to identify the regulatory elements which were involved in IL-6 gene activation by cAMP, prostaglandin and LPS.

They have compared the effects of PGs and their second messenger cyclic AMP with the effect of LPS on IL 6 gene expression. The authors demonstrate that secretion of IL-6 is induced by cAMP in murine monocytic PU5 -1.8 cells, even though to a lesser extent than LPS.

Nevertheless, cAMP and PGs of E series in the presence of theophylline induced transcription of the IL -6 promoter more strongly than LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations within four regulatory elements, namely, the multiple response element (MRE), AP -1, NF-IL6 and NFκB sites significantly reduced but did not completely abrogate, inducibility by cAMP and PGE 1 ,

whereas

alterations of four additional sites had no effects. LPS induced promoter activity was completely abolished by mutations in the NF-κB site, suggesting that a single regulatory element is crucial for inducibility by LPS.

Stimulation by cAMP correlated with the binding of inducible factors to AP-1, NF-IL6 and NF-κB elements, whereas

factors

binding

to

MRE

were

constitutively

expressed. Recombinant cAMP response element binding

22

protein did bind to MRE, indicating a potential role for this factor in the cAMP response.

In conclusion their results indicated that the activity of at least four transcriptional factors is simultaneously required to maximally induce IL-6 gene transcription upon stimulation with cAMP or LPS. Despite redundancy of regulatory sites, each of the sites appears to contribute a necessary signal to the transcriptional machinery, and different sites vary in their responsiveness to cAMP or LPS.

Signal

transmission

most

likely

occurs

through

protein-protein interactions, and it is becoming increasingly apparent

that

ATF/CREB,

the DNA binding proteins of Fos/Jun, C/EBP

and

Rel/NF-κB

families

contain

structural motifs involved in homo and heterodimerization necessary for cooperative protein -protein interactions. Joseph P. Portanova et al, 1996 2 9 the role of prostaglandin E 2 (PGE 2 ) in the development of inflammatory symptoms and cytokine production was evaluated in vivo using a neutralizing

anti-PGE 2

monoclonal

antibody

2B5.

In

carrageenan-induced paw inflammation, pre-treatment of rats with 2B5 substantially prevented the development of tissue edema and hyperalgesia in affec ted paws.

23

The antibody was shown to bind the majority of PGE 2 produced at the inflammatory site. Anti-PGE 2 treatment also reduced paw levels of IL-6 RNA and serum IL-6 protein without modifying tumor necrosis factor RNA levels in the same tissue. In each model, the anti-inflammatory efficacy of 2B5 was indistinguishable from that of the non steroidal

anti-inflammatory

drug

indomethacin,

which

blocked the production of all PGs.

These results indicate that PGE 2 plays a major role in tissue oedema, hyperalgesia, and IL-6 production at sites of inflammation and they suggest that selective pharmacologic modulation of PGE 2 synthesis or activity may provide a useful means of mitigating the symptoms of inflammatory disease. Roger M. Hinson et al, 1996 5 6 in this study the authors have shown that injected mineral oils such as pristine into peritoneal cavities of BALB/c mice resulting in chronic peritonitis was associated with high tissue levels of interleukin-6.Levels of both PGE 2 and IL-6 were elevated in inflammatory

exudates

from

pristine

treated

mice

as

compared to lavage samples from untreated mice. The COX-2 gene was found to be induced in the peritoneal macrophage fraction isolated from the mice.

24

A cause and effect relationship between increased macrophage PGE 2 and IL-6 production was also shown in vitro. When peritoneal macrophages were activated with an inflammatory stimulus (polymerized albumin), the COX-2 gene was induced and secretion of PGE 2 and IL-6 increased, with elevated PGE 2 appearing before IL-6. Treatment with 1µM indomethacin inhibited PGE 2 production by the cells and reduced induction of IL-6 mRNA but had no effect on COX-2 mRNA, which is consistent with the fact that the drug inhibits catalytic activity of cyclooxygenase but does not affect expression of the gene.

Addition of exogenous PGE 2 to macrophages induced IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression.

PGE 2

stimulated

IL-6

production

was

unaffected by addition of indomethacin. These results showed that indomethacin diminishes the elevation of IL-6 in pristine treated mice and PGE 2 can induce IL-6 production in vivo and implicate expression of the COX-2 gene in the regulation of this cytokine. Takuya Miyawaki et al, 1996 6 1 the purpose of their study was to evaluate the response of peripheral IL -6 associated with oral and maxillofacial surgery. Fifteen patients were taken as the sample size in which they studied the change in plasma IL-6 levels during and after surgery and the 25

correlation between plasma IL-6 and the surgical procedure performed.

They detected elevated IL-6 levels 2 to 6 hours after incisions and observed to reach a peak level within 6 hours after the end of the surgery. The results suggested that IL-6 would be released by surgical injury and elevation of plasma IL-6 level would be associated with magnitude of tissue damage in the oral and maxillofacial surgical procedure. Walter L. Biffl et al, 1996 6 6 the authors performed a review of current literature to find out the effects of IL -6 on

patients

in

an

attempt

to

clarify

the

potential

pathophysiologic role of IL-6 in response to injury.

They found out that IL-6 is a multifunctional cytokine expressed by a variety of cells after a multitude of stimuli, under the complex regulatory control mechanisms. The IL -6 response to injury was uniquely consistent and related to magnitude of the insult. Moreover the early post injury IL -6 response correlates with complications as well as mortality.

In conclusion they stipulated that IL-6 appeared to play an active role in the post injury immune response, making it an attractive therapeutic target in attempts to control hyper inflammatory provoked organ injury.

26

Wolfgang Scholz et al, 1996 6 9 IL-6 belongs to a group of helical cytokines that bind to a specific receptor to mediate biological activity. Human IL-6 is encoded by a 1.3 kilobase (kb) mRNA and the protein was predicted to be 184 amino acids with a calculated molecular weight of 21 kDa. It is now believed that the mature protein consists of 184-186 amino acids with an apparent molecular weight of 19-30 kDa due to post translational modifications including glycosylation and phosphorylation.IL-6 is produced by almost all cell types in response to a vari ety of different stimuli including LPS or cytokines such as IL -1.

The high affinity receptor for IL-6 consists of 2 chains, a 80 kDa protein (IL-6R) and a 130 kDa protein (gp130). IL-6 binds directly to the 80 kDa protein which is expressed on lymphoid and non-lymphoid cells, but not gp130.

The first 28 N-terminal amino acids of IL-6 are not important for function and the carboxyl terminus is mainly responsible for biological actions. However, as shown more recently two distinct parts in a new region pri marily comprising the loop between helix A and helix B are essential for receptor and signal transduction.

There is substantial evidence that IL -6 plays a role in the immune hemostasis in healthy individuals. It is readily

27

induced in response to a wide sp ectrum of stimuli, including cytokines that are produced in an immune response during viral and bacterial infections. Furthermore IL-6 might play a role as a stress response gene, leading to acute phase protein production. IL-6 is found in complexes with its soluble receptor and other proteins that clearly have that activity and soluble gp130 found in human serum was shown to modulate signaling through gp130.

A

substantial

overproduction

of

amount IL-6

of

data

occurs

demonstrates

either

that

experimentally

induced or in human diseases, in particular plasmacytoma s and myelomas. Thus IL-6 antagonists might be beneficial for patients with multiple myeloma. However anti IL -6 antibodies

treatments

have

limited

success

and

are

complicated, if not rendered useless , because of depot effect and the paradoxical increased circulating IL -6 levels following “neutralizing” antibody treatment.

Taking

advantage

of

the

regulatory

effect

for

thrombocyte differentiation IL-6 therapy has become a focus

of

interest

in

cancer

pa tients

who

underwent

chemotherapy and suffer from thrombocytopenia. Increased platelet counts and a dose related increase of acute phase protein was reported in phase I/I -II studies in cancer patients. Furthermore induction of IL -6Ra and release of soluble TNF-R observed in an earlier study might play a

28

part in the anti-inflammatory pathway of IL-6.In other conditions, such as inflammatory conditions a causative relationship has not been clearly established and IL -6 appears to be more of diagnostic value. I n some diseases with unknown etiology such as RA, IL-6 might well be the cause of symptoms that are hallmarks of the disease such as autoantibody production, while not being the cause of disease.

How vital is IL-6 for a normal functioning immune system? The answer is somewhat disappointing: IL -6 knockout mice are viable and fertile and show only “minor” immunological

defects

and

disease.

Two

important

messages are that a cytokine redundancy exists which can compensate for IL-6 in some inflammatory situatio ns and that at least some human B cell malignancies could benefit from treatments which inhibit B cell activity. Carlos A. Camargo et al, 1997 1 1 Interleukin-6 is an acute reactant cytokine with anti -inflammatory properties, which has been found to prevent injury in a model of acute hepatitis in mice through down regulation of tumor necrosis factor alpha. In this study the authors investigated the role of

IL-6

in

rodent

models

of

hepatic

warm

ischemia/reperfusion (WI/Rp) injury. IL -6 deficient (-/-) were

subjected

to

hepatic

recombinant IL-6(rIL-6).

29

WI

and

compared

with

The effects of rIL-6 following various periods of ischemia were further studied in models of hepatic ischemia in rats. IL-6 -/- mice had increased reperfusion injury as assessed by transaminase levels and a tissue necrosis scoring system when compared with controls, an effect prevented by pretreatment with rIL-6. Similarly, rats pretreated with rIL-6 had reduced reperfusion injury and better survival than controls in each respective WI group. Tissue TNF-α expression measured by Northern Blot analysis and serum C-reactive protein (CRP) levels, a marker of inflammation, were significantly reduced in animals pretreated with rIL-6. Administration of antibodies to

TNF-α

reproduced

the beneficial

effect

of

r IL-6.

Hepatocyte infiltration, as assessed by a scoring method for mitotic

index

and

proliferating

nuclear

cell

antigen

staining, was markedly increased in rIL -6 treated rats when compared with controls.

In conclusion, this study suggested that IL -6 could play

an

important

role

in

limiting

hepatic

warm

ischemia/reperfusion(WI/Rp) injury, probably through its anti-inflammatory properties, modulation of TNF -α and/or promotion of liver regeneration. rIL -6 might become an important cytokine in clinical situat ions associated with WI/Rp injury.

30

Claude Martin et al, 1997 1 3 the objective of the study was to

compare

the

patterns

of

evolution

of

two

proinflammatory cytokines (TNF-α and IL-6) in two major clinical entities associated with systemic inflammatory response: septic shock and multiple traumas (with and without hemorrhagic shock).

Twenty five non trauma patients with septic shock and 60 multiple trauma patients formed the study group on which the study was undertaken. Serial blood samples were collected in each patient for determination of serum cytokine concentrations. Samples were obtained over 7 days in septic shock patients and 11 days in trauma patients. High concentrations of circulating TNF -α and IL-6 were found in patients with septic shock. High IL -6 concentrations,

but

normal

TNF -α

concentration

were

detected in trauma patients. TNF -alpha concentrations were higher in non-survivor septic shock than in non-survivor trauma patients. During the whole study period, non survivor

shock

concentrations

patients

than

maintained

non-survivor

higher

trauma

TNF -α

patients.

In

survivors in both groups, normal values for TNF -α were detected during the whole study pe riod.

IL-6 concentrations were significantly higher in non survivor septic shock patients than in non -survivor trauma

31

patients. During the whole study period, much higher concentrations of IL-6 were detected in septic shock patients

than

in

trauma

patien ts.

In

survivors,

IL-6

concentrations were much higher in septic shock patients than in trauma patients. In septic shock patients, changes in both TNF-α and IL-6 were correlated with outcome, higher values being found in patients likely to die. Neither TNF -α nor IL-6 values were of any significance in predicting the outcome of trauma patients.

In the conclusion it was postulated that in septic shock patients, high amounts of circulating T NF-α and IL-6 are found and they correlated with fatal outcome. In trauma patients much less increased concentrations of IL -6 are detected while normal TNF -α circulating concentrations are measured. In these patients, cytokine concentrations do not correlate with outcome. This finding suggests a much higher degree of activation of the immune inflammatory cascade in septic shock than in multiple trauma pat ients. Increased IL-6 values were also an indicator of the development of nosocomial infection in trauma patients. Decker D et al, 1997 1 6 authors

examined

the

in this prospective study the C-reactive

protein

level,

the

production of the cytokines IL-6, IL-8 and IL-1 RA in 25 laparoscopic and 21 conventional cholecystectomies. In addition the cell surface markers CD25 and CD30 on

32

different cell populations and HLA-DR on monocytes were measured.

They found out that the humoral markers showed a more

distinct

increase

in

patients

operated

on

conventionally at two and 24 hours after surgery. Th e cell surface markers CD25 and CD30 showed the same reaction. The HLA-DR expression on monocytes was found to be significantly lower in patients operated on conventionally.

In conclusion it was apparent that elective surgical approaches cause changes in the immune system, which can be evaluated by the reaction of cytokines and cell surface markers. activation

Laparoscopic of

the

cholecystectomies

immune

system

than

cause d

less

conventional

operations. Mark T.R. et al, 1997 43 their study compared the effect of oral administration of NSAID flurbiprofen, to placebo, on tissue levels of immunoreactive prostaglandin E 2 (iPGE 2 ), leukotriene B 4 (iLTB 4 ) and (S)-flurbiprofen within the surgical wound using implanted microdialysis probes in the dental impaction pain model.

They

recruited

24

healthy

patients

in

need

of

extraction of partial to complete bony mandibular third molars for a randomized, double-blind, placebo controlled

33

study. Immediately following completion of surgery, two semi-permeable microdialysis probes were implanted into each mandibular surgical site. The patients were then taken to the recovery room and microdialysis samples and patient pain reports (VAS) were collected at 30 min intervals for 4 hrs. Patients randomly received either flurbiprofen (200mg orally) or placebo at the onset of post -operative pain. Diasylate samples were collected, frozen and later assayed for iPGE 2 , iLTB 4 , and (S)-flurbiprofen levels. The results of this study showed that flurbiprofen decreased

post-operative

compared

to

the

pain

placebo

by

treated

approximately patients.

It

70% also

significantly reduced peak tissue levels of iPGE 2 (9.2+2.6 vs. 0.4+0.86 nM), without having a significant effect on peak tissue levels of iLTB 4 (2.5+1.4 vs. 1.9+0.86 nM) compared to placebo treatment. Levels of (S)-flurbiprofen significantly increased within the surgical wound exceeding therapeutic levels by 60 min after administration.

The data obtained from this study revealed that NSAIDs selectively alter eicosanoid levels within the surgical

wound

and

evoke

analgesia

at

time

points

coincident with elevated wound levels of the drug. Takuya Miyamaki et al, 1998 6 2

they did a study

comprising of 10 patients undergoing elective oral and maxillofacial surgical procedures in which they inv estigated

34

whether there were any changes in plasma cytokine level s of IL-6 , IL -1 beta and TNF-α involved in post-operative fever. Plasma cytokine levels were investigated using Enzyme Linked Immunosorbent Assay and also measured the core temperature and peripheral vasoconstriction after the surgery.

The relationships between the changes in plasma cytokine levels and post-operative fever were statistically evaluated using Spearman’s rank correlation coefficients. The results of the study was tha t elevated plasma IL-6 levels were found significantly co relating with the increase in core temperature after surgery and with the degree of post-operative shivering and vasoconstriction, whereas the changes in plasma IL-1 beta or TNF-α levels were not.

Thereby the authors concluded that elevation of plasma IL-6 levels was probably involved in post-operative fever following oral and maxillofacial surgical procedures. Dimitris A. Papanicolaou et al, 1998 1 7 Interleukin-6, an inflammatory cytokine is characterized by pleiotropy and redundancy of action. Apart from its hematologic, immune, and hepatic effects it has many endocrine and metabolic actions.

Specifically

it

is

a

potent

stimulator

of

hypothalamic-pituitary-adrenal axis and is under the tonic negative control of glucocorticoids. It acutely stimulates

35

the

secretion

of

growth

hormone,

inhibits

thyroid

stimulating hormone secretion, and decreases serum lipid concentrations. Furthermore it is secreted during stress and is positively controlled by catecolamines. Administration of interleukin-6 results in fever, anorexia and fatigue.

Elevated levels of circulating IL-6 have been seen in the

steroid

withdrawal

syndrome

and

in

the

severe

inflammatory, infectious and traumatic states pot entially associated with the inappropriate secretion of vasopressin. Levels of circulating IL-6 are also elevated in several inflammatory diseases such as rheumatoid arthritis. IL -6 is negatively controlled by estrogens and androgens and it plays a central role in the pathogenesis of osteoporosis seen in conditions characterized by increased bone resorption, such as sex-steroid deficiency and hyperparathyroidism.

Overproduction of IL-6 may contribute illness during aging

and

chronic

stress.

Finally

admini stration

of

recombinant human interleukin -6 may serve as a stimulation test for the integrity of the hypothalamic -pituitary-adrenal axis. Panagiotis V. Giannoudis et al, 1998 4 9 the authors studied 31 blunt trauma victims, Injury Severity Score (ISS) mean 14 (9-57), for the pattern of release of C -reactive protein (CRP) and cytokine interleukin -6 (IL-6). Blood samples

36

were taken on admission (within 6 hours of injury), as well as at 24 hours, and 3, 5 and 7 days. Serum CRP and IL-6 were measured by ELISA. Subsequent surgical events and sepsis were noted.

Serum IL-6 levels on admission were considerably higher (median 135 pg /mL) than the laboratory reference range (< 5 pg/ mL), slowly returning towards reference values during the study. Serum CRP levels wer e similar to laboratory normal values on admission (median 8.5 mg/ L vs. 7.5 mg/ L), reaching peak values (median 110 mg/ L) after 3 days. There was a correlation between IL -6 release and ISS but not between CRP and ISS. Patients undergoing surgery showed further increases in IL-6 and CRP levels post-operatively. Of 24 surgical patients, 9 developed postoperative sepsis.

In conclusion in patients with blunt trauma, early assessment of the markers CRP or IL-6 were not useful for the diagnosis of sepsis. Levels of CRP following accidental or surgical trauma should be assessed with caution. Vane J.R. et al, 1998 6 5 salicylic acid and salicylates, obtained from natural sources, have long been used as medicaments. Salicylic acid was chemically synthesized in 1860 and was used as an antiseptic, an antipyretic, and an antirheumatic. Almost 40 years later, aspirin was developed

37

as a more palatable form of salicylate. Soon after, other drugs having similar actions to aspirin were discovered, and the

group

was

termed

the

"aspirin-like

drugs"

the

nonsteroidal anti-inflammatory drugs [NSAIDs]. Twentyfive years ago, it was proposed that the mechanism of action

of

NSAIDs

was

through

their

inhibition

of

prostaglandin biosynthesis. Since then, there has been general acceptance of the concept that these drugs work by inhibition of the enzyme cyclooxygenase (COX), which we now know to have at least two distinct isoforms: the constitutive isoform, COX-1, and the inducible isoform, COX-2.

COX-1

has

clear

physiologic

functions.

Its

activation leads, to the production of prostacyclin, which when released by the endothelium is antithrombogenic and when released by the gastric mucosa is cytoprotective.

COX-2, discovered

6

years

ago, is

induced by

inflammatory stimuli and cytokine s in migratory and other cells. It is therefore attractive to suggest that the anti inflammatory actions of NSAIDs are due to inhibition of COX-2,

whereas

the

unwanted

side-effects,

such

as

irritation of the stomach lining, are due to inhibition of COX-1. Drugs that have the highest COX-2 activity and a more favorable COX-2:COX-1 activity ratio will have a potent anti-inflammatory activity with fewer side -effects than drugs with a less favorable COX -2:COX-1 activity

38

ratio. The identification of selective in hibitors of COX-2 will therefore lead to advances in therapy. Zhou Xing et al, 1998 73

in this study, the role of IL-6 in

acute inflammatory responses were investigated in animal models of endotoxic lung or endotoxemia by using IL-6+/+ IL-6-/- mice. Aerosol exposure of endotoxin induced increased IL-6 and proinflammatory cytokines TNF -α and MIP-2 and a neutrophilic response in the lung of IL-6+/+ mice. However, the levels of TNF-α and MIP-2 and neutrophilia were significantly higher in the lung of IL -6-/mice. The rate of neutrophil apoptosis in the se mice was similar to that of IL-6 +/+ mice. A low constitutive level of anti-inflammatory cytokine IL-10 was not enhanced by endotoxin and remained similar in the lung in both IL -6 -/mice.

Systemically , intraperitoneal delivery of endotoxin resulted in much more pronounce circulating levels of TNF α , MIP-2 , GM–CSF , and IFNγ in IL-6-/- mice than IL6+/+ mice and the administration of recombinant IL -6 to IL-6 -/- mice abolished

these differences. In con trast,

circulating IL-10 levels were induced to a similar degree in both IL-6+/+ and IL-6 -/- mice.

Thus, their studies revealed that endogenous IL-6 plays a crucial anti-inflammatory role in both local and

39

systemic acute inflammatory responses by contro lling the level of proinflammatory , but not the anti -inflammatory , cytokines and that these anti -inflammatory activities by IL6 cannot be compensated for by IL-10 or other IL-6 family members. Mauro Bianchi et al, 1999 4 4 they examined the ability of the analgesic drug tramadol to affect the development of inflammation

in

rats.

They

found

out

that

acute

administration of tramadol significantly reduced the edema and the hyperalgesia induced by yeast injection in the paw.

In subcutaneous carrageenan induced inflammation, tramadol not only reduced the amount of the exudate, but also reduced the prostaglandin E 2 like bio and immune activity in it. The leukotriene B 4 concentrations were unchanged in the exudate. However they also found out that tramadol did not affect the ability of the macrophages to migrate

towards

the

chemotactic

peptide

N-formyl-L-

methionile-L-leucly-L-phenylalanine (FMLP).

Their results suggested that tramadol is able to inhibit the development of different types of inflammation in the rat without affecting immune mechanisms and contribute to explain the efficacy of this drug in the treatment of inflammatory pain.

40

Buccellati C. et al, 2000 8 the authors evaluated the putative activity of tramadol to suppress prostaglandin endoperoxide synthase

(PGHS-1)

and

prostaglandin

endoperoxide

synthase-2 (PGHS-2) activities in human whole blood on vitro. Platelet thromboxane B 2 (TXB 2 ) production and monocyte PGE 2 production in LPS stimulated blood were also

measured

in

samples

incubated

with

diffe rent

concentrations (300ng/ml, 3micr og/ml, and 30microg/ml) of tramadol or its enantiomers. Neither tramadol nor its enantiomers inhibited the formation of arachidonic acid metabolites.

These results suggested that the anti -inflammatory effect of tramadol demonstrated in some models is not related to a direct inhibitory effect on the formation of prostanoids. Florian Gebhard et al, 2000 2 1 the authors designed a prospective study starting as early as at the scene of the unintentional injury to determine t he trauma-related release of plasma IL-6 in multiple injured patients. They enrolled 94 patients with different injuries following trauma. (Injury Severity Score (ISS) median, 19 , Range, 3-75).

They collected the first blood sample at the scene of the

unintentional

injury

before

cardiopulmonary

resuscitation, when appropriate. Then, blood samples were

41

collected in hourly to daily intervals. Interleukin 6 plasma levels were determined using a commercial enzyme -linked immunosorbent assay test. The short-term acute phase protein, C-reactive protein, was measured to characterize the extent of trauma and to relate these results to IL-6 release. They found out, elevated IL-6 plasma levels as early as immediately after the trauma had occurred. This phenomenon was pronounced in patients with major trauma (ISS>32).

Patients

with

minor

injury

had

elevated

concentrations as well but to a far lesser extent. In surviving patients, IL-6 release correlated with the ISS values best during the first 6 hours after hospital admission. All patients revealed increased C-reactive protein levels within 12 hours following trauma, reflecting the individual injury severity. This was most pronounced in patients with the most severe (ISS >32) trauma.

The

results

revealed

an

early

inc rease

of

IL-6

immediately after trauma. Moreover, patients with the most severe injuries had the highest IL-6 plasma levels. There is strong evidence that systemic IL-6 plasma concentrations correlated with ISS values at hospital admission. Therefore, IL-6 release can be used to evaluate the impact of injury early regardless of the injury pattern. J. Riese et al, 2000 3 2 the author suggested surgical trauma is followed both by a transient increase of interleukin 6 (IL -

42

6) concentrations in the serum and impa ired function of circulating leukocytes. They investigated the relationship of IL-6 concentrations in the serum with lipopolysaccharide (LPS) stimulated production peri-operatively in whole blood of patients undergoing elective major abdominal surgery.

The authors found out that in 50 patients there was a transient increase in IL-6 concentrations in the serum. Six hours after the incision, the in vitro stimulated production of IL-6 concentrations and TNF-α was diminished by 72%. A significant increase in cytokine production was observed three days postoperatively which was found to be 63% below

the

pre-operative

values.

Patients

with

h igh

concentrations of circulating IL-6 showed a significantly lower stimulated IL-6 production than patients with low serum concentrations.

In conclusion the authors hypothesized that two opposing

effects

are

associated

with

surgery

i.e.

an

activation leading to IL-6 release into the circulation and a prolonged hypo responsiveness of circulating leukocytes. Jeffery L. Ebersole et al, 2000 2 8 the acute phase reaction represents an early and highly complex reaction of the organism to a variety of injuries s uch as bacterial, viral or parasitic infection, mechanical or thermal trauma, ischemic

43

necrosis or malignant growth. It refers to the physiological and metabolic alterations that ensue immediately after inset of infection or tissue injury.

A variety of changes in the organism act in concert to neutralize the inflammatory agent and foster healing of damaged tissues. In contrast with the specificity of cellular and humoral immunity, the acute phase changes are non specific and occur in response to many con ditions. The purpose of these responses is to restore homeostasis and to remove the cause of its disturbance.

The author reviewed the various steps and molecular mechanisms of acute phase response and also the role of acute phase cytokines in the acute p hase reaction. Gerd Dannhardt et al, 2001 2 4 prostaglandins are formed from arachidonic acid by the action of cyclooxygenase and subsequent downstream synthetases. Two closely related forms of the cyclooxygenase have been identified which are now known as COX-1 and COX-2. Both isoenzymes transform arachidonic acid to prostaglandins, but differ in their distribution and their physiological roles. Meanwhile, the responsible genes and their regulation have been clarified. COX-1, the pre-dominantly constitutive form of the enzyme, is expressed throughout the body and performs a number of homeostatic functions such as maintaining

44

normal gastric mucosa and influencing renal blood flow and platelet aggregation.

In contrast, the inducible form is expressed in response to inflammatory and other physiological stimuli and growth factors, and is involved in the production of the prostaglandins

that

mediate

pain

and

support

the

inflammatory process. All the classic NSAIDs inhibit both COX-1 and COX-2 at standard anti-inflammatory doses. The beneficial anti-inflammatory and analgesic effects are based on the inhibition of COX-2, but the gastrointestinal toxicity and the mild bleeding diathesis are a result of the concurrent inhibition of COX-1.

Agents that inhibit COX-2 while sparing COX-1 represent a new attractive therapeutic development and could represent a major advance in the treatment of rheumatoid arthritis and osteoarthritis. Apart from its involvement in inflammatory processes, COX -2 seems to play a role in angiogenesis, colon cancer and Alzheimer’s disease, based on the fact that it is expressed during these diseases. The benefits of specific and selective COX -2 inhibitors are currently under discussion and offer a new perspective for a further use of COX -2 inhibitors. Myung Hee Kim et al, 2001 4 7 performed a prospective study comprising of 22 patients who underwent abdominal

45

hysterectomy. The purpose of this study was to determine the

differences

between

morphine

patient

controlled

analgesia and a combination o f morphine and ketorolac in IL-6 and IL-10 responses and in analgesia and morphine related side effects. They divided the patients into two groups a) patient – controlled analgesia with morphine b) patient controlled analgesia with a combination of morphi ne and ketorolac. Blood samples to measure cytokines were collected at pre operatively, immediately post operatively and 2 hours, 4 hours and 24 hours postoperatively.

The authors found out that in two groups, IL -6 increased immediately postoperatively a nd its concentration remained consistent till 24 hours. IL-10 peaked at 2 hours post

operatively

and

progressively

decreased.

Total

analgesic use was not different, but morphine consumption was significantly different.

They

concluded

ketorolac

plus

cytokine

response

that

the

administration and

may

supplementa tion of

morphine

contribute

to

using

modifies immune

augmentation during post-operative periods. Simon A. Jones et al, 2001 6 0 Interleukin 6 (IL-6) performs a prominent role during disease and has bee n described as

46

both a pro- and anti-inflammatory cytokine. A key feature in

the

regulation

of

IL-6

responses

has

been

the

identification of a soluble interleukin 6 receptor (sIL -6R), which forms a ligand–receptor complex with IL-6 that is capable of stimulating a variety of cellular responses including proliferation, differentiation and activation of inflammatory processes. Elevated sIL -6R levels have been documented in numerous clinical conditions indicating that its production is coordinated as part of a disease response. Thus, sIL-6R has the potential to regulate both local and systemic IL-6-mediated events.

This review by the authors outlined the central role of sIL-6R in the coordination of IL-6 responses. Details relating to the mechanisms of sIL-6R production were provided, and the potential significance of sIL-6R during the development of clinical conditions was emphasized.

The authors want to convey, therefore, that when thinking about the inflammatory capability of IL-6, it is essential to consider not only the action of IL-6 itself, but also the effect sIL-6R may have on cellular processes. A.M. Mahdy et al, 2002 4 in a randomized, double – blind placebo controlled study the authors investigated the effect of diclofenac in patients undergoing majo r surgery. 24 patients were randomized to receive either diclofenac 50 mg

47

orally every 8 hrs the day before surgery and 75 mg i.m. every 12 hrs on the day of surgery or placebo.

They measured serum IL-6, IL-10 and cortisol before surgery and 30 min, 2, 6, 12, 24 hrs after giving skin incision along with temperature, leucocyte count and C – reactive protein concentration .

They found

out

that

IL-6,

IL-10

concentrations

increased after surgery reaching peak levels at 12 hours and 6 hours respectively. At 12 hours, the IL-6 concentrations were significantly lower in patients receiving diclofenac than

in

those

receiving

placebo.

In

contrast,

IL -10

concentration at 6 hours was higher in diclofenac treated patients and was associated with less pyrexia, a lower leucocyte

count

and

a

lower

C-reactive

protein

concentration. Serum cortisol concentration was found to be similar in the two groups of patients until 24 hours and then it was lower in patients who had received diclofenac. Cortisol concentration correlated w ith IL-6 concentration at 24 hours.

The authors concluded that diclofenac was associated with

lower

IL-6

concentration

and

higher

IL-10

concentration and lower leucocyte count, C -reactive protein concentrations and temperature. Hence they concluded that

48

diclofenac may have an anti-inflammatory role in major surgery. Daniel G. Remick et al, 2002 1 4 the authors sought to determine whether IL-6 measured 6 hours after surgery could predict the outcome of excessive inflammation resulting from experimental sepsi s.

79 Adult female BALB/c mice were subjected to cecal ligation and puncture with a 21 gauge needle and treated with imipenem in D5W every 12 hours for 5 days, resulting in a homogenous population at the outset. Six hours after surgery, 20 µl of blood was obtained from tail vein to measure IL-6. Mortality was followed for 21 days. Overall 3 day survival as 77% and 21 day mortality was 56%. Plasma levels > 2000 pg/ml was determined to predict mortality within the first 3 days with a sensitivity of 58% and specificity of 97%. To further refine the mortality prediction, body weight and a complete blood count were performed 24 hours after cecal ligation and puncture.

Discriminate

analysis

indicated

that

a

weighted

formula combining body mass, lymphocyte and platelet count would predict death with a sensitivity of 83% and a specificity of 79%. The authors tested the value of the IL-6 prediction by surgically resecting the cecum in those animals with IL-6 > 2000 pg/ml, which resulted in a

49

significant

improvement

in

survival.

These

data

demonstrate that IL-6 measured 6 hours after injury accurately predicts mortality resulting from experimental sepsis.

This measurement may be determined quickly so that therapy may be targeted only to those individuals at significant risk of dying and initiated within sufficient time to be effective. Kuniyasu Soda et al, 2003 4 0 the authors investigated the effect of IL-6 increase on the rate of morbidity and mortality or if IL-6 is just a bystander that only indicates the severity of the injury.

They investigated the effect of IL-6 on a multibacterial infection, which is one of the most common post injury complications. CDFI male mice were administered recombinant human IL-6 (hIL-6) continuously at a dose of 0, 1, or 10 µg/day. The mice then underwent cecal ligation without

puncture

that

induced

slow

multi -bacterial

infection. The survival rate of mice receiving 10 µg/day of hIL-6 was significantly lower (38.5%) than the rate of those receiving 0 (83.3) or 1 (92.3) µg/day of hIL-6.

The results of this study showed that only excessive increases in serum IL-6 to levels that were observed among

50

patients who underwent severe injury or extensive surgery with high incidence of post-injury infection, jeopardized the hosts defence against bacterial infection. K. Sakamoto et al, 2003 3 8 the authors wanted to investigate the alternations in post-operative levels of IL-6 and soluble IL-6 receptor (sIL-6R). They examined the levels of IL-6 and sIL-6R in serum and drainage fluids from patie nts who underwent thoracoabdominal surgery.

Serum IL-6 levels reached maximum within the first post-operative and decreased thereafter. The IL -6 levels were significantly correlated with surgical trauma as defined by the operation time and volume of bloo d loss during operation. The IL-6 levels in the drainage fluid were much higher than in the serum in the early post -operative phase. IL-6 mRNA was detected in leukocytes from drainage fluid, but not from the peripheral blood. A large quantity of sIL-6R concentrations in the drainage fluid was found significantly lower than in serum in the early post operative phase.

Based on the above results the authors proposed that IL-6 is produced in the operative field and enters the peripheral blood stream to induce elevation of serum IL-6. The levels of IL-6 were influenced by surgical trauma. sIL6 was being constantly produced in areas other than the

51

operative

field,

while

sIL-6R

level

was

reduced

by

consumption in the operative field.

Hence supplement

they

concluded

mechanism

to

that cope

t here with

exists

sIL-6R

surgical

stress,

together with its ligand IL-6. Raf F. De Jongh et al, 2003 5 4 surgeries are followed by acute phase response, including hypothalamo-pituitaryadrenal (HPA) - axis activation and fever. Considering its physiological properties and its behaviour in plasma after stress and surgery, the pro-inflammatory cytokine IL-6 is a putative

candidate

symptoms.

in

However,

eliciting

evidence

these for

this

stress

related

hypothesis

is

lacking.

Rats subjected to individual psychological stress for 1 hour were injected intraperitoneally with saline or 3.33 micro gram per 100 gm rat neutralizing antibodies against rat

IL-6.

Thereafter

the

single

housed

rats

were

anaesthetized for 25 minutes, with or witho ut laparotomy. Intermittently oesophageal temperatures were measured at defined time points. A parallel group of rats undergoing the same study were decapitated, at time points when body temperature differed, to obtain blood for measurement of plasma

adrenocorticopic

hormone

and

corticosterone.

Individual housing resulted in hyperthermia. Anti -bodies

52

against IL-6 accelerated accentuated hyperthermia after anaesthesia alone. Anti-body administration was not able to significantly influence the plasma hormone levels during any experiment.

The

present

thermoregulatory

study

indicates

factor

that

during

IL-6

is

a

psychological,

anaesthesiological and surgical stress, but the cytokine does not participate in HPA – axis activation until 6h after anaesthesia or surgery. A dose finding study wit h antibodies against IL-6 is required to further identify the degree

of

contribution

of

IL-6

to

perioperative

thermoregulation. Ground S. et al, 2004 2 5 Tramadol, a centrally acting analgesic is structurally related to codei ne and morphine and consists of two enantiomers, both of which contribute to

analgesic

activity

via

different

mechanisms.

(+) -

Tramadol and the metabolite (+) -O-desmethyl-tramadol (M1) are agonists of the µ opioid receptor. (+) -Tramadol inhibits

serotonin

reuptake

and

(-)-tramadol

inhibits

norepinephrine reuptake, enhancing inhibitory effects on pain transmission in the spinal cord.

The complementary and synergistic actions of the two enantiomers tolerability

improved profile

of

the the

53

analgesic racemate.

efficacy

and

After

oral

administration, tramadol was rapidly and almost completely absorbed.

Sustained-release

tablets

release

ingredient

over

of

hours,

a

period

12

the

active

reach

peak

concentrations after 4.9 hours and have a bioavailability of 87-95% compared with capsules. Tramadol is rapidly distributed in the body. Plasma protein binding is about 20%. Tramadol is mainly metabolised by O - and Ndemethylation

and

by

conjugation

reactions

forming

glucuronides and sulphates. Tramadol and its metabolites are mainly excreted via the kidneys. The mean elimination half-life is about 6 hours. The O-demethylation of tramadol to M1, the main analgesic effective metabolite, is catalysed by cytochrome P450 (CYP) 2D6, whereas N -demethylation to M2 is catalysed by CYP2B6 and CYP3A4.

The wide variability in the pharmacokinetic properties of tramadol can partly be ascribed to CYP polymorphism. O- and N-demethylation of tramadol as well as renal elimination

are

stereoselective.

Pharmacokinetic -

pharmacodynamic characterisation of tramadol is difficult because of differences between tramadol concentrations in plasma

and

at

the

site

of

action,

and

because

of

pharmacodynamic interactions between the two enantiomers of tramadol and its active metabolites. The analgesic potency of tramadol is about 10% of that of morphine following parenteral administration.

54

Tramadol

provides

postoperative

pain

relief

comparable with that of pethidine, and the analgesic efficacy

of

tramadol

can

further

be

improved

by

combination with a non-opioid analgesic. Tramadol may prove particularly useful in patients with a risk of poor cardiopulmonary function, after surgery of the thorax or upper

abdomen

and

when

non-opioid

analgesics

are

contraindicated.

Tramadol is an effective and well tolerat ed agent to reduce pain resulting from trauma, renal or biliary colic and labour, and also for the management of chronic pain of malignant or non-malignant origin, particularly neuropathic pain. Tramadol appears to produce less constipation and dependence than equianalgesic doses of strong opioids. John R. Zuniga et al, 2004 3 1 the authors conducted a 3 arm parallel , single blind , placebo -controlled , randomized study to compare the analgesic efficacy and tolerability of a single

dose

of

100mg

diclofenac

potassium,

100mg

diclofenac soft gels and placebo in 75 patients experiencing severe post-operative pain after third molar extraction surgery.

The

patients

received

a

single

dose

of

study

medication and analgesic efficacy measures included the time to meaningful pain relief measured using a stopwatch

55

and time to rescue medication. Pain relief (PR) and Pain Intensity (PI) ratings were recorded at 0.25, 0.5, 0.75,1, 1.5,2, 3, 4,5,6,8 and 24 hours post dosing. Summary analgesic measures, including Summed Pai n Relief Score (TOTPAR) and Summed Pain Intensity Differences (SPID), were calculated from the 0.25 to 6 hour response. The time between pain relief and rescue and a global evaluation for the effectiveness of the study medications were recorded at the end of the study. They also scheduled blood samples from each patient for determining the plasma concentrations of diclofenac anion. They found out that both diclofenac sodium soft gels and tablet forms were more effective than placebo.

The average overall pain relief was substantially better from diclofenac softgels than

tablet

form but

difference was not statistically significant. In patients taking diclofenac sodium softgel, 50% of the patients experienced time to onset of analgesic activity within 18 minutes and the median analgesic duration was 5 hours (302 minutes). Fifty percent of the patients taking tablet form had time to onset of action within 38 minutes, and the median duration of analgesia was 4.5 hours (272 minutes).

In conclusion, soft gelatin capsules of diclofenac sodium formulated using Prosorb technology demonstrated more rapid and possible more complete absorption from the

56

GI tract than an equivalent dosage in a conventional oral formulation. Diclofenac sodium was well tolerated in the study. This study suggested that diclofenac sodium soft gel is a safe and efficacious analgesic for acute post -operative dental pain. K.S.Ong et al, 2004 35 in the present study the authors evaluated the efficacy of preemptive ketorolac in a cross over design in patients undergoing bilateral mandibular third molar surgery. They conducted a double blind, randomized, placebo-controlled study where 34 patients had each of their identical impacted mandibular third molars removed under local anaesthesia on two occ asions.

Each patient acted as their own control with one side pre-treated with intravenous ketorolac 30 mg before surgery followed by placebo injection after surgery and for the other side, the patient was given placebo injection before surgery and post treated with intravenous ketorolac 30 mg after surgery. The difference in post-operative pain between pre-treated and post treated side in each patient was then assessed by four primary end-points i.e. pain intensity as measured by 100mm visual analogue sc ale hourly for 12 hours, time to rescue analgesic , post -operative analgesic consumption , and patients global assessment.

57

Through the 12 hour investigative period, patients reported significantly lower pain intensity scores in the ketorolac pretreated sides when compared with the post treated sides. Patients also reported a significantly longer time to rescue analgesic, lesser post -operative analgesic consumption and better global assessment for the ketorolac pretreated sides. Pretreatment with intravenous ketorolac has a preemptive effect for post -operative third molar surgery and extended the analgesia by approximately 2 hours. K.S. Ong et al, 2004 36 the objective of this study was to compare analgesic efficacy of a single dose of preoperative intravenous tramadol versus ketorolac in preventing pain after third molar surgery. Sixty four patients undergoing elective third molar surgery were randomly assigned into one of the two groups with 32 patients in each group.

Group 1 received tramadol 50 mg a nd group 2 received ketorolac 30 mg intravenously preoperatively before surgery. After injection of the study drugs, a standard intravenous sedation technique was administered and the impacted third molars were removed under local anaesthetic. The difference in post-operative pain was assessed by four primary end points: pain intensity as measured by a 100 mm visual analogue scale hourly for 12 hours, median time to rescue analgesic, post-operative

58

acetaminophen

consumption,

and

patient’s

global

assessment.

Throughout the 12 hour investigative period, patients reported significantly lower pain intensity scores in the ketorolac versus tramadol group. Patients also reported significantly longer median time to rescue analgesic, lesser post-operative

acetaminophen

consumption

and

better

global assessment for the ketorolac versus tramadol group.

They

concluded

that

pre-operative

intravenous

ketorolac 30 mg is more effective than tramadol 50 mg in the prevention of postoperative dental pain. Babatunde O.B. et al, 2005 6 the authors conducted a prospective randomized double blind study with a total of 100 patients divided randomly into two groups

of

dexamethasone (prophylactic 8mg and postoperative 4 mg IV) and diclofenac K (50mg oral before and after surgery ) and diclofenac K alone. They assessed the overall analgesic efficacy of the drug combinations postoperatively by determination of pain intensity using a category rating scale. They measured facial swelling using a tape measure placed from tragus to gonion to tragus and interincisal mouth

opening

using

a

vernier

postoperatively.

59

calliper

pre -

and

They

found

out

that

the

co-administration

of

dexamethasone and diclofenac K was significantly superior to diclofenac alone for the relief of pain (p3000pg/ml. Both IL-KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 hours. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 hours of severe sepsis routinely observed in the WT mice.

These data demonstrate that IL-6 serves as a marker of disease

severity

in

sepsis

and

does

modulate

some

physiologic responses, but complete lack of IL-6 does not alter mortality due to sepsis. Kenichi Miyaoka et al, 2005 3 4 in this prospective study the authors Response

assessed

the

Syndrome

risk

of

(SIRS)

Systemic and

Inflammatory

Multiple

Organ

Dysfunction Syndrome (MODS) after orthognathic surgery by

measuring

the

circulation

levels

of

inflammatory

cytokines such as IL-6, IL-10, CRP, neutrophil count and neutrophil function from 21 patients with mandibular

62

prognathism at 2 days before and at 1 and 3 days after orthognathic

surgery.

The

neutrophil

function

was

estimated by superoxide production and elastase release under the stimulation of FMLP.

8 of 21 patients were applicable to SIRS criteria 1 day post operatively and all of the subjects were excluded from SIRS criteria 3 days post operatively. Even though IL -6 and IL-10 levels were raised 1 day post operatively, increased cytokine concentrations were decreased in most patients. The IL-6 concentrations and the ratio of IL-6 and IL-10 were higher in the SIRS matched group as compared with the non – SIRS matched group. Neutrophil priming for superoxide production and elastase release was discovered 1 day after orthognathic surgery and differences in those values could not be distinguished between the groups.

The results suggested that a few patients in whom high levels of circulating inflammatory cytokine and neutrophil derived toxic factor continue may have a possibility of contracting severe diseases such as SIRS and MODS after orthognathic surgery. Hence the authors concluded that the ratio of IL-6 to IL10 may be a predictive factor in SIRS. Wang G. et al, 2005 6 8 the objective of this study was to investigated the effect of Tramadol on the product ion of

63

serum IL-6, IL-10 and IL-2 and soluble IL-2 receptors, thereby evaluating its effect on the pro–inflammatory and anti–inflammatory responses and immune function in cancer patients undergoing conventional pulmonary lobectomy.

They conducted a randomized, double blind, placebo controlled study and included 40 patients with ASA–I and ASA–II

physical

status

who

were

scheduled

for

conventional pulmonary lobectomy.

Patients were randomly divided into 2 groups and administered IV tramadol (group I) and IV normal saline (group-2). Venous samples for measurement of serum cytokine concentration were taken before anaesthesia at set intervals until 24 hours after operation. Serum IL -6 and IL10 in both groups were increased significantly during and after operation compared with baseline levels.

They did not find any statistica l differences between the groups in terms of IL-6 and IL-10 concentration. Levels of IL-2 were significantly elevated at 4 hours after operation in group I as compared with baseline levels whereas they remained low at 4 and 24 hours after operation in group II. They were significant increases in levels of sIL-2 R at 4 and 24 hours after operation in group II as compared with baseline levels and at 24 ho urs after operation in group- I.

64

They concluded the IV infusion of tramadol does not seem to alter IL-6/IL-10 cytokine response to pulmonary lobectomy. As tramadol was associated with increased IL -2 and delayed enhancement of sIL-2R in their study, it may attenuate to some extent an impaired immune response in pulmonary lobectomy. Young-Soo Jung et al, 2005 7 2 the goal of their study was to compare

analgesic

effects

of

a

non -steroidal

anti-

inflammatory drug for oral surgical pain according to 3 different administration times.

This study was conducted with 80 healthy patients undergoing a surgical removal of a n impacted third molar requiring bone removal 25 patients were assigned to the preemptive group,26 to the post treatment group and 29 to the

no

treatment

administered

1

group. hour

The

oral

NSAID

preoperatively,

or

was

first

1

hour

postoperatively, or on scheduled administration pre-or postsurgery. Whenever patients felt at least moderate pain (score >= 5 on a 10 –point scale) scale, they were instructed to take the same drug. Pain intensities and times to the first and second onsets of post-operative pain from the end of surgery were assessed for 24 hours. The mean time to first onset of postoperative pain was not significantly prolonged in the post treatment group and the no treatment group. The

65

mean time to second onset of post-operative pain was not significantly different among the 3 groups.

No significant statistical difference was found among the mean pain intensities at the first and second onsets of post-operative in the 3 groups.

The results in this population implied that scheduled postoperative

analgesics

before

pain

development

are

adequate for postoperative analgesia without preoperative administration. Asokumar Buvanendran et al, 2006 5 the primary aim of their study was to determine whether hip replacement surgery causes an upregulation of PGE 2 in the CSF. The secondary objective was to determine if perioperative oral COX-2 inhibitor administration modulates CSF PGE 2 levels. Thirty osteoarthritis patients undergoing hip arthroplasty with spinal anaesthesia were randomly divided into 3 groups: placebo for 4 days before surgery and on the morning of surgery; placebo for 4 days before surgery and oral rofecoxib 50 mg on the morning of surgery; oral rofecoxib 50 mg 4 days before surgery and the morning of surgery. Cerebrospinal fluid and plasma were collected before surgery and up to 30 hrs after incision for measurement

of

PGE 2

and

interleukins.

When

hip

replacement was complete, a drain was placed in the hip

66

wound and exudates were collected at 3 to 30 hrs after incision.

Cerebrospinal fluid showed an initial increase in IL-6 and a later rise in PGE 2 , concentration after surgery. Interleukin-1β

and

tumor

necrosis

factor

α

were

undetectable. Hip surgical site fluid evidenced an increase in PGE 2 , IL-6, IL-8 and IL-1β; TNF-α decreased at 24 hrs and 30 hrs. Preoperative administration of cyclooxygenase 2 inhibitor rofecoxib reduced the cerebrospinal and surgical site

prostaglandin

E2

and

cerebrospinal

fluid

IL-6.

Cerebrospinal fluid PGE 2 was positively correlated with postoperative pain and cerebros pinal fluid IL-6 with sleep disturbance. Poorer functional recovery was positively correlated with increased surgical site PGE 2 . The primary conclusion of their study was that the hip replacement surgery was associated with postoperative upregulation of PGE 2 , IL-6 and IL-8 both centrally and at the

surgical

site.

CSF

PGE 2

was

associated

with

postoperative pain, and tissue PGE 2 was associated with functional recovery. Preoperative administration of a COX 2 inhibitor reduced CSF and surgical site PGE 2 levels as well as CSF IL-6 levels. Carmen Lopez Carriches et al, 2006 1 0 conducted this study to determine if there is a release of certain cytokines,

67

such as Interleukin-6 (IL-6) after surgical removal of lower third molars and to compare the amount of IL -6 in patients treated

with

NSAID

and

in

those

treated

with

glucocorticoids. The level of IL-6 in plasma increases after different operations and therefore, they assume that a large amount of cytokines (IL-6 among them) are released locally after surgical removal of lower third molars, and that to a great

extent

these

are

responsible

for

postoperative

complications.

It is a Prospective study on 73 patients who needed surgical removal of their lower third molars. These patients were separated into two groups: the diclofenac group and the methylprednisolone group. A record card was completed with preoperative and postoperative epidemiological and clinic data. Samples of gingival crevicular fluid were collected in order to assess the release of interleukin -6 after surgery.

Levels of IL-6 were higher after surgical extraction of lower third molars and remained high until the seventh day after. Levels were higher in the diclofenac group 24 hours after surgery; the difference was significant (0.008). The high increase of IL-6 24 hours after lower third molar surgery indicates that this procedure involves significant surgical trauma. Since this cytokine is one of the most important ones involved in the physiological response to

68

trauma, inflammation and infection, a great investigative effort is being made in order to find methods to regulate the systemic effects of IL-6 and improve postoperative control. Glucocorticoids have already been tested in experimental and

clinical

studies.

The

endogenous

and

exogenous

increase in the levels of glucocorticoids in blood diminishes the production of IL-6 in human and animal studies.

Authors concluded that the IL-6 is higher after surgical

extraction

of

lower

third

molars,

behaving

differently in each of the groups. Cem Gabay et al, 2006 1 2 Interleukin-6 is produced at the site of inflammation and plays a key role in the acute phase response as defined by a variety of clinical and biological features such as the production of acute phase proteins. IL 6 in combination with its soluble receptor sIL-6Rα, dictates the transition from acute to ch ronic inflammation by changing

the

nature

of

leucocyte

infiltrate

from

polymorphonuclear neutrophils to monocyte/macrophages.

In addition, IL-6 exerts stimulatory effects on T- and B-cells, thus favoring chronic inflammatory responses. Strategies targeting IL-6 and IL-6 signaling may lead to effective prevention and treatment of models of rheumatoid arthritis and other chronic inflammatory diseases.

69

Eman A. El-Sharrawy et al, 2006 19 the anti–inflammatory effects of ibuprofen and tramadol were investigated by measuring C-reactive protein concentrations after removal of an impacted lower third molar. Forty fiv e ASA-I patients were randomly categorized into 3 equal groups according to post-operative analgesic medication.

The first group received tramadol (100mg every 8 hourly), the second group received ibuprofen (400 mg every 8 hours), and the last group received half doses of both drugs in combination (50 mg tramadol every 8 hours and 200 mg ibuprofen every 8 hours). C-reactive protein was measured before surgery to exclude the presence of any preexisting inflammatory condition that might interfere with the

study.

C-reactive

protein

was

also

determined

immediately after surgery and 72 hours p ost-operatively. At 72 hours, C-reactive protein had increased over postsurgery base line by 123 % in the tramadol group, 84 % in the ibuprofen group and only 37 % in the combined analgesic group.

These results suggested that tramadol may produce supra-additive anti-inflammatory effects with ibuprofen after third molar extractions. Keskinbora K. et al, 2006 3 3 tramadol is a centrally acting analgesic, consists of two enantiomers, both of which

70

contribute to analgesic activity via different mechanisms. (+) Tramadol and the metabolite (+) -O- desmethyltramadol (M1) are agonists of the µ opioid receptor. (+) Tramadol also stimulates presynaptic release of serotonin and

inhibits

serotonin

reuptake whereas

( -)

tramadol

inhibits norepinephrine reuptake. Thus t ramadol enhances inhibitory effects on pain transmission both by opioid and monoaminergic

mechanisms.

The

complementary

and

synergistic actions of the two enantiomers improve the analgesic efficacy and tolerability profile of the racemate. Following

oral

administration

the

bioavailability

of

tramadol is high and with new slow release preparations twice daily administration enables effective pain control. The recommended maximum daily dose of tramadol is 400 mg/day.

Tramadol is characterised by low plasma protein binding and quite extensive tissue distribution. Elimination is primarily by the hepatic route (metabolism by CYP2D6) and partly by the renal route. It is effective in different types

of

moderate-to-severe

acute

and

chronic

pain,

including neuropathic pain, low back pain, osteoarthritis pain and breakthrough pain. It also causes fewer opioid type adverse effects, e.g. nausea, drowsiness, vomiting, dry mouth

and

constipation.

Although

trials

in

literature

demonstrate immune-stimulating effects of tra madol, there

71

are also trials suggesting immunosuppressive effects that are lesser than morphine.

Owing to its pharmacologi cal properties, tramadol was more appropriate than NSAIDs for patients suffering from gastrointestinal and renal problems. Besides i ts proven clinical efficacy tramadol is a safe drug as respiratory depression, cardiovascular side effects, drug abuse and dependence are of minor clinical relevance, unlike some other opioids. Amaury J. Pozos et al, 2007 2 a total of 48 patients were recruited

in

a

double

blind,

randomized,

placebo–

controlled, clinical trial in patients after extraction of an impacted mandibular third molar. Patients were randomized into 4 treatment groups, each with 12 patients : group A, tramadol 50 mg intramuscular (IM) 1 hour before surgery; group B, tramadol 50 mg into the surgical site, injected submucosally immediately after local anaesthesia; group C , tramadol through both routes of administration 50 mg IM 1 hour before surgery and 50 mg into the surgical site ; grou p D , placebo. To assess the possible redistribution of tramadol from the surgical site to the general blood stream, 6 hour blood sampling (at 0, 0.5,1,2,3,4,5 and 6 hours after drug administration) was done. A 10 cm VAS was used to assess pain.

72

The

duration

of

the

anaesthetic

effect

was

significantly longer in the groups receiving tramadol injection into the surgical site (group B and group C). Administration of 50 mg tramadol systemically and locally suppressed the pain intensity values in comparison wi th control group and also in comparison with all groups.

There was no difference in pain intensity values in the in the groups receiving systemic and local administration of tramadol (group A and group C). Amaury Pozos-Guillen et al, 2007 3 in a prospective, randomized, controlled, double-blind pilot study the authors compared

the

efficacy

immediately after mandibular

third

of

surgical molar

tramadol extraction

under

local

given of

an

before

or

impacted

anaesthesia.

The

intensity of pain and analgesic con sumption was evaluated in 3 groups of 20 patients each where they administered: tramadol preoperative, 100 mg intramuscularly (im) 1 hour before surgery (group A), tramadol postoperative, 100 mg im immediately after surgery (group B), and sali ne (group C).

The analgesic efficacy measured as comp lete relief of pain at 2 hours was 86% in the preemptive tramadol compared with 70% and 36% for post -operative tramadol administration and control group.

73

A significant reduction

in the consumption of analgesics wa s seen in preoperative group as compared with the post-operative and control groups.

This study suggested that preemptive use of tramadol as an alternative for the acute pain treatment after removal of an impacted mandibular third molar carried out under local anaesthesia. Mauro Bianchi et al, 2007 4 5 the purpose of their study was to measure the concentrations of PGE 2 and TNFα in the CSF of rats after intraplantar administration of complete Freund’s adjuvant (CFA) in the hindpaw of mice. They also investigated

whether

prevent

biochemical

the

different

analgesic

changes

at

the

drugs

could

spinal

level

associated with peripheral inflammation.

They used Randall-Selitto paw withdrawal test to measure the inflammatory hyperalgesia. Tramadol (7.5 mg/kg), paracetamol (65 mg/kg), tramadol plus paracetamol and nimuselide (5mg/kg) were administered orally twice a day, starting from the first day after the CFA infection. PGE 2 in CSF was measured by enzyme immunoassay and TNFα by ELISA. Behavioural and biochemical parameters were measured on Day 7 after injection with CFA or saline. They found out that withdrawal thresholds to mechanical stimuli decreased markedly in the CFA treat ed paw. In

74

those

animals

the

quantification

of

proinflammatory

mediators in the CSF revealed a significant increase in both PGE 2 and TNF-α concentrations. All

the

pharmacologic

treatments

prevented

the

development of the hyperalgesia as well as the PGE 2 increase in the CSF. A prevention of the increase in TNF-α levels was observed only in rats treated with nimesulide or tramadol and paracetamol in combination.

The results suggested that peripheral inflammatory hyperalgesia was associated with significan t changes in the proinflammatory increase

in

hyperalgesia

mediators

TNF-α

in

the

CSF.

concentrations

represent

two

in

However the

phenomena

CSF

that

can

the and be

dissociated from pharmacological point of view. Sota Omoigui et al, 2007 5 9 the authors describe the inflammation

pathway

from

Cholesterol

to

Aging.

Interleukin 6 mediated inflammation is implicated in age related

disorders

including

Atherosclerosis,

Peripheral

Vascular Disease, Coronary Artery Disease, Osteoporosis, Type 2 Diabetes, Dementia and Alzheimer's disease and some

forms

of

bisphosphonates

Arthritis

and

Cancer.

inhibit

Interleukin

Statins 6

and

mediated

inflammation indirectly through regulation of endogenous cholesterol synthesis and isoprenoid depletion.

75

Polyphenolic compounds found i n plants, fruits and vegetables inhibit Interleukin 6 mediated inflammation by direct

inhibition

of

the

signal

transduction

pathway.

Therapeutic targets for the control of all the above diseases should

include

inhibition

of

Interleukin -6

mediated

inflammation. Yong-Min Liu et al, 2008 7 1 forty two Sprague-Dawley (SD) rats were scheduled for plantar incision after being divided into 7 groups to evaluate the effects of tramadol on the proinflammatory responses in a rat model of incisional pain by investigating its effects on nociceptive thresholds and serum interleukin-6 and interleukin-2 levels. Rats in group 1 received general anesthesia with no incision were served as control. At 30 minutes before skin incision, groups 2 to 5 were given 5 ml normal saline o r 1, 10, and 20 mg/kg tramadol respectively, intraperitoneally (i.p.). Group 6 received 10 mg/kg tramadol after operation. Group 7 received 10 mg/kg tramadol before incision, followed by 200 µg/kg naloxone after operation.

They measured mechanical allodynia by electronic von Frey filament to evaluate the nociceptive thre sholds 1 hour before incision and 1 hour and 2 hour after operation. The authors found out that mechanical thresholds decreased significantly and serum IL-6 levels increased significantly after operation in group 2 as compared with the control and

76

these changes were reversed respectively by tramadol in a dose dependent manner. IL-2 remained unchanged after operation in group 2, but decreased in group 3, then gradually returned to the normal level in group 4 and 5. The intra peritonially injected tramadol (10 and 20 mg/kg) produced a potent and dose-dependent antinociceptive effect on the lesioned paw. The antinociceptive effects of tramadol

were

partially

antagonized

by

naloxone

(200µg/kg) suggesting an additional non-opioid mechanism.

In conclusion the results suggested that tramadol could be a good choice for the treatment of pain under the conditions that immunosuppression may be particularly contraindicated.

Also the present results suggest that tramadol has some beneficial effects on plantar incisional pain conditions in rats, e.g., suppression of IL-6 production. Tramadol was associated with decreased IL-6 and unchanged IL-2 levels, suggesting that it may suppress the inflammation ind uced by incision and has a beneficial role in the modulation of IL-2 associated with cell-mediated immunity. Durval Campos Kraychete et al, 2009 18 the objective of their

study

was

to

evaluate

plasma

levels

of

proinflammatory cytokines before and after tre atment with tramadol in patients with herniated intervertebral disks and

77

carpal tunnel syndrome and to compare them with normal individuals.

Thirty eight patients with neuropathic pain secondary to herniated intervertebral disks or carpal tunnel syndrome participated in this study. All patients were treated with controlled release tramadol (100mg every 12 hrs) for 10 days. Venous blood was collected in the morning, before treatment and on the 11 t h day and stored at -70 degrees until analysis. ELISA was used to determine the plasma levels of cytokines TNFα, IL-1, and IL-6 and receptors sTNF-R1. Plasma levels of cytokines of 10 healthy volunteers were also determined. The authors found out the concentration of TNFα before (5.8+2.8pg/ml) was significantly higher than after treatment with tramadol (4.8 +2.1pg/ml). The levels of IL1β, IL-6 and sTNF-R1 before and after treatment with tramadol showed no significant differences. Plasma levels of

TNFα

(healthy

individuals

1.4 +0.5;

pain

patients:

3.5+2.6 pg/ml) and IL-6 (healthy individuals: 1.2 +0.8; pain patients 3.5+2.6 pg/ml) were significantly higher in patients with neuropathic pain.

The authors concluded that in patients with herniated intervertebral disks and carpal tunnel syndrome, plasma levels of TNFα an IL-6 were higher than in healthy

78

individuals. Also differences in the concentrations of sTNF R and IL-1β were not observed. Plasma levels of TNFα but not of IL-6, sTNF-R, and IL-1β decreased after treatment with tramadol. May Hamza et al, 2009 4 6 non-opioid analgesics including both selective and non-selective cyclooxygenase (COX) inhibitors

and

acetaminophen

are

most

widely

used

treatment of pain. Inhibition of COX is thought to be largely responsible for both the therapeutic and adverse effects of this class of drugs. Accumulating evidence over the past decades has demonstrated effects of non -opioids beyond the inhibition of COX and prostaglandin synthesis that might explain their therapeutic and adverse effects.

These include their interaction with endo cannabinoids, nitric oxide, monoaminergic, and cholinergic systems. The recent development of microarray technology that allows the study of human gene expression suggest ed multiple pathways that may be related to the analgesic and anti inflammatory effects of non-opioids.

In their article the authors have discussed the multiple actions of non-opioids and their interactions with these systems during inflammation and pain, suggesting that COX inhibition is an incomplete explanation for the actions of

79

non-opioids and proposed the involvement of multiple targets for their analgesic as well as their adverse effects. King Kim et al, 2009 39 the use of medication to relieve pain and inflammation after removal of third molars has been

explored

analgesics,

thoroughly

in

non-steroidal

the

l iterature.

Narcotic

anti-inflammatory

drugs

(NSAIDs), corticosteroids, and combinations of these all have a role in the post-operative management of pain and swelling within this group of patients.

The authors

addressed

the use

of NSAIDs

and

corticosteroids after third molar surgery along with a review of the literature. Liu Y.M. et al, 2009 4 2 the authors investigated the effect of tramadol on c-fos expression in spinal cord dorsa, horn and serum IL-6 levels induced by plantar incision in rats. The Brennan pain model was induced by incision on the planter surface of left hind paw in rats. Forty eight rats were randomly divided into six groups: Sham group (group C), control group (group 1, pre-treatment with saline 5ml), three tramadol pre-treatment groups (group T1, T10 and T 20,

pre-treated

with

1mg/kg,

10mg/kg

and

20mg/kg

respectively) and one tramadol treatment group (group PT10, treated with tramadol 10mg/kg immediately after operation).

80

Pain behaviour was assessed by withd rawal threshold to von Frey filament stimulation intensity, response latency of the hind paw to radiant thermal and cumulative pain score 2 hrs after incision. Fos -positive neurons in spinal cord

were

identified

by

the

immunohistochemical

technique. Serum IL-6 levels were measured by ELISA.

They found out that the withdrawal threshold to von Frey filament stimulation intensity and response latency of the hind paw to thermal in group1 were significantl y lower than those in group C. Cumulative pain score in group 1 was significantly higher than in group C. In groups T10 and T20, withdrawal threshold to von Frey filament intensity and response latency of the hind paw to radiant thermal were significantly higher than those in group 1, cumulative pain scores were significantly lower than those in group 1 in a dose dependant manner and were also those in group PT10. The greatest density of Fos -positive neurons was located in lamine I-II in group 1. Serum IL-6 levels were significantly elevated

in

group 1. Pre -treatment

with

tramadol showed a dose dependant inhibitory effect on c fos expression and serum IL-6 production, but not in group T1.

Administration

of

tramadol

postoperatively

also

suppressed the c-fos expression and serum IL-6 production as shown in PT10 but were weaker than those in group T10.

81

The

authors

concluded

that

pre-treatment

with

tramadol produced a dose dependant inhibitory effect on c fos expression in spinal cord dorsal horn and then supress the inflammatory response to the trauma. George M. Grant et al , 2010 2 3 the authors did a double blind, randomised, placebo–controlled study on 80 patients to evaluate the safety and efficacy of intranasal (IN) Ketorolac in patients who had third molar extraction surgery with bony impactions. Patients we re randomly assigned to receive IN Ketorolac 31.5 mg or IN placebo. Safety was assessed from spontaneously reported adverse events and measurement of vital signs.

Efficacy assessment included pain intensity which was measured on 0 to 10 mm visual analogue scale, total pain relief and global pain evaluation up to 8 hours after dosing or until patients required rescue analgesia.

The primary efficacy variable was the summed pain intensity difference score over the first 8 hours after dosing.

The

summed

pain

intensity

values

were

significantly higher in the Ketorolac group compared with placebo. Total pain relief scores were significantly higher in the Ketorolac group compared with placebo group at all times. Eight patients in the placebo group had adverse events, none of which were serious.

82

The authors concluded that IN ketorolac 31.5 mg dose was well tolerated and provided rapid and effective pain relief in oral surgical patients for a period up to 8 hours. Pu Wang et al, 2010 5 3 elevated levels of prostaglandin E 2 (PGE 2 ) and interleukin-6 have been reported in the cartilage and synovial fluid from patients with arthritic disorders. PGE 2 regulates IL-6 production in numerous different cells including macrophages and synovial fibroblasts. Although there

is

PGE 2

stimulated

IL-6

expression

in

human

chondrocytes, the underlying signalling pathway of this process is yet to be delineated. In this study the authors have investigated the mechanism of IL -6 induction in human T/C-28a2 chondrocytes treated with exogenous ly added PGE 2 . PGE 2 induces IL-6 mRNA and protein expression via a cAMP dependant pathway reaching maximal levels after 60 min of stimulation before declining to baseline levels at 6 hrs.

Forskolin,

an

adenylyl

cyclase

inhibitor,

also

stimulates IL-6 expression in human chondrocytes in a dose and time dependant fashion. Inhibition of downstream effectors of cAMP activity such as protein kinase A (PKA) or phosphatidylinositol 3 kinase (PI3K) blocks PGE 2 and forskolin

induced

IL-6

upregulation.

Simultaneous

inhibition of PKA and PI3K reduces IL -6 expression in

83

stimulated chondrocytes well below the basal levels of untreated cells.

Gel

shift,

supershift

and

chromatin

immunoprecipitation assays revealed the activation and binding of the nuclear factor NF -κB p65 subunit to the IL-6 promoter, which is markedly suppressed by selective PI3K or

PKA

pharmacological

inhibitors.

P65

knockdown

completely abrogated the IL-6 mRNA synthesis in PGE 2 and forskolin primed chondrocytes.

The authors hence showed that PGE 2 and forskolin induce

IL-6

expression

in

human

chondrocytes

via

cAMP/PKA and PI3K dependent pathways which in turn regulate the activation and binding of p65 to the IL -6 promoter. Selma Sijercic Avdagic et al, 2010 5 8 the aim of their research was to determine the influence of continuous opiate and intermittent non -opiate postoperative analgesia on thoracic surgical patients acute phase response, based on acute phase response protein serum values (IL -6 and Creactive protein) 24, 48 and 72 hrs after surgery and also to analyze the acute phase responses in those thoracic surgical patients in which the postoperative complications have developed and in those in which they haven’t.

84

The authors studied 60 patients prospectively by dividing them into two groups of 30 patients each. The first group was administered non-opioid methamisol natrium and to the second group tramadol chloride opiate analgesia was applied after thoracic surgical procedures.

They obtained the following results: CRP values enhanced in both groups in all three measurements with no statistical differences. The IL -6 values enhanced in all measurements in group I, in group II they remained within the reference range, with a significant statistical difference.

This study suggested that CRP values increased in both groups as a result from surgery. The enhanced IL -6 values in group I and maintained IL-6 values within the reference range (0-20pg/ml) in group II were the result of continuous tramadol chloride opiate analgesia. Hidir Esme et al, 2011 2 7 the aim of their study was to evaluate the serum levels of acute phase reactants such as CRP, and cytokines (IL-6) and TNFα in patients undergoing thoracotomy and to investigate the effects of flurbiprofen on post-operative inflammatory response.

Forty patients undergoing posterolateral thoracotomy were randomly divided into 2 groups of 20 each. Control group received tramadol (4X100mg) IV for four days and

85

flurbiprofen group received both tramadol (4X100mg) and flurbiprofen (2X100mg). Blood sampl es were collected before surgery and at 3 r d and 168 t h hours after surgical procedure to measure serum CRP, IL-6 and TNFα. Pain visual analogue scales were recorded daily during the first four postoperative days. Spirometric measurements of forced expiratory volume in the first second (FEV1) was done before and four days after the operation. They found out that serum IL-6, CRP and TNFα levels in both groups increased significantly at 3 r d hour after thoracotomy.

Serum

TNFα

levels

did

not

differ

significantly between the groups at postoperative 4 t h day. However

IL-6

and

CRP

were

significantly

lower

in

flurbiprofen group than in control group at the same day (p250 µl/well Wash Buffer with soaking period of approximately 1 minute and blotted with absorbent paper to remove any residual buffer. 3. Part 5X Assay Diluent was diluted with 4 parts Millipore water (mp) and the wells were blocked with 200 µl/well of assay diluent. It was then incubated at room temperature for 1 hour. 4. The plates were washed as in step 2 for a total of 5 washes. 5. Using 1X assay diluent , 100 µl/well of standard was added to the appropriate wells. 2 fold - serial dilutions of the top standards was done to make the standard curve. 100 µl/well of the samples to be tested were added to the appropriate wells. The plates were sealed and incubated at room temperature for 2 hours. 7. The wells were washed as in step 2 for a total of 5 washes. 8. 100 µl/well of detection antibody diluted in 1X Assay Diluent was added and the plates sealed and incubated at room temperature for 1 hour.

126

9. The plates were washed as in step 2 for a total of 5 washes. 10. 100 µl/well of Avidin-HRP diluted in 1X Assay Diluent was added to the plates and the plates sealed and incubated at room temperature for 30 minute s. 11. The plates were aspirated and washed in step 2 for a total of 5 washes. 12. 100 µl/well of substrate solution was added to each well and the plates incubated at room temperature for 15 minutes. 13. 50 µl/well of stop solution was added to each well 14. The plates were read at 450 nm. 15. Sensitivity of the kit was 2pg/ml and standard curve range was 2-200pg/ml.

Standard curve for Human IL-6Ready-Set-Go

127

COMPONENTS OF THE IL-6 KIT

Capture Antibody and Detection Solution

Assay Diluent and TMB Substrate Solution

128

COMPONENTS OF THE IL-6 KIT

Coating Buffer and Avidin-HRP

Standard Human IL-6

129

96 well Corning Costar 9018 ELISA plate s

Reagents added with micropipettes

130

Serum Samples added to the plates

Blue colour can be seen after addition of substrate solution to the wells

131

Yellow colour can be seen after addition of stop solution

The plate inside the ELISA reader being rea d at 450 nm

132

Statistical Analysis: the data was available in tables after appropriate calculations with IL-6 values in pg/ml and SPSS software version 15 was used to produce a detailed description of data, frequencies tables, bivariate graphics, t Tests and repeated measures analysis of varian ce.

133

In a randomised double blind prospective study a total of 57 patients with symptomatic lower third molars were taken up for serum IL-6 estimation. They were divided into 3 groups. Group 1 consisted of 21 patients who were given Tab Diclofenac 50mg BD for 5 days post-operatively. Group 2 consisted of 20 patients who were given Tab Ketorolac 10mg TDS for 5 days post -operatively and Group 3 consisted of 16 patients who were given Tab Tramadol 50 mg BD for 5 days post-operatively. Cap Amoxicillin 500mg TDS was administered to all 3 groups as antibiotic and Tab Rantac 150mg BD was given to group 1 and 2 as antacid. Tab Emeset 4mg BD was given to Group 3 as antiemetic. Blood samples were collected at specific intervals and Intereukin-6 was estimated from serum using Human IL-6 ELISA kits. All the surgeries were done by a single surgeon.

134

Table 1 shows the overall sex distribution of 57 patients taking part in the study. A total of 33 patients were male (57.9%) and 24 patients were female (42.1%).

Table 1

Graph 2 shows the overall sex distribution of 57 patients in the study

Graph 2

135

Table 2 shows the different types of impactions encounter in the study.

Radiographic analysis of the type of impaction showed that mesioangular impaction constitu ted 35.1% of the total cases, followed by distoangular impactions 28.1%, then vertical 21.1% and least were horizontal 15.8% in the complete study group.

Table 2 Graph

3

shows

the

different

encountered in the study group.

Graph 3 136

types

of

impactions

Table

3

shows

the

sex

distribution

of

the

patients

distributed in the 3 groups.

Out of 21 patients in Diclofenac group 13 were males (39.4%) and 8 (33.3%) were females.

In the Ketorolac group among the 20 patients 14 (42.4%) were males and 6 (25%) were f emales.

In the Tramadol group out of 16 patients 6 (18.2%) were males and 10 (41.7%) were females.

Table 3

137

Graph 4 shows the sex distribution of the patients in the different drug groups.

Graph 4

138

Table

4

shows

the

different

types

of

impactions

encountered in the 3 drug groups.

In the diclofenac group 7(33.3%) impactions were distoangular

type,

5(23.8%)

were

of

horizontal

type,

4(19%) were of mesioangular type and 5(23.85) were of vertical type.

In the ketorolac group 4 (20%) of impact ions were of distoangular type, 2(10%) were of horizontal type, 7(35%) were of mesioangular and vertical type each.

In the tramadol group 5(31.3%) of impactions were of distoangular type, 2(12.3%) were of horizontal type and 9(56.3%) were of mesioangular type.

Table 4

139

Graph 5 shows the different types of impaction in the 3 different drug groups.

Graph 5

140

Chart 1 represents the value of IL-6 in patient’s serum of the 3 different groups: Drug Sample Values

Values

Values

Values

code

for

for

for

for

preop

24hrs

72 hrs

1week

(pg/ml)

(pg/ml)

(pg/ml)

(pg/ml)

no

D

25

7.954545 8.522727

4.318182

5.056818

T

14

7.670455 19.20455

5

7.897727

D

15

6.193182 9.204545

4.602273

6.306818

T

7

5

8.409091

3.181818

4.715909

D

26

4.943182

7.5

3.409091

4.659091

T

47

5.170455 8.920455

3.409091

4.602273

K

43

5.852273 8.181818

4.090909

5.170455

K

45

8.863636 11.98864

7.329545

11.42045

K

44

6.647727 10.56818

8.181818

8.75

K

28

6.931818 11.53409

4.545455

7.102273

K

32

7.727273 10.45455

5.397727

7.5

T

27

5.795455 9.545455

4.261364

4.715909

K

17

5.965909 7.954545

4.375

4.715909

K

39

7.443182

3.522727

5.511364

D

16

5.056818 9.431818

5.511364

5.227273

T

31

8.011364 9.545455

5.852273

7.045455

K

29

5.795455 6.988636

3.863636

4.886364

T

19

5.170455 8.352273

3.522727

5.227273

D

34

6.534091 8.465909

3.181818

5.738636

D

8

7.045455 9.034091

3.693182

6.420455

5

141

T

10

7.272727 20.28409

6.25

7.159091

K

9

8.522727 9.602273

3.863636

6.988636

D

1

5.795455 8.238636

3.238636

5.397727

D

4

5.568182 9.204545 3.068182

5.113636

K

13

5.852273 8.693182 3.068182

5.738636

T

11

5.795455 8.920455 3.011364

5.909091

D

12

5.738636 9.545455 2.897727

5.738636

D

5

5.738636 8.693182 3.068182

5.568182

D

57

4.545455 6.818182

0.00

0.00

D

23

3.977273 6.590909

6.590909

8.693182

T

18

6.022727 14.09091

9.431818

12.21591

D

6

4.431818 6.988636

6.704545

8.522727

T

3

2.954545 6.022727

4.829545

7.897727

D

24

5.625

8.522727

T

46

2.897727 6.420455

5.681818

7.5

K

48

3.295455 5.852273

5.625

7.386364

K

42

7.613636

8.068182

12.32955

K

41

3.977273 7.613636

7.556818

8.409091

K

30

3.522727 7.272727

4.829545

6.931818

K

33

4.602273 5.909091

4.204545

7.727273

T

22

3.636364 8.068182

5.284091

5.738636

K

2

3.352273

5.340909

6.420455

K

38

3.181818 4.659091

4.829545

7.159091

D

20

3.693182 6.647727

6.477273

7.784091

T

21

5.965909 7.556818

7.5

9.090909

K

35

6.25

5.795455

3.125

6.25

2.5

4.829545

4.375

4.602273

142

T

49

D

3.125

5.227273

4.829545

5.909091

40

2.784091 5.454545

4.772727

6.534091

D

50

3.181818 5.454545

5.511364

5.454545

T

56

3.068182 15.73864

7.102273

7.670455

K

37

5.795455 7.443182

5.625

10.11364

D

53

3.181818 4.488636

3.977273

5.113636

D

52

2.784091 5.056818

3.75

6.704545

K

35

2.727273 4.488636

3.863636

4.772727

T

51

2.386364 4.943182

4.318182

5.625

D

36

2.840909 5.397727

4.034091

5.454545

D

54

2.954545 4.431818

3.977273

5.113636

Chart 1

D-Tab Diclofenac 50mg K-Tab Ketorolac 10mg T-Tab Tramadol 50mg

143

ANOVA test and Paired T-test were performed on the following sets of values and Graphs tabulated.

Table 5 showing the comparison between the pre-op, 24 hrs, 72hrs and 1 week values of all the 3 drugs administered with mean values and standard deviation.

Table 5

144

Table 6 showing the results after ANOVA analysis showing statistically significant p value of 0.014 in 24 hours group.

Table 6

145

Table 7 shows the values at 24 hours for the IL -6 levels of Ketorolac and Diclofenac are significantly lower than those of Tramadol.(p=.005 for Diclofenac vs. Tramadol and p=.022 for Ketorolac vs. Tramadol)

MULTIPLE COMPARISONS

Table 7

146

Tables 8 and 9

show the comparison with the various

values of IL-6 at

different time intervals with each other

within the Diclofenac group.

PAIRED SAMPLE STATISTICS FOR DICLOFENAC

Table 8

PAIRED SAMPLE TEST FOR DICLOFENAC

Table 9

147

Tables 10 and 11 show the comparison with the various values of IL-6 at different time intervals with each other within the Ketorolac group.

PAIRED SAMPLE STATISTICS FOR KETOROLAC

Table 10

PAIRED SAMPLE TEST FOR KETOROLAC

Table 11

148

Tables 12 and 13 show the comparison with the various values of

IL-6 at different time intervals with each other

within the Tramadol group.

PAIRED SAMPLE STATISTICS FOR TRAMADOL

Table 12

PAIRED SAMPLE TEST FOR TRAMADOL

Table 13 149

Graph 6 shows the mean of Pre-op values of IL-6 of all three drug groups combined.

Graph 6

150

Graph 7 shows the mean of 24 hours values of IL-6 in all 3 drug groups

Graph 7

151

Graph 8 shows the mean values of IL-6 at 72 hours in all three drug groups.

Graph 8

152

Graph 9 shows the mean values of IL-6 in all the 3 drug groups at 1 week interval

Graph 9

153

Graph 10 and 11 show the mean values of IL-6 during the time periods i.e. pre-op, 24 hrs, 72hrs, and 1 week of all the 3 different drug groups.

Graph 10

154

Graph 11

155

Graphs 12 and 13 shows the individual variation in the levels of IL-6 in the 3 groups of drugs over given the time periods i.e. pre-op, 24 hrs, 72 hrs and 1 week.

Graph 12

156

5.00 4.0

4.0

Graph 13

157

4.0

Graph 14 shows the variations in Il -6 values over the 4 time intervals of the three drugs administered in a single graph.

Graph 14

158

Oral surgical procedures vary in difficulty and in degree of trauma caused to the surrounding tissue. The greater amount of tissue injury leads to an increased amount of inflammation in the peri surgical area.

The classic signs of inflammation are pain, edema, erythema and loss of function. The inflammatory process is necessary if healing is to occur. But often excessive inflammation causes the patients unnecessary pain, edema and trismus.

There are many mediators of inflammation which include serotonin.

prostaglandins, Bradykinin

inflammatory

histamine, has

pharmacology

wide

bradykinin spectrum

including

and

of

pro -

p otent

pain

producing properties.

Prostaglandins are derived from precursor arachidonic acid. Arachidonic acid is a major compone nt of mammalian cell membrane phospholipids and is released from these phospholipids via cellular phospholipases that have been activated by various mechanical, chemical or physical stimuli.

159

Arachidonic acid metabolism can proceed along one of two major pathways  The Cyclooxygenase pathway  The Lipooxegenase pathway

Interleukin-6 is one of the most important cytokines involved

in

the

physiological

response

to

trauma,

inflammation and infection.

Acute phase response is the answer of the organism to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth, or immunological disorders.

The acute phase response is thought to be beneficial to the injured organism with the aim of restoring the disturbed physiological status.

160

The following chart sums up the local and systemic reaction of an acute phase response of an organism(Fig-1):

Fig-1 (Courtesy Peter C. Heinrich et al 5 1 , Biochem. J. 1990; 265: 621-636)

161

Interleukin-6 is the major regulator of acut e phase response in human hepatocytes. IL-6 has turned out to be the most potent inducer of acute phase response in the human hepatocytes.

The

monokines

IL-1,

and

TNF-α

also

stimulate

fibroblasts, endothelial cells and keratinocytes to synthesise IL-6 thus amplifying its biological effects. IL-6, IL-1 and TNF-α have been shown to stimulate the release of ACTH from the pituitary cells, leadin g to an increased secretion of glucocorticoids by adrenocortical cells.

The

glucocorticoids

on

one

hand,

increase

the

stimulatory effect of monokines on the synthesis of acute phase proteins by hepatocytes, and on the other hand, inhibit the synthesis of monokines.

162

A simplified scheme of role of IL-6 is shown on the following diagram(Fig-2):

Fig-2 (Courtesy Peter C. Heinrich et al 5 1 , Biochem. J. 1990; 265: 621-636)

Initially

it

was

thought

that

IL-6

is

a

pro-

inflammatory cytokine, recent fi ndings have suggested that IL-6 has many anti-inflammatory and immunosuppressive activities.

163

Zhou Zing et al 7 3 also investigated the role of IL-6 in acute inflammatory response in the animal model.

Most, if not all nucleated cells have been shown to express and synthesize IL-6 in vitro. The most prominent source appears to be stimulated monocytes/macrophages, and cytokine stimulated stromal cells . IL-6 is clearly an interleukin that mediates communication between a large number of cell types by playing a role in the proliferation and differentiation of B lymphocytes, plasmacytomas and hybridomas , hematopoietic progenators , hepatocytes and T-lymphocytes.

Thus

IL-6

is

considered

a

major

immune

and

inflammatory mediator. Trauma or infection causes plasma level of IL-6 to rise. As an early alarm cytokine it recruits adjacent stromal cells to release high levels of IL -6 and other mediators. The sequential activation and cytokine cascade leads to raised IL-6 levels in inflammation. Since IL-6 can induce cortisol levels and cortisol is required for hepatic acute phase response, IL-6 plays an inductive role as a second signal to the liver. A.M. Mahdy et al 4 found out that diclofenac is associated with lower IL-6 concentrations and hence may have an anti inflammatory role to play after major surgeries.

164

The

authors

found

out

that

the

IL-6,

IL-10

concentrations increased after surgery reaching maximum levels at 12 hrs and 6 hrs respectively. At 12 hours the IL -6 concentration was significantly lower in patients receiving diclofenac than the placebo group.

In a prospective study of 22 patients Myung Hee Kim et al 4 7 compared the effect of morphine and morphine ketorolac administered to 2 groups of patients on the concentrations of IL-6 post surgically.

They

found

lower

IL-6

response

in

morphine-

ketorolac group as compared to morphine group 24 hrs post operatively. It may indicate that cyclooxygenase inhibitor ketorolac may have an important role in the attenuation of IL-6 response via inhibition of prostaglandin E 2 . In the study conducted by Wang G. et al 6 8 the authors investigated the effect of Tramadol on the production of serum IL-6, IL-10 and IL-2 and soluble IL-2 receptors in two groups receiving IV tramadol (group-I) and IV saline (group – II). Serum IL-6 and IL-10 increased in both groups significantly during and after operation as compared with baseline levels.

Also tramadol IV did not alter serum IL -6/IL-10 cytokine response but it was associated with increased IL -2

165

and delayed enhancement of soluble IL-2 receptors in patients undergoing pulmonary lobectomy.

In addition to its well-recognized stimulation of µopioid receptors and inhibition of biogenic amine reuptake, tramadol

reduces

prostaglandin

E2

concentrations

in

inflammatory exudates as demonstrated by Mauro Bianchi et al 4 5 in their study on rats. In their study Eman A. Sharrawy et al 1 9 concluded that the combination of tramadol and NSAID ibuprofen appeared

to

produce

supra-additive

anti-inflammatory

effects that may provide clinical advantages i n safety or efficacy for treating postsurgical dental pain. In a study conducted on rats Yong-Min Liu et al 7 1 concluded that tramadol was associated with decreased IL -6 and unchanged IL-2 levels. Thus suggesting that tramadol may supress the inflammation induced by incision and has beneficial role in the modulation of IL -2 associated cell mediated immunity.

The presence of cyclooxygenase inhibitors at the surgical site may limit the production of prostaglandins and prostacyclins associated with hyperalges ia and edema.

166

Post-operative swelling and edema may be in part due to the conversion of phospholipids to arachidonic acid by phospholipase

A2

resulting

in

the

production

of

leukotrienes, prostacyclins , prostaglandins an thromboxane A 2 which are mediators of the inflammatory response. By pharmacologically controlling the extent of the inflammatory process the intensity and the severity of the post-operative sequelae such as pain, swelling and trismus may be reduced. Prostaglandins play a major role in the induction of pain, inflammation and fever. As evident in their study done by Babatunde O. et al 6 the

reduction

of

biosynthesis

of

prostaglandins

by

inhibition of cyclooxygenase enzyme system is considered an important mechanism of action of NSAIDs.

Prostaglandin

E2

is

the

predominant

eicosanoid

released after surgical trauma and has been associated with inflammation, pain and fever. Surgery leads to a complex systemic response in plasma PGE 2 and interleukin-6 despite perioperative neuronal blockade. May Hazma et al 4 6 have reported downregulation of gene and protein expression of phosphodiesterase type IV enzyme

by

rofecoxib

and

ketorolac

in

oral

biopsies, 3hrs after third molar tooth extraction.

167

mucosal

NFκB also plays an important role in the upregulation of IL-6 in response to several inflammatory mediators. It is known that different non-opioids produce variable effects on

the

activation

of

NFκB.

NFκB

inhibition

lowers

peptidoglycan- and PGE 2 induced IL-6 production in RAW 264.7 macrophages.

The authors have shown in a study that selective COX-2

inhibitor

NS398,

inhibited

the

peptidoglycan-

induced NFκB specific DNA protein complex formation from 2-12 hrs of treatment, but not in the f irst 60 min, suggesting that NFκB activation may be PGE 2 / cAMP dependant. Furthermore the authors have reported an increase in gene and protein expression of SOCS3 in response to both rofecoxib and ibuprofen in the oral mucosa, 48 hrs after tissue injury and acute inflammation in the oral surgery model. Over expression of SOCS3 blocks the proinflammatory effects of IL-6 signalling through gp130.

Thus even if non-opioids under certain conditions might upregulate IL-6 production, an accompanying over expression

of

SOCS3

might

proinflammatory effects.

168

in

fact

block

its

A hypothetical diagram of possible sites of action of non-opioids in the regulatory pathway of IL -6(Fig-3). 

IL-6 binds to IL-6 receptor forming a hexadimer with intracellular gp-130 molecule.



This activates (phosphorylates) JAK -1 (Janus kinase 1), which leads to the phosphorylation of gp -130 and subsequently the activation of STAT1 and STAT 3 (signal transducers and activators of transcription).



The activation of this signaling cascade results in the induction of SOCS3 formation that ultimately inhibits the signaling transduction of IL-6.



PGE2 via EP2 and EP4 receptors activates adenylyl cyclase leading to the formation of cAMP that activates (protein

kinase

A)

PKA

and

subsequently

NFkB

(nuclear factor kappa B).



NFkB activation results in further IL -6 expression and it also interferes with activation of STAT. Non -opioids interfere with synthesis of PGE2, the degradation of c AMP and also regulate SOCS3 and NFkB.

169

Fig-3 (Courtesy: May Hamza et al 4 6 , Curr Mol Pharmacol. 2009 January 1; 2(1): 1–14)

170

Proposed cascade of signaling events in human T/C -28a2 chondrocytes stimulated with PGE 2 or forskolin(Fig-4). 

PGE 2 stimulates cAMP formation, which in turn up regulates PI3K/Akt and PKA activities, leading to NF κB activation.



Binding of NF-κB to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes.

Fig-4 (Courtesy: Pu Wang et al 5 3 , Am J Physiol Cell Physiol. 2010 June; 298(6): C1445–C1456)

171

In our study we have compared the IL-6 values after commonly performed oral surgical procedure i.e. surgical removal of lower third molars and observed the variations after administering two different NSAIDs Diclofenac and Ketorolac along with one semi -synthetic opioid Tramadol over a period of 7 days.

Table 5 shows the mean concentrations of IL-6 in the 3 drug groups at different time intervals .

The mean concentrations of IL-6 in diclofenac group were 4.66+1.55pg/ml in pre-op serum sample, 7.14+1.7 pg/ml in 24 hrs serum sample, 4.42 +1.2 pg/ml in 72 hrs serum sample and 6.15+1.27 in 1 week serum sample.

The mean concentrations of IL-6 in ketorolac group were 5.31+1.89 pg/ml in pre-op serum sample, 7.66+2.31 pg/ml in 24 hrs serum samples, 5.22+1.53pg/ml in 72 hrs serum samples and 7.24+2.12pg/ml in 1 week serum sample.

The mean concentrations of IL-6 in tramadol group were 4.99+1.8 pg/ml in pre-op serum samples, 10.07+4.72 pg/ml in 24 serum samples, 5.2+1.74pg/ml in 72 hrs serum samples and 6.80+1.97 in 1 week serum sample.

172

The laboratory reference for IL-6 in a study done by Panagiotis V. Giannoudis et al 4 9 was