Download Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. NAME OF THE CANDIDATE
AND
ADDRESS :
Dr. AKSHATHA. B.K.
POST GRADUATE STUDENT
DEPT.OF ORAL AND MAXILLOFACIAL PATHOLOGY
AND MICROBIOLOGY,
VYDEHI INSTITUTE OF DENTAL SCIENCES AND
RESEARCH CENTER,
2. NAME OF THE INSTITUTION:
#82, EPIP AREA, NALLURAHALLI POST, WHITEFIELD,
BANGALORE-560066.
VYDEHI INSTIUTE OF DENTAL SCIENCES AND
RESEARCH CENTRE,
#82, EPIP AREA, NALLURAHALLI POST, WHITEFIELD,
BANGALORE-560066.
3. COURSE OF THE STUDY AND MASTER OF DENTAL SURGERY IN ORAL AND
MAXILLOFACIAL PATHOLOGY AND MICROBIOLOGY.
SUBJECT:
4
DATE OF ADMISSION TO
COURSE
26- MAY-2010
5. TITLE OF THE TOPIC:
“EXPRESSION OF INDUCIBLE NITROUS OXIDE SYNTHASE IN ODONTOGENIC
KERATOCYST, DENTIGEROUS CYST AND RADICULAR CYST”; AN
IMMUNOHISTOCHEMICAL STUDY.
6.0 BREIF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY
The three most common Odontogenic cysts are Radicular cysts (RCs), Dentigerous cysts
(DCs) and Odontogenic Keratocysts(OKCs).
Nitric oxide synthases (NOS) are a family of eukaryotic enzymes that catalyze the production
of nitric oxide (NO). NOS has three isoforms of which, inducible nitrous oxide synthase
(iNOS), is found in various cells including macrophages and natural killer cells in immune
system. iNOS is a calcium independent cytosolic enzyme induced mainly by cytokines such as
Interleukin-1β, tumor necrosis factor and Interferon-γ (INF-γ) at sites of inflammation. Also,
iNOS and VEGF has been shown to play role in cell proliferation, tumor growth, angiogenesis and
metastasis.
Increased nitric oxide production in OKCs, DCs, and RCs may participate in bone resorption
and cystic enlargement. This is because matrix metalloprotienases(MMPs) can be activated by
nitric oxide and MMPs play key role in destruction of bone matrix.
iNOS plays important role in pathology of both inflammatory and developmental cysts.
The epithelial lining cells of OKCs ,DCs, and RCs can produce their own cytokines such as ILα and IL-6; These cytokines may activate iNOS expression of the epithelial cells in autocrine
fashion.
OKC is now designated by WHO as a “Keratocystic Odontogenic Tumor” (KCOT) because
of its aggressive behavior, recurrence risk, and malignant potential. The epithelial lining of OKC
appears to have intrinsic growth potential not present in other types of Odontogenic cysts.
The present study is to investigate role of iNOS in the pathogenesis of OKCs, DCs and
RCs by evaluating the iNOS expression in the epithelial lining of these cysts.
6.2 REVIEW OF LITERATURE:

A study was conducted to investigate immunohistochemical expression of iNOS and
p53 in OKCs, DCs and RCs and to determine the correlation of these two proteins
within each cyst type. The expression of p53 positive cells was significantly found to
be higher in OKC when compared to DC and RC; but expression of iNOS positive
cells was found to be lower in RC when compared to OKC and DC, hence indicating
no correlation between expression of p53 and iNOS in OKCs , DCs and RCs 1.

An immunohistochemical Study was conducted to study the expression and distribution
of iNOS and heat shock proteins (HSPs) in periapical inflammatory lesions. The results
showed increased epithelial staining intensity of iNOS, HSP60 and HSP70 in Radicular
cyst compared to periapical granuloma, and residual radicular cyst, suggesting their
involvement in inflammatory process and may play a role in the activation and
proliferation of lining epithelium, leading to progression of periapical inflammatory
lesions2.

A study was carried out to determine immunohistochemical correlation of iNOS with
the level of inflammation in 30 specimens of periapical cysts. The underlying fibrous
connective tissue wall of periapical cysts were inflamed with variable degree of
inflammatory cell infiltration; 4 (15%) , 9(30%) and 17(57%) cases present with mild,
moderate and severe inflammation respectively. These results showed statistically
significant increase of iNOS expression in periapical cysts with higher levels of
inflammation when compared to cysts with
lower level of inflammatory infiltrate3.

Another study was carried out immunohistochemically to investigate the interaction of
(IFN-γ) and iNOS producing cells in human radicular cysts . The results revealed that
iNOS producing cells were localized adjacent to IFN-γ producing cells and epithelial
cells of RCs showed significant levels of iNOS production but not IFN-γ .These data
showed that iNOS production could be controlled by autocrine or paracrine effects of
IFN-γ producing cells in radicular cysts and might play pivotal role in periapical
lesions4.

A Study using immunohistochemistry was done to examine the expression of iNOS
and Vascular Endothelial Growth Factor (VEGF) in ameloblastoma and to investigate
the relationship of this expression to angiogenesis and clinical and biological behavior of
the tumor. The results showed that microvessel density counts increased with increasing
expression of iNOS and VEGF in ameloblastoma indicating that their expression may
be closely related to angiogenesis and invasive biological behavior of ameloblastoma5.
6.3 AIMS AND OBJECTIVES OF THE STUDY:

To evaluate expression of iNOS in the lining cells of Odontogenic Keratocyst (OKCs),
Dentigerous cyst (DCs) and Radicular cyst (RCs).

To compare the iNOS expression in lining cells of OKCs, DCs and RCs.

To evaluate the iNOS expression in epithelial lining cells of OKCs to determine its
malignant potentiality.
7. MATERIALS AND METHODS:
7.1 SOURCE OF DATA
Archived paraffin embedded tissues histopathologically diagnosed as Odontogenic Keratocyst,
Dentigerous cyst and Radicular cyst, obtained from the Department of Oral and Maxillofacial
Pathology , Vydehi Institute of Dental Science and Research Center, Bangalore.
7.2 METHOD OF COLLECTION OF DATA
After obtaining the patient consent, biopsy for routine diagnosis is taken. The specimen is then
processed, embedded in paraffin wax and stored at room temperature.

Sample size – 60

group 1- Odontogenic Keratocyst(n=20)

group 2-Dentigerous cyst(n=20)

group 3- Radicular cyst(n=20)
INCLUSION CRITERIA

Archived tissues histopathologically diagnosed as Odontogenic Keratocyst, Dentigerous
cyst and Radicular cyst.
EXCLUSION CRITERIA:

Systemic diseases.
7.3 METHOD

Hematoxylin and Eosin staining will be performed to confirm the clinical diagnosis for
OKC, DC and RC.

Immunohistochemistry will be done on 4μm thick formalin fixed and paraffin embedded
tissue sections mounted on 3- aminopropyl-tri-ethoxysilane(APES) coated slides.

Sections will be dewaxed with xylene and rehydrated through graded series of alcohol and
finally in distilled water.

Sections will be immersed in 0.4% pepsin in 0.01M HCL at 37oC and antigen retrieval will
be done using heat induced antigen retrieval method.

Blocking will be performed with 3% hydrogen peroxide in absolute methanol for 10 minutes
to quench endogeneous peroxidase activity.

Washing will be done with phosphate buffered saline ( PBS) .Sections will be incubated
with 2% bovine serum albumin for 30 mins to block non specific binding.

The sections will be treated with 1:200 dilution of mouse anti-human iNOS monoclonal
antibody (biogenex) for 2 hours at room temperature.

Sections will be treated with secondary detection system (HRP polymer/ label system) for
30 minutes.

The sections will be stained with chromogen 3.3’ diaminobenzidine, counterstained with
heamtoxylin and mounted.

Phosphate buffered saline will be used throughout for washing and rinsing of slides.

Negative controls will be kept with phosphate buffered saline instead of primary antibody.

The expression of iNOS in epithelial lining cells of Odontogenic keratocyst, Dentigerous
cyst and Radicular cyst will be assessed and data obtained will be compared and statistically
analysed.
7.4 STATISTICAL ANALYSIS
Statistical analysis will be made using chi- square test.
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE
CONDUCTED ON PATIENTS OR OTHER HUMAN OR ANIMALS? IF SO, PLEASE
DESCRIBE BRIEFLY.
Yes, biopsy specimen of lesional tissue obtained for routine diagnosis will be processed and used.
7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
YES
8.
LIST OF REFFERENCES :
1. Poomsawat S, Punyasingh J, Vejchapipat P. Immuno-histochemical Expression of p53
Protein and iNOS in Odontogenic Cysts. J Med Assoc Thai 2009; 92 (7):952-60.
2. Suzuki T, Kumamoto H, Ooya K, Motegi K. Expression of inducible nitrous oxide synthase
and heat shock proteins in periapical inflammatory lesions. J Oral Path Med 2002; 31: 48893.
3. Matsumoto MA and Ribeiro DA .Inducible Nitrous Oxide Expression
Correlates with the Level of Inflammation in Periapical Cysts Eur J Dent. 2007 October;
1(4):212-215.
4. Takeichi O et al. Inducible nitrous oxide synthase activity by interferon-gamma- producing
cells in human radicular cysts. Int endodontic j 1999 ;32:124-130.
5. Chen WL, Ouyang KX, Li HG, Huang ZQ, Li JS, Wang JG. Expression of inducible nitrous
oxide synthase and vascular endothelial growth factor in ameloblastoma. J Craniofac Surg.
2009 Jan;20(1):171-5; discussion 176-7
.
9. SIGNATURE OF THE
CANDIDATE:
10. REMARKS OF THE GUIDE:
11. 11.1 NAME AND
DESIGNATION OF GUIDE:
Dr.K.KARPAGASELVI
PROFESSOR AND HOD,
DEPARTMENT OF ORAL AND MAXILLOFACIAL
PATHOLOGY AND MICROBIOLOGY,
VYDEHI INSTITUTE OF DENTAL SCIENCES AND
RESEARCH CENTER.
#82, EPIP AREA, NALLURAHALLI POST, WHITEFIELD,
BANGALORE-560066.
11.2 SIGNATURE
11.3 CO-GUIDE:
11.4 SIGNATURE
Dr.K.KARPAGASELVI
11.5 HEAD OF THE
DEPARTMENT:
PROFESSOR AND HOD,
DEPARTMENT OF ORAL AND MAXILLOFACIAL
PATHOLOGY AND MICROBIOLOGY,
VYDEHI INSTITUTE OF DENTAL SCIENCES AND
RESEARCH CENTER,
#82, EPIP AREA, NALLURAHALLI POST, WHITEFIELD,
BANGALORE-560066.
11.6 SIGNATURE
12. 12.1 Remarks of the Chairman
and The Principal
12.2 Signature