Research Title: Oxidative Damage to Limbal Stem Cells
of the Eye in Response to Bright and Ultraviolet Light
and its Associated Mechanisms
Grant proposal presentation
Deeksha K, JRF
Dept. of Ophthalmology/Yenepoya
Research Centre
Yenepoya University
Funding agency applying for:
Indian Council for Medical Research (ICMR)

Principle investigator
Dr. (Lt. Col)Cynthia Arunachalam,
Professor and head , Dept. of
Ophthalmology, YU
Co- Principle investigators
Dr. Bipasha Bose,
Asst. Professor
Yenepoya Research Centre, YU
Co- Principle investigators
Deeksha K
Research scholar
Yenepoya University
Introduction

Sight is our most important sense
The surface of eye is constantly exposed to the
harshness of external environment.

As a homeostatic mechanism for replenishing the
worn off cells of the ocular surface, a balance is
maintained via the role of ocular stem cells.

Under extreme conditions of harshness like exposure
to bright and/or ultraviolet light, the ocular stem cells
are unable to do the repair mechanisms resulting in
severe impairment of vision and disturbances in the eye.

Eye care scenario in India

15-18.6 million blind people

1/4th of world’s blind population (Eye care in India,2011).

National Survey records- Madhya Pradesh, Rajasthan and
Jammu & Kashmir - high blindness prevalence (2% and
above).

Females - higher preponderance of blindness than males.

World Health Organization- Global bilateral blindness in 45
million people worldwide, ≥ 20% are secondary to corneal
diseases.

LSCD - most severe and difficult-to-treat clinical entities
(Chan et al.; 2013).

National Programme of Blindness(NPCB) estimatescurrently 120,000 corneal blind person in India.

25,000-30,000 corneal blindness cases every year in the
country(Gupta et al.;2013)

Limbal Stem Cell Deficiency(LSCD) is a severe disease,
multifactorial in nature, caused by damage or depletion of
the corneal stem cells in the limbus region of the eye.

This study attempts to discern the molecular mechanisms
responsible for limbal stem cell depletion in response to UV
and bright light
Medicine .academic.nu/137051/limbus/cornea
Rationale of the study



Limbal stem cells play a major role in corneal
epithelial regeneration, and
There are no studies done on the UV and bright
light induced oxidative damages in limbal stem
cells.
Thus the study will help in finding the
mechanisms involved in limbal stem cells during
oxidative damages.
Aim of the study

To investigate the cellular and molecular changes on
Limbal Epithelial Stem Cells(LESCs) and limbal stromal
stem cells of the eye
in response to bright
ultraviolet light and their associated mechanisms.
and
Research Objectives
1.
Successful isolation of limbal epithelial stem cells and
limbal stromal stem cells from the eyes of mice and
human.
2.
Characterization and expansion of limbal epithelial
and stromal stem cells.
3.
Exposure of limbal epithelial stem cells and limbal
stromal stem cells with bright light and ultraviolet
light.
4.
Assessment of cellular and molecular changes and
signaling mechanisms in limbal epithelial stem cells and
limbal stromal stem cells in response to bright and
ultraviolet light
Methodology
 Isolation of limbal epithelial stem cells from eyes:

Limbal tissues from mice and human will be dissected
and further processed.

The limbal tissues will be isolated as per the protocol
and further processed and cultured for study of effect
of UV and bright light on limbal stem cells.
Characterization and expansion of limbal epithelial
and limbal stromal stem cells:
a)Primary Culture for Limbal Stem Cells
b) Isolation of pure limbal stem cells- FACS
 Cell size and Hoechst 33342 dye efflux ability.
 For cell sorting as Hoechst negative fraction and
positive for p63, ABCG2 , PAX6, etc. markers for limbal
stem cells
 Expansion of limbal stem cells
Characterization of limbal stem cells
c)Western Blot Analysis
d)Gene expression analysis
 Exposure dosage of limbal stem cells to UV and
bright light
 Assessment of changes in limbal epithelial and
stromal stem cells in response to bright and
ultraviolet light and its associated mechanisms:

Measurement of ROS Production

Antioxidant Enzyme Activities

Cell signaling pathways associated with oxidative
stress: Changes in the cell signaling molecules -
assessed - qRT-PCR, western blots, flow cytometry
and ELISA.
Work plan
Months
Work plan
6
Objective 1
Objective 2
Objective 3
Objective 4
Data analysis
12
18
24
36
Expected outcome

Findings of this study will give clues to modulate the
signalling pathways to overcome Limbal Stem Cell
Deficiency that can be used as a therapy.
Budget estimates
Expenses
JRF– 16000 INR /
First year
Second year
Third year
Total
1,92,000/-
1,92,000/-
2,16,000/-
6,00,000/-
2,00,000/-
2,00,000/-
1,00,000/-
5,00,000/-
month
Recurring
Mice, Culture media,
disposable pipettes
and culture plates
,antibodies for
western blot and
immunohistochemistry
, antioxidant enzyme
assay kits, PCR kits:
Primers & Probes
Budget estimates
Expenses
First year
Second year
Third year
Total
Non- recurring
UV and bright light
source, Micropipette,
1,00,000/-
-
-
1,00,000/-
Contingency
5000/-
5000/-
5000/-
15,000/-
Travel
10,000
10,000
30,000
50,000
20,000/-
15,000/-
15,000/-
50,000/-
5,27,000/-
4,22,000/-
3,66,000/-
13,15,000
tips, glass slides, media
filtration unit, plastic
ware, glassware
Overhead Charges
Grand Total (Rs.)
Ethical clearance

For animal experimentation: C57BL6J mice will be
used for isolating the limbal epithelial and stromal
stem cells. The use of mice will require ethical
clearance from the Institutional Animal Ethics
Committee.

For obtaining human samples: The limbal tissues for
isolating limbal stem cells will be obtained from
patients visiting Department of Ophthalmology for
cataract surgery. This will require ethical clearance
from the Human ethics committee.
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
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