1. 1: GLAUCOMA TREATMENTS AND ORIGINS
GLAUCOMA TREATMENTS AND ORIGINS
ARIEL CHOU
CAPS RESEARCH/INTERNSHIP

2. 2: GLAUCOMA TREATMENTS AND ORIGINS
Glaucoma Treatments and Origins
Glaucoma is a condition in which the optic nerve is subject to damage because of
unusually high intraocular pressure(IOP). If left untreated, permanent vision loss is likely. Vision
damage starts with blind spots in the field of vision, advancing to what is known as tunnel vision,
and then to blindness. More than two million people in the U.S. have glaucoma, and over 80
thousand are legally blind (Bekker, 2006). Glaucoma is the second most common eye disease
that leads to blindness in the world, over 60 million people have it ( Boland et al., 2013).
There are two types of Glaucoma, open-angle and closed-angle, judged based on whether
the drainage canal for the aqueous humor is open or closed. In a normal eye, the aqueous humor
is produced and drained. But in an abnormal eye, the aqueous humor builds up and,
consequently, the intraocular pressure goes up. High intraocular pressure is what leads to damage
of the optic nerve. Studies have shown that reducing IOP has decreased the chances of glaucoma
occurring in patients and the rate of new damage in glaucoma patients ( Boland et al., 2013).
Open-angle glaucoma is more prevalent, comprising of 90% of all glaucoma cases. In this case,
the angle between the iris and cornea is open and the progression of glaucoma is slow. Narrow
angle glaucoma has a small angle, therefore when the drainage canal is blocked sudden attacks
may occur. Another form is normal tension glaucoma(NTG), in which patients have normal
intraocular pressure but still having glaucoma (Bekker, 2006). Most of the medication on the
market today target the reduction of aqueous humor production and decreasing IOP.
Learning more about the genetic basis of what causes glaucoma will aid in the
development of new treatments for heritable eye conditions (Fingert, 2011). So far, large
pedigree and twin studies have shown that glaucoma is hereditary. In the past 20 years, two types
of gene mutations have been singled out that cause primary open-angle glaucoma. The first type

3. 3: GLAUCOMA TREATMENTS AND ORIGINS
is capable of causing glaucoma by itself with little help from environmental factors. Individuals
that carry this type of mutation almost always develop glaucoma. For example, mutations in the
myocilin and optineurin genes are cases of this first mutation. The second type of mutations need
environmental factors to assist it in causing glaucoma. These types of genetic risk factors are
more common in POAG(primary open angle glaucoma) patients and controls (Fingert, 2011).
The glaucoma causing mutation in the gene myocilin was first detected when observing
large pedigrees with juvenile open angle glaucoma. A large percent of people with JOAG also
have the myocilin mutation, 8-36% of patients in some published case series. Myocilin mutation
causes higher IOP because in normal situations, the myocilin protein is secreted by many cell
types in the eye for no known reason. However mutated myocilin produces an abnormal protein
that is poorly secreted and stuck within the trabecular meshwork cells. The trabecular meshwork
cells are tissue located at the base of the cornea that drain the aqueous humor via the anterior
chamber, located behind the cornea. The accumulation of these cells can be toxic and lead to
decreased aqueous outflow, high IOP, and glaucoma (Fingert, 2011). By observing large normal
tension glaucoma pedigree, Optineurin was identified as a glaucoma-associated gene. It was
shown that a GLU50LYS( From Glutamate (E) to Lysine (K) at position 50) mutation was what
caused this. These mutations are different in that they are not associated with high IOP like the
myocilin gene in most but not all populations. The affects of this gene depends on what
population is being observed. Experiments with transgenic mice have shown that optineurin
leads to the apoptosis of retinal ganglion cells. By observing African-American NTG(normal
tension glaucoma) pedigrees it has been shown that copy number variations of TBK1(TANK-
binding kinase 1) can cause a dysregulation of NF-kB(nuclear factor kappa-light-chain-enhancer
of activated B cells), which is a protein complex that controls the transcription of DNA

4. 4: GLAUCOMA TREATMENTS AND ORIGINS
signaling, that ultimately causes the apoptosis, programmed cell death, of retinal ganglion cells
and the development of NTG. Mutations in the WDR36(WD repeat-containing protein 36) gene
also caused some cases of POAG to occur although what role it plays is still in debate (Fingert,
2011). These glaucoma genes are together responsible for only less than 5 percent of POAG. The
rest are either caused by a group of genes or added environmental factors.
There are two main types of treatments for Open-Angle Glaucoma. One category is non-
invasive treatment which involves "topical or oral agents that decrease aqueous humor
production or augment outflow (Boland et al., 2013)." Gene therapy is also being studied. By
targeting the basis of the disease, scientists can predict if a person is susceptible to glaucoma
from viewing the person's gene code. Treatments involving the inhibition of myocilin production
could be used not just to treat IOP but also before glaucoma sets in. Taking vitamin supplements
such as Vitamin C, vitamin B1, chromium, zinc, and rutin may reduce IOP. Other treatments
include invasive procedures, such as eye surgeries like trabeculectomy, laser peripheral
iridotomy, and argon laser trabeculoplasty. Trabeculectomy is aimed at increasing the outflow of
aqueous humor by opening the drainage canals or making an opening in the iris. Laser peripheral
iridotomy is used to treat narrow angle glaucoma by essentially opening up the narrow angle
between the cornea and the iris. Argon laser trabeculoplasty is used as a last resort or when the
patient cannot use other glaucoma treatments or eyedrops. In this procedure, 180 degrees of
trabecular meshwork is hit with lasers, increasing the outflow of aqueous humor (Bekker, 2006).
Although these treatments are all used on patients with hopes of helping them get better. An
overall evaluation of 22 potentially relevant studies has shown that not a big difference was
observed on the neuroprotective effects of oral and topical medical therapy (Sena, Ramchand, &
Lindsley, 2010). A comparative experiment for the effectiveness of these treatments conducted

5. 5: GLAUCOMA TREATMENTS AND ORIGINS
by the U.S. Preventive Services Task Force showed that trabeculectomy was better at decreasing
IOP compared with trabeculoplasty or medical therapy but led to greater risk for adverse
outcomes. Overall, these surgical treatments all had a positive effect on the patients, they
prevented progressive visual field loss and optic nerve damage. However, they were unable to
find sufficient evidence comparing these treatments to one another (Boland et al., 2013).
Picture of Trabecular Meshwork:
One of the studies that the lab is conducting is a human clinical study identifying
biomarkers for retinal vein occlusions, which is essentially a stroke to the eye. There is currently
no known treatment. One of the risk factors involve people with diabetes. The lab took 20 blood
samples from patients with RVO and had control patients with and without diabetes. Serum was
extracted from these blood samples. This serum sample was dotted onto microcellulose
membrane to be tested for AGE proteins (Advanced Glycation End-Product). A dot blot works
by dotting a mixture containing the molecule to be detected directly onto the membrane. This is
then followed by detection using nucleotide probes (Dot blot, 2013). We used a system called
densitometry to measure the intensity of the AGE signal for each serum sample. We found that
there was no difference between the groups. Below is a picture of our dotblot and comparison of
the intensity of AGE serum levels in our two samples.

6. 6: GLAUCOMA TREATMENTS AND ORIGINS
(Kaja, 2013)
Another experiment that we had done at the lab was we were trying to see FRET or
Forster Resonance Energy Transfer take place. We performed cell transformation to insert a
certain segment of DNA into bacterial cells. Then we grew these bacteria on plates and picked
colonies and streaked them on plates. We were trying to see if fluorescence could be seen after
we performed this cell transformation. Essentially, in the absence of glutamate, CFP and YFP are
in such proximity and orientation for FRET to occur. When excited at 435 nm, CFP acts as
energy donor to YFP. Emission is maximal at the peak emission wavelength of YFP, 530 nm.
Upon binding of glutamate, the ybeJ protein undergoes a conformational change, preventing
FRET from occurring. Light is emitted at the peak emission of CFP, 477 nm. Below is a picture
that explains FRET (Kaja, 2013)

7. 7: GLAUCOMA TREATMENTS AND ORIGINS
(Kaja,2013)
Besides these two experiments, I also assisted with a western blot experiment, learned
about confocal microscopy and fluorescent molecules/probes. The western blot aims to use gel
electrophoresis to separate native proteins. " Confocal microscopy is an optical imaging
technique used to increase optical resolution and contrast of a micrograph by using point
illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker
than the focal plane. It enables the reconstruction of three-dimensional structures from the
obtained images (Confocal microscopy, 2013)." Dr. Kaja showed me cells with fluorescence in
them under the microscope. Additionally, I was given the opportunity to culture and maintain
certain cell lines. We grew and maintained cell lines under controlled conditions (Cell culture,
2013). We had to spray everything with alcohol before it could be put under the tissue culture
hood.
FRET is an experiment involving the energy transfer between two
chromophores. A donor chromophore, in its excited state, can transfer
energy to an acceptor chromophore through dipole-dipole coupling.
FRET testing can determine if two fluorophores are within a certain
distance of each other. (FRET, 2009)

8. 8: GLAUCOMA TREATMENTS AND ORIGINS
(Kaja, 2013)
All in all, great strides have been made to try and find a cure for glaucoma. Right now,
treatments are focused in preventing further increase in intraocular pressure because that is the
main cause of damage to the optic nerve. With better technology and conduction of more studies
it won't be long before we can find a treatment that can treat and hopefully cure glaucoma
patients.
GFAP Phalloidin
Hoechst Merge
Backscatter
DIC
Astrocyte cell isolated from Brown
Norway rat, 6 weeks of age
GFAP is a marker protein for
astrocytes, phalloidin labels actin
filaments, Hoechst stains DNA in
nuclei.
DIC = differential interference
contrast
Scale 25 µm

9. 9: GLAUCOMA TREATMENTS AND ORIGINS
References
Bekker, M. (2006). Glaucoma. In J. L. Longe (Ed.), The Gale Encyclopedia of Nursing and
Allied Health (2nd ed., Vol. 2, pp. 1194-1199). Detroit: Gale. Retrieved from
http://go.galegroup.com/ps/i.do?id=GALE%7CCX2584700384&v=2.1&u=over30272
&it=r&p=GPS&sw=w
Boland, M. V., Ervin, A., Friedman, D. S., Jampel, H. D., Hawkins, B. S., Vollenweider,
D., Chelladurai, Y., Ward, D., Suarez-Cuervo, C., & Robinson, K. A. (2013).
Comparative Effectiveness of Treatments for Open-Angle Glaucoma: A Systematic
Review for the U.S. Preventive Services Task Force. Annals Of Internal Medicine,
158(4), 271-279.
Burroughs, S., Kaja, S., & Koulen, P. (2011, June 01). Quantification of deficits in spatial
visual function of mouse models for glaucoma. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantification of Deficits in spacial
visual function of Mouse models for glaucoma
Cell culture. (2013, March 28). In Wikipedia, The Free Encyclopedia. Retrieved 04:04,
April 22, 2013,
from http://en.wikipedia.org/w/index.php?title=Cell_culture&oldid=547442158
Confocal microscopy. (2013, March 13). In Wikipedia, The Free Encyclopedia. Retrieved
03:53, April 22, 2013,
from http://en.wikipedia.org/w/index.php?title=Confocal_microscopy&oldid=5438891
83

10. 10: GLAUCOMA TREATMENTS AND ORIGINS
Dot blot. (2013, March 26). In Wikipedia, The Free Encyclopedia. Retrieved 03:41, April
22, 2013, from http://en.wikipedia.org/w/index.php?title=Dot_blot&oldid=547134584
Fingert, J. H. (2011). Primary open-angle glaucoma genes. Eye, 25(5), 587-595.
doi:10.1038/eye.2011.97
FRET. (2009, October 7). In Wikipedia, The Free Encyclopedia. Retrieved 03:37, April 22,
2013, from http://en.wikipedia.org/w/index.php?title=FRET&oldid=318463164
Kaja, S. (2013). Work in UMKC School of Medicine Vision Research Center Labs
[Powerpoint Slides]. Retrieved from
https://mail.google.com/mail/u/0/#imp/13dadc5ef9e4eb92
Sena, D. F., Ramchand, K., & Lindsley, K. (2010, Feb 17). Neuroprotection for treatment of
glaucoma in adults. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20166085
(DF, K & K, 2010)