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6 Gulani Refractive Descemet’s Endothelial Keratoplasty (REFDEK) Classification
Targeting Emmetropia in Endothelial Keratoplasty
Fig. 13.7 Poorly done DSAEK or failing endothelial keratoplasty can be rejuvenated and
enhanced using REFDEK principles
Fig. 13.8 Endothelial
in a case of radial
A. Excimer Laser ASA (advanced surface ablation) can be used to correct residual
refractive errors to emmetropic outcomes (Fig. 13.9).
B. Lens implant-based optical manipulations can be undertaken, i.e., piggyback
lens, toric lens rotation and only if needed, lens exchange surgery too as we
would in any patient expectant of refractive outcomes (Fig. 13.10).
C. Miscellaneous: Given proper indications, I do not see a contraindication to even
using collagen cross-linking in cases of endothelial keratoplasty in RK cases
once refractively stable or touch of procedures like astigmatic keratotomy, Femto
laser astigmatic keratotomy, conductive keratoplasty, etc.
In summary then, excited as we are about the surgical evolution of endothelial
keratoplasty from DSEK to DMEK and PDEK, let us also raise the bar on ourselves
and deliver vision at the highest level we can through this exciting procedure once
again not only in restoring anatomic function but also vision outcomes.
With technologies today ranging from new-generation excimer lasers, femtosecond technology, lens implants with individualized optics, refractively fine tuning procedures, and permanizing techniques like collagen cross-linking, we can
truly live up to ever-enchanting properties of the cornea which not only provides
Fig. 13.9 The patient in
Fig. 13.4 at 6 months
underwent laser vision
surgery to 20/20
Fig. 13.10 Toric lens implant with endothelial keratoplasty planned to emmetropic outcome
transparency to see the outside world but also has the optical power to focus our
May I take this opportunity then to reiterate my respects to our cornea by calling
it our “Vision rehabilitative platform!”
Targeting Emmetropia in Endothelial Keratoplasty
My Personal Techniques
Minimal DSAEK Surgery: Gulani Key Hole Transplant
(i) Specifics: topical anesthesia, sutureless superior incision, single instrument
• Since most of the referred cases of PBK have had cataract surgery with a
temporal incision, my preferred approach is a superior limbal incision which
allows me to operate in an untouched area with good self-sealing incision
architecture. This area also is cosmetically under the upper lid and therefore
next day postop these patients look like they have had no surgery.
• Single instrument (Gulani scorer and peeler) allows for less tissue manipulation and elegant working in a closed system, double paracentesis arena (until
the main incision is entered).
• Topical anesthesia allows for faster recovery and ocular medication toward
an encouraging outcome.
• Sealant option if needed (Fig 13.11).
Fig. 13.11 ReSure sealant was used in this case for excellent refractive and anatomic outcome
1. Facts about the cornea and corneal diseases. National Eye Institute. National Institutes of
Health, 1 May 2013. Web. 28 Oct 2014. http://www.nei.nih.gov/health/cornealdisease/.
2. Cassin B, Solomon S. Dictionary of eye terminology. Gainesville: Triad Publishing Company;
3. Sayegh F. The correlation of corneal refractive power, axial length, and the refractive power of
the emmetropizing intraocular lens in cataractous eyes. Ger J Ophthalmol. 1996;5(6):328–31.
PubMed. Web. 28 Oct 2014. http://www.ncbi.nlm.nih.gov/pubmed/9479513.
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Bowman's layer in some mammals relationships with other constituent corneal structures. Eur
J Anat. 2002;6(3):133–40.
5. Corneal conditions. Cornea Research Foundation of America. Web. 5 Oct 2014. http://www.
6. Stodola E. Cornea surgeons compare thin DSAEK and DMEK as options for endothelial keratoplasty procedures. EyeWorld. 1 Jan 2013. Web. 28 Oct 2014. http://www.
7. Fernandez M, Natalie A. How to perform Descemet’s stripping automated endothelial keratoplasty. American Academy of Ophthalmology. EyeNet Magazine, 1 Jan 2007. Web. 28 Oct
8. Shaw J. DMEK: A new contender for corneal transplantation. American Academy of
Ophthalmology. EyeNet Magazine, 1 Sept 2012. Web. 28 Oct 2014. http://www.aao.org/publications/eyenet/201209/cornea.cfm.
9. Agarwal A. Pre Descemet’s Endothelial Keratoplasty (PDEK): A novel method of endothelial transplantation. EyeWorld. 1 Jan 2014. Web. 28 Oct 2014. http://www.eyeworld.org/
10. Agarwal A, Priya N. PDEK: a revolution in corneal transplantation. Ophthalmology
Management. 1 Mar 2014. Web. 28 Oct 2014. http://www.ophthalmologymanagement.com/
11. Gulani AC. Tips, insights, and techniques from other surgeons. In: Price F, editor. Textbook –
DSEK what you need to know about endothelial keratoplasty, vol. 9. Thorofare: SLACK Inc;
2009. p. 113–5.
12. Gulani AC. Key hole corneal transplant. Surgical techniques in ophthalmology corneal surgery. JP Publ. 2009;25:154–6.
13. Gulani AC. Corneoplastique™: art of vision surgery. Indian J Ophthalmol. 2014;62:3–11.
14. Gulani AC. Evaluating the impact of femto laser-assisted capsulotomy. Cataract Refract Surg
Today Eur. 2014;9(2):36–50.
15. Gulani AC. Decoding corneal scars: straight to 20/20. Ophthalmol Times. 2014;39(4):6–12.
16. Gulani AC. Corneoplastique. Tech Ophthalmology. 2007;5(1):11–20.
Rhokinase Inhibitors for Endothelial
Dhivya Ashok Kumar
14.3 Rhokinase Enzyme and Its Role
14.4 In Vitro Studies with Monoclonal Endothelial Cells (MCECs)
14.5 In Vivo Rhokinase Inhibitors
14.6 Human Trials
Cornea is a unique structure with varied physiological mechanism working for its
transparency and integrity. Orientation of collagen, arrangements of keratocytes,
fluid transport mechanism, endothelial cell function and proteoglycan proportions
are some of the factors which contribute to its physical nature. Conditions which
hinder the above normal systematic functions are known to affect corneal transparency. Corneal endothelial cell is one vital structure which is highly efficient in preserving the vitality of the corneal layers.
D.A. Kumar, MD, FICO
Consultant and Head R&D, Dr. Agarwal’s Refractive and Cornea Foundation,
Dr. Agarwal’s Eye Hospital and Eye Research Centre, 19, Cathedral Road,
Chennai, TN 600086, India
© Springer India 2016
S. Jacob (ed.), Mastering Endothelial Keratoplasty,
Corneal endothelium is a 5-μm thick single monolayer of cell spread along the posterior most part of the cornea. It has active pumps for ionic and fluid transport across
it. Conditions like trauma, congenital cell damage (Fuchs’ endothelial dystrophy or
posterior polymorphous dystrophy) and post-surgery (cataract or vitreoretinal) can
affect its routine function. Abnormal cell morphology in normal cell count or low
pre-operative cell count or endothelial damage intra-operatively can cause this. It is
known that endothelial cell has less potential to regenerate due to the following
reasons: (1) cell-to-cell contact inhibition, (2) G1 phase arrest, (3) less response to
microenvironment stimulators, and (4) TGF-B2 suppression of S phase . Though
there are varied surgical treatment options like penetrating and endothelial keratoplasty in advance cases of corneal endothelial decompensation, only few medical
management options have been tried in early cases.
Rhokinase Enzyme and Its Role
The Rho/ROCK pathway is known to be involved in regulating the cytoskeleton,
cell migration, cell apoptosis, and cell proliferation. ROCK inhibitor Y27632 specifically blocks ROCK1 (P160ROCK). Chemically, it is trans-4-(1-aminoethyl)-N(4-pyridyl) cyclohexane carboxamide dihydrochloride. It has shown to have some
promising effects in corneal endothelial decompensation [2–4]. ROCK regulates the
formation of actin stress fibres assembly and cell contraction (Fig. 14.1). In addition
Fig 14.1 Schematic picture showing the functions of rhokinase pathway
Rhokinase Inhibitors for Endothelial Decompensation
to the primary function in cytoskeleton remodelling and migration, Rho signalling
pathway has been shown to be involved in the regulation of other biological processes like gene transcription, G1 cell cycle progression and apoptosis . It has
been demonstrated that ROCK inhibitors use both cyclin D and p27 through PI
3-kinase signalling to promote corneal endothelial cell proliferation . The ROCK
pathway is involved in regulating various cell functions such as migration, apoptosis, differentiation, and proliferation which are cell-type dependent [7–9]. Because
the ROCK pathway is involved in a variety of diseases, ROCK inhibitors have been
developed as therapeutic drugs for endothelial dysfunction .
In Vitro Studies with Monoclonal Endothelial Cells
Okumura et al. immunostained MCECs for the cell cycle population marker Ki67
. MCECs cultured with Y-27632 showed the presence of a larger number of
Ki67-positive cells compared with the controls. Quantitative flow cytometric analysis revealed an increased number of Ki67-positive cells in MCECs cultured with
Y-27632. Additionally, BrdU-labelling assays were conducted to confirm the effect
of Y- 27632 on cell proliferation. They revealed a significantly greater number of
BrdU-positive MCECs among the Y-27632-treated cells compared with the control
cells. This demonstrated that Y-27632 plays a central role in the proliferation of
In the same study, MCECs were cultured to confluence over a 14-day period and
were scraped with a plastic pipette tip to create linear defect sites, and the culture
was then continued for a further 24 h in fresh medium with or without 10 mM
Y-27632. The mean wound distance was noted to be significantly shorter in the
Y-27632 group than in the control group . These findings suggested that Y-27632
promoted wound healing in the in vitro model. A study by Pipparelli et al. showed
that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours . It induces
changes in cell shape, increases cell adhesion, and enhances wound healing ex vivo
and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in
human therapy .
In Vivo Rhokinase Inhibitors
In an in vivo animal experiment, a corneal endothelial wound was initially made,
then 10 mM Y-27632 was applied topically six times daily in the form of eye drops
over a 2-day period in rabbits . Slit-lamp microscopic examination showed that
corneal transparency and corneal thickness recovered faster in the Y-27632 group
compared to the control group. Ultrasound pachymetry revealed that the corneal
thickness was significantly thinner in the Y-27632 group compared to the control
group after 48 h of treatment. Rabbits were then euthanized, and the wound area of
the corneal endothelium was evaluated by Alizarin red staining following enucleation after 48 h. The mean wound area of the Y-27632 group was significantly
smaller than that of the control group. These results demonstrated that the topical
administration of Y-27632 eye drops enhances endothelial wound healing .
In an animal model, Okumura compared rhokinase inhibitors with endothelial
cell injection in vivo in rabbit eyes . Rabbit eyes were injected with cultivated
rabbit corneal epithelial cells (RCECs) with the selective ROCK inhibitor Y-27632
and those cultivated RCECs without Y-27632. A control corneal endothelial dysfunction model was also kept. The eyes were assessed after 48 h. Rabbit eyes
injected with cultivated RCECs combined with Y-27632 recovered complete transparency of the cornea. In contrast, eyes injected with cultivated RCECs without
Y-27632 and control eyes exhibited a hazy cornea with severe oedema. Injection of
cultured MCECs with a ROCK inhibitor regenerated healthy corneal endothelium
and recovered corneal transparency in the monkey model, similar to the findings in
the rabbit model . The first human clinical trial has now been initiated.
Koizumi et al. reported the effect of rhokinase inhibitor in human eye in a patient
with Fuchs’ corneal dystrophy . The patient was treated by a corneal endothelial
denudation in the prepupillary region followed by the topical administration of a
selective ROCK inhibitor, Y-27632, as eye drops for 1 week (50 mL of 10 mM
ROCK inhibitor, Y-27632, was applied topically as eye drops, repeated six times
daily for 7 days) and followed up for 24 months. Corneal clarity was noted to
improve after 2 weeks of treatment and endothelial function was observed to sustain
for 24 months. In a trial by Okumura et al., the effect of Y-27632 eye drops after
transcorneal freezing was evaluated in eight corneal endothelial dysfunction
patients: four central corneal oedema patients and four diffuse corneal oedema
patients . The clinical study showed that Y-27632 eye drops effectively improved
corneal oedema of corneal endothelial dysfunction patients with central oedema.
Although corneal transplantations provide considerable clinical benefits, graft
rejection, primary graft failure, and the shortage of donor corneas are problems that
still need to be overcome. Rhokinase inhibitors are in the early stage of its functional assessment in human eyes, and reports exhibit that ROCK inhibitor converts
corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue . Further investigations are necessary; the topical instillation of the
ROCK inhibitor might be a clinically applicable and a less invasive therapeutic
modality for the treatment of corneal endothelial dysfunction in future.