Ординатура / Офтальмология / Учебные материалы / Clinical Diagnosis and Management of ocular trauma

206

Clinical Diagnosis and Management of Ocular Trauma

cells compared with those treated with anti-SDF-1.

Corneal epithelial stem cells reside in the basal region

Antibody treatment generally reduced the degree of

of the limbus, and are involved in renewal and

gfp+ stem cell recruitment and incorporation into the

regeneration of corneal epithelium. Following injury,

CNV lesions, compared with the control. Treatment with

basal stem cells are stimulated to divide and undergo

either antibody also significantly reduced the size of the

differentiation to form transient amplifying cells (TAC).

CNV lesions. These results indicate that homing and

Subsequent cell divisions result in non-dividing post-

adhesion of progenitor cells to CNV may be targeted

mitotic cells (PMC),which migrate towards the central

differentially or in combination to prevent CNV.

cornea and superficially forming terminal differentiated

cells (TDC).

Role of Corneal Stem Cell in

The first step is the isolation of healthy corneal

epithelial cell, and these are collected from the

Pediatric

unaffected eye of the individual to be treated if the

problem is in one eye only, or from donor material

Stem cell research offers hope to countless patients

if the problem is bilateral. These cells are then cultivated

whose conditions have here to fore been deemed

in the laboratory for a couple of weeks under carefully

incurable. Better understanding of stem cell behaviors

monitored conditions, so that they grow into a nicely

and functions will lead to insights into biological

stratified cellular multi-layer, which resembles corneal

mysteries encompassing the fields of angiogenesis,

epithelium, in situ. This can then be used for the surgical

development, tissue homeostasis, wound healing, and

repair of the ocular surface. One problem, however

carcinogenesis. Clarity of vision requires smooth ocular

is that cells grown in this way on regular cell culture

surface on which the corneal epithelial cells undergo

dishes are difficult to detach from the underlying plastic

continuous turnover every 3 to 10 days.Tragically,

without damaging them in some way. Now, however,

many patients are blinded and devastated by severe

evolving research has provided a way to obtain intact,

ocular surface diseases due to limbal stem cell

isolated corneal epithelial cell sheets with minimal

deficiency even though, besides opaque cornea, their

harm. The secret lies in the use of a novel temperature-

eyes are otherwise healthy. Corneal stem cell

responsive culture surface, which readily supports cell

transplantation offers hope by creating clear windows

growth, but also allows easy cell release.

for these eyes; unfortunately, the long-term successful

outcome remains limited. The nature of corneal

OCULAR SURFACE STEM CELLS (FIG. 32.1)

epithelial stem cell is poorly understood, but many

More than two decades ago, Thoft and Friend described

circumstantial evidences suggest the presence of

the X,Y and Z hypothesis which explained how corneal

“source cells”

in the limbal region of the eye.

epithelial cells are continuously shed from the surface

Nonetheless, the precise biomarker of corneal stem

and regenerated by cells from the periphery. The limbal

cell remains elusive. The stem cell puzzle can be solved

stem cell residing in the alisades of Vogt are now

with application of the fundamental scientific method-

believed to be the never-ending source of corneal

asking salient questions at the right time and finding

epithelial cells. Adult corneal and conjunctival stem cells

answers using keen observations and proper tools.

represent a small, quiescent subopulation of epithelial

Readily accessibility and structural simplicity of the

cells of the ocular surface. The limbus is a 1.5 mm-

cornea lend themselves to study of the stem cell biology.

to-2 mm-wide area that straddles the cornea and

The ability to identify and isolate corneal stem cell will

be a gateway to meaningful investigation into its

biology. This advance will also have direct impact on

improving the efficacy of promising stem-cell-based

therapies, including limbal stem cell transplantation.

Technical advances in the surgical reconstruction

of severely injured or diseased ocular surfaces have

improved the quality of the life for many of those

affected. An ever-present hurdle, however, is the

limited availability of suitable donor material, partly

because of this, the concept has emerged of fabricating

transplantable tissue constructs by expanding small

biopsies of corneal epithelial cells taken from the limbs.

Conjunctival stem cells represent a small quiescent

Fig. 32.1: Ocular surface stem cells

subpopulation of epithelial cells of ocular surface.

Applications of Stem Cell Therapy in Ophthalmology

207

bulbar conjunctiva. Corneal epithelial stem cells reside

HOPE OF STEM CELLS FOR UNTREATABLE

in the basal region of the limbus, and are involved

EYE DISEASE

in the renewal and regeneration of the corneal

There are practical and ethical issues to the use of

epithelium. Following injury, these limbal basal stem

embryonic stem cells and the alternative of using

cells are stimulated to divide and undergo

somatic or adult stem cells has major advantage in

differentiation to form transient amplifying cells (TACs)

terms of immediate clinical application. The adult

(Fig. 32.2). Subsequent cell divisions result in non-

human eye harbors stem cells in the limbal region,

dividing post-mitotic cells (PMCs), which then

in the conjunctiva, the pars plana and plicata of the

terminally differentiate and migrate towards the central

retinal ciliary margin and adult human retina.

cornea and superficially, taking on the final corneal

IDENTIFICATION OF STEM CELLS

phenotype as terminal differentiated cells (TDCs). Their

presence allows continued replacement and

Several putative stem cells markers have been

regeneration of tissues following injury, thereby

proposed, although no single molecular marker that

maintaining a steady-state population of healthy cells.

is specific for stem cells has been identified.

Conjunctival and corneal epithelial cells have been

Taking advantage of the slowcycling characteristic

shown to belong to two separate, distinct lineages.

of stem cells, an indirect method of labelling stem cells

was developed.

Unlike corneal epithelium, conjunctival epithelium

Another characteristic of stem cells is their capacity

consists of both non-goblet epithelial cells as well as

to remain highly proliferative in vitro. Cells that have

mucin-secreting goblet cells. Wei et al showed that both

the highest proliferative capacity (holoclones-with less

these populations of cells arise from a common

than 5% of colonies being terminal) are considered

bipotent progenitor cell. The conjunctival forniceal

stem cells. Pelligrini et al showed by clonal analysis that

region appears to be the site that is enriched in

nuclear P63 was abundantly expressed by epidermal

conjunctival stem cell, although stem cells are also likely

and limbal holocolones, but were undetectable in

to be present in other regions of the conjunctiva.

paraclones, suggesting that P63 might be a marker

of keratinocyte stem cells.

Disease Arising from Stem Cell Deficiency due to

Ocular Surface Causes

Ocular surface failure may be of two types according

to Tseng et al depending on the epithelial phenotype

as identified by impression cytology. Type 1 failure

characterized by squamous metaplasia where the non-

keratinized corneal epithelium is converted to a

keratinized epithelium. Type 2 failure is characterized

by limbal stem cell deficiency where the normal corneal

epithelial is replaced by conjunctival epithelium. Limbal

stem cell deficiency can be caused by the variety of

hereditary or acquired disorders. Inherited disorders

include aniridia keratitis and kratitis associated with

multiple endocrine deficiency, in which limbal stem

cells may be congenitally absent or dysfuntional.

Acquired conditions that may result in limbal stem cell

deficiency include Stevens-Johnson syndrome,

chemical injuries, ocular cicatricial pemphigoid, contact

lens-induced keratopathy, multiple surgeries or

cryotherapies to the limbal region, neurotrophic

keratopathy and peripheral ulcerative keratitis.

Acquired disorders from the majority of cases seen in

the clinical setting.

Limbal stem cell deficiency is characterized by

persistent or recurrent epithelial defects, ulceration,

Fig. 32.2: Limbal epithelial stem cell therapy

corneal vascularization, chronic inflammation, scarring,

Applications of Stem Cell Therapy in Ophthalmology

209

CULTIVATED VS DIRECT LIMBAL

Hisatomi T et al have provided direct and novel

TRANSPLANTATION

evidence for the migration of BM and HSC cells into

The potential problems with direct limbal trans-

the sclera differentiating into macrophages and

plantation include harvesting a relatively large amount

dendritic cells.Vast infiltration of BM and HSC cells was

of limbal tissue (upto 6 clock hours) from healthy

found to be part of inflammatory process in EAU.

donor eyes. Pellegrini et al were the first to describe

Corneal epithelial stem cells are known to be

culturing of limbal stem cells ex vivo thereby spear-

localized to the basal layer of the limbal epithelium,

heading ophthalmology into the field of regenerative

providing a model system for epithelial stem cell

medicine. Technically cultivated stem cells have the

biology; however the mechanisms regarding the

disadvantages of the long procedure.

maintenance of these stem cells in their specialized

1.

It takes 10 days to 2 weeks for the cells to grow.

niche remain poorly understood. N-cadherin is a

2.

A dedicated stem cell laboratory is required.

member of the classical cadherin family and has

previously been demonstrated to be expressed by

Bioengineered Ocular Surface Equalents for

hematopoietic stem cells. In the present study Hayashi

Transplantation (Fig. 32.5)

R, et al have demonstrated that N-cadherin is expressed

Limbal autograft overcomes the problem of

by putative stem/progenitor cells as well as melanocytes

in the human limbal epithelial stem cell niche.

immunologic rejection, but because large segments are

Additionally, they have demonstrated that upon in vitro

required this places the donor eye at the risk of surgically

culture using 3T3 feeder layers, loss of N-cadherin

induced donor stemcell deficiency. The use of

expression occurs with cell proliferation. These results

autologus cultivated limbal stemcell has overcome this

indicate that N-cadherin may be a critical cell-to-cell

problem. for this procedure only a small limbal biopsy

adhesion molecule between corneal epithelial stem/

is needed (approximately 2mm2),which minimizes the

risk of damage to the donor eye. This is then cultivated

progenitor cells and their corresponding niche cells in

the limbal epithelium.

on various substrates, such as human amniotic

Sun CC demonstrated that intact human AM may

membrane or fibrin based substrates, which results in

prevent cultured human limbal epithelial cells from

a composite graft which is then transplanted on the

undergoing apoptosis. IL-1RA might be a candidate

diseased eye. With this procedure reasonable success

mediator to exert as an anti-apoptotic molecule during

upto one year of follow-up has been achieved. More

the interaction between human limbal epithelial cells

recently Tan DT et al demonstrated the development

and intact human AM.

of serum-free derived conjunctival tissueequalents.

Li W et al after dissolution of original amniotic BM,

The use of bioengineered tissue replacements

new BM formed by ex vivo expanded human limbal

represents the future for replacement and regeneration

corneal epithelial cells on iAM deposits much faster

of various tissues and organs.

and is more mature,resulting in regeneration of a limbal

epithelial phenotype. In contrast, BM deposition is

delayed and remains immature on dAM, resembling

wound healing by a corneal epithelial phenotype. Thus,

BM resynthesis may be used as another objective

readout for assessing the success of ex vivo expansion

of limbal epithelial progenitor cells on AM.

Recently, Prof Yuichi Mori, dr Hiroshi Yoshioka of

Waseda and Dr Samuel JK Abraham (Cardiac surgeon,

Yamanashi University Hospital, Japan) in collaboration

with M/S Nichi-In Biosciences (P) Ltd has started a

joint venture program in india with G Sitalakshmi from

Sankara Nethralaya and are in the process of

collaborating with dr Rajpal from RP Centre using non-

biological (Totally synthetic) material called

Mebiol Gel.

First time in the world:

This is the first time in the world that a nonbiological

(Totally synthetic) material has been used as a substrate

Fig. 32.5

to grow (cultivate) corneal limbal stem cells thereby

210

Clinical Diagnosis and Management of Ocular Trauma

paving a way for avoiding biological materials for this

normal rabbit:dose-dependent effects on ERG and retinal

purpose and avoiding viral and other dangerous

histology. Invest Ophthalmol Vis Sci 2004;45:2420-30.

known and unknown contamination in such biological

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filed.

Invest Ophthalmol Vis Sci 1991;32:2219-33.

19. Coffey PJ, Girman S, Wang SM et al. Long-term

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Primary Globe Repair

215

• Identification of any factors that could confound

removing the loose or necrotic tissue and then apply

the management.

the thin layer of cyanoacrylate glue with help of cotton

• Development and execution of the therapeutic

tip applicator. The glue is allowed to get dry and if

plan.

required another thin layered film of glue is applied

on the previously layered glue. Bandage contact lens

COMPLETE EVALUATION OF THE

needs to be applied on surface of glue and cornea

EYEANDADNEXA

following tissue adhesive application.

Detailed history including details about the incidence

SURGICAL MANAGEMENT

and objects causing the injury should be elucidated

from the patient or informant and documented in the

The different surgical options which can be contemp-

file. Relative afferent papillary defect or consensual

lated depending on the case to case basis:

reflex of the light in fellow eye should be assessed for

•

Corneal laceration repair

optic nerve integrity. External examination and diffuse

•

Corneoscleral laceration repair

slitlamp examination should be carried out to assess

•

Scleral laceration repair

the complete extent of damage to the eye without

•

Limbal laceration repair

causing further trauma to the eye and intraocular

•

Patch grafts

structures. Fundus examination should be carried out

• With or without iris abscission or repositioning

at the earliest sitting if fundus view is not precluded

•

With or without lens aspiration

because of media haze.

•

With or without vitrectomy and intraocular

antibiotic injections

ANCILLARY TESTINGAS NECESSARY

•

With complete pars plana vitrectomy with other

vitreoretinal procedures

Ultrasound examination of the eye with gentle standoff

The management of these injuries is thus a thought

technique can be employed to assess the posterior

out process, rather than a reflexive response to an

segment status and to rule out intraocular foreign body

obvious injury. The present chapter will highlight on

in suspected cases. X-rays or CT scans can be employed

the surgical principles for corneal and scleral laceration

in selective cases to rule out intraocular foreign body

repair.

and/or bony status of the orbit/optic canal.

IDENTIFICATION OF ANY FACTORS THAT

Anesthesia

The recommended anesthesia in all open globe injuries

COULD CONFOUND THE MANAGEMENT

should be general anesthesia, but the cases with small

To watch out for infection, intraocular foreign bodies

lacerations can be managed under local anesthesia.

or other factors which can have impact on final visual

The patient is prepared for surgery as soon as possible

outcome.

and should be medically and neurosurgically cleared.

DEVELOPMENT AND EXECUTION OF THE

For general anesthesia, the time of the last meal or

drink determines when surgery is scheduled. To prevent

THERAPEUTIC PLAN

aspiration, at least 6 hours should have elapsed since

The planning in cases of open globe injury could be

the last meal. Once the physician decides to repair the

of broad spectrum comprises.

laceration, the patient should be kept NPO. Anesthesia

should be achieved without any increase in intraocular

Surgical vs Nonsurgical

pressure, which can occur during intubation or because

of anesthetic agents. Depolarizing agents (e.g. succinyl-

Management

choline) are not used. Although succinylcholine

possesses several advantages, it contracts extraocular

NONSURGICAL MANAGEMENT

muscles and increases intraocular pressure. External

Nonsurgical management can be considered in cases

pressure from the mask can also increase intraocular

with self sealed corneal laceration or those which can

pressure.

be sealed with help of tissue adhesives and small

Preparing the Eye

conjunctival lacerations. Cyanoacrylate glue is the tissue

adhesive which can be of great help in providing

The eye should be prepared and draped with care.

support lasting for several days to several weeks.

Pressure should not be applied to the globe. The eye

Cyanoacrylate glue on exposure to air starts getting

is irrigated with a sterile balanced salt solution (BSS)

polymerized. Dry the surface of the cornea after

to remove any superficial foreign bodies. The eye is