Ophthalmic Drug Delivery Systems - part 8 docx

keratinizedmucosaehavebeenusedinstudyingtheinvitrorateofpenetra-
tionofdrugsthroughthebuccaltissue.Invivoabsorptionofpeptides/
proteinsfromthebuccalcavityislikelytobeinfluencedbythepresence
ofmucosalsecretionsandimmunologicalreactionsamongotherfactors.
Molecularsizemaynotbethelimitingfactorinthebuccaldeliveryof
peptides(64).GandhiandRobinson(65)reportedthataminoacidpenetrate
thebuccalmembranebyanactiveprocess,whereaspeptidedrugspermeate
passively.Thebuccalcavityexhibitsgreaterproteolyticenzymeactivitythan
thenasalorvaginalmucosa(64).Themetabolicactivityisshowntoreside
primarilyintheepithelium(67).AungstandRogers(8,68)studiedavariety
ofabsorptionenhancerstodeterminetheireffectsonbuccalabsorptionand
showedthatsignificantchangesinthemorphologyofthismucosalbarrier
takeplacefollowingexposuretotheabsorptionenhancers.
D.Pulmonary
Deliveryofproteinandpeptidedrugsviathepulmonaryroutehasalso
receivedsignificantattentioninrecentyears.Thewallsofthealveoliare
thinnerthantheepithelial/mucosalmembrane;thesurfaceareaofthelung
ismuchgreaterandthelungsreceivetheentirebloodsupplyfromtheheart,
allofwhichworkinfavorfortheabsorptionofproteindrugsmorerapidly
andtoagreaterextent.Ofcourse,thelungsarerichinenzymes,andover-
comingthisbarrierisnoeasytask.Peptidehydrolases,peptidases,anda
widevarietyofproteinasesarepresentinthelungcells(69).However,some
proteinasesinhibitorsarealsopresentatconcentrationsvaryingwiththe
diseasestate,whichmightworktopreventthedestructionofadministered
peptides(70).Liposomaldeliveryofpeptideandproteindrugsthroughthe
pulmonaryroutehavebeenattempted(71).Molecularmodificationshave
alsobeenundertakentoexplorethisrouteofproteinandpeptidedelivery
(72).
E.OcularRoute
Leereviewedthefactorsaffectingcornealdrugpenetration(73).
Rojanasakuletal.showedthatpolylysinepermeatedthroughepithelialsur-

facedefectsviaanintracellularpathwaywhenadministeredtotheeye,
whereasinsulinpredominatesinthesurfacecellsofthecornea(23).They
notedthattherewasasignificantamountofaminopeptidaseactivitypresent
intheocularfluidsandtissues.Figure1summarizestheresultsofthe
metabolism of topically applied enkephalins to the eye (74). Pretreatment
with the peptidase inhibitor bestatin had a significant protease inhibitory
effect, albeit in the tears only.
500 Dey et al.
Copyright © 2003 Marcel Dekker, Inc.
of insulin could be improved in the following descending order by coadmi-
nistration of the permeation enhancers: polyoxyethylene-9-lauryl
ether > sodium deoxycholate > sodium glycoch olate $ sodium taurocho-
late.
IV. OCULAR DELIVERY OF PEPTIDE AND PROTEIN
DRUGS
Peptides and proteins may be instilled into the eye for local/topical use.
Instillation of a topical dose of a drug to the eye leads to absorption of a
drug mainly through the conjunctival and corneal epithelia. For drugs
meant for topical use, it must be minimally absorbed systemically as it
can lead to undesirable side effects. Absorption into the systemic circulation
may occur across the conjunctiva and sclera. However, for local delivery the
cornea presents a significant barrier to the introcular penetration of peptide
drugs in view of their high molecular weight and low lipophilicity. Lee et al.
(75) reported that the penetration of inulin through the rabbit cornea was
probably occurring via a paracellular route rather than a transcellular route.
Systemic absorption of peptide and protein drugs following topical
administration to the eye could occur through contact with the conjunctival
and nasal mucosae, the latter occurring as a result of drainage through the
nasolacrimal duct. When systemic effects are desired, absorption through
the conjunctival and nasal mucosae needs to be maximized. One also must

consider other competing processes present in the ocular tissues. Of these
processes, absorption by the avascular cornea is important, since a large
portion of the drug thus absorbed is distributed to adjacent ocular tissues.
Ahmed and Patton (80) found that noncorneal (scleral) absorption
accounted for about 80% absorption of inulin, a highly hydrophilic macro-
molecule, into the iris-ciliary body. This observation is important, since
most therapeutic peptides act locally in the iris-ciliary body, which is con-
502 Dey et al.
Table 4 Penetration Enhancers Used to Improve Ocular Absorption
Enhancer Effect
Azone Threefold increase in cyclosporine absorption
Cetrimide, cytochalasin B Increased absorption of inulin
EDTA Threefold increase in glycerol absorption
Taurocholate, taurodeoxycholate Increased permeation of insulin and
FITC-dextran
Copyright © 2003 Marcel Dekker, Inc.
tiguous with the sclera. Therefore, macromolecular drug absorption would
benefit from scleral absorption.
Beside the transport barrier, another factor severely limiting the ocular
absorption of peptide drugs is metabolism by ocular enzymes, specifically
peptidases. Endopeptidases, like plasmin and collagenase, and exopepti-
dases, like aminopeptidases, are present in the ocular fluids and tissues.
The endopeptidase levels are usually low unless the eye is inflamed (81,82)
or injured (83) and are of little concern relative to the stability of topically
applied doses. Lee et al. (74) reported that within 5 minutes postinstillation,
about 90% of leucine enkephalin and almost 100% of methionine enkepha-
lin (pentapeptides) was recovered in the rabbit corneal epithelium in a
hydrolyzed form. Therefore, aminopeptidase activity must be inhibited to
facilitate ocular peptide absorption. Controlling these enzymes in the target
tissues may not be practical given the fact that the same enzymes might be

necessary for the homeostasis in the eye.
Cyclosporin A has been shown to improve the prognosis for corneal
allograft rejection. It was found that when administered by nonocular routes
in rabbits, it was detected in the systemic circulation but not in the ocular
tissues (20,84,85). Also, topical administration of cyclosporin A did not
produce any significant penetration within the eye beyond the cornea or
the conjunctiva. This may be because cyclosporin A was bound to corneal
and conjunctival epithelial cell membranes. Cyclosporin A eyedrops formu-
lated in absolute ethanol did produce higher levels in intraocular tissues,
which may be due to damage to corneal epithelium by alcohol.
Growth factors, especially epidermal growth factor (EGF), have been
found to stimulate cell proliferation in the corneal epithelium, thus stimu-
lating epithelialization during wound healing. Growth factors are mostly
used in accelerating the wound-healing process, and it would be of great
importance in corneal wounds since the cornea is an avascular organ. Many
in vitro corneal preparations have been used to demonstrate the wound-
healing process. Human EGF promotes endothelial wound healing (84).
Many other growth factors also play a major role in corneal wound healing,
including transforming growth factor  (TGF-) (87) and platelet-derived
growth factor (PDGF). Basic fibroblast growth factor (bFGF) and insulin-
like growth factor I (IGF-I) have been found in higher levels in patients
suffering from diabetic retinopathy (88–90). IGF-I and bFGF can also
induce fibrovascular changes in the retinal vessels.
A more practical strategy for circumventing the enzymatic barrier
would be to administer peptide analogs that are resistant to the principal
peptidases but possess equivalent biological activity [D-Ala
2
]methionine
enkephalinamide (DAMEA), which resists aminopeptidase-mediated clea-
vage, falls in this category of peptide analogs (74). The permeation and

Peptides and Proteins as Therapeutic Agents 503
Copyright © 2003 Marcel Dekker, Inc.
metabolic degradation of DAMEA in the albino rabbit cornea, conjunctiva,
and scler a has been studied (91). DAMEA was administered with and with-
out peptidase inhibitors bestatin (aminopeptidase inhibitor) and SCH 39370
(enkephalinase inhibitor). It was found that sclera was the most permeable
membrane to DAM EA, while cornea was almost impermeable to DAMEA.
Without inhibitors, the permeability coefficients of DAMEA were 2:7Â
10
À8
cm/s, 3:1 Â 10
À6
cm/s, and 12:5 Â 10
À6
cm/s across the cornea, con-
junctiva, and sclera, respectively. When inhibitors were co-administered with
DAMEA, the corneal permeability of intact DAMEA increased 15 times,
conjunctival permeability increased 5.5 times, while scleral permeability
remained practically unaltered.
The corneal and conjunctival penetration of 4-phenylazobenzyloxy-
carbonyl-l-Pro-l-Leu-Gly-l-Pro-d-Arg (Pz-peptide) and its effect on the
corneal and conjunctival penetration of hydrophilic solutes as well as on
the ocular and systemic absorption of topically applied atenolol and pro-
pranolol in the rabbit have been evaluated (92). The conjunctiva was 29
times more permeable than the cornea to 3 mM Pz-peptide. Conjunctival
Pz-peptide transport was 1.7 times greater in the mucosal-to-serosal than
in the opposite direction, whereas corneal Pz-peptide transport showed no
directionality. The apparent permeability coefficients of Pz-peptide across
the cornea and the conjunctiva increased over the 1–5 mM range, which
suggests that Pz-peptide enhanced its own transport across both epithelial

tissues. The cornea was more sensitive than the conjunctiva to the pene-
tration-enhancement effect of Pz-peptide. Pz-peptide elevated the corneal
transport of mannitol, fluorescein, and FD4 by 50, 57, and 106%, respec-
tively, but it did not affect the conjunctival transport of mannitol and
fluorescein. While Pz-peptide enhanced the ocular absorption of topically
applied hydrophilic atenolol, it did not affect the ocular absorption of
lipophilic propranolol. Interestingly, Pz-peptide did not affect the systemic
absorption of either -adrenergic antagonist. Pz-peptide appeared to facil-
itate its own penetration across the cornea and the conjunctiva and
increase the ocular absorption of topically applied hydrophilic but not
lipophilic drugs, while not affecting the systemic absorption of either
type of drug.
In addition, the presence of sites beyond the absorbing epithelia that
are capable of degrading peptides and protein and the availability of multi-
ple peptidases in a given site further decrease the absorption potential of
such compounds. While the ocular route has been widely accepted for the
use of topical application, its use in systemic delivery of peptides and pro-
teins will be rather limited.
504 Dey et al.
Copyright © 2003 Marcel Dekker, Inc.
V. SYSTEMIC ADMINISTRATION OF PEPTIDES AND
PROTEINS THROUGH THE OCULAR ROUTE
Systemic absorption of polypeptides and proteins primarily occur through
contact with the conjunctival and nasal mucosae. Table 5 lists some of the
peptides that could be administered through the ocular route (93). Almost
all the studies involving the absorption of peptides and proteins in animal
models have been carried out using labeled peptide samples (94–96). Apart
from monitoring the blood concentrations for pharmacokinetic evaluation,
pharmacodynamic studies have also been extensively pursued. Some of the
biological response parameters include reduction in blood sugar by insulin,

increase in blood glucose by glucagon, analgesic effects by enkephalins, and
increase in blood pressure by vasopressin.
Systemic pep tide availability following ocular administration has been
related to biological response. The study by Christie and Hanzal (97)
showed that insulin instilled into the conjunctiva is absorbed rapidly, giving
rise to a fairly constant and consistent lowering of blood sugar levels in
rabbits. Another study with somatostatin and its analog revealed that
there was an attenuation of the miotic response to noiceptive stimuli by
these agents, whereas intracameral injection of 1–50 mg met-enkephalin
had no effect on the miotic response (98).
Lee et al. (99) found that enkephalinamide and inulin are absorbed
into the blood stream following topical ocular administration, the former to
a greater extent than the latter. The authors proposed that depending on the
Peptides and Proteins as Therapeutic Agents 505
Table 5 Therapeutically Useful Peptides that Could Be
Administered Through the Ocular Route
Peptide Application
ACTH Antiallergic, decongestant anti-inflammatory
-Endorphin Analgesic
Calcitonin Paget’s disease, hypercalcemia
Glucagon Hypoglycemic crisis
Insulin Diabetes mellitus
Leu-enkephalin Analgesic
Met-enkephalin Immunostimulant
Oxytocin Induce uterine contractions
Somatostatin Attenuate miotic responses
TRH Diagnosis of thyroid cancer
Vasopressin Diabetes insipidus
VIP Secretion of insulin
Copyright © 2003 Marcel Dekker, Inc.

molecularsize,lipophilicity,andsusceptibilitytoproteolysis,otherpeptides
andproteinsmayalsobeabsorbedtovaryingextents.Similarly,Chiouand
Chuang(94)demonstratedthefeasibilityofeffectivesystemicdeliveryof
topicallyinstilledpeptidesintheeye.Theirfindingssuggestthatsystemic
deliveryofpeptidedrugsissuperiortotheparenteralroute,especiallywhen
thedrugispotentanddosesrequiredarelow.Enkephalincouldeffectively
beabsorbedsystemicallythroughtheeyewiththeuseofanabsorption
enhancer(95).Thisocularroutewasfoundtobesuperiortoadministering
thepeptidebyanintravenousroute.Similarresultshavebeenobtainedwith
otherpeptideslikethyrotropin-releasinghormone(TRH),luteinizinghor-
mone–releasinghormone(LHRH),glucagon,andinsulin(94).Spantide,a
tachykininantagonist,isreadilytakenupintotherabbiteyefollowing
topicalapplication.Measurableconcentrationsofthepeptidewereobserved
intheaqueoushumoraswellasinthegeneralcirculation.Similarly,insulin
couldbeabsorbedeffectivelyintothesystemiccirculationthroughocular
instillation(100).Thesystemicabsorptionof1%insulinthroughtheeyes
canbeenhancedatleastsevenfoldwhen1%saponin,asurfactant,was
addedtothesolution.Thisabsorptionenhancementwasnotaffectedby
aminopeptidaseinhibition.Recently,calcitonin,apolypeptidehormone,
wasfoundtobepoorlyabsorbedintothesystemiccirculationthroughthe
ocularrote(101).InclusionofpermeationenhancerslikeBrij-78andBL-9
markedlyimproveditssystemicabsorption.
Insummary,smallpolypeptidessuchasTRH(MW300),enkephalins
(MW$600),LHRH(MW1200),andglucagon(MW3500)areabsorbedto
asignificantextentthroughtheeyes,almosttotheextentof99%(94).
Polypeptideswithlargermolecularweightsuchas-endorphin
(MW$5000)andinsulin(MW$6000)arealsoabsorbed,buttoamuch
lesserextent.Theabsorptionofsuchlargemolecularweightcompounds
can,however,beimprovedbysimultaneoususeofabsorptionenhancers
(78).

VI.ENHANCEDSYSTEMICABSORPTIONWITH
PERMEATIONENHANCERS
Oneofthemajorproblemsassociatedwiththeoculardeliveryofpeptide
drugsistheirpoorsystemicbioavailability.Thismaybeovercomebyusing
penetrationenhancers.Mostpermeationenhancersneedtobeevaluated
withcaution,sincemostoftheseagentscauselocalirritationtotheeye.
AmongthemthemosteffectiveareBrij-78andBL-9,becausethesecom-
poundshavebeenshowntoenhanceinsulinabsorptiontoasignificant
extentwithoutcausinganynoticeableirritation(78).Table6liststhepene-
506 Dey et al.
Copyright © 2003 Marcel Dekker, Inc.
VII. CONCLUSIONS
With breakthroughs in biotechnology, newer and more potent peptide and
protein drugs are emerging in the market. The majority of these polypep-
tides require special delivery systems. However, since most of these com-
pounds are very potent, require low doses, and are well absorbed from the
mucous membrane, their delivery via the ocular ro ute may be viable.
However, one of the principal problems in the ocular delivery of peptide
and protein drugs is that of relatively low bioavailability to the ocular
tissues. This problem may be circumvented by the use of penetration enhan-
cers. The conjunctival administration of this class of compounds to achieve
therapeutic levels in the systemic circulation may well be possible in the near
future. We hope that novel drug delivery systems will be developed to
deliver potent polypeptide drugs through the ocular route.
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17
RetinalDiseaseModelsfor
DevelopmentofDrugandGene
Therapies
LeenaPitka
¨
nen
UniversityofKuopioandKuopioUniversityHospital,Kuopio,Finland
LottaSalminen
UniversityofTampereandTampereUniversityHospital,Tampere,
Finland
ArtoUrtti
UniversityofKuopio,Kuopio,Finland
I.INTRODUCTION
Inhumanstheretinaistheinnermostlayeroftheeye,whichconsistsof
retinalpigmentepithelium(RPE)andneuralretina.Theneuralretinahas
severallayersandvariouscelltypes,whichareillustratedinFigure1.RPEis
a single layer of hexagonal cells that maintains the homeostasis of neural
retina. It has essential biochemical, physiological, physical, and optical func-
tions in maintaining the visual system, including phagocytosis of rod outer
segments, transport of substances between photo receptors and choriocapil-
laries, and uptake and co nversion of the retinoids, which are needed in
visual cycle. Together with endothelial cell linings of retinal capillaries,
RPE forms the blood-retinal barrier. The neural retina is a complicated
and delicate multilayer. The thickness of neu ral retina varies from 0.4 mm
near the optic nerve to about 0.1 mm anteriorly at the ora serrata. The

photoreceptors are the light-sensing part of retina. The electric impulses
are amplified and integrated by bipolar, horizontal, amacrine, and ganglion
cells. The principal glial cell of the retina is the Mu
¨
ller cell. The bipolar cells
515
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from the vitreous cavity through a retinal hole or tear. Extravasation may
originate from choroid or retina and results in secondary retinal detach-
ment. Retinal detachment caused by the traction of fibrous bands in vitreous
is called traction retinal detachment. Traumas, intraocular inflammations,
retinal or vitreal degeneration, or vitreal bleeding are etiological factors of
retinal detachment. Proliferative vitreoretinopathy (PVR) is found in about
5% of retinal detachments. It is characterized by the formation of vitreal,
epiretinal, or subretinal membranes after retinal reattachment surgery or
ocular trauma. In some cases the membranes cause traction and distortion
of retina. Severe postoperative PVR is the most common cause of failed
retinal detachment surgery.
Retinoblastoma is a malignant retinal tumor with an incidence of
about 1 : 20,000. The genetic abnormality of this disease located to 13q14.
Both genes in this locus must be abnormal before this malignancy develops.
In the nonhereditary form, mutation occurs only in the retinal cells. In the
hereditary form the patient has inherited the first mutation from his or her
parents, and 90% of these patients develop a clinical retinoblastoma.
In this chapter we present some recent development in the retinal
disease models of animals. Models of retinal degeneration, proliferative
diseases, and neo vascularization are presented. These models are important
tools in current research, since various growth factors, gene therapies, and
transplantation strategies have demonstrated possibilities for treating severe
retinal diseases.

II. RETINAL DEGENERATION—GENETIC MODELS
Retinal degeneration leads to impaired function of the photoreceptors and
consequently gradual loss of vision., Many types of degeneration are based
on genetic factors, e.g., retinitis pigmentosa, a common term for various
mutations causing retinal degeneration. In addition to genetic factors, envir-
onmental factors (e.g., light exposure) may lead to retinal degeneration.
Macular degeneration is the most common type of retinal degeneration,
being the leading cause of vision loss in the industrial world. In the following
sections we present some genetic and environmental animal models of ret-
inal degeneration.
A. Natural Mutation Mouse Models
The naturally occurring mouse rd (retinal degeneration) and rds (retinal
degeneration slow) photoreceptor dystrophies are recessively inherited.
The mice have defects in the cGMP phosphodiesterase beta subunit gene
Retinal Disease Models 517
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(1,2) and in the peripherin gene (3,4). The rd mouse is a model of retinitis
pigmentosa in which a mutation of a rod-specific photophodiesterase leads
to the rapid loss of photoreceptors during early postnatal life. Very little is
known about the associated changes in the inner retinal neurons. Bipolar
and horizontal cells of the rd mouse retina undergo dramatic morphological
changes accompanying photoreceptor loss, demonstrating a dependence of
second-order neurons on photoreceptors (5).
The rds phenotype is considered to be an appropriate model for per-
ipherin 2/rds-mediated retinitis pigmentosa. Peripherin 2 glycoprotein is
needed for the formation of photoreceptor outer discs. The photoreceptor
cell is the primary site of the genetic defect that results in retinal dystrophy
in the rds mouse model (6).
The protective effect of a number of survival factors on degenerating
photoreceptors in mutant mice with naturally occurring inherited retinal

degenerations, including retinal degeneration (rd/rd), retinal degeneration
slow (rds/rds), nervous (nr/nr), and Purkinje cell degeneration (pcd/pcd), in
three different forms of mutant rhodopsin transgenic mice and in light
damage in albino mice were examined by La Vail et al. (7). The slowing
of degenerat ion in the rd/rd and Q344ter (a naturally occurring stop codon
mutation that removes the last five amino acids of rhodopsin) mu tant mice
demonstrated that intraocularly injected survival factors can protect photo-
receptors from degenerating. Importantly, these animal models have the
same or similar genetic defects as those in human inherited retinal degen-
erations (7). Such models have also been used to improve the condition of
photoreceptors by adeno-associated virus-mediated peripherin 2 gene ther-
apy (8). The outcome of the gene therapy was dependent on the timing of
the therapy (9).
B. Transgenic Mouse Models
To generate transgenic animals, whole genes are injected into a fertilized egg
pronucleus. The genes associate randomly into the genome, and their
expression is controlled by their own regulatory sequences. Due to the
complexity of the photoreceptor biology, several genes can be used to gen-
erate transgenic mouse models of retinal degeneration.
The VPP mouse carries three mutations (P23H, V20G, P27L) near the
N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment.
These animals have slowly progressive degeneration of the rod photorecep-
tors and subsequent changes in retinal function. Thes e change s mimic auto-
somal dominant retinitis pigmentosa of humans, which results from a point
mutation (P23H) in opsin (10). The rate of photoreceptor degeneration in
VPP mice seems to be adversely affected by the existence of the albino
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phenotype (11). Light deprivation affects the rate of degeneration in pig-
mented transgenic VPP mice (12).
To establish a transgenic mouse line with a mutated mouse opsin gene
in addition to the endogenous opsin gene, a mutated mouse opsin gene was
introduced into the germ line of a normal mouse. Simultaneous expression
of mutated and normal opsin genes induces a slow degeneration of both rod
and cone photoreceptors. The time course mimics the course of human
autosomal dominant retinitis pigmentosa (13).
The biochemical, morphological, and physiological analyses of a
transgenic mouse model for retinal degeneration slow (RDS) retinitis pig-
mentosa have been carried out. RDS retinitis pigmentosa is caused by a
substitution of proline 216 to leucine (P216L) in rds/peripherin. The phe-
notype in P216L-transgenic mice probably caused by a combination of two
genetic mechanisms: a dominant effect of the P216 substituted protein and a
reduction in the concentration of normal rds/peripherin. The expression of
the normal and mutant genes is similar to that predicted for humans with
RDS-mediated autosomal-dominant retinitis pigmentosa. These mice may
be used as an animal model for this disease (14).
The W70A transgenic mouse carries a point mutation (W70A) in the
gene that encodes for the gamma-subunit of rod cGMP phosphodiesterase .
This mouse represents a new model of stationary nyctalopia that can be
recognized by its unusual ERG (electroretinogram) features (15).
Another transgenic mouse model with defective expression of the
alpha subunit of the rod cGMP-gated channel was reported recently (16).
Expression was reduced by antisense RNA. The low expression of the rod
cGMP-gated channel causes a disease model that can be used to test thera-
pies designed to slow down or cure retinal degenerations (16).
Mice (Pdegtm1/Pdegtm1) that are homozygous for a mutant allele of
the gamma subunit of retinal cyclic guanosine monophosphate phospho-
diesterase (PDE gamma) have a severe photoreceptor degeneration.

Interestingly, the transgene that encodes the BCL2 protein was introduced
by mating into the mutant background. Antiapoptotic transgene BCL2
delayed temporarily the degeneration of photoreceptors in this murine
model of retinal degeneration (17).
C. Knockout Mouse Models
Knockout mutation is created by transferring a gene that is inactivated by
mutation to pluripotent embryonal stem cells. They often find their copy in
the genome and settle beside it and then change places by recombination.
The cells with wanted recombination are transferred to blastocysts to pro-
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duce chimeric animals. Homozy gous animals with the mutation can be
produced by mating.
A retinitis pigmentosa GTPase regulator-deficient mouse model for X-
linked retinitis pigmentosa has been created by gene knockout. In the
mutant mice, cone photoreceptors exhibit ectopic localization of cone
opsins. Rod photoreceptors have a reduced level of rhodopsin, and subse-
quently photoreceptors degenerate (18).
Likewise, rhodopsin knockout (opsinÀ/À) mice have been generated
as an animal model of retinitis pigmentosa. In that case a gene encoding
ciliary neurotrophic factor (CNTF) was delivered subretinally with adeno-
associated virus-vector. CNTF gene therapy delayed the death of photo re-
ceptors (19).
Homozygous rhodopsin knockout (RhoÀ/À) mice have a mutation in
exon 2 of the rhod opsin gene. They show a complete absence of functional
rhodopsin and do not build rod outer segments. The Rho(À/À) mice can
serve during postnatal weeks 4–6 as a model for pure cone function (20).
These mice do not elaborate rod outer segments, and the photoreceptors are
lost in 3 months. No rod ERG response is seen in 8-week-old animals. In
contrast, Rhoþ=À animals retain most of their photoreceptors, although the

inner and outer segments of the cells display some structural disorganiza-
tion. These animals may be a useful genetic background on which other
mutant opsin transgenes can be expressed. (21).
Knockout mice with arrestin gene defect have been generated.
Excessive light accelerated the cell death in pigmented arrestin knockout
mice. Human patients with mutations leading to nonfunctional arrestin
and rhodopsin kinase have Oguchi disease. This disease is a form of sta-
tionary night blindness (22).
D. Rat Models
The Royal College of Surgeons (RCS) rat is the first animal model with
inherited retinal degeneration. Although the genetic defect is actually not
known, the RCS rat is widely used as a model of photoreceptor degenera-
tion with relevance to retinitis pigmentosa and hereditary retinal dystrophies
(23,24). Experiments with RCS rats have been used to demonstrate the
beneficial effects of growth factors (like basic fibroblast growth factor,
bFGF) on retinal degeneration (25).
Adenovirus-mediated gene transfer has been used to develop a rat
model for photoreceptor degeneration. Recombinant adenovirus-mediated
downregulation of cathepsin S (CatS) in the retinal pigment epithelium and/
or neural retina was achieved. These results demonstrate that the transient
modulation of gene expression in RPE cells induced changes in the retina.
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Despite the low expression of endogenous CatS in RPE cells, this enzyme
appears to play an important role in the maintenance of normal retinal
function (26).
Transgenic rat P23H have been used as a model of autosomal domi-
nant retinitis pigmentosa. Substitution of proline by histidine in position 23

in rhodopsin (P23H) is the most common human mutation in RP in the
United States, with a prevalance of 15%. Several sublines of this strain have
been developed. These lines have a similar genotype, but the rate of retinal
degeneration varies. In line 1, almost complete degeneration is seen in 2
months, but in line 2 similar degeneration develops in one year. Similarly,
there are many sublines of transgenic rats that carry a rhodopsin mutation
S334ter with different rates of retin al degeneration. Ribozyme-directed clea-
vage of mutant mRNAs slows the rate of photoreceptor degeneration in this
rat model (27). d-cis-Diltiazem did not rescue photoreceptors of Pro23His
rhodopsin mutation line 1 rats treated according to the protocol used in rd
mouse (28). Extended photoreceptor viability by light stress has been
detected in RCS rats but not in opsin P23H mutant rats (29). The photo-
receptors of transgenic rats expressing either a P23H or an S334ter rhodop-
sin mutation were protected from apoptosis by recombinant adeno-
associated virus-mediated production of fibroblast growth factors fgf-2,
fgf-5, and fgf-18 (30,31), while lens epithelium-derived growth factor pro-
moted photoreceptor survival in light-damaged and RCS rats, but not in
P23H rats (32).
In addition to biochemical measures, these disease state models can be
monitored on the basis of retinal morphology (number of outer nuclear
layers) and ERG (a and b waves).
E. Cat Models
Abyssinian cats with recessively inherited rod-cone degeneration have been
introduced (33). Photoreceptor allografts were examined to determine the
viability and influence of such transplants on the host retina of the cats.
Also, clinical and pathological features, light and electron microscopy, and
the electrophysiology of an autosomal dominant, early-onset feline model of
rod/cone dysplasia (Rdy cats) have been documented (34–36). The immu-
nohistochemical changes in the retina and photoreceptor cell death of this
model have also been studied (37).

F. Transgenic Pig Model
Transgenic pigs that express a mutated rhodopsin gene (Pro347Leu) were
generated (38). These transgenic pigs provide a large animal model to study
Retinal Disease Models 521
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the protracted phase of cone degeneration in retinitis pigmentosa and for
preclinical treatment trials.
G. Dog Models
Canine rcd1 model of retinitis pigmentosa is caused by a null mutation in
the PDE6B gene. Treatment of rcd1-affected dogs with d-cis-diltiazem did
not modify the photoreceptor disease (39).
Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (red2; collie) in
dogs are early-onset forms of progressive retinal atrophy, which serve as
models of retinitis pigmentosa in humans (40).
Swedish Briard dogs have a very slowly progressive retinal dystrophy
that is inherited in an autosomal recessive manner. The lipid and fatty acid
compositions of plasma, retina, and retinal pigment epithelium were ana-
lyzed in this model (41). These studies provide evidence for yet another
animal model of inherited retinal degeneration with a defect in retinal poly-
unsaturated fatty acid metabolism. The fatty acid pattern in affected dogs
resembles that in the retina in n-3 fatty acid deficiency.
III. RETINAL DEGENERATION—LIGHT-INDUCED MODELS
Retinal damage by light has two distinct action spectra. One peaks in the
ultraviolet A (UVA) and the other in the midvisible wavelength. It was
shown in the Long Evans rat that UVA and green light can produce histo-
logically dissimilar types of damage. UVA light in particular produces
severe retinal damage at low irradiation levels (42).
Albino rats were continuously exposed to blue light for 1–7 days.
Continuous exposure of albino rats to moderate blue light for 2–5 days
selectively eliminated most of the photoreceptors while leaving the RPE

intact (43).
Monocularly aphakic gray squirrels were exposed for 10 minutes to
monochromatic near-ultraviolet radiation to determine if their yellow pig-
mented lens protected retinal tissue from photochemical damage. In aphakic
eyes the retinas revealed irreversible lesions to the photoreceptors. Eyes
exposed to ultraviolet radiation with their lenses intact were devoid of sig-
nificant retinal lesions. This study represents a model system for studying
the potential damaging effects of near-UV radiation to the aphakic eyes of
humans (44).
Constant fluorescent light can also be used to generate light-induced
degeneration model. Albino rats of the F344 strain were exposed to 1 or 2
weeks of constant light, either with or without intravitreal or subretinal
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bFGF solution injected 2 days before the start of light exposure. Constant
light exposure causes a decrease in the thickness of the outer nuclear layer
and blocks ERG responses. The results indicated that the photoreceptor
rescue activity of bFGF is not restricted to inherited retinal dystrophy in
the rat. The light damage is an excellent model for studying the normal
function of bFGF and its survival-promoting activity (45). It has been
shown that, in the retina, basic fibroblast growth factor delays photorecep-
tor degeneration in Royal College of Surgeons rats with inherited retinal
dystrophy. bFGF also reduces or prevents the rapid photoreceptor degen-
eration produced by constant light in the rat. This light-damage model was
used to assess the survival-promoting activity in vivo of a number of growth
factors and other molecules. Photoreceptors can be significantly protected
from the damaging effects of light by intravitreal injection of eight different
growth factors, cytokines, and neurotrophins. They act through several

distinct receptor families. In addition to basic fibroblast growth factor,
effective photoreceptor rescue was obtained with brain-derived neurotrophic
factor, ciliary neurotrophic factor, interleukin 1 beta, and acidic fibroblast
growth factor. Less activity was seen with neurotrophin 3, insulin-like
growth factor II, and tumor necrosis factor alpha, while nerve growth fac-
tor, epidermal growth factor, platelet-derived growth factor, insulin, insulin-
like growth factor I, heparin, and laminin did not show any protection (25).
IV. PROLIFERATIVE VITREORETINOPATHY
Proliferative vitreoretinopathy (PVR) is found in about 5% of retinal
detachments. The cellular evens of PVR include migration of glial cells,
pigment epithelial cells, and fibrocytes into the vitreous cavity, where they
proliferate and transform and dedifferentiate. The cells may interact with
endogenous membranous components of the vitreous. This leads to the
formation of vitreal, epiretinal, and subretinal membranes and traction ret-
inal detachment (47). Severe postoperative PVR is the most common cause
of failed retinal detachment surgery. Animal models of PVR are based on
environmental injuries.
A. Cell Injection
Injury can be caused by intraocular injection of fibroblasts into rabbit eye.
This was used as a model to test treatment of PVR by gene therapy. A
classification of severity of PVR in this model has been published (48–50).
The extent of PVR by cells that do or do not express the receptors for
platelet-derived growth factor (PDGF) was investigated. Mouse embryo
Retinal Disease Models 523
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fibroblasts was derived from PDGF receptor knock-out embryos. They do
not express PDGF receptors and induced PVR poorly when injected into
the eyes of rabbits. PDGF made an important contribution to the develop-
ment of PVR in this animal model. Furthermore, there was a marked dif-
ference between the two receptors from PDGF. PDGF  receptor was

capable of inducing PVR (51).
B. Dispase
PVR can be induced by injecting dispase intravitreally to rabbits (Dutch
belted, New Zealand white). Proliferative vitreoretinopathy developed in
response to subretinal or intravitreal dispase, with or without retinal
break. Severity of PVR was correlated with increasing doses of dispase.
The dispase model of PVR is easy to perform, and it permits a clear view
of the retina. This model showed a high success rate in development of PVR
(52), and intravitreally administered prinomastat decreased development of
PVR in this experimental model (53).
C. Combined Models
A proliferative vitreoretinopathy model was generated in albino rabbits by
combing some factors that probably cause the disease. The eyes were
injected with platelet-rich plasma, and in additio n they underwent cryother-
apy or vitrectomy or both procedures. Total retinal detachment and giant
holes were obtained more often in experimental eyes than in controls.
Microscopic investigation showed intravitreal or preretinal proliferation
of fibroblast-like cells (54).
Another combination model involves retinotomy with removal of vitr-
eous, cryotherapy, and platelet-rich plasm injection. This is an efficient
model of PVR: retinal detachments were produced in 100% of rabbit eyes
(55).
In a similar model (56) combined therapy of systemic methylpredni-
solone, sodium diclofenac, and colchicine was combined with topical atro-
pine, adrenaline, and dexamethasone phosphate. The therapies were useful
in treating experimental PVR.
D. Laser
Laser-induced retinal injury can be used to provoke PVR formation. For
example, pigmented rabbits underwent argon laser panretinal photocoagu-
lation in one eye. Then cultured fibroblasts were implanted into the intact

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vitreous of both eyes. More severe PVR developed in the eye with prior
panretinal photocoagulation than in the controls (57).
E. Other Models
Platelet-rich plasma causes PVR after injection into the vitreous in rabbit
eyes (59). It contributed more effectively to the development of an experi-
mental porcine PVR than PDGF. The efficacy depends on the platelet con-
centration of the plasma. It seems that other growth factors and plasma
components may interact synergistically with PDGF in the pathogenesis of
PVR (58).
V. NEOVASCULARIZATION
Neovascularization is involved in various diseases (e.g., cancer, psoriasis),
and the mechanism of angiogenesis has intensely been studied.
Neovascularization complicates the treatment of many retinal diseases,
and, therefore, appropriate animal models are needed.
A. Laser-Induced Neovascularization
Subretinal neovascularization (NV) can be induced by intense laser photo-
coagulation in monkey eyes (60). In pigs, the laser-induced branch of retinal
venous obstruction with rose bengal develops neovascularization of the
optic nerve head and retina (612). This process was assisted by photody-
namic thrombosis. A model of retinal ischemia and associated NV estab-
lished by venous thrombosis was produced. After anesthesia, eyes of
pigmented rats received an intraperitoneal injection of sodium fluorescein
prior to laser treatment. With a blue-green argon laser , selected venous sit es
next to the optic nerve head were photocoagulated (64).
B. Angiogenic Factor
Several ocular NV models are based on exposing the retina to excess of

angiogenic compounds. The effect of increased vascular endothelial growth
factor (VEGF) expression in the retina was investigated using transgenic
mice in which bovine rhodopsin promoter is coupled with the gene for
human VEGF. This study demonstrated that overexpression of VEGF in
the retina was sufficient to cause intraretinal and subretinal NV and pro-
vided a valuable new animal model (62).
Retinal Disease Models 525
Copyright © 2003 Marcel Dekker, Inc.
Controlled-release systems have been developed in order to provide a
long-term supply of angiogenic factors to the retina at defined levels.
Ethylene–vinyl acetate copolymer pellets release VEGF slowly into the vitr-
eous cavity of rabbits and primates. This induces neovascularization.
Sustained intravitreal release of VEGF caused widespread retinal vascular
dilation and breakdown of the blood-retinal barrier. Retinal NV seems to
require persistent high levels of VEGF at the retinal surface. This can be
achieved in rabbits more easily than in primates (63).
In alternative controlled-release models, subretinal implantation of
bFGF-impregnated gelatin microspheres is used to induce subretinal neo-
vascularization in the rabbit (65).
C. Ischemia
Relative hypoxia triggers formation of neovessels in the retina. When one-
week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then
to room air, the retinal ne ovascularization occurs between postnatal days 17
and 21. This model can then be used to study the therapeutic strategies (66).
In a similar ischemia-induced ocular neovascularization model, the expres-
sion of Flk-1 and neuropilin-1 was restricted on neovascularized vessels,
suggesting that these molecules may play important roles in retinal NV (67).
NV studies with ischemic models suggest that PaO
2
fluctuation is more

important than extended hyperoxia for retinal neovascular response in rats
(68). Indeed, a cycled hypoxia/hyperoxia (10–50% O
2
) protocol followed by
normoxia (20% O
2
) has been used as a retinal model of retinopathy of
prematurity to induce neovascularization in rat pups (69,70).
The time course and degree of proliferative vascular response after
hyperoxic insult were examined in dogs after oxygen-induced retinopathy.
In the neonatal dog, revascularization after hyperoxic insult involves a per-
iod of marked vasoproliferation peaking 3–10 days after a return to room
air. Oxygen-induc ed changes in the extravascular milieu pro bably affect the
pattern of reforming vasculature and possibly restrict the growth anteriorly
(71).
D. Genetic Models
NV of the RPE occurs earlier in a line of P23H mutant rhodopsin transgenic
mice than in most other mice and rats. The temporal course of RPE NV in
P23H mice was compared with that of two other retinal degeneration
mutants with a similar time course of photoreceptor cell loss. The findings
suggest that the P23H mutant rhodopsin transgenic mouse may be a useful
model for studying the regulation of NV in the outer retina (72).
526 Pitka
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Copyright © 2003 Marcel Dekker, Inc.
E. Other Models
NH4Cl gavage in the neonatal rat produced a metabolic acidosis–induced
retinopathy that may be a model for retinopathy of prematurity. Acidosis is
induced by high-dose acetazolamide. Independently of hyperox emia or

hypoxemia, the treatment is associated with preretinal neovascularization
in the neonatal rat (73).
A consistent model of preretinal NV in the rabbit was developed by
partially digesting the posterior virtreous with repeated injection of hyalur-
onidase. Then 250,000 homologous dermal fibroblasts were injected intravi-
treally (74). Neovascular events observed in this model agree with those
previously described for diabetic retinopathy and retinopathy of prematur-
ity in humans (75).
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