A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid

A new experimental model of glaucoma in rats through intracameral

injections of hyaluronic acid

Marıa Cecilia Morenoa, Hernan J. Aldana Marcosb, J. Oscar Croxattoc, Pablo H. Sandea,

Julieta Campanellia, Carolina O. Jaliffaa, Jorge Benozzia, Ruth E. Rosensteina,*

aLaboratorio de Neuroquımica Retiniana y Oftalmologıa Experimental, Departamento de Bioquımica Humana, Facultad de Medicina,

Universidad de Buenos Aires, Paraguay 2155, 5th floor, 1121 Buenos Aires, ArgentinabLaboratorio de Histologıa, Facultad de Medicina, Universidad de Moron, Pcia. de Buenos Aires, Buenos Aires, Argentina

cLaboratorio de Patologıa Ocular, Fundacion Oftalmologica Argentina ‘Jorge Malbran’, Buenos Aires, Argentina

Received 18 June 2004; accepted in revised form 13 January 2005

Available online 23 February 2005

Abstract

An experimental model of pressure-induced optic nerve damage would greatly facilitate the understanding of the cellular events leading to

ganglion cell death, and how they are influenced by intraocular pressure and other risk factors associated to glaucoma. The aim of the present

report was to study the effect of a long-term increase of intraocular pressure in rats induced by intracameral injections of hyaluronic acid with

respect to electroretinographic activity and retinal and optic nerve histology. For this purpose, hyaluronic acid was injected weekly in the rat

anterior chamber of one eye, whereas the contralateral eye was injected with saline solution. The results showed a significant decrease of

oscillatory potentials and a- and b-wave amplitude of the scotopic electroretinogram after 3 or 6 weeks of hyaluronic acid administration,

respectively. These parameters were further reduced after 10 weeks of treatment with hyaluronic acid. No significant changes in anterior

chamber angle structures from hyaluronic acid- and vehicle-injected eyes were observed, whereas a significant loss of ganglion cell layer

cells and of optic nerve axons were detected in animals that received hyaluronic acid for 10 weeks, as compared to eyes injected with saline

solution. In summary, present results indicate that the chronic administration of hyaluronic acid induced a significant decrease in the

electroretinographic activity and histological changes in the retina and optic nerve that seem consistent with some features of chronic open-

angle glaucoma. Therefore, this could be an experimental model to study the cellular mechanisms by which elevated intraocular pressure

damages the optic nerve and the retina.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: hyaluronic acid; intraocular pressure; electroretinography; glaucoma; ganglion cells; optic nerve

1. Introduction

Although the pathogenesis of optic neuropathy in

glaucoma is still elusive, increased intraocular pressure

(IOP) is probably the most important risk factor in primary-

angle open glaucoma. In fact, nearly all glaucoma therapy

relies on lowering IOP. The trabecular meshwork in the

anterior chamber angle is thought to play a key role in

0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.exer.2005.01.008

* Corresponding author. Dr Ruth E. Rosenstein, Laboratorio de

Neuroquımica Retiniana y Oftalmologıa Experimental, Departamento de

Bioquımica Humana, Facultad de Medicina, Universidad de Buenos Aires,

Paraguay 2155, 5th floor, 1121 Buenos Aires, Argentina.

E-mail address: [email protected] (R.E. Rosenstein).

the regulation of aqueous outflow and IOP. It is composed of

sheets of trabecular beams that contain lamellae made of

extracellular matrix materials, which comprise a significant

portion of this tissue and probably of the outflow barrier.

Among the materials of the trabecular extracellular matrix,

glycosaminoglycans (GAGs) profile (i.e. hyaluronic acid

(HA), keratan sulfate, heparan sulfate, and hybrid dermatan

sulfate–chondroitin sulfate) has been identified in rabbit

(Knepper et al., 1981), monkey, and human eyes (Acott

et al., 1985). Trabecular GAGs, particularly, have been

implicated in the modulation of outflow resistance as well as

in the development of glaucoma. In the trabecular mesh-

work obtained from patients with primary open-angle

glaucoma, several studies have noted excessive

Experimental Eye Research 81 (2005) 71–80

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M.C. Moreno et al. / Experimental Eye Research 81 (2005) 71–8072

accumulation of extracellular matrix materials (Segawa,

1975; Rohen, 1983). In particular, a depletion of HA and the

accumulation of chondroitin sulfates were detected in

primary open-angle glaucoma trabecular meshwork

(Knepper et al., 1996). An abnormal accumulation of acid

mucopolysaccharides in the anterior chamber angle was

also reported in steroid-induced ocular hypertension

(Francois et al., 1984). Intense histochemical staining for

GAGs in the human trabecular meshwork suggests that a

substantial amount of HA is present in the outflow pathway

(Lerner et al., 1997) and a quantitative analysis indicated

that it is the most abundant GAG of the human trabecular

meshwork (Acott et al., 1985). In a previous report we

showed that brimonidine, a highly selective a2-adreno-

receptor agonist that decreases IOP, significantly increases

hyaluronidase activity in rabbit trabecular meshwork

(Benozzi et al., 2000). Based on these results, we suggested

that the effect of brimonidine in increasing outflow could be

mediated, at least in part, by its stimulation of hyaluronidase

activity, e.g. by increasing GAGs clearance. If a decrease of

GAGs content reduces IOP, it seems likely that an increase

of intracameral HA levels could induce the opposite effect.

In this sense, it was shown that the injection of HA into the

anterior chamber of rabbits and owl monkeys significantly

increases IOP (Harooni et al., 1998; Schubert et al., 1984).

However, while in these species the effect of HA lasts for

relatively short periods (70 and 24 hr post-injection in rabbit

and owl monkeys, respectively), we showed that a single

injection of HA maintains elevated levels of IOP for 8 days,

while an injection performed once a week induces a

sustained and significant hypertension that lasts all along

the duration of the study (10 weeks) (Benozzi et al., 2002).

The longer-lasting effect of a single injection of HA on IOP

could be attributed to the fact that the rat anterior chamber

being very shallow, the actual intracameral concentration of

HA is higher than that achieved in other species. In addition,

a poorer ability of HA washout in rats cannot be ruled out.

These results indicate that the injection of HA could be a

new model of ocular hypertension in rats.

It is well known that ocular hypertension plays a causal,

albeit not necessarily exclusive, role in glaucomatous visual

loss. In fact, not all models of ocular hypertension cause the

same disease at the level of the retina or the optic nerve.

Therefore, the aim of the present study was to examine the

effect of a long-term increase of IOP in rats induced by the

intracameral injection of hyaluronic acid (HA) on retinal

function and histology.

2. Materials and methods

2.1. Animals

Male Wistar rats (average weight 200G40 g) were

housed in a standard animal room with food and water

ad libitum under controlled conditions of humidity and

temperature (218G28C). The room was lighted by fluor-

escent lights, that were turned on and off automatically

every 12 hr, with the lights on from 6:00 a.m. to 6:00 p.m.

Rats were anaesthetized with ketamine hydrochloride

(50 mg kgK1) and xylazine hydrochloride (0.5 mg kgK1)

administered intraperitoneally. Using a Hamilton syringe

with a 30-gauge needle, 25 ml of HA (obtained from Sigma

Chemical Co., St Louis, MO, USA, catalog # H1751,

10 mg mlK1 in sterile saline solution) were injected weekly

into one eye of anaesthetized rats, while an equal volume of

vehicle (saline solution) was injected in the fellow (control)

eye. The eyes were focused under a binocular Colden

surgical microscope with coaxial light. The needle moved

through the corneoscleral limbus to the anterior chamber

with the bevel up. When the tip of the bevel reached the

anterior chamber, the liquid progressively increased the

chamber’s depth, separating the needle from the iris and

avoiding needle–lens contact. Injections were applied at the

corneoscleral limbus beginning from hour 12 and changing

the site of the next injection from hour to hour by rotating

the head to achieve better access to the limbus. The

injections and IOP assessments were performed after

applying one drop of 0.5% proparacaine hydrochloride to

each eye. Of 48 rats, three showing cataract were excluded

from the experiments. In addition, almost all the animals

developed a localized corneal edema at the site of the

injection that lasted less than 24 hr.

2.2. IOP assessment

A TonoPen XL (Mentor, Norwell, MA) tonometer was

used to assess IOP in conscious, unsedated rats as described

by Moore et al. (1995). All IOP determinations were

assessed by operators who were blind with respect to the

treatment applied to each eye. Animals were wrapped in a

small towel and held gently, with one operator holding the

animal and another making the readings. Five IOP readings

were obtained from each eye by using firm contact with the

cornea and omitting readings obtained as the instrument was

removed from the eye. Differences among reading were less

than 10% (standard error). The mean of these readings was

recorded as the IOP for this eye and this day. Mean values

from each rat were averaged, and the resultant mean value

was used to compute the groups mean IOPGSE. HA was

injected once a week in one eye, whereas the other eye was

injected with saline solution. IOP was assessed weekly,

before the new injection. IOP measurements were per-

formed at the same time each day or week (between 11:00

and 12:00 hr) to correct for diurnal variations in IOP.

2.3. Electroretinography

Electroretinographic activity was assessed in dark-

adapted rats as follows: after 6 hr of dark adaptation, rats

were anaesthetized with an intraperitoneal injection of

ketamine hydrochloride and xylazine hydrochloride under

M.C. Moreno et al. / Experimental Eye Research 81 (2005) 71–80 73

dim red illumination. Phenylephrine hydrochloride (2.5%)

and 1% tropicamide (Alcon Laboratories, Argentina) were

used to dilate the pupils, and the cornea was intermittently

irrigated with balanced salt solution (Alcon Laboratories,

Argentina) to maintain the baseline recording and to prevent

exposure keratopathy. Rats were placed facing the stimulus

at a distance of 20 cm. All recordings were completed

within 20 min of the induction of anaesthesia and animals

were kept warm during and after the procedure. A reference

electrode was placed through the ear, a grounding electrode

was attached to the tail, and a gold electrode was placed in

contact with the central cornea. A 15 W red light was used

to enable accurate electrode placement. This maneuver did

not significantly affect dark adaptation and was switched off

during the electrophysiological recordings. Electroretino-

grams (ERGs) were recorded from both eyes simultaneously

and 10 responses to flashes of unattenuated white light

(5 ms, 0.2 Hz) from a photic stimulator (light-emitting

diodes) set at maximum brightness (350 cd sec mK2 with-

out a filter) were amplified, filtered (1.5-Hz low-pass filter,

1000 high-pass filter, notch activated) and averaged (Akonic

BIO-PC, Akonic, Argentina). The a-wave was measured as

the difference in amplitude between the recording at onset

and the trough of the negative deflection and the b-wave

amplitude was measured from the trough of the a-wave to

the peak of the b-wave. Electrophysiological responses were

averaged for each run. Runs were repeated three times with

5 min intervals, to confirm consistency and the mean of

these three runs was used for subsequent analysis. The mean

peak latencies and peak-to-peak amplitudes of the responses

from each group of rats were compared. Oscillatory

potentials (OPs) were assessed as described by Bayer

et al. (2001a). Briefly, the same photic stimulator with a

0.2 Hz frequency and filters of high (300 Hz) or low

(100 Hz) frequency were used. The amplitudes of the OPs

were estimated by measuring the heights from the baseline

drawn between the troughs of successive wavelets to their

peaks. The sum of three OPs was used for statistical

analysis. ERGs were performed 3 days after the injection of

HA or vehicle and they were assessed by operators who

were blind with respect to the treatment applied to each eye.

2.4. Light microscopy

At 10 weeks of treatment with HA or vehicle, eight

eyes/group were analyzed by light microscopy. Eyes were

enucleated after anaesthetic overdose and immersed

immediately in a fixative containing 4% paraformaldehyde

and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2)

for 1 hr. The nictitans membrane was maintained in each

eye to facilitate orientation. The cornea and the lens were

carefully removed, and the posterior portions were fixed for

an additional 12-hr period in the same fixative. A cross-

section of the optic nerve from control and HA-treated eyes

was removed 1.5 mm posterior to the globe and post-fixed in

1% osmium tetroxide in phosphate buffer. Nerves were

processed into epoxy resin, sectioned at 1 mm, and stained

with 1% toluidine blue. Eyecups were dehydrated in an

alcohol series, and embedded in paraffin. Sections (4 mm

thick) were cut along the horizontal meridian through the

optic nerve head and stained with haematoxylin and eosin.

In addition, three whole eyes were fixed for 12 hr in B4-G

(6% HgCl2 and 0.1% glutaraldehyde in 1% sodium acetate).

The latter mixture was considered to be a good fixative for

tissues containing glycoconjugates (Spicer and Schulte,

1992). The eyes were routinely processed with paraffin for

histological and histochemical studies of the anterior

segment. For histological details Masson trichrome staining

was used. The histochemical characterization of HA was

performed using Alcian Blue 8G stain (Kiernan, 2003). A

hyaluronidase digestion technique was used to detect HA.

For the hyaluronidase digestion procedure, 50 mg of bovine

testicular hyaluronidase (Sigma Chemical Co., St Louis,

MO, USA) were diluted in 100 ml phosphate-buffered

saline solution. Each section was incubated in this solution

for 1 hr at 378C. Then, sections were stained with Alcian

Blue (pH 2.5). HA was detected by comparing the sections

without digestion with those with digestion.

2.5. Image analysis

Microscopic images were digitally captured with a Nikon

Eclipse E400 microscope (illumination: 6-V halogen lamp,

20 W, equipped with a stabilized light source) via a Sony

SSC-DC50 camera. The microscope was set up properly for

Koehler illumination. The camera output was digitized into

a 520!390 pixel matrix (each pixel with 0–255 grey levels)

with a Leadteckw WinView 601 video capture card,

displayed on a computer monitor and saved as an image

of 24 bit RGB in BMP format. The digitalized images were

transferred to a Scion Image for Windows analysis system

(Scion Corporation Beta 4.0.2).

2.6. Eye cups analysis

Retinal morphometry was evaluated as described by

Takahata et al. (2003) with minor modifications. Three

sections were randomly selected from each eye. Nine

microscopic images at 1 mm from the temporal edge of the

optic disc were digitally analyzed. The light microscope was

adjusted to level 4 and a 40! CF E achromat objective was

used. At the magnification utilized, each pixel of the image

corresponds to 0.31 mm, and each field in the monitor

represented a tissue area of 19 318.7 mm2. The thickness

(in mm) of the inner plexiform layer (IPL), inner nuclear

layer (INL), outer nuclear layer (ONL), and total retina was

measured. The number of cells in the ganglion cell layer

(GCL) was calculated by linear cell density (cells per

100 mm). For each eye, results obtained from three separate

sections were averaged and the mean of five eyes was

recorded as the representative value for each group. No

attempt was made to distinguish cell types in the GCL for

0

5

10

15

20

25

intr

aocu

lar

pre

ssu

re (

mm

Hg

)

vehicle HA

6 weeks 10 weeks

vehicle vehicleHA HA

3 weeks

Fig. 1. Tonopen measurements of IOP in rats injected with HA in one eye

and vehicle in the contralateral eye for 3, 6, or 10 weeks. At all these

intervals, HA induced a significant increase in this parameter. No

differences were observed in control eyes among 3, 6, and 10 weeks of

treatment (ANOVA). Data are meanGSE (n, 20 eyes/group), **p!0.01,

by Student’s test.


enumeration of cell number. The morphometric analysis

was performed by observers masked to the protocol used in

each eye.

2.7. Optic nerve morphometry

Optic nerve axon counting was evaluated as described by

Levkovitch-Verbin et al. (2002) with minor modifications.

To measure the density and fibre diameter distributions,

images were captured with a 100! achromat objective from

10 spaced nerve regions (two central and eight peripheral in

a clockwise manner). The light microscope was adjusted to

level 6. At the magnification utilized, each pixel of the

image corresponds to 0.123 mm, and each field in the

monitor represented a tissue area of 3090.99 mm2. These

captured images were edited to eliminate non-axonal

images, and the size of each axon with its myelin sheath

(diameter) and the density of axons per square millimetre

were calculated for each image. The axon number/field in

the HA-treated groups was compared with the control

fellow eye. The number of axons counted in 10 images from

each nerve was approximately 10.6% of the total optic nerve

area. The counting process was performed by observers

masked to the protocol used in each nerve.

2.8. Statistical analysis

Statistical analysis of results was made by a Student’s

t-test or by a two-way analysis of variance (ANOVA)

followed by Student’s t-test or Dunnett’s test, as stated.

All animal use procedures were in strict accordance with

the ARVO Statement for the Use of Animals in Ophthalmic

and Vision Research.

3. Results

Fig. 1 shows the average IOP of 20 rats injected weekly

with HA (in one eye) or vehicle in the other during 3, 6, or

10 weeks. At all these intervals, a significant increase of IOP

was observed in eyes injected with HA, as compared with

the respective controls. The injection itself did not affect

IOP (data not shown), and no differences in the IOP of

vehicle-injected eyes were detected among these time-

points. In order to analyze the functional state of the retinas

from HA-injected eyes, scotopic ERGs were performed.

Representative scotopic ERG traces from rats injected with

vehicle or HA for 6 weeks are shown in the left panel of

Fig. 2A. The average amplitudes of scotopic ERG a- and

b-waves of 20 rats injected with vehicle in one eye and with

HA in the contralateral eye during the same interval are

depicted in the right panel of the same figure. These

parameters were significantly reduced in eyes receiving HA.

Similar studies were performed after 10 weeks of treatment

with HA or vehicle (Fig. 2B). Also in this case, a significant

reduction of ERG a- and b-waves amplitude was observed in

the eyes injected with HA. The treatment with HA for 6 or

10 weeks did not affect a- and b-wave latencies, as

compared with animals injected with vehicle. After

3 weeks of HA or vehicle treatment, no changes in ERG

a- and b-waves amplitude were detected (data not shown).

In contrast, the sum of the scotopic ERG OP amplitudes was

significantly reduced at all times of treatment with HA, as

shown in Fig. 3. The injection itself did not affect

the electroretinographic response, since no changes in

ERG a- and b-waves or OPs amplitude were observed

between vehicle-injected and intact eyes (Figs. 2 and 3).

Fig. 4A shows a light microscopy analysis of a

representative anterior chamber angle from rats treated

during 10 weeks with HA. The anterior chamber angle and

outflow structures were conserved and looked similar in

both HA-injected and control animals. In all eyes, the

anterior chamber angle was open. No significant differences

in the morphology of the iris, ciliary body, and ciliary

processes were observed between HA-injected and control

eyes. The histochemical staining method for HA revealed

the presence of irregular granular masses alcian blue

positive associated with the trabecular meshwork in the

eyes of rats treated with HA (Fig. 4B) but not in vehicle-

injected eyes (data not shown). The positive reaction in the

granular masses was abolished with the previous digestion

treatment with hyaluronidase, indicating the presence

of HA.

The only significant changes were observed in the retina

and the optic nerve from rats injected with HA for 10 weeks.

Fig. 2. Scotopic ERGs in rats injected with HA or vehicle. (A) Animals were weekly injected with HA in one eye or saline solution in the contralateral eye

during 6 weeks. (B) Effect of a 10-weeks treatment with HA or vehicle. In both cases left panels, representative scotopic ERG traces; right panels, average

amplitudes of scotopic ERG a- and b-waves. A significant reduction in the amplitude of both scotopic ERG a- and b-wave amplitude was observed in eyes

injected with HA for 6 and 10 weeks. No changes between intact and vehicle injected eyes were observed. Data are meanGSE (n, 20 eyes/group), *p!0.05,

**p!0.01 by Student’s test.


Although total retinal, IPL, INL and ONL thickness did not

change between vehicle- or HA-injected eyes (control

132G7, 32.2G5, 22.2G3.5, 37.1G3; HA 123G12, 30.6G3.5, 19.4G2.3, 33.5G4.7 for total retinal, IPL, INL and

ONL width, respectively) a significant loss of GCL cells in

HA-injected eyes for 10 weeks was observed (Fig. 5). The

mean number/100 mmGSE of GCL cells from the control

group was 8.7G0.7, whereas in HA-injected eyes, it was

5.2G0.6 (p!0.01). The optic nerve from eyes treated with

HA for 10 weeks exhibited an overall loss of staining

uniformity and integrity (Fig. 6). Axons in control optic

nerve were generally uniform in shape (rounded) and

showed a variable size. In contrast, in HA-treated eyes,

spacing between bundles of axons increased by an apparent

development of glial profiles, glial nuclei, and axon loss.

Individual axons of treated optic nerve also exhibited

distention and distortion that resulted in a departure from the

circular morphology of normal axons. This loss of

uniformity at the level of individual axons and the

prominence of glial profiles and nuclei probably underlies

the overall loss of staining uniformity and nerve integrity.

To compare the loss of axons by diameter, the number of

Fig. 3. OPs in intact rats or injected with HA or vehicle for 3, 6, or 10 weeks. Left panels, representative OPs traces; right panels, average amplitudes of OPs in

intact animals or injected with HA or vehicle. The differences in sum of scotopic ERG OP amplitudes between vehicle and HA-injected eyes were significant at

all times of measurement. No changes between intact and vehicle injected eyes were observed. Data are meanGSE (n, 10 eyes/group), *p!0.05, **p!0.01 by

Student’s test.

Fig. 4. (A) Section of the anterior portion of an eye injected with HA for 10 weeks showing the cornea (C), iris (I), ciliary body with ciliary processes (CB) and

zonule filaments (Z). Note that the anterior chamber including the trabecular meshwork had not signs of damage. Masson trichrome. (B) The histochemical

staining method for HA revealed the presence of irregular granular masses alcian blue positive associated with the fine connective strands of the trabecular

meshwork (arrows). Alcian blue.

Fig. 5. Light micrographs of transverse sections of a retina from a rat, in which one eye was injected with saline solution (A) and the contralateral eye was

injected with HA during 10 weeks (B). Note the diminution of ganglion cell layer (GCL) cells in the eye injected with HA. The other retinal layers showed a

normal appearance. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Haematoxylin and eosin.


Fig. 6. Low- and high-magnification images of optic nerve cross-sections in control eye (A, C) and HA-treated eye (B, D). (A) Healthy, intact optic nerve. Note

the homogeneity of the staining. In (C) individual axons were generally uniform in shape (arrowhead), generally rounded and packed together tightly to form

the fibres of the healthy nerve. In the nerve of the treated eye (B) a less stained area (arrowhead) indicates a nerve alteration. At high-magnification (D) this area

is characterized by a prominence of glial profiles. Disease in individual axons was characterized by axonal distention and distortion that resulted in a departure

from the circular morphology of normal axons (arrowheads). There is a predominance of small-diameter axons. Toluidine blue.


axons in each size group from 0.2 to 5 mm was compared in

both experimental groups (Fig. 7). A significant loss of large

and medium but not of small-diameter axons was observed.

4. Discussion

The foregoing results indicate that chronic injections of

HA in the rat anterior chamber induced significant retinal

changes both at functional and histological level, as

compared with the contralateral eye injected with vehicle.

In this sense, a significant decrease in the scotopic ERG

activity as well a significant damage confined to the GCL

and optic nerve axons were observed in HA-injected eyes.

In a previous report, we postulated that the intracameral

injection of HA could be a new model of ocular

hypertension in rats (Benozzi et al., 2002). Several groups

have developed different ways to increase IOP in the rat eye:

by injection of hypertonic saline into aqueous humour

collecting veins (Moore et al., 1993), by cautery and

occlusion of two or three of the episcleral/extraorbital veins

(Shareef et al., 1995), or by blockage of aqueous pathway

caused by photocoagulation after injection of India ink into

anterior chamber of the eye (Ueda et al., 1998). Several

advantages support the usefulness of our model: (1) it is

inexpensive and easy to perform, (2) a highly consistent

intraocular hypertension may be achieved, (3) it may have a

reasonably long course, and (4) daily variations in IOP

persisted in HA-injected eyes. In contrast to the model of

veins cautery, in all likelihood, HA does not impede the

blood flow out of the eye. Furthermore, we have demon-

strated that this model may also be useful for pharmaco-

logical studies with drugs that affect aqueous outflow,

formation, or a combination of both (Benozzi et al., 2002).

Although multiple injections of HA may be needed to obtain

a sustained hypertension, we have demonstrated that the

procedure of injection itself does not affect IOP.

To assess retinal function, scotopic flash ERGs were

performed in the eyes from animals injected chronically

with HA or vehicle since the rat has a predominantly

0

200

400

600

nu

mb

er o

f ax

on

s

axon diameter (µm)

0.05 - 0.2 0.2 - 0.4 0.4- 0.6

vehicle HA vehicle HA vehicle HA

Fig. 7. Axonal diameter distribution in eyes injected with vehicle or HA

during 10 weeks. A significant decrease in the number of middle and large

but not small axons was observed in HA-injected eyes. Data are meanGSE

(n, 8 eyes/group), **p!0.01, by Student’s test.


rod-retina. As shown herein, a significant reduction of OPs

as well as the a- and b-wave amplitude but not of their

latencies was observed in HA-injected eyes. The pattern

electroretinogram (PERG), a direct indicator of retinal

ganglion cell function, has become accepted as a sensitive

test for detecting early functional changes in glaucoma. In

contrast to the PERG, the flash ERG has been viewed

traditionally as reflecting activity of retinal neurons distal to

the ganglion cells and to be relatively unaffected in

glaucoma (Korth, 1997; Graham and Klistorner, 1998).

However, more recently, flash ERG was successfully

introduced as a non-invasive procedure to monitor the

progression of pathophysiology in a rat glaucoma model

(Bayer et al., 2001a). From a practical standpoint, the

uniform field ERG is easier to record than the PERG,

because it does not require refractive correction or exact

foveal placement. Together with similar reports on ERG

findings in subjects with advanced open-angle glaucoma

(Fazio et al., 1986; Holopigian et al., 1990; Korth et al.,

1994; Velten et al., 2001a,b), several observations support

the fact that in experimental models of glaucoma some

components of the flash ERG, such as scotopic ERG

amplitude, can be affected. In this sense, in the three-vein

episcleral/limbal vein occlusion model for inducing glau-

comatous pathology in the rat eye, a significant decrease of

OPs and the a- and b-wave amplitude was observed (Bayer

et al., 2001a). Similar changes in ERG a- and b-waves were

described in DBA/2 mouse with angle-closure glaucoma

(Bayer et al., 2001b). Furthermore, Viswanathan et al.

(2000) showed changes in the uniform field ERG that

correlate with PERG responses produced by a laser-induced

glaucoma in monkeys. More recently, a significant

reduction of scotopic b-wave amplitude was described in

Brown Norway rats with hypertonic saline injection into an

episcleral vein (Chauhan et al., 2002). The retinal dysfunc-

tion described herein seems to be time of hypertension-

dependent. Only OPs decreased at 3 weeks of ocular

hypertension, whereas changes in a- and b-wave amplitude

began at 6 weeks of treatment with HA. In both cases, a

higher decrease in these parameters was observed at 10

weeks as compared to 6 weeks of ocular hypertension. It

should be noted that before the injections of HA or vehicle,

in control experiments we had ascertained that flash ERGs

in the two eyes of the rats were very similar. Thus, the

different records between treated and control eyes are valid

indicators of the effects of the chronic ocular hypertension.

The present findings on flash ERG changes in rats with

chronic ocular hypertension strengthens the suggestions

from previous studies that the full-field ERG could serve as

a useful alternative in the assessment of glaucomatous

neuropathy, particularly in experimental models. Moreover,

in agreement with Bayer et al. (2001a) these results further

support that OPs constitute the earliest significant change,

and therefore it probably could be the best predictor of

glaucomatous neuropathy.

As for the histological analysis, the eye’s general

architecture was relatively unaffected by the treatment

with HA. Conceivably, chronic intracameral injections per

se could cause some degree of chamber angle constriction.

However, the histopathologic examination indicated that

it was not the case in our experimental setting, since a

normal angle was observed in both HA- and vehicle-

injected eyes. In contrast, differences were observed at

retinal level at 10 weeks of treatment with HA. While no

changes were observed in IPL, INL, and ONL, a significant

loss (y40%) was observed in GCL cells. These data on

ganglion cell loss are in agreement with results obtained by

other groups using different methodologies to induce

experimental glaucoma (Naskar et al., 2002; Grozdanic

et al., 2003).

In control eyes, morphology compatible with a healthy,

intact optic nerve was observed. Individual axons were

generallyuniforminshapeandpackedtogether tightly toform

the fibres of the nerve. In contrast, an overall loss of nerve

integrity as well as an alteration of axons shape was observed

in hypertensive eyes. In addition, a significant decrease in

axon number was evident, being large axons significantly

more vulnerable than small-diameter fibres. Since no shift of

axonal diameter distribution to smaller sizes was evident in

HA-injected eyes, it seems unlikely that this result may be

attributable to axons shrinkage. This loss of uniformity at the

levelof individual axons probablyunderlies the overall loss of

uniformity and nerve integrity. In agreement, a preferential

loss of large optic nerve axons was demonstrated in other

modelsof rat andmonkeyexperimentalglaucoma(Glovinsky

et al., 1991; Levkovitch-Verbin et al., 2002).


The correlation between retinal histology and ERG

response in the eyes injected with HA is not completely well

understood. The a-wave of the flash ERG is classically

thought to represent photoreceptor activity, whereas the

b-wave reflects bipolar and Muller cells function. Although

glaucoma is known to cause primary death of ganglion cells,

there is also morphological evidence for damage to the outer

retina (Panda and Jomas, 1992). Swelling and loss of

photoreceptors were found in human and monkey glauco-

matous eyes (Nork et al., 2000). In addition, changes in

Muller cells in glaucomatous eyes were reported (Wang

et al., 2002). Since, as mentioned before, the components of

the ERG have been shown to be affected in some patients

with glaucoma, it seems possible either that, at least in some

cases, glaucoma affects retinal cells besides ganglions cells

or that there is a component of the ERG that reflects the

activity of these cells. Although no significant major

morphological changes of photoreceptors were found in

the eyes injected with HA, this evidence does not detract the

possibility of a functional impairment in these cells that

could explain the ERG findings described herein. Glutamate

is the main excitatory neurotransmitter in the retina but it

can be toxic to retinal ganglion cells in animal models when

administered at non-physiological concentrations (Vorwerk

et al., 1996). Several lines of evidence support that

abnormalities of glutamate metabolism play a causal role

in glaucomatous neurotoxicity (Dreyer et al., 1996; Brooks

et al., 1997). Since glutamate is necessary for light-elicited

signalling between photoreceptors and their target cells, it

seems likely that an alteration of glutamatergic neurotrans-

mission may affect other retinal layers besides its effect on

retinal ganglion cells. This hypothesis is under current

investigation. Moreover, since measuring the scotopic

threshold response (STR) in addition to the standard

a- and b-waves could provide information on the magnitude

of functional damage to third-order retinal neurons, the

assessment of this parameter in HA-treated eyes will be

performed in the near future.

Whether the histologic and functional retinal changes

observed are merely consequence of a toxic effect of HA per

se or, alternatively, they are a hypertension triggered

process is not clear. However, while HA was detected in

the anterior chamber of HA-injected eyes, no histological

evidences were found that support the possibility that this

GAG injected in the anterior chamber may reach the retina

or the optic nerve head. In addition, since no loss of the

midretinal architecture (the area supplied by retinal arteries)

was evident, it seems improbable that the HA-induced

hypertension could provoke a significant retinal ischaemia.

These evidences together with the fact that the changes

described herein are similar to those observed in other

glaucoma experimental models strongly suggest that they

are mostly attributable to the HA-induced hypertension.

Although considered the major risk factor for glaucoma

neuropathy, the cellular mechanisms by which elevated IOP

damages the optic nerve remain unknown. Acquiring this

knowledge relies heavily on the use of animal models of

chronically elevated IOP. The use of rodents allows

experimentation with sufficient number of animals and

could provide a complete picture of both ultrastructural

consequences and cell biology of pressure-induced optic

nerve damage. Based on both functional and histomorpho-

logic evidences, present results indicate that the intracam-

eral injection of HA appears to mimic some features of

primary open-angle glaucoma, and therefore it may be a

useful tool for understanding this ocular disease.

Acknowledgements

The authors wish to thank Drs Rafael Iribarren and Diego

Golombek, for the helpful discussion of this manuscript.

This research was supported by grants from Agencia

Nacional de Promocion Cientıfica y Tecnologica

(ANPCyT), Sigma Xi, Fight for Sight Research Division

of Prevent Blindness America, and by the ‘Ramon Carrillo-

Arturo Onativia’ fellowship from the National Health

Office, Argentina.

References

Acott, T.S., Westcott, M., Passo, M.S., Van Buskirk, E.M., 1985.

Trabecular meshwork glycosaminoglycans in human and cynomolgus

monkey eye. Invest. Ophthalmol. Vis. Sci. 26, 1320–1329.

Bayer, A.U., Danias, J., Brodie, S., Maag, K.P., Chen, B., Shen, F.,

Podos, S.M., Mittag, T.W., et al., 2001a. Electroretinographic

abnormalities in a rat glaucoma model with chronic elevated intraocular

pressure. Exp. Eye Res. 72, 667–677.

Bayer, A.U., Neuhardt, T., May, A.C., Martus, P., Maag, K.P., Brodie, S.,

Lutjen-Drecoll, E., Podos, S.M., Mittag, T., et al., 2001b. Retinal

morphology and ERG response in the DBA/2NNia mouse model of

angle-closure glaucoma. Invest. Ophthalmol. Vis. Sci. 42, 1258–1265.

Benozzi, J., Jaliffa, C.O., Firpo Locoste, F., Weinberg Llomovatte, D.,

Keller Sarmiento, M., Rosenstein, R.E., 2000. Effect of brimonidine on

rabbit hyaluronidase activity. Invest. Ophthalmol. Vis. Sci. 41,

2268–2272.

Benozzi, J., Nahum, L.P., Campanelli, J.L., Rosenstein, R.E., 2002. Effect

of hyaluronic acid on intraocular pressure in rats. Invest. Opthalmol.

Vis. Sci. 43, 2196–2200.

Brooks, D.E., Garcia, G.A., Dreyer, E.B., Zurakowski, D., Franco-

Bourland, R.E., 1997. Vitreous body glutamate concentration in dogs

with glaucoma. Am. J. Vet. Res. 58, 864–867.

Chauhan, B.C., Pan, J., Archibald, M.L., LeVatte, T.L., Kelly, M.E.,

Tremblay, F., 2002. Effect of intraocular pressure on optic disc

topography, electroretinography, and axonal loss in a chronic pressure-

induced rat model of optic nerve damage. Invest. Ophthalmol. Vis. Sci.

43, 2969–2976.

Dreyer, E.B., Zurakowski, D., Schumer, R.A., Podos, S.M., Lipton, S.A.,

1996. Elevated glutamate levels in the vitreous body of humans and

monkeys with glaucoma. Arch. Ophthalmol. 114, 299–305.

Fazio, D.T., Heckenlively, J.R., Martin, D.A., Christensen, R.E., 1986. The

electroretinogram in advanced open-angle glaucoma. Doc. Ophthalmol.

63, 45–54.

Francois, J., Benozzi, J., Victoria-Troncoso, V., Bohyn, W., 1984.

Ultraestructural and morphometric study of corticosteroid glaucoma

in rabbits. Ophthalmic Res. 16, 168–178.


Glovinsky, Y., Quigley, H.A., Dunkelberger, G.R., 1991. Retinal ganglion

cell loss is size dependent in experimental glaucoma. Invest.

Ophthalmol. Vis. Sci. 32, 484–491.

Graham, S.L., Klistorner, A., 1998. Electrophysiology. A review of signal

origins and applications to investigating glaucoma. Aust. NZ

J. Ophthalmol. 26, 71–85.

Grozdanic, S.D., Betts, D.M., Sakaguchi, D.S., Allbaugh, R.A.,

Kwon, Y.H., Kardon, R.H., 2003. Laser-induced mouse model of

chronic ocular hypertension. Invest. Ophthalmol. Vis. Sci. 44,

4337–4346.

Harooni, M., Freilich, J.M., Abelson, M., Refojo, M., 1998. Efficacy of

hyaluronidase in reducing increases in intraocular pressure related to

the use of viscoelastic substances. Arch. Ophthalmol. 116, 1218–1221.

Holopigian, K., Seiple, W., Mayron, C., Koty, R., Lorenzo, M., 1990.

Electrophysiological and psychophysical flicker sensitivity in patients

with primary open-angle glaucoma and ocular hypertension. Invest.


Kiernan, J.A., 2003. Carbohydrate histochemistry, Third Ed., In: Histo-

logical and Histochemical Methods: Theory and Practice Arnold,

London pp. 213–242.

Knepper, P.A., Farbman, A.I., Telser, A., 1981. Aqueous outflow pathway

glycosaminoglycans. Exp. Eye Res. 32, 265–277.

Knepper, P.A., Goossens, W., Palmberg, P.F., 1996. Glycosaminoglycan

stratification of the juxtacanalicular tissue in normal and primary open-

angle glaucoma. Invest. Ophthalmol. Vis. Sci. 37, 2414–2425.

Korth, M., 1997. The value of electrophysiological testing in glaucomatous

diseases. J. Glaucoma 6, 331–343.

Korth, M., Nguyen, N.X., Horn, F., Martus, P., 1994. Scotopic threshold

response and scotopic PII in glaucoma. Invest. Ophthalmol. Vis. Sci.

35, 619–625.

Lerner, L.E., Polansky, J.R., Howes, E.L., Stern, R., 1997. Hyaluronan in

the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 38,

1222–1228.

Levkovitch-Verbin, H., Quigley, H.A., Martin, K.R., Valenta, D.,

Baumrind, L.A., Pease, M.E., 2002. Translimbal laser photocoagulation

to the trabecular meshwork as a model of glaucoma in rats. Invest.


Moore, C., Milne, S., Morrison, J.C., 1993. A rat model of pressure-induced

optic nerve damage [ARVO Abstract]. Invest. Ophthalmol. Vis. Sci. 34,

1141 (Abstract nr 2158).

Moore, C.G., Epley, D., Milne, S.T., Morrison, J.C., 1995. Long-term non-

invasive measurement of intraocular pressure in the rat eye. Curr. Eye

Res. 14, 711–717.

Naskar, R., Wissing, M., Thanos, S., 2002. Detection of early neuron

degeneration and accompanying microglial responses in the retina of a

rat model of glaucoma. Invest. Ophthalmol. Vis. Sci. 43, 2962–2968.

Nork, T.M., Ver Hoeve, J.N., Poulsen, G.L., Nickells, R.W.,

Davis, M.D., Weber, A.J., Vaegan, Sarks, S.H., Lemley, H.L.,

Millecchia, L.L., et al., 2000. Swelling and loss of photoreceptors in

chronic human and experimental glaucomas. Arch. Ophthalmol. 118,

235–245.

Panda, S., Jonas, J.B., 1992. Inner nuclear layer of the retina in eyes with

secondary angle-block glaucoma. Ophthalmologe 89, 468–471.

Rohen, J.W., 1983. Why is intraocular pressure elevated in chronic simple

glaucoma? Ophthalmology 90, 758–769.

Schubert, H.D., Denlinger, J.L., Balazs, E.A., 1984. Exogenous Na-

hyaluronate in the anterior chamber of the owl monkey and its effect on

the intraocular pressure. Exp. Eye Res. 39, 137–152.

Segawa, K., 1975. Ultraestructural changes of the trabecular meshwork in

primary open glaucoma. Jpn. J. Ophthalmol. 19, 317–322.

Shareef, S.R., Garcia-Valenzuela, E., Salierno, A., Walsh, J., Sharma, S.C.,

1995. Chronic ocular hypertension following episcleral venous

occlusion in rats. Exp. Eye Res. 61, 379–382.

Spicer, S.S., Schulte, B.A., 1992. Diversity of cell glycoconjugates shown

histochemically: a perspective. J. Histochem. Cytochem. 40, 1–38.

Takahata, K., Katsuki, H., Kume, T., Nakata, D., Ito, K., Muraoka, S.,

Yoneda, F., Kashii, S., Honda, Y., Akaike, A., et al., 2003. Retinal

neuronal death induced by intraocular administration of a nitric oxide

donor and its rescue by neurotrophic factors in rats. Invest. Ophthalmol.

Vis. Sci. 44, 1760–1766.

Ueda, J., Sawaguchi, S., Hanyu, T., Yaoeda, K., Fukuchi, T., Abe, H.,

Ozawa, H., et al., 1998. Experimental glaucoma model in the rat

induced by laser trabecular photocoagulation after an intracameral

injection of India ink. Jpn. J. Ophthalmol. 42, 337–344.

Velten, I.M., Horn, F.K., Korth, M., Velten, K., 2001a. The b-wave of the

dark adapted flash electroretinogram in patients with advanced

asymmetrical glaucoma and normal subjects. Br. J. Ophthalmol. 85,

403–409.

Velten, I.M., Korth, M., Horn, F.K., 2001b. The a-wave of the dark adapted

electroretinogram in glaucomas: are photoreceptors affected? Br.

J. Ophthalmol. 85, 397–402.

Viswanathan, S., Frishman, L.J., Robson, J.G., 2000. The uniform field and

pattern ERG in macaques with experimental glaucoma: removal of

spiking activity. Invest. Ophthalmol. Vis. Sci. 41, 2797–2810.

Vorwerk, C.K., Lipton, S.A., Zurakowski, D., Hyman, B.T., Sabel, B.A.,

Dreyer, E.B., 1996. Chronic low dose glutamate is toxic to retinal

ganglion cells: toxicity blocked by memantine. Invest. Ophthalmol.

Vis. Sci. 37, 1618–1624.

Wang, X., Tay, S.S., Ng, Y.K., 2002. An electron microscopic study of

neuronal degeneration and glial cell reaction in the retina of

glaucomatous rats. Histol. Histopathol. 17, 1043–1052.

A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid

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