Q-Switched Neodymium-YAG Laser
Trabeculopuncture in Monkeys
Shlomo Melamed, MD; Jean Pei, MD; Carmen A. Puliafito, MD; David L. Epstein, MD
\s=b\ In nine cynomolgus monkeys, trabeculopuncture was performed with a Q-switched neodymium-YAG laser, using a pulse
energy of 5 to 7 millijoules and an exposure time of 14
nanoseconds. A penetration into Schlemm's canal was successfully achieved with two to four pulses; this penetration was
accompanied by intraocular pressure (IOP) reduction and blood
reflux into the anterior chamber. However, after eight days, IOP
returned to baseline level, while white tissue was observed
gonioscopically to fill in the puncture sites. Histologically, one
hour after laser treatment, a blasting effect on the trabecular
meshwork was observed with no signs of necrosis. Ater eight
days, a hypertrophic scar formed, with the corneal endothelium
extending over the scarred surface. At eight weeks and at six
months after laser treatment, further shrinkage of the scar and
the formation of a membrane over it was evident. Attempts to
control scar formation by preventing blood reflux or injecting
fluorouracil subconjunctivally for two weeks were unsuccessful.
Scar formation at the trabecular puncture site severely limits the
applicability of this potentially simple glaucoma treatment.
(Arch Ophthalmol 1985;103:129-133)
hole through the trabecular mesh¬
Puncturing
work into Schlemm's canal has been attractive to
a
several investigators113 as a potentially simple treat¬
ment for lowering intraocular pressure (IOP) in
glaucomatous eyes. With various laser systems
(ruby,1"4 argon,510 and neodymium-glass,12·13) a pene¬
tration into Schlemm's canal has been successfully
achieved, but such "holes" have eventually closed
with the subsequent return of IOP to baseline level.
Scar formation at the puncture site has been corre¬
lated histologically with the failure of the proce¬
dure.5·6·712
Q-switched neodymium-YAG lasers have only reAccepted for publication Nov 20, 1984.
From the Howe Laboratory of Ophthalmology, Massachusetts
Eye and Ear Infirmary, Harvard Medical School, Boston.
Reprint requests to the Massachusetts Eye and Ear Infirmary,
243 Charles St, Boston, MA 02114 (Dr Epstein).
cently been introduced. They have the special prop¬
erties of long wavelength (1,064 nm) and ultrashort
exposure time (nanoseconds), which make them
capable of nonthermal cutting, as opposed to the
coagulative thermal effects produced by the con¬
tinuous wave argon laser.11 The aim of our study was to
describe the morphologic changes that occur following
Nd-YAG laser trabeculopuncture and to determine
whether such a nonthermal disruptive effect
could help prevent scar formation at the puncture site.
Also, pharmacologie attempts were made to affect
the healing process following trabeculopuncture.
MATERIALS AND METHODS
Nine cynomolgus monkeys were used in our study. Prior
to laser treatment, all monkeys were anesthetized with
intramuscular (IM) ketamine hydrochloride (10 mg/kg),
followed by intravenous (IV) sodium phénobarbital (35
mg/kg). Intraocular pressure measurements (Digilab
Pneumotonograph) and gonioscopy were performed before
and at various intervals after laser treatment. A Kauf¬
man-Wallow monkey laser goniolens, a single mirror with
antireflecting glass (Ocular Instruments, Ine, Redmond,
Wash) was used.
Penetrating Lesions
In each of four monkeys, four penetrating lesions into
Schlemm's canal were created (one penetration per quad¬
rant) in the right eye; the left eye served as a control.
Inferior quadrants were initially treated to prevent inter¬
ference of blood reflux in subsequent treatments. For
penetration, a YAG-100 system (American Medical Optics)
was used, delivering a pulse energy of 5 to 7 millijoules,
with an exposure time of 14 nanoseconds and a spot size of
20 µ (the calculated power density was 11 to 16 X 10'°
W/sq cm per pulse). Perforation into Schlemm's canal was
considered successful when blood reflux into the anterior
chamber, followed by a large decrease in IOP, was
observed. The treated eyes were enucleated and processed
one hour, eight days, eight weeks, and six months after
laser treatment.
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Intraocular Pressure (IOP) Changes Following
Neodymium-YAG Laser Trabeculopuncture (TP)
IOP, mm Hg
Eye
1
2
3
4
5
6
7
8
Procedure
Penetrating TP
Penetrating TP
Penetrating TP
Penetrating TP
Nonpenetrating TP
Nonpenetrating TP
Penetrating TP and
fluorouracil
Penetrating TP and
fluorouracil
Sodium hyaluronate
injection into
anterior chamber
followed by
penetrating TP
Before After Before Enucleation
Laser Laser (Postlaser Interval)
12
12 (1 hr)
10
19 (8 days)
22 (8 wk)
20
10
21 (6 mo)
18
17
17 (1 hr)
20
18
22 (8 wk)
21
11
20 (3 wk)
22
19
21
21 (6 wk)
20
35
25*
18 (3 wk)
'The IOP fell to 8 mm Hg the following day.
Fig 1.—Floor of crater one hour after neodymium-YAG trabec¬
ulopuncture. Outer wall of Schlemm's canal is ruptured (ar¬
rows), exposing scierai collagen (S), as shown by transmission
electron microscopy (X3,660).
Nonpenetrating Lesions
In two monkeys, nonpenetrating lesions in the trabecu¬
lar meshwork were created, using energy pulse levels of 3
to 5 millijoules. The treated eyes were enucleated and
processed immediately and eight weeks following treat¬
ment.
Sodium Hyaluronate Injection
Into the Anterior Chamber
In an attempt to prevent blood reflux into the anterior
chamber, 0.2 mL of sodium hyaluronate was injected into
the right eye of one monkey prior to trabeculopuncture.
Intraocular pressure increased to 35 mm Hg. Penetrating
energy (5 to 7 millijoules) was then delivered to four sites
in each of four quadrants of the angle.
Subconjunctival Fluorouracil Injections
Fig 2.—Gonioscopic view eight days after neodymium-YAG
trabeculopuncture. White area represents heavily scarred
puncture site. Scarred sites vary from containing gray "webby"
tissue to dense white material.
In two monkeys, an attempt to inhibit scar formation
was made by injecting 10 mg (0.2 mL) of fluorouracil
subconjunctivally immediately following penetrating tra¬
beculopuncture. This procedure was followed by daily
injections of 5 mg (0.1 mL) of fluorouracil for a week and
the same dose every alternate day for another week. Eyes
were enucleated and processed three weeks following
treatment.
The monkey eyes were fixed in vivo at various intervals
after trabeculopuncture with the IOP at 15 mm Hg, using
anterior chamber perfusion with 3% glutaraldehyde in
0.1N cacodylate buffer for 60 minutes. After enucleation,
radial sections of the iridocorneal angle were incised,
postfixed in 1% osmium tetroxide, and passed through
graded alcohol concentrations. For scanning electron
microscopy, tissues were critical-point dried (Samdri PVT3), sputter-coated with gold-palladium (Polaron E5000),
and observed under the scanning electron microscope
(JEOL-35). For light microscopy, tissues were embedded in
epoxy resin (EPON); 1-^m thick sections were stained with
méthylène blue and fuchsin. For transmission electron
microscopy, thin sections (silver-golden, 500 A) were
stained with uranyl acetate and lead citrate and observed
under the electron microscope (JEOL JEM 7).
RESULTS
A summary of IOP changes in all treated eyes is
given in the Table.
Penetrating Lesions
Using a pulse energy of 5 to 7 millijoules, two to
four pulse applications were required to perforate
the trabecular meshwork into Schlemm's canal.
Immediate Effect.—In all four eyes, blood reflux
into the anterior chamber was followed by a dramat¬
ic decrease of IOP from a mean of 20 mm Hg to a
of 10 mm Hg. Morphologically, a blasting
effect on the trabecular meshwork was strikingly
evident, with crater formation and distortion of the
remaining trabecular meshwork and Schlemm's
canal. In transmission electron microscopy, the "microknife action" of the Nd-YAG laser can be seen
cutting through the cytoplasm of cells, with no signs
of coagulation or necrosis. The floor of the crater
formed by the blast reached beyond the posterior
mean
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Fig 3.—Light microscopic view eight days after neodymiumYAG trabeculopuncture. Hypertrophie scar is formed at crater
site (T). Fibrocytes are filling in lasered area (arrows), with total
loss of trabecular meshwork structure. Note dilated, thrombosed vessels (V) of iris. CB indicates ciliary body (méthylène
blue and fuchsin, X180).
wall of Schlemm's canal, exposing scierai collagen
(Fig 1).
Eight Days After Laser Treatment.—Intraocular
pressure returned to the pre-laser-treatment level,
and white tissue was gonioscopically observed filling
the puncture site over an eight-day period following
trabeculopuncture (Fig 2). When compared with the
Fig 4.—Eight days after neodymium-YAG trabeculopuncture.
Trabecular meshwork is totally scarred (T), and processes from
corneal endothelium (CE) and operculum are sliding over scar
surface, as shown by scanning electron microscopy. CB
indicates ciliary body (X220) (tilted 60°).
control eye, hypertrophie scar formation in the
treated area was observed, with total loss of
Schlemm's canal and trabecular meshwork struc¬
ture, and infiltration of the tissue by fibrocytes
(Fig 3). Also, a thin "membranelike" extension
was observed over the scarred meshwork originat¬
ing from the corneal endothelium and operculum
(Fig 4).
Long-term Follow-up.—At eight weeks and at six
months after laser treatment, further extension of
the "membrane" and shrinkage of the scar were the
two most striking findings (Fig 5). Transmission
electron microscopy disclosed the "membrane" to be
composed of scar surface fibrocytes, with very thin,
elongated cytoplasmic extensions in continuation
with the corneal endothelium (Fig 6). In some
treated areas, a Descemet's membrane-like struc¬
ture was clearly seen extending into the scar tissue,
without accompanying corneal endothelium.
Nonpenetrating Lesions
Using
pulse of 3 or 5 millijoules, there was no
blood reflux into the anterior chamber and no change
in IOP.
Immediate Effect.—Fractured beams and tissue
debris were observed via scanning electron microsco¬
py. Also, extensive circular damage to the corneal
endothelium was evident, indicating a shock-wave
impact in this region (Fig 7).
Eight Weeks After Laser Treatment.—Despite the
absence of blood reflux following nonpenetrating
treatment, the trabecular meshwork was similarly
one
Fig 5.—Light microscopic view eight weeks after neodymiumYAG trabeculopuncture. Thinned, elongated cytoplasmic pro¬
cesses (arrows) form a "membrane" over scarred, shrunken
trabecular meshwork (T). Note fibrosis of Schlemm's canal
area (SC). CC indicates collecting channel; AC, anterior cham¬
ber (méthylène blue and fuchsin, X720).
scarred and covered by the extensive growth of a
Descemet's membrane-like structure over its sur¬
face.
Sodium Hyaluronate Injection
Into the Anterior Chamber
There was no blood reflux into the anterior cham¬
ber following trabeculopuncture. Intraocular pres¬
sure decreased to 8 mm Hg the following day, but six
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Fig 6.—Transmission electron microscopic view six months
after neodymium-YAG trabeculopuncture. Superficial fibrocyte
(F), with thinned cytoplasmic processes (arrows) covers dense
collagenous scar. Descemet's membrane-like structure (D) is
deeply embedded in scar, without accompanying corneal
endothelium (X2.450).
Fig 7.—Scanning electron microscopic view of nonpenetrating
lesion immediately following one pulse of 5 millijoules. Lasered
lesion (L) can be seen at trabecular meshwork. Note extensive
circular damage to corneal endothelium presumably caused by
shock-wave (arrows). CE indicates corneal endothelium; I, Iris
(X140).
days later returned to baseline level, and white tissue
brane" was observed to slide over the trabecular
scar, consisting of corneal endothelial cells connected
to thinned, elongated cytoplasmic processes of sur¬
face fibrocytes. Growth and progression of this sheet
of cells, along with Descemet's membrane-like struc¬
ture, over the shrunken, densely collagenous scarred
lesion were seen at eight weeks and at six months
following the treatment.
Three mechanisms might be involved in this clo¬
sure of the trabeculopuncture site. First, there is the
growth of a "membrane" over the lasered area. The
corneal endothelium, and thereby Descemet's mem¬
brane-like structure, are stimulated to grow
because of a direct laser (or direct shock-wave) effect
on the corneal endothelium itself in the operculum
region. This tissue then connects with scar surface
fibrocytes to form an impermeable sheet over the
trabecular meshwork. In the monkey, Schlemm's
canal lies more anteriorly than in the human, mak¬
ing it necessary to direct the laser beam closer to the
corneal endothelium to penetrate the trabecular
meshwork. However, such stimulation and growth of
corneal endothelium has been similarly shown to
occur in humans after argon laser trabeculoplasty in
glaucomatous eyes14 and after Nd-YAG trabeculo¬
puncture in normal eyes.12 The exact mechanism for
this laser-induced cellular stimulation is still
unknown. It could conceivably be a secondary phe¬
nomenon occurring subsequent to trabecular mesh¬
work tissue breakdown and scarring.
The second possible mechanism is blood reflux into
the anterior chamber, with thrombocytes and clot¬
ting factors initiating fibrosis. We made two differ-
was observed to fill the
puncture sites.
Subconjunctival Fluorouracil Injections
Despite treatment with fluorouracil subconjunctivally for two weeks, IOP returned to baseline level
eight to ten days following trabeculopuncture, and
scar tissue filled the puncture site. Histologically,
three weeks after laser treatment, pigment-laden
macrophages with fibrosis were evident.
COMMENT
Two to four pulses of Q-switched Nd-YAG laser (5
to 7 millijoules each) were sufficient to create a hole
through the trabecular meshwork into Schlemm's
canal. They were also associated with blood reflux
into the anterior chamber and notable IOP reduction
in all treated eyes. The calculated power density for
such penetration was in the range of 22 to 64 X 1010
W/sq cm, which is slightly higher than the power
density calculated for Q-switched ruby laser report¬
ed by Bonney et al4 to cause extensive damage to the
trabecular meshwork. A blasting effect on the tra¬
becular meshwork and a direct communication into
Schlemm's canal were achieved by the nonthermal
tissue disruption and microknife action of the NdYAG laser, with sharply demarcated cutting planes
through cellular cytoplasm. Despite this technically
successful penetration, the use of the Nd-YAG laser
did not prevent scar formation at the puncture site.
Eight days following trabeculopuncture, a hypertro¬
phie scar had already formed, and IOP had already
returned to baseline level. Concurrently, a "mem-
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ent attempts to eliminate blood reflux, thereby
determining its importance in scar formation. In the
first attempt, nonpenetrating lesions were made
with the Nd-YAG laser, using a single pulse applica¬
tion of 3 to 5 millijoules. Despite the resulting lack of
blood reflux into the anterior chamber, superficial
scarring of the trabecular meshwork occurred (with
Schlemm's canal remaining intact) and a Descemet's
membrane-like structure was again observed to have
grown over the trabecular meshwork region.
In the second attempt, sodium hyaluronate was
injected into the anterior chamber prior to Nd-YAG
laser trabeculopuncture, the IOP was thereby ele¬
vated, and blood reflux (due to contact lens compres¬
sion of limbal vessels) was not observed. However, as
with the nonpenetrating lesions, scar formation was
still observed six days after trabeculopuncture, with
a return of IOP to baseline level. These two experi¬
ments suggest that blood reflux into the anterior
chamber does not play a major role in scar formation
at the laser puncture site.
The third mechanism that might be responsible
for the closure of the trabeculopuncture site is the
tissue breakdown caused by the Nd-YAG laser, with
liberation of factors that trigger a healing process in
the trabecular meshwork. We hypothesize this to be
the major mechanism involved in the occurrence of
fibrosis at the laser puncture site. As mentioned
above, trabecular tissue breakdown products may
also be responsible for a secondary downgrowth of a
Descemet's membrane-like structure over the
scarred laser lesion. In an attempt to inhibit fibro¬
cytic proliferation within the trabecular meshwork
following trabeculopuncture, fluorouracil1517 was
injected subconjunctivally for two weeks, using a
dosage sufficient for a calculated therapeutic concen¬
tration to occur in the anterior chamber.18 However,
despite this antimetabolite therapy, after eight to
ten days, scar tissue once again was observed to fill
the puncture sites, and IOP returned to the prelaser-treatment level. The failure of fluorouracil to
inhibit scar formation might be explained by the
infiltration of nonproliferating fibrocytes from the
vascular system or adjacent tissue (ciliary body or
iris) into the lasered tissue. Alternately, it is possi¬
ble, despite our calculations, that a great enough
therapeutic level of the drug was not sustained in the
trabecular meshwork region.
We conclude that the Nd-YAG laser is technically
effective in penetrating the trabecular meshwork
into Schlemm's canal, but despite its nonthermal
cutting effect, the tissue response is such that scar
formation at the puncture site occurs. Such scar
formation remains a severe obstacle to the applica¬
tion of this potentially simple treatment for glauco-
ma. Neither preventing blood reflux nor injecting
fluorouracil appeared to alter this fibrotic process in
the trabecular meshwork. Further studies are
needed to better understand the biologic response to
such laser energy absorption in the trabecular mesh¬
work before Nd-YAG laser trabeculopuncture can be
utilized therapeutically in open-angle glaucoma.
This study was supported in part by National Eye Institute
grant EY01894 and National Glaucoma Research, a program of
the American Health Assistance Foundation.
The authors wish to acknowledge the technical veterinary
assistance of Susan Bassett-Chu.
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