Download Dissecting ocular tissue for intraocular drug studies.

Investigative Ophthalmology
March 1976
216 Reports
munologic stimuli such as corneal allografts1 as
well as prolong graft survival.1"4
However, in considering the adoption of this
technique to human corneal transplantation, several considerations make a homologous antibody
preparation more desirable than a heterologous
preparation. There are now available techniques
for measuring the number and quantity of various
antibodies against human transplantation antigens. Thus, it would be possible to repeatedly
make a given antibody preparation for use in
corneal transplantation. A homologous preparation is less cytotoxic and therefore carries less
potential for damage to the graft. Likewise, it is
not necessary to chemically modify the homologous preparation to avoid this problem.
The results of the present experiments indicate
that homologous antibody against transplantation
antigens is successful in prolonging rabbit corneal
allograft survival. Based on these studies, it
would appear that similar pretreatment of human
corneal allografts might be successful in prolonging graft survival and even, perhaps, in reducing
the incidence of allograft rejection.
The author acknowledges the technical assistance of Ms. Patricia Skahen and Ms. Karen
Vedder.
From the Department of Ophthalmology University of Washington School of Medicine, Seattle,
Wash. 98195. Supported by Grant EY-01282
from the National Eye Institute. Submitted for
publication Sept. 17, 1975. Reprint requests: Dr.
J. Chandler.
REFERENCES
1. Burde, R. M., Waltman, S. R., and Berrios,
J. H.: Homogaft rejection delayed by treatment of donor tissue in vitro with antilymphocyte serum, Science 173: 921, 1971.
2. Chandler, J. W., Gebhardt, B. M., and Kaufman, H. E.: Immunologic protection of corneal allografts: preparation and in vitro
testing of heterologous "blocking" antibody,
INVEST. OPHTHALMOL. 12: 646, 1973.
3. Chandler, J. W., Gebhardt, B. M., Sugar,
J., et al.: Immunologic protection of rabbit
corneal allografts: survival of corneas pretreated with succynilated anti-lymphocyte
globulin, Transplantation 17: 146, 1974.
4. Binder, P. S., Gebhardt, B. M., Chandler,
J. W., et al.: Immunologic protection of rabbit corneal allografts with heterologous blocking antibody, Am. J. Ophthalmol. 79: 949,
1975.
5. Kaliss, N.: Micromethod for assaying immune
cytolysis by the release of 51Cr, Transplantation 8: 526, 1969.
6. Khodadoust, A. A.: Penetrating keratoplasty
in the rabbit, Am. J. Ophthalmol. 66: 899,
1968.
7. Khodadoust, A. A., and Silverstin, A. M.:
Transplantation and rejection of individual
cell layers of the cornea, INVEST. OPHTHALMOL. 8: 180, 1969.
8. Guttman, R. D., Carpenter, C. B., Lindquist,
R. R., et al.: Renal transplantation in the
inbred rat. III. A study of heterologous antithymocyte sera, J. Exp. Med. 126: 1099,
1967.
9. Wilson, W. E. C , Kirkpatrick, C. H., and
Talmage, D. W.: Immunologic studies in
human organ transplantation. III. The relationship of delayed cutaneous hypersensitivity to the onset of attempted kidney allograft rejection, J. Clin. Invest. 43: 1881, 1964.
10. Sell, S.: Antilymphocytic antibody: effects in
experimental animals and problems in human
use, Ann. Intern. Med. 71: 177, 1969.
Dissecting ocular tissue for intraocular
drug studies. ROBERT ABEL, JR.* AND
GERARD L. BOYLE.
This report describes a convenient reproducible
ocular dissection technique which has important
applications for ocular antimicrobial penetration
studies. Different ocular tissues can be sectioned
while frozen and then plated directly on culture
medium containing the test organism; after the
zones of bacterial inhibition are measured at 18
hours following incubation, the tissue specimens
are weighed providing more reliable evidence
regarding drug concentrations. In such a fashion,
a drug can be administered topically, subconjunctivally, or systemically, and assayed, from the
cornea to the optic nerve at various time intervals.
Analysis of antibiotic in the vitreous body, which
has important application in the therapy of endophthalmitis, can be routinely performed in the experimental model.
The judicious use of antimicrobial therapy is
based on controlled ocular penetration studies.
Traditionally, these evaluations are limited to
analyzing drug concentrations in the aqueous
humor and serum. Recently, certain authors1-3
have incorporated tissue dissection, in addition to
performing anterior chamber paracentesis in order
to study drug uptake in the eye. The purpose of
this report is to present a convenient method of
ocular dissection which facilitates antibiotic
analysis of separate tissues with minimal contamination. For this reason, cefazolin sodium was
selected as a sample antibiotic to illustrate the
information that can be provided by ocular dissection.
Methods. In a previous report,4 albino rabbits
received subconjunctival (one eye only), intramuscular, and intravenous cefazolin sodium at
various time intervals before sacrifice. The discussion will be confined to the group of 46 rabbits (two to four animals for each time period)
L
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Volume 15
Number 3
which received unilateral subconjunctival antibiotic injection. Painless eardiorespiratory arrest
was accomplished by the administration of sufficient intravenous sodium pentobarbital. Samples
of primary aqueous humor were immediately obtained from each eye by anterior chamber paracentesis under sterile conditions using a 25-gauge
needle mounted on a disposable syringe. Simultaneously, 10 ml. of blood was also collected by
intracardiac aspiration.
The sterile containers of aqueous humor and
serum samples were frozen at -70° C. until the
time of the bioassay. The eyes of each rabbit
were enucleated removing as much episcleral and
leptomeningeal tissue as possible leaving at least
5 mm. of optic nerve. Each eye was positioned,
cornea-side up, in a sterile disposable capped
tube, which was immediately frozen by immersion
in a mixture containing solid carbon dioxide and
acetone. The frozen eye samples were then stored
in a deep freeze at -70° C. until the time of
dissection.
The tube dilution technique"' was employed in
the determination of the aqueous humor and
serum levels of ccfaznlin sodium with tin; lest
organism, Staphyhcoccus aureus (ATCC 12(300).
The cefazolin sodium activty of the other ocular
tissues and fluids was determined by the agar
diffusion assay technique employing Mueller-Hinton agar and the same test organism. The test
organism was grown and standardized, after which
the sensitivity plates were seeded with a 10-a
dilution of this culture. The seeded plates were
then dried at 37° C. for 15 minutes.
A standard curve for determining antibiotic
concentration in ocular tissue and vitreous humorsamples was prepared from data obtained when
sterile filter paper discs (6.35 mm. in diameter)
were immersed in known cefazolin concentration
in trypticasc soy broth and then placed in
triplicate on the inoculated plates containing the
test organism. After overnight incubation at 37°
C, the zones of bacterial inhibition were measured
and results averaged in order to plot the standard
curve for the known concentrations of cefazolin
sodium. In such a way, concentrations of antibiotic in ocular tissue could be expressed in micrograms per 100 mg. of tissue, whereas the vitreous
humor antibiotic concentration was determined in
mierograms per milliliter of fluid.
The frozen eyes were removed for dissection
using sterile technique. Each globe was bisected
just through the back of the lens in order to
provide an anterior and posterior vitreous compartment, by using a No. 11 Bard-Parker blade
and scalpel in a sawing movement (Fig. 1). This
step was facilitated by the use of an adjustable
wire clamp which limits excursion of the globe
during the initial dissection. The anterior and
posterior vitreous samples were rapidly eviscerated
Reports
217
Fig. 1. Bisected eye showing the frozen lens in
the anterior half of the globe.
Fig. 2. Dissection of the corneoseleral cap, iris,
and ciliary bod)' away from the lens and anterior
vitreous. Frozen aqueous can be seen lying on the
sterile Petri dish between the solera and iris.
(Fig. 2) and placed in separate sterile tubes;
it is essential to complete this step before thawing
occurs. Lens, iris, cornea, sclcra, and optic nerve
tissue samples were rinsed in sterile saline (to
eliminate surface contaminants), drained, and
placed on the previously dried agar plates inoculated with the test organisms. At a later time
they were weighed and measured. Sterile filter
paper discs were absorbed with anterior and
posterior vitreous samples and then placed on the
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Investigative Ophthalmology
March 1976
218 Reports
Table I. Comparison of in vitro susceptibilities of various organisms to cefazolin sodium with
the aqueous and vitreous humor levels attained after subconjunctival injection (25 mg.)
Average antibiotic concentration (ng/ml.)
Vitreous level
Pathogen
Citrobacter sp.
Herellea sp.
Ps. aeruginosa
Serratia sp.
Prot. sp. (ind. pos.)
Enterobacter sp.
Enterococcus
D. pneumoniae
Pr. mirabilis
Shigella sp.
Salmonella sp.
Bacteroides
H. influenza
Esch. coli
Kleb. pneumoniae
Coryn. diphtheriae
Str. viridans
S. aureus
N. gonorrhoeae
Str. pyogenes
Cl. perfringens
Cl. tetani
M.I.C. (ng/ml.)
Cefazolin (5)
Aqueous level
0.25 hr.
1.0 hr.
Anterior
0.25 hr.
Posterior
1.0 hr.
0.25 hr.
1.0 hr.
>128
> 128
> 128
>128
32->128
8-M28
32.0
0.12-25
8.0
8.0
4.0
2-4.0
2-4.0
1-4.0
2.0
2.0
0.5-1
0.25-5
0.25
0.12
0.06- .12
0.06
30.8
surface of the inoculated plates. Following overnight incubation at 37° C. for 18 hours, zones
of bacterial inhibition (i.e., a clear zone surrounding the various ocular samples) were measured.
Controls included similar ocular tissue samples
obtained from uninjected rabbits.
Results. Table I indicates the aqueous and
vitreous humor antibiotic concentrations at 0.25
and 1 hour following 25 mg. subconjunctival injection of cefazolin sodium juxtaposed on a list
of the in vitro susceptibilities of many potential
pathogens to this agent. Fig. 3 illustrates the
cefazolin concentrations achieved at varying time
intervals in aqueous humor and anterior and
posterior vitreous following subconjunctival administration. The results of bioassay of cornea and
sclera samples are shown in Fig. 4; peak concentrations are attained in 30 minutes.
Discussion. Investigations of the intraocular
penetration of new antimicrobial agents have
usually been based on aqueous humor and serum
determinations. Employing ocular tissue dissection
in such experimental studies can provide meaningful information about antibiotic distribution in
other ocular fluids and tissues whatever the route
of drug administration.
Immediate freezing of the eye permits the later
analysis without complication of biodegradation
of drug or difficulty of keeping tissues separate.
Freezing also allows the investigators to dissect
9.0
18.0
15.3
2.92
1.08
all of the eyes at one time without being disturbed
by other procedures. More importantly, the
gelatonoid vitreous humor can now be conveniently
obtained and analyzed.
The concentrations of antibiotic in solid tissue
were calculated by comparing the zone of bacterial inhibition from weighed tissue specimens
to the zones obtained from filter paper discs immersed in known cefazolin concentrations, in a
manner similar to the technique of Barza and
co-workers.3 However, these tissue levels of antibiotics must be considered relative rather than
absolute because diffusion of drug from tissue
samples does not necessarily coincide with diffusion from filter paper. It would be disadvantageous to employ tissue homogenization because
the dilutional factor would affect the quantitation
of small amounts of antibiotic and many separate
blenders would have to be used to eliminate
contamination.
Significant antibiotic penetration was demonstrated in sclera, cornea, and iris tissue samples
after subconjunctival injection (as might be expected), but also following intramuscular and
intravenous cefazolin administration. 4 There is
some antibiotic absorption into the anterior and
posterior vitreous humor (Fig. 3) and even the
optic nerve after subconjunctival injection of antimicrobial agents. Perhaps even higher drug levels
in the vitreous, retina, and choroid could be at-
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Volume 15
Number 3
Reports 219
A Aqueous
o Anterior Vitreous
• Posterior Vitreous
A
30
n.a.a.
A
O
A
20
A
A
A
0
t
No antibiotic assayable
A
A
0
10.0
o
7.5
0
A
5.0
•
o
-.
2.5
0 o
o
o
•
0
A A A
O O O O
A A
0
15
m
a
30
45
0
2
1
MINUTES
3
HOURS
Fig. 3. Comparison of aqueous and vitreous humor
drug (cefazolin sodium 25 mg.) levels following
subconjunctival injection.
A CORNEA
3000
2000
A
,
• SCLERA
A
n.a.a.
1000
No antibiotic assayable
•
*•
200
80 :
60
'A
40
•
20.0
A
A
A
io.o;
A
A
5.0
REFERENCES
•
2.5
•
0
n.a.a.
A
••
0
15 30 45
MINUTES
1
3
From the Departments of Ophthalmology,
Mount Sinai School of Medicine of the City University of New York, and Temple University
Hospital, Philadelphia, Pa. This study was supported in part by Grant No. EY-00340 of the
National Eye Institute, National Institutes of
Health. Submitted for publication Aug. 13, 1975.
Reprint requests: Dr. R. Abel. ^Present address:
1300 Harrison St., Wilmington, Dela. 19806.
Key words: ocular tissue dissection technique,
antibiotic penetration studies, cefazolin sodium,
cryodissection, aqueous humor drug level, vitreous
body drug level.
A
7.5
hematogenous distribution. Although our method
of assaying cefazolin sodium was not sensitive
enough to quantitate the aqueous humor drug
level in the contralateral eye after subconjunctival
injection, another study demonstrated adequate
bactericidal concentrations of gentamicin in the
aqueous humor of the opposite eye.7
This ocular cryodissection technique is especially useful for analyzing antibiotic activity in anterior and posterior vitreous tumor, in addition to
the other ocular tissue samples. In the cefazolin
study, significant vitreous absorption was present
from five minutes to three hours after subconjunctival inoculation of 25 mg. of antibiotic; also,
minimal cefazolin levels were detected in the
contralateral eye for 15 to 45 minutes after the
injection. The ability to achieve high antibiotic
concentrations in the vitreous humor is an extremely important aspect in the prevention and
treatment of intraocular infections.
Such an ocular tissue dissection method can be
applied to various avenues of drug delivery, can
include retina and choroid specimens, and can
provide meaningful information regarding drug
penetration in the inflamed or postoperative eye.
4
5
HOURS
Fig. 4. Antibiotic (cefazolin sodium 25 mg.)
activity in cornea and sclera following subconjunctival administration.
tained after retrobulbar or intracameral administration. Orally administered agents, such as amoxicillin, a new semisynthetic penicillin, have also
been found to adequately penetrate ocular tissues
with this dissection technique.6
Interestingly, significant antibiotic content can
be found in the cornea and sclera of the contralateral eye after subconjunctival administration.4
This can probably be explained on the basis of
1. Salminen, L., Jarvinen, H., and Toiva.nen, P.:
Distribution of tritiated penicillin in the rabbit
eye, Acta Ophthalmol. 47: 115, 1969.
2. Bloom, M. A., Golden, B., and McKee, A. P.:
Antibiotic concentration in ocular tissues, Arch.
Ophthalmol. 83: 78, 1970.
3. Barza, M., Baum, J., Birkby, B., et al.: Intraocular penetration of carbenicillin in the rabbit, Am. J. Ophthalmol. 75: 307, 1973.
4. Abel, R., Boyle, G. L., Furman, M., et al.:
Intraocular penetration of cefazolin sodium in
rabbits, Am. J. Ophthalmol. 78: 779, 1974.
5. Schneierson, S. S., and Torarsky, B.: A method for the determination of Aureomycin in the
blood, J. Bacteriol. 57: 483, 1949.
6. Faigenbaum, S. J., Boyle, G. L., Prywes,
A. S., et al.: Intraocular penetration of
amoxicillin, submitted for publication.
7. Abel, R., Jr., Mascuilli, L., and Boyle, G. L.:
Subconjunctival gentamicin prophylaxis against
post-operative staphylococcus endophthalmitis
in the rabbit, submitted for publication.
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