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Immunocharacterization of conjunctival mucins with impression cytology
X ICER Abstracts
Tuesday, 5:30-7:00 P.M., Sep 22, 1992 Palazzo Dei Congressi
342
34 ULTRASTRUCTURAL STUDY OF DYSTROPHIC AND CORNEAS BY OUICK-FREEZE, DEEP-ETCH Hirsch,M.,Dagonet,F.,Savoldelli.M.,LeVarl~t,B.Malek,N~ and Pouliquen, Y. INSERM U.86,Hdtel Dieu,75181 Paris, Cedex
SCARRED METHOD
04,
HUMAN
France.
The fine structure of the human cornea1 stroma has investigated with conventional been essentially methods for electron microscopy. In this study, we used the quick-freeze, deep-etch, and rotary-shadow replication method to analyze the 3-D organization of the extracellular matrix in some freshly removed dystrophic and scarred corneas.Controls were taken from the transparent periphery of centrally scarred corneas. Results showed the following abnormalities : In macular Groenouw's II dystrophy, thin filaments forming meshworks with globular entities, rounded inclusions with smooth surface, and disordered arrays granular Groenouw' s of the major collagen fibrils.In I dystrophy, linearly disposed small varicosities, and patches of branched fine beaded filaments.In BiberHaab-Dimmer amyloidosis, packs of relatively thick filaments, and bundles of major collagen fibrils without interfibrillar bridges.In opaque scars, very irregular arrangements of the major collagen fibrils, and rare interfibrillar attachments. This cryomethod of investigation which adds further information to that obtained from classicallyprocessed specimens, may be used to study the 3-D structure of pathologic corneas at the macromolecular level nearer to the native state.
343
35
THE EFFECT OFTOPICALLY APPLJED AUI’OLOGOUS ON CORNEAL EPlT?IELIAL HEALING FOLLOWING WOUNDS. Choi. C-H and Chump. Department of Ophthalmology and Eye Research Koryo General Hospital, Seoul, Korea.
EVALUATION OF CORNEAL EPITHELIAL CELL REPLICATION AFTER ALKALI INJURY BY IMHUNOCYTOCHEMlSTRY FOR PROLIFERATING CELL NUCLEAR ANTIGEN. Keiko Yamaguchi, Yoshiko Numazaki, Hakoto Taeai. Department of Ophthalmology, Tohoku University School of Medicine, Sendai. Jaoan. Cornea1 epithelial cell replication after alkali injury was evaluated immunocytochenistry for proliferating cell nuclear antigen (PCNA/ cyclin). an endogenous marker for detecting proliferating cells. PCNA. a cell-cycle regulated protein is present in varying amounts only in proliferating cells. Central cornea1 alkali injuries were produced in New Zealand white rabbits aged 2 months by 7 mm filter paper discs soaked in 1N NaOH uniformly. Animals were examined at l-. 3-. 55. ?-. 14-day after the injury by slit lamp and photography and eyes The cornea1 epithelial defect were processed for light microscopy. healed with single layer of the epithelial cells in 1 day in all animals spite of severe cornea1 swelling. Immunocytocheoistry for PCNA revealed rapid cell replication at areas between the alkali burn site and intact epitheliun at l- and J-day after the injury. lamunoreactive cells were observed centripetally from the limbus at 5-. 'I-day after the injury. Although the central cornea1 epitheliun showed layered structure at ll-day after the injury, opacifications were still observed and inmunoreactive cells were not observed neither in limbus nor for evaluating
central
area.
cell
PCNA
replication
would
in
be
a
promissing
The therapeutic effect of the topically applied autologous serum on the epithelial healing was examined in the rabbit eyes following alkali wounds. The corneal alkali wounds were produced by applying 5.5 mm filter paper soaked in Alkali IN NaOH on the central cornea for 60 seconds. wounded corneas were examined morphometrically at 0 hour, 6 hour, 18 hours, 30 hours, 42 hours, and then 2 days intervals after initial damage. Autologous serum in treated group and the BSS solution in control group were given topically 4 times a day for 3 weeks. The initial epithelial resurfacing in the treated group and in the con&r01 group were 0.80 t 0.14, 0.80 t 0.17, respectively. No statistical significance was observed in the initial epithelial healing phase. During the late healing phase, there was no significant difference between the treated gruop and control group on epithelial healing. (supported by Hyosuck Med. Research Fund and Kabi Pharmacia, Seoul)
indicator
344 ENCAPSULATED
GENTAMICIN
THERAPY
OF
Nos-Barber& Sz, M,Portol6s’-, A.Parento’, (1)Dept. Genet its t Microbiology. AUTbNOMA DEO;ARCELONA. Spain. (2)Dept. Rarcel ona. Spain. (*) present adress INSTITUTE, Harvard University. Boston,
PSEUDOMONAL
IMMUNOCHARACTERlW IMPRESSION
JM.B.Antbn’.
UNIVERSITFIT R&D, LIPOTEC : EYE RESEARCH Ma. USA.
of Burgundy,
agents of choice I” the keratitis. At “fortif ied” compliance and toxicological effects are importance. In a model of P.aeruQinosa pismented rabbits (n=6 per group) we compared treatment with Qentamicin (0.141) multilamellar liposomes applied topically twice a day to eentamicin (0.3%) eye drops applied every four hours ; control eye* received PBS. Liposome @Ye irritancy was tested by a) the "in viva" Draize test
Irritancy 1 owest (0.7fO.l) 1 ipo*omes
uentamicin keratitls.
and tests
grade
control demonstrated
of and
the
represent
eye
Impression
bacterial
drops
15.0f2.5(24h)/ that
toxicity X0/P
a good in the
in (4.020.5).
the Thus
alternative treatment
of
21000
Dijon
cytology n~ucua
characterize
co&nc!ival We have
of mucins
a specific
MUCINS
cells.
and
studied
Viiejuif
as a reliable
Classical mucina
WITH
but
they
Dpt, University
France.
and
histochemical
immunochemical
non invasi~
method
methods
allowed
suffered
from
their
stainIngs
of mucus
to
lack cells
of with
peptidiccoreendpoIysacohaMic
as well.
sensitive
1. Ophthalmology 2. IRS,
monoclonalantibodiesdirectedagainst moieties
CONJUNCTIVAL
G. Gu& France,
is considered
for studying
specificity.
monoclonal
These method
to hameat
antibodies mucin
provide
chains.
us with
Quantitative
assessmentwithimageanalysisassociatedtoaqualitativecharacterization of mucins patterns
21.5f3.0(9.5h).
liposomes both
OF
CYTOLOCY.
T.Baraa. A&on’.
C.Caroher’.
.
the
are
347
39
KERATITIS.
Aminogl ycosides treatment of dosages 11.4%) of considerable keratitis in
Lab
tissues.
36 LIPOSOME
SERUM ALKALI
in mucus ocouring
immunoohemicai
have the Draize test gentamicin to fortified P.aerusin0e.a
s.104
mucus
layer
ocular
dryness
with
disorders
are
of help
in the understanding
of wild
in dry eye syndroms. The fact we could applied these technics to impti seems to be a good way to collect a non Invasive conditions.
and reproducible
method
speciaUy
under
Tuesday, 5:30-7:00 P.M., Sep 22, 1992 Palazzo Dei Congressi
342
34 ULTRASTRUCTURAL STUDY OF DYSTROPHIC AND CORNEAS BY OUICK-FREEZE, DEEP-ETCH Hirsch,M.,Dagonet,F.,Savoldelli.M.,LeVarl~t,B.Malek,N~ and Pouliquen, Y. INSERM U.86,Hdtel Dieu,75181 Paris, Cedex
SCARRED METHOD
04,
HUMAN
France.
The fine structure of the human cornea1 stroma has investigated with conventional been essentially methods for electron microscopy. In this study, we used the quick-freeze, deep-etch, and rotary-shadow replication method to analyze the 3-D organization of the extracellular matrix in some freshly removed dystrophic and scarred corneas.Controls were taken from the transparent periphery of centrally scarred corneas. Results showed the following abnormalities : In macular Groenouw's II dystrophy, thin filaments forming meshworks with globular entities, rounded inclusions with smooth surface, and disordered arrays granular Groenouw' s of the major collagen fibrils.In I dystrophy, linearly disposed small varicosities, and patches of branched fine beaded filaments.In BiberHaab-Dimmer amyloidosis, packs of relatively thick filaments, and bundles of major collagen fibrils without interfibrillar bridges.In opaque scars, very irregular arrangements of the major collagen fibrils, and rare interfibrillar attachments. This cryomethod of investigation which adds further information to that obtained from classicallyprocessed specimens, may be used to study the 3-D structure of pathologic corneas at the macromolecular level nearer to the native state.
343
35
THE EFFECT OFTOPICALLY APPLJED AUI’OLOGOUS ON CORNEAL EPlT?IELIAL HEALING FOLLOWING WOUNDS. Choi. C-H and Chump. Department of Ophthalmology and Eye Research Koryo General Hospital, Seoul, Korea.
EVALUATION OF CORNEAL EPITHELIAL CELL REPLICATION AFTER ALKALI INJURY BY IMHUNOCYTOCHEMlSTRY FOR PROLIFERATING CELL NUCLEAR ANTIGEN. Keiko Yamaguchi, Yoshiko Numazaki, Hakoto Taeai. Department of Ophthalmology, Tohoku University School of Medicine, Sendai. Jaoan. Cornea1 epithelial cell replication after alkali injury was evaluated immunocytochenistry for proliferating cell nuclear antigen (PCNA/ cyclin). an endogenous marker for detecting proliferating cells. PCNA. a cell-cycle regulated protein is present in varying amounts only in proliferating cells. Central cornea1 alkali injuries were produced in New Zealand white rabbits aged 2 months by 7 mm filter paper discs soaked in 1N NaOH uniformly. Animals were examined at l-. 3-. 55. ?-. 14-day after the injury by slit lamp and photography and eyes The cornea1 epithelial defect were processed for light microscopy. healed with single layer of the epithelial cells in 1 day in all animals spite of severe cornea1 swelling. Immunocytocheoistry for PCNA revealed rapid cell replication at areas between the alkali burn site and intact epitheliun at l- and J-day after the injury. lamunoreactive cells were observed centripetally from the limbus at 5-. 'I-day after the injury. Although the central cornea1 epitheliun showed layered structure at ll-day after the injury, opacifications were still observed and inmunoreactive cells were not observed neither in limbus nor for evaluating
central
area.
cell
PCNA
replication
would
in
be
a
promissing
The therapeutic effect of the topically applied autologous serum on the epithelial healing was examined in the rabbit eyes following alkali wounds. The corneal alkali wounds were produced by applying 5.5 mm filter paper soaked in Alkali IN NaOH on the central cornea for 60 seconds. wounded corneas were examined morphometrically at 0 hour, 6 hour, 18 hours, 30 hours, 42 hours, and then 2 days intervals after initial damage. Autologous serum in treated group and the BSS solution in control group were given topically 4 times a day for 3 weeks. The initial epithelial resurfacing in the treated group and in the con&r01 group were 0.80 t 0.14, 0.80 t 0.17, respectively. No statistical significance was observed in the initial epithelial healing phase. During the late healing phase, there was no significant difference between the treated gruop and control group on epithelial healing. (supported by Hyosuck Med. Research Fund and Kabi Pharmacia, Seoul)
indicator
344 ENCAPSULATED
GENTAMICIN
THERAPY
OF
Nos-Barber& Sz, M,Portol6s’-, A.Parento’, (1)Dept. Genet its t Microbiology. AUTbNOMA DEO;ARCELONA. Spain. (2)Dept. Rarcel ona. Spain. (*) present adress INSTITUTE, Harvard University. Boston,
PSEUDOMONAL
IMMUNOCHARACTERlW IMPRESSION
JM.B.Antbn’.
UNIVERSITFIT R&D, LIPOTEC : EYE RESEARCH Ma. USA.
of Burgundy,
agents of choice I” the keratitis. At “fortif ied” compliance and toxicological effects are importance. In a model of P.aeruQinosa pismented rabbits (n=6 per group) we compared treatment with Qentamicin (0.141) multilamellar liposomes applied topically twice a day to eentamicin (0.3%) eye drops applied every four hours ; control eye* received PBS. Liposome @Ye irritancy was tested by a) the "in viva" Draize test
Irritancy 1 owest (0.7fO.l) 1 ipo*omes
uentamicin keratitls.
and tests
grade
control demonstrated
of and
the
represent
eye
Impression
bacterial
drops
15.0f2.5(24h)/ that
toxicity X0/P
a good in the
in (4.020.5).
the Thus
alternative treatment
of
21000
Dijon
cytology n~ucua
characterize
co&nc!ival We have
of mucins
a specific
MUCINS
cells.
and
studied
Viiejuif
as a reliable
Classical mucina
WITH
but
they
Dpt, University
France.
and
histochemical
immunochemical
non invasi~
method
methods
allowed
suffered
from
their
stainIngs
of mucus
to
lack cells
of with
peptidiccoreendpoIysacohaMic
as well.
sensitive
1. Ophthalmology 2. IRS,
monoclonalantibodiesdirectedagainst moieties
CONJUNCTIVAL
G. Gu& France,
is considered
for studying
specificity.
monoclonal
These method
to hameat
antibodies mucin
provide
chains.
us with
Quantitative
assessmentwithimageanalysisassociatedtoaqualitativecharacterization of mucins patterns
21.5f3.0(9.5h).
liposomes both
OF
CYTOLOCY.
T.Baraa. A&on’.
C.Caroher’.
.
the
are
347
39
KERATITIS.
Aminogl ycosides treatment of dosages 11.4%) of considerable keratitis in
Lab
tissues.
36 LIPOSOME
SERUM ALKALI
in mucus ocouring
immunoohemicai
have the Draize test gentamicin to fortified P.aerusin0e.a
s.104
mucus
layer
ocular
dryness
with
disorders
are
of help
in the understanding
of wild
in dry eye syndroms. The fact we could applied these technics to impti seems to be a good way to collect a non Invasive conditions.
and reproducible
method
speciaUy
under