Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo L. F. Rich, J. M. Hatfield & I. Louiselle To cite this article: L. F. Rich, J. M. Hatfield & I. Louiselle (1992) The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo, Current Eye Research, 11:8, 719-725 To link to this article: http://dx.doi.org/10.3109/02713689209000746
Published online: 02 Jul 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [Stockholm University Library]
Date: 11 September 2015, At: 18:04
Current Eye Research
Volume 1 1 number 8 1992, 719-725
The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo L.F. Rich, J .M .Hatfield and I .Louiselle
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
Casey Eye Institute, Oregon Health Sciences University, 3375 S.W. Terwilliger Boulevard, Portland, OR 97201-4197, USA
ABSTRACT Using standardized freeze wounds in cat corneas, we tested the efficacy of basic Fibroblast Growth Factor (bFGF) solubilized in phosphate buffered saline (PBS) to promote endothelial healing when injected intraocularly at doses ranging from 0.01 pg to 10 pg. After 6 days, animals were humanely sacrificed and corneal tissues were fixed and stained for light microscopy and computation of remaining wound areas. A significant dose-response relationship was found between the dosages of 0.01, 0.1, and 1.0 pg of bFGF/eye and the stimulation of more completely healed endotheliurn six days after transcorneal freeze wounding. Significantly larger endothelial wounds were present six days after wounding when the eyes were treated with 10 pg of bFGF/eye compared with controls treated with PBS only.
maintenance of laboratory animals; throughout the experiment, animal subjects were handled according to the ARVO Resolution on the care and use of animals in research. Our protocols for preoperative prophylaxis with ophthalmic gentamicin, surgical anesthesia and freezewounding have been described in detail elsewhere (7). Briefly, each animal was treated twice daily for one week preoperatively with gentamicin eyedrops; for freezewounding, each animal was anesthetized by intramuscular injection and both eyes received topical anesthetic to suppress corneal sensation. A 3mm-diameter cryoprobe, chilled to -120°C by liquid nitrogen, was applied to the
INTRODUCTION Numerous studies have been reported which document the ability of basic fibroblast growth factor (bFGF) to stimulate the proliferation of corneal fibroblasts and
central cornea of both eyes of each animal for 15 seconds, producing standardized transcorneal freeze wounds of
5.5mm diameter f 0.4mm (meanfSEM). Immediately after wounding, each eye received an
endothelial cells in vitro (1-4). However, relatively few
injection of bFGF or PBS vehicle into the anterior cham-
studies (5,6) substantiate a wound healing effect of bFGF
ber. Recombinant human basic FGF was provided by
on the corneal endothelium in vivo.
Drs. Kiorpes and Gooding of Ethicon, Inc., Somerville NJ.
This study was conducted to determine whether bFGF
Both eyes of a given animal received the same solution,
dissolved in phosphate buffered saline (PBS) would en-
which was delivered slowly from an injection site near the
hance healing of cat corneal endothelium in vivo in re-
limbus, and directed at mid-anterior chamber to avoid
sponse to freeze injury. Various concentrations of bFGF
disruptive currents near the wounded area. The needle
were evaluated for their relative influence, if any, on
remained in place for 60 seconds following completion of
wound healing in this cell layer.
the injection, to permit equilibration of anterior chamber pressure, and then was quickly withdrawn. The relatively
MATERIALS AND METHODS
Domestic short-haired adult cats were obtained from
small volume (100 p l ) injected, the equilibration period and the fine 30-gauge needle used all helped avoid large
licensed breeders and quarantined 10 days on standard
increases or decreases in intraocular pressure; the anterior
rations in accordance with PHS/USDA guidelines on the
chamber remained formed throughout the procedure.
-~
Received on January 28, 1991; accepted on July 6, 1992
@ Oxford University Press
719
Current Eye Research Each cat received the same concentration of bFGF in
consists of four general regions (7-9). Zone 1, the area
PBS, or PBS alone, in both eyes; all injected volumes
outside the original wound boundary, contains normal,
were 100 pl. Nine animals (18 eyes) received PBS place-
hexagonal cells of consistent size. Zones 2, 3, and 4 are
bo alone; six animals (12 eyes) received 0.01 pg of bFGF
shown in Figure la. Zone 2, just inside the original
/eye; six cats (12 eyes) received 0.1 pg of bFGF/eye; nine
wound boundary, consists of larger and more irregularly
animals (18 eyes) received 1.0 pg of bFGF/eye; and four
sized cells, fewer of which are hexagonal, compared with
cats (8 eyes) received 10.0 pg of bFGF/eye.
the unwounded endothelium. However, their basic ap-
Beginning at six hours post-wounding, all cats received
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
ophthalmic gentamicin sulfate drops (0.3%) in each eye twice daily for three days, then once daily for two days.
Six days after wounding, all cats were humanely sacrificed by anesthetic overdose and their corneas excised immediately to Carnoy's fixative. The six-day timepoint was chosen based on preliminary experiments in which numerous growth factor-treated eyes were tested using this freeze-wound model, and examined daily for seven days post-op. We realized that if a growth factor stimulated endothelial repair so profoundly that no discernible wound was visible under dissecting microscope magnification and the remaining wound area had to be counted as zero at seven-day or later timepoints, data analysis would be skewed. In order to be certain that all wounds being evaluated were still in the process of healing and had not completely closed, we found that six days post-injury was the optimal time for examination using this freeze-wound
Figure la: Whole mount of a typical remaining wound in a cat cornea 6 days after freeze wounding and treatment with 0.1 pg bFGF. 22: wounded, healed endothelium; 23: multilayered "activated zone"; and 24: central area covered by very polymegathic and pleomorphic cells. Zone 1 consists of unwounded endothelium peripheral to original wound edge, and lies beyond the boundary of this photograph. (Hematoxylin, 3 0 X bar = 1.0mm).
technique (7,8). The tissue was fixed at room temperature for six hours, then transferred to 70% ethanol. Our protocols for Ehrlich's hematoxylin staining, mounting and photography of the corneal specimens have been described previously
(7). Briefly, the fixed corneal tissue was stained on the endothelial surface, then the area surrounding the original wound was dissected using a 9mm corneal trephine and the resulting whole-mount disc was aqueous mounted and photographed at 2.5X using a Zeiss Axioskop microscope and camera. Standard-focus enlargements at 5x7" were made for wound tracing. Using the transcorneal freeze-wounding technique we have found that, six days after wounding, the endothelium
720
Figure lb: Sagittal section of cat cornea 6 days after freeze wounding and bFGF treatment. 22: healed endothelium; 23: multilayered "activated zone"; Z4: polymegathic and pleomorphic zone. Note intact Descemet's membrane ( t ) (hematoxylin, 300X; bar = loop).
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
Current Eye Research pearance, including the kidney-shaped nucleus and mono-
vehicle control group were performed using Dunnett’s test
layered arrangement, is like that of the cells in the un-
(16,17). A t-test of the slope of the regression line was
wounded area (8). The third zone inward, called the
used to determine if there was a significant logarithmic
activated zone, consists of a roughly circular, multilayered,
dose response relationship between the 0, 0.01, 0.1 and 1
irregularly arranged mat of cells which are fibroblastic in
pg/eye doses of bFGF and remaining wound areas (18).
appearance. In this zone, one can distinguish up to 10
A bFGF concentration of 0.001 pg/eye was substituted for
different layers of cells in sagittal section (Figure lb) (8).
the 0 pgleye value of the placebo group during regression
The fourth, innermost zone consists of pleomorphic and
analysis because the logarithmic transformation of 0 is
polymegathic cells, ranging from small fibroblastic figures
mathematically meaningless.
to enormous polygonal, multinucleated cells. At six days
It should be noted that the values presented in this
after freeze injury, there is no large central area of denud-
study must be multiplied by 2.3 for direct comparison to
ed Descemet’s membrane as found in Weimar’s 2-day
our earlier MGF study (7),since 8x10 enlargements were
specimens; rather, numerous smaller open spaces between
used for the MGF study and 5x7” enlargements were used
cells are visible at high magnification. The most distin-
for the present study.
guishable border between the healed and still-regenerating areas of these freeze wounds, and the one we use in
RESULTS
our wound-healing assessments, is the outer edge of the
Differential endothelial freeze wound area was ob-
activated zone. Peripheral to this, the healed endotheli-
served six days after injury and anterior chamber injection
um consists of a confluent monolayer of primarily hexago-
of 0.0, 0.01, 0.1, 1.0 or 10.0 pg of bFGF per eye (Table 1).
nal cells with characteristic kidney-shaped nuclei.
Smaller mean areas for the remaining endothelial wounds
As stated above, we consider a wound closed (healed)
were found in cats that received bFGF in PBS, compared
if there is no activated zone remaining and the original
with cats that received an equal volume of PBS alone.
wound area is covered with a monolayer of endothelial
The 1.0 pg dose of bFGF produced significantly smaller
cells. Descemet’s membrane remains intact after a 15-
wounds compared to PBS alone (p
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo L. F. Rich, J. M. Hatfield & I. Louiselle To cite this article: L. F. Rich, J. M. Hatfield & I. Louiselle (1992) The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo, Current Eye Research, 11:8, 719-725 To link to this article: http://dx.doi.org/10.3109/02713689209000746
Published online: 02 Jul 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icey20 Download by: [Stockholm University Library]
Date: 11 September 2015, At: 18:04
Current Eye Research
Volume 1 1 number 8 1992, 719-725
The influence of basic fibroblast growth factor on cat corneal endothelial wound healing in vivo L.F. Rich, J .M .Hatfield and I .Louiselle
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
Casey Eye Institute, Oregon Health Sciences University, 3375 S.W. Terwilliger Boulevard, Portland, OR 97201-4197, USA
ABSTRACT Using standardized freeze wounds in cat corneas, we tested the efficacy of basic Fibroblast Growth Factor (bFGF) solubilized in phosphate buffered saline (PBS) to promote endothelial healing when injected intraocularly at doses ranging from 0.01 pg to 10 pg. After 6 days, animals were humanely sacrificed and corneal tissues were fixed and stained for light microscopy and computation of remaining wound areas. A significant dose-response relationship was found between the dosages of 0.01, 0.1, and 1.0 pg of bFGF/eye and the stimulation of more completely healed endotheliurn six days after transcorneal freeze wounding. Significantly larger endothelial wounds were present six days after wounding when the eyes were treated with 10 pg of bFGF/eye compared with controls treated with PBS only.
maintenance of laboratory animals; throughout the experiment, animal subjects were handled according to the ARVO Resolution on the care and use of animals in research. Our protocols for preoperative prophylaxis with ophthalmic gentamicin, surgical anesthesia and freezewounding have been described in detail elsewhere (7). Briefly, each animal was treated twice daily for one week preoperatively with gentamicin eyedrops; for freezewounding, each animal was anesthetized by intramuscular injection and both eyes received topical anesthetic to suppress corneal sensation. A 3mm-diameter cryoprobe, chilled to -120°C by liquid nitrogen, was applied to the
INTRODUCTION Numerous studies have been reported which document the ability of basic fibroblast growth factor (bFGF) to stimulate the proliferation of corneal fibroblasts and
central cornea of both eyes of each animal for 15 seconds, producing standardized transcorneal freeze wounds of
5.5mm diameter f 0.4mm (meanfSEM). Immediately after wounding, each eye received an
endothelial cells in vitro (1-4). However, relatively few
injection of bFGF or PBS vehicle into the anterior cham-
studies (5,6) substantiate a wound healing effect of bFGF
ber. Recombinant human basic FGF was provided by
on the corneal endothelium in vivo.
Drs. Kiorpes and Gooding of Ethicon, Inc., Somerville NJ.
This study was conducted to determine whether bFGF
Both eyes of a given animal received the same solution,
dissolved in phosphate buffered saline (PBS) would en-
which was delivered slowly from an injection site near the
hance healing of cat corneal endothelium in vivo in re-
limbus, and directed at mid-anterior chamber to avoid
sponse to freeze injury. Various concentrations of bFGF
disruptive currents near the wounded area. The needle
were evaluated for their relative influence, if any, on
remained in place for 60 seconds following completion of
wound healing in this cell layer.
the injection, to permit equilibration of anterior chamber pressure, and then was quickly withdrawn. The relatively
MATERIALS AND METHODS
Domestic short-haired adult cats were obtained from
small volume (100 p l ) injected, the equilibration period and the fine 30-gauge needle used all helped avoid large
licensed breeders and quarantined 10 days on standard
increases or decreases in intraocular pressure; the anterior
rations in accordance with PHS/USDA guidelines on the
chamber remained formed throughout the procedure.
-~
Received on January 28, 1991; accepted on July 6, 1992
@ Oxford University Press
719
Current Eye Research Each cat received the same concentration of bFGF in
consists of four general regions (7-9). Zone 1, the area
PBS, or PBS alone, in both eyes; all injected volumes
outside the original wound boundary, contains normal,
were 100 pl. Nine animals (18 eyes) received PBS place-
hexagonal cells of consistent size. Zones 2, 3, and 4 are
bo alone; six animals (12 eyes) received 0.01 pg of bFGF
shown in Figure la. Zone 2, just inside the original
/eye; six cats (12 eyes) received 0.1 pg of bFGF/eye; nine
wound boundary, consists of larger and more irregularly
animals (18 eyes) received 1.0 pg of bFGF/eye; and four
sized cells, fewer of which are hexagonal, compared with
cats (8 eyes) received 10.0 pg of bFGF/eye.
the unwounded endothelium. However, their basic ap-
Beginning at six hours post-wounding, all cats received
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
ophthalmic gentamicin sulfate drops (0.3%) in each eye twice daily for three days, then once daily for two days.
Six days after wounding, all cats were humanely sacrificed by anesthetic overdose and their corneas excised immediately to Carnoy's fixative. The six-day timepoint was chosen based on preliminary experiments in which numerous growth factor-treated eyes were tested using this freeze-wound model, and examined daily for seven days post-op. We realized that if a growth factor stimulated endothelial repair so profoundly that no discernible wound was visible under dissecting microscope magnification and the remaining wound area had to be counted as zero at seven-day or later timepoints, data analysis would be skewed. In order to be certain that all wounds being evaluated were still in the process of healing and had not completely closed, we found that six days post-injury was the optimal time for examination using this freeze-wound
Figure la: Whole mount of a typical remaining wound in a cat cornea 6 days after freeze wounding and treatment with 0.1 pg bFGF. 22: wounded, healed endothelium; 23: multilayered "activated zone"; and 24: central area covered by very polymegathic and pleomorphic cells. Zone 1 consists of unwounded endothelium peripheral to original wound edge, and lies beyond the boundary of this photograph. (Hematoxylin, 3 0 X bar = 1.0mm).
technique (7,8). The tissue was fixed at room temperature for six hours, then transferred to 70% ethanol. Our protocols for Ehrlich's hematoxylin staining, mounting and photography of the corneal specimens have been described previously
(7). Briefly, the fixed corneal tissue was stained on the endothelial surface, then the area surrounding the original wound was dissected using a 9mm corneal trephine and the resulting whole-mount disc was aqueous mounted and photographed at 2.5X using a Zeiss Axioskop microscope and camera. Standard-focus enlargements at 5x7" were made for wound tracing. Using the transcorneal freeze-wounding technique we have found that, six days after wounding, the endothelium
720
Figure lb: Sagittal section of cat cornea 6 days after freeze wounding and bFGF treatment. 22: healed endothelium; 23: multilayered "activated zone"; Z4: polymegathic and pleomorphic zone. Note intact Descemet's membrane ( t ) (hematoxylin, 300X; bar = loop).
Downloaded by [Stockholm University Library] at 18:04 11 September 2015
Current Eye Research pearance, including the kidney-shaped nucleus and mono-
vehicle control group were performed using Dunnett’s test
layered arrangement, is like that of the cells in the un-
(16,17). A t-test of the slope of the regression line was
wounded area (8). The third zone inward, called the
used to determine if there was a significant logarithmic
activated zone, consists of a roughly circular, multilayered,
dose response relationship between the 0, 0.01, 0.1 and 1
irregularly arranged mat of cells which are fibroblastic in
pg/eye doses of bFGF and remaining wound areas (18).
appearance. In this zone, one can distinguish up to 10
A bFGF concentration of 0.001 pg/eye was substituted for
different layers of cells in sagittal section (Figure lb) (8).
the 0 pgleye value of the placebo group during regression
The fourth, innermost zone consists of pleomorphic and
analysis because the logarithmic transformation of 0 is
polymegathic cells, ranging from small fibroblastic figures
mathematically meaningless.
to enormous polygonal, multinucleated cells. At six days
It should be noted that the values presented in this
after freeze injury, there is no large central area of denud-
study must be multiplied by 2.3 for direct comparison to
ed Descemet’s membrane as found in Weimar’s 2-day
our earlier MGF study (7),since 8x10 enlargements were
specimens; rather, numerous smaller open spaces between
used for the MGF study and 5x7” enlargements were used
cells are visible at high magnification. The most distin-
for the present study.
guishable border between the healed and still-regenerating areas of these freeze wounds, and the one we use in
RESULTS
our wound-healing assessments, is the outer edge of the
Differential endothelial freeze wound area was ob-
activated zone. Peripheral to this, the healed endotheli-
served six days after injury and anterior chamber injection
um consists of a confluent monolayer of primarily hexago-
of 0.0, 0.01, 0.1, 1.0 or 10.0 pg of bFGF per eye (Table 1).
nal cells with characteristic kidney-shaped nuclei.
Smaller mean areas for the remaining endothelial wounds
As stated above, we consider a wound closed (healed)
were found in cats that received bFGF in PBS, compared
if there is no activated zone remaining and the original
with cats that received an equal volume of PBS alone.
wound area is covered with a monolayer of endothelial
The 1.0 pg dose of bFGF produced significantly smaller
cells. Descemet’s membrane remains intact after a 15-
wounds compared to PBS alone (p
