Effects Of Topical Heterologous Blood Serum And Bovine Thrombin Activated Platelet Rich Plasma On Experimental Corneal Stromal Ulcers In Rabbit

Abstract

Nowadays there is an increasing prevalence of the use of autologous blood products to facilitate healing in a variety of applications. Corneal ulcers due to trauma and burns are one of the most common ocular disorders in animals that if not cured might progress to vision impairments, ocular perforation and blindness. In this research we tried to evaluate a better method to replace the current therapies in purpose to decrease the healing period, and to improve the quality of vision retained after healing. We decided to evaluate the effects of platelet derived growth factors in combination with heterologous serum droplets on the rate and quality of healing. We adopted of negative control (5) positive control (10) treatment (10) groups totally comprised of 25 adult male rabbits weighing 2±200 g. Then by means of a probe able to produce uniform corneal ulcers in cornea made an ulcer on central cornea of each animal. Then we placed pre prepared activated PRP on the ulcer and administered daily heterologous serum. In positive control group we used chloramphenicol, ciprofloxacin and nafazoline ophthalmic drops. In negative control group stilled water drops were administered. The remaining days every 3 groups were evaluated for 35 days for the vision retainement and took daily photos. Finally in all photos ulcer diameter and ulcerated area opacity, vision, conjunctivitis and corneal edema were evaluated. Statistical analysis showed that treatment group in compare with other two groups significantly has repaired and shortened diametrically sooner and gained better vision and least oedema and least scar production. It seems this new method for treating ulcers in the body specially those on the cornea will do better than other traditional methods and help our animals back to sweet life with clearer vision.

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Advances in Environmental Biology, 6(1): 95-99, 2012
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE
Corresponding Author Kaffashi Elahi, R, Assistant Professor of Small Animal Internal Medicine, Department of Clinical
Sciences, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz-Iran
Effects Of Topical Heterologous Blood Serum And Bovine Thrombin Activated Platelet
Rich Plasma On Experimental Corneal Stromal Ulcers In Rabbit
1Kaffashi Elahi, R, 2Mousavi, Gh, 3Mohajeri, D
1Assistant Professor of Small Animal Internal Medicine, Department of Clinical Sciences, Faculty of Veterinary
Medicine, Tabriz Branch, Islamic Azad University, Tabriz-Iran
2Assistant Professor of Veterinary Surgery, Department of Clinical Sciences, Faculty of Veterinary Medicine,
Tabriz Branch, Islamic Azad University, Tabriz-Iran
3Associate Professor of Veterinary Pathology, Department of Pathobiology, Faculty of Veterinary Medicine,
Tabriz Branch, Islamic Azad University, Tabriz-Iran
Kaffashi Elahi, R, Mousavi, Gh, Mohajeri, D: Effects Of Topical Heterologous Blood Serum And
Bovine Thrombin Activated Platelet Rich Plasma On Experimental Corneal Stromal Ulcers In Rabbit
ABSTRACT
Nowadays there is an increasing prevalence of the use of autologous blood products to facilitate healing in a
variety of applications. Corneal ulcers due to trauma and burns are one of the most common ocular disorders in
animals that if not cured might progress to vision impairments, ocular perforation and blindness. In this research
we tried to evaluate a better method to replace the current therapies in purpose to decrease the healing period,
and to improve the quality of vision retained after healing. We decided to evaluate the effects of platelet derived
growth factors in combination with heterologous serum droplets on the rate and quality of healing. We adopted
of negative control (5) positive control (10) treatment (10) groups totally comprised of 25 adult male rabbits
weighing 2±200 g. Then by means of a probe able to produce uniform corneal ulcers in cornea made an ulcer on
central cornea of each animal. Then we placed pre prepared activated PRP on the ulcer and administered daily
heterologous serum. In positive control group we used chloramphenicol, ciprofloxacin and nafazoline
ophthalmic drops. In negative control group stilled water drops were administered. The remaining days every 3
groups were evaluated for 35 days for the vision retainement and took daily photos. Finally in all photos ulcer
diameter and ulcerated area opacity, vision, conjunctivitis and corneal edema were evaluated. Statistical analysis
showed that treatment group in compare with other two groups significantly has repaired and shortened
diametrically sooner and gained better vision and least oedema and least scar production. It seems this new
method for treating ulcers in the body specially those on the cornea will do better than other traditional methods
and help our animals back to sweet life with clearer vision.
Key words: corneal ulcer, platelet rich plasma, PRP
Introduction
In Europe and the United States, there is an
increasing prevalence of the use of autologous blood
products to facilitate healing in a variety of
applications. Recently, we have learned more about
specific growth factors, which play a crucial role in
the healing process. With that knowledge there is
abundant enthusiasm in the application of
concentrated platelets, which release a supra-
maximal quantity of these growth factors to stimulate
recovery in non-healing injuries [2,20]. The
application of PRP has been documented in many
fields. First promoted by M. Ferrari in 1987 [7] as an
autologous transfusion component after an open
heart operation to avoid homologous blood product
transfusion. There are now over 5200 entries in fields
from orthopedics, sports medicine, dentistry,
ophthalmology etc. Corneal ulcers especially with
traumatic causes are one of important and severe
disorders in small animal paediatric ophthalmology
that if not cured might progress to vision
impairments, ocular perforation and blindness due to
added secondary infections and other deteriorative
factors. There are many effective therapeutic trials
[21] but in veterinary medicine, Patient Corporation
and hard practicality of treatment, results in low
functional healing. It seems that topical application
of heterologous platelet jelly and blood serum may
shorten the healing period of ulcerated area and
causes healing of better quality. A study from Chen
et al. demonstrated that PRP might potentially play a
role in prevention of disc degeneration. They
demonstrated that PRP can act as a growth factor
cocktail to induce proliferation and differentiation
and promote tissue-engineered nucleus formation

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Adv. Environ. Biol., 6(1): 95-99, 2012

regeneration via the same pathway [4]. Autologous
platelet-rich plasma promoted healing of dormant
corneal ulcers even in eyes threatened by corneal
perforation and was accompanied by a reduction in
pain and inflammation[1]. Tsubota k, et al was
applied daily topical blood serum drops over
superficial corneal excoriations and reported its
positive effects on their healing [22,23]. The aim of
this research was to identify a possible better method
to help cornea with healing of better quality and
retaining vision faster with least scar formation and
finally more clear vision
Materials and Methods
25 healthy adult male rabbits weighing 2±200 kg
were randomly selected, fed with standard ration and
water ad libitum. After 2 weeks adaptation period,
randomly divided into 3 groups of negative control
(n=5) positive control (n=10) and treatment (n=10)
respectively. All animals had access to free walking
and fresh air 1 hour per day.
A fresh whole blood in Citrate Phosphate
Dextrose (CPD) bags was centrifuged (200 G for 5
minutes) by double centrifugation method [15],
packed cell portion were extracted and second
centrifugation (1000 G for 15 minutes) were done to
concentrate platelets as much as possible. Platelet
concentrate were kept with no harsh handling for
preventing accidental platelet degranulation. Platelet
aggregate was derived and 20 microliter activator
(1000 IU/ml bovine thrombin mixed with 1 ml
0.05calcium chloride) added to each 200 microliter
of platelet aggregate.
All rabbits handled with extreme caution to
decrease environmental stress and given oral
diazepam (0.5 mg/kg) 2 hour before anesthesia, each
animal were anesthetized by ketamine (80mg/kg)
diazepam (0.5 mg/kg) mixture. To produce uniform
ulcers, by means of a probe, on central corneal area
on left eye of each animal a round stromal ulcer was
created.
In Treatment group, 0.05cc of pre-prepared
thrombin activated PRP placed on the ulcer and
administered daily heterologous serum drops (2
drops 3 times per day). The remaining days we
evaluated the repair period and captured daily
standard photographs. In positive control group we
used chloramphenicol, ciprofloxacin and nafazoline
ophthalmic drops each one 2 drops 3 times per day.
In Negative control group we used distilled water
drops 2 drops 3 times per day. The remaining days
every 3 groups were evaluated for 35 days for the
vision retainement and took daily photos (standard
and fixed settings by fixed distance between cornea
and camera lens, by means of a specially designed
photography tuning apparatus and a canon G7 digital
camera).
Finally in all photos ulcer diameter evaluated by
computer and using Corel software. Degree of vision
evaluated by means of obstacle course and motion
detection tests. All parameters were categorized in
four degrees: loss of vision=0, weak vision=1, semi
complete vision=2, complete vision=3. This
experiment was conducted as blind and in all parts of
research period, all animals treated with extreme
gentle, mixed with healthy females, 1 hour daily
access to fresh air and free walking.
Statistical analysis made by variance analysis
(ANOVA) and Tukey test, at a significance level of
5% (p<0.05).
Results:
Statistical analysis showed that treatment group
in compare with negative and positive control groups
had significantly achieved the desired vision in
shorter period of time, in other words, in day 16,
treatment group achieved full vision, while in
negative control vision score in the same day was not
desirable and around day30 has been able to reach
more than 90 percent of that vision. At the end of
day35, this situation has continued. Negative control
group until the end of research did not reach the
complete vision (figure 4). And between groups,
treatment group had significantly less inflammatory
reaction and infectious process (figure 1and 3) and
negative control group had maximum inflammation
and purulent discharge. Corneal opacity of non-
ulcerated area (figure 2) and ulcerated area (figure 6)
are of important factors denoting better corneal
wound healing and goes parallel with sooner vision
retainment. Also Corel software aided ulcerated area
diameter measurements showed significant diameter
reduction in treatment group in compare to other two,
but also positive control group showed better
performance in compare to negative control group
(figure5). Statistical analysis made by variance
analysis (ANOVA) and Tukey test, at a significance
level of 5% (p<0.05).
Discussion and Conclusion:
This study showed that vision retaining, certainly
is faster, clearer with minimum scar production,
minimum local inflammation and least infectious
complications when using PRP Jelly and ophthalmic
serum drops. Zhijie et al [24] in a study after cutting
and removing a 6 mm diameter circle in the rat
cornea, without inducing damage to limbus and
stroma (superficial epithelial excoriation),
investigated the re-epithelialization status and
invasion of neutrophils. They found that platelets
accumulate in limbal vessels and neutrophils
emigrated to the damaged place. They found that the
induction of thrombocytopenia in mice significantly
endangered the healing process and re-transfusion of
platelets acts conversely. The results show that local
platelets in corneal limbus and near the wound are
essential in epithelialization. The research subject of

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this study is similar that both impacts on importance
and role of platelet in the corneal healing. Reid and
colleagues [19] found that local administration of
autologous blood serum droplets are effective in the
treatment of ocular superficial disorders it seems our
research findings are similar with their results. Man
D and colleagues [13] and Federico and colleagues
[6] used of PRP sticky properties in tissue
transplantation, in the present research also
characteristics of stickiness and actions of remaining
platelet gel products and exerting there local effects
were evaluated. Pierce and colleagues [18] in a study
found that the administration of PRP can reduce the
final wound diameter; our results showed the same
results. Nishijima and colleagues [16] and [17] found
that the major role of the platelet in recalling and
localization of leukocytes in acute corneal
inflammation, this can be the reason for lesser local
inflammation and purulent discharges in our study.
Cerwinka and colleagues [3] and Tailor and
colleagues [21] found that anti-neutrophilic serum,
administration interferes with platelet localization in
mesenteric venules up to 50 percent, it shows the
important role of platelets in corneal healing and
shows that our research may had concomitant results
with mentioned research. Cooper D et al [5] also
found that the leukocytes themselves also play a role
to platelet aggregation. So in our research recalled
neutrophils and PRP and serum droplets have done
two important role, first, leukocytes play role in fight
with microorganisms (so that treatment group had
minimal to lack of any local infection) and secondly
more recalling of platelets into the wounded area(5).
Gawaz [9] and Kuligowski [12] and Tailor [21],
found sharing and cooperation of platelet releasates
in adjustment of inflammatory response and
acceleration of healing process. In our study showed
that the inflammation and other complications of
ocular inflammation was in the lowest degree in
treatment group (early elimination of corneal edema
and neurological reflexes) are results that concluded
from this article. In another study, two groups were
chosen; one of them received routine treatment of
corneal ulcers and other group received routine
treatment with subcutaneous autologous platelet
concentrates, Significant differences between the two
mentioned groups occurred in shorter treatment
period and time of discharge that reduced the time
needed to complete the treatment group after
discharging from hospital [14]. during a study found
that the applying other growth factors derived from
platelets on the corneal epithelium, increase their
development and trophism, these findings are
consistent with our results that showed rapid wound
healing and fast return of corneal transparency
[11,16,17]. Far has not been established that blood
platelet isolates are the only materials that contain
corneal epithelial growth incentives, and Goto [10],
Tsubota [22] and Fox [8] found that the other blood
segments also contain corneal growth incentives, also
this reveals that serum droplet have been effective in
rapid wound healing. It seems that autologous serum
as the non-platelet segment, have role in ocular
surface damage regeneration and epithelialization.
Finally with the above interpretations and study of
vision ranks in different days after PRP
administration results shows that treatment group
gained faster and sooner healing in compare with
other two groups.

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It seems that there might be better indications for
PRP effects on corneal ulcer healing that might do
better for proving our results, like pathologic
evaluations, but it is second step for next research
project. Before knowing PRP positive effects we are
not allowed to induce remaining defects on an
animal’s cornea.
Acknowledgement
This research project has been accomplished by
the financial support of, Tabriz branch, Islamic Azad
University, Iran-Tabriz.
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