Intra-stromal injection of honey-treated keratocytes as a cell-based therapy for experimental corneal laceration.

OBJECTIVES: This study aimed to investigate the potential of honey-supplemented medium (HSM) for expanding corneal keratocytes and its transplantation in a model of corneal laceration. METHODS: Keratocytes were cultured in 1 % HSM- or 10 % fetal bovine serum (FBS)-supplemented medium for 24 h. The effect of HSM on keratocyte proliferation was evaluated using the MTT assay. The relative expression of Lum, Kera, and ALDH3A1, known markers of native keratocytes, was quantified by real-time PCR. The safety and efficacy of HSM-treated keratocyte intrastromal injection in a rabbit model of corneal laceration were also evaluated. RESULTS: The MTT assay showed that HSM treatment did not significantly affect cell viability compared to FBS-supplemented medium (84.71 ± 2.38 vs. 100.08 ± 10.92, respectively; p=0.076). Moreover, HSM-treated keratocytes had significantly increased expression of Lum, Kera, and ALDH3A1 compared to cells treated with FBS, while the expression of the proliferation biomarker Thy-1 did not significantly differ between the two treatments. Intrastromal injection of HSM-treated keratocytes in the laceration animal model was safe and uneventful, resulting in less stromal inflammation and neovascularization, and consequently, better final architecture with less residual haze compared to the group injected with FBS-treated keratocytes. CONCLUSIONS: These findings suggest that honey is a suitable supplement for keratocyte treatment and corneal cell therapy. The use of HSM may have potential applications in the treatment of corneal injuries and diseases.

Autologous Activated Omental versus Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells in Corneal Alkaline Injury: An Experimental Study.

PURPOSE: To compare the effects of two types of mesenchymal stem cells (MSCs), activated omental cells (AOCs), and adipose tissue-derived stem cells (ADSCs) in the healing process of animal model of ocular surface alkali injury. METHODS: An alkaline burn was induced on the ocular surfaces of eighteen rats divided randomly into three groups. The first and second groups received subconjunctival AOCs and ADSCs, respectively. The control group received normal saline subconjunctival injection. On the 90th day after the injury, the eyes were examined using slit-lamp biomicroscopy. Corneal neovascularization and scarring were graded in a masked fashion. Histological evaluation of the corneal scar was performed, and the number of inflammatory cells was evaluated. RESULTS: Corneal neovascularization scores revealed higher neovascularization in the control (0.49 ± 0.12) than the AOC (0.80 ± 0.20, P = 0.01) and ADSC groups (0.84 ± 0.24, P = 0.007). There were no statistically significant differences between the neovascularization score of the AOC and ADSC groups (P > 0.05). According to histologic evaluation, stromal infiltration was significantly more in the control group compared to AOC and ADSC groups (P < 0.05). CONCLUSIONS: Our results suggest that MSCs, even with different sources, can be used to promote wound healing after corneal chemical burns. However, the ease of harvesting ADSC from more superficial fat sources makes this method more clinically applicable.

Non-medial infectious orbital cellulitis: etiology, causative organisms, radiologic findings, management and complications.

BACKGROUND: Orbital cellulitis is an ophthalmic emergency, which is associated with vision-threatening adverse effects. The purpose of this study is investigating etiology, radiologic findings, management and complications of patients with non-medial orbital cellulitis. METHOD: A retrospective medical record and radiologic file review of patients with infectious orbital cellulitis was performed to detect all patients with non-medial orbital cellulitis who referred to Khalili hospital from 2016 to 2019. Age, sex, origin of infection, size of collection or abscess, medical or surgical management, microbiology, first and final best-corrected visual acuity, duration of admission, and complications was recorded. Patients divided into two groups; medical management and surgical management groups and all of data compared between in this groups. RESULTS: Of ninety-six patients with infectious orbital cellulitis, 23 cases (14 male, 9 female) were included. Five patients (21.7%) were managed medically and 18 patients (78.3%) were managed surgically. Patients' age range was 5-70 years old. Most common location for non-medial cellulitis was superior space (66.7% in surgical and 40% in medical group; p = 0.511). In 13 cases of surgical group (72.3%) were detected microorganisms. The mean ± SD of collection volume in medical group were 476.5 ± 290.93 mm3 and 2572.94 ± 1075.75 mm3 in surgical group (p < 0.001). Ten patients in surgical group had compressive optic neuropathy. The mean ± SD of collection volume was 3204.97 ± 879.88 mm3 in patient with compressive optic neuropathy and 1280.43 ± 880.68 mm3 in patient without compressive optic neuropathy (P < 0.001). One case complicated by subdural empyema and another case progressed to necrotizing fasciitis. CONCLUSION: Non-medial orbital cellulitis is an uncommon but sight-threatening and life-threatening condition. Timely diagnosis and accurate management reduce morbidity and mortality. Combined surgery for patients with superior or supra-temporal and large non-medial abscess is recommended.

Propagation of limbal stem cells on polycaprolactone and polycaprolactone/gelatin fibrous scaffolds and transplantation in animal model.

Introduction: This study was conducted to compare the effect of nanofibrous polycaprolactone (PCL) and PCL/gelatin (PCL/Gel) on limbal epithelial stem cell (LESC) and its efficiency for transplantation in animal model. Methods: PCL and PCL/Gel with a mass ratio of 70:30 and 50:50 was fabricated by electrospinning method. Human LESCs were cultured on PCL and PCL/Gel scaffolds and the effect of each scaffold on LESC proliferation, attachment and corneal epithelial regeneration in an animal model was evaluated, considering ease of use of scaffold and final transparency of the cornea. Results: Our data showed that PCL was more suitable than PCL/Gel for LESCs adherence, induction of epithelial morphology and proliferation. Histopathologic analysis of corneal sections from transplanted animals showed that epithelium was regenerated almost similar in PCL and PCL/Gel groups; however, vascularization and inflammation were significantly lower in the group receiving PCL. Conclusion: The represented data indicated the priority of PCL to PCL/Gel for the LESC attachment, proliferation and final outcome in an animal model of alkaline injury. This finding might be promising for cell therapy of corneal diseases.

Five Years Pathological Evaluation of Corneal Regrafts: A Study from Southern Iran.

PURPOSE: Corneal regrafts sometimes needed to restore the transparency after graft failure. The aim of the study is five years epidemiologic and histopathological evaluation of corneal regrafts. METHODS: In this cross-sectional study, all corneal regrafts during 5 years (2012-2016) were assessed in the Khalili Ophthalmology Center at Shiraz city. Demographic data including age, area of residence, primary disease, type of graft, cause of regraft, interval between primary and subsequent grafts (IPSG), associated eye diseases or surgeries, and systemic diseases were recorded. Also, microscopic findings of corneas were reviewed. RESULTS: Among a total of 1190 corneal grafts, 76 of them (6.38%) were regrafts. The most common type of grafting was penetrating keratoplasty (PK). The shortest IPSG was observed in fungal keratitis. Main causes of graft failure were endothelial dysfunction, infection, immunologic rejection, technical problems, and recurrence of primary disease, respectively. The most common histopathological finding in failed grafts was severe endothelial cell loss (89.8%). Also, more than half and one-third of cases had Descemet membrane changes and stromal ingrowth, respectively. CONCLUSION: Endothelial cell loss was the major cause of failure in our study. Also, recurrence rate in infective cases, especially fungal keratitis, was very high. Considerable presence of histopathological changes such as doubling of Descemet membrane and retrocorneal fibrous ingrowth need further investigations. Perhaps, modification in techniques of corneal grafting and assessment of donor tissue and recipient bed along with any need for longer medical treatment are the basis for future studies in order to increase graft survival.

Caffeic Acid Alkyl Amide Derivatives Ameliorate Oxidative Stress and Modulate ERK1/2 and AKT Signaling Pathways in a Rat Model of Diabetic Retinopathy.

The purpose of this study was to examine the neuroprotective effects of caffeic acid hexyl (CAF6) and dodecyl (CAF12) amide derivatives on the early stage of retinopathy in streptozotocin-induced diabetic rats. Animals were divided in five groups (n=8/group); one group consisted of non-diabetic rats as control, while the other four were diabetic animals either non-treated or treated with CAF6, CAF12 or resveratrol intravitreally for four weeks. Retinal superoxide dismutase (SOD) activity and 8-iso-prostaglandin F2α (iPF2α ) levels were evaluated by an ELISA assay. Phosphorylation of ERK1/2 and AKT was determined by immunoblotting in retinal homogenates. Retinal morphology was also examined using light microscopy. Treatment with CAF6 and CAF12 increased retinal SOD activity, while it decreased iPF2α levels in diabetic rats. Phosphorylation of ERK1/2 was increased, while AKT phosphorylation was decreased in diabetic rats compared to normal control and these alterations were significantly reversed in diabetic rats treated with CAF6 and CAF12. Furthermore, thickness of the whole retinal layer, outer nuclear layer, and ganglion cell count were decreased in diabetic rats compared to control and CAF6 and CAF12 treatments prevented these changes. CAF6 and CAF12 seem to be effective agents for treatment of diabetic retinopathy via attenuation of retinal oxidative stress and improvement of neuronal survival signaling.

Expression of interleukin-6 in ocular surface squamous neoplasia.

PURPOSE: Interleukin-6 (IL-6) is an important cytokine in the cascade of inflammation and cancer progression. The aim of this study was to identify IL-6 expression in ocular surface squamous neoplasia (OSSN) in comparison with non-neoplastic conjunctival tissue. METHODS: Twenty paraffin-embedded tissue sections of conjunctiva from patients with OSSN including conjunctival intraepithelial neoplasia (CIN) in all grades of severity and squamous cell carcinoma (SCC) were assessed by immunohistochemistry staining for IL-6. Twenty non-neoplastic conjunctival sections from age matched patients were selected as the control group. Tissues with more than one focus of inflammatory cell infiltration were excluded from the study. The mean area of positive staining was recorded and the intensity of staining was scored in both groups and compared by statistical methods. RESULTS: The mean staining area in the dysplasia group was significantly more than non-neoplastic conjunctival tissue (63.5±25.96 and 30±15.98 percent respectively; P-value of <0.0001). Nuclear staining was observed in both groups and the difference was not statistically significant. CONCLUSION: IL-6 expressed more in the dysplastic group in compare to non-neoplastic conjunctiva and can therefore be used to diagnose dysplastic state of the conjunctiva; however, in our study, intensity of staining does not correlate with the severity of dysplasia statistically; most probably because of a low sample size in each category. The role of nuclear staining is not clear. Our findings can be an introduction toward targeted treatment of ocular surface neoplasia by the aim of newer anti-IL agents. Further investigation is needed.

Evaluation of intravitreal injection of pentoxifylline in experimental endotoxin-induced uveitis in rabbits.

The objective of the present study was to investigate the clinical and histopathological effects of intravitreal injection of pentoxifylline (PTX) the management of an experimental model of uveitis. Fifty-two rabbits were divided randomly into six intravitreal treated groups as below: 1) Balanced salt solution (BSS), 2) Salmonella typhimurium lipopolysaccharide endotoxin (LPS) + BSS, 3) LPS + PTX 100 µg, 4) LPS + PTX 500 µg, 5) BSS + PTX 100 µg and 6) BSS + PTX 500 µg. Inflammation was evaluated by clinical examinations using slit lamp on days 1, 3, 5 and 7 post injections and histopathological examinations were also performed at the end of the study. Clinical examinations demonstrated a statistically significant difference between group 1 and group 2 on day 5 and day 7. Moreover, the comparison of clinical severity scores of group 1 with groups 3, 4, 5 and 6, on third, fifth and seventh post-injection days showed statistically significant differences. The mean histopathological inflammation intensity score in groups 5 and 6 was significantly higher than group 1. The mean histopathological inflammation intensity score in groups 3, 4, 5 and 6 was significantly higher than group 2. Intravitreal injection of PTX in an experimental model of uveitis in rabbits not only does not reduce inflammation but also leads to inflammation when used alone or in combination with LPS.

Congenital Orbital Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common malignant tumor of the orbit in children, but it is rarely present at birth. We report a large congenital orbital RMS with intracranial extension in a newborn baby. A newborn baby girl was referred to our hospital due to severe right-eye exophthalmia. Imaging studies showed an orbital mass with intracranial extension. Treatment was started with a course of neoadjuvant chemotherapy followed by right orbital exenteration and intracranial resection of the tumor. Histologic examination of the mass showed undifferentiated malignant small-cell tumor. Immunohistochemical study proved it to be RMS. The patient was categorized as intermediate-risk RMS and chemotherapy was continued accordingly with VAC regimen. In spite of treatment, the infant developed intracranial recurrence at the age of 6 months and died 1 month later. Congenital orbital RMS has a poor prognosis. Reconstruction surgery should be deferred due to high rate of recurrence and low chance of survival. A multidisciplinary approach might increase the survival of these patients.

Intra-vitreal injection of methotrexate in experimental endotoxin-induced uveitis in rabbit.

Uveitis is a major cause of vision loss. Methotrexate (MTX) has been widely used in uveitis due to its relatively safe profile. The purpose of this study was to evaluate the effects of two different dosages of MTX via intra-vitreal administration for treatment of endotoxin induced uveitis (EIU) in an experimental model. Thirty-five healthy rabbits were randomly divided into four groups and all animals were tolerated intra-vitreal injections. The first group received normal saline (NS), the second group received normal saline plus Salmonella typhimurium lipopolysaccharide endotoxin (LPS), (NS+LPS), the third group received 400 µg MTX plus LPS (LPS+MTX 400) and the fourth group received 800 µg MTX plus LPS (LPS+MTX 800). Intra-ocular inflammation was evaluated by clinical examination scoring during 7 post-injection days and histopathological examination at the end of study. Kruskal-Wallis and Mann-Whitney U tests were used to compare the histopathological and clinical scores. According to the clinical examinations, all groups demonstrated higher uveitis score than group 1 on first post-injection day. Also, groups 2 and 3 showed greater uveitis score than group 4. On the third, fifth and seventh post-injection days, clinical uveitis score in groups 2, 3 and 4 was significantly higher than group 1. The mean histopathological inflammation intensity scores in groups 2, 3 and 4 were significantly higher than group 1. Single intra-vitreal injection of 400 µg and 800 µg of MTX did not show significant anti-inflammatory effects on EIU in rabbits.

Optic nerve glioma with complete intraocular extension.

PURPOSE: To present a rare presentation of optic nerve glioma (ONG) with total intraocular extension. METHODS: A 44-year-old man with a history of loss of vision since childhood and recent development of progressive disfiguring proptosis was referred. RESULTS: The vision in the affected eye was no light perception, and a proptosis of about 2-3 mm was present. Computed tomography and Magnetic Resonance Imaging (MRI) of brain/orbit showed a fusiform enlargement of the left optic nerve and total filling of vitreous cavity by a mass with high signal intensity in T2-weighted MRI. There was no extension into the intracranial cavity. The patient also had no signs of neurofibromatosis. Histopathology confirmed the diagnosis of ONG of the left orbit with extension into the globe and filling vitreous cavity. CONCLUSION: ONG may extend to the vitreous cavity with no simultaneous intracranial involvement.

Expression of maspin and ezrin proteins in periocular Basal cell carcinoma.

Background. The aim of this study was to investigate maspin and ezrin expression in different subtypes of periocular basal cell carcinoma (BCC). Methods. Tissue samples from 43 patients with periocular BCC. Our cases were comprised of 10 morpheaform, 25 nodular, and 8 adenoid type BCCs. Immunohistochemical staining for maspin and ezrin was performed by Envision detection system. Results. There was no difference between different subtypes of BCC in maspin expression regarding positivity, intensity, and pattern of expression. Ezrin was expressed in all subtypes of BCC but the intensity was significantly higher in morpheaform BCC compared to nodular and adenoid types (P < 0.001 and P = 0.012, resp.); ninety percent of morpheaform samples showed strong ezrin intensity, while this strong intensity was only present in 25% and 12% of adenoid and nodular subtypes, respectively. There was no correlation between age, sex, or tumor margin involvement and expression of neither maspin nor ezrin. There was no correlation between maspin and ezrin expression except in nodular type, in which an inverse correlation was found (P = 0.004). Conclusion. Ezrin is expressed intensely in morpheaform BCC of periocular region. Further studies are needed to show the significance of this finding in prognosis of morpheaform BCC.

Omental transposition in treatment of severe ocular surface alkaline burn: an experimental study.

Since alkaline substances can rapidly penetrate into the cornea and subsequently damage limbal stem cells, another source of stem cells may be necessary to reconstruct the ocular surface. Omentum has some such characteristics like ability to regenerate tissue as well as anti-inflammatory capacity. Presence of adult stem cells and pluripotent embryonic cell markers make it suitable in wound healing; therefore, it seems reasonable to evaluate whether omentum can be helpful to restoration of ocular surface in severe alkaline burn. In this experimental trial, two groups of dogs (5 in each) were assigned. Following ethics approval, ocular surface alkaline burn was induced in both groups by placing filter papers soaked with NaOH (0.5 mol/l) on the cornea of one eye. Subsequently, group 1 (n=5) was treated only by conventional therapy; group 2 (n=5) was treated with omental elongation and transposition to the injured eye immediately following injury. Both groups were followed for six months. Ocular surface was evaluated by slit lamp microscope and corneal clarity was assessed and graded. At the end of six months, corneal opacity and vascularization were significantly reduced in group 2 (p-values of 0.009, 0.049, and 0.032 for corneal opacity, fluorescein staining, and vascularization grades, respectively). We have concluded that transposition of omental pedicle may be an effective treatment for severe ocular surface alkaline burn although more studies might be required.

Morphologic changes of corneal epithelial flap: ethanol-mediated versus mechanical removal.

PURPOSE: To evaluate morphologic changes in human corneal epithelial flap removed mechanically or after ethanol application. METHOD: Epithelial corneal flap was removed after ethanol application (20 eyes) or mechanically (19 eyes). Any changes were studied by transmission electron microscopy. RESULTS: Thirty-nine eyes were enrolled in the study. The following changes were found in the alcohol-applied group: apoptotic cells, membrane-bound blebs with marked dilatation of endoplasmic reticulum, and short intercellular cleavage with approximately one-third of cell length depth. In mechanical debridement, cleavages extended more than half of the cell length by tearing hemidesmosomes. CONCLUSION: Alcohol application leads to cell damage in basal epithelial cells but cleavage plane remains smooth. Generally, none of the methods caused trauma to the basement membrane.

Effect of different treatment regimen with dexamethasone and acetylcysteine on corneal wound healing in rabbits.

BACKGROUND: The effect of corticosteroid therapy on corneal wound healing is controversial. The objective of this study was to evaluate the effects of combination therapy with dexamethasone and acetylcysteine at different times and durations on experimentally-induced corneal wounds and haze in rabbits. METHODS: Eighteen adult New Zealand white rabbits were divided into three groups of six each. Under anesthesia corneal wounds were created surgically in the center of all eyes. The right eyes of rabbits in group 1 were treated topically with acetylcysteine and dexamethasone immediately after surgery, those in group 2 were treated with acetylcysteine from day 1 and with acetylcysteine and dexamethasone from day 8, and those in group 3 were treated with acetylcysteine from day 1 and with acetylcysteine and dexamethasone from day 15. The left eyes were assigned as controls and were treated with normal saline. All eyes were treated six times a day for 28 days. Corneal wounds were measured by fluorescein staining every day. RESULTS: The combination of acetylcysteine and dexamethasone in group 1 significantly increased mean healing time, but did not change that in groups 2 and 3. Clinical and histopathologic examinations revealed that one month after the ulceration in groups 1 corneal haze was greater in treated than in the control eyes. Moreover, there was no significant difference between the control and treated eyes of group 1, 2, or 3 in terms of corneal haze at two or three months after the ulceration. CONCLUSIONS: The findings of the present study show that the association of 3% concentration of NAC and 0.1% concentration of dexamethasone immediately after corneal ulceration can delay corneal wound healing, and consequently produce more corneal haze. Thus, the use of 0.1% concentration of dexamethasone should be delayed at least until the completion of the epithelial defects.

Intrasclera schwannoma.

Schwannomas located in the eye are extremely rare and mainly arise from ciliary nerves. Ocular schwannoma usually present in the choroid, ciliary body or rarely sclera as a benign solitary amelanotic lesions. A 41-year-old woman presented with a history of decreased vision in her right eye of 3 months duration. She had an amelanotic lesion in the nasal side of left fundus. After all work-ups, malignant melanoma was suspected and enucleation was done. In clinicopathologic examination, findings were in favour of intrascleral schwannoma. Despite new modern diagnostic modalities, there are still some fundus lesions that are easily mistaken for amelanotic melanoma. Diagnostic procedures such as fine needle aspiration should be considered for equivocal lesions in the fundus that are not definitely diagnosed before surgery.

Choroidal neovascular membranes express toll-like receptor 3.

BACKGROUND: Recent evidence has suggested a role for toll-like receptor 3 (TLR3) in experimental models of age-related macular degeneration (AMD). To date, however, few data exist about TLR3 in human AMD. The purpose of this study was to investigate the expression of TLR3 in human choroidal neovascular (CNV) membranes. METHODS: Immunostaining for TLR3 was performed on sections of CNV membranes from 8 AMD patients and eyes from 4 donors without CNV. RESULTS: All CNV membranes expressed TLR3 in retinal pigment epithelial (RPE) cells. One was classified as having strong intensity, 5 as having moderate intensity and 2 as having weak intensity. All cases had ≥30% of the RPE cells staining for TLR3, ranging from 30 to 90%. No expression of TLR3 was observed in vascular endothelial cells or fibroblasts in any CNV membrane. In the donor eyes, the RPE cells near the ora serrata stained stronger than those at the posterior pole, where no staining was observed in 3 out of 4 cases. CONCLUSION: TLR3 was found in all CNV membranes and was expressed exclusively in RPE cells. The observed difference in RPE staining for TLR3 in donor eyes and CNV membranes suggests a possible role for this receptor in human neovascular AMD.

Aspergillus endophthalmitis in orthotopic liver transplantation.

In this report, we describe a patient with drug-induced liver failure who developed endogenous endophthalmitis after liver transplantation. Our patient's clinical course was so fulminant that the eye was lost in less than 1 month, without any response to therapy. Recognition of this infection is important because many patients die of disseminated Aspergillus infection, which may be detected early with bedside funduscopic examination by an ophthalmologist. Probably if the patient had referred to us earlier, it may have been possible to save the eye.

Effect of intravitreal triamcinolone acetonide in the treatment of experimental Staphylococcus epidermidis endophthalmitis.

PURPOSE: To investigate the effect of intravitreal injection of triamcinolone acetonide (TA) in the treatment of experimental Staphylococcus epidermidis endophthalmitis. MATERIALS AND METHODS: Experimental endophthalmitis induced in one eye of 38 rabbits by intravitreal injection of Staphylococcus epidermidis. The infected rabbits were randomly divided into three groups. The rabbits in group 1 (n = 15) received intravitreal injection of 1 mg vancomycin hydrochloride. Group 2 eyes (n = 15) received intravitreal TA (4 mg) plus vancomycin hydrochloride (1 mg). Group 3 eyes (8 eyes) received normal saline. Clinical examination in each group was performed two, seven, and 15 days after inoculation of the bacteria. The eyes were enucleated for histopathologic evaluation on the 15th day. Clinical and histological scoring systems were used to evaluate clinical and histological severity of the intraocular infection. RESULTS: The mean clinical scores of three study groups at the 7th day were 5.8 +/- 2.3, 3.3 +/- 1.5, and 9.5 +/- 2.1, respectively. The mean of this value at 15th day were 6.2 +/- 2.6, 3.9 +/- 2.1, and 10 +/- 2.2, respectively. At the 7th and 15th day, the mean clinical score of group 2 was significantly lower when compared with the other two groups. Mean histopathologic scores of group 1, group 2, and group 3 were 11 +/- 2.7, 7.9 +/- 2.9, and 14.0 +/- 3.1, respectively. Histopathologic studies revealed less tissue destruction for eyes treated with TA. There was a significant positive correlation between the severity of clinical signs and pathologic damage in all three experiment groups; however, it was weakest in the IVTA treated group. CONCLUSION: A standard clinical dose of intravitreal TA attenuates the clinical signs and tissue destruction in a rabbit model of Staphylococcus epidermidis endophthalmitis.