Ahmad Kheirkhah

Amniotic Membrane Transplantation as a New Therapy for the Acute Ocular Manifestations of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis

Stevens-Johnson syndrome and its more severe variant, toxic epidermal necrolysis, have relatively low overall incidence; however, this disease presents with high morbidity and mortality. The majority of patients develop ocular inflammation and ulceration at the acute stage. Due to the hidden nature of these ocular lesions and the concentration of effort toward life-threatening issues, current acute management has not devised a strategy to preclude blinding cicatricial complications. This review summarizes recent literature data, showing how sight-threatening corneal complications can progressively develop from cicatricial pathologies of lid margin, tarsus, and fornix at the chronic stage. It illustrates how such pathologies can be prevented with the early intervention of cryopreserved amniotic membrane transplantation to suppress inflammation and promote epithelial healing at the acute stage. Significant dry eye problems and photophobia can also be avoided with this intervention. This new therapeutic strategy can avert the catastrophic ophthalmic sequelae of this rare but devastating disease.

Role of conjunctival inflammation in surgical outcome after amniotic membrane transplantation with or without fibrin glue for pterygium.

Purpose: To determine the clinical significance of postoperative conjunctival inflammation noted at the third or fourth week after intraoperative application of mitomycin C and amniotic membrane transplantation for pterygium. Methods: This retrospective study included 27 eyes of 23 patients with primary (n = 12) or recurrent (n = 15) pterygia. All cases were operated by extensive removal of subconjunctival fibrovascular tissue and intraoperative application of 0.04% mitomycin C in the fornix, followed by amniotic membrane transplantation by using either fibrin glue (14 eyes) or sutures (13 eyes). Main outcome measures included development of conjunctival inflammation, pyogenic granuloma, and pterygium recurrence after surgery. Results: For a follow-up of 29.6 ± 17.2 months (range, 6-56 months), 16 (59.3%) eyes without postoperative conjunctival inflammation resulted in favorable outcomes. Conjunctival inflammation around the surgical site was noted in the remaining 11 (40.7%) eyes and was significantly more common in eyes with sutures than those with fibrin glue (61.5% vs. 21.4%, respectively; P = 0.05). Among those with this inflammation, 7 eyes receiving subconjunctival injection of triamcinolone resulted in complete resolution and a good aesthetic outcome. Four eyes without this injection gradually developed conjunctival (n = 1) or corneal (n = 1) recurrence and/or pyogenic granuloma (n = 3). Conclusions: Host conjunctival inflammation is still common after intraoperative application of mitomycin C and amniotic membrane transplantation, especially when sutures are used in pterygium surgery. If left untreated, persistent inflammation may lead to a poor surgical outcome.

Human Amniotic Epithelial Cells as Novel Feeder Layers for Promoting Ex Vivo Expansion of Limbal Epithelial Progenitor Cells

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan‐cytokeratins (pan‐CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan‐CK(+)/Vim(+) or pan‐CK(+)/Vim(‐) were maintained in serum‐free media with high calcium. In contrast, all HAECs became pan‐CK(+)/Vim(+) in serum‐containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C‐arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC‐supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell‐associated markers such as p63, Musashi‐1, and ATP‐binding cassette subfamily G2 than those of 3T3‐supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63‐positive epithelial progenitor cells as well as nestin‐positive neuronal‐like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus.

Characterization and Comparison of Intercellular Adherent Junctions Expressed by Human Corneal Endothelial Cells in Vivo and in Vitro

PURPOSE Human corneal endothelial cell (HCEC) proliferation is controlled by HCEC junctions, but the mechanism of proliferation remains unknown. The authors sought to characterize adherent junction components of in vivo HCECs and to compare their gene expression and their proliferative potential with those of in vitro counterparts. METHODS Stripped human Descemet membranes were digested with collagenase A, and the resultant HCEC aggregates were cultured for 7, 14, and 21 days in supplemented hormonal epithelial medium (SHEM). The growth of HCEC monolayers was monitored by BrdU labeling performed 24 hours before termination. In vivo and in vitro HCECs were subjected to immunostaining to FITC-phalloidin and antibodies to different junction components and BrdU. Their mRNA expressions were determined by RT-PCR. RESULTS In vivo HCECs expressed transcripts of N-, VE-, E-, and P-cadherins, alpha-, beta-, gamma-, and p120-catenins, and p190. In vitro HCEC counterparts also expressed all these mRNAs except P-cadherin. In vivo HCECs displayed continuous circular F-actin, N-cadherin, beta- and p120-catenins, and p190, discontinuous circular VE-cadherin bands at or close to cell junctions, and E-cadherin in the cytoplasm. Such an in vivo pattern was gradually achieved by in vitro HCECs at day 21 and was correlated with a progressive decline of BrdU labeling. CONCLUSIONS In vivo and in vitro HCECs displayed distinct protein cytolocalization of N-, VE-, and E-cadherins, beta- and p120-catenins, and p190. Progressive maturation of adherent junctions was associated with a decline of the proliferative potential. This information allows us to devise new strategies to engineer in vitro HCECs by targeting these components.

Surgical Strategies for Fornix Reconstruction Based on Symblepharon Severity

PURPOSE To identify surgical strategies of fornix reconstruction for symblepharon graded according to the length from the limbus to the lid margin, to the width, and to associated inflammation. DESIGN Retrospective, comparative, interventional case series. METHODS In 61 eyes with symblepharon, cicatrix lysis and amniotic membrane transplantation (AMT) were performed with sutures (n = 34) or fibrin glue (n = 27) together with (n = 47) or without (n = 14) intraoperative mitomycin C (MMC), plus fornix reconstruction using anchoring sutures without (n = 30) or with (n = 7) oral mucosal graft or with conjunctival autograft (n = 4). Overall, success was defined as an outcome of complete success (restoration of an anatomically deep fornix) or partial success (focal recurrence of scar), and failure was defined as the return of symblepharon. RESULTS For a follow-up of 25 +/- 10.8 months, the overall success was achieved by the first attempt in 52 eyes (85.2%) and failure resulted in nine eyes (14.8%); however, the success rate improved to 59 eyes (96.7%) with additional attempts. At the first attempt, AMT alone achieved overall successes in 92.8% of grade I eyes and in 100% of grade II eyes. Additional anchoring sutures achieved successes in 100% of grade I eyes, 70% of grade II eyes, and 71.4% of grade III/IV eyes. Additional oral mucosa or conjunctival autograft achieved successes in 100% of grade III/IV eyes. The complete success was correlated positively with lower grades of symblepharon or intraoperative use of MMC, but negatively correlated with younger ages, canthal involvement, or use of anchoring sutures. Anatomic improvement was accompanied by reduction of preoperative conjunctival inflammation (n = 40), improved visual acuity (n = 14), improved ocular motility (n = 18), improved eyelid closure (n = 3), and feasibility of contact lens wear (n = 10). CONCLUSIONS Successful outcome can be achieved by selectively deploying cicatrix lysis and AMT, intraoperative MMC, anchoring sutures, and oral mucosal or conjunctival autograft based on the severity of pathogenic symblepharon.

Topical 0.005% tacrolimus eye drop for refractory vernal keratoconjunctivitis

PurposeTo evaluate the efficacy and safety of topical 0.005% tacrolimus eye drop for treatment of refractory vernal keratoconjunctivitis (VKC).MethodsThis prospective study included 20 eyes of 10 patients with refractory VKC, who had active symptomatic disease despite conventional medications including topical steroids. After discontinuing all other medications, patients were treated with topical 0.005% tacrolimus eye drop four times a day. Changes in subjective symptoms and objective signs after treatment were evaluated, and development of possible complications was assessed.ResultsMean age of patients was 21.3±7.4 years and mean duration of VKC was 12.1±5.8 years. After starting tacrolimus eye drop, patients were followed for a mean duration of 10.7±3.7 months (range, 6–15 months). All symptoms including itching, redness, photosensitivity, foreign body sensation, and mucus discharge improved after the treatment; itching was the first symptom to show dramatic relief. In addition, there was improvement in objective signs including conjunctival hyperaemia, conjunctival papillary hypertrophy, giant papillae, limbal hypertrophy, corneal punctate epithelial erosions, and corneal pannus; conjunctival hyperaemia was the first sign to show improvement. No patient required addition of other medications including steroids for further relief. Any attempt to discontinue tacrolimus eye drop was associated with recurrence of patients’ symptoms and signs, necessitating continued use of the medication during the entire follow-up time. No ocular complication related to tacrolimus was noted.ConclusionTopical 0.005% tacrolimus eye drop seemed to be a safe and effective treatment for steroid-resistant refractory VKC; however, long-term use was needed to control the disease.

Minimal Conjunctival Limbal Autograft for Total Limbal Stem Cell Deficiency

Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA+ cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA+ cells were found to be primarily CD68− and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-γ, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.PURPOSE To report the results of one 60 degrees conjunctival limbal autograft (CLAU) combined with amniotic membrane (AM) transplantation for an eye with total limbal stem cell deficiency (LSCD). METHODS One eye of a patient with chronic total LSCD and symblepharon caused by chemical burn was subjected to symblepharon lysis, removal of pannus from corneal surface, AM transplantation to cover the conjunctival and corneal surfaces as a permanent graft, one 60 degrees CLAU to the superior limbal area, and insertion of ProKera as a temporary AM patch to cover the CLAU. RESULTS After surgery, corneal epithelialization over the AM was evident adjacent to the CLAU on day 6, progressed to pass the horizontal midline by day 11, and was completed by day 18. During a follow-up of 1 year, the corneal surface remained stable and smooth, and the stroma considerably regained clarity with regression of midstromal vascularization. The best-corrected visual acuity improved from 20/400 to 20/50. The conjunctival inflammation completely resolved, and the fornices were deep. CONCLUSIONS One 60 degrees CLAU combined with AM transplantation as both a permanent graft and a temporary patch can restore the entire corneal surface in an eye with total LSCD caused by chemical burn.

Fluorescein dye improves microscopic evaluation and counting of demodex in blepharitis with cylindrical dandruff.

Purpose: To show whether fluorescein dye helps detect and count Demodex embedded in cylindrical dandruff (CD) of epilated eyelashes from patients with blepharitis. Methods: Two eyelashes with CD were removed from each lid of 10 consecutive patients with blepharitis and subjected to microscopic examination with and without fluorescein solution to detect and count Demodex mites. Results: Of 80 eyelashes examined, 36 (45%) lashes retained their CD after removal. Before addition of the fluorescein solution, the mean total Demodex count per patient was 14.9 ± 10 and the mean Demodex count per lash was 3.1 ± 2.5 and 0.8 ± 0.7 in epilated eyelashes with and without retained CD, respectively (P < 0.0001). After addition of the fluorescein solution, opaque and compact CD instantly expanded to reveal embedded mites in a yellowish and semitransparent background. As a result, the mean total Demodex count per patient was significantly increased to 20.2 ± 13.8 (P = 0.003), and the mean count per lash was significantly increased to 4.4 ± 2.8 and 1 ± 0.8 in eyelashes with and without retained CD (P < 0.0001 and P = 0.007), respectively. This new method yielded more mites in 8 of 10 patients and allowed mites to be detected in 3 lashes with retained CD and 1 lash without retained CD that had an initial count of zero. Conclusions: Addition of fluorescein solution after mounting further increases the proficiency of detecting and counting mites embedded in CD of epilated eyelashes.

Autologous Serum Tears for Treatment of Photoallodynia in Patients with Corneal Neuropathy: Efficacy and Evaluation with In Vivo Confocal Microscopy

OBJECTIVE Patients suffering from corneal neuropathy may present with photoallodynia; i.e., increased light sensitivity, frequently with a normal slit-lamp examination. This study aimed to evaluate the efficacy of autologous serum tears (AST) for treatment of severe photoallodynia in corneal neuropathy and to correlate clinical findings with corneal subbasal nerve alterations by in vivo confocal microscopy (IVCM). METHODS Retrospective case control study with 16 patients with neuropathy-induced severe photoallodynia compared to 16 normal controls. Symptom severity, clinical examination and bilateral corneal IVCM scans were recorded. RESULTS All patients suffered from extreme photoallodynia (8.8±1.1) with no concurrent ocular surface disease. Subbasal nerves were significantly decreased at baseline in patients compared to controls; total nerve length (9208±1264 vs 24714±1056 μm/mm(2); P<.0001) and total nerve number (9.6±1.4 vs 28.6±2.0; P<.0001), respectively. Morphologically, significantly increased reflectivity (2.9±0.2 vs 1.8±0.1; P<.0001), beading (in 93.7%), and neuromas (in 62.5%) were seen. AST (3.6±2.1 months) resulted in significantly decreased symptom severity (1.6±1.7; P=.02). IVCM demonstrated significantly improved nerve parameters (P<.005), total nerve length (15451±1595 μm/mm(2)), number (13.9±2.1), and reflectivity (1.9±0.1). Beading and neuromas were seen in only 56.2% and 7.6% of patients. CONCLUSION Patients with corneal neuropathy-induced photoallodynia show profound alterations in corneal nerves. AST restores nerve topography through nerve regeneration, and this correlated with improvement in patient-reported photoallodynia. The data support the notion that corneal nerve damage results in alterations in afferent trigeminal pathways to produce photoallodynia.

Human ocular onchocerciasis caused by Onchocerca lupi (Spirurida, Onchocercidae) in Iran.

Cases of canine onchocerciasis caused by Onchocerca lupi are increasingly reported from Europe and the western United States of America. The zoonotic role of this parasite had already been suspected in Europe as the clinical signs and histopathology seen in two ocular cases from Albania and the Crimean region were very similar to those of canine ocular onchocerciasis. In the most recent reports of human onchocerciasis, O. lupi has been morphologically and molecularly identified as the causative agent of ocular infestation in two patients from Turkey, and one patient from Tunisia. Here, we report an additional case of nodular lesions involving two, and possibly more, immature worms in a patient from Iran. The parasite was found to belong to the genus Onchocerca based on morphological features and the species was confirmed as O. lupi from a partial sequence analysis of 12S ribosomal DNA.