Mohammad Javed Ali

Establishing Human Lacrimal Gland Cultures with Secretory Function

Purpose Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in–vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures. Methods Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA. Results Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15–20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed ‘spherules’ with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons). Conclusion The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

Purpose: To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. Design: Laboratory investigation. Methods: Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1–0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4′,6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. Results: The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. Conclusions: The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Primary canaliculitis: clinical features, microbiological profile, and management outcome.

Purpose: To describe the demographic profile, clinical presentation, microbiological profile, and management outcome of primary canaliculitis. Methods: Single-center, retrospective, interventional case series. Clinical records of all patients diagnosed with primary canaliculitis and treated at the Department of Ophthalmic Plastic Surgery, LV Prasad Eye Institute, Hyderabad, India, between 1987 and 2010 were reviewed. Retrospective data analysis included demographic profile, clinical presentation, microbiological profile, and management outcomes. The management outcome was further analyzed regarding conservative medical treatment alone, versus punctoplasty with canalicular curettage. Results: Of the 74 patients, 40 (54%) were women. Mean age at presentation was 48 years. Right eye was involved in 38 (51%) patients, left eye in 34 (46%) patients, and both eyes in 2 (3%) patients. The mean delay in diagnosis was 10 months. Lower canaliculus was involved in 48 (65%) patients, upper canaliculus in 17 (23%) patients, and both canaliculi in 9 (12%) patients. The most common presenting symptom was epiphora, noted in 63 (85%) patients, and the most common clinical sign was thickening of canalicular portion of eyelid seen in 53 (72%) patients. Microbiological workup was available in 54 patients, of whom 49 (91%) yielded positive results. The most common isolate was staphylococcus species in 19 (39%) patients. Conservative medical therapy (punctal dilatation, canalicular expression, and topical antibiotics) resulted in resolution in 35 of 51 (69%) patients, whereas punctoplasty with canalicular curettage resulted in resolution in all 39 (100%) patients. Of the 74 patients, 57 (70%) resolved completely with single intervention, 14 (19%) with 2 interventions, 6 (8%) with 3 interventions, and 2 (3%) with 4 interventions. Recurrence was noted in 2 (3%) patients that subsequently resolved with treatment. Conclusion: Primary canaliculitis is predominantly a unilateral disease with a significant delay in diagnosis. The microbiological profile of canaliculitis is evolving, with staphylococcus species emerging as the most common pathogen. Although conservative medical therapy is beneficial, punctoplasty with canalicular curettage combined with topical antibiotic therapy is the gold standard treatment for canaliculitis.

Acquired nasolacrimal duct obstructions secondary to naso-orbito-ethmoidal fractures: patterns and outcomes.

Purpose: This study reports a series of 14 consecutive patients of nasolacrimal duct obstruction secondary to naso-orbito-ethmoid fractures (NOE), their presentations, surgical interventions, and outcomes. Methods: Fourteen consecutive patients presenting with secondary acquired nasolacrimal duct obstructions following naso-orbito-ethmoid fractures were included in a retrospective, noncomparative case series. Medical records were reviewed for demographic data, mode of trauma, types of NOE fractures, type of fracture repair, symptoms at presentation, duration of symptoms, areas of lacrimal obstructions, surgical interventions, lengths of follow up, and outcomes. Success was defined as both anatomical patency of the lacrimal passages and resolution of symptoms. Results: The mean age at presentation was 26.4 years (range: 4–46). The most common mode of trauma was a road traffic accident (71.4%), and the most common type of fracture was NOE type II (64.2%). The majority of the fractures (71.4%) were repaired by open reduction and internal fixation. All patients presented with epiphora, and 50% of them also presented with a swelling below the medial canthus. The mean duration of symptoms were 19.5 months (range: 1–48). All patients underwent an external DCR with mitomycin C and silicone intubation. All stents were removed at 12 weeks following surgery. The mean follow up after stent removal was 6 months (range 3–24 months) with a minimum follow up of 3 months. At the last follow up, the success rate was 92.8%. There was one failure, which was due to cicatricial closure of the ostium. Conclusion: External dacryocystorhinostomy with Mitomycin-C and intubation is effective in treatment of acquired nasolacrimal duct obstructions following NOE fractures. A delayed dacryocystorhinostomy did not appear to influence the outcomes.

A comprehensive, sensitive and economical approach for the detection of mutations in the RB1 gene in retinoblastoma

Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. It is brought about by the mutational inactivation of both alleles of RB1 gene in the developing retina. To identify the RB1 mutations, we analysed 74 retinoblastoma patients by screening the exons and the promoter region of RB1. The strategy used was to detect large deletions/duplications by fluorescent quantitative multiplex PCR; small deletions/insertions by fluorescent genotyping of RB1 alleles, and point mutations by PCR-RFLP and sequencing. Genomic DNA from the peripheral blood leucocytes of 74 Rb patients (53 with bilateral Rb, 21 with unilateral Rb; 4 familial cases) was screened for mutations. Recurrent mutations were identified in five patients with bilateral Rb, large deletions in 11 patients (nine with bilateral Rb and two with unilateral Rb), small deletions/insertions were found in 12 patients all with bilateral Rb, and point mutations in 26 patients (14 nonsense, six splice site, five substitution and one silent change). Three mutations were associated with variable expressivity of the disease in different family members. Using this method, the detection rates achieved in patients with bilateral Rb were 44/53 (83%) and with unilateral Rb, 5/21 (23.8%). This approach may be feasible for clinical genetic testing and counselling of patients.

Circumostial injection of mitomycin C (COS-MMC) in external and endoscopic dacryocystorhinostomy: efficacy, safety profile, and outcomes.

Purpose: To evaluate the safety and efficacy of a new technique of circumostial mitomycin C (COS-MMC) injection in dacryocystorhinostomy (DCR) and to evaluate the outcomes. Methods: A prospective, interventional consecutive case series involving 110 DCR surgeries performed over a period of 1 year using the COS-MMC technique were evaluated. All patients were operated by a single surgeon (M.J.A.). After fashioning of the mucosal flaps, all patients underwent an intramucosal injection of 0.02% MMC at 4 points (0.1 ml at each point) along the edges of the freshly created ostium. All patients were intubated, and stent removal was done at 12 weeks following the surgery. All patients underwent regular monitoring of the ostium to look for any complications. A minimum follow up of 6 months after stent removal was considered for final analysis. The outcome measures analyzed were anatomical and functional success, complications, and ostium. Results: The mean age at presentation was 39.91 years. The diagnosis was primary acquired nasolacrimal duct obstruction in 60.9% (67/110) and persistent congenital nasolacrimal duct obstruction in 9.1% (10/110) of the patients. Twenty-six patients were known cases of failed DCR and underwent a revision DCR. Of the total patients, 19.1% (21/110) had a history of acute dacryocystitis, of which, 73% (80/110) of the surgeries were performed by an external approach and 27% (30/110) by endoscopic endonasal route. Anatomical success was noted in 97.3% (107/110) and functional success in 96.4% (106/110). All the 4 failures were due to cicatricial closure of the ostium. None of the patients were noted to have mucosal burns in the area where MMC was injected, and none of the Ostia showed any features of necrosis, infection, or any other complications. Conclusions: COS-MMC is a safe and effective adjunctive modality in DCR. No adverse effects on the ostium were noted. The technique appears to be promising for the success of cases with high risk of failures like revision DCR and traumatic secondary acquired nasolacrimal duct obstruction.

Long-term outcomes in primary powered endoscopic dacryocystorhinostomy

Aim To assess the long-term anatomical and functional outcomes of primary powered endoscopic dacryocystorhinostomy (DCR). Methods Retrospective review of all primary powered endoscopic DCRs performed over a 10-year period from 2003 to 2012. A minimum of 12 months follow-up after stent removal was required for inclusion into this study. Patient records were reviewed for demographic data, clinical and surgical profiles, adjunctive procedures, complications and success rates at last follow-up. Anatomical success was defined as a patent ostium on irrigation, and functional success as free flow of dye into ostium, and resolution of epiphora. Results Ninety powered endoscopic DCRs were performed on 69 patients. The mean age at surgery was 58.7 years (range 4–91 years). All patients presented with epiphora. 50.7% (35/69) patients underwent adjunctive endonasal procedures. The mean follow up was 21.8 months (range: 12–103 months). No intraoperative complications were noted. Postoperative complications noted included postoperative bleeding, ostium granuloma and membrane over internal common opening in one patient each. At last follow-up, the final anatomical success was achieved in 97.7% and functional success in 95.5% of the cases. Conclusions Powered endoscopic DCR is a safe procedure and offers excellent long-term results. Adjunctive endonasal procedures should be performed where indicated.

Dacryocystorhinostomy ostium: parameters to evaluate and DCR ostium scoring.

Aim This study aims to provide a systematic protocol for the evaluation of a dacryocystorhinostomy (DCR) ostium and to propose a scoring system to standardize the assessment. Methods Retrospective evaluation of 125 consecutive lacrimal ostia post-DCR was performed. Medical records were screened, and photographs and videos were assessed to note the details of various ostial parameters. The major time points in evaluation were 4 weeks, 6 weeks, 3 months, and 6 months post-DCR. The ostia were defined and parameters like shape, size, location, and evolution of ostium were noted. Evaluation parameters were defined for internal common opening (ICO), ostium stents, and ostium granulomas. Ostium cicatrix and synechiae were graded based on their significance. Surgical success rates were computed and ostium characteristics in failed cases were studied. Results A total of 125 ostia were evaluated on the aforementioned ostium parameters. Because five ostia showed a complete cicatricial closure with no recognizable features, the remaining 120 ostia were studied. The ostium location was anterior to the axilla of middle turbinate in 85.8% (103/120) of the cases. Moreover, 76.6% (92/120) of the ostia were circular to oval in shape, with a shallow base. The ostium size was >8×5 mm in 78.3% (94/120) of the cases. The ICO was found to be located in the central or paracentral basal area in 75.8% (91/120). The anatomical and functional success rates achieved were 96% and 93.6%, respectively. All the five cases with anatomical failures showed a complete cicatrization and the ICO movements were poor in all the three cases of functional failure. Conclusion The article attempts to standardize the postoperative evaluation of a DCR ostium and provides a systematic protocol and scoring system for possible use by surgeons and researchers alike.

Long-term outcomes in revision powered endoscopic dacryocystorhinostomy.

Long‐term outcomes of revision dacryocystorhinostomy are less known. The aim of this study was to assess the long‐term outcomes of revision powered endoscopic dacryocystorhinostomy (DCR).Background Long-term outcomes of revision dacryocystorhinostomy are less known. The aim of this study was to assess the long-term outcomes of revision powered endoscopic dacryocystorhinostomy (DCR). Methods Retrospective review of all consecutive revision powered endoscopic DCRs performed over a period of 10 years from 2003 to 2012 were included. All patients completed a minimum of 12 months follow-up following stent removal. Patient records were reviewed for demographic data, type of past surgery, causes of failure, clinical and surgical profiles, adjunctive procedures, complications, and success rates at last follow-up. Results Twenty-three revision powered endoscopic DCRs were performed on 18 patients who had failed primary DCR. The mean age at surgery was 61 years (range, 28–84 years). All patients presented with epiphora. Nearly 67% (66.7%; 12/18) were failed endoscopic DCRs and 33.3% (6/18) were failed external DCRs. The most common cause of failure was from cicatricial closure of ostium in 55.5% (10/18) of the cases. Over 44% (44.4%; 8/18) of patients underwent adjunctive endonasal procedures at the time of revision endoscopic DCR. The mean follow-up was 26.4 months (range, 12–66 months). A complication of ostium granuloma in 1 patient was noted, which was successfully managed. At the last follow-up, anatomical success was achieved in 91.3% of the cases and functional success in 86.9%. Conclusion Powered endoscopic DCR is a suitable option for revising failed DCRs and offers good long-term results. Meticulous evaluation to identify causative factors of failure and addressing them is crucial for successful outcomes.

Clinical Profile and Management Outcome of Acute Dacryocystitis: Two Decades of Experience in a Tertiary Eye Care Center

Abstract Aim: To report the demographic profile, clinical presentation, and management outcome of acute dacryocystitis. Methods: Retrospective study of 347 eyes of 320 patients, who presented to the Ophthalmic Plastic Clinic of a tertiary eye care center over a period of 22 years from January 1990 to March 2012 with acute dacryocystitis, were reviewed for demographic profile, clinical presentation, and management outcome. The numbers of patients with lacrimal disorders during the same period were retrieved to assess the incidence of acute dacryocystitis. Successful anatomical outcome was defined as patency on irrigation and a successful functional outcome was defined as resolution of infection and epiphora. Results: The mean age at presentation was 37 years. The female to male ratio was 2:1. There was no difference in the laterality between the right and the left eyes. Bilateral disease was noted in 8.4% (27/320) patients. 23% (80/347) eyes presented with lacrimal abscess while 2.8% (10/347) eyes had orbital cellulitis. Intensive medical care with hospital admission was needed in 4.4% (14/320) patients. The mean time to resolution of acute symptoms was 10 days. 5.6% (18/320) patients developed a fistula, among which 83% (15/18) were following a spontaneous rupture of the lacrimal abscess. Dacryocystorhinostomy was performed in 82.5% (264/320) patients with an anatomical success of 94.5% and a functional success of 93.5%. Conclusions: Acute dacryocystitis comprises 2.4% of all patients presenting with lacrimal system disorders. Fistula formation is a sequel more commonly seen with spontaneous rupture of a lacrimal abscess. The long-term outcomes in patients presenting with acute dacryocystitis are good with a surgical success rate of 94.3%.