Jignesh G. Parikh

Postoperative use of bevacizumab as an antifibrotic agent in glaucoma filtration surgery in the rabbit.

PURPOSE To evaluate the efficacy of bevacizumab as an antifibrotic agent after trabeculectomy in rabbits. METHODS Forty-two rabbits underwent trabeculectomy and were randomly assigned to receive a postoperative course of seven subconjunctival injections of bevacizumab (1.25 mg, 25 mg/mL), 5-fluorouracil (5-FU; 5 mg, 50 mg/mL), or balanced salt solution (BSS; 0.1 mL, control). Bleb survival and characteristics were evaluated over a 30-day period. The animals were killed on postoperative day (PD)10, PD20, and PD30. Histology and immunohistochemistry of the surgical eyes was performed to evaluate and grade the amount of scarring and fibrosis in each group. RESULTS Bevacizumab significantly improved the outcome of filtration surgery in this model. Bevacizumab prolonged bleb survival compared with the 5-FU and control groups (16.0 +/- 1.3 days vs. 6.9 +/- 0.6 and 7.4 +/- 0.85 days, respectively; P < 0.001). Bevacizumab-treated eyes had significantly larger and higher blebs than the control and 5-FU-treated groups (P < 0.05). Histologic analysis revealed that eyes treated with bevacizumab had significantly less postoperative scarring at the microscopic level at PD10 and PD20 (P = 0.009). CONCLUSIONS Postoperative subconjunctival injection of bevacizumab is associated with improved trabeculectomy bleb survival in the rabbit model. Bevacizumab may be a useful agent for improving success and limiting scar tissue formation after trabeculectomy.

Immunohistochemical Study of Chronic Nongranulomatous Anterior Uveitis in Juvenile Idiopathic Arthritis

PURPOSE To provide a detailed immunohistochemical analysis of juvenile idiopathic arthritis (JIA)-associated anterior uveitis. DESIGN Interventional case report. PARTICIPANT One patient. INTERVENTION A 12-year-old patient had recurrent pauciarticular JIA and smoldering anterior uveitis in the right eye. Despite treatment with local and systemic corticosteroids and an anti-tumor necrosis factor agent, the right eye became hypotonous and painful and eventually was enucleated. The clinical history and histopathologic and immunohistochemical analyses of the enucleated globe were reviewed. MAIN OUTCOME MEASURES Histopathologic and immunohistochemical features of JIA-associated anterior uveitis. RESULTS The iris and ciliary body showed nongranulomatous chronic inflammation predominantly made up of plasma cells, Russell bodies, and plasmacytoid lymphocytes. The ciliary processes and pars plana ciliaris showed focal aggregates of CD20-positive cells with several CD3- and CD8-positive cells and occasional CD4- and CD68-positive cells. Pancytokeratin stain showed ciliary epithelial proliferation admixed with lymphocytes. The iris revealed kappa-positive cells within the stroma and lambda-positive cells on the surface. The iris infiltrate primarily was made up of immunoglobulin (Ig) G-positive cells with occasional IgA- and IgM-positive cells. The anterior chamber exudate was mainly positive for IgG and IgA. CONCLUSIONS The immunohistochemical findings suggest that JIA-associated nongranulomatous iridocyclitis is a primarily B-cell-infiltrative process.

The role of TLR4 activation in photoreceptor mitochondrial oxidative stress.

PURPOSE Herein the authors investigated whether the activation of Toll-like receptors (TLRs) in the innate immune response causes retinal photoreceptor oxidative stress and mitochondrial DNA (mtDNA) damage. METHODS On day 5 after injection of complete Freunds adjuvant containing heat-killed Mycobacterium tuberculosis (CFA), retinas were submitted to polymerase chain reaction (PCR) array focused on the TLR signaling, or apoptosis, pathway. CFA-mediated TLR4 activation, oxidative stress, and mtDNA damage were determined in B10.RIII and knockout (KO) mice (recombination activation gene [Rag] 1(KO), TLR4(KO), myeloid differentiation primary response gene 88 [MyD88](KO), tumor necrosis factor [TNF]-α(KO), or caspase 7(KO) mice) using quantitative real-time PCR, enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry. The mycobacterial DNA load on the retina, brain, liver, and spleen was determined by real-time PCR after intracardiac perfusion. RESULTS PCR array demonstrated the upregulation of TLRs and their signaling molecules in retinas of CFA-injected mice compared with those of control animals without inflammatory cell infiltration in the retina and uvea. Mycobacterial DNA was detected in the retinas of CFA-injected mice. Retinas of CFA-injected animals showed oxidative stress and mtDNA damage, primarily in the photoreceptor inner segments. Upregulated TLR4 was localized with CD11b(+)MHCII(+) cells but not with GFAP(+) astrocytes. This oxidative stress/damage was similar in CFA-injected Rag1(KO) mice compared with wild-type controls. Such damage was absent in the retinas of CFA-injected TLR4(KO), MyD88(KO), and TNF-α(KO) mice. CFA-mediated inducible nitric oxide synthase expression in the retina was significantly decreased in TNF-α(KO) mice. CONCLUSIONS Retinal photoreceptors are susceptible to mitochondrial oxidative stress/mtDNA damage in robust TLR4-mediated innate immune response.

Immunopathology of necrotising scleritis

Aim: To detect the immunohistopathology of necrotising scleritis. Methods: Immunohistochemical staining was performed on two groups of enucleated eyes with necrotising scleritis (systemic autoimmune disease and idiopathic scleritis). Deparaffinised sections were stained with CD3, CD20, CD68, CD8, CD4 and dendritic reticulum cells (DRC). Results: Within the autoimmune group, about 43% of inflammatory cells stained positive with CD20, 35% with CD68, 17% with CD3, 8% with CD8, 4% with DRC and less than 1% with CD4. Within the idiopathic group of eyes, about 43% of cells stained positive for CD68, 23% for CD3, 17% for CD20, 7% for CD8, 1% for DRC and less than 1% for CD4. Conclusions: The infiltrate within the group of eyes with systemic autoimmune disease suggests that the inflammation may be driven by B cells. However, the large numbers of CD68 cells found in both groups of eyes indicate that macrophages could play a role in the necrotising process.

Mitochondrial Oxidative DNA Damage in Experimental Autoimmune Uveitis

PURPOSE In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress early in uveitis, before macrophage cellular infiltration. The purpose of this study was to determine whether oxidative mitochondrial DNA damage occurs early in EAU, before leukocyte infiltration. METHODS Lewis rats were immunized with S-antigen mixed with complete Freund adjuvant (CFA) to induce EAU. Nonimmunized animals and animals injected with CFA served as controls. Animals were killed on days 3, 4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was assessed using a novel long quantitative polymerase chain reaction technique. TUNEL staining to detect apoptosis and immunohistochemical detection of leukocyte infiltration in EAU retinas were also performed at these times. RESULTS Mitochondrial DNA damage occurred early in EAU, from day 4 to day 12. In the early phase of EAU (days 4-7), there was no inflammatory cell infiltration. On day 12 inflammatory cells infiltrated the retina and uvea. Nuclear DNA damage occurred later in EAU at day 12. Neither mitochondrial nor nuclear DNA damage was detected in the controls. TUNEL-positive staining for apoptosis was detected only at day 12 in EAU retina. CONCLUSIONS Oxidative mitochondrial DNA damage begins at day 4 in EAU, supporting the view that oxidative stress selectively occurs in the mitochondria in the early phase of EAU, before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal degeneration in EAU.

Expression of heat shock protein 27 and alpha-crystallins in human retinoblastoma after chemoreduction

Aim: Small heat shock proteins (sHSP) play an important role in the resistance to anticancer drugs. We examined the expression of the sHSP family, HSP27 and α-crystallins, in human retinoblastoma with and without preoperative chemotherapy. Methods: Eighteen enucleated eyes from patients with retinoblastoma were used. Six patients had undergone chemotherapy before enucleation. Formalin-fixed, paraffin-embedded tissue sections were processed for H&E staining and examined by immunohistochemistry using anti-HSP27 and α-crystallins antibodies. Results: Eleven of 12 cases with no history of preoperative chemotherapy showed weakly positive or negative staining for HSP27, whereas six and five cases were strongly positive for αA and αB-crystallin, respectively. In the six cases with a history of chemotherapy, several viable retinoblastoma cells remained. Immunoreactivity for HSP27 and αB-crystallin was strongly detected in the cytoplasm of viable retinoblastoma cells, while αA-crystallin immunoreactivity was less marked. Immunoreactivity for HSP27 was significantly higher in retinoblastoma cases with preoperative chemotherapy than in those without chemotherapy (p<0.0001). In contrast, immunoreactivity for αA-crystallin was significantly lower in cases with chemotherapy than in cases without chemotherapy (p<0.01). Conclusions: HSP27 and αB-crystallin, but not αA-crystallin, were highly expressed in viable tumour cells after chemotherapy, suggesting that HSP27 and αB-crystallin may protect tumour cells from apoptotic signals produced by anticancer drugs in retinoblastoma.

Photoreceptor oxidative damage in sympathetic ophthalmia.

PURPOSE To determine photoreceptor oxidative stress and damage in sympathetic ophthalmia (SO). DESIGN Immunohistologic study. METHODS Eight formalin-fixed and paraffin-embedded human globes with typical histologic features of SO and five age-matched globes without intraocular inflammation (controls) were retrieved from the Doheny Eye Institute ophthalmic pathology files. Deparaffinized sections of the globes were processed to localize tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor-1 (TNF-R1), acrolein, inducible nitric oxide synthase (iNOS), and nitrotyrosine by immunolocalization method. The latter two were localized to photoreceptor mitochondria using anti-cytochrome C antibody. Apoptotic cells were detected by Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) assay and were localized to the site of oxidative stress using antinitrotyrosine antibody. RESULTS Increased expression of TNF-alpha can be seen in the photoreceptor nuclear layer in all SO globes, whereas no such expression was observed in control globes. TNF-R1, iNOS, acrolein, and nitrotyrosine were immunolocalized to the inner segments of the photoreceptors in all SO globes, but only mild focal staining was observed in the control retinas. Both nitrotyrosine and iNOS immunolocalization revealed positive staining restricted primarily to mitochondria at the inner segments of the photoreceptors. Most of the TUNEL-positive cells were detected in the photoreceptors at the site of nitrotyrosine staining. In contrast, the age-matched control globes showed negative results. CONCLUSIONS In SO, photoreceptor mitochondrial oxidative stress occurs in the absence of leukocytic infiltration of the retina and may lead to photoreceptor apoptosis and subsequent vision loss. The oxidative stress seems to be mediated by iNOS and TNF-alpha. The current anti-inflammatory therapy combined with agents that could prevent oxidative stress may prevent photoreceptor damage in SO and may preserve vision.

Expression of alpha-crystallin in retinoblastoma.

OBJECTIVE To examine the expression of alpha-crystallin, a small heat-shock protein family, and apoptosis in retinal neoplastic cells. METHODS Thirteen enucleated globes were included in this study, 1 with retinocytoma and 12 with retinoblastoma. Formalin-fixed paraffin-embedded tissue sections were processed for immunohistochemistry with alpha-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS In the retinocytoma, alphaA-crystallin was expressed in the cytoplasm of all tumor cells, whereas alphaB-crystallin immunoreactivity was only weakly positive. Apoptotic cells were rarely noted in retinocytoma cells; the apoptotic index was 0.29. Examination of the retinoblastoma globes revealed 6 cases (50%) that were strongly positive for alphaA-crystallin. The mean (SD) apoptotic indices in the strongly and weakly positive cases were 3.55 (2.61) and 7.50 (2.61), respectively. The apoptotic index was significantly higher in those cases that were weakly positive for alphaA-crystallin than in those that were strongly positive (P < .05). No correlation was observed between apoptotic index and alphaB-crystallin immunoreactivity, although 50% of retinoblastomas were strongly positive for alphaB-crystallin. CONCLUSIONS The alphaA- and alphaB-crystallins are expressed in retinoblastomas, and alphaA-crystallin expression may prevent apoptosis of neoplastic cells. Clinical Relevance Suppression of alphaA-crystallin may be useful in controlling tumor growth.

Expression of α-Crystallin in Retinoblastoma

OBJECTIVE To examine the expression of alpha-crystallin, a small heat-shock protein family, and apoptosis in retinal neoplastic cells. METHODS Thirteen enucleated globes were included in this study, 1 with retinocytoma and 12 with retinoblastoma. Formalin-fixed paraffin-embedded tissue sections were processed for immunohistochemistry with alpha-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS In the retinocytoma, alphaA-crystallin was expressed in the cytoplasm of all tumor cells, whereas alphaB-crystallin immunoreactivity was only weakly positive. Apoptotic cells were rarely noted in retinocytoma cells; the apoptotic index was 0.29. Examination of the retinoblastoma globes revealed 6 cases (50%) that were strongly positive for alphaA-crystallin. The mean (SD) apoptotic indices in the strongly and weakly positive cases were 3.55 (2.61) and 7.50 (2.61), respectively. The apoptotic index was significantly higher in those cases that were weakly positive for alphaA-crystallin than in those that were strongly positive (P < .05). No correlation was observed between apoptotic index and alphaB-crystallin immunoreactivity, although 50% of retinoblastomas were strongly positive for alphaB-crystallin. CONCLUSIONS The alphaA- and alphaB-crystallins are expressed in retinoblastomas, and alphaA-crystallin expression may prevent apoptosis of neoplastic cells. Clinical Relevance Suppression of alphaA-crystallin may be useful in controlling tumor growth.

Transforming Growth Factor β in Retinoblastoma-Related Cataract

OBJECTIVE To analyze the histopathology and expression of transforming growth factor beta (TGF-beta) in retinoblastoma with and without cataractous changes. METHODS Twenty patients with unilateral retinoblastoma underwent enucleation. None of these patients had received preoperative chemotherapy or radiotherapy. Formalin-fixed, paraffin-embedded tissue sections were examined histologically for the presence of morgagnian globules or liquefaction of lens fibers; TGF-beta was immunolocalized using an anti-TGF-beta antibody. RESULTS Two globes showed several morgagnian globules and liquefaction of the lens fibers, representing cataractous changes. One patient had posterior subcapsular cataract; the other, anterior polar cataract. In both cases, prominent cytoplasmic immunoreactivity for TGF-beta was detected in retinoblastoma cells. In contrast, 3 patients showed histologic evidence of minor cataractous changes. The globes with either minor or no cataractous changes revealed minimal to no expression of TGF-beta. CONCLUSIONS These results suggest that TGF-beta produced by retinoblastoma cells may induce cataract formation. Clinical Relevance The growth factors produced by retinoblastoma cells may lead to associated pathologies, such as cataracts, in the ocular structures. This study implies that when a child presents with a unilateral cataract, retinoblastoma should be excluded as the primary diagnosis.