RESUMO
IMPORTANCE: Transient corneal endothelial changes are routinely noted on slit-lamp examination immediately following selective laser trabeculoplasty (SLT). BACKGROUND: To determine the mechanism of transient corneal endothelial changes observed following SLT. DESIGN: University laboratory-based observational study. SAMPLES: Ten corneas from six human cadaveric donors. METHODS: Corneas were treated with SLT, direct laser or peroxide, or used as controls. Haematoxylin and eosin staining and immunolabelling for zonula occludens-1 (ZO-1) and beta-catenin were performed. MAIN OUTCOME MEASURES: Histological appearance; ZO-1 and beta-catenin immunostaining. RESULTS: There were no differences in histological features between SLT-treated and control corneas. Corneas treated with SLT or peroxide showed reduced and less regular ZO-1 immunofluorescence along cell membranes compared with ZO-1 expression in controls. These changes were generalized across the endothelium. There was no effect on the ZO-1 immunostaining after direct laser. There was no difference in beta-catenin immunostaining patterns between control, SLT and peroxide-treated corneas. CONCLUSIONS AND RELEVANCE: Altered ZO-1 immunostaining may represent disassembly of tight junctions between corneal endothelial cells. The similarity of our findings between SLT-treated and peroxide-treated corneas suggests that both conditions trigger changes at the level of endothelial tight junctions, perhaps triggered by liberation of free radicals as previously proposed.
Assuntos
Endotélio Corneano/patologia , Glaucoma de Ângulo Aberto/cirurgia , Pressão Intraocular/fisiologia , Terapia a Laser/métodos , Trabeculectomia/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Feminino , Glaucoma de Ângulo Aberto/diagnóstico , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Adulto JovemRESUMO
PURPOSE: Conjunctival autografting is a technically difficult step of pterygium surgery, and novice surgeons have limited opportunities to develop and practice their surgical skills. The porcine eye model closely approximates the human eye in tissue consistency when preparing conjunctival and limbal-conjunctival grafts. This study assessed the efficacy of a cadaveric porcine model in teaching and improving a novice's skills of conjunctival autograft creation. METHODS: A novice was taught to prepare 5 × 5 mm conjunctival grafts and created 58 grafts on fresh porcine eyes. The conjunctival graft thickness was measured using standard histological techniques. RESULTS: Between grafts 1-10 and grafts 49-58, there was a statistically significant difference in both thickness (P < 0.0001; mean thickness, 133 ± 27 and 87 ± 23 µm, respectively) and time of creation (P = 0.037; median time, 191 and 126 seconds, respectively). CONCLUSIONS: The cadaveric porcine model may be a useful method for teaching this important technique to novice surgeons.
Assuntos
Túnica Conjuntiva/transplante , Modelos Animais , Procedimentos Cirúrgicos Oftalmológicos/educação , Pterígio/cirurgia , Ensino , Transplante Autólogo/educação , Animais , Autoenxertos , Educação de Pós-Graduação em Medicina , Feminino , Internato e Residência , Masculino , Duração da Cirurgia , Retalhos Cirúrgicos , Técnicas de Sutura , SuínosRESUMO
PURPOSE: The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. METHODS: We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). RESULTS: Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. CONCLUSION: This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.
Assuntos
Olho/metabolismo , Sistema Renina-Angiotensina/fisiologia , Idoso , Angiotensinogênio/biossíntese , Linhagem Celular , Feminino , Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/biossíntese , Receptor Tipo 1 de Angiotensina/biossíntese , Receptores de Superfície Celular , Renina/biossíntese , Retina/metabolismo , Úvea/metabolismo , Receptor de Pró-ReninaRESUMO
PURPOSE: To describe findings of light microscopic examination of trabeculectomy blebs. METHODS: Histologic and immunohistochemical features of blebs including cell types, and distribution of apoptotic cells and proapoptotic death messengers were examined in six eyes 6.0 to 25.9 years after trabeculectomy. RESULTS: All specimens showed a channel opening into a rectangular cleft in the middle layer of sclera. The channels showed degenerative changes with disintegration and loss of collagen fibers and few pyknotic cells. Changes were extensive on the upper surface of the cleft forming irregular channels extending through and around the edges of the superficial scleral flap into moderately cellular and vascular oedematous conjunctiva covered by intact epithelium. Immunohistochemical staining demonstrated migration of histiocytes from superficial blood vessels into the deeper layers where they apoptosed and disintegrated. Fas ligand+ proapoptotic death messengers were concentrated in the superficial conjunctival regions of blebs. CONCLUSIONS: These results indicated that the normal cell processes in functioning trabeculectomy blebs were similar to that of Molteno implant capsules. These processes involved migration of cells from superficial blood vessels into perivascular spaces and the deeper tissues where apoptosis occurred. Apoptosis was associated with breakdown of tissue matrix and release of proapoptotic death messengers that were transported by aqueous to the superficial layers where they suppressed collagen synthesis by inducing apoptosis in metabolically active cells.
Assuntos
Vesícula/metabolismo , Vesícula/patologia , Implantes para Drenagem de Glaucoma , Glaucoma/cirurgia , Trabeculectomia/métodos , Biomarcadores/metabolismo , Túnica Conjuntiva/cirurgia , Glaucoma/metabolismo , Glaucoma/patologia , Humanos , Imuno-Histoquímica , Limbo da Córnea/cirurgia , Proteínas Nucleares/metabolismoRESUMO
PURPOSE: To determine putative limbal epithelial stem cell marker expression in human limbal dermoids compared to stem cell niches in normal limbus and hair follicles of normal human dermis. METHODS: Human limbal dermoids (n = 7), normal skin (n = 2) and normal limbal (n = 7) tissue were examined. Immunohistochemistry was performed on paraffin embedded specimens using automated and manual immunostaining with primary antibodies to CK15, CK14, Cadherin-P (CDH3), Wnt-3, Wnt-4, Wnt-5a, Dickkopf (DKK)-3, Sox-2, Sox-10, Sox-13, PEDF, NGFR p75 and ß-catenin. RESULTS: Positive immunostaining was found for CK15, CK14, CDH3, NGFR p75, PEDF, Sox-2, Sox-10 and Wnt 4 in the basal dermoid epithelium, limbus and hair follicles. Suprabasal epithelium was immunostained with PEDF, Sox-2 and Wnt-4 in these tissues. The sebaceous and sweat glands, vascular endothelium and nerves of the limbal dermoid immunostained with PEDF and Sox-2. Sebaceous and sweat glands stained for Sox-10. DKK-3 immunostaining occurred in the dermoids' suprabasal epithelium and vascular endothelium but not in the limbus or hair follicle. CONCLUSION: Human limbal dermoids share a similar antigenic expression profile similar to the basal limbal epithelium and to the stem cell niche of hair follicles. This supports the notion that limbal dermoids could have properties in common with limbal and/or dermal epithelial stem cells.
Assuntos
Cisto Dermoide/patologia , Epitélio Corneano/citologia , Neoplasias Oculares/patologia , Cabelo/citologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Antígenos/metabolismo , Biomarcadores/metabolismo , Cisto Dermoide/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Neoplasias Oculares/metabolismo , Cabelo/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Imuno-Histoquímica , Limbo da Córnea/metabolismo , Fenótipo , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
CXCR3 and its ligands are important for the trafficking of activated CD4(+) T(H)1 T cells, CD8(+) T cells, and natural killer cells during inflammation. Recent functional studies demonstrate a more diverse role of CXCR3 in inflammatory diseases of the central nervous system (CNS). We examined the impact of CXCR3 on a less complex interferon-γ-dependent, type 1 cell-mediated immune response in the CNS, induced in mice by the transgenic production of glial fibrillary acidic protein IL-12 (GF-IL12) by astrocytes and retinal Müller cells. GF-IL12 mice develop ataxia because of severe cerebellar inflammation but have little overt ocular disease. Surprisingly, CXCR3-deficient GF-IL12 mice (GF-IL12/CXCR3KO) have drastically reduced ataxia but developed cataracts, severe ocular inflammation, and eye atrophy. Most GF-IL12/CXCR3KO mice had minimal cerebellar inflammation but severe retinal disorganization, loss of photoreceptors, and lens destruction in the eye. The number of CD3(+), CD11b(+), and natural killer 1.1(+) cells were reduced in the CNS but highly increased in the eyes of GF-IL12/CXCR3KO compared with GF-IL12 mice. High levels of interferon-γ, IL-1, tumor necrosis factor α, CXCL9, CXCL10, and CCL5 were found in GF-IL12 cerebelli and GF-IL12/CXCR3KO eyes. Our findings demonstrate key but paradoxical functions for CXCR3 in IL-12-induced immune disease in the CNS, promoting inflammation in the brain yet restricting it in the eye. We conclude that the function of CXCR3 in cellular immune disease is driven by a common trigger and is controlled by tissue-specific factors.
Assuntos
Astrócitos/metabolismo , Ataxia Cerebelar/imunologia , Oftalmopatias/imunologia , Imunidade Celular/imunologia , Interleucina-12/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Receptores CXCR3/deficiência , Animais , Cegueira/imunologia , Ataxia Cerebelar/patologia , Encefalite/imunologia , Encefalite/patologia , Endoftalmite/imunologia , Endoftalmite/patologia , Oftalmopatias/patologia , Genótipo , Proteína Glial Fibrilar Ácida , Interferon gama/metabolismo , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Receptores de Interleucina-12/metabolismo , Fator de Transcrição STAT4/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND OBJECTIVES: To demonstrate the feasibility of a novel, thin film, laser-activated adhesive in sealing penetrative corneal wounds with a view to replacing sutures in ophthalmic operations. METHODS: A previously described thin film adhesive composed of chitosan and indocyanine green activated by infrared laser (808 nm) was used to seal penetrating corneal wounds ranging from 1 to 6 mm in size in enucleated bovine eyes. The excised corneas were subjected to pressure tests to evaluate the strength of the corneal repairs and compared to sutures and commercial fibrin glue, Tisseel®. Temperatures at the adhesive-tissue interface were measured and histological examinations of the repairs performed to investigate potential tissue damage. Biodegradability of the films was monitored in lysozyme solutions at concentrations reported in tears. RESULTS: The adhesive effectively sealed corneal wounds, withstanding pressures of 140-320 mmHg, far in excess of the normal intraocular pressure. In contrast, pressures of 40-80 mm Hg were determined using a combination of sutures with Tisseel® as a sealant. The laser-activation process was 1.5-5 times faster than other procedures studied and required no curing time. A transient, mean temperature of 56 ± 2°C was measured at the adhesive-tissue interface while histology showed no tissue damage as a consequence of the irradiation process. Irradiation had no significant influence on adhesive biodegradation in vitro, which lost approximately 30% of their initial weight in a lysozyme solution (6 mg ml(-1)). CONCLUSIONS: The thin film adhesive was found to be an effective in sealing corneal wounds with considerable advantages over sutures, including speed of application and sealing strength and biodegradability.
Assuntos
Quitosana , Lesões da Córnea , Perfuração da Córnea/terapia , Verde de Indocianina , Terapia a Laser , Adesivos Teciduais , Animais , Bovinos , Estudos de ViabilidadeRESUMO
OBJECTIVE: The anastomosis of peripheral nerves is a demanding procedure that has potential complications due to foreign body reactions elicited by sutures. In this study, the sutureless in vivo anastomosis of rat tibial nerves was successfully performed, using for the first time a chitosan-based laser-activated adhesive. The nerve thermal damage caused by the laser irradiation was quantitatively assessed. MATERIALS AND METHODS: A novel adhesive composed of chitosan, indocyanine green, acetic acid, and water, was fabricated in thin sheets. Its adhesive strength was tested in vitro by bonding strips (surface area approximately 20 mm2, thickness approximately 20 microm) onto rat sciatic nerves and sheep intestine by laser activation with low fluence ( approximately 50 J/cm2), using a fiber-coupled diode laser (n = 13). The tensile strength of the adhesive/tissue bonds was measured after tissue repair. The chitosan adhesive was then used to perform sutureless anastomosis of tibial nerves in vivo (n = 6). Adhesive strips were also bonded in vivo onto intact rat sciatic nerves (n = 6) in order to quantitatively assess, by counting myelinated axons, the thermal damage induced by the laser. RESULTS: The adhesive bonded well to tissue with a tensile strength of 12.5 +/- 2.6 KPa (mean +/- SD; n = 13). The in vivo anastomosed nerves were in continuity 3 d after surgery. Axon counting showed the number and morphology of myelinated axons were normal proximally ( approximately 96%) compared with intact nerves (100%). Axon demyelination was observed at the operation site ( approximately 49%) and distally ( approximately 27%), and was attributed to laser-induced thermal damage. CONCLUSIONS: Nerve anastomosis, performed by the laser-adhesive procedure, was successful 3 d postoperatively. Proximal myelinated axons were not significantly damaged by the low laser fluence.
Assuntos
Quitosana/farmacologia , Quitosana/efeitos da radiação , Lasers , Nervos Periféricos/cirurgia , Adesivos Teciduais/farmacologia , Adesivos Teciduais/efeitos da radiação , Animais , Projetos Piloto , Ratos , Nervo Isquiático/fisiologia , Ovinos , Resistência à TraçãoRESUMO
Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n=5) and foetal (n=3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n=10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n=4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.
Assuntos
Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Receptor de Fator de Crescimento Neural/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Células Epidérmicas , Epiderme/metabolismo , Células Epiteliais/citologia , Epitélio Corneano/metabolismo , Feminino , Feto/citologia , Feto/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Neural/metabolismo , Transporte Proteico , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
We describe mutations in the PML nuclear body protein Sp110 in the syndrome veno-occlusive disease with immunodeficiency, an autosomal recessive disorder of severe hypogammaglobulinemia, combined T and B cell immunodeficiency, absent lymph node germinal centers, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusive disease.
Assuntos
Hepatopatia Veno-Oclusiva/genética , Síndromes de Imunodeficiência/genética , Mutação , Proteínas Nucleares/genética , Feminino , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , LinhagemRESUMO
The nm23 gene is a putative tumour and metastasis suppressor gene. A number of studies have found that reduction of its expression is associated with increased metastatic potential in several human malignancies. Similarly, clinical studies have shown correlation between reduced nm23 protein expression and a poor prognosis. The aim of this study was to determine if a relationship exists between nm23 expression in primary cutaneous melanomas with or without cerebral metastases. Paraffin-embedded tissues were retrieved from 21 patients with primary cutaneous melanomas (n=21) and who subsequently developed cerebral metastases (n=24). Primary cutaneous melanomas with no regional or organ metastases within a 10 year period were used as control cases (n=34). Nm23 staining was localized in the cytoplasm of the tumour cells. Most of the control cases showed strong expression, whereas the majority of the primary melanomas with cerebral metastases showed no or weak expression of nm23. The cerebral metastases mostly showed strong expression. In summary, the results of this study may have significant prognostic implications for patients presenting with cutaneous melanoma. Patients with cutaneous melanomas with a low expression of nm23 appear to be more at risk of developing brain metastases.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/secundário , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Neoplasias Cutâneas/metabolismo , Citoplasma/metabolismo , Feminino , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Nucleosídeo NM23 Difosfato Quinases , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Neoplasias Cutâneas/patologiaRESUMO
AIM: Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma. METHODS: Paraffin-embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n=46), some of which were accompanied by tissue from areas with high-grade dysplasia (n=24) and from metastasis in regional lymph nodes (n=16) were studied. Histologically normal oesophageal glandular tissue (of cardiac-type) (n=32) obtained from areas of the resections located away from the primary tumour masses and archival tissues of Barrett's metaplasia obtained from endoscopic biopsies (n=77) were used as non-neoplastic controls. Sections were immunohistochemically stained by the labelled streptavidin-biotin method using NCL-nm23 antibody. RESULTS: The total or overall amount of nm23 protein expression paralleled that of cytoplasmic expression and was increased in oesophageal adenocarcinomas (36/46 cases, 78%) when compared with normal oesophageal glandular epithelium (2/32, 6%), Barrett's metaplasia (8/77, 10%) and dysplasia (14/24, 58%). In metastatic carcinoma in regional lymph nodes, overall nm23 expression was similar in proportion (13/16, 81%) to that seen in primary carcinoma. In the analysis of the sequential development of oesophageal adenocarcinoma based on non-neoplastic, preneoplastic and neoplastic archival material, it was found that a high level of overall nm23 expression occurred firstly at the transition from Barrett's metaplasia (8/77, 10%) to dysplasia (14/24, 58%). Nuclear nm23 expression was low in dysplastic tissue (with none of the cases having a high level of nuclear nm23 expression) followed by increased levels as the lesion progressed to invasive adenocarcinoma (13/46, 28%) and metastatic carcinoma in regional lymph nodes (10/16, 63%). However, nm23 expression did not appear to correlate with sex, age, tumour size, extent of tumour infiltration into the oesophageal wall, presence of lymph node metastasis or overall patient survival. CONCLUSION: An overall increase in nm23 expression or increase in nm23 expression in the cytoplasm of cells may be important in the early development of oesophageal adenocarcinoma but increased levels of nuclear nm23 occur in its progression to metastatic disease.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/metabolismo , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Idoso , Progressão da Doença , Intervalo Livre de Doença , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/cirurgia , Feminino , Humanos , Técnicas Imunoenzimáticas , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Nucleosídeo NM23 Difosfato QuinasesRESUMO
Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system (CNS) characterized by demyelination associated with progressive disability. The mechanisms underlying the pathogenesis of MS remain a mystery. The highly pleiotropic syndrome known as ataxia telangiectasia (A-T) overlaps with MS in that it also presents with demyelination in the CNS. Whether demyelination in MS or in A-T is initiated through neuronal degeneration or immune dysfunction is not yet known. However, unlike MS, the underlying cause of A-T is known to result from mutations in the A-T gene (ATM) that often result in the complete loss of ATM protein and loss/gain of function. ATM is implicated in neurological degeneration, particularly in the cerebellum, cellular apoptosis, immunodeficiency, double stranded deoxyribonucleic acid (DNA) rejoining, VDJ antibody recombination, tumour suppression, particularly T-lymphoid malignancies, signal transduction, cell-cycle control and cellular radiohypersensitivity. In this study, we describe a case of MS in a family with cellular radiosensitivity and abnormally low postinduction levels of the ATM protein. Defective DNA repair/rejoining may impact on autoimmunity.