[Technique for separating Descemet membrane for corneal endothelial cells transplantation: experimental study in rabbits]. / Técnica de separação da membrana de Descemet para transplante de células endoteliais da córnea: estudo experimental em coelhos.

PURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemet's membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS: The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06%, the Group two showed an index of 3.58% and the control group an index of 0.18% of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 microm thickness of stroma remained attached to the Descemet's membrane. CONCLUSION: This technique should be better investigated because it is a viable and efficient alternative of Descemet's membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06%. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58%.

Técnica de separação da membrana de Descemet para transplante de células endoteliais da córnea: estudo experimental em coelhos / Technique for separating Descemet membrane for corneal endothelial cells transplantation: experimental study in rabbits

OBJETIVO: Avaliar a porcentagem de dano endotelial induzido por uma técnica cirúrgica para a separação da membrana de Descemet contendo endotélio sadio, analisar a viabilidade e eficácia desta técnica, e avaliar a porcentagem de dano endotelial causado pela inversão da córnea em câmara anterior artificial. MÉTODOS: As córneas de três grupos de 12 coelhos da linhagem Nova Zelândia foram avaliadas. O grupo 1 foi usado como controle; portanto, as córneas foram analisadas após coletadas e trepanadas. O grupo 2 foi analisado após a inversão da córnea (endotélio para cima na posição convexa), montada em câmara anterior artificial, para o cálculo da porcentagem do dano endotelial induzido por esta inversão. O grupo 3 foi avaliado após a separação entre a membrana de Descemet e o estroma com o uso de substância viscoelástica em córneas invertidas e montadas em câmara anterior artificial. O dano endotelial foi avaliado por meio de fotografias digitais tiradas no microscópio após impregnar o endotélio com vermelho de alizarina. Amostras do grupo 3 foram processadas para avaliação histopatológica. RESULTADOS: O grupo 3 (separação viscoelástica) apresentou um índice de lesão celular endotelial de 10,06 por cento, o grupo 2 apresentou um índice de 3,58 por cento e o grupo controle um índice de 0,18 por cento de lesão celular endotelial (p<0,05). A avaliação histológica das córneas do grupo 3 revelou que aproximadamente 120 µm de espessura estromal manteve-se aderido à membrana de Descemet. CONCLUSÃO: Esta técnica deve ser melhor investigada, pois é uma alternativa viável e eficaz de separação da membrana de Descemet para transplante celular endotelial, já que o porcentual de dano celular induzido é de 10,06 por cento. A porcentagem de dano celular causado pela inversão da córnea em câmara anterior artificial foi de 3,58 por cento.

PURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex shape) mounted on an artificial anterior chamber to calculate the percentage of endothelial cell damage caused by this inversion. The Group three was evaluated after the separation between the Descemet's membrane and the stroma using viscoelastic substance in corneas inverted and mounted on an artificial anterior chamber. The endothelial cell damage was analyzed by digital photographs taken under a microscope after staining the endothelium with alizarin red. Group three samples were processed for histologic evaluation. RESULTS: The Group three (viscoelastic separation) showed an index of endothelial cell damage of 10.06 percent, the Group two showed an index of 3.58 percent and the control group an index of 0.18 percent of endothelial cell damage (p<0.05). Histological evaluation of the Group three corneas revealed that approximately a 120 µm thickness of stroma remained attached to the Descemet's membrane. CONCLUSION: This technique should be better investigated because it is a viable and efficient alternative of Descemet's membrane separation for endothelial cells transplantation, since the percentage of induced cell damage is 10.06 percent. The percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber was 3.58 percent.

Visual Arrestin 1 contributes to cone photoreceptor survival and light adaptation.

PURPOSE: To evaluate morphologic and functional contributions of Arrestin 1 (Arr1) and Arrestin 4 (Arr4) in cone photoreceptors, the authors examined the phenotypes of visual arrestin knockout mice (Arr1(-/-), Arr4(-/-), Arr1(-/-)Arr4(-/-) [Arr-DKO]) reared in darkness. METHODS: Retinal rods and cones were evaluated in wild-type (WT), Arr1(-/-), Arr4(-/-), and Arr-DKO mice using quantitative morphologic analysis, immunoblot, immunohistochemistry, TUNEL, and electroretinographic (ERG) techniques. RESULTS: Compared with either Arr4(-/-) or WT, Arr1(-/-) and Arr-DKO mice had increased apoptotic nuclei in their retinal outer nuclear layer (ONL) at postnatal day (P) 22. By P60, cone density was significantly diminished, but the ONL appeared normal. After 1 minute of background illumination, cone ERG b-wave amplitudes were similar in WT and all Arr KO mice. However, by 3 minutes and continuing through 15 minutes of light adaptation, the cone b-wave amplitudes of WT and Arr4(-/-) mice increased significantly over those of the Arr1(-/-) and Arr-DKO mice, which demonstrated no cone b-wave amplitude increase. In contrast, ERG flicker analysis after the 15-minute light adaptation period demonstrated no loss in amplitude for either Arr1(-/-) or Arr4(-/-) mice, whereas Arr-DKO had significantly lower amplitudes. When Arr1 expression was restored in Arr1(-/-) mice (+p48(Arr1-/-)), normal cone density and light-adapted ERG b-wave amplitudes were observed. CONCLUSIONS: In the adult dark-reared Arr1(-/-) and Arr-DKO mice, viable cones diminish over time. Arr1 expression is essential for cone photoreceptor survival and light adaptation, whereas either Arr1 or Arr4 is necessary for maintaining normal flicker responses.

A novel, local technique for studying rabbit lacrimal gland secretion in situ.

AIM: To develop a local approach to study rabbit lacrimal secretion in situ by administering specific secretagogues directly onto the lacrimal gland (LG). METHODS: After the rabbit has been anesthetized, the inferior bulbar conjunctiva and underlying connective tissue are blunt dissected. A polyethylene tube, for drug delivery, is inserted through the fibrous membrane overlying the inferior surface of the orbital cavity beneath which the LG resides. Lacrimal fluid is collected by cannulating the lacrimal duct. RESULTS: Pilocarpine induced robust lacrimal fluid secretion from the LG that had been bathed with either pilocarpine or phenylephrine, with no detectable effect on the contralateral LG and salivary secretion. By next giving pilocarpine or phenylephrine to the control LG while the experimental gland used before served as control, we duplicated the results exactly. CONCLUSION: This novel approach avoids the unwanted systemic effects of intravenous injection and is a promising technique to study rabbit LG secretion in situ.

Technique for preparation of the corneal endothelium-Descemet membrane complex for transplantation.

PURPOSE: Replacing diseased corneal endothelium with a preparation of Descemet membrane carrying functional endothelium and no stroma may be a feasible method for treating corneal endothelial decompensation. To obtain a viable donor of a Descemet membrane endothelium disc, we modified the Descemet membrane stripping technique and monitored the percentage of endothelial damage to the donor tissue preparation. METHODS: Forty-eight human corneas were used. Cornea buttons were mounted on an artificial anterior chamber, endothelial side up. Endothelia were stained with alizarin red, examined under the microscope, and photographed at 5 different sites (microscope, x100; digital magnification, x2.83). A 6 x 7-mm rectangular piece of endothelium-Descemet membrane complex was obtained using a Grieshaber microsurgical knife and Kelman-McPherson forceps. Digital photographs of endothelia were analyzed with a computer, and the percentage of endothelial damage was calculated. Specimens were processed for hematoxylin-eosin staining. RESULTS: Forty of 48 endothelium-Descemet membrane preparations (83.3%) were complete peels with minimal endothelial damage. Endothelial damage before and after the surgery was 1.57 +/- 2.11% and 2.61 +/- 1.77%, respectively. Eight preparations (16.7%) failed because of tearing. Multiple hematoxylin-eosin-stained sections showed the presence of endothelium with intact Descemet membrane and no stromal tissue. CONCLUSION: We modified the technique of Melles and obtained a sheet of Descemet membrane and endothelium with minimal endothelial damage and with no remaining stroma observed. This simple technique can be used to obtain the endothelium-Descemet membrane complex in minutes. It may be useful for corneal endothelium transplantation.

Carboxypeptidase E is required for normal synaptic transmission from photoreceptors to the inner retina.

Defects in the gene encoding carboxypeptidase E (CPE) in either mouse or human lead to multiple endocrine disorders, including obesity and diabetes. Recent studies on Cpe-/- mice indicated neurological deficits in these animals. As a model system to study the potential role of CPE in neurophysiology, we carried out electroretinography (ERG) and retinal morphological studies on Cpe-/- and Cpe fat/fat mutant mice. Normal retinal morphology was observed by light microscopy in both Cpe-/- and Cpe(fat/fat) mice. However, with increasing age, abnormal retinal function was revealed by ERG. Both Cpe-/- and Cpe fat/fat animals had progressively reduced ERG response sensitivity, decreased b-wave amplitude and delayed implicit time with age, while maintaining a normal a-wave amplitude. Immunohistochemical staining showed specific localization of CPE in photoreceptor synaptic terminals in wild-type (WT) mice, but in both Cpe-/- and Cpe fat/fat mice, CPE was absent in this layer. Bipolar cell morphology and distribution were normal in these mutant mice. Electron microscopy of retinas from Cpe fat/fat mice revealed significantly reduced spherule size, but normal synaptic ribbons and synaptic vesicle density, implicating a reduction in total number of vesicles per synapse in the photoreceptors of these animals. These results suggest that CPE is required for normal-sized photoreceptor synaptic terminal and normal signal transmission to the inner retina.