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Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

ABSTRACT: Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head.

SUBMITTER: Kimball EC

PROVIDER: S-EPMC9202842 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

Kimball Elizabeth C EC Quillen Sarah S Pease Mary E ME Keuthan Casey C Nagalingam Aru A Zack Donald J DJ Johnson Thomas V TV Quigley Harry A HA

PloS one 20220616 6

Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, ...[more]

PMID: 35709078

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Project description:Purpose: Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. The authors report the first study of global and ECM-focused gene transcription differentials between GFAP-negative negative lamina cribrosa (LC) cells from normal and POAG human donors. Methods: GFAP-negative LC cell lines were generated from the optic nerve tissue of three normal (n=3) and three POAG (n=3) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was carried out using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data. Results: 285 genes were up regulated by greater than 1.5 fold and 413 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, SPARC, periostin, thrombospondin, CRTL-1, CTGF and collagen types I, III, V and VIII. Downregulated ECM genes in POAG included MMP-1, fibulin, decorin and tenacsin XB. All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells. Conclusions: This study reports for the first time that POAG LC cells in-vitro demonstrate up regulated ECM and pro-fibrotic gene expression compared with normal LC cells. This may be a pathological characteristic of this cell type in POAG in-vivo. We believe that the LC cell may be a pivotal regulator of optic nerve head ECM remodeling and an attractive target for future therapeutic strategies in POAG.

Project description:PURPOSE: Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. We report the first study of global and ECM-focused gene transcription differentials between GFAP-negative lamina cribrosa (LC) cells from normal and POAG human donors. METHODS: GFAP-negative LC cell lines were generated from the optic nerve tissue of four normal (n=4) and four POAG (n=4) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was performed using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data. RESULTS: 183 genes were upregulated by greater than 1.5 fold and 220 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, transforming growth factor beta 1 (TGFbeta1), secreted acid protein cysteine rich (SPARC), periostin (POSTN), thrombospondin-1 (THBS1), cartilage linking protein-1 (CRTL-1), and collagen type I (COL1A1), collagen type V (COL5A1), and collagen type XI (COL11A1). Downregulated ECM genes in POAG included fibulin 1 (FBLN1), decorin (DCN), and collagen type XVIII (COL18A1). All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells. CONCLUSIONS: This study reports for the first time that POAG LC cells in-vitro demonstrate upregulated ECM and pro-fibrotic gene expression compared with normal LC cells. This may be a pathological characteristic of this cell type in POAG in-vivo. We believe that the LC cell may be a pivotal regulator of optic nerve head ECM remodeling in POAG and an attractive target for molecular therapeutic strategies in the future.

Project description:Glaucoma is among the leading causes of blindness worldwide that is characterized by irreversible damage to the retinal ganglion cell axons in the lamina cribrosa (LC) region of the optic nerve head (ONH), most often associated with elevated intraocular pressure (IOP). The LC is a porous, connective tissue structure that provides mechanical support to the axons as they exit the eye and the biomechanics of the LC microstructure likely play a crucial role in protecting the axons passing through it. There is a limited knowledge of the IOP-driven biomechanics of the LC microstructure, primarily due to its small size and the difficulty with imaging the LC both in vitro and in vivo. We present finite element (FE) models of three human eye posterior poles that include the LC microstructure and interspersed neural tissues (NT) composed of retinal axons that are constructed directly from segmented, binary images of the LC. These models were used to estimate the stresses and strains in the LC and NT for an acute IOP elevation from 0 to 45 mmHg and compared with identical models except that the LC was represented as a homogenized continuum material with either homogeneous isotropic neo-Hookean properties or heterogeneous properties derived from local connective tissue volume fraction (CTVF) and predominant LC beam orientation. Stresses and strains in the LC and NT microstructure were investigated, and results were compared against those from the models wherein the LC was represented as a homogenized continuum. The regionalized volumetric average stresses and strains showed that the microstructural model yielded similar patterns to our prior approach using an LC continuum representation with mapped LC CTVF/anisotropy, but the microstructural modeling approach allows analysis of the stresses and strains in the LC and NT separately. As expected, the LC beams carried most of the IOP load in the microstructural models but exhibited less strain, while the encapsulated NT exhibited lower stresses and much higher strains. Results also revealed that the continuum models underestimate the maximum strains in the LC beams and NT by a factor of 2-3. Microstructural modeling should provide greater insight into the biomechanical factors driving damage to the axons (NT) and LC connective tissue remodeling that occur in glaucoma. The methods presented are ideal for modeling any structure with a complex microstructure composed of different materials, such as trabecular bone, lung, and tissue engineering scaffolds such as decellularized LC. Matlab code for mesh generation from a segmented image stack of the microstructure is included as Supplemental Material. STATEMENT OF SIGNIFICANCE: Glaucoma is among the leading causes of blindness worldwide that is characterized by axon damage in the lamina cribrosa (LC) region of the eye. We present a new approach for finite element modeling the entire eye-specific 3D LC microstructure and the interspersed neural tissues, incorporated into an eye-specific posterior eye model that provides appropriate boundary and loading conditions. Results are presented for three human donor eyes, showing that prior modeling approaches underestimate the stresses and strains in the laminar microstructure. We constructed models from image stacks of the segmented microstructure (Matlab code included) using an approach that is ideal for modeling any structure with a complex microstructure composed of different materials, such as trabecular bone, lung, and tissue engineering scaffolds.

Dataset Information

Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

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Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

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