A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes

Ing, Eliesa; Lozano, Diana C; Cepurna, William O.; Chan, Fountane; Yang, Yue; Morrison, John C.; Keller, Kate E.

doi:10.1016/j.exer.2022.109367

Cited by 3 publications

(6 citation statements)

References 43 publications

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“…TM and ONH dissection occurred as described in previous publications. 27, 28, 37 Following dissection, the tissues were placed in individual tubes with 200 ul TriZol and RNA was isolated as described. 28 The NanodropOne (ThermoScientific, Waltham, MA) was used to measure RNA concentration.…”

Section: Methodsmentioning

confidence: 99%

“…27, 28, 37 Following dissection, the tissues were placed in individual tubes with 200 ul TriZol and RNA was isolated as described. 28 The NanodropOne (ThermoScientific, Waltham, MA) was used to measure RNA concentration. RNA was isolated and mean RNA concentration was determined to be 61 ng/TM and 60 ng/ONH.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, we have recently described a method to microdissect the TM from rat eyes and isolated sufficient RNA for gene expression analysis. 28 This produced relatively pure TM that was not substantially contaminated with ciliary body tissue, as defined by quantitative PCR using biomarkers of the ciliary body. With these advances we are now able to investigate IOP-related gene response in the TM in a live rodent model for the first time.…”

Section: Introductionmentioning

confidence: 99%

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Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

Lozano,

Yang,

Cepurna

et al. 2024

Preprint

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Purpose: The rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naive animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the 'CEI-50 vs. CEI-20' and 'CEI-50 vs. naive' comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGFβ pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the 'CEI-50 vs. naive' comparison. Pathway analysis identified 'defense response' and 'immune response' as two significantly upregulated biological process pathways. Conclusions: This study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFβ pathway genes suggest that TM responses may prevent TGFβ-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

Lozano,

Yang,

Cepurna

et al. 2024

Preprint

Self Cite

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show abstract

“…TM and ONH dissection occurred as described in previous publications. 27 , 30 , 39 Following dissection, the tissues were placed in individual tubes with 200 µL Invitrogen TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), and RNA was isolated as described. 30 The NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to measure RNA concentration.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, we have recently described a method to microdissect TM from rat eyes and isolated sufficient RNA for gene expression analysis. 30 This produced relatively pure TM that was not substantially contaminated with ciliary body tissue, as defined by qPCR using biomarkers of the ciliary body. 30 With these advances, we are now able to investigate IOP-related gene response in the TM in a live rodent model for the first time.…”

mentioning

confidence: 99%

Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure

Lozano,

Yang,

Cepurna

et al. 2024

Invest. Ophthalmol. Vis. Sci.

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The rat controlled elevation of intraocular pressure (CEI) model allows study of in vivo responses to short-term exposure to defined intraocular pressures (IOP). In this study, we used NanoString technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. METHODS.Male and female rats (N = 35) were subjected to CEI for 8 hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5× IOP (CEI-50). Naïve animals that received no anesthesia or surgical interventions served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA was isolated, and samples were analyzed with a NanoString panel containing 770 genes. Postprocessing, raw count data were uploaded to ROSALIND for differential gene expression analyses. RESULTS.For the TM, 45 IOP-related genes were significant in the CEI-50 versus CEI-20 and CEI-50 versus naïve comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and transforming growth factor beta (TGFβ) pathways to be the most up-and downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. For ONH, 22 significantly differentially regulated genes were identified in the CEI-50 versus naïve comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. CONCLUSIONS.This study demonstrated the ability to assay short-term IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFβ pathway genes suggests that TM responses may reduce TGFβ-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

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The Trabecular Meshwork

Mao

2024

Reference Module in Neuroscience and Biobehavioral Psychology

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A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes

Cited by 3 publications

References 43 publications

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure

The Trabecular Meshwork

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