Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Summary

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Abstract

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Introduction

Glaucoma is a group of diseases characterized by optic neuropathy and retinopathy that ultimately leads to irreversible blindness^1,2. It is estimated that by 2020 over 70 million people worldwide will be living with some form of the disease^3,4,5,6,7. Primary open angle glaucoma (POAG), the most prevalent type of glaucoma, is characterized by a decrease in aqueous humor (AH) outflow leading to increased intraocular pressure (IOP)^{8,9,10,11,12,13,143,15,16,17,18}. Left untreated, chronically elevated IOP leads to progressive and irreversible damage to the retina and optic nerve head causing radial blindness^1,2,19. All current methods for slowing the progression of glaucoma focus on reducing IOP, either by decreasing the rate of production of AH by the ciliary body or enhancing it's outflow^{1,8,9,10,11,12,13,14}. The trabecular meshwork (TM) plays a vital role in actively regulating the primary AH outflow pathway and its improper function is a causative factor for hypertensive glaucoma^1,2,19. However, the molecular mechanisms associated with TM dysfunction and how it regulates AH drainage are not yet fully understood and is currently a major focus of glaucoma research^1,2,19,20. While several genome-wide association studies (GWAS) have linked a number of genes to glaucoma and increased resistance to AH outflow facility at the TM, the exact molecular mechanisms that lead to disease are not yet fully understood^{21,22,23,24,25}.

Animal models have greatly enhanced our current knowledge of disease progression in glaucoma (extensively reviewed in^{3,15,16,26,27,28,29,30,31,32,33}). Several pioneering methods have been developed to study the TM^34,35,36 and these methods have been widely used to advance our current understanding of normal and diseased tissue. One area that has not been extensively explored is the use of genetically modified mouse models to study the molecular mechanisms of TM failure. Transgenic knock-in and knock-out mouse studies of TM associated genes, such as Myocilin (Myoc)^37,38 and Cyp1b1³⁹, have been the primary tools for studying the molecular mechanisms of TM function. Understandably, the small size of the TM in mice represents a serious hurdle that must be overcome in order to begin to study this tissue. Mouse models represent a powerful tool for studying the genetics and molecular mechanisms of disease, while advances in LCM technologies provide the necessary tools to empower the study of the smallest and most delicate tissues, including the TM.

In this report, a robust and reproducible method is described for the LCM of highly enriched TM from mouse eyes along with subsequent RNA isolation, and amplification for downstream expression analysis. Similar methods have been used successfully in mice to isolate other types of eye tissues^{40,41,42,43,44}, the methodology reported herein can be applied to other discrete tissues of the eye to study RNA, microRNA, DNA, and proteins. Importantly, this technique enables the use of genetically modified mice to better understand the molecular pathogenesis of TM impairment in glaucoma and ocular disease^{3,15,16,17,18,26,31,45,46}. The ability to isolate the TM of mouse eyes by LCM will be a useful technique in obtaining further insights into the molecular mechanisms of several ocular diseases.

Protocol

The National Institute of Environmental Health Sciences (NIEHS) Animal Care and Usage Committee (ACUC) approved all methodology of this study under the NIEHS Animal Study Proposal IIDL 05-46.

1. Optimal Tissue Collection for Laser Microdissection

1. Obtain 2 to 3-month-old mice, male or female C57BL/6. Euthanize with CO₂ for a minimum of 1 min or until respiration has ceased. Remove the animal from the cage and assure death by either cervical dislocation, decapitation, or thoracotomy.
2. Before dissection, ensure all dissection tools are clean and sterilized.
   NOTE: For the removal of the eyes curved scissors and forceps with serrated tip (preferred tip size: 0.5 x 0.4 mm) will be needed.
3. Lay the mouse down on a flat surface on its side so that the eye is easily accessible. Grasp onto the canthus (corner of the eye) with the forceps to stabilize the mouse, then using the curved scissors, facing the curve away from the eye, cut around the eye using the eye socket as a guide until eyeball can easily be taken from the socket (use the curve not the tips of the scissors).
4. Gently pull and, using the curved scissors, cut the optic nerve, which releases the eye from the orbital socket. Carefully remove non-ocular tissue from the eye and rinse in 1x phosphate buffered saline (PBS). Repeat 1.3 - 1.4 for the contralateral eye.
5. Blot the eye with a tissue wipe to remove any moisture before embedding. Place the eye into specimen mold (25 x 20 x 5 mm³) so that the lens and optic nerve are parallel to the bench top in order to obtain sagittal sections. Embed eyes in Optimal Cutting Temperature (O.C.T.) compound and place on the dry ice to freeze. Once frozen, store the blocks at -80 °C.

2. Frozen Section Preparation for Laser Microdissection

1. Before use incubate polyethylene terephthalate (PET) membrane slides in UV cross-linker at maximum power for 45 min.
2. Spray RNase decontamination solution onto the brush, forceps, coupling jars, and mounting chuck. Wipe thoroughly. Rinse with nuclease-free water and dry. Wipe down the cryostat with 100% ethanol to avoid cross-contamination.
3. Adjust cryostat to -18 °C. Remove one frozen block from -80 °C freezer at a time and deliver to the cryostat with dry ice. Allow frozen block to equilibrate with the temperature of the cryostat for a minimum of 10 min.
4. Squeeze a small amount of O.C.T. onto the mounting chunk (Figure 1A) and remove the tissue block from the mold and carefully place the block on the mounting chuck (Figure 1B). Once the tissue block on the mounting chuck is frozen solid, insert onto the specimen holder of the cryostat (Figure 2A).
5. Adjust the cryostat to cut 8 µm sections and carefully section through the O.C.T. block to reach the tissue sample. Once the tissue is observed, stop sectioning, trim the frozen blocks on all sides using a clean razor blade and leave 3 - 4 mm of O.C.T. surrounding the tissue to enable handling with the paint brush (Figure 2B). Use a new clean paint brush instead of the roll plate between each sample change to avoid the sample cross-contamination.
6. Section through the eye working quickly. Stain every third section by flooding the slide with a quick one-step hematoxylin and eosin (H&E) stain for 60 s, rinse with tap water, air dry, clear in clearing agent and mount on a charged glass slide with a coverslip. View under the microscope and continue sectioning until the TM is evident in the cut sections.
7. Once the TM starts to appear, cut 2 sections and mount onto a charged glass slide for routine H&E staining to form a map for cutting with LCM.
   NOTE: See Section 3 for map and staining details.
8. After the first H&E map slide, cut and line up 6 serial 8-µm thick sections in the cryostat and simultaneously mount onto the PET membrane slide (Figure 3A, B). Adhere the tissue by briefly sliding gloved thumb along the back of the membrane. After mounting, place the PET membrane slide immediately into a slide box on dry ice.
   NOTE: Fewer sections may be mounted, however, mounting multiple sections onto the slide at once is recommended to reduce RNA degradation by limiting the amount of time each section is exposed to the room temperature air.
9. Continue to section the eye, after every two PET membrane slides with 6 sections each, collect two sections on a charged glass slide to create another map slide for H&E staining. Continue sectioning, approximately 100 serial 8-µm thick sections, until TM is no longer visible. Confirm by quickly staining one section on a charged glass slide (See 2.6).
10. Store all PET membrane slides at -80 °C for later LCM use.

3. H&E Map Slide Staining Protocol and Morphological Review

1. To visualize and review the map slides on the charged glass slides, fix the tissue by transferring immediately into a staining jar filled with 100 mL of the rapid fixing solution for 7 s. Rinse the slide by dipping it into distilled water 10 - 20 times, then transfer the slide to a coupling jar with clean distilled water.
2. Remove the slide from water and blot dry edges with tissue wipe. Pipette 200 µL modified filtered Harris Hematoxylin onto each section and incubate for 30 s at room temperature. Carefully rinse the slide under running tap water until water runs clear. Blot the end of the slide with a tissue wipe between each step (blot between each step from 3.2 - 3.5) to remove excess liquid.
3. Place slide in 1x PBS at room temperature for 30 s. Then, carefully rinse the slide under running tap water for 30 s.
4. Dip the slide 3 - 4 times into 95% ethanol bath. Apply enough Eosin Y dye solution to cover the tissue on the slide and incubate for 15 s at room temperature.
5. Rinse the slide in 95% ethanol bath two times with 10 - 20 quick dips each. Rinse the slide in 100% ethanol bath two times with 10 - 20 quick dips each. Then, rinse slide two times in xylene bath (10 - 20 quick dips each).
6. Mount by applying resinous mounting medium to the tissue, add coverslip carefully to avoid air bubbles and let it dry in the hood overnight.
7. Review H&E slides visually or by using a digital slide scanner. Identify map slides with TM clearly visible and accessible for cutting. Use the PET membrane slides between these map slides for LCM.

4. Polyethylene Terephthalate (PET) Membrane Slides Processing and Staining Protocol

1. Before removing PET slides from freezer first prepare cresyl violet stock solution by dissolving 0.3 g cresyl violet acetate in 20 mL of 75% ethanol and place onto shaker for 2 h. Filter the solution with 0.22 µm sterile filter unit and store at 4 °C for no longer than 6 months.
2. Prepare eosin Y alcoholic solution with fresh 75% ethanol at a 1:4 ratio. Prepare fixation (75% ethanol) and dehydration solutions (75%, 95%, & 100% ethanol) in the clean, nuclease-free, 50 mL conical polypropylene tubes on ice. Add RNase Inhibitor to each solution.
3. Remove the slide box containing frozen tissue sections from -80 °C freezer and place it on dry ice. Process one slide at time. Fix slides by placing the slide immediately into cold 75% ethanol for 30 s.
4. Gently dip the slide in nuclease-free water for 10 - 15 s to dissolve O.C.T. without interfering with the tissue sections.
5. Carefully pipette 300 µL of 1.5% cresyl violet acetate solution directly onto the sections and incubate for 45 s at room temperature. Then, wash one time by placing it into 75% ethanol bath for 30 s. Pipette 200 µL eosin Y solution onto the sections and incubate for 3 - 5 s.
6. Dehydrate tissue by placing the slide subsequently in 75% ethanol, 95% ethanol, and finally, 100% ethanol bath for 30 s each. Blot the end of the slide with a tissue wipe between every step to remove excess liquid. Let the slide air dry completely under the hood for 5 min. Once dry, perform LCM immediately thereafter.
   NOTE: It is recommended to avoid the use of xylene for this step, it can cause tissue sections to inadequately bond to the membrane slide and reduces the efficiency of tissue capture.

5. Laser Microdissection with UV Laser

1. Turn on the computer and allow to boot. Turn on the microscope white light power supply. Turn on the UV laser key and a yellow LED will illuminate. Start the LCM software, allow it to load fully and a live video will display. Press the laser button on the controller box and a green LED will illuminate.
2. Load a new glass slide onto the microscope stage, then place the PET membrane slide with the stained tissue side facing down directly on the top of the glass slide. Make sure the membrane and the glass slide are paired tightly on the microscope stage.
3. Start LCM by first setting the slide limits. Choose the lowest 4X magnification in the objective panel (Figure 4). Then define the work area of the membrane slide by moving the microscopes xy-stage until the upper left corner where the metal and membrane meet is visible in the live video feed, press the "Limit 1" button and a red rectangle will appear on the roadmap. Next, move to the xy-stage to the opposite corner and press the "limit 2" button. Press the "scan" button to create a roadmap image of the membrane and tissues between the set limits.
4. Double click near the TM of one of the eye sections within the roadmap, the TM can be located near the apex of the iridocorneal angle (Figure 5A). Once the TM has been identified, increase the magnification to 10X and manually focus on the TM. Repeat with 20X and 40X objectives. When the TM is in focus with the 40X objective (Figure 5B), navigate into a blank area nearby (focus may need to be slightly adjusted), select the "freehand drawing tool" and draw a long line on a blank area of tissue.
5. Set the laser parameters so that the "cut velocity" is 34%, "focus" is 58%, and "power" is 70% (Figure 4), then press the "cut" button. Make fine laser adjustments to the speed, focus, and power parameters until a clear and fine cutting line is observed (See Figure 5C). For precise cutting, ensure cutting action follows the drawn line.
6. Load the isolation tube lid into the cap holder and open the tube. Attach the cap-holder to the cap-lift and make sure the tube is inverted. Assure that the adhesive cap material protrudes beyond the tip of the cap to ensure that the desired tissue is properly captured.
7. Select the "Manual" dissection mode in the software panel. Carefully review that the TM (Figure 5B) is directly beneath the isolation tube. Set the white balance and adjust brightness to acquire optimal image quality (Figure 4).
8. To begin cutting, manually adjust the focus and draw a line using the freehand tool, be sure that the collection cap remains in the up position not yet touching the membrane. Draw partial circles near where the TM meets the sclera (Figure 5C), then press "cut." Once the first cuts are done, position the cap into the down position and re-adjust the focus, then draw two lines in the open or free space to connect the two partial circles completing the circle around the TM, then press "cut" and collect tissue from section, ensure that the TM was picked up by the microdissection cap (Figure 5D-F).
9. Position the cap back into the up position and repeat (5.2 - 5.8) on the remaining sections. Slightly adjust the cap position so all the isolated samples will fit onto the cap and not overlap (Figure 5G). For consistency, the time window for one slide is 20 min. Use fewer sections per PET slide if more time is needed for isolating TM from all sections. For the next PET membrane, insert a new isolation tube and repeat section 5.

6. Lysis of Microdissected TM Tissue

1. Freshly prepare RNA lysis buffer to lyse LCM isolated tissue. Add 10 µL β-mercaptoethanol to each 1 mL of the lysis buffer. Store the lysis buffer with β-mercaptoethanol at room temperature for no longer than 1 month.
2. Remove the isolation tube from cap holder within 20 min from the start of the microdissection. Visualize the cap surface by eye to ensure the capture of TM.
3. Carefully pipette 10 µL lysis buffer onto the lid of the collection tube ensuring the lysis buffer completely covers the microdissected TM. Gently close adhesive cap and incubate collection tube upside-down at room temperature for 10 min. After incubation, centrifuge at 800 x g for 2 min and place lysate on dry ice. Store lysates at -80 °C for later purification and analysis.

7. RNA Isolation and Analysis of Quality

1. Analyze the RNA quality by thawing samples at room temperature. Pool together all samples isolated from a single mouse into one RNase-free 1.5 mL microfuge tube.
   NOTE: For example, for each biological replicate, 10 - 12 tubes with microdissected caps in 10 µL lysate were generated and all lysates were transferred into a single tube for each biological replicate (100 - 120 µL lysate).
2. Add one volume of freshly prepared 70% ethanol (made with RNase-free water) to each lysate solution. Then, purify total RNA following the RNA isolation kit user guidelines. Ensure the removal of genomic DNA from each RNA sample.
   NOTE: Genomic DNA can interfere with downstream RNA analysis.
3. Measure the quality of RNA isolated from the TM by pooling together samples from each mouse and analyzing the ribosomal RNA integrity (Figure 6A).
   NOTE: Due to the small size of the TM, ribosomal RNA peaks may not be observable (Figure 6B) on the RNA quality profile (two peaks observed between 1,000 - 4,000 bp in Figure 6A, C) which is used to calculate the RNA integrity number (RIN)⁴⁷.
   1. Obtain a more accurate measure of RNA integrity by isolating RNA from the more abundant remaining tissue on the membrane slide. To obtain a representative RIN, isolate RNA from the ocular-tissue on the membrane slide by pipetting 10 µL lysis buffer onto one tissue section and perform RNA isolation. Analyze RNA quality and calculate RIN (Figure 6C).
4. For downstream RNA applications, use a low input RNA kit for sequencing and cDNA library generation to yield reproducible results from as little as 80 sections of LCM isolated TM.

8. Analysis

1. To verify that the tissue collected is in fact enriched in TM-associated genes, collect multiple additional RNA from microdissected whole eye, sclera, iris, retina, cornea, and lens from 3 separate mice. Use this RNA in combination with LCM collected RNA from isolated TM and ciliary body collected from 4 separate mice.
2. Convert the RNA from the microdissected samples was to cDNA using a cDNA reverse transcription kit. Amplify the RNA from LCM isolated samples and convert to cDNA using a low input RNA to cDNA kit.
3. Analyze all RNA for trabecular meshwork expressing genes, myocilin (MYOC) and alpha-actin-2 (ACTA2), and normalize using the HSP90a1 housekeeping gene by digital PCR (Figure 7A-B).

Results

LCM collected RNA from the TM and ciliary body from 4 different mice was isolated in order to be able to analyze gene expression and compare the expression with that in whole eye, sclera, iris, retina, cornea, and lens isolated from three separate mice. TM expressing genes, MYOC⁴⁸ and ACTA2⁴⁹ were analyzed in all the collected tissues to confirm that the isolated TM samples were indeed highly enriched in TM. Due to the extr...

Discussion

The TM plays a vital role in actively maintaining homeostatic IOP and its dysfunction is widely accepted as the main causative factor for hypertensive glaucoma^1,2,19. A number of single nucleotide polymorphisms in several genes identified by GWAS analysis have been linked to increased glaucoma risk and increased resistance to AH outflow facility at the TM; however, the precise molecular mechanisms that give rise to this disease ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACTA2 ddPCR Primers (dMmuCPE5117282)	BioRad	10031252	FAM
Agilent 2100 Bioanalyzer	Agilent Technologies	G2946-90004
Agilent RNA 6000 Pico kit	Agilent Technologies	5067-1513
BioRad QX200 Droplet Digital PCR System	BioRad
Small Paint Brush
Charged Glass Microscope Slide	Thermo scientific	4951PLUS-001
Cresyl Violet Acetate	Sigma Aldrich	C5042
Curved Scissors
Eosin Y dye	Thermo scientific	71204
Ethanol
Forceps			Curved and Serrated tip (preferred tip size: 0.5 x 0.4 mm)
HemaCen	American MasterTech	STHEM30
High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems	4368814
Hsp90a ddPCR Primers(dMmuCPE5097465)	BioRad	10031255	VEX
Leica CM1850 Cryostat	Leica
Millex-GS filter unit	EMD Millipore	SLGS033SB	0.22 µm
MMI CellCut UV Cutting Model	Molecular Machines & Industries		LCM intrument
MMI CellTools Software	Molecular Machines & Industries	50202	LCM software
Sample Tube for Laser Capture Microdisssection	ASEE Products	ST-LMD-M-500	Isolation Cap Tube/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products
Sample Tube for Laser Capture Microdisssection (Alternative)	Molecular Machines & Industries
modified Harris Hematoxylin	Thermo scientific	7211	FAM
MYOC ddPCR Primers (dMmuCPE5095712)	BioRad	10031252
PBS
Memebrane Slides, RNase Free	ASEE Products	FS-LMD-M-50r	Polyethylene terephthalate (PET) membrane/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products
Memebrane Slides, RNase Free (Alternative)	Molecular Machines & Industries	50102
Rapid Fix	Thermo scientific	6764212	H&E staining
RLT Buffer	Qiagen	79216	lysis bufffer used for LCM samples
RNAseZap	Sigma	R2020	RNase decontamination solution
Protect RNA RNAse Inhibitor	Sigma Aldrich	R7397
RNeasy Micro Kit	Qiagen	74004	RNA isolation kit
SMART-Seq v4 Ultra Low Input RNA Kit	Takara Clontech	634888	low input RNA to cDNA kit for LCM samples
SuperMix (no dUTP)	BioRad	1863023	digital PCR master mix
Tissue-Tek Cryomold (25mm x 20mm x5mm)	Sakura	4557
Tissue-Tek O.C.T. Compound	Sakura	4583
Stratalinker UV Crosslinker	Stratagene	400075
Xylene	Macron	8668