Quantification of Immunostained Caspase-9 in Retinal Tissue

Summary

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.

Abstract

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Introduction

The caspases are a family of proteases that regulate developmental cell death, immune responses, and aberrant cell death in disease^1,2. While it is well known that members of the caspase family are induced in a variety of neurodegenerative diseases, understanding which caspase drives disease pathology is more challenging³. Such studies require tools to identify, characterize, and validate the function of individual caspase family members. Parsing out the relevant individual caspases is important both from a mechanistic and a therapeutic standpoint, as the literature has multiple studies providing evidence of the diverse roles of caspases^4,5. Thus, if the goal is to target a caspase in a disease for a therapeutic benefit, it is critical to have specific targeting of the relevant family member(s). Traditional techniques to detect caspase levels in tissue include western blotting and enzymatic and fluorometric approaches^3,6. However, none of these measures allow for cell-specific detection of caspase levels, and in some scenarios, cleaved caspases often cannot be detected by traditional protein analysis measures. It is known that caspases can play different apoptotic and non-apoptotic roles in the same tissue⁷, therefore careful characterization of cell-specific caspase levels is needed for accurate understanding of developmental and disease pathways.

This study shows caspase activation and function in a model of neurovascular hypoxia-ischemia - retinal vein occlusion (RVO)^7,8. In a complex tissue such as the retina, there are multiple cell types that can be affected by the hypoxia-ischemia induced in RVO, including glial cells, neurons, and vasculature⁷. In the adult mouse retina, there is very little expression of caspases evident in healthy tissue, as measured by immunohistochemistry (IHC)⁷, but that is not the case during development⁹ or in models of retinal disease^10,11. IHC is a technique that is well established in biomedical research and has allowed validation of disease and pathological targets, identification of new roles through spatial localization, and quantification of proteins. In cases where cleaved caspase products cannot be detected by western blot or fluorometric analysis, nor the specific cell location of distinct caspases or interrogation of caspase signaling pathways through localization, then IHC should be used.

In order to determine the caspase(s) functionally relevant in RVO, IHC was used with validated antibodies for caspases and cellular markers. The previous studies performed in the lab showed that caspase-9 was rapidly activated in a model of ischemic stroke and inhibition of caspase-9 with a highly specific inhibitor protected from neuronal dysfunction and death¹². Because the retina is part of the central nervous system (CNS), it serves as a model system to query and further investigate the role of caspase-9 in neurovascular injuries¹³. To this end, the mouse model of RVO was used to study the cell-specific location and distribution of caspase-9 and its implication in neurovascular injury. RVO is a common cause of blindness in working aged adults that results from vascular injury¹⁴. It was found that caspase-9 was expressed in a non-apoptotic manner in endothelial cells, but not in neurons.

As a tissue, the retina has the advantage of being visualized as either a flatmount, which allows appreciation of the vascular networks, or as cross-sections, which highlights the neuronal retinal layers. Quantification of caspase protein expression in cross-sections provides context, regarding which caspase is potentially critical in retinal neuronal connectivity and vision function by identifying the localization of the caspase(s) in the retina. After identification and validation, targeting of the caspase of interest is achieved using inducible cell specific deletion of the caspase identified. For potential therapeutic inquiries, the relevance of the caspases of interest was tested using specific tools to inhibit the activated caspase. For caspase-9 a cell permeant highly selective inhibitor^7,15, Pen1-XBIR3 was used. For this report, 2-month-old, male C57BL/6J strain and tamoxifen-inducible endothelial caspase-9 knockout (iEC Casp9KO) strain with a C57BL/6J background were used. These animals were exposed to the mouse model of RVO and C57BL/6J were treated with the caspase-9 selective inhibitor, Pen1-XBir3. The described methodology can be applied to other models of disease in the central and peripheral systems^7,15.

Protocol

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.

1. Preparation of retinal tissue and cryosectioning

1. Euthanize the animals by administration of intraperitoneal anesthesia (ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg)), and perfuse the mice subjected to RVO (see⁸ for details) with 4% paraformaldehyde (PFA) using a peristaltic pump.
   NOTE: Toe-pinch the animal to confirm depth of anesthesia before proceeding with euthanization and perfusion. Details about perfusion can be found in¹⁶.
2. Harvest the eyes by careful enucleation using forceps and put the globe in 1 mL of 4% PFA. Leave the eyes overnight at 4 °C. Wash the eyes three times for 10 min, adding 1 mL of 1x PBS and placing them in the shaker.
3. Immerse the eyes in 1 mL of 30% sucrose for 3 days, until they settle to the bottom of tube, indicating absorption of the sucrose.
4. Fill the eyes with optimal cutting temperature compound using a 30 G syringe, until the eye has a rounded appearance (approximately 50 µL).
5. Embed the eyes in a cryomold with optimal cutting temperature compound until the eyes are covered and freeze at -80 °C until ready to cryosection.
6. Section embedded the eyes at 20 µm onto glass slides using a cryostat.
   1. Remove the optimal cutting temperature compound block from the cryomold.
   2. Place it on the cryostat chuck by adding optimal cutting temperature compound and placing the block on the chuck. Leave it inside the cryostat until it freezes.
   3. Proceed to cut the block at 20 µm until the retinal tissue is seen.
      NOTE: This can be confirmed with a light microscope at 30x magnification (3x objective, 10x eyepieces). Tissues can be distinguished from the OCT media by color and shape.
   4. Collect the retinal tissue in microscope slides in a series of four sections per slide.
      NOTE: See Figure 1A for suggested placing of retinal sections.
7. Label the slides with an ID that de-identifies the treatment and genotype.
8. Store the slides at -20 °C until staining.

2. Immunohistochemistry

NOTE: Use fixed cryopreserved tissue for immunohistochemistry to maintain cell morphology. Pick sections that are at the level of the optical coherence tomography (OCT) images acquired in vivo⁷. Use the first two series of slides collected from the cryostat or sections from 150 µm into the retinal tissue.

1. Place the slides in a dark and humid slide chamber.
2. Permeabilization and blocking: Wash the retinal cross-sections with 300 µL of 1x PBS for 5 min to remove the OCT and discard after.
3. Permeabilize the tissue by adding 300 µL of 1x PBS with 0.1% Triton X-100 for 2 h at room temperature (RT) and discard after.
4. Block the tissue by adding 300 µL of blocking buffer (10% Normal Goat Serum (NGS), 1% Bovine Serum Albumin (BSA) in 1x PBS, filtered) and leave overnight at 4 °C.
5. Primary antibodies: Dilute primary antibodies in blocking buffer. Primary antibodies used include anti-cl-caspase-9 at 1:800, anti-CD31 at 1:50, and anti-caspase-7-488 (direct labeled antibody) at 1:150.
   NOTE: Follow manufacturer's recommendation for the appropriate dilution of primary antibodies.
   1. Pour the blocking buffer off sections and apply 100 µL of the primary antibody cocktail to the retinal slides.
   2. Incubate the cross-sections overnight at 4 °C.
6. Wash the sections four times for 5 min with 300 µL of 1x PBS.
   NOTE: If the tissue sections are very fragile, washes should be removed using capillary action by applying a tissue to the corner of the slide to avoid displacing the retinal sections.
7. To avoid cross-labeling of primary antibodies raised in the same host species, complete the staining with secondary antibodies prior to applying the directly labeled antibodies.
8. Secondary antibodies: Dilute the secondary antibodies in blocking buffer at a concentration of 0.1%. For example, to detect anti-cl-caspase-9, an antibody raised in rabbit, use goat-anti-rabbit-568 secondary.
   1. Apply 200 µL of the secondary antibody cocktail to the retinas.
   2. Incubate the tissue for 2 h at RT.
   3. Wash the sections four times for 5 min with 300 µL of 1x PBS.
   4. Stain the nuclei for 5 min with 300 µL of DAPI at a dilution of 0.02%.
9. Wash once for 5 min with 300 µL of 1x PBS.
10. Place a coverslip on the retinal sections using 500 µL of fluoromount-G media, which preserves the fluorescent signal, and carefully place the coverslip on the top of the slide, avoiding bubbles.

3. Confocal imaging

1. Acquire images of the stained sections with confocal microscopy.
   1. Turn on the confocal microscope.
   2. Place the slide on the stage.
   3. Adjust the focus to see the retinal section clearly.
      NOTE: Take at least four images per section and image four sections per retina. See the Retinal imaging schematic for suggested imaging areas (Figure 1B, C).
2. Image caspase, vascular, and nuclear staining using a confocal microscope that has Z-stack acquisition, using a 20x or 40x objective.
   NOTE: The imaging parameters and software setup should be constant for all imaging in an experiment. The 20x objective provides an overview of the retinal layers, which are well-defined by the nuclear staining. The 40x objective provides more cellular detail.
   1. Set appropriate exposure and laser intensity times per channel by clicking acquire settings.
   2. Set the Z-stack to visualize the whole section by clicking Z-series and set up and down parameters to cover the depth of the tissue.
3. Save the images following the masked ID assigned during cryosectioning.

4. Quantification of caspase levels

1. Drag image files of 405, 470, 555, and 640 channels to FIJI's console.
2. Click Image > Color > Merge Channels.
3. Assign colors to the files as follows: red to 555, green to 470, gray to 405, cyan to 640.
4. Compress the Z-stack by clicking Image > Stack > Z Project.
5. Click Projection Type: Max Intensity.
6. Open the Channels Interface by clicking Image > Color Channels Tool.
7. Open the Brightness and Contrast interface.
8. Select parameters per channel.
   1. Go to Channels and select one channel.
   2. Put the cursor on top of the caspase expressed cell and annotate the pixel value.
   3. Put the cursor on top of the background and annotate the pixel value.
9. Test parameters
   1. Go to the Brightness and Contrast interface.
   2. Click Select.
   3. Plug in the minimum displayed value (annotated pixel value of the caspase expressed cell).
   4. Plug in the maximum displayed value (annotated pixel value of the background).
   5. Click Ok.
10. Repeat steps 4.8-4.9 for all channels.
11. Test the parameters by choosing random images from blinded tissue.
    NOTE: Edit brightness and contrast parameters only if the background value is not adequate for other images.
12. Once the parameters are set, open the image files and compress the Z-stack.
13. Add in the brightness and contrast parameters for the caspase channel and the isolectin, vascular marker channel.
14. Use the point tool to quantify the number of vascular positive areas using colocalization of the vascular marker with caspase expression as the readout of positive areas.
15. Repeat step 4.14 but using Hoechst as the marker of neuronal positive areas.
16. Annotate the values on a spreadsheet per image.
17. Average the values by section.
18. Average the section values-this will be the readout per eye.

5. Genetic confirmation of the relevance of the endothelial cell caspase-9

1. Use retinas obtained from the Casp9FL/FL-VECad-CreERT2 mice that were subjected to RVO⁷.
2. Immunostain the retinas for caspase-9 (the deleted caspase), caspase-7 (a down-stream effector caspase), vascular markers, and DAPI as described above in section 2.
3. Image and quantify as described above in sections 3 and 4.

6. Targeting caspase-9 in RVO

1. Obtain eyes from the mice subjected to RVO followed by application of the caspase-9 inhibitor Pen1-XBir3 topically to eyes as in⁷.
2. Immunostain the retinas for caspase-7 (a down-stream effector caspase), vascular markers, and DAPI as described above in section 2.
3. Image and quantify as described above in sections 3 and 4.

Results

The described protocol allows the user to analyze and quantify caspase-9 levels in the retinal tissue. Additionally, it present tools to further identify, validate, and specifically target caspase-9 and downstream substrates. The summarized steps allow quantifiable analysis of caspase levels and cellular specificity in fluorescent photomicrographs. All figures show representative photomicrographs and quantification of the indicated caspase levels in the total retina, endothelial cells, and neurons in uninjured and 1-day ...

Discussion

Caspases are a multi-membered family of proteases best studied for their roles in cell death and inflammation; however, more recently a variety of non-death functions have been uncovered for some family members^4,5. Much of our understanding of caspase function is derived from work in cell culture and from inferential data from human disease. While it is appreciated that there is aberrant induction, activation, or inactivation of caspases in disease, it has been c...

Materials

Name	Company	Catalog Number	Comments
anti-Caspase-7 488	Novus Biologicals	NB-56529AF488	use at 1:150
anti-cl-Caspase-9	Cell Signaling	9505-S	use at 1:800
anti-CD31	BD Pharmingen	553370	use at 1:50
Confocal Spinning Disc Microscope	Biovision
FIJI 2.3.0	open source
Fluormount G	Fisher	50-187-88
Forcep	Roboz	RS-5015
iCasp9FL/FL X VECad-CreERT2 mice	lab generated		see Avrutsky 2020
Isolectin (594, 649)	Vector	DL-1207	use at 1:200
Ketamine Hydrochloride	Henry Schein	NDC: 11695-0702-1
Perfusion pump	Masterflex
Pen1-XBir3	lab generated		see Avrutsky 2020
Prism 9.1	GraphPad
Tissue-Tek O.C.T.	Fisher	14-373-65
Vis-a-View 4.0	Visitron Systems
Xylazine	Akorn	NDCL 59399-110-20