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.

Representative 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 P-RVO retina cross-sections. The retinal cross-section allows visualization of retinal nuclei in the retinal ganglion layer (RGL), inner nuclear layer (INL), and outer nuclear layer (ONL) when stained with Hoechst. In addition, retinal blood vessels are visible in the retinal plexiform layers or between the RGL and INL, and between the INL and ONL. Because of the histological nature of blood vessels, when the retina is sectioned, blood vessels will appear as disconnected and separate, supplying the plexiform layers. Figure 2 identifies caspase-9 as highly regulated 1-day P-RVO. Figure 3 validates the functional relevance of endothelial caspase-9 by using inducible endothelial cell knockout mice. Figure 4 shows that targeting active caspase-9 pharmacologically blocks the induction of a downstream target of caspase-9-caspase-7. The protocol identifies the cellular localization of the caspases and the neuronal retinal layers in which the caspases are expressed.

Figure 1: Retinal imaging scheme. (A) Recommended placing of retinal sections in the microscope slide. (B) Overview of the imaging areas in the retina. (C) Representation of retinal view at 20x objective. Retinal layers: RGL = retinal ganglion layer, INL = inner nuclear layer, ONL = outer nuclear layer. Please click here to view a larger version of this figure.

Figure 2: RVO induces caspase-9 in endothelial cells and neurons. (A) Representative retinal cross-sections from uninjured and 1-day P-RVO stained with cl-caspase-9 1:800 (green), isolectin 1:200 (red), and DAPI (white). (B) Quantification of the number of neurons with cl-caspase-9 (see Table of Materials for details on the antibodies). (C) Quantification of the number of endothelial cells with cl-caspase-9. (D) Total number of cells expressing cl-caspase-9. Uninjured, n = 6; 1-day P-RVO, n = 5. Error bars represent the mean ± SEM; One-way ANOVA, Fisher's LSD test. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 3: Endothelial cell deletion of endothelial caspase-9 blocks RVO-induction of caspase-7. (A) Representative retinal cross-sections from iEC Casp9WT and iEC Casp9KO littermate mice 1-day P-RVO stained with caspase-7 (green), cl-caspase-9 (blue), CD31, a vascular marker (red), and DAPI (white). (B) Quantification of the number of cells in the inner retina with cl-caspase-9 and with caspase-7. (C) Quantification of the number of endothelial cells with cl-caspase-9 and with caspase-7. (D) Total number of cells expressing cl-caspase-9 or caspase-7. iEC Casp9WT, n = 6-12; iEC Casp9KO, n = 3-8. Error bars represent the mean ± SEM; One-way ANOVA, Fisher's LSD test. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 4: Inhibition of caspase-9 activity inhibits caspase-7 expression P-RVO. (A) Representative retinal cross-sections from uninjured and 1-day P-RVO treated or untreated with Pen1-XBIR3 stained with caspase-7 (green), isolectin (red), and DAPI (white). (B) Quantification of the number of endothelial cells with caspase-7. (C) Quantification of the number of leukocytes with caspase-7. (D) Quantification of the number of neurons per neuronal layer with caspase-7. (E) Total number of cells expressing caspase-7. Uninjured Pen1 Saline, n = 6; uninjured Pen1-XBIR3, n = 5; 1-day P-RVO Pen1 Saline, n = 5; 1-day P-RVO Pen1-XBIR3, n = 3. Error bars represent the mean ± SEM; One-way ANOVA, Fisher's LSD test. Abbreviations: RGL = retinal ganglion layer, INL = inner nuclear layer, and ONL = outer nuclear layer. Scale bar = 50 µm. Please click here to view a larger version of this figure.

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 challenging to functionally determine if the caspases are driving disease pathology. Many tools used to assess specific caspases detect multiple family members, undercutting the functional relevance of data obtained with these reagents¹⁷. Here we provide a protocol to study caspases in complex tissue, using the retina as an example. In the nervous system, caspase expression is down-regulated pre- and postnatally. This allows the use of specific antibodies to test changes in caspase levels in a disease model.

While the focus of the protocol is on image processing and analysis, the success of the technique also relies on careful tissue preparation, as well as IHC and microscopic imaging to ensure consistency, validity, and reliability of the data. Care must be taken to utilize the same parameters to image an entire data set. Poor quality images, low contrast, and blurry focus will not provide reliable data. When sectioning and imaging P-RVO regions of the retina, tears and folding should be avoided as these areas may result in artifacts. Areas of direct damage should also be avoided. The investigator performing the imaging needs to be blinded to the treatment of the tissue. For analysis, it is recommended that two independent observers, blinded to the treatment group, perform the analysis. In addition, primary mouse antibodies should not be used in mouse injury models as the secondary antibodies bind non-specifically to the vasculature. An alternative is to use directly conjugated primary antibodies.

An alternative approach to cell counting is quantification of the percentage of the area of expression. This can be used when cell counting is not possible (e.g., if caspase expression is localized to neuronal processes or photoreceptor segments). Central and peripheral retina are differentially affected in various injury models. The method can be adapted to specifically analyze different retinal regions. If performing BRVO, then sections should be selected from the injured portion of the eye. The protocol also provides for staining of different cell markers to identify where the caspase is expressed.

Limitations of the method include that it does not differentiate between high and low levels of caspase signal in a given cell. Additionally, using a nuclear stain to identify neuronal layers is convenient but does not definitively indicate neuronal expression (glia and leukocytes are present in these layers also). Additional neuronal markers can be used to validate neuronal expression. There are also a variety of antibodies available to evaluate caspase expression; these have been best defined for human caspase-9, where there are antibodies for full length/cleaved (cl) caspase-9, autocleaved caspase-9, and caspase-3-cleaved caspase-9.

The mean intensity of the IHC signal is frequently reported as a quantification method. However, background noise or autofluoresence from red blood cells can produce inaccurate results. Cell specific quantification allows for determination and discrimination of background, non-specific staining, and autofluoresence.

This protocol can be used to measure tissue wide changes in the expression of individual caspases, changes in expression in a specific cell type, such as an endothelial cell, and changes in specific cell types in specific locations, such as neurons in different retinal layers. This flexibility allows the experimenter to query how the disease state is altering individual caspase levels. Existing/alternative methods use western blotting analysis for semiquantitative comparison of caspase signaling, although methods will not provide the clear separation of cellular localization that this protocol acheives. Notably, in Figure 3D, caspase-9 inhibition is more effective at reducing neuronal caspase-9 in the INL and ONL, compared to the RGL. This kind of resolution is challenging to achieve by other methods. Additionally, when a whole tissue is analyzed biochemically, the levels of caspases that change may be too low to pick up, since there are many different cell types in a complex tissue.

The antibodies, which must be validated by the user, provide specificity for the caspase probed. Caspases can act in cascades (i.e., initiator activating effector and then leading to outcome - death, inflammation, cell signaling). This protocol can be used in samples collected at different time points post-damage; in the examples presented here, samples are collected at different times post-RVO. In previously published work, it has been found that caspase-9 was increased within 1 h post-RVO⁷, which lead to further validation and targeting studies. After identifying endothelial caspase-9 as a potential driver of RVO pathology, a mouse with inducible endothelial cell caspase-9KO to validate endothelial caspase-9 was used. The use of inducible cell specific caspase KO mice provides another level of specificity and avoids the developmental death seen with constitutive caspase-9KO¹⁸. It also avoids compensatory changes in other family members that have been shown to occur in constitutive caspase KO mice¹⁹. Moreover, the use of cell specific inducible caspase KO mice allows the identification of the cell type in which the caspase is regulating pathology in the tissue. The protocol also provides the steps to interrogate the efficacy of a therapeutic approach, targeting active caspase-9 in RVO. This approach can also be applied to other tissues, including the brain.

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