Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.
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.
The caspases are a family of proteases that regulate developmental cell death, immune responses, and aberrant cell death in disease1,2. 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 caspases4,5. 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 approaches3,6. 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 disease10,11. 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 inhibitor7,15, 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 systems7,15.
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
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.
3. Confocal imaging
4. Quantification of caspase levels
5. Genetic confirmation of the relevance of the endothelial cell caspase-9
6. Targeting caspase-9 in RVO
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.
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 members4,5. 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.
The authors have nothing to disclose.
This work was supported by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) grant DGE – 1644869 and the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH), award number F99NS124180 NIH NINDS Diversity Specialized F99 (to CKCO), the National Eye Institute (NEI) 5T32EY013933 (to AMP), the National Institute of Neurological Disorders and Stroke (RO1 NS081333, R03 NS099920 to CMT), and the Department of Defense Army/Air Force (DURIP to CMT).
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 |