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).

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
<ol>
	<li>Euthanize the animals by administration of intraperitoneal anesthesia (ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg)), and perfus...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+retinal+vein+occlusion+mouse+model+to+limit+variability.">Optimization of the retinal vein occlusion mouse model to limit variability.</a> Journal of Visualized Experiments. (174), e62980 (2021).</li><li>Tisch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-8+modulates+physiological+and+pathological+angiogenesis+during+retina+development.">Caspase-8 modulates physiological and pathological angiogenesis during retina development.</a> The Journal of Clinical Investigation. 129 (12), 5092-5107 (2019).</li><li>Chi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB1+promotes+the+activation+of+NLRP3+and+caspase-8+inflammasomes+via+NF-kappaB+pathway+in+acute+glaucoma.">HMGB1 promotes the activation of NLRP3 and caspase-8 inflammasomes via NF-kappaB pathway in acute glaucoma.</a> Journal of Neuroinflammation. 12, 137 (2015).</li><li>Thomas, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-2+mediates+site-specific+retinal+ganglion+cell+death+after+blunt+ocular+injury.">Caspase-2 mediates site-specific retinal ganglion cell death after blunt ocular injury.</a> Investigative Ophthalmology &amp; Visual Science. 59 (11), 4453-4462 (2018).</li><li>Akpan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> Journal of Neuroscience. 31 (24), 8894-8904 (2011).</li><li>London, A., Benhar, I., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retina+as+a+window+to+the+brain-from+eye+research+to+CNS+disorders.">The retina as a window to the brain-from eye research to CNS disorders.</a> Nature Reviews Neurology. 9 (1), 44-53 (2013).</li><li>Song, P., Xu, Y., Zha, M., Zhang, Y., Rudan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epidemiology+of+retinal+vein+occlusion:+a+systematic+review+and+meta-analysis+of+prevalence,+incidence,+and+risk+factors.">Global epidemiology of retinal vein occlusion: a systematic review and meta-analysis of prevalence, incidence, and risk factors.</a> Journal of Global Health. 9 (1), 010427 (2019).</li><li>Akpan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> The Journal of neuroscience. 31 (24), 8894-8904 (2011).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>McStay, G. P., Salvesen, G. S., Green, D. 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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 c...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-Caspase-7 488</td>
			<td>Novus Biologicals</td>
			<td>NB-56529AF488</td>
			<td>use at 1:150</td>
		</tr>
		<tr>
			<td>anti-cl-Caspase-9</td>
			<td>Cell Signaling</td>
			<td>9505-S</td>
			<td>use at 1:800</td>
		</tr>
		<tr>
			<td>anti-CD31</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>use at 1:50</td>
		</tr>
		<tr>
			<td>Confocal Spinning Disc Microscope</td>
			<td>Biovision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI 2.3.0</td>
			<td>open source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluormount G</td>
			<td>Fisher</td>
			<td>50-187-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>iCasp9FL/FL X VECad-CreERT2 mice</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Isolectin (594, 649)</td>
			<td>Vector</td>
			<td>DL-1207</td>
			<td>use at 1:200</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump&#160;</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen1-XBir3</td>
			<td>lab generated</td>
			<td></td>
			<td>see Avrutsky 2020</td>
		</tr>
		<tr>
			<td>Prism 9.1</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T.</td>
			<td>Fisher</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Vis-a-View 4.0</td>
			<td>Visitron Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDCL 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of immunostained caspase-9 in retinal tissue

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 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 ...

Watch this Scientific Journal Video about Quantification of Immunostained Caspase-9 in Retinal Tissue at JoVE.com

Quantification of Immunostained Caspase-9 in Retinal Tissue

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 ...

This protocol provides a detailed immunohistochemical method to identify, validate, and target functionally relevant caspases in complex tissues. The main advantage of this technique is that 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.Begin quantifying the caspase levels using confocal microscopy images of stained retinal sections. To do so, drag the image files of 405, 470, 555 and 640 channels to Fiji's console. Click image, color, and merge channels tabs to assign colors to the files.Then compress the Z stack by clicking the image stack and Z project. Select the projection type as max intensity. Open the channel's interface by clicking the image followed by color channel's tool.Next, open the brightness and contrast interface. To select the parameters per channel, go to the channel's tab and choose one channel. Put the cursor on top of the background and annotate the pixel value.Then put the cursor on the caspase expressed cell and annotate the pixel value. To test the parameters, go to the brightness and contrast interface and click select. Plug in the minimum displayed value or the caspase expressed cell's pixel value.Then plug in the maximum displayed value or the background pixel value before clicking okay. Choose the random images from blinded tissue and repeat the selection of parameters per channel to test the parameters for all the channels. Once the parameters are set, open the image files, and compress the Z stack.Add the brightness and contrast parameters for the caspase channel and the isolectin for the vascular marker channel. Use the point tool and quantify the number of positive vascular areas using colocalization of the vascular marker with caspase expression as the readout of positive areas. Then using hooks as the marker of positive neuronal areas, quantify the number of positive vascular areas.Annotate the values on a spreadsheet per image and average the values by section. Average the section values, which will be the readout per eye. The protocol identified the cellular localization of the caspases and the neuronal retinal layers in which the caspases are expressed.The retinal cross section allowed the visualization of retinal nuclei in the retinal ganglion layer or RGL, Internuclear layer or INL, and the outer nuclear layer or ONL when stained with hooks. In the cross section, the retinal blood vessels appeared disconnected and separate supplying the plexiform layers between the RGL and INL and between the INL and ONL. Retinal cross sections also identified the highly regulated caspase-9, one day post retinal vein occlusion.The caspase-9 inhibition was more effective at reducing neuronal caspase-7 expression in endothelial cell knockout mice which validated the functional relevance of endothelial caspase-9. The representative analysis showed that the inhibition of caspase-9 activity pharmacologically blocked the caspase-7 expression post retinal vein occlusion. The success of the technique relies on careful tissue preparation as well as IHC and microscopic imaging to ensure consistency, validity, and reliability of the data.This method could be combined with western blotting analysis for semi-quantitative comparison of caspase signaling. The technique provided 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.

quantification-of-immunostained-caspase-9-in-retinal-tissue

Research

JoVE Journal

Neuroscience

1.2K visualizações.  Columbia University. Este protocolo fornece um método imuno-histoquímico detalhado para identificar, validar e direcionar caspases funcionalmente relevantes em tecidos complexos. A principal vantagem desta técnica é que permite a interrogação de funções específicas da caspase-9 apoptótica e não apoptótica do tipo celular e pode ser aplicada a outros tecidos complexos e caspases de interesse. Compreender as funções das caspases pode ajudar a expandir o conhecimento atual em biologia celular e também...