Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord

Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord Radovan Holota, Viktória Buľková, Anna Alexovič Matiašová, Ján Košuth, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2854960/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2023 Read the published version in Histochemistry and Cell Biology → Version 1 posted 8 You are reading this latest preprint version Abstract Cell death is an essential process occurring during the development of the central nervous system. Despite the availability of wide range of commercially produced antibodies against various apoptotic markers, data regarding the apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. This study aimed to investigate the apoptosis in the rat spinal cord at different stages of ontogenesis (8, 29 and 90 postnatal days). For this purpose, we used immunofluorescent detection of two widely used apoptotic markers, activated caspase-3 (aC3) and cleaved PARP (cPARP). Surprisingly, we found significant discrepancy between the amounts of aC3 + cells and PARP + cells, varying with ratio around 500:1–5,000:1 in the rat spinal cord in all postnatal time points. Majority of aC3 + cells were glial cells and did not exhibit apoptotic phenotype. In contrast with the results of in vivo study, in vitro analysis of primary cell culture derived from neonatal rat spinal cord, treated with apoptotic inductor staurosporine, revealed similar onset of occurrence of both markers in cells subjected to apoptosis. Gene expression analysis of spinal cord tissue revealed elevated expression of Birc4 (XIAP) , Birc2 and Birc5 (Survivin) genes, which are known as potent inhibitors of apoptosis. Our data indicates that the activated caspase-3 is not an exclusive marker of apoptosis, especially in glial cells, due its possible presence in inhibited forms and/or its participation in other, non-apoptotic roles. Therefore, in the light of our recent results, cPARP appears to be more appropriate marker for detection of apoptosis. apoptosis activated caspase-3 cleaved PARP spinal cord rat development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Caspase-3 is a member of cysteine-aspartate proteases considered as important effectors of apoptosis representing the process of regulated cell death (Asadi et al. 2022 ; Taatjes et al. 2008 ). The very first member of this family of proteases active in cell death, termed cell death protein 3 (CED-3), was identified in Caenorhabditis elegans , and was reported as homolog to murine ICE - related proteases (later known as caspases) (Yuan et al. 1993 ). The involvement of CED-3 and its homologs in cell death ensured that caspases became considered as apoptotic proteins (Fraser and Evan 1997 ; Miura et al. 2004 ). During the apoptotic cascade, inactive zymogen of caspase-3 is activated by initiator caspase-8 and/or caspase-9 (Taatjes et al. 2008 ). Subsequently, activated caspase-3 (aC3) interacts with numerous substrates present in cell, including cytoplasmic and nuclear structural proteins, molecules involved in protein synthesis and protein modification, as well as with the proteins of multiple signaling pathways controlling proliferation, differentiation or cell adhesion (Fischer et al. 2003 ). Activity of aC3 and other effector molecules of apoptosis results in changes of cellular morphology typical for apoptotic cells, e.g. chromatin condensation, DNA fragmentation, formation of plasma membrane protrusions and externalization of phosphatidylserine (Taatjes et al. 2008 ; Ziegler and Groscurth 2004 ). One of the most studied substrates of aC3 is Poly (ADP-ribose) polymerase (PARP), the protein important for DNA repair and for maintenance of chromatin and genome stability (Dantzer et al. 1999 ). Due to the catalytic activity of aC3, PARP protein is cleaved to two fragments: 24-kD fragment (p24) and 89-kD fragment (p89), which are specific for apoptosis, unlike the other forms of cell death (Duriez and Shah 1997 ). Afterwards, the p24 fragment of cleaved PARP (cPARP) enables progression of apoptosis by inhibition of DNA repair, whereas the p89 fragment localized in nucleoplasm dimerizes and inactivates uncleaved PARP protein (Soldani and Scovassi 2002 ). Ultimately, the activity of effector caspases leads to final disintegration of cell to membrane-bound apoptotic bodies, which are subsequently phagocyted by immune cells (Taatjes et al. 2008 ). Although the aC3 is not an exclusive effector-protease of apoptosis in mammals (Wilson and Kumar 2018 ), due to its ability to interact with a wide spectrum of substrates (Walsh et al. 2008 ) it is considered as the main apoptotic protease. Thus, various assays designed for identification of apoptotic cells in tissue samples or in cell culture, often focus on detection of activated effector caspases, especially the aC3 (Fox and Aubert 2008 ; Gown and Willingham 2002 ; Hanson and Finkelstein 2019 ; Takano et al. 2014 ). On the other hand, presence of aC3 has been reported in cells without typical apoptotic hallmarks in various cell types (Krajewska et al. 1997 ). Until now, caspase-3 activity has been linked to several non-apoptotic processes. It has been reported, that activated form of this protein is required for the differentiation of various cell types, including bone marrow stromal stem cells, osteoclasts (Szymczyk et al. 2006 ), skeletal muscle fibers (Fernando et al. 2002 ) or embryonic cells (Fujita et al. 2008 ), as well as for regulation of proliferation of B-lymphocytes (Woo et al. 2003 ). In nervous system, apoptosis has been extensively studied due to its crucial role in embryonic development, tissue formation, function of neuronal circuits (Yuan and Yankner 2000 ), as well as its involvement in various pathological events linked to the neurodegenerative disorders (Erekat 2022 ) and tissue injury (Springer 2002 ). In spite of the evidence on involvement of aC3 in non-apoptotic events, it is commonly used as an apoptotic marker in both older and recent studies on spinal cord, especially under pathological conditions (Dai et al. 2019 ; Gülmez et al. 2022 ; Mirzaie et al. 2022 ; Nesic et al. 2001 ; Shen et al. 2022 ). On the other hand, despite the availability of wide range of commercially produced antibodies against various apoptotic markers (including both, aC3 and cPARP), data regarding the apoptosis in intact spinal cord during postnatal development or in adulthood are still missing. Thus, present study focuses on the analyses of aC3 + and cPARP + populations in the intact spinal cord tissue at selected stages of ontogenesis, with the ambition to verify the validity of aC3 as an apoptotic marker in the intact nervous tissue. To accomplish our goal, we first compared the abundance of both aC3 + and cPARP + cell populations in the spinal cords of neonatal, preadolescent and adult rats. Second, employing an in vitro model with synchronously induced apoptosis by staurosporine treatment, we acquired the data necessary for deciphering the pattern of presence of aC3 and cPARP in cells undergoing apoptosis in spinal cord tissue samples. Finally, employing the gene expression profile analysis, we identified potential regulatory mechanisms that cells may employ to control caspase-3 activity under normal conditions in both, developing and mature nervous tissue. 2 Material and methods 2.1 Experimental animals All experiments on rats were performed in accordance with ARRIVE guidelines, the European Community Council (Directive 2010/63/EU) and in compliance with current national legislation, conducted with approval from the National Food Administration of the Slovak Republic under no. Ro-3051-5/2021 − 220 and the Animal Care Committee of the Pavol Jozef Šafárik University in Košice. Wistar albino rats (strain Crl:WI, RRID:RGD_2308816) were obtained from Velaz (Prague, Czech Republic). Standard laboratory conditions with a 12-hour light/dark cycle was used for housing the animals and each animal was fed a complete and balanced standard laboratory diet (Altromin International, Lage, Germany) and had ad libitum access to food and water. Experiments were performed on rats at the age of 8 postnatal days (P8, neonatal period, n = 11), 29 postnatal days (P29, preadolescent period, n = 6) and 90 postnatal days (P90, young adult period, n = 6). 2.2 Tissue isolation Experimental animals in each time point were terminally anesthetized with i.p . overdose of sodium thiopental (500 mg/kg). Animals (n = 3 animals per time point/experimental group) were either i) transcardially perfused with heparinized saline for gene expression analyses, or ii) transcardially perfused with heparinized saline followed by freshly prepared solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for immunofluorescent analyses. For immunofluorescent analyses, isolated lumbar segments (L3-L5) of spinal cord were postfixed in the same fixative at 4°C for 24 hours, followed by cryoprotection in 30% sucrose. Spinal cord samples were stored at 4°C until further processing. Isolated tissue samples were cut to 40 µm-thick coronal sections using freezing microtome (Leica CM1850, Leica Microsystems, Mannheim, Germany). For gene expression analyses, isolated segments of lumbar spinal cord tissue (20–50 mg) were immediately immersed in TRI Reagent™ Solution (Invitrogen, Vilnius, Lithuania, #AM9738) and stored at -80°C until further analysis. 2.3 Primary cell culture preparation Primary cell culture was derived from spinal cords of P8 rats (n = 5). Rats were terminally anaesthetized, decapitated and spinal cord was dissected. After meninges removal, spinal cords were cut to small pieces and transferred into a Papain Dissociation System solution (Worthington Biochemical Corporation, Lakewood, New Jersey, #LK003150) containing 0.01% papain and 0.01% DNase in line with the isolation protocol at 37°C with intermittent gentle shaking for 45 min. After digestion, excisions were further mechanically dissociated into cell suspension and centrifuged (300 g, 21 ℃, 10 min). Cell pellet was resuspended in DNase dilute albumin-inhibitor solution and discontinuous density gradient (70 g, 21 ℃, 10 min) was used to remove membrane fragments. Subsequently, cells were seeded into 24-well tissue culture plates with laminin-coated cover glasses on the bottom (TPP, Trasadingen, Switzerland) and cultivated in culture medium composed of Dulbecco’s Modified Eagle Medium (#L0102-500) and Ham’s F12 (#L0135-500; 1/1 v/v) (both Biosera, Nuaille, France) supplemented with 5% fetal bovine serum (Biowest, Nuaillé, France, #S1400), antibiotics (Penicillin/Streptomycin 10.000 U/10.000 mg/ml, Biochrom AG, Berlin, Germany, #2213B), 1% B-27 supplement (#17504044) and 0.5% N-2 supplement (#17502048) (both Gibco, Invitrogen, Carlsbad, California). Cells were cultivated under standard conditions in an incubator at 37 ℃, 5% CO 2 , and 95% humidity. In all wells, half of the medium volume was replaced every third day. 2.4 Establishment of in vitro model of apoptotic neural cell population Apoptotic effect of staurosporine on primary cell culture (7x10 4 cells/well) was analyzed on the thirteenth day of cultivation. 1mM staurosporine (Cell Signalling Technology, Leiden, Netherlands, #9953) diluted in DMSO was added to culture medium (1:1000 dilution ratio, final concentration = 1 µM.dm − 3 ) along with Incucyte® Caspase-3/7 Dye for Apoptosis (Sartorius AG, Göttingen, Germany, #4440) and Incucyte® Annexin V Dye for Apoptosis (Sartorius AG, #4641) at 1:1500 and 1:200 dilutions, respectively. The progress of development and distribution of apoptotic markers in a staurosporine-induced apoptotic population of cells was monitored using the IncuCyte™ ZOOM (Essen BioScience, Ann Arbor, Michigan) at Ex/Em 500/530 nm for Caspase-3/7 Dye and Ex/Em 593/614 nm for Annexin V Dye. Analysis was performed using IncuCyte ZOOM 2016B analysis software (Essen BioScience, RRID:SCR_019874) on nine microphotographs for each well (n = 4 wells) taken with a 10x objective lens (Supplemental Table 1) every 30 min for a total of 24 hours. For both markers, individual analysis procedure was prepared and uniformly used for every microphotograph captured (Supplemental Table 2). Results were compared with control data obtained in a similar manner from primary cell culture that was cultivated simultaneously in the staurosporine-free medium. 2.5 Collection of samples for in vitro time-dependent analysis of apoptotic markers On the fourteenth day of cultivation, time-dependent analysis of presence of apoptotic markers was conducted using primary cell culture treated with staurosporine diluted in culture medium (final concentration = 1 µM.dm − 3 ). After the addition of staurosporine to cells, primary cell cultures were fixed each hour during the cultivation, starting at time point 0h (no staurosporine effect) until 7 h time point. Cells were fixed by freshly prepared 4% paraformaldehyde in 0.1 M PB at room temperature for 20 min. Fixed primary cell cultures were then washed with 0.1 M phosphate buffered saline (PBS) and immunofluorescently labelled. For selection of antibodies both, staurosporine-free primary cell culture (negative control) and primary cell culture five hours after staurosporine administration (positive control), were used. 2.6 Immunofluorescent labelling Coronal sections of lumbar spinal cord and fixed primary cell cultures derived from spinal cord (henceforth referred as samples) were rinsed in 0.1 M PBS. If necessary, antigen retrieval was applied by immersion of samples to 10 mM citrate buffer (pH 6.0) at 95.5°C (± 0.5°C) for 10 min. After cooling down to room temperature, samples were washed by 0.1 M PBS. Non-specific protein activity was blocked by incubating samples with 5% solution of normal donkey serum (NDS, Jackson Immunoresearch, West Grove, Pennsylvania, #017-000-12) in 0.1 M PBS with 0.3% Triton-X 100 at 4°C overnight. Subsequently, samples were incubated in mixture of primary antibodies (Table 1 ) diluted in 0.1 M PBS containing 1% NDS and 0.3% Triton-X 100 at 4°C overnight. Subsequently, samples were washed with 0.1 M PBS and incubated with corresponding secondary antibodies (Table 1 ) diluted in 0.1 M PBS containing 1% NDS and 0.3% Triton-X 100 at room temperature in dark for 2 hours. Afterwards, samples were washed with 0.1 M PBS. To visualize cell nuclei, far red dye DRAQ5 (1:500, Cell Signalling Technology, #4084) diluted in 0.1 M PBS was applied to tissue samples for 20 min. Afterwards, tissue samples were washed in 0.1 M PBS, mounted on glass slides, dried and cover-slipped using ProLong Gold with DAPI (Invitrogen, #P36930). Table 1 List of antibodies used in the study Name Host Clonality Manufacturer Catalogue number Lot. number RRID Dilution Cleaved Caspase-3 (Asp175) Rabbit Polyclonal Cell Signaling Technology 9661S GR309480-1 AB_2341188 1:500 anti- Cleaved Caspase-7 (Asp198) (D6H1) Rabbit Monoclonal Cell Signaling Technology 8438S Lot 3 AB_1117837 1:100 anti-Cleaved PARP (Asp214) (D6X6X) Rabbit Monoclonal Cell Signaling Technology 94885 Lot 1 AB_2800237 1:100 anti-AIF (D39D2) Rabbit Monoclonal Cell Signaling Technology 5318 Lot 3 AB_10634755 1:250 anti-EndoG Rabbit Polyclonal Abcam ab9647 GR3181009-13 AB_2098770 1:250 anti-Fractin Rabbit Polyclonal Merck Millipore AB3150 2976523 AB_262159 1:1000 anti-actin (clone C4) Mouse Monoclonal Merck Millipore MAB1501 2951837 AB_2223041 1:200 anti-Olig2 (clone 211F1.1) Mouse Monoclonal Merck Millipore MABN50 3128845 AB_1080741 1:200 anti-NeuN (clone A60) Mouse Monoclonal Merck Millipore MAB377 2279235 AB_2298772 1:200 anti-S100 β Mouse Monoclonal Proteintech Group 66616-1-Ig 10004814 AB_2881976 1:100 anti – APC (Ab-7) (CC-1) Mouse Monoclonal Calbiochem OP80 D00150228 AB_2057371 1:200 anti-Tubulin ß 3 (TUBB3) (clone TUJ1) Mouse Monoclonal Covance Biolegend 801202 B233555 AB_1006340 1:500 anti-Rabbit IgG (H + L) conjugated s AlexaFluor 488 Donkey Polyclonal Abcam ab150073 GR3248726-1 AB_2636877 1:500 anti-Rabbit IgG (H + L) conjugated s AlexaFluor 555 Donkey Polyclonal Abcam ab150074 GR3241278-8 AB_2636997 1:500 anti-Mouse IgG (H + L) conjugated s AlexaFluor 555 Donkey Polyclonal Abcam ab150110 GR3446637-1 AB_2783637 1:500 anti-Mouse IgG (H + L) conjugated s AlexaFluor 594 Donkey Polyclonal Abcam ab150108 GR3360080-5 AB_2732073 1:500 2.7 Microscopic analysis and microphotographs preparation To analyze immunofluorescently labelled tissue samples, Leica TCS SP5X confocal system equipped with LAS AF software (Leica Microsystems) and Leica Thunder Imager DMi8 epifluorescent microscope equipped with LAS X software (Leica Microsystems, RRID:SCR_013673) using 10x, 40x and 100x objective lens (Supplemental Table 1) were used. Confocal imagining was done in XYZ mode (resolution 8 bits, 1024 x 1024 pixels, scanning speed 100 Hz, gain 600–750 V). Following excitation (Ex) and emission (Em) wavelengths were used for visualization of fluorophores: AlexaFluor 488 (Ex 488 nm, Em 500–540 nm); AlexaFluor 555 (Ex 555 nm, Em 565–590 nm); DRAQ5 (Ex 643, Em 655–690). Epifluorescent imaging was done in XYZ mode using Leica DFC9000 GTC camera (resolution 24 bits, 2048 x 2048 pixels). Following excitation and emission wavelength were used for visualization of fluorophores: DAPI (LED_405 filter cube Ex 405/60 nm, Em 470/40 nm); AlexaFluor 488 (GFP filter cube, Ex 470/40 nm, Em 525/50 nm). For analysis of aC3 + population of cells in spinal cord, five optical tissue sections (Z-stacks) per animal of P8, P29 and P90 spinal cords (n = 3 animals per time point) were captured using 40x objective lens. Stereological quantification of aC3 + cells was performed in ventral, lateral and dorsal funiculi, dorsal and ventral horns, and in the central grey matter (henceforth referred to as regions of interest - ROIs) using ConfoCounter software (Institute of Experimental Physics, SAS, Košice, Slovakia, available for free download from Microsoft Store at https://apps.microsoft.com/store/detail/confocounter/9PNSHZPXKHMM?hl=sk-sk&gl=sk&rtc=1 ). Densitometry analysis of the positivity of aC3 + nuclei was evaluated as relative intensity of fluorescence using Ellipse 2.0 software (ViDiTo, Košice, Slovak Republic). Abundance of cPARP + cells in spinal cord tissue was counted using 40x objective lens on ROIs of 20 tissue slices per animal of P8, P29 and P90 spinal cords (n = 3 animals per time point). To analyze primary cell cultures IncuCyte™ ZOOM system (Essen BioScience) using 10x and 20x objective lens (Supplemental Table 1) was used. Following parameters for imaging were used: resolution 24 bits, 1392 x 1040 pixels, Ex 460/40 nm and Em 524/40 nm; Ex 585/40 nm and Em 665/80 nm filter. Microphotographs were assembled to figures by ImageJ 1.53t (NIH, Bethesda, Maryland, RRID:SCR_003070); Adobe Illustrator (RRID:SCR_010279) and Adobe Photoshop (RRID:SCR_014199) (both Adobe Systems, San Jose, California). All modifications were limited to cropping, adjustment of brightness and contrast. 2.8 Identification of aC3 and cPARP after staurosporine-induced apoptosis in in vitro model To analyze presence of aC3 and cPARP in neuronal and oligodendroglial populations of primary cell culture treated by staurosporine, immunofluorescent co-localization using anti-TUJ1 and anti-APC antibodies was realized. Uniform analysis procedure (Supplemental Table 2) was prepared by IncuCyte ZOOM 2016B analysis software (Essen BioScience) and used to estimate the number of aC3 + and cPARP + cells per mm 2 in 14 microphotographs taken with a 10x objective lens from each well (n = 6 wells per experimental group). Colocalization of apoptotic (aC3, cPARP) and phenotypic markers (TUJ1, APC) in cells was identified as signal overlap and performed on n = 3 wells per experimental group. Co-localization was evaluated as percentage of neuronal or oligodendroglial cells. 2.9 Analysis of gene expression Gene expression analysis was performed to evaluate the abundance of inhibitors of apoptosis (IAPs, encoded by Birc genes) in the spinal cord tissue of P8, P29 and P90 rats (n = 3 per time point). The absolute amount of the studied gene transcripts was determined by digital PCR (dPCR). The analyzed cDNA was prepared by reverse transcription (RT) of total RNA, isolated by TRI Reagent™ Solution. Anchored oligo dT primer and RevertAid Reverse Trancriptase (Thermo Scientific, Vilnius, Lithuania, #EP0441) was used for the RT. Both protocols were performed according to manufacturer’s instructions. dPCR was performed by nanoplate-based technology QIAcuity Digital system (Qiagen, Hilden, Germany); QIAcuity One 5-plex instrument, 8.5K QIAcuity™ Nanoplates (#250021) and QIAcuity™ EG PCR Kit (#2500112) for amplification and detection of the products. Gene specific PCR primers (Supplemental Table 3) were designed according to the reference sequence of rat Birc1-7 genes by Primer-Blast (NIH, RRID:SCR_003095). Amplification of the desired product and lack of primer-dimers was confirmed by agarose gel electrophoresis and RT-qPCR. Depending on the gene, 10 to 50 ng of RNA/cDNA per 12µL reaction was used and amplified by 2-step PCR; 35 cycles (15 s – 95°C, 15 s – 60°C) preceded by denaturation at 95°C for 2 min. To verify the detection/quantification at linear range, half dilution of each sample was prepared and analyzed together with the undiluted sample. RNA/cDNA isolated from testes of P29 rats was used as a positive control for transcripts with low amount or absent PCR product in the spinal cord samples. Finally, the detected number of the individual Birc -gene transcripts was normalized to normalization factor (NF) based on expression of two reference genes ( Gapdh and eEF1a1 ), which were shown to be stably expressed during postnatal development (Košuth et al. 2020 ). The NF was calculated as geometric mean of copies of both reference genes. For genes with very low or absent transcripts in spinal cord, limit of quantification (LOQ) was determined. Serial dilutions of RNA/cDNA isolated from rat testes (tissue with high Birc genes expression) were used for the purpose. Then the LOQ of Birc1 , Birc3 and Birc7 transcripts per NF were 3.1–3.7x10 − 5 , 2.2–2.6x10 − 4 and 1.5–1.6x10 − 4 , respectively. 2.10 Statistics For statistical analysis and graph assembly, GraphPad Prism (version 9.0.0, RRID:SCR_002798) was used. Obtained data were analyzed using unpaired t-test; one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons; and repeated-measure two-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. A difference between groups or time points was considered statistically significant with a P value of p < 0.05 (*), 0.01 (**) and 0.001 (***). 3 Results 3.1 Selection of antibodies against apoptotic markers In order to explore the populations of cells undergoing apoptosis in intact spinal cord during postnatal development and adulthood, we decided in the first step to select the appropriate apoptotic marker(s) which should be targeted with commercially available antibodies. With respect to the requirement of unambiguous interpretation of immunofluorescence labelling, two basic criteria should meet the optimal apoptotic markers: specificity to the apoptotic cells and discrete appearance of the signal restricted optimally to the nucleus. Verified and widely used antibodies against AIF, EndoG, Fractin, aC3, aC7 and cPARP (Table 1 ) were tested in following samples: (i) intact primary in vitro culture derived from spinal cord (negative control), (ii) primary in vitro culture treated with inductor of apoptosis - staurosporine (positive control) and (iii) coronal sections of intact adult rat spinal cord (Fig. 1 A - J). Both AIF and EndoG were localized in cytoplasm of cells in the primary cell culture with or without apoptotic morphology ( i.e. pyknotic, semilunar, and shattered nucleus) (Fig. 1 A, B, D, E). However, AIF and EndoG were also detected in the cytoplasm of cells in the spinal cord tissue sections, most notably in motor neurons (Fig. 1 G, H). Similarly, anti-fractin antibody, which detects the cleaved form of actin as a result of caspase activity in apoptosis, was identified in both, cleaved (specific signal) and uncleaved (non-specific signal) forms of actin in cells of primary cell culture (Fig. 1 C, F). However, we also detected non-specific signal (colocalization of anti-fractin and anti-actin antibodies in the ependymal cells of the central canal) in spinal cord samples (Fig. 1 I). Also, aC7 staining was found in both, axons in white matter and cytoplasm of motor neurons (Fig. 1 J). Contrary to abovementioned apoptotic markers, presence of aC3 and cPARP signal was located in cell nuclei. aC3-antibody-stained nuclei in cell culture and tissue samples with various intensity with or without apoptotic morphology (Fig. 2 A - D). Based on the results of our densitometric analysis, we classified the nuclei to two categories: the “low intensity signal” and the “high intensity signal” with relative fluorescent intensity ranging in 9.23% ± 1.39% and 26.3% ± 6.92%, respectively (Supplemental Fig. 1A, B). For the purpose of the study, only the nuclei with the high intensity of signal were considered as positive. Immunofluorescent signal of antibody against 89 kDa fragment of protein PARP (cleaved PARP; cPARP) was identified mostly as perinuclear ring only in cells with apoptotic hallmarks, with no or only low background signal (Fig. 2 E - H). Taken together, our analysis revealed that cPARP could represent a proper marker of apoptosis in spinal cord, giving strong signal in fragmented cells, while aC3 seems to be present in broad population of cells with morphology ranging from normal to fragmented. This could be in line with the assumption that unlike cPARP, aC3 should occur earlier the cell progressing through the apoptotic cascade. Therefore, in the subsequent analyses we focused on characterization of aC3 + and cPARP + cell populations. 3.2 aC3 positive population of cells in spinal cord is much more abundant in comparison to cPARP positive cells In next step, we focused on detection of apoptotic cells in intact spinal cord tissue at subsequent stages of ontogenesis: neonatal period (P8), preadolescent period (P29) and adulthood (P90). According to our results, immunofluorescence analysis of aC3 + cells and cPARP + cells shows a large discrepancy between studied populations as well as changes within population abundance during rat ontogenesis (Fig. 2 A - J). Based on the results of quantification analysis, we calculated that the ratios of aC3 + to cPARP + in the spinal cord tissue were 593:1 (P8), 4490:1 (P29), and 487:1 (P90), respectively (Fig. 2 I). The aC3 + cell population was the most prevalent in P8 rats (307,212.83 ± 20,710.03 cells/mm 3 ) and diminished significantly in the later phases of ontogenesis (P29: 80,166.35 ± 6,428.40 cells/mm 3 ; P90: 47,518.84 ± 20,7410.03 cells/mm 3 ). cPARP + cells were also most prevalent in P8 spinal cord tissue (613.40 ± 70.936 cells/mm 3 ), contrary to P29 (24.33 ± 7.37 cells/mm 3 ) and P90 (97.51 ± 16.85 cells/mm 3 ) intervals. In addition to differences in abundance of aC3 + and cPARP + populations, we also observed differences between distributions of both populations in analyzed areas of the spinal cord on coronal sections (also called as region of interest, ROI) (Fig. 2 J). At P8, the majority of aC3 + cells were identified in the white matter (mainly in the ventral and lateral funiculus). In P29 and P90 rats, aC3 + cells were most abundant in the grey matter, mainly in the dorsal horn and the central and lateral grey matter. On the contrary, cPARP + cells were mostly found in the dorsal horn at P8. The abundance of these cells decreased in all ROIs during the later stages of ontogenesis, with the exception only in the dorsal funiculus of P90 rats. Significant differences in the abundance and distribution of the aC3 + and cPARP + cells confirm the presumption that unlike cPARP, aC3 should occur earlier and possibly longer in the cell progressing through the apoptotic cascade. Therefore, aC3 + population could represent more abundant group of cells with early caspase-3 activation contrary to a significantly smaller population of cells with late cleavage of PARP during the apoptosis. The other explanation for significantly larger population of aC3-positive cells could be either the inhibition of aC3, or its possible non-apoptotic functions in cells of spinal cord. In order to decide which hypothesis is more relevant, in the next step we studied the presence of both, aC3 and cPARP in cells after apoptotic stimulus employing in vitro model of primary cell culture isolated from P8 rat spinal cord. 3.2 Employment of in vitro apoptotic model for determination of involvement of aC3 and cPARP throughout the progression of apoptotic cascade Previous analyses revealed significant difference between the number of aC3 + and cPARP + cells in postnatal rat spinal cord. In the next step, we focused on determination, whether this discrepancy between the size of both populations reflects the discrepancies in the time windows, when these markers are detectable in the cells undergoing apoptosis. In order to identify the period, when aC3 and cPARP are detectable in apoptotic cells, we performed in vitro test on the caspase-3 activation and PARP cleavage using primary cell culture isolated from P8 spinal cord. To obtain the synchronous populations of apoptotic cells, primary cell culture was treated by staurosporine, which should trigger the apoptosis. In order to get the very first overview about the effect of staurosporine on the primary cell culture, we took the advantage of real-time live-cell imaging and analysis using platform IncuCyte™ ZOOM systems. Effect of staurosporine treatment on primary cell culture in the 24 hour period was analyzed by Casp3/7 dye (substrates indicating activation of both caspase-3 and caspase-7) and by Annexin V dye (substrate indicating the externalization of phosphatidylserine) (Fig. 3 A - C). Compared to interval 0 hours ( i.e. the time just before induction of apoptosis), we found a significant increase in the number of cells with active effector caspases, as well as cells with externalized phosphatidylserine 3.5 hours after staurosporine treatment. Numbers of aC3/7 + cells reached plateau at ninth hour while number of Annexin V + cells continuously raised in all intervals with significant overgrow of aC3/7 + population at 7.5 hours after the staurosporine administration (two-way ANOVA, p < 0.05). Based on results of previous in vitro studies, we presume that higher numbers of Annexin V + cells in later intervals indicate the presence of alternative forms of regulated and/or unregulated cell death (Shlomovitz et al. 2019 ). Therefore, for reduction of the impact of other forms of cell death on our measurements, we decided to investigate possible time delay between the activation of caspase-3 and cleavage of PARP in main experiment during first seven hours after administration of staurosporine. Subsequently, we investigated temporal relationship between caspase-3 activation and PARP cleavage in primary cell culture during period 0–7 hours after the induction of apoptosis with staurosporine. According to our results, number of aC3 + (in line with our previous experimental data) and cPARP + cells significantly increased during the fourth hour after induction of apoptosis (Fig. 4 A). Thus, our data indicate the onset of presence of detectable levels of aC3, as well as cPARP in cells in approximately similar time, 3.5 hour since the beginning of induction of apoptosis. In the next step, we decided to investigate, whether there are any differences between the induction of apoptosis and onset of presence of aC3 and cPARP in two major phenotypes, present in the primary cell culture sensitive to staurosporine; neurons (identified by anti-Tuj-1 antibody) and oligodendroglial cells (identified by anti-APC antibody) (Fig. 4 B, C). Our analyses revealed that TUJ-1 + neurons show similar distribution of both apoptotic markers as the whole primary cell culture in examined time points after staurosporine treatment (Fig. 4 B, D, E). On the other hand, we noticed high number of aC3 + /APC + oligodendrocytes even before the induction of apoptosis and during the subsequent period until third hour (Fig. 4 C - E). During fourth hour the number of aC3 + /APC + oligodendrocytes increased only moderately contrary to PARP + oligodendrocytes, which levels followed similar pattern as the whole population and TUJ-1 + neurons (Fig. 4 A, B). Based on the data obtained from in vitro model, we assume that both, temporal activation of caspase-3 and cleavage of PARP in studied populations, occur at similar time points after induction of apoptosis. Our results indicate that due to the similar time windows, when aC3 and cPARP are present in cells undergoing apoptosis, the size of aC3 + and cPARP + populations should be also similar in tissues, where both markers are involved solely in the apoptotic cascade. Thus, due to the significant discrepancy between the size of aC3 + and cPARP + populations in the spinal cord tissue, it can be concluded that aC3 may be inhibited, or involved in other, non-apoptotic processes. 3.3 Caspase-3 activation occurs mostly in glia of postnatal rat spinal cord Since in vitro analyses revealed possible activation of caspase-3 in non-apoptotic cells, especially in glial cells, we decided to examine, whether phenotypic representation of aC3 + population identified in in vitro experiments resembles to real conditions in spinal cord tissue. Colocalization study of aC3 and selected phenotypic markers of neurons, oligodendrocytes and astrocytes proved that the aC3 + population of the spinal cord tissue is primarily composed of glial cells (Fig. 5 A - H). Surprisingly, unlike the aC3 + populations in the spinal cords of P8 and P90 rats, which predominantly consist of S100β + astrocytes, the aC3 + population in P29 rats is composed mostly of Olig2 + oligodendrocytes (Fig. 5 A). Single ROI aC3 + composition analysis revealed the same result as whole tissue analysis. Just a few exceptions were discovered. Less aC3 + /Olig2 + oligodendrocytes were seen in the P8 ventral and lateral funiculus. On the other hand, there was a huge amount of aC3 + oligodendrocytes in the ventral horns of P29 and P90. In comparison to glial cells, NeuN + neurons represent smaller portion of the spinal cords aC3 + population. At all ages studied, aC3 + /NeuN + neurons were identified mostly in the dorsal horns (Fig. 5 B). Furthermore, examining the abundance of aC3 + cells in individual populations provided even more intriguing results. In S100β + astroglial population, substantial portion of all analyzed cells was aC3 positive. At P8, most of S100β + cells colocalized with aC3, and were highly abundant in all analyzed ROIs. In the central and lateral grey matter, almost all analyzed astrocytes were aC3 + positive. Even though the number of aC3 + astrocytes significantly decreased in the later stages of ontogenesis, they still represented most of all astrocytes in almost all analyzed ROIs (Fig. 5 C). A significant number of aC3 + cells can also be seen in the Olig2 + oligodendroglial population. Around a quarter of all cells of P8 and P90 rat spinal cord are positive for activated caspase-3. While the proportion of aC3 + /Olig2 + oligodendrocytes is comparable within the ROIs studied at P29 and P90, there is a greater proportion of positive aC3 + /Olig2 + cells in the ROIs of grey matter of the spinal cord at P8 (Fig. 5 D). Interestingly, only 9.44% of all Olig2 + oligodendrocytes in the P8 ventral funiculus were also positive for aC3. In NeuN + neuronal population, we identified only a small portion of aC3 + cells, with significantly higher representation only in dorsal horn of grey matter (Fig. 5 E). Taking together, our data indicate that caspase-3 is activated in only small population of neurons, while most of glial cells, especially astrocytes, contain detectable levels of aC3. In comparison to aC3, cPARP + population may be difficult to co-localize to phenotypic markers due to the fact that morphology in most of cPARP + cells of spinal cord resembled to apoptotic phenotype and phenotypic markers were often missing due to degree of fragmentation of the cell body. In contrast to cPARP + cells, most of the aC3 + cells have normal morphology, indicating that activation of caspase-3 in rat spinal cord could play an important role in other processes, especially in glia. 3.4 Activity of aC3 in rat spinal cord may be regulated by specific IAP proteins With respect to the number of cells with detectable levels of aC3 present in the spinal cord tissue on one side, and the diminutive number of cells with apoptotic phenotype on the other side, we presumed that there could be some mechanisms regulating the caspase activity in those aC3 + cells, which obviously do not undergo the apoptosis. One possible explanation could be the inhibition of aC3 activity by specific proteins known as Inhibitors of Apoptosis (IAPs), which could maintain the aC3 in steady state in the cells. Therefore, we decided to examine the expression of all known Birc genes ( Birc1-7 ), encoding the members of IAP protein family in the rat spinal cord tissue at all selected ontogenetic stages (P8, P29 and P90). Absolute amounts of their transcripts were determined by digital PCR (dPCR). We found that majority of the IAPs ( Birc1 , 3 , 6 and 7 ) are only slightly expressed in the rat spinal cord (Fig. 6 A - C). On the other hand, the most abundant gene transcripts represented Birc4 , Birc2 , and Birc5 . Collectively, these three IAPs constituted more than 90% of the whole IAP gene family (98% at P8, 96% at P29 and 91% at P90). The most highly expressed IAP represented Birc4 ( XIAP ). mRNA transcripts of XIAP comprised more than 50% of all IAP gene transcripts at P8 and raised to more than 70% at P29 and P90. mRNA level of Birc4 was approximately two times (P8) or even four times (in both, P29 and P90) higher than the level of Birc2 . The second IAP gene with highest level of expression was Birc2 , with more than 20% and 15% share on all AIP transcripts at P8 and P29 and P90 spinal cord, respectively. The amount of this, relatively abundant gene transcript, seems to be very steady in spinal cord, without apparent changeover during postnatal life. Similarly, high copy number as Birc2 was recorded also by Birc5 ( Survivin ), but only in neonatal spinal cord. In older individuals, the activity of Birc5 transcription gradually decreased (2.5-fold at P29 and 4.5-fold at P90). In comparison with other IAPs, Survivin represented the only gene with negative regulation during ontogenesis. Taken together, our analysis proved the presence of considerable levels of several IAPs ( Birc4 , Birc2 and Birc5 ) in the spinal cord tissue, which could be responsible for inhibition of caspase-3 activity. This could be the possible explanation of the discrepancy between the amounts of PARP + and aC3 + cells in the rat spinal cord during postnatal life. 4 Discussion Due to critical role in development and pathological states of spinal cord (Erekat 2022 ; Springer 2002 ; Yuan and Yankner 2000 ) both apoptosis and caspase-3 are intensively investigated topics of neuroscience. aC3, regarded as a crucial protein of apoptosis (Lakhani et al. 2006 ; Walsh et al. 2008 ), interact with numerous proteins leading to characteristic changes in morphology and biochemistry of cell (Taatjes et al. 2008 ). At the same time, multiple studies reported activity of caspase-3 in non-apoptotic processes (Fernando et al. 2002 ; Fujita et al. 2008 ; Miura et al. 2004 ; Szymczyk et al. 2006 ; Woo et al. 2003 ). Nevertheless, numerous experimental studies have investigated apoptosis in the spinal cord, especially under pathological conditions, by detecting caspase-3 activity (Dai et al. 2019 ; Gülmez et al. 2022 ; Mirzaie et al. 2022 ; Nesic et al. 2001 ; Shen et al. 2022 ); On the other hand, studies investigating the apoptotic processes in the intact developing nervous system employing the commercially available antibodies are lacking. In our study, we provided a comprehensive analysis of the aC3 + cell populations in rat spinal cord at selected stages of postnatal life and challenged the relevance of aC3 as an apoptotic marker during non-pathological conditions utilizing in vivo and in vitro models. To revise the relevance of aC3 protein as an apoptotic marker in intact spinal cord tissue, we compared population of aC3 + cells with cPARP immunoreactive cells in various experimental contexts. Prior to the analyses, we tested several commercially available antibodies designed against the common apoptotic markers AIF, EndoG, Fractin, aC3, aC7 and cPARP. In the analysis, the optimal apoptotic marker(s) should meet two important criteria: specificity to the apoptotic cells and discrete appearance of the signal restricted optimally to the nucleus. In comparison to other markers, cPARP proved to be a reliable marker of apoptotic cells, probably due to its ability to bind with newly generated epitopes produced by activated effector caspase during the apoptosis (Soldani and Scovassi 2002 ). Contrary to anti-cPARP antibody, antibodies against other apoptotic markers did not show same level of specificity. In primary cell culture, AIF and EndoG were detected in both cells with and without morphological changes associated with apoptosis. This can be explained by the fact that during apoptosis, AIF and EndoG only change their localization in intracellular space and do not generate new epitopes in their structure (Joza et al. 2009 ; Li et al. 2001 ). We also focused on detection of fractin, a cleaved form of actin produced by caspase activity (Rossiter et al. 2000 ). Although we were able to identify fractin + cells with hallmarks of apoptotic morphology, it was revealed that anti-fractin antibody is at least partially immunoreactive to uncleaved actin. Furthermore, we tested an antibody against aC7, another effector caspase involved in apoptosis. According to our results, we detected aC7 immunoreactivity primarily in the cytoplasm and neural processes of cells without clear signs of apoptosis. This observation was in line with previous study, which confirmed the general activity of effector caspases 3, 6 and 7 in neural processes (D’amelio et al. 2010 ). Thus, massive immunoreactivity in cytoplasm disqualified anti-aC7 antibody from detection of apoptotic cell. According to our results on quantification of both, aC3 + and cPARP + cells, spinal cord tissue contains massive population of aC3 + cells with non-homologous distribution of cells, neither between the studied ROIs of spinal cord, nor during the studied spinal cord ontogenesis phases. aC3 + population was most abundant in the neonatal spinal cord (mainly in white matter) and its number declined significantly in later stages of ontogenesis. Majority of observed aC3 + cells did not display any apoptotic morphological changes. On the contrary, we identified only a small population of cells containing p89 fragment of PARP, which is specifically generated during apoptosis (Soldani and Scovassi 2002 ). In addition, the cPARP population differs from aC3 + cells not only in size, but also in the distribution of cells across ROIs in neonatal spinal cord, with the majority of the cPARP + cells present in the grey matter, especially in the dorsal horns. The localization of the cPARP + cells is consistent with the previously identified population of apoptotic neurons (Lawson et al. 1997 ), which undergo programmed cell death as the result of their inability to be involved into the functional neuronal circuits in dorsal horns during early postnatal period (Prasad et al. 2008 ). For similar reasons, ablation of superfluous neurons could be expected also in other areas of grey matter. Intriguingly, we discover a substantial proportion of cPARP + glial cells showing nuclear alterations in the dorsal funiculus of P90 rats. However, more investigation is required to determine the role of apoptosis in the dorsal funiculi in adulthood. In general, majority of cPARP + cells displayed hallmarks of apoptosis (semilunar, condensed or fragmented nucleus) similarly, as described in Taatjes et al. ( 2008 ), and significant subgroup of these cells was absent of the phenotypic markers. This prevented us from specifying phenotypic characteristics of cPARP + cells in our study. The accumulation of aC3 + cells in the spinal cord (mostly without apoptotic morphology), as well as striking differences in size between cPARP + and aC3 + populations, could be explained in two ways, as (i) significant time-delay in caspase-3 activation followed by the PARP cleavage; or (ii) the aC3 involvement in non-apoptotic processes. Based on previous experimental studies, aC3 directly cleaves PARP in the apoptotic pathways (Duriez and Shah 1997 ; Soldani and Scovassi 2002 ). Therefore, the presence of cPARP in cells is considered as the proof of the aC3 activity (Li et al. 2000 ). At the same time, PARP cleavage prevents necrosis during apoptosis, ensuring appropriate execution of caspase-mediated programmed cell death. Therefore, cleavage of PARP by caspases is considered to be a hallmark of apoptosis (Herceg and Wang 1999 ). The cleavage of PARP has been observed in early stages of apoptosis, even before DNA degradation in multiple type of tumor cells (Soldani et al. 2001 ) and cPARP has been referred to as an early apoptotic marker (Kaufmann et al. 1993 ). However, to our knowledge, the dynamics of aC3 and cPARP in neural cell population has not been studied before. In order to test our hypothesis, we investigated the time-dependent relationship between the presence of both markers in the apoptotic cells. Primary cell culture prepared from neonatal spinal cord was used to simulate heterogeneity of spinal cord tissue containing cell populations at various stages of cell cycle and/or the level of cell differentiation. In the primary cell culture, apoptosis was triggered by the administration of staurosporine, well established apoptotic inductor commonly used in experimental in vitro studies of neuronal apoptosis (Negishi et al. 2003 ; Prince and Oreland 1997 ; Yu et al. 2007 ). Compared to other apoptotic inductors, which action is based on inducing oxidative stress (Simon et al. 2000 ), or inhibiting of specific enzymes involved in cell proliferation (Park et al. 1997 ; Wang et al. 2000 ), staurosporine triggers apoptosis through both mechanisms: kinase inhibition (Karaman et al. 2008 ), as well as induction of oxidative stress (Kruman et al. 1998 ). Thus, staurosporine should induce apoptosis in cells regardless of the state of differentiation or cell cycle phase (Bertrand et al. 1994 ). Due to staurosporine ability to induce caspase-dependent apoptosis (Belmokhtar et al. 2001 ) resulting in the activation of aC3 and cPARP proteins, which could be detected by antibodies, our model proved to be a useful tool for studying the aC3 - cPARP time-dependent relationship. On the other hand, higher abundance of Annexin V + cells compared to aC3/7 + cells in primary cell culture 7.5 hours after staurosporine induction was caused probably by its ability to induce also caspase-independent apoptosis (Belmokhtar et al. 2001 ), or by an increase of the amount of necrotic and necrotic-like cells that are falsely positive for Annexin V in the cell culture (Shlomovitz et al. 2019 ). Therefore, our model does not offer an universal instrument for studying apoptosis in neural cells, but rather a specific method for caspase-substrate dynamic research. In line with data from an in vitro analysis of staurosporine impact on the activity of effector caspases 3/7 and the abundance of Annexin V + cells, we found a significant increase in the number of both, aC3 + cells as well as cPARP + cells at the fourth hour after synchronous induction of apoptosis in the primary culture. Therefore, we assume that consecutive activation of both, aC3 followed by the cleavage of PARP should results in similar sizes of aC3 + and cPARP + populations in the nervous tissue containing asynchronous populations of apoptotic cells in vivo . Thus, discrepancy in the size of aC3 + and cPARP + populations may be caused by the role of aC3 + cells in non-apoptotic processes during which PARP cleavage does not occur. Similar observations were already made in neural cells of murine neutrospheres, in which aC3 endogenous activity without PARP cleavage was present during differentiation of nestin + precursors to astrocytes, oligodendrocytes and neurons (Fernando et al. 2005 ). Elevated number of aC3 + oligodendrocytes in employed in vitro model in intervals even before apoptosis induction, as well as surprisingly abundant number of aC3 + cells in astroglial and oligodendroglial populations in spinal cord tissue indicate that aC3 may play important non-apoptotic role mainly in glial cells. Presence of aC3 in astrocytes and radial glia like cells was already reported in multiple studies. Non-apoptotic functions of aC3 were observed during process of astrogliosis and astrocyte cytoskeletal remodelation induced by various pathological conditions (Acarin et al. 2007 ; Aras et al. 2012 ; Guyenet et al. 2013 ), or even after non-pathological stimuli like exercise (Stevenson et al. 2018 ) in various parts of the rodent brain. Activity of caspase-3 was also observed during differentiation of Bergman glia of cerebellum in rats (Oomman et al. 2006 ). In small population of aC3 + neurons, aC3 may as well participate in several non-pathological processes required for remodelation and the establishment of neural networks. Caspase-3 proved to be active during long-term potentiation of synapses in the CA1 region of the rat hippocampus (Gulyaeva et al. 2003 ), during the long-term phase of synaptic input sensitization in a terrestrial snail Helix lucorum (Bravarenko et al. 2006 ), and in the auditory forebrain of the Zebra finch during long-term habituation to tape-recorded birdsongs (Huesmann and Clayton 2006 ). More intriguing, it has been proposed that caspase-3 is already present in active form in Zebra finch postsynaptic neurites and its activity is regulated by the interaction with the apoptotic inhibitor XIAP (encoded by Birc4 gene) (Huesmann and Clayton 2006 ). This is consistent with our investigation of IAPs gene expression in the rat spinal cord, which revealed that XIAP is the most expressed inhibitor of apoptosis in rats at all studied ages. We observed increased levels of mRNAs encoding rIAP-1 protein (encoded by Birc2 ), whose presence may be explained by fact, that the protein is involved in regulation of multiple signal pathways besides the inhibition of apoptosis (Saleem et al. 2013 ). We also observed elevated levels of Survivin ( Birc5 ) expression in P8, which is associated with proliferation and also should be capable to bond with aC3 (Shin et al. 2001 ). Higher expression levels can be explained by higher proliferation rate of cells during the early stages of postnatal development in spinal cord tissue, which is attenuated in later ages (Alexovič Matiašová et al. 2017 ). Due to the ability of both XIAP and Survivin to establish bonds with aC3 (Riedl et al. 2001 ), we hypothesize that aC3 could be in activated, but still inhibited state. Dynamic dissociation and re-binding of XIAP or Survivin to active site of aC3 probably allow to regulate the activity of aC3 spatially and temporally (Huesmann and Clayton 2006 ). However, to confirm our hypothesis, further research is necessary. In conclusion, we observed a large population of aC3 + cells in the spinal cord during neonatal and preadolescent development and in adulthood that do not correspond to the size of population of cPARP + cells, which are considered to be truly apoptotic cell population. Based on data from in vitro analysis of the time-dependent interaction between aC3 and cPARP, we assume that aC3 may be inhibited and/or involved in other non-apoptotic processes. One of the possible explanations is that the activity of aC3 is regulated by Birc4 (XIAP) , Birc2 and/or Birc5 (Survivin) genes by forming the “stand-by” complex with aC3. However, further investigation is required to determine the definitive nature of the interaction between IAPs and aC3 in spinal cord tissue. Our data also demonstrated that aC3 + should not be considered as an exclusive apoptotic marker and other markers/methods should be considered for the detection of apoptotic cells, especially in intact nervous tissue. Declarations Acknowledgements: Authors thank to Bc. Ladislav Pačut, Viera Balážová and Eva Pastorová for technical assistance. This study was supported by VEGA no. 1/0760/20, VEGA no. 2/0101/22 and APVV-19-0279. Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Brain Res Bull 43:515–523. https://doi.org/10.1016/S0361-9230(97)00328-6 Riedl SJ, Renatus M, Schwarzenbacher R, et al (2001) Structural basis for the inhibition of caspase-3 by XIAP. Cell 104:791–800 https://doi.org/10.1016/S0092-8674(01)00274-4 Rossiter JP, Anderson LL, Yang F, Cole GM (2000) Caspase-cleaved actin (fractin) immunolabelling of Hirano bodies. Neuropathol Appl Neurobiol 26:342–346 https://doi.org/10.1046/j.1365-2990.2000.00252.x Saleem M, Qadir MI, Perveen N, et al (2013) Inhibitors of apoptotic proteins: new targets for anticancer therapy. Chem Biol Drug Des 82:243–251 https://doi.org/10.1111/cbdd.12176 Shen X, Zhang Y, Zhu H, et al (2022) Triad1 promotes the inflammatory response and neuronal apoptosis to aggravate acute spinal cord injury in rats. Comput Math Methods Med 2022: https://doi.org/10.1155/2022/2025756 Shin S, Sung B-J, Cho Y-S, et al (2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and-7. 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Supplementary Files Supplemetarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 08 Nov, 2023 Read the published version in Histochemistry and Cell Biology → Version 1 posted Editorial decision: Major revision 05 Jun, 2023 Reviews received at journal 16 May, 2023 Reviewers agreed at journal 14 May, 2023 Reviewers agreed at journal 25 Apr, 2023 Reviewers invited by journal 25 Apr, 2023 Editor assigned by journal 25 Apr, 2023 Submission checks completed at journal 25 Apr, 2023 First submitted to journal 24 Apr, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2854960","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":194875372,"identity":"3d677ebb-a459-45b4-a617-09775ea6fc5d","order_by":0,"name":"Radovan Holota","email":"","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":false,"prefix":"","firstName":"Radovan","middleName":"","lastName":"Holota","suffix":""},{"id":194875373,"identity":"402f4a3f-45e6-442f-af70-2a0b4e9ddeb6","order_by":1,"name":"Viktória Buľková","email":"","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":false,"prefix":"","firstName":"Viktória","middleName":"","lastName":"Buľková","suffix":""},{"id":194875374,"identity":"fdaf4bfe-7624-4c98-9100-eb83d09bba5c","order_by":2,"name":"Anna Alexovič Matiašová","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"Alexovič","lastName":"Matiašová","suffix":""},{"id":194875375,"identity":"a91a249a-b81f-4b67-afa9-a8b731207c1b","order_by":3,"name":"Ján Košuth","email":"","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":false,"prefix":"","firstName":"Ján","middleName":"","lastName":"Košuth","suffix":""},{"id":194875376,"identity":"26886d23-8e13-4c18-b8f9-df9d53acd023","order_by":4,"name":"Lucia Slovinská","email":"","orcid":"","institution":"Associated Tissue Bank, Faculty of Medicine of P. J. Šafárik University in Košice and L.Pasteur University Hospital, Tr. SNP 1, 040 11 Košice","correspondingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"","lastName":"Slovinská","suffix":""},{"id":194875377,"identity":"301fa83a-a488-4acd-8868-cff48c2dee7f","order_by":5,"name":"Zoltán Tomori","email":"","orcid":"","institution":"Institute of Experimental Physics, Slovak Academy of Sciences, 040 01 Košice","correspondingAuthor":false,"prefix":"","firstName":"Zoltán","middleName":"","lastName":"Tomori","suffix":""},{"id":194875378,"identity":"da58eb34-0e95-43b7-9605-e01f592044a8","order_by":6,"name":"Zuzana Daxnerová","email":"","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":false,"prefix":"","firstName":"Zuzana","middleName":"","lastName":"Daxnerová","suffix":""},{"id":194875379,"identity":"38db6012-d734-4db6-a01b-3209b37a8b58","order_by":7,"name":"Juraj Ševc","email":"","orcid":"","institution":"Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice","correspondingAuthor":false,"prefix":"","firstName":"Juraj","middleName":"","lastName":"Ševc","suffix":""}],"badges":[],"createdAt":"2023-04-24 13:29:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2854960/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2854960/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00418-023-02249-7","type":"published","date":"2023-11-08T15:01:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36393262,"identity":"acbc0d92-c0d5-4511-959b-eb94745d992f","added_by":"auto","created_at":"2023-04-27 19:28:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3813813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of apoptotic markers in untreated and staurosporine treated primary cell cultures and in intact spinal cord. \u003c/strong\u003eMicrophotographs of primary cell cultures without staurosporine treatment (A – C; negative control) and five hours after staurosporine treatment (D – F; positive control) immunolabelled by anti-AIF (A, D), anti-endoG (B, E) and anti-fractin (C, F) antibodies (magenta) with highlighted nuclei (DAPI, gray). Apoptotic morphology is indicated by arrows. Microphotographs of of P90 rat spinal cord labelled with anti-AIF (G) and anti-EndoG (H) antibodies (magenta) and anti-NeuN antibody (cyan). White squares indicate motor neurons. Microphotographs of P90 rat spinal cord labelled with anti-fractin (magenta) and anti-actin (cyan) antibodies (I), with detailed pictures of fractin\u003csup\u003e+\u003c/sup\u003e cell with fragmented nucleus (DRAQ5, grey) (I1) and apical cytoplasm of ependymal cells positive both for presence of fractin and actin (I2). Cross section of aC7\u003csup\u003e+\u003c/sup\u003e nerve fibers in lateral funiculus (J) and motor neurons (J4).\u003c/p\u003e\n\u003cp\u003eScale bar in A – F and G –J (insets) = 25 µm; scale bar in G – J (main) = 100 µm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/909cfc60002cd6776c6e8988.png"},{"id":36394307,"identity":"1456abd6-fa61-4eaa-94d8-ace6177a31a5","added_by":"auto","created_at":"2023-04-27 19:52:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1511887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of aC3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and cPARP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e populations in postnatal rat spinal cord. \u003c/strong\u003eRepresentative microphotographs of single optical section of spinal cords of P8 (A), P29 (B) and P90 (C) rats with detail microphotographs of selected ROIs showing abundance and distribution of aC3\u003csup\u003e+\u003c/sup\u003e population (magenta) in spinal cords (1 - 6). Detailed microphotograph shows typical morphology of aC3\u003csup\u003e+\u003c/sup\u003e (magenta) and DRAQ5\u003csup\u003e+\u003c/sup\u003e nucleus (grey) (D). Representative microphotographs of single optical section of spinal cord of P8 (E), P29 (F), P90 (G) rats with detail microphotographs of selected ROIs showing abundance and distribution of cPARP\u003csup\u003e+\u003c/sup\u003e population (cyan) in spinal cords (7 - 12). Detailed microphotograph shows fragmented cPARP\u003csup\u003e+\u003c/sup\u003e (cyan) and DRAQ5\u003csup\u003e+\u003c/sup\u003e nucleus (grey) (H). A comparison of the populations studied in the P8, P29, and P90 rat spinal cords (I). The overall number of cells in both groups was expressed as a weighted average ± SD of the individual ROIs. Statistical comparisons were made between the aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations within analyzed age interval (dashed black line; unpaired t-test). The changes in the population of aC3\u003csup\u003e+\u003c/sup\u003e cells (black line) and cPARP\u003csup\u003e+\u003c/sup\u003e cells (grey line) during ontogenetic development were statistically analyzed using one-way ANOVA. Heatmaps of distribution of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations in analyzed ROIs in spinal cord tissue with corresponding mean value of cells/mm\u003csup\u003e3 \u003c/sup\u003e(J).\u003c/p\u003e\n\u003cp\u003eVF – ventral funiculus, LF – lateral funiculus, DF – dorsal funiculus, DH – dorsal horn, CLGM – central and lateral grey matter, VH – ventral horn. Statistical significance: ns – non significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Scale bar in A, B, C, E, F, G = 100 µm; scale bar in D, H = 10 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/268ff482ab990b534212d682.png"},{"id":36394206,"identity":"738ce8ba-08e7-4cba-9916-3f7e9839824b","added_by":"auto","created_at":"2023-04-27 19:44:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7203770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of staurosporine effect on primary cell culture derived from P8 spinal cord\u003c/strong\u003e. Effect of staurosporine treatment on primary cell cultures in twenty-four-hour period of time was measured by activation of caspase-3/7 (A) and by externalisation of phosphatidylserine (Annexin V) (B). Primary cell culture without staurosporine was used as a negative control. In both, abundance is expressed as mean value of cells/mm\u003csup\u003e2\u003c/sup\u003e ± SD. Changes in abundance of cells were compared to interval 0 hours of staurosporine treatment (= unaffected by staurosporine; repeated measures two-way ANOVA). Representative microphotograph of primary cell cultures in selected time points (C). Microphotographs show phase contrast, aC3/7\u003csup\u003e+\u003c/sup\u003e positive cells (magenta) and Annexin V\u003csup\u003e+\u003c/sup\u003e cells (cyan).\u003c/p\u003e\n\u003cp\u003eStatistical significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Scale bar = 300 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/f9fa7ad6519404526c05cf6b.png"},{"id":36393891,"identity":"5e6aa060-eaac-4bb7-9081-bf00294b27a6","added_by":"auto","created_at":"2023-04-27 19:36:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1292147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis of presence of aC3 and cPARP in apoptotic cells on fixed, staurosporine treated primary cell culture. \u003c/strong\u003eTime-dependent analysis of abundance of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cells in staurosporine treated primary cell culture (A). Abundance of both markers is expressed as mean value of cells/mm\u003csup\u003e2\u003c/sup\u003e ± SD. In comparison to interval 0 (interval without staurosporine effect, ANOVA), we observed increased number of aC3\u003csup\u003e+\u003c/sup\u003e cells and cPARP\u003csup\u003e+\u003c/sup\u003e cells in the same time point. Time dependent analysis of changes in proportion of aC3\u003csup\u003e+\u003c/sup\u003e/TUJ-1\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e/TUJ-1\u003csup\u003e+\u003c/sup\u003e neurons (B) and aC3\u003csup\u003e+\u003c/sup\u003e/APC\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e/APC\u003csup\u003e+\u003c/sup\u003e oligodendrocytes (C) in the whole TUJ-1\u003csup\u003e+\u003c/sup\u003e neuronal or APC\u003csup\u003e+ \u003c/sup\u003eoligodendroglial populations (ANOVA). Abundance of cells is expressed as mean percentage of neurons or oligodendrocytes in the whole aC3\u003csup\u003e+\u003c/sup\u003e or cPARP\u003csup\u003e+\u003c/sup\u003e population ± SD. TUJ-1+ neurons show similar distribution of both apoptotic markers as the whole primary cell culture in examined time points after staurosporine treatment. While APC\u003csup\u003e+\u003c/sup\u003e/cPARP\u003csup\u003e+\u003c/sup\u003e oligodendrocytes show similar distribution as whole primary cell culture, we observed higher representation APC\u003csup\u003e+\u003c/sup\u003e/aC3\u003csup\u003e+\u003c/sup\u003e cells from beginning of experiment. The increase of the abundance is indicated with black arrow (aC3\u003csup\u003e+\u003c/sup\u003e cells) and grey arrow (cPARP\u003csup\u003e+\u003c/sup\u003e cells), respectively. Representative microphotographs of primary cells cultures O hours (D) and 4 hours (E) after staurosporine administration. Apoptotic markers (aC3, cPARP) are shown in magenta, phenotypic markers (TUJ-1, APC) are shown in cyan and arrowheads show the colocalizations in neuronal and oligodendroglial cells.\u003c/p\u003e\n\u003cp\u003eStatistical significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/1fa8152c5ac93996b9a1cc0b.png"},{"id":36393894,"identity":"4aec4fe0-54a0-4297-89a8-578399867297","added_by":"auto","created_at":"2023-04-27 19:36:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":839307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of phenotypical composition of aC3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e population. \u003c/strong\u003eComparison of the phenotypical representation of aC3\u003csup\u003e+\u003c/sup\u003e population studied in P8, P29 and P90 rat spinal cord (A). Phenotypical representation is expressed as percentage ± SD of all analyzed aC3\u003csup\u003e+\u003c/sup\u003e cells and was statistically compared within analyzed ages (one-way ANOVA). Heatmaps displaying distribution of analyzed phenotypes of aC3\u003csup\u003e+\u003c/sup\u003e population within ROIs (B). A comparison of the abundance of aC3\u003csup\u003e+\u003c/sup\u003e cells within the whole tissue and distribution of aC3\u003csup\u003e+\u003c/sup\u003e cells in ROIs within population of S100\u003csup\u003e+\u003c/sup\u003e astrocytes (C), Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes (D), and NeuN\u003csup\u003e+\u003c/sup\u003e neurons (E). The abundance of aC3\u003csup\u003e+\u003c/sup\u003e cells is presented as a percentage of all S100\u003csup\u003e+\u003c/sup\u003e astrocytes, Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes, and NeuN\u003csup\u003e+\u003c/sup\u003e neurons ± SD, respectively (one-way ANOVA). Representative microphotographs showing aC3\u003csup\u003e+\u003c/sup\u003e/S100β\u003csup\u003e+ \u003c/sup\u003eastrocyte (F) and aC3\u003csup\u003e+\u003c/sup\u003e/Olig2\u003csup\u003e+ \u003c/sup\u003eoligodendrocyte (G) and aC3\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+ \u003c/sup\u003eneuron (H).\u003c/p\u003e\n\u003cp\u003eStatistical significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Scale bar = 5 µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/b32069da67dccd04b58b66e7.png"},{"id":36393264,"identity":"c50d5255-ec91-4ba1-af83-a395bb39f502","added_by":"auto","created_at":"2023-04-27 19:28:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":152013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBirc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes encoding inhibitors of apoptosis in rat spinal cord\u003c/strong\u003e. Absolute quantities of \u003cem\u003eBirc\u003c/em\u003e mRNAs in spinal cord tissue at P8 (A), P29 (B) and P90 (C) detected by dPCR. Normalized copy number represents the number of \u003cem\u003eBirc\u003c/em\u003emRNAs per normalization factor derived from the mean of expression of two reference genes – \u003cem\u003eGapdh\u003c/em\u003e and \u003cem\u003eeEF1a1\u003c/em\u003e. Data are expressed as mean values of three animals ± SD. LOQ – under the limit of quantification, ND - non-detected. Statistical comparison was performed by one-way ANOVA with statistical significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/3e0e05bf29540c90c76b1e55.png"},{"id":46349601,"identity":"1cd11e81-54f8-4429-a071-c3909c58bc00","added_by":"auto","created_at":"2023-11-13 15:11:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4355308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/65fc595b-27fc-4fd0-8c87-dc871f637cc4.pdf"},{"id":36393892,"identity":"1e530926-9e04-4b56-9d8b-700a4961f2f2","added_by":"auto","created_at":"2023-04-27 19:36:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":507771,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemetarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2854960/v1/0b822fe0f7a0e9469bfdfa41.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCaspase-3 is a member of cysteine-aspartate proteases considered as important effectors of apoptosis representing the process of regulated cell death (Asadi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Taatjes et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The very first member of this family of proteases active in cell death, termed cell death protein 3 (CED-3), was identified in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, and was reported as homolog to murine ICE - related proteases (later known as caspases) (Yuan et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The involvement of CED-3 and its homologs in cell death ensured that caspases became considered as apoptotic proteins (Fraser and Evan \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Miura et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). During the apoptotic cascade, inactive zymogen of caspase-3 is activated by initiator caspase-8 and/or caspase-9 (Taatjes et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Subsequently, activated caspase-3 (aC3) interacts with numerous substrates present in cell, including cytoplasmic and nuclear structural proteins, molecules involved in protein synthesis and protein modification, as well as with the proteins of multiple signaling pathways controlling proliferation, differentiation or cell adhesion (Fischer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Activity of aC3 and other effector molecules of apoptosis results in changes of cellular morphology typical for apoptotic cells, \u003cem\u003ee.g.\u003c/em\u003e chromatin condensation, DNA fragmentation, formation of plasma membrane protrusions and externalization of phosphatidylserine (Taatjes et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ziegler and Groscurth \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). One of the most studied substrates of aC3 is Poly (ADP-ribose) polymerase (PARP), the protein important for DNA repair and for maintenance of chromatin and genome stability (Dantzer et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Due to the catalytic activity of aC3, PARP protein is cleaved to two fragments: 24-kD fragment (p24) and 89-kD fragment (p89), which are specific for apoptosis, unlike the other forms of cell death (Duriez and Shah \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Afterwards, the p24 fragment of cleaved PARP (cPARP) enables progression of apoptosis by inhibition of DNA repair, whereas the p89 fragment localized in nucleoplasm dimerizes and inactivates uncleaved PARP protein (Soldani and Scovassi \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Ultimately, the activity of effector caspases leads to final disintegration of cell to membrane-bound apoptotic bodies, which are subsequently phagocyted by immune cells (Taatjes et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the aC3 is not an exclusive effector-protease of apoptosis in mammals (Wilson and Kumar \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), due to its ability to interact with a wide spectrum of substrates (Walsh et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) it is considered as the main apoptotic protease. Thus, various assays designed for identification of apoptotic cells in tissue samples or in cell culture, often focus on detection of activated effector caspases, especially the aC3 (Fox and Aubert \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gown and Willingham \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hanson and Finkelstein \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Takano et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). On the other hand, presence of aC3 has been reported in cells without typical apoptotic hallmarks in various cell types (Krajewska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Until now, caspase-3 activity has been linked to several non-apoptotic processes. It has been reported, that activated form of this protein is required for the differentiation of various cell types, including bone marrow stromal stem cells, osteoclasts (Szymczyk et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), skeletal muscle fibers (Fernando et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) or embryonic cells (Fujita et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), as well as for regulation of proliferation of B-lymphocytes (Woo et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn nervous system, apoptosis has been extensively studied due to its crucial role in embryonic development, tissue formation, function of neuronal circuits (Yuan and Yankner \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), as well as its involvement in various pathological events linked to the neurodegenerative disorders (Erekat \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and tissue injury (Springer \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In spite of the evidence on involvement of aC3 in non-apoptotic events, it is commonly used as an apoptotic marker in both older and recent studies on spinal cord, especially under pathological conditions (Dai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; G\u0026uuml;lmez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mirzaie et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nesic et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, despite the availability of wide range of commercially produced antibodies against various apoptotic markers (including both, aC3 and cPARP), data regarding the apoptosis in intact spinal cord during postnatal development or in adulthood are still missing. Thus, present study focuses on the analyses of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations in the intact spinal cord tissue at selected stages of ontogenesis, with the ambition to verify the validity of aC3 as an apoptotic marker in the intact nervous tissue. To accomplish our goal, we first compared the abundance of both aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cell populations in the spinal cords of neonatal, preadolescent and adult rats. Second, employing an \u003cem\u003ein vitro\u003c/em\u003e model with synchronously induced apoptosis by staurosporine treatment, we acquired the data necessary for deciphering the pattern of presence of aC3 and cPARP in cells undergoing apoptosis in spinal cord tissue samples. Finally, employing the gene expression profile analysis, we identified potential regulatory mechanisms that cells may employ to control caspase-3 activity under normal conditions in both, developing and mature nervous tissue.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e \u003cp\u003e All experiments on rats were performed in accordance with ARRIVE guidelines, the European Community Council (Directive 2010/63/EU) and in compliance with current national legislation, conducted with approval from the National Food Administration of the Slovak Republic under no. Ro-3051-5/2021\u0026thinsp;\u0026minus;\u0026thinsp;220 and the Animal Care Committee of the Pavol Jozef Šaf\u0026aacute;rik University in Košice. Wistar albino rats (strain Crl:WI, RRID:RGD_2308816) were obtained from Velaz (Prague, Czech Republic). Standard laboratory conditions with a 12-hour light/dark cycle was used for housing the animals and each animal was fed a complete and balanced standard laboratory diet (Altromin International, Lage, Germany) and had \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Experiments were performed on rats at the age of 8 postnatal days (P8, neonatal period, n\u0026thinsp;=\u0026thinsp;11), 29 postnatal days (P29, preadolescent period, n\u0026thinsp;=\u0026thinsp;6) and 90 postnatal days (P90, young adult period, n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Tissue isolation\u003c/h2\u003e \u003cp\u003eExperimental animals in each time point were terminally anesthetized with \u003cem\u003ei.p\u003c/em\u003e. overdose of sodium thiopental (500 mg/kg). Animals (n\u0026thinsp;=\u0026thinsp;3 animals per time point/experimental group) were either i) transcardially perfused with heparinized saline for gene expression analyses, or ii) transcardially perfused with heparinized saline followed by freshly prepared solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for immunofluorescent analyses.\u003c/p\u003e \u003cp\u003eFor immunofluorescent analyses, isolated lumbar segments (L3-L5) of spinal cord were postfixed in the same fixative at 4\u0026deg;C for 24 hours, followed by cryoprotection in 30% sucrose. Spinal cord samples were stored at 4\u0026deg;C until further processing. Isolated tissue samples were cut to 40 \u0026micro;m-thick coronal sections using freezing microtome (Leica CM1850, Leica Microsystems, Mannheim, Germany). For gene expression analyses, isolated segments of lumbar spinal cord tissue (20\u0026ndash;50 mg) were immediately immersed in TRI Reagent\u0026trade; Solution (Invitrogen, Vilnius, Lithuania, #AM9738) and stored at -80\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Primary cell culture preparation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePrimary cell culture was derived from spinal cords of P8 rats (n\u0026thinsp;=\u0026thinsp;5). Rats were terminally anaesthetized, decapitated and spinal cord was dissected. After meninges removal, spinal cords were cut to small pieces and transferred into a Papain Dissociation System solution (Worthington Biochemical Corporation, Lakewood, New Jersey, #LK003150) containing 0.01% papain and 0.01% DNase in line with the isolation protocol at 37\u0026deg;C with intermittent gentle shaking for 45 min. After digestion, excisions were further mechanically dissociated into cell suspension and centrifuged (300 g, 21 ℃, 10 min). Cell pellet was resuspended in DNase dilute albumin-inhibitor solution and discontinuous density gradient (70 g, 21 ℃, 10 min) was used to remove membrane fragments. Subsequently, cells were seeded into 24-well tissue culture plates with laminin-coated cover glasses on the bottom (TPP, Trasadingen, Switzerland) and cultivated in culture medium composed of Dulbecco\u0026rsquo;s Modified Eagle Medium (#L0102-500) and Ham\u0026rsquo;s F12 (#L0135-500; 1/1 v/v) (both Biosera, Nuaille, France) supplemented with 5% fetal bovine serum (Biowest, Nuaill\u0026eacute;, France, #S1400), antibiotics (Penicillin/Streptomycin 10.000 U/10.000 mg/ml, Biochrom AG, Berlin, Germany, #2213B), 1% B-27 supplement (#17504044) and 0.5% N-2 supplement (#17502048) (both Gibco, Invitrogen, Carlsbad, California). Cells were cultivated under standard conditions in an incubator at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% humidity. In all wells, half of the medium volume was replaced every third day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of \u003cem\u003ein vitro\u003c/em\u003e model of apoptotic neural cell population\u003c/h2\u003e \u003cp\u003eApoptotic effect of staurosporine on primary cell culture (7x10\u003csup\u003e4\u003c/sup\u003e cells/well) was analyzed on the thirteenth day of cultivation. 1mM staurosporine (Cell Signalling Technology, Leiden, Netherlands, #9953) diluted in DMSO was added to culture medium (1:1000 dilution ratio, final concentration\u0026thinsp;=\u0026thinsp;1 \u0026micro;M.dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) along with Incucyte\u0026reg; Caspase-3/7 Dye for Apoptosis (Sartorius AG, G\u0026ouml;ttingen, Germany, #4440) and Incucyte\u0026reg; Annexin V Dye for Apoptosis (Sartorius AG, #4641) at 1:1500 and 1:200 dilutions, respectively.\u003c/p\u003e \u003cp\u003eThe progress of development and distribution of apoptotic markers in a staurosporine-induced apoptotic population of cells was monitored using the IncuCyte\u0026trade; ZOOM (Essen BioScience, Ann Arbor, Michigan) at Ex/Em 500/530 nm for Caspase-3/7 Dye and Ex/Em 593/614 nm for Annexin V Dye. Analysis was performed using IncuCyte ZOOM 2016B analysis software (Essen BioScience, RRID:SCR_019874) on nine microphotographs for each well (n\u0026thinsp;=\u0026thinsp;4 wells) taken with a 10x objective lens (Supplemental Table\u0026nbsp;1) every 30 min for a total of 24 hours. For both markers, individual analysis procedure was prepared and uniformly used for every microphotograph captured (Supplemental Table\u0026nbsp;2). Results were compared with control data obtained in a similar manner from primary cell culture that was cultivated simultaneously in the staurosporine-free medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Collection of samples for \u003cem\u003ein vitro\u003c/em\u003e time-dependent analysis of apoptotic markers\u003c/h2\u003e \u003cp\u003eOn the fourteenth day of cultivation, time-dependent analysis of presence of apoptotic markers was conducted using primary cell culture treated with staurosporine diluted in culture medium (final concentration\u0026thinsp;=\u0026thinsp;1 \u0026micro;M.dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). After the addition of staurosporine to cells, primary cell cultures were fixed each hour during the cultivation, starting at time point 0h (no staurosporine effect) until 7 h time point. Cells were fixed by freshly prepared 4% paraformaldehyde in 0.1 M PB at room temperature for 20 min. Fixed primary cell cultures were then washed with 0.1 M phosphate buffered saline (PBS) and immunofluorescently labelled. For selection of antibodies both, staurosporine-free primary cell culture (negative control) and primary cell culture five hours after staurosporine administration (positive control), were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescent labelling\u003c/h2\u003e \u003cp\u003eCoronal sections of lumbar spinal cord and fixed primary cell cultures derived from spinal cord (henceforth referred as samples) were rinsed in 0.1 M PBS. If necessary, antigen retrieval was applied by immersion of samples to 10 mM citrate buffer (pH 6.0) at 95.5\u0026deg;C (\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) for 10 min. After cooling down to room temperature, samples were washed by 0.1 M PBS. Non-specific protein activity was blocked by incubating samples with 5% solution of normal donkey serum (NDS, Jackson Immunoresearch, West Grove, Pennsylvania, #017-000-12) in 0.1 M PBS with 0.3% Triton-X 100 at 4\u0026deg;C overnight. Subsequently, samples were incubated in mixture of primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) diluted in 0.1 M PBS containing 1% NDS and 0.3% Triton-X 100 at 4\u0026deg;C overnight. Subsequently, samples were washed with 0.1 M PBS and incubated with corresponding secondary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) diluted in 0.1 M PBS containing 1% NDS and 0.3% Triton-X 100 at room temperature in dark for 2 hours. Afterwards, samples were washed with 0.1 M PBS. To visualize cell nuclei, far red dye DRAQ5 (1:500, Cell Signalling Technology, #4084) diluted in 0.1 M PBS was applied to tissue samples for 20 min. Afterwards, tissue samples were washed in 0.1 M PBS, mounted on glass slides, dried and cover-slipped using ProLong Gold with DAPI (Invitrogen, #P36930).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies used in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClonality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCatalogue number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLot. number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRRID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCleaved Caspase-3 (Asp175)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9661S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR309480-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2341188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti- Cleaved Caspase-7 (Asp198) (D6H1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8438S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLot 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_1117837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Cleaved PARP (Asp214) (D6X6X)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLot 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2800237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-AIF (D39D2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLot 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_10634755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-EndoG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab9647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR3181009-13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2098770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Fractin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMerck Millipore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB3150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2976523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_262159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-actin (clone C4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMerck Millipore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMAB1501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2951837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2223041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Olig2 (clone 211F1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMerck Millipore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMABN50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3128845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_1080741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-NeuN (clone A60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMerck Millipore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMAB377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2279235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2298772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-S100 β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProteintech Group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66616-1-Ig\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10004814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2881976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti \u0026ndash; APC (Ab-7) (CC-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCalbiochem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOP80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD00150228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2057371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Tubulin \u0026szlig; 3 (TUBB3) (clone TUJ1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonoclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCovance Biolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e801202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB233555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_1006340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Rabbit IgG (H\u003csup\u003e+\u003c/sup\u003eL) conjugated s AlexaFluor 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab150073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR3248726-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2636877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Rabbit IgG (H\u003csup\u003e+\u003c/sup\u003eL) conjugated s AlexaFluor 555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab150074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR3241278-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2636997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Mouse IgG (H\u003csup\u003e+\u003c/sup\u003eL) conjugated s AlexaFluor 555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab150110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR3446637-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2783637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanti-Mouse IgG (H\u003csup\u003e+\u003c/sup\u003eL) conjugated s AlexaFluor 594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab150108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGR3360080-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB_2732073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Microscopic analysis and microphotographs preparation\u003c/h2\u003e \u003cp\u003eTo analyze immunofluorescently labelled tissue samples, Leica TCS SP5X confocal system equipped with LAS AF software (Leica Microsystems) and Leica Thunder Imager DMi8 epifluorescent microscope equipped with LAS X software (Leica Microsystems, RRID:SCR_013673) using 10x, 40x and 100x objective lens (Supplemental Table\u0026nbsp;1) were used. Confocal imagining was done in XYZ mode (resolution 8 bits, 1024 x 1024 pixels, scanning speed 100 Hz, gain 600\u0026ndash;750 V). Following excitation (Ex) and emission (Em) wavelengths were used for visualization of fluorophores: AlexaFluor 488 (Ex 488 nm, Em 500\u0026ndash;540 nm); AlexaFluor 555 (Ex 555 nm, Em 565\u0026ndash;590 nm); DRAQ5 (Ex 643, Em 655\u0026ndash;690). Epifluorescent imaging was done in XYZ mode using Leica DFC9000 GTC camera (resolution 24 bits, 2048 x 2048 pixels). Following excitation and emission wavelength were used for visualization of fluorophores: DAPI (LED_405 filter cube Ex 405/60 nm, Em 470/40 nm); AlexaFluor 488 (GFP filter cube, Ex 470/40 nm, Em 525/50 nm).\u003c/p\u003e \u003cp\u003eFor analysis of aC3\u003csup\u003e+\u003c/sup\u003e population of cells in spinal cord, five optical tissue sections (Z-stacks) per animal of P8, P29 and P90 spinal cords (n\u0026thinsp;=\u0026thinsp;3 animals per time point) were captured using 40x objective lens. Stereological quantification of aC3\u003csup\u003e+\u003c/sup\u003e cells was performed in ventral, lateral and dorsal funiculi, dorsal and ventral horns, and in the central grey matter (henceforth referred to as regions of interest - ROIs) using ConfoCounter software (Institute of Experimental Physics, SAS, Košice, Slovakia, available for free download from Microsoft Store at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://apps.microsoft.com/store/detail/confocounter/9PNSHZPXKHMM?hl=sk-sk\u0026amp;gl=sk\u0026amp;rtc=1\u003c/span\u003e\u003cspan address=\"https://apps.microsoft.com/store/detail/confocounter/9PNSHZPXKHMM?hl=sk-sk\u0026amp;gl=sk\u0026amp;rtc=1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Densitometry analysis of the positivity of aC3\u003csup\u003e+\u003c/sup\u003e nuclei was evaluated as relative intensity of fluorescence using Ellipse 2.0 software (ViDiTo, Košice, Slovak Republic). Abundance of cPARP\u003csup\u003e+\u003c/sup\u003e cells in spinal cord tissue was counted using 40x objective lens on ROIs of 20 tissue slices per animal of P8, P29 and P90 spinal cords (n\u0026thinsp;=\u0026thinsp;3 animals per time point).\u003c/p\u003e \u003cp\u003eTo analyze primary cell cultures IncuCyte\u0026trade; ZOOM system (Essen BioScience) using 10x and 20x objective lens (Supplemental Table\u0026nbsp;1) was used. Following parameters for imaging were used: resolution 24 bits, 1392 x 1040 pixels, Ex 460/40 nm and Em 524/40 nm; Ex 585/40 nm and Em 665/80 nm filter.\u003c/p\u003e \u003cp\u003eMicrophotographs were assembled to figures by ImageJ 1.53t (NIH, Bethesda, Maryland, RRID:SCR_003070); Adobe Illustrator (RRID:SCR_010279) and Adobe Photoshop (RRID:SCR_014199) (both Adobe Systems, San Jose, California). All modifications were limited to cropping, adjustment of brightness and contrast.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Identification of aC3 and cPARP after staurosporine-induced apoptosis in \u003cem\u003ein vitro\u003c/em\u003e model\u003c/h2\u003e \u003cp\u003eTo analyze presence of aC3 and cPARP in neuronal and oligodendroglial populations of primary cell culture treated by staurosporine, immunofluorescent co-localization using anti-TUJ1 and anti-APC antibodies was realized. Uniform analysis procedure (Supplemental Table\u0026nbsp;2) was prepared by IncuCyte ZOOM 2016B analysis software (Essen BioScience) and used to estimate the number of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cells per mm\u003csup\u003e2\u003c/sup\u003e in 14 microphotographs taken with a 10x objective lens from each well (n\u0026thinsp;=\u0026thinsp;6 wells per experimental group). Colocalization of apoptotic (aC3, cPARP) and phenotypic markers (TUJ1, APC) in cells was identified as signal overlap and performed on n\u0026thinsp;=\u0026thinsp;3 wells per experimental group. Co-localization was evaluated as percentage of neuronal or oligodendroglial cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Analysis of gene expression\u003c/h2\u003e \u003cp\u003eGene expression analysis was performed to evaluate the abundance of inhibitors of apoptosis (IAPs, encoded by \u003cem\u003eBirc\u003c/em\u003e genes) in the spinal cord tissue of P8, P29 and P90 rats (n\u0026thinsp;=\u0026thinsp;3 per time point). The absolute amount of the studied gene transcripts was determined by digital PCR (dPCR). The analyzed cDNA was prepared by reverse transcription (RT) of total RNA, isolated by TRI Reagent\u0026trade; Solution. Anchored oligo dT primer and RevertAid Reverse Trancriptase (Thermo Scientific, Vilnius, Lithuania, #EP0441) was used for the RT. Both protocols were performed according to manufacturer\u0026rsquo;s instructions. dPCR was performed by nanoplate-based technology QIAcuity Digital system (Qiagen, Hilden, Germany); QIAcuity One 5-plex instrument, 8.5K QIAcuity\u0026trade; Nanoplates (#250021) and QIAcuity\u0026trade; EG PCR Kit (#2500112) for amplification and detection of the products. Gene specific PCR primers (Supplemental Table\u0026nbsp;3) were designed according to the reference sequence of rat \u003cem\u003eBirc1-7\u003c/em\u003e genes by Primer-Blast (NIH, RRID:SCR_003095). Amplification of the desired product and lack of primer-dimers was confirmed by agarose gel electrophoresis and RT-qPCR. Depending on the gene, 10 to 50 ng of RNA/cDNA per 12\u0026micro;L reaction was used and amplified by 2-step PCR; 35 cycles (15 s \u0026ndash; 95\u0026deg;C, 15 s \u0026ndash; 60\u0026deg;C) preceded by denaturation at 95\u0026deg;C for 2 min. To verify the detection/quantification at linear range, half dilution of each sample was prepared and analyzed together with the undiluted sample. RNA/cDNA isolated from testes of P29 rats was used as a positive control for transcripts with low amount or absent PCR product in the spinal cord samples. Finally, the detected number of the individual \u003cem\u003eBirc\u003c/em\u003e-gene transcripts was normalized to normalization factor (NF) based on expression of two reference genes (\u003cem\u003eGapdh\u003c/em\u003e and \u003cem\u003eeEF1a1\u003c/em\u003e), which were shown to be stably expressed during postnatal development (Košuth et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The NF was calculated as geometric mean of copies of both reference genes. For genes with very low or absent transcripts in spinal cord, limit of quantification (LOQ) was determined. Serial dilutions of RNA/cDNA isolated from rat testes (tissue with high \u003cem\u003eBirc\u003c/em\u003e genes expression) were used for the purpose. Then the LOQ of \u003cem\u003eBirc1\u003c/em\u003e, \u003cem\u003eBirc3\u003c/em\u003e and \u003cem\u003eBirc7\u003c/em\u003e transcripts per NF were 3.1\u0026ndash;3.7x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 2.2\u0026ndash;2.6x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e and 1.5\u0026ndash;1.6x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistics\u003c/h2\u003e \u003cp\u003eFor statistical analysis and graph assembly, GraphPad Prism (version 9.0.0, RRID:SCR_002798) was used. Obtained data were analyzed using unpaired t-test; one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons; and repeated-measure two-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. A difference between groups or time points was considered statistically significant with a P value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), 0.01 (**) and 0.001 (***).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Selection of antibodies against apoptotic markers\u003c/h2\u003e \u003cp\u003eIn order to explore the populations of cells undergoing apoptosis in intact spinal cord during postnatal development and adulthood, we decided in the first step to select the appropriate apoptotic marker(s) which should be targeted with commercially available antibodies. With respect to the requirement of unambiguous interpretation of immunofluorescence labelling, two basic criteria should meet the optimal apoptotic markers: specificity to the apoptotic cells and discrete appearance of the signal restricted optimally to the nucleus. Verified and widely used antibodies against AIF, EndoG, Fractin, aC3, aC7 and cPARP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were tested in following samples: (i) intact primary \u003cem\u003ein vitro\u003c/em\u003e culture derived from spinal cord (negative control), (ii) primary \u003cem\u003ein vitro\u003c/em\u003e culture treated with inductor of apoptosis - staurosporine (positive control) and (iii) coronal sections of intact adult rat spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA - J). Both AIF and EndoG were localized in cytoplasm of cells in the primary cell culture with or without apoptotic morphology (\u003cem\u003ei.e.\u003c/em\u003e pyknotic, semilunar, and shattered nucleus) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, D, E). However, AIF and EndoG were also detected in the cytoplasm of cells in the spinal cord tissue sections, most notably in motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). Similarly, anti-fractin antibody, which detects the cleaved form of actin as a result of caspase activity in apoptosis, was identified in both, cleaved (specific signal) and uncleaved (non-specific signal) forms of actin in cells of primary cell culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, F). However, we also detected non-specific signal (colocalization of anti-fractin and anti-actin antibodies in the ependymal cells of the central canal) in spinal cord samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Also, aC7 staining was found in both, axons in white matter and cytoplasm of motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Contrary to abovementioned apoptotic markers, presence of aC3 and cPARP signal was located in cell nuclei. aC3-antibody-stained nuclei in cell culture and tissue samples with various intensity with or without apoptotic morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA - D). Based on the results of our densitometric analysis, we classified the nuclei to two categories: the \u0026ldquo;low intensity signal\u0026rdquo; and the \u0026ldquo;high intensity signal\u0026rdquo; with relative fluorescent intensity ranging in 9.23% \u0026plusmn; 1.39% and 26.3% \u0026plusmn; 6.92%, respectively (Supplemental Fig.\u0026nbsp;1A, B). For the purpose of the study, only the nuclei with the high intensity of signal were considered as positive. Immunofluorescent signal of antibody against 89 kDa fragment of protein PARP (cleaved PARP; cPARP) was identified mostly as perinuclear ring only in cells with apoptotic hallmarks, with no or only low background signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE - H). Taken together, our analysis revealed that cPARP could represent a proper marker of apoptosis in spinal cord, giving strong signal in fragmented cells, while aC3 seems to be present in broad population of cells with morphology ranging from normal to fragmented. This could be in line with the assumption that unlike cPARP, aC3 should occur earlier the cell progressing through the apoptotic cascade. Therefore, in the subsequent analyses we focused on characterization of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cell populations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 aC3 positive population of cells in spinal cord is much more abundant in comparison to cPARP positive cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn next step, we focused on detection of apoptotic cells in intact spinal cord tissue at subsequent stages of ontogenesis: neonatal period (P8), preadolescent period (P29) and adulthood (P90). According to our results, immunofluorescence analysis of aC3\u003csup\u003e+\u003c/sup\u003e cells and cPARP\u003csup\u003e+\u003c/sup\u003e cells shows a large discrepancy between studied populations as well as changes within population abundance during rat ontogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA - J). Based on the results of quantification analysis, we calculated that the ratios of aC3\u003csup\u003e+\u003c/sup\u003e to cPARP\u003csup\u003e+\u003c/sup\u003e in the spinal cord tissue were 593:1 (P8), 4490:1 (P29), and 487:1 (P90), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). The aC3\u003csup\u003e+\u003c/sup\u003e cell population was the most prevalent in P8 rats (307,212.83\u0026thinsp;\u0026plusmn;\u0026thinsp;20,710.03 cells/mm\u003csup\u003e3\u003c/sup\u003e) and diminished significantly in the later phases of ontogenesis (P29: 80,166.35\u0026thinsp;\u0026plusmn;\u0026thinsp;6,428.40 cells/mm\u003csup\u003e3\u003c/sup\u003e; P90: 47,518.84\u0026thinsp;\u0026plusmn;\u0026thinsp;20,7410.03 cells/mm\u003csup\u003e3\u003c/sup\u003e). cPARP\u003csup\u003e+\u003c/sup\u003e cells were also most prevalent in P8 spinal cord tissue (613.40\u0026thinsp;\u0026plusmn;\u0026thinsp;70.936 cells/mm\u003csup\u003e3\u003c/sup\u003e), contrary to P29 (24.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.37 cells/mm\u003csup\u003e3\u003c/sup\u003e) and P90 (97.51\u0026thinsp;\u0026plusmn;\u0026thinsp;16.85 cells/mm\u003csup\u003e3\u003c/sup\u003e) intervals.\u003c/p\u003e \u003cp\u003eIn addition to differences in abundance of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations, we also observed differences between distributions of both populations in analyzed areas of the spinal cord on coronal sections (also called as region of interest, ROI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). At P8, the majority of aC3\u003csup\u003e+\u003c/sup\u003e cells were identified in the white matter (mainly in the ventral and lateral funiculus). In P29 and P90 rats, aC3\u003csup\u003e+\u003c/sup\u003e cells were most abundant in the grey matter, mainly in the dorsal horn and the central and lateral grey matter. On the contrary, cPARP\u003csup\u003e+\u003c/sup\u003e cells were mostly found in the dorsal horn at P8. The abundance of these cells decreased in all ROIs during the later stages of ontogenesis, with the exception only in the dorsal funiculus of P90 rats.\u003c/p\u003e \u003cp\u003eSignificant differences in the abundance and distribution of the aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cells confirm the presumption that unlike cPARP, aC3 should occur earlier and possibly longer in the cell progressing through the apoptotic cascade. Therefore, aC3\u003csup\u003e+\u003c/sup\u003e population could represent more abundant group of cells with early caspase-3 activation contrary to a significantly smaller population of cells with late cleavage of PARP during the apoptosis. The other explanation for significantly larger population of aC3-positive cells could be either the inhibition of aC3, or its possible non-apoptotic functions in cells of spinal cord. In order to decide which hypothesis is more relevant, in the next step we studied the presence of both, aC3 and cPARP in cells after apoptotic stimulus employing \u003cem\u003ein vitro\u003c/em\u003e model of primary cell culture isolated from P8 rat spinal cord.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Employment of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e \u003cb\u003eapoptotic model for determination of involvement of aC3 and cPARP throughout the progression of apoptotic cascade\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious analyses revealed significant difference between the number of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cells in postnatal rat spinal cord. In the next step, we focused on determination, whether this discrepancy between the size of both populations reflects the discrepancies in the time windows, when these markers are detectable in the cells undergoing apoptosis. In order to identify the period, when aC3 and cPARP are detectable in apoptotic cells, we performed \u003cem\u003ein vitro\u003c/em\u003e test on the caspase-3 activation and PARP cleavage using primary cell culture isolated from P8 spinal cord. To obtain the synchronous populations of apoptotic cells, primary cell culture was treated by staurosporine, which should trigger the apoptosis. In order to get the very first overview about the effect of staurosporine on the primary cell culture, we took the advantage of real-time live-cell imaging and analysis using platform IncuCyte\u0026trade; ZOOM systems. Effect of staurosporine treatment on primary cell culture in the 24 hour period was analyzed by Casp3/7 dye (substrates indicating activation of both caspase-3 and caspase-7) and by Annexin V dye (substrate indicating the externalization of phosphatidylserine) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA - C). Compared to interval 0 hours (\u003cem\u003ei.e.\u003c/em\u003e the time just before induction of apoptosis), we found a significant increase in the number of cells with active effector caspases, as well as cells with externalized phosphatidylserine 3.5 hours after staurosporine treatment. Numbers of aC3/7\u003csup\u003e+\u003c/sup\u003e cells reached plateau at ninth hour while number of Annexin V\u003csup\u003e+\u003c/sup\u003e cells continuously raised in all intervals with significant overgrow of aC3/7\u003csup\u003e+\u003c/sup\u003e population at 7.5 hours after the staurosporine administration (two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Based on results of previous \u003cem\u003ein vitro\u003c/em\u003e studies, we presume that higher numbers of Annexin V\u003csup\u003e+\u003c/sup\u003e cells in later intervals indicate the presence of alternative forms of regulated and/or unregulated cell death (Shlomovitz et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, for reduction of the impact of other forms of cell death on our measurements, we decided to investigate possible time delay between the activation of caspase-3 and cleavage of PARP in main experiment during first seven hours after administration of staurosporine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we investigated temporal relationship between caspase-3 activation and PARP cleavage in primary cell culture during period 0\u0026ndash;7 hours after the induction of apoptosis with staurosporine. According to our results, number of aC3\u003csup\u003e+\u003c/sup\u003e (in line with our previous experimental data) and cPARP\u003csup\u003e+\u003c/sup\u003e cells significantly increased during the fourth hour after induction of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Thus, our data indicate the onset of presence of detectable levels of aC3, as well as cPARP in cells in approximately similar time, 3.5 hour since the beginning of induction of apoptosis.\u003c/p\u003e \u003cp\u003eIn the next step, we decided to investigate, whether there are any differences between the induction of apoptosis and onset of presence of aC3 and cPARP in two major phenotypes, present in the primary cell culture sensitive to staurosporine; neurons (identified by anti-Tuj-1 antibody) and oligodendroglial cells (identified by anti-APC antibody) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Our analyses revealed that TUJ-1\u003csup\u003e+\u003c/sup\u003e neurons show similar distribution of both apoptotic markers as the whole primary cell culture in examined time points after staurosporine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D, E). On the other hand, we noticed high number of aC3\u003csup\u003e+\u003c/sup\u003e/APC\u003csup\u003e+\u003c/sup\u003e oligodendrocytes even before the induction of apoptosis and during the subsequent period until third hour (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC - E). During fourth hour the number of aC3\u003csup\u003e+\u003c/sup\u003e/APC\u003csup\u003e+\u003c/sup\u003e oligodendrocytes increased only moderately contrary to PARP\u003csup\u003e+\u003c/sup\u003e oligodendrocytes, which levels followed similar pattern as the whole population and TUJ-1\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003eBased on the data obtained from in \u003cem\u003evitro\u003c/em\u003e model, we assume that both, temporal activation of caspase-3 and cleavage of PARP in studied populations, occur at similar time points after induction of apoptosis. Our results indicate that due to the similar time windows, when aC3 and cPARP are present in cells undergoing apoptosis, the size of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations should be also similar in tissues, where both markers are involved solely in the apoptotic cascade. Thus, due to the significant discrepancy between the size of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations in the spinal cord tissue, it can be concluded that aC3 may be inhibited, or involved in other, non-apoptotic processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Caspase-3 activation occurs mostly in glia of postnatal rat spinal cord\u003c/h2\u003e \u003cp\u003eSince \u003cem\u003ein vitro\u003c/em\u003e analyses revealed possible activation of caspase-3 in non-apoptotic cells, especially in glial cells, we decided to examine, whether phenotypic representation of aC3\u003csup\u003e+\u003c/sup\u003e population identified in \u003cem\u003ein vitro\u003c/em\u003e experiments resembles to real conditions in spinal cord tissue.\u003c/p\u003e \u003cp\u003eColocalization study of aC3 and selected phenotypic markers of neurons, oligodendrocytes and astrocytes proved that the aC3\u003csup\u003e+\u003c/sup\u003e population of the spinal cord tissue is primarily composed of glial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA - H). Surprisingly, unlike the aC3\u003csup\u003e+\u003c/sup\u003e populations in the spinal cords of P8 and P90 rats, which predominantly consist of S100β \u003csup\u003e+\u003c/sup\u003e astrocytes, the aC3\u003csup\u003e+\u003c/sup\u003e population in P29 rats is composed mostly of Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Single ROI aC3\u003csup\u003e+\u003c/sup\u003e composition analysis revealed the same result as whole tissue analysis. Just a few exceptions were discovered. Less aC3\u003csup\u003e+\u003c/sup\u003e/Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes were seen in the P8 ventral and lateral funiculus. On the other hand, there was a huge amount of aC3\u003csup\u003e+\u003c/sup\u003e oligodendrocytes in the ventral horns of P29 and P90. In comparison to glial cells, NeuN\u003csup\u003e+\u003c/sup\u003e neurons represent smaller portion of the spinal cords aC3\u003csup\u003e+\u003c/sup\u003e population. At all ages studied, aC3\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e neurons were identified mostly in the dorsal horns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFurthermore, examining the abundance of aC3\u003csup\u003e+\u003c/sup\u003e cells in individual populations provided even more intriguing results. In S100β \u003csup\u003e+\u003c/sup\u003e astroglial population, substantial portion of all analyzed cells was aC3 positive. At P8, most of S100β \u003csup\u003e+\u003c/sup\u003e cells colocalized with aC3, and were highly abundant in all analyzed ROIs. In the central and lateral grey matter, almost all analyzed astrocytes were aC3\u003csup\u003e+\u003c/sup\u003e positive. Even though the number of aC3\u003csup\u003e+\u003c/sup\u003e astrocytes significantly decreased in the later stages of ontogenesis, they still represented most of all astrocytes in almost all analyzed ROIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eA significant number of aC3\u003csup\u003e+\u003c/sup\u003e cells can also be seen in the Olig2\u003csup\u003e+\u003c/sup\u003e oligodendroglial population. Around a quarter of all cells of P8 and P90 rat spinal cord are positive for activated caspase-3. While the proportion of aC3\u003csup\u003e+\u003c/sup\u003e/Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes is comparable within the ROIs studied at P29 and P90, there is a greater proportion of positive aC3\u003csup\u003e+\u003c/sup\u003e/Olig2\u003csup\u003e+\u003c/sup\u003e cells in the ROIs of grey matter of the spinal cord at P8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Interestingly, only 9.44% of all Olig2\u003csup\u003e+\u003c/sup\u003e oligodendrocytes in the P8 ventral funiculus were also positive for aC3. In NeuN\u003csup\u003e+\u003c/sup\u003e neuronal population, we identified only a small portion of aC3\u003csup\u003e+\u003c/sup\u003e cells, with significantly higher representation only in dorsal horn of grey matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Taking together, our data indicate that caspase-3 is activated in only small population of neurons, while most of glial cells, especially astrocytes, contain detectable levels of aC3. In comparison to aC3, cPARP\u003csup\u003e+\u003c/sup\u003e population may be difficult to co-localize to phenotypic markers due to the fact that morphology in most of cPARP\u003csup\u003e+\u003c/sup\u003e cells of spinal cord resembled to apoptotic phenotype and phenotypic markers were often missing due to degree of fragmentation of the cell body. In contrast to cPARP\u003csup\u003e+\u003c/sup\u003e cells, most of the aC3\u003csup\u003e+\u003c/sup\u003e cells have normal morphology, indicating that activation of caspase-3 in rat spinal cord could play an important role in other processes, especially in glia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Activity of aC3 in rat spinal cord may be regulated by specific IAP proteins\u003c/h2\u003e \u003cp\u003eWith respect to the number of cells with detectable levels of aC3 present in the spinal cord tissue on one side, and the diminutive number of cells with apoptotic phenotype on the other side, we presumed that there could be some mechanisms regulating the caspase activity in those aC3\u003csup\u003e+\u003c/sup\u003e cells, which obviously do not undergo the apoptosis. One possible explanation could be the inhibition of aC3 activity by specific proteins known as Inhibitors of Apoptosis (IAPs), which could maintain the aC3 in steady state in the cells. Therefore, we decided to examine the expression of all known \u003cem\u003eBirc\u003c/em\u003e genes (\u003cem\u003eBirc1-7\u003c/em\u003e), encoding the members of IAP protein family in the rat spinal cord tissue at all selected ontogenetic stages (P8, P29 and P90). Absolute amounts of their transcripts were determined by digital PCR (dPCR).\u003c/p\u003e \u003cp\u003eWe found that majority of the IAPs (\u003cem\u003eBirc1\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e and \u003cem\u003e7\u003c/em\u003e) are only slightly expressed in the rat spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA - C). On the other hand, the most abundant gene transcripts represented \u003cem\u003eBirc4\u003c/em\u003e, \u003cem\u003eBirc2\u003c/em\u003e, and \u003cem\u003eBirc5\u003c/em\u003e. Collectively, these three IAPs constituted more than 90% of the whole IAP gene family (98% at P8, 96% at P29 and 91% at P90). The most highly expressed IAP represented \u003cem\u003eBirc4\u003c/em\u003e (\u003cem\u003eXIAP\u003c/em\u003e). mRNA transcripts of \u003cem\u003eXIAP\u003c/em\u003e comprised more than 50% of all IAP gene transcripts at P8 and raised to more than 70% at P29 and P90. mRNA level of \u003cem\u003eBirc4\u003c/em\u003e was approximately two times (P8) or even four times (in both, P29 and P90) higher than the level of \u003cem\u003eBirc2\u003c/em\u003e. The second IAP gene with highest level of expression was \u003cem\u003eBirc2\u003c/em\u003e, with more than 20% and 15% share on all AIP transcripts at P8 and P29 and P90 spinal cord, respectively. The amount of this, relatively abundant gene transcript, seems to be very steady in spinal cord, without apparent changeover during postnatal life. Similarly, high copy number as \u003cem\u003eBirc2\u003c/em\u003e was recorded also by \u003cem\u003eBirc5\u003c/em\u003e (\u003cem\u003eSurvivin\u003c/em\u003e), but only in neonatal spinal cord. In older individuals, the activity of \u003cem\u003eBirc5\u003c/em\u003e transcription gradually decreased (2.5-fold at P29 and 4.5-fold at P90). In comparison with other IAPs, \u003cem\u003eSurvivin\u003c/em\u003e represented the only gene with negative regulation during ontogenesis. Taken together, our analysis proved the presence of considerable levels of several IAPs (\u003cem\u003eBirc4\u003c/em\u003e, \u003cem\u003eBirc2\u003c/em\u003e and \u003cem\u003eBirc5\u003c/em\u003e) in the spinal cord tissue, which could be responsible for inhibition of caspase-3 activity. This could be the possible explanation of the discrepancy between the amounts of PARP\u003csup\u003e+\u003c/sup\u003e and aC3\u003csup\u003e+\u003c/sup\u003e cells in the rat spinal cord during postnatal life.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eDue to critical role in development and pathological states of spinal cord (Erekat \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Springer \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Yuan and Yankner \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) both apoptosis and caspase-3 are intensively investigated topics of neuroscience. aC3, regarded as a crucial protein of apoptosis (Lakhani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Walsh et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), interact with numerous proteins leading to characteristic changes in morphology and biochemistry of cell (Taatjes et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). At the same time, multiple studies reported activity of caspase-3 in non-apoptotic processes (Fernando et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Miura et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Szymczyk et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Woo et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Nevertheless, numerous experimental studies have investigated apoptosis in the spinal cord, especially under pathological conditions, by detecting caspase-3 activity (Dai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; G\u0026uuml;lmez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mirzaie et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nesic et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); On the other hand, studies investigating the apoptotic processes in the intact developing nervous system employing the commercially available antibodies are lacking. In our study, we provided a comprehensive analysis of the aC3\u003csup\u003e+\u003c/sup\u003e cell populations in rat spinal cord at selected stages of postnatal life and challenged the relevance of aC3 as an apoptotic marker during non-pathological conditions utilizing \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e \u003cp\u003eTo revise the relevance of aC3 protein as an apoptotic marker in intact spinal cord tissue, we compared population of aC3\u003csup\u003e+\u003c/sup\u003e cells with cPARP immunoreactive cells in various experimental contexts. Prior to the analyses, we tested several commercially available antibodies designed against the common apoptotic markers AIF, EndoG, Fractin, aC3, aC7 and cPARP. In the analysis, the optimal apoptotic marker(s) should meet two important criteria: specificity to the apoptotic cells and discrete appearance of the signal restricted optimally to the nucleus. In comparison to other markers, cPARP proved to be a reliable marker of apoptotic cells, probably due to its ability to bind with newly generated epitopes produced by activated effector caspase during the apoptosis (Soldani and Scovassi \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Contrary to anti-cPARP antibody, antibodies against other apoptotic markers did not show same level of specificity. In primary cell culture, AIF and EndoG were detected in both cells with and without morphological changes associated with apoptosis. This can be explained by the fact that during apoptosis, AIF and EndoG only change their localization in intracellular space and do not generate new epitopes in their structure (Joza et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). We also focused on detection of fractin, a cleaved form of actin produced by caspase activity (Rossiter et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Although we were able to identify fractin\u003csup\u003e+\u003c/sup\u003e cells with hallmarks of apoptotic morphology, it was revealed that anti-fractin antibody is at least partially immunoreactive to uncleaved actin. Furthermore, we tested an antibody against aC7, another effector caspase involved in apoptosis. According to our results, we detected aC7 immunoreactivity primarily in the cytoplasm and neural processes of cells without clear signs of apoptosis. This observation was in line with previous study, which confirmed the general activity of effector caspases 3, 6 and 7 in neural processes (D\u0026rsquo;amelio et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, massive immunoreactivity in cytoplasm disqualified anti-aC7 antibody from detection of apoptotic cell.\u003c/p\u003e \u003cp\u003eAccording to our results on quantification of both, aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e cells, spinal cord tissue contains massive population of aC3\u003csup\u003e+\u003c/sup\u003e cells with non-homologous distribution of cells, neither between the studied ROIs of spinal cord, nor during the studied spinal cord ontogenesis phases. aC3\u003csup\u003e+\u003c/sup\u003e population was most abundant in the neonatal spinal cord (mainly in white matter) and its number declined significantly in later stages of ontogenesis. Majority of observed aC3\u003csup\u003e+\u003c/sup\u003e cells did not display any apoptotic morphological changes. On the contrary, we identified only a small population of cells containing p89 fragment of PARP, which is specifically generated during apoptosis (Soldani and Scovassi \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition, the cPARP population differs from aC3\u003csup\u003e+\u003c/sup\u003e cells not only in size, but also in the distribution of cells across ROIs in neonatal spinal cord, with the majority of the cPARP\u003csup\u003e+\u003c/sup\u003e cells present in the grey matter, especially in the dorsal horns. The localization of the cPARP\u003csup\u003e+\u003c/sup\u003e cells is consistent with the previously identified population of apoptotic neurons (Lawson et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), which undergo programmed cell death as the result of their inability to be involved into the functional neuronal circuits in dorsal horns during early postnatal period (Prasad et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For similar reasons, ablation of superfluous neurons could be expected also in other areas of grey matter. Intriguingly, we discover a substantial proportion of cPARP\u003csup\u003e+\u003c/sup\u003e glial cells showing nuclear alterations in the dorsal funiculus of P90 rats. However, more investigation is required to determine the role of apoptosis in the dorsal funiculi in adulthood. In general, majority of cPARP\u003csup\u003e+\u003c/sup\u003e cells displayed hallmarks of apoptosis (semilunar, condensed or fragmented nucleus) similarly, as described in Taatjes et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and significant subgroup of these cells was absent of the phenotypic markers. This prevented us from specifying phenotypic characteristics of cPARP\u003csup\u003e+\u003c/sup\u003e cells in our study.\u003c/p\u003e \u003cp\u003eThe accumulation of aC3\u003csup\u003e+\u003c/sup\u003e cells in the spinal cord (mostly without apoptotic morphology), as well as striking differences in size between cPARP\u003csup\u003e+\u003c/sup\u003e and aC3\u003csup\u003e+\u003c/sup\u003e populations, could be explained in two ways, as (i) significant time-delay in caspase-3 activation followed by the PARP cleavage; or (ii) the aC3 involvement in non-apoptotic processes. Based on previous experimental studies, aC3 directly cleaves PARP in the apoptotic pathways (Duriez and Shah \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Soldani and Scovassi \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Therefore, the presence of cPARP in cells is considered as the proof of the aC3 activity (Li et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). At the same time, PARP cleavage prevents necrosis during apoptosis, ensuring appropriate execution of caspase-mediated programmed cell death. Therefore, cleavage of PARP by caspases is considered to be a hallmark of apoptosis (Herceg and Wang \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The cleavage of PARP has been observed in early stages of apoptosis, even before DNA degradation in multiple type of tumor cells (Soldani et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and cPARP has been referred to as an early apoptotic marker (Kaufmann et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, to our knowledge, the dynamics of aC3 and cPARP in neural cell population has not been studied before. In order to test our hypothesis, we investigated the time-dependent relationship between the presence of both markers in the apoptotic cells. Primary cell culture prepared from neonatal spinal cord was used to simulate heterogeneity of spinal cord tissue containing cell populations at various stages of cell cycle and/or the level of cell differentiation. In the primary cell culture, apoptosis was triggered by the administration of staurosporine, well established apoptotic inductor commonly used in experimental \u003cem\u003ein vitro\u003c/em\u003e studies of neuronal apoptosis (Negishi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Prince and Oreland \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Compared to other apoptotic inductors, which action is based on inducing oxidative stress (Simon et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), or inhibiting of specific enzymes involved in cell proliferation (Park et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), staurosporine triggers apoptosis through both mechanisms: kinase inhibition (Karaman et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), as well as induction of oxidative stress (Kruman et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Thus, staurosporine should induce apoptosis in cells regardless of the state of differentiation or cell cycle phase (Bertrand et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Due to staurosporine ability to induce caspase-dependent apoptosis (Belmokhtar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) resulting in the activation of aC3 and cPARP proteins, which could be detected by antibodies, our model proved to be a useful tool for studying the aC3 - cPARP time-dependent relationship. On the other hand, higher abundance of Annexin V\u003csup\u003e+\u003c/sup\u003e cells compared to aC3/7\u003csup\u003e+\u003c/sup\u003e cells in primary cell culture 7.5 hours after staurosporine induction was caused probably by its ability to induce also caspase-independent apoptosis (Belmokhtar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), or by an increase of the amount of necrotic and necrotic-like cells that are falsely positive for Annexin V in the cell culture (Shlomovitz et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, our model does not offer an universal instrument for studying apoptosis in neural cells, but rather a specific method for caspase-substrate dynamic research.\u003c/p\u003e \u003cp\u003eIn line with data from an \u003cem\u003ein vitro\u003c/em\u003e analysis of staurosporine impact on the activity of effector caspases 3/7 and the abundance of Annexin V\u003csup\u003e+\u003c/sup\u003e cells, we found a significant increase in the number of both, aC3\u003csup\u003e+\u003c/sup\u003e cells as well as cPARP\u003csup\u003e+\u003c/sup\u003e cells at the fourth hour after synchronous induction of apoptosis in the primary culture. Therefore, we assume that consecutive activation of both, aC3 followed by the cleavage of PARP should results in similar sizes of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations in the nervous tissue containing asynchronous populations of apoptotic cells \u003cem\u003ein vivo\u003c/em\u003e. Thus, discrepancy in the size of aC3\u003csup\u003e+\u003c/sup\u003e and cPARP\u003csup\u003e+\u003c/sup\u003e populations may be caused by the role of aC3\u003csup\u003e+\u003c/sup\u003e cells in non-apoptotic processes during which PARP cleavage does not occur. Similar observations were already made in neural cells of murine neutrospheres, in which aC3 endogenous activity without PARP cleavage was present during differentiation of nestin\u003csup\u003e+\u003c/sup\u003e precursors to astrocytes, oligodendrocytes and neurons (Fernando et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Elevated number of aC3\u003csup\u003e+\u003c/sup\u003e oligodendrocytes in employed \u003cem\u003ein vitro\u003c/em\u003e model in intervals even before apoptosis induction, as well as surprisingly abundant number of aC3\u003csup\u003e+\u003c/sup\u003e cells in astroglial and oligodendroglial populations in spinal cord tissue indicate that aC3 may play important non-apoptotic role mainly in glial cells. Presence of aC3 in astrocytes and radial glia like cells was already reported in multiple studies. Non-apoptotic functions of aC3 were observed during process of astrogliosis and astrocyte cytoskeletal remodelation induced by various pathological conditions (Acarin et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Aras et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Guyenet et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), or even after non-pathological stimuli like exercise (Stevenson et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in various parts of the rodent brain. Activity of caspase-3 was also observed during differentiation of Bergman glia of cerebellum in rats (Oomman et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In small population of aC3\u003csup\u003e+\u003c/sup\u003e neurons, aC3 may as well participate in several non-pathological processes required for remodelation and the establishment of neural networks. Caspase-3 proved to be active during long-term potentiation of synapses in the CA1 region of the rat hippocampus (Gulyaeva et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), during the long-term phase of synaptic input sensitization in a terrestrial snail \u003cem\u003eHelix lucorum\u003c/em\u003e (Bravarenko et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and in the auditory forebrain of the Zebra finch during long-term habituation to tape-recorded birdsongs (Huesmann and Clayton \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). More intriguing, it has been proposed that caspase-3 is already present in active form in Zebra finch postsynaptic neurites and its activity is regulated by the interaction with the apoptotic inhibitor \u003cem\u003eXIAP\u003c/em\u003e (encoded by \u003cem\u003eBirc4\u003c/em\u003e gene) (Huesmann and Clayton \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This is consistent with our investigation of IAPs gene expression in the rat spinal cord, which revealed that \u003cem\u003eXIAP\u003c/em\u003e is the most expressed inhibitor of apoptosis in rats at all studied ages. We observed increased levels of mRNAs encoding rIAP-1 protein (encoded by \u003cem\u003eBirc2\u003c/em\u003e), whose presence may be explained by fact, that the protein is involved in regulation of multiple signal pathways besides the inhibition of apoptosis (Saleem et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We also observed elevated levels of \u003cem\u003eSurvivin\u003c/em\u003e (\u003cem\u003eBirc5\u003c/em\u003e) expression in P8, which is associated with proliferation and also should be capable to bond with aC3 (Shin et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Higher expression levels can be explained by higher proliferation rate of cells during the early stages of postnatal development in spinal cord tissue, which is attenuated in later ages (Alexovič Matiašov\u0026aacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Due to the ability of both \u003cem\u003eXIAP\u003c/em\u003e and \u003cem\u003eSurvivin\u003c/em\u003e to establish bonds with aC3 (Riedl et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), we hypothesize that aC3 could be in activated, but still inhibited state. Dynamic dissociation and re-binding of \u003cem\u003eXIAP\u003c/em\u003e or \u003cem\u003eSurvivin\u003c/em\u003e to active site of aC3 probably allow to regulate the activity of aC3 spatially and temporally (Huesmann and Clayton \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, to confirm our hypothesis, further research is necessary.\u003c/p\u003e \u003cp\u003eIn conclusion, we observed a large population of aC3\u003csup\u003e+\u003c/sup\u003e cells in the spinal cord during neonatal and preadolescent development and in adulthood that do not correspond to the size of population of cPARP\u003csup\u003e+\u003c/sup\u003e cells, which are considered to be truly apoptotic cell population. Based on data from \u003cem\u003ein vitro\u003c/em\u003e analysis of the time-dependent interaction between aC3 and cPARP, we assume that aC3 may be inhibited and/or involved in other non-apoptotic processes. One of the possible explanations is that the activity of aC3 is regulated by \u003cem\u003eBirc4 (XIAP)\u003c/em\u003e, \u003cem\u003eBirc2\u003c/em\u003e and/or \u003cem\u003eBirc5 (Survivin)\u003c/em\u003e genes by forming the \u0026ldquo;stand-by\u0026rdquo; complex with aC3. However, further investigation is required to determine the definitive nature of the interaction between IAPs and aC3 in spinal cord tissue. Our data also demonstrated that aC3\u003csup\u003e+\u003c/sup\u003e should not be considered as an exclusive apoptotic marker and other markers/methods should be considered for the detection of apoptotic cells, especially in intact nervous tissue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e Authors thank to Bc. Ladislav Pačut, Viera Bal\u0026aacute;žov\u0026aacute; and Eva Pastorov\u0026aacute; for technical assistance. This study was supported by VEGA no. 1/0760/20, VEGA no. 2/0101/22 and APVV-19-0279. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcarin L, Villapol S, Faiz M, et al (2007) Caspase-3 activation in astrocytes following postnatal excitotoxic damage correlates with cytoskeletal remodeling but not with cell death or proliferation. 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Physiology 19:124\u0026ndash;128 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/nips.01519.2004\u003c/span\u003e\u003cspan address=\"10.1152/nips.01519.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"apoptosis, activated caspase-3, cleaved PARP, spinal cord, rat, development","lastPublishedDoi":"10.21203/rs.3.rs-2854960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2854960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell death is an essential process occurring during the development of the central nervous system. Despite the availability of wide range of commercially produced antibodies against various apoptotic markers, data regarding the apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. This study aimed to investigate the apoptosis in the rat spinal cord at different stages of ontogenesis (8, 29 and 90 postnatal days). For this purpose, we used immunofluorescent detection of two widely used apoptotic markers, activated caspase-3 (aC3) and cleaved PARP (cPARP). Surprisingly, we found significant discrepancy between the amounts of aC3\u003csup\u003e+\u003c/sup\u003e cells and PARP\u003csup\u003e+\u003c/sup\u003e cells, varying with ratio around 500:1\u0026ndash;5,000:1 in the rat spinal cord in all postnatal time points. Majority of aC3\u003csup\u003e+\u003c/sup\u003e cells were glial cells and did not exhibit apoptotic phenotype. In contrast with the results of \u003cem\u003ein vivo\u003c/em\u003e study, \u003cem\u003ein vitro\u003c/em\u003e analysis of primary cell culture derived from neonatal rat spinal cord, treated with apoptotic inductor staurosporine, revealed similar onset of occurrence of both markers in cells subjected to apoptosis. Gene expression analysis of spinal cord tissue revealed elevated expression of \u003cem\u003eBirc4 (XIAP)\u003c/em\u003e, \u003cem\u003eBirc2\u003c/em\u003e and \u003cem\u003eBirc5 (Survivin)\u003c/em\u003e genes, which are known as potent inhibitors of apoptosis. Our data indicates that the activated caspase-3 is not an exclusive marker of apoptosis, especially in glial cells, due its possible presence in inhibited forms and/or its participation in other, non-apoptotic roles. Therefore, in the light of our recent results, cPARP appears to be more appropriate marker for detection of apoptosis.\u003c/p\u003e","manuscriptTitle":"Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-27 19:28:40","doi":"10.21203/rs.3.rs-2854960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-06-05T10:15:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-05-16T07:41:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42db4bd5-94ce-45ab-a2ab-f97fffd721ca","date":"2023-05-14T16:08:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44d2a3d3-bd6e-45b3-a48b-09606cf20994","date":"2023-04-25T23:02:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-25T16:36:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-25T08:15:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-04-25T06:59:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2023-04-24T13:18:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"700b2ca5-f863-4a2d-a674-847e1006342b","owner":[],"postedDate":"April 27th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-11-13T15:10:46+00:00","versionOfRecord":{"articleIdentity":"rs-2854960","link":"https://doi.org/10.1007/s00418-023-02249-7","journal":{"identity":"histochemistry-and-cell-biology","isVorOnly":false,"title":"Histochemistry and Cell Biology"},"publishedOn":"2023-11-08 15:01:28","publishedOnDateReadable":"November 8th, 2023"},"versionCreatedAt":"2023-04-27 19:28:40","video":"","vorDoi":"10.1007/s00418-023-02249-7","vorDoiUrl":"https://doi.org/10.1007/s00418-023-02249-7","workflowStages":[]},"version":"v1","identity":"rs-2854960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2854960","identity":"rs-2854960","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}