Manuscript details
Manuscript number DN_2016_30
Title Continuous monitoring of caspase-3
activation induced by propofol in developing mouse brain
Article type Research Paper
Abstract The neurotoxicity of anesthetics
on the developing brain has drawn the attention of anesthesiologists.
Several studies have shown that apoptosis is enhanced by exposure
to anesthesia during brain development.
Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain.
If it starts during the early stage of anesthesia, even short-term anesthesia could harm the brain.
Therefore, apoptogenesis should
be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe
the development of a continuous
monitoring system to detect caspase-3 activation using an in vivo model.
Brain slices from postnatal days 0-4 SCAT3 transgenic mice with a heterozygous genotype (n = 20) were used for the monitoring of caspase-3 cleavage. SCAT3 is a fusion protein of ECFP and Venus
connected by a caspase-3 cleavable peptide, DEVD. A specimen from the hippocampal CA1 sector was
mounted on a confocal laser microscope and was continuously superfused
with artificial cerebrospinal fluid, propofol (2,6-diisopropylphenol,
þÿ1 ¼M or 10 ¼M), and dimethyl sulfoxide.
Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio > 1 indicated the number of pixels from caspase-3-activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase-3 activation. This right-ward shift dramatically changed at five hours þÿin the propofol 1 ¼M and 10 ¼M groups and was obviously different from that in the control group.
Thus, real-time fluorescence energy transfer (FRET) imaging was capable of identifying the onset of apoptosis triggered by propofol in neonatal brain slices. This model may be
a useful tool for monitoring apoptogenesis in the developing brain.
Keywords Propofol anesthesia; Caspase-3;
FRET; SCAT3; neonatal mouse
Manuscript region of origin Asia Pacific
Corresponding Author Akiko Nishimura
Corresponding Author's Institution Showa University, School of Dentistry
Order of Authors Ayumi Konno, Akiko Nishimura, Shiro Nakamura, Ayako Mochizuki, Atsushi Yamada, Ryutaro Kamijo, Tomio Inoue, Takehiko Iijima
Suggested reviewers Tobias Back, Konstantin-Alexander Hossmann
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Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain
Ayumi Konno,1 Akiko Nishimura,1,* Shiro Nakamura,2 Ayako Mochizuki,2 Atsushi Yamada,3 Ryutaro Kamijo,3 Tomio Inoue,2 Takehiko Iijima1
1 Department of Perioperative Medicine, Division of Anesthesiology, Showa University School of Dentistry
2 Department of Physiology, Showa University School of Dentistry
3 Department of Biochemistry, Showa University School of Dentistry
*Corresponding author:
Akiko Nishimura, DDS, PhD
Department of Perioperative Medicine, Division of Anesthesiology, Showa University School of Dentistry, Tokyo, Japan
2-1-1 Kitasenzoku, Ota-ku, Tokyo, Japan Phone: +81-33787-1151
FAX: +81-33787-0248
Email: [email protected]
Abbreviations:
FRET, fluorescence energy transfer; ACSF, artificial cerebrospinal fluid; TNFα, tumor necrosis factor-α; CHX, cycloheximide; DMSO, dimethyl sulfoxide.
Abstract
The neurotoxicity of anesthetics on the developing brain has drawn the attention of anesthesiologists. Several studies have shown that apoptosis is enhanced by exposure to anesthesia during brain development. Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain. If it starts during the early stage of anesthesia, even short-term anesthesia could harm the brain. Therefore, apoptogenesis should be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe the development of a continuous monitoring system to detect caspase-3 activation using an in vivo model. Brain slices from postnatal days 0-4 SCAT3 transgenic mice with a heterozygous genotype (n = 20) were used for the monitoring of caspase-3 cleavage. SCAT3 is a fusion protein of ECFP and Venus connected by a caspase-3 cleavable peptide, DEVD. A specimen from the hippocampal CA1 sector was mounted on a confocal laser microscope and was continuously superfused with artificial cerebrospinal fluid, propofol (2,6-diisopropylphenol, 1 μM or 10 μM), and dimethyl sulfoxide. Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio > 1 indicated the number of pixels from caspase-3-activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase-3 activation. This right-ward shift dramatically changed at five hours in the propofol 1 μM and 10 μM groups and was obviously different from that in the control group. Thus, real-time fluorescence energy transfer (FRET) imaging was capable of identifying the onset of apoptosis triggered by propofol in neonatal brain slices. This model may
be a useful tool for monitoring apoptogenesis in the developing brain.
Keywords:
Propofol anesthesia; Caspase-3; FRET; SCAT3; Neonatal mouse
1. Introduction
Exposure to some anesthetics and sedative drugs can reportedly have a damaging effect on the developing brain. Such neurotoxicity has been confirmed histopathologically, presenting as apoptosis in the developing brain. Previous reports have demonstrated that apoptotic changes occur several hours after exposure to anesthetics (Brambrink et al. 2010;
Istaphanous et al. 2011; Satomoto et al. 2009; Zou et al. 2011). Creeley et al. confirmed the presence of obvious histopathological changes, suggesting the apoptosis of neurons and oligodendrocytes, at 3 hours after exposure to propofol for 5 hours (Creeley et al. 2013). The late appearance of apoptosis has also been confirmed in neuronal cultures. Pearn et al.
confirmed the presence of apoptosis at six hours after exposure to propofol in a primary neuronal culture (Pearn et al. 2012). However, all of these previous studies confirmed the presence of apoptosis histopathologically, based on the appearance of neuron shrinkage or TUNEL-positive neurons or Western blotting findings for cleaved caspase; these methods visualize the consequences of apoptosis, not the actual process of apoptosis. Information on when apoptosis is initiated after exposure to anesthetics and also the ability to differentiate normal physiological apoptosis in the developing brain and pathological apoptosis induced by drugs are needed. Determining whether apoptosis induced by anesthetics requires a prolonged exposure or if the switch to the apoptotic cascade commences at an early stage and may require a long time to reach the final execution of apoptosis is also important. Such information would help us to determine whether short periods of exposure to anesthetics are safe for neonatal and fetal brains.
Fluorescence energy transfer (FRET) technology can provide real-time information about the dynamics and pattern of endogenous enzyme activation in vivo (Tyas et al. 2000;
Takemoto et al. 2003). Briefly, FRET is based on the phenomenon of distance-dependent energy
transfer from a donor molecule to an acceptor molecule. If the donor and acceptor are linked by a molecular sequence that is cleaved by the action of an enzyme, the FRET disappears when the linker is cleaved. Using this phenomenon, the cleavage of linkers can be monitored by observing changes in the wavelengths of the emissions. SCAT3 is a probe for detecting caspase cleavage that utilizes Venus and ECFP as a fluorescent donor and acceptor, respectively.
Previous studies have shown that this probe can actually detect the real-time cleavage of caspase-3 in transgenic mice and transfected cells (Yamaguchi et al. 2011; Nakazawa et al.
2013). Thus, this biosensor was expected to be useful for monitoring the activation of caspase-3 in response to anesthetic exposure in vivo. Here, we describe a real-time imaging system for monitoring caspase-3 activation in mouse brain slices to identify the initiation of apoptosis.
2. Materials and methods 2. 1. Animal care and use
Postnatal day 0-4 C57BL/6J mice were used (n = 20). Animal care and all the experimental procedures were approved by the Institutional Animal Research Committee of Showa University and were in accordance with Japanese Government Law No. 105. All efforts were made to minimize the number of animals to be used and their suffering.
2.2. SCAT3 transgene
SCAT3 is a fluorescence resonance energy transfer (FRET) probe that consists of a donor (enhanced cyan fluorescent protein [ECFP]) and an acceptor (Venus, a mutant of yellow fluorescent protein). The donor and the acceptor are linked with a caspase-3 recognition and cleavage sequence (DEVD). With this probe, activated caspase-3 cleaves the linker peptide (DEVD) and abolishes the FRET, resulting in a marked increase in the lifetime of ECFP.
Therefore, the dissociation of ECFP and Venus upon cleavage of the linker increases the
ECFP/Venus ratio.
The EcoRI fragment of pJC13-1 was removed and replaced with a NotI site, and the BamHI fragment of pJC13-1 was then removed and replaced with the SCAT3 expression cassette (CAG-SCAT3-pA) to create a 2×HS4-CAG-SCAT3-pA-2×HS vector.
HeLa cells were plated on polyethyleneimine-coated glass coverslips and were transfected with 0.5 μg of plasmid vector for 6 hours, then maintained in growth medium for another 12 hours.
A transgenic mouse strain with the caspase3 active detection indicator SCAT3 (Takemoto et al. 2003) under CAG promoter control (Yamaguchi et al. 2011) was used. This mouse strain was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan.
2.2. HeLa cell culture and imaging analysis
HeLa cells were maintained in DME (Sigma-Aldrich) supplemented with 10% FBS.
Transfections of the HeLa cells were performed using the SuperFect reagent (QIAGEN).
SCAT3-expressing HeLa cells were exposed to 50 ng/mL of tumor necrosis factor-α (TNFα) and 10 μg/mL of cycloheximide (CHX) for three hours at room temperature.
2.3. Preparation of slices
The experiments were performed with brain slices from postnatal days (P) 0-4 SCAT3 transgenic mice with a heterozygous genotype (n = 20). The day of birth was defined as P0. The animals were anesthetized deeply with isoflurane and then decapitated. Each brain was removed rapidly and placed in ice-cold (4C) artificial cerebrospinal fluid (ACSF) containing the following (in mM): 130 NaCl, 3 KCl, 2.0 CaCl2, 2.0 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose. The ACSF was continuously bubbled with a 95% O2-5% CO2 gas mixture to establish a
pH of 7.4. The brain was cut into 350- to 400-µm coronal sections that included the hippocampal CA1 region using a vibrating microslicer (VT1200S; Leica Microsystems Japan, Tokyo, Japan). The slices were allowed to recover in a holding chamber containing ACSF at 34C for one hour and then were maintained at room temperature (22-25C) in ACSF.
2. 4. Administration of agents and observation
Twenty slices from twenty P0-4 animals were transferred into a recording chamber that was mounted on a confocal laser microscope (A1R; Nikon, Tokyo, Japan) equipped with a water immersion ×40 (0.8 NA) objective (Nikon Instruments), and the slices were superfused continuously with ACSF at a rate of 2.0 mL/min at 37C using a peristaltic pump and a warming device (Thermo Plate; Warner Instruments, Hamden, CT, USA). Propofol (2,6- diisopropylphenol; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in an organic solvent (dimethyl sulfoxide [DMSO]; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and applied to the ACSF at a final concentration of 1 μM or 10 μM at 37C. DMSO without propofol was added to the ACSF in the DMSO group.
SCAT3 is a fusion protein of ECFP and Venus connected by a caspase-3 cleavable peptide, DEVD. Cleavage of DEVD impairs the intracellular fluorescence resonance energy transfer (FRET) between the two fluorescent proteins and changes the emission wavelength from 525 nm for Venus to 475 nm for ECFP. We used a 457.9-nm excitation filter, a 465- to 500-nm emission filter (ECFP), and a 525- to 555-nm emission filter (Venus). The wavelength changes in CA1 neurons expressing SCAT3 were captured using an imaging scanner and were imaged at a frame rate of 4 Hz using an image acquisition and analysis system (NIS-Elements, Nikon Instruments). We used imaging software (NIS-Elements, Nikon, Tokyo, Japan), and the hippocampal CA1 neurons were scanned in region of interest (ROI)-integrated Z-axis images of
all the layers. Caspase-3-activated cells were evaluated as pseudocolor changes from blue to red on the C/V images. The z stacks (5-8 μm) and time-lapse images of the hippocampal CA1 neurons were recorded every 30 minutes for 5 hours.
To visualize the living hippocampal CA1 neurons from SCAT3 transgenic neonatal mice, we set up a live-imaging system using a fast-scanning confocal microscope, which allowed us to scan the slice regions within a short time and to reduce photobleaching and phototoxicity as much as possible.
A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio > 1 indicated the number of pixels from caspase-3-activated CA1 neurons.
Furthermore, a rightward shift of the histogram and an increase in the area with a ratio > 1 indicated the loss of FRET and an increase in caspase-3 activation.
2. 5. Statistical Analysis
Each value represents the mean ± standard error of the mean (SEM). Data obtained before and after drug application and differences between groups were compared using a multivariate analysis of variance (MANOVA). The MANOVA was followed by a Dunnett t-test for multiple comparisons (post-hoc test), when appropriate. Probability values of less than 0.05 were considered significant. Statistical analyses were conducted using SPSS ver. 17 (SPSS Inc.) and Microsoft Excel 2008.
3. Results
3. 1. Confirmation of SCAT3 expression in apoptotic model
The SCAT3 probe was effectively expressed in HeLa cells. The FRET disappeared after treatment with 50 ng/mL of TNFα and 10 μg/mL of CHX. The ECFP/Venus ratio of the
HeLa cells started to increase from the baseline value after approximately 90 min of exposure to TNFα/CHX (Fig. 1A), but the increase was not accompanied by any morphological changes (Fig. 1B). The area of fluorescence suddenly changed after 105 min of exposure to TNFα/CHX, suggesting the occurrence of cell shrinkage as an early apoptotic morphological change (Fig.
1A). The trend for the ECFP and Venus intensities in this cell showed a conversion of the ratio after 100 min (Fig. 1B)
3. 2. Visualization of caspase-3 activation in living neurons from the hippocampal CA1 sector We also confirmed the occurrence of FRET changes in brain slices from SCAT3 transgenic mice. The ECFP/Venus ratio increased in a single cell at 100 min after the start of observation in an ACSF bath without the application of any apoptogenic agent. The activated cells changed from blue-green to yellow-red (Fig. 2A). This change was thought to correspond to physiological apoptosis in the developing brain. The trend for the ECFP and Venus intensities in this cell showed a conversion of the ratio after 100 min (Fig. 2B).
3.3. Caspase-3 activation in the CA1 region induced by propofol
We observed mostly FRET-positive cells in the control CA1 sector mixed with a few yellow FRET-negative cells (white arrowheads), indicating the cleavage of SCAT3 protein by activated caspase-3 (Fig. 3 Ab). In the control group, the number of yellow FRET-negative cells did not increase even after five hours. The histograms of fluorescence intensity in the control group also did not shift significantly during the five-hour observation period (Fig. 3B). The number of yellow cells increased after propofol application (Fig. 4A). The peak of the histogram shifted toward the right, and the number of activated cells (ECFP/EYFP ratio of more than 1.0) increased significantly in the propofol group (Fig. 4B). In some samples, this shift started as
early as one hour after the addition of propofol.
The area of the histogram with an ECFP/Venus ratio of more than 1.0, indicating the ratio of caspase-activated area to inactivated area, was compared among four groups (control, 1 μM of propofol, 10 μM of propofol, and DMSO) (Fig. 5). Each group showed an increase over time. The 1 μM of propofol and the 10 μM of propofol groups showed significant increases from the baseline values after five hours (24.2% ± 7.2% for the 1 μM group and 36.5% ± 11.4%
for the 10 μM group, P < 0.05). The histograms of the fluorescence intensity in the control and DMSO groups did not change significantly and only shifted slightly to the right after five hours.
4. Discussion
4.1. Advantage of FRET analysis
Many fluorescent labeling methods for endogenous enzymes have been developed to detect their locations, although such methods are unable to confirm the timing of enzymatic changes. Fluorescence resonance energy transfer (FRET) technology enables the detection of not only the location of an enzyme, but also the real-time activation of the enzyme. Venus is a variant of EYFP that exhibits a fast and efficient maturation at 37C (Nagai et al. 2002). The SCAT3 probe has been used to detect the local initiation of caspase-3 activation for the normal development and maintenance of tissue homeostasis in embryos (Takemoto et al. 2007;
Yamaguchi et al. 2011). The development of this indicator has enabled researchers to monitor caspase-3 activation at the single-cell level in real time. FRET-based evaluations of caspase activation have been performed in transfected cell lines, human umbilical vein endothelial cells (Zhu, Fu, and Luo 2012), human lung adenocarcinoma cells (Wu, Xing, and Chen 2006), and human neuroblastoma SH-SY5Y cells (Figueroa et al. 2011). However, in cultured neurons such as hippocampal cells, the transfection is far less efficient, and the change in FRET is not as
easily observed because of the weak signal. In the present study, we used a SCAT3 transgenic mouse model, which enabled the clear visualization of changes in fluorescence.
Caspase-3 activation rapidly reaches a maximum in 10 min or less in HeLa cells treated with TNFα/CHX, and the nuclear activation of caspase-3 precedes nuclear apoptotic morphological changes (Takemoto et al. 2003). We also observed that the fluorescent intensity changed in 10-20 min, followed by morphological changes in the HeLa cells (Fig. 1). We confirmed that this biosensor was actually functioning in the transgenic mice.
4.2. Initiation of apoptotic cascade after anesthetic exposure
We demonstrated that caspase-3 was linearly activated over time in the control and DMSO groups, while the activation increased exponentially after five hours. This time-sequence profile of apoptosis induced by propofol has not been previously reported. Previous studies have shown that prolonged inhalational and intravenous anesthetic exposure causes altered dendritic spine morphology and synaptic loss in the developing brains of several animal species (Creeley et al. 2013; Satomoto et al. 2009; Istaphanous et al. 2011; Zou et al. 2011; Yang et al. 2014).
Since these reports were in vitro studies, however, the time-sequence profile could not be confirmed.
Repeated exposure to sevoflurane led to a greater loss of synapses in rat pups, compared with a single exposure. This reduction was correlated with the total anesthetic exposure, rather than the frequency of exposure (Amrock et al. 2015). In recent studies, caspase activation was confirmed to occur after more than five hours of exposure (Brambrink et al.
2010). Thus, apoptosis induced by anesthetic exposure may require more than 5 hours. Our study confirmed this hypothesis. Exposure to 3% sevoflurane for 6 hours reportedly impaired the long term potential (LTP), a surrogate marker of synaptic plasticity, and induced structural
damage in P7 rat hippocampus samples (Xiao et al. 2016). Our study also observed hippocampal neurons; thus, the effect of anesthetics on the electrophysiological activity may be correlated with cell death.
Propofol (2,6-diisopropylphenol) is an intravenous general anesthetic commonly used for the induction and maintenance of anesthesia. Most studies investigating the neurotoxicity of anesthetics have used inhalational anesthetics, not intravenous anesthetics. Recently, prolonged propofol anesthesia was reported to induce caspase-3 activation and apoptotic changes in a histological study of developing animals. More than five hours of propofol exposure is also required to induce neuroapoptosis, as demonstrated in subcortical and caudal regions during the fetal period and in neocortical regions during the neonatal period (Creeley et al. 2013). In neonatal rodents and cell cultures, 5-6 hours of propofol anesthesia are required to induce the apoptotic degeneration of neurons (Krzisch et al. 2013; Pearn et al. 2012). At least four hours were required to activate caspase-3 after the single administration of propofol at 25 mg/kg to rat pups (Pesic et al. 2009). Our study also confirmed a 5-hour latent period before the start of apoptosis.
The latent period before caspase activation may involve a preceding process of apoptosis. Cheng et al. reported that only 1 hour of exposure to 2% isoflurane induced cytochrome c release from mitochondria, triggering the activation of caspase-3 in the developing retina (Cheng et al. 2015). The apoptotic process may actually commence even earlier than several hours. We have also observed caspase activation as early as after 1 hour of exposure in some examples (Fig. 3). Thus, our model was suitable for detecting the very early phase of caspase activation.
4.3. Clinically relevant concentration of propofol and its effect on vulnerability
Clinical induction doses of propofol (2-3 mg/kg intravenously) correspond to peak plasma concentrations of 60-80 μM (Dowrie et al. 1996; Fan et al. 1995), and the maintenance concentration is reported to be approximately 30 μM (Hirota et al. 1999; Handa-Tsutsui and Kodaka 2005). The effective concentration of propofol in plasma-free media is assumed to be approximately as low as 1 μM, because 97%-98% of propofol binds to plasma protein (Gleason et al. 2010). Therefore, we employed 1 μM of propofol, which should be relevant to clinical concentrations in the brain. Propofol at clinically relevant plasma concentrations (1-10 μM) applied to a primary hippocampal neuronal culture actually reduced the extracellular signal- regulated protein kinase phosphorylation (Kozinn et al. 2006). Therefore, even a bath application of 1 μM should be sufficient to expect a pharmacological effect of propofol.
Previous immunohistological studies have suggested that neurons in the developing brain are vulnerable to propofol anesthesia ( Liang et al., 2012; Milanovic et al., 2014; Pesic et al., 2009). The mechanism of propofol-mediated apoptosis in the developing brain is still unclear, but it is likely to share common mechanisms with the effects of inhalational general anesthetics, such as GABA receptor activation (Zhao et al. 2011), P75 neurotrophin receptor activation (Pearn et al. 2012), and the overactivation of inositol trisphosphate (InsP3) receptors (Y. Peng 2011; Zhao et al. 2010; Wei et al. 2008). Further study is needed to clarify these mechanisms, and our model may be useful for exploring the mechanism of propofol-induced neuroapoptosis.
4.4. Analysis of caspase-3 activation using the FRET technique
Our monitoring system enables the detection of even single cell changes (Fig. 2).
However, a quantitative analysis method using this system has not yet been established.
Nakazawa et al. used a probe constructed using CFP and YFP with Bid that contained a tandem
caspase-8-specific cleavage site for the detection of apoptosis in cell cultures. They focused on areas of high-intensity fluorescence in single cells and depicted histograms of the intensity. A FRET shift expresses the appearance of caspase activation without excluding non-significant background signals (Nakazawa et al. 2013). We applied this technique to the tissue of the hippocampus, where the cells are regularly aligned. We also confirmed a shift in the histogram in response to the appearance of caspase-3 activation. We defined a ratio > 1 in the histograms of the ECFP/Venus ratio as indicating caspase-3 activation, and we confirmed the turnover of the intensity of ECFP and Venus in a single apoptotic cell.
4.5. Limitation
Some limitations of this study should be acknowledged. Propofol was applied to a bath and was used to superfuse the slices examined in our study, rather than being administered intravenously or intraperitoneally. Anesthetic-induced neuroapoptosis in the developing brains of rodents and monkey was also examined using whole animals, and propofol was administrated intravenously or intraperitoneally. Our model may be difficult to extrapolate to other vital organs. However, our model may be appropriate for the detection of the early onset of apoptosis, since this model enabled the continuous monitoring of apoptosis activation.
5. Conclusion
In conclusion, we have developed a continuous monitoring system for detecting caspase-3 activation in neonatal brain slices. This system revealed that caspase-3 activation induced by exposure to propofol (1-10 μM) at clinical relevant concentrations increased significantly at five hours, which was a distinctly different profile from the non-specific activation of caspase. This monitoring system could be a useful tool for examining the
apoptogenic properties of anesthetics.
Acknowledgments
This work was supported by a MEXT grants-in-aid for scientific research (Nos.
23792131 and 25861974). The authors declare that they do not have any potential conflicts of interest with respect to the authorship and/or publication of this article.
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Figure legends
Fig. 1. Single cell imaging analysis of caspase-3 activation using SCAT3. (A) ECFP/Venus (C/V) ratio and emission intensity images of SCAT3-expressing HeLa cells exposed to 50 ng/mL of TNFα and 10 μg/mL of CHX. HeLa cells were transfected with 0.5 μg of pcDNA- SCAT3. The imaging analysis was started at 18 h after transfection. The yellow circle indicates a region of interest (ROI). Bar: 20 μm. (B) Emission intensity profiles of ECFP and Venus, and ECFP/Venus emission ratio of the SCTA3-expressing HeLa cells examined in A.
Fig. 2. Visualization of caspase-3 activation in a living neuron. (A) ECFP/Venus (C/V) ratio and emission intensity images of a hippocampal CA1 neuron in a SCAT3 transgenic mouse. The neuron (white arrowhead) began undergoing an apoptotic change at 120 min. Bar: 10 μm. (B) Emission intensity profiles of ECFP and Venus, and ECFP/Venus emission ratio of the SCTA3- expressing neuron examined in A.
Fig. 3. Real-time detection of caspase-3 activation in hippocampal CA1 neurons of SCAT3 transgenic mice. (A) Ratio images of the hippocampal CA1 region in a coronal section from a SCAT3 transgenic mouse (Aa). Caspase-3 activation is represented as pseudocolors corresponding to the ECFP/Venus ratio (0.2-1.8). The region inside the white square in (Aa) is shown magnified in the images shown in (Ab). The images in the top row are superimposed images of all the Z-axis layers from top to bottom. The white arrowheads showed neurons in which the amount of ECFP exceeded the amount of Venus, causing the ratio to exceed 1.0 and resulting in the disappearance of FRET. Bars: 500 µm in (Aa), 100 µm in (Ab). (B) Histogram analysis of the ECFP/Venus ratios shown in the images in Ab. The loss of FRET induced an increase in the ECFP/Venus ratio above 1.0. The rightward shift of the histograms and the
increase in the area above 1.0 indicated an increase in caspase-3 activation.
Fig. 4. Caspase-3 activation induced by propofol in living hippocampal CA1 neurons of SCAT3 transgenic mice. (A) Ratio images of hippocampal CA1 region treated with propofol.
Caspase-3 activation is represented as pseudocolors corresponding to the ECFP/Venus ratio (0.2-1.8). The images in the top row are superimposed images of all the Z-axis layers from top to bottom. The white arrowheads showed neurons in which the amount of ECFP exceeded the amount of Venus, causing the ratio to exceed 1.0 and resulting in the disappearance of FRET.
Bar: 100 µm. (B) Histogram analysis of the ECFP/Venus ratios shown in the images in A. The loss of FRET induced an increase in the ECFP/Venus ratio above 1.0. Note that a rightward shift of the histograms and an increase in the area above 1.0 is visible even one hour after exposure (arrowhead). Compare with Fig. 3
Fig. 5. Summary of effects of propofol and DMSO on hippocampal CA1 neurons in SCAT3 mice. The areas of the histograms for an ECFP/Venus ratio of more than 1.0, corresponding to the cleavage of the SCAT3 protein by activated caspase-3 significantly increased from baseline at 5 hours in the propofol 1 µM, 10 µM and DMSO groups (P < 0.05).
Highlights
・ SCAT3 transgene continuously monitored caspase-3 cleavage in neonatal mouse brain.
・ Caspase-3 pathologically activated at five hours after exposure of propofol.
・ Real-time FRET imaging identified the onset of apoptosis by propofol anesthesia.
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