Unilateral Blood Flow Decrease Induces Bilateral and Symmetric Responses in the Immature Brain
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.090257
ISSN1525-2191
AutoresSonia Villapol, Philippe Bonnin, Sébastien Fau, Olivier Baud, Sylvain Renolleau, Christiane Charriaut‐Marlangue,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe effects of hemodynamic changes in the developing brain have yet to be fully understood. The aim of this study was to explore the relationship between perturbations of the cerebral blood flow in the developing brain via unilateral hypoperfusion in P7 rats. As expected, nuclear caspase-3 (casp3) cleavage and DNA fragmentation were detected at 48 hours after stroke in the injured cortex. Surprisingly, casp3 was also cleaved in the contralateral cortex, although without cell death markers. Delayed (48 hours) casp3 cleavage without DNA fragmentation was also identified after unilateral common carotid artery occlusion, both in the hypoperfused cortex and the unaffected cortex, producing mirror images. Upstream calpain activation, caspase-2 cleavage, and mitochondrial cytochrome c release initiated casp3 cleavage, but did not produce preconditioning. The neuronal marker NeuN co-localized with cleaved casp3 in cortical layers II–III and VI and with gaba-amino butyric acid in layer III. Indeed, collateral supply was provided from the opposite side during carotid artery occlusion but not after reperfusion, and the number of cleaved casp3-positive cells significantly negatively correlated with the common carotid artery immediate reperfusion percentage. In summary, unilateral hypoperfusion, while insufficient to induce cell death, may active bilateral and symmetric casp3 in the P7 rat brain. Additionally, the opposite healthy hemisphere is altered due to the injury and thus should not be used as an internal control. The effects of hemodynamic changes in the developing brain have yet to be fully understood. The aim of this study was to explore the relationship between perturbations of the cerebral blood flow in the developing brain via unilateral hypoperfusion in P7 rats. As expected, nuclear caspase-3 (casp3) cleavage and DNA fragmentation were detected at 48 hours after stroke in the injured cortex. Surprisingly, casp3 was also cleaved in the contralateral cortex, although without cell death markers. Delayed (48 hours) casp3 cleavage without DNA fragmentation was also identified after unilateral common carotid artery occlusion, both in the hypoperfused cortex and the unaffected cortex, producing mirror images. Upstream calpain activation, caspase-2 cleavage, and mitochondrial cytochrome c release initiated casp3 cleavage, but did not produce preconditioning. The neuronal marker NeuN co-localized with cleaved casp3 in cortical layers II–III and VI and with gaba-amino butyric acid in layer III. Indeed, collateral supply was provided from the opposite side during carotid artery occlusion but not after reperfusion, and the number of cleaved casp3-positive cells significantly negatively correlated with the common carotid artery immediate reperfusion percentage. In summary, unilateral hypoperfusion, while insufficient to induce cell death, may active bilateral and symmetric casp3 in the P7 rat brain. Additionally, the opposite healthy hemisphere is altered due to the injury and thus should not be used as an internal control. Perinatal hypoxia-ischemia followed by reperfusion is an important cause of neonatal brain injury. In approximately 8 out of 100,000 children affected, it results in cerebral palsy, learning disabilities, visual field deficits, and epilepsy.1Ferriero DM Neonatal brain injury.N Engl J Med. 2004; 351: 1985-1995Crossref PubMed Scopus (933) Google Scholar However, recent data suggest a higher incidence of focal ischemia in neonates compared with the incidence of global cerebral ischemia arising from systemic asphyxia,2Golomb MR Garg BP Saha C Azzouz F Williams LS Cerebral palsy after perinatal arterial ischemic stroke.J Child Neurol. 2008; 23: 279-286Crossref PubMed Scopus (70) Google Scholar, 3Lynch JK Hirtz DG DeVeber G Nelson KB Report of the National Institute of Neurological Disorders and Stroke workshop on perinatal and childhood stroke.Pediatrics. 2002; 109: 116-123Crossref PubMed Scopus (535) Google Scholar whereas mechanisms of arterial ischemic injury without the confounding effect of hypoxia are not fully understood.To investigate cell death pathways and neuroprotection, the most commonly used model of hypoxia-ischemia (HI)4Rice 3rd, JE Vannucci RC Brierley JB The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1902) Google Scholar and others stroke models5Derugin N Ferriero DM Vexler ZS Neonatal reversible focal cerebral ischemia: a new model.Neurosci Res. 1998; 32: 349-353Crossref PubMed Scopus (81) Google Scholar, 6Renolleau S Aggoun-Zouaoui D Ben-Ari Y Charriaut-Marlangue C A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.Stroke. 1998; 29 (discussion 1461): 1454-1460Crossref PubMed Scopus (144) Google Scholar have been designed in the developing brain (age range from P3 up to P12) in both rats and mice. In all of these models, injury is only seen in the ipsilateral hemisphere and in a great number of studies the contralateral hemisphere was used as an internal control. However, two recent studies have pointed out that multiple molecular/cellular mediators were also activated in the contralateral hemisphere. In the first work, HI in P12 rats produced increased cytokine expression, hypoxic-inducing factor (HIF-1) α, and phospho (p)-Akt to the same extent in both hemispheres, although injury was unilateral. Conversely, hypoxia alone was sufficient to regulate these mediators, but not sufficient to induce long-term damage.7van den Tweel ER Kavelaars A Lombardi MS Nijboer CH Groenendaal F van Bel F Heijnen CJ Bilateral molecular changes in a neonatal rat model of unilateral hypoxic-ischemic brain damage.Pediatr Res. 2006; 59: 434-439Crossref PubMed Scopus (49) Google Scholar In the second study, we demonstrated that after stroke (electrocoagulation of the left middle cerebral artery (MCA) in association with transient homolateral common carotid artery occlusion) in P7 rats,6Renolleau S Aggoun-Zouaoui D Ben-Ari Y Charriaut-Marlangue C A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.Stroke. 1998; 29 (discussion 1461): 1454-1460Crossref PubMed Scopus (144) Google Scholar cell injury was visible in the ipsilateral (IL) parietal cortex, but cell death also affected the contralateral white matter during the first 24 hours of recovery followed by delayed repair mechanisms in both the IL and contralateral (CL) hemispheres.8Spiegler M Villapol S Biran V Goyenvalle C Mariani J Renolleau S Charriaut-Marlangue C Bilateral changes after neonatal ischemia in the P7 rat brain.J Neuropathol Exp Neurol. 2007; 66: 481-490Crossref PubMed Scopus (10) Google Scholar Because the molecular mechanisms of cell death/survival in remote regions and particularly in the CL hemisphere are still poorly understood, the aim of this study was to explore the relationship between the molecular pathways and perturbations of the cerebral blood flow (CBF) in the developing brain.Arterial occlusion causes hemodynamic perturbations and a redistribution of blood flow. The circulatory system can compensate for this by various processes, for example, using collateral circulation and/or autoregulatory mechanisms.9Liebeskind DS Collateral circulation.Stroke. 2003; 34: 2279-2284Crossref PubMed Scopus (766) Google Scholar However, the reserve capacity of the circulatory system is limited, and when these processes offer insufficient compensation, inevitably tissue will enter a state of hypoperfusion. This study highlights that unilateral hypoperfusion, not sufficient to induce cell death, may active cellular mechanisms bilaterally and symmetrically producing images in mirror in P7 rat brain.Materials and MethodsPerinatal IschemiaAll animal experimentation was conducted in accordance with the French and European Community guidelines for the care and use of experimental animals. Ischemia was performed on 7-day-old rat pups (17 to 21 g, both sexes), as described previously.6Renolleau S Aggoun-Zouaoui D Ben-Ari Y Charriaut-Marlangue C A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.Stroke. 1998; 29 (discussion 1461): 1454-1460Crossref PubMed Scopus (144) Google Scholar Rat pups were anesthetized with an i.p. injection of chloral hydrate (350 mg/kg). The anesthetized rat was positioned on its back, and a median incision was made in the neck to expose the left common carotid artery (CCA). The rat was then placed on its right side and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then the left MCA, exposed just after its appearance over the rhinal fissure, was electrocoagulated (MCAo) at the inferior level of the cerebral vein. After this procedure, a vascular clip (18055-04; Fine Science Tools, Heidelberg, Germany) was placed to occlude the left common carotid artery (tCCAo). Rats were then placed in an incubator to avoid hypothermia. After 50 minutes, the clip was removed. Carotid blood flow restoration was verified with the aid of a microscope. This stroke model is referred to as model M1 (see Table 1). Both neck and cranial skin incisions were then closed. During the surgical procedure, external body temperature was maintained at 37–37.5°C. After recovery, pups were transferred to their mothers. In other sets of experiments, animals were only subjected to one (left or right) or both transient (50 minutes) CCAo, whereas others underwent definitively ligation of one or both CCA (referred to as model M2, M3, or M4 respectively; see Table 1). Sham animals were only subjected to carotid isolation and did not receive MCA electrocoagulation or transient CCA occlusion. Of 68 animals, 1 died in model M1, and 3 died in model M4 during the first 24 hours of recovery.Table 1Description of the Models UsedAnimal modelArtery occlusionModel descriptionNo. of animals*Tissues from animals were used either for immunohistochemistry or Western blotting analyses.M1pMCAo + uni-tCCAoLeft middle cerebral artery occlusion (MCAo) and unilateral transient left Common Carotid Artery occlusion (tCCAo)n = 27M2Uni-t/pCCAoUnilateral transient or permanent†Animals with permanent CCAo provided either from animals, who after CCAo release, did not show recanalization (determined after CBF measurements) or from animals with unilateral ligated CCA. left or right Common Carotid Artery occlusion (t/pCCAo)n = 21M3Bi-tCCAoBilateral transient common carotid artery occlusionn = 6M4Bi-pCCAoBilateral permanent common carotid artery occlusionn = 8M5Uni-tCCAo 24 hours after pMCAo + uni-tCCAoUnilateral transient common carotid artery occlusion (uni-tCCAo) and 24 hours after Left middle cerebral artery occlusion (MCAo) with uni-tCCAon = 6* Tissues from animals were used either for immunohistochemistry or Western blotting analyses.† Animals with permanent CCAo provided either from animals, who after CCAo release, did not show recanalization (determined after CBF measurements) or from animals with unilateral ligated CCA. Open table in a new tab Arterial Blood Flow Monitoring Using Ultrasound ImagingRats subjected to model M2 (n = 8) were analyzed using ultrasound measurements via an echocardiograph (Vivid 7; GE Medical Systems Ultrasound, Horten, Norway) equipped with a 12-MHz linear transducer (12L) as reported previously.10Bonnin P Debbabi H Mariani J Charriaut-Marlangue C Renolleau S Ultrasonic assessment of cerebral blood flow changes during ischemia-reperfusion in 7-day-old rats.Ultrasound Med Biol. 2008; 34: 913-922Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Doppler spectral recordings in the right and left internal carotid arteries, basilar trunk, and posterior (P2 segment) cerebral arterie,11Hilger T Niessen F Diedenhofen M Hossmann KA Hoehn M Magnetic resonance angiography of thromboembolic stroke in rats: indicator of recanalization probability and tissue survival after recombinant tissue plasminogen activator treatment.J Cereb Blood Flow Metab. 2002; 22: 652-662Crossref PubMed Scopus (28) Google Scholar were evaluated 1) before surgery, 2) during ischemia, 3) after reperfusion, and 4) 24 hours later (n = 8). Data were then transferred to an ultrasound image workstation for subsequent analysis (PC EchoPAC; GE Medical Systems Ultrasound). The repeatability coefficient values for intraobserver repeatability were 1.5 cm s−1 for the peak systolic, 1.7 cm s−1 for the end-diastolic, and 1.7 cm s−1 for the mean blood flow velocity (mBFV) in ICA.Regional CBF MonitoringCortical regional CBF (rCBF) was monitored in the MCA territory by laser Doppler flowmetry in the same animals that were monitored using ultrasound imaging (n = 8, group M2). A laser-Doppler probe (MP7b, Moor Instruments Ltd, Axminster, UK) was positioned on the parietal cortex, and rCBF was monitored using a Moor CBF device in each animal before (basal) and during arterial occlusion and 15 minutes after reperfusion (n = 8). The relative changes in rCBF were expressed as percentage of the average basal level recorded over 15 minutes.Assessment of Cell DamageAt 12, 48, and 72 hours after injury, the brains were removed, fixed and cryoprotected and cut into 20-μm-thick coronal sections. Sections through the dorsal hippocampus (bregma – 3.3 mm) were stained with cresyl-violet or processed either for terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining according to the manufacturer’s instructions (Roche, Meylan, France) or for Fluoro-Jade B (Histochem, Jefferson, AR) as reported previously.12Joly LM Mucignat V Mariani J Plotkine M Charriaut-Marlangue C Caspase inhibition after neonatal ischemia in the rat brain.J Cereb Blood Flow Metab. 2004; 24: 124-131Crossref PubMed Scopus (48) Google ScholarImmunohistochemistryImmunohistochemistry was performed on cryostat coronal sections (at 4, 12, 24 (M1), 48 (M1 to M4) and 72 (M2) hours after injury) as previously described,13Benjelloun N Joly LM Palmier B Plotkine M Charriaut-Marlangue C Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia-ischaemia in the rat brain.Neuropathol Appl Neurobiol. 2003; 29: 350-360Crossref PubMed Scopus (59) Google Scholar using anti-cleaved caspase-3 (casp3) (Asp175; Cell Signaling Technology, Ozyme, St-Quentin-en-Yvelines, France). Casp3- labeled cells were counted (in a blind manner) in both IL- and CL cortex in three to four coronal sections for each animal at 48 hours of recovery (n = 4–6) and in age-matched control (n = 2) rats, using a ×20 objective. Cleaved casp3 is found in the cytosol and becomes active after translocation in the nucleus.14Marks N Berg MJ Recent advances on neuronal caspases in development and neurodegeneration.Neurochem Int. 1999; 35: 195-220Crossref PubMed Scopus (100) Google ScholarSimple and Double ImmunofluorescenceImmunofluorescent staining was performed on free-floating sections (30 μm thick) as previously described,15Villapol S Acarin L Faiz M Castellano B Gonzalez B Survivin and heat shock protein 25/27 colocalize with cleaved caspase-3 in surviving reactive astrocytes following excitotoxicity to the immature brain.Neuroscience. 2008; 153: 108-119Crossref PubMed Scopus (34) Google Scholar using anti-cleaved casp3 (rabbit polyclonal ALX-210-807; Alexis Biochemicals, Coger, Paris, France), NeuN (VMA377; AbCys, Paris, France) to label neurons, glial fibrillary acidic protein (clone G-A-5; Sigma-Aldrich) to stain astrocytes, nestin (clone Rat 401; Chemicon, Euromedex, Souffelweyersheim, France), and vimentin (clone VIM 13.2; Sigma-Aldrich) to detect progenitor cells, and finally GABA (MAB351; Chemicon, Millipore, St-Quentin-en-Yvelines, France) to identify GABA interneurons. Selected sections were photographed using a Nikon Eclipse E800M microscope (Paris, France) and DFC 300 FX camera (Leica Microsystems, Rueil-Malmaison, France) interfaced with IM50 imaging software. 4′,6′-Diamidino-2-phenylindole labeling was used to visualize nuclear morphology. Double immunofluorescence (cell marker and cleaved casp3) staining was analyzed using a Leica TCS SP5 AOBS confocal laser microscope with a ×63 lens, and z-series stack (2-μm-optical slice thickness) images were obtained and imported into Adobe Photoshop (version 7.0) and ImageJ (1.38x; National Institutes of Health, Bethesda, MD) for image processing and analysis.Western Blot AnalysisSham (n = 2), model M1 (n = 6), and model M2 (n = 6) animals were sacrificed at 48 hours after injury and brains rapidly dissected out on a cold plate as reported previously.16Renolleau S Fau S Goyenvalle C Joly LM Chauvier D Jacotot E Mariani J Charriaut-Marlangue C Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.J Neurochem. 2007; 100: 1062-1071Crossref PubMed Scopus (141) Google Scholar Cortical tissues were harvested and stored to −70°C until use. To each tissue piece, 9 volumes of ice-cold homogenization buffer [15 mmol/L Tris-HCl (pH 7.6), 320 mmol/L sucrose, 3 mmol/L EDTA, 1 mmol/L DTT, and the protease inhibitor mixture (Roche)] were added. Cell lysates were then centrifuged at 800 ×g for 10 minutes. at 4°C, given a supernatant (S1) and a pellet (P1, nuclear fraction). S1 was then centrifuged at 9200 ×g for 20 minutes at 4°C, producing a crude cytosolic (S2) fraction in the supernatant and a mitochondrial fraction (M1). P1 and S2 fractions were solubilized, and an equal amount of protein (40 μg) was submitted to Western blotting. The primary antibodies used in this study were anti-casp2 (ALX-804-356; Alexis Biochemicals), casp8 (sc-5263), and cytochrome c (sc-13560) both from Santa Cruz Biotechnology (Tebu-Bio, Le Perray-en-Yvelines, France), cleaved casp9 (Asp353) and casp3 (Asp175), -Akt (number 9272) and -p-Akt (Ser473), glycogen synthase kinase-3β (27C10) and -p-GSK-3β (Ser9), Erk1/2 (number 9102), and -p-Erk1/2 (Thr202/Tyr204) all from Cell Signaling Technology. Antibodies against αII-spectrin (FG 6090; BIOMOL, Coger, Paris, France), poly(ADP-ribose) polymerase-1 (11835238001; Roche), and β-actin (clone AC-15; Sigma-Aldrich) were also used. The blots were semiquantified using gel densitometry and ImageJ analysis system. An absence of cyclophilin D (AP1035; Calbiochem, Merck Chemicals, Nottingham, UK) immunostaining in the cytosolic S2 fraction indicated that S2 was not contaminated with mitochondrial material.Statistical AnalysisAll results are expressed as mean ± SD. One-way analysis of variance and post hoc Bonferronni test were used to analyze differences between the groups (Western blotting analysis, laser Doppler measurements). A Kruskal-Wallis nonparametric test was used to analyze differences in peak systolic, end-diastolic, and time-average mBFVs measured in the right and left cerebral arteries. Pearson correlation analysis was used to reveal the correlation among the number of cleaved casp3-positive cells and the percentage of reperfusion. StatView 6.0 software was used in all statistical analyses.ResultsNeonatal Ischemia Triggers Cleavage of Casp3 in Both HemispheresNo p17 cleaved casp3 immunoreactivity was found in the cortex of sham animals (data not shown). In contrast, casp3 labeling was detected in both IL and CL cortices in ischemic animals from the group M1, at 48 hours. In the CL cortex, casp3 was detected in the more caudal region (dorsal hippocampus) and exclusively in the cytosol of numerous cells present in layers IV to VI (Figure 1). However, DNA fragmentation in these regions was undetectable as evidenced by TUNEL assay (Figure 1, A–C and I). In contrast, cleaved casp3 was detected in a large number (∼82–84%) of TUNEL-positive cells throughout cortical layers with a cytosolic and/or nuclear localization in the IL cortex (Figure 1, D–F and I). The number of cleaved casp3-positive cells increased from 4 to 48 hours (peak) in both IL and CL cortex with 861 ± 231 and 442 ± 154 positive cells at 48 hours, respectively (Figure 1G). The number of casp3-positive cells remained elevated in the IL cortex but not in the CL cortex at 3 days postinjury. Western blot analysis demonstrated that p17-cleaved casp3 was found in both cytosolic and nuclear fractions from IL cortical tissues and only in cytosolic fractions from CL cortices at 48 hours postinjury (Figure 1H); cleaved casp3 expression in the CL cortex only represented 15 to 25% (n = 8) of that detected in the IL cortex. It should be noted that sections from ischemic (model M1) animals treated with the pan-caspase inhibitor Q-VD-OPh (used in our previous study16Renolleau S Fau S Goyenvalle C Joly LM Chauvier D Jacotot E Mariani J Charriaut-Marlangue C Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.J Neurochem. 2007; 100: 1062-1071Crossref PubMed Scopus (141) Google Scholar) and analyzed 48 hours later demonstrated a very significant reduction in casp3-positive cells in the IL hemisphere and no labeling in the CL hemisphere (data not shown).CCA Occlusion Triggers Casp3 Cleavage in Both HemispheresTo determine the depth of hypoperfusion sufficient and required to produce cleaved casp3 without DNA fragmentation in the CL cortex, we tested blood flow perturbation induced by unilateral (uni) or bilateral (bi-) transient (t) or permanent (p) CCA occlusion (model M2 to M4; Table 1). As shown in supplemental Figure S1 (see http://ajp.amjpathol.org) with cresyl-violet staining at 48 hours postinjury, uni-tCCAo (supplemental Figure S1, A and B; n = 7), uni-pCCAo (data not shown, n = 5), and bi-tCCAo (supplemental Figure S1, C and D; n = 5) did not induce significant cell death. In contrast, a pale zone in cortical layer VI and white matter (external capsule; supplemental Fig. S1, E and F, see http://ajp.amjpathol.org; n = 7) was observed after Bi-pCCAo. Additionally, a pale zone in all cortical layers and white matter was observed after MCAo with uni-tCCAo (model M1; supplemental Figure S1, G and H, see http://ajp.amjpathol.org; n = 6). All ischemic procedures induced cleavage of casp3 in both IL and CL cortices, and the labeling was observed in layers II–III and VI for the different models studied (Figure 2). No cell death (TUNEL and Fluoro Jade B staining) was found to be associated with the former (uni-tCCAo or uni-pCCAo, M2; Figure 2, A–D), whereas TUNEL-positive nuclei were detected to a lesser (bi-tCCAo, M3, Figure 2, E–H) or greater (bi-pCCAo, M4; Figure 2, I–L) extent. Furthermore, cleaved casp3 displayed a cytosolic (Figure 2H) and a nuclear (Figure 2L) location after bi-tCCAo (M3) and bi-pCCAo (M4), respectively. Interestingly, a pattern of cleaved casp3 was found as images in mirror (Figure 2, A, E, and I) in all of the studied models. Although brain injury and apoptosis in the immature brain has been previously shown to depend on gender,16Renolleau S Fau S Goyenvalle C Joly LM Chauvier D Jacotot E Mariani J Charriaut-Marlangue C Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.J Neurochem. 2007; 100: 1062-1071Crossref PubMed Scopus (141) Google Scholar, 17Hagberg H Wilson MA Matsushita H Zhu C Lange M Gustavsson M Poitras MF Dawson TM Dawson VL Northington F Johnston MV PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury.J Neurochem. 2004; 90: 1068-1075Crossref PubMed Scopus (234) Google Scholar, 18Johnston MV Hagberg H Sex and the pathogenesis of cerebral palsy.Dev Med Child Neurol. 2007; 49: 74-78Crossref PubMed Scopus (212) Google Scholar subanalysis according to sex demonstrated that the presence of cleaved casp3 in both hemispheres did not depend on gender in the present study.Figure 2Casp3 cleavage and DNA fragmentation in several models of carotid occlusion at 48 hours after injury. Spatial distribution (A, E, and I) of cleaved casp3 (red), TUNEL-positive (green) cells, and lesion area (gray) after uni-CCAo (model M2), bi-tCCAo (model M3) and bi-pCCAo (model M4), respectively. Note that images in mirror were detected in all three ischemic conditions. Double fluorescent staining in the CL cortex for cleaved casp3 (B, F, and J) and TUNEL (C, top, G, and K) or Fluorojade B (FluJB) labeling (C, bottom). After uni-CCAo, only cytosolic cleaved casp3 (enlarged image in D) without TUNEL or FluJB staining was observed. In contrast, several cells displayed cytosolic cleaved casp3 co-localized with TUNEL staining (F–H, note chromatin clumps typical of apoptotic cells, enlarged panel in H) after model M3. A large number of cells exhibited both cytosolic and nuclear cleaved casp3 and DNA fragmentation after model M4 (J–L). Some sections were counterstained with 4′,6′-diamidino-2-phenylindole (overlay in D and L). Scale bar represents 50 and 20 μm (enlarged panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then only focused our analysis on animals subjected to unilateral CCAo (model M2) to determine which type of cells displayed cleaved casp3 without death features at 48 hours postinjury for comparison with CL hemisphere of M1 animals. Indeed, the number of cleaved casp3-positive cells increased from 12 to 48 hours (peak) and moderately declined at 72 hours later. Using double immunofluorescence and confocal analysis, we observed that mainly neurons (labeled with NeuN; Figure 3, A and B) and immature cells (labeled with nestin, Figure 3, C and D; and vimentin, Figure 3, E and F) expressed cleaved casp3, but not astrocytes (data not shown), and in a similar manner both in IL and CL cortex as in the CL side of model M1 animals. Specific labeling was detected in cortical layers II–III and VI. Interestingly, most of cleaved casp3-positive cells in the neocortical layer III expressed GABA (Figure 3, G–I).Figure 3Unilateral transient CCA occlusion mainly triggers casp3 cleavage in neurons and undifferentiated cells in the P7 rat brain. Contralateral cortex from uni-CCAo (model M2) and MCAo + tCCAo (model M1) animals contain most cleaved casp3 colocalized with neurons (NeuN) (A, B), nestin- (C, D), and vimentin (E, F) positive cells Arrows indicate location magnified in insets. Co-localization of cleaved casp3 was observed in GABA-immunostained neurons in cortical layer III (ly III in G, and enlarged panels in H, indicated by the asterisk in G). Cleaved casp3 labeling is shown in red, whereas NeuN, nestin, vimentin and GABA markers are shown in green. Scale bar represents 50 and 20 μm (enlarged panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Molecular Pathways Involved in Cytosolic Casp3 CleavageCasp3 cleavage (and activation) has been reported in apoptosis13Benjelloun N Joly LM Palmier B Plotkine M Charriaut-Marlangue C Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia-ischaemia in the rat brain.Neuropathol Appl Neurobiol. 2003; 29: 350-360Crossref PubMed Scopus (59) Google Scholar, 19Manabat C Han BH Wendland M Derugin N Fox CK Choi J Holtzman DM Ferriero DM Vexler ZS Reperfusion differentially induces caspase-3 activation in ischemic core and penumbra after stroke in immature brain.Stroke. 2003; 34: 207-213Crossref PubMed Scopus (144) Google Scholar and preconditioning.20Tanaka H Yokota H Jover T Cappuccio I Calderone A Simionescu M Bennett MV Zukin RS Ischemic preconditioning: neuronal survival in the face of caspase-3 activation.J Neurosci. 2004; 24: 2750-2759Crossref PubMed Scopus (120) Google Scholar We therefore first evaluated by western blotting casp3 cleavage and subsequent cleavage of its substrates, such as α-II spectrin (fodrin) and poly (ADP-ribose) polymerase-1. At 48 hours after uni-tCCAo (Figure 4A), p17-cleaved casp3 and the 150-kDa α-II spectrin breakdown product, a marker of Ca2+-mediated calpain activation, were detected (Figure 4C). This later appeared slightly (by ∼25%) increased compared with sham tissues. Casp3-dependent spectrin cleavage, yielding p120 kDa breakdown product,21Nath R Huggins M Glantz SB Morrow JS McGinnis K Nadimpalli R Wanga KK Development and characterization of antibodies specific to caspase-3-produced αII-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.Neurochem Int. 2000; 37: 351-361Crossref PubMed Scopus (46) Google Scholar was only detected in the IL ischemic cortex but neither in the CL cortex after ischemia nor in both cortex tissues after uni-tCCAo. We then investigated upstream caspase (casp2, casp8, and casp9) cleavage, which could be involved in casp3 cleavage. Data reported in Figure 4B demonstrated that casp2 underwent cleavage until its small unit (17 kDa) after ischemia, in association with casp8 and casp9 cleavage, in the IL hemisphere. In the CL cortex after ischemia and in both cortex after uni-tCCAo, casp2 underwent an incomplete cleavage (absence of the 17-kDa fragment but presence of the 37- and 25-kDa fragments) without casp8 and casp9 cleavage. After ischemia, release of cytochrome c in the cytosol was more abundant that the one observed after uni-tCCAo. In contrast, cytochrome c release has not been detected in the CL cortex. It should be noted that no significant endogenous caspase inhibitor (survivin and c-IAP-2) proteins were up-expressed in cortical tissues from the CL ischemic brain (supplemental Figure S2, see http://ajp.amjpathol.org), whereas an increase was observed in the IL penumbra close t
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