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Apoptotic and necrotic cell death after photothrombotic ring stroke: characterization of a stroke model and its morphological and molecular consequences
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine. Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cerebral ischemic cell death is a major cause of disability and death among stroke patients. The brain cell demise can occur through apoptosis or necrosis or as a continuum of both. This study aimed at establishing a dual setup of a photothrombotic ring stroke model and exploring its morphological and molecular consequences.

    Photothrombotic ring stroke was induced in adult male Wistar rats by a ring shaped laser irradiation beam (514.5nm, outer diameter 5mm, thickness 0.35 mm) for 120 seconds focused on the exposed intact skull bone with simultaneous intravenous infusion of the photosensitizer erythrosin B (17 mg/kg). By using otherwise identical experimental conditions, high intensity irradiation (1.94 W/cm2) resulted in consistent lack of reperfusion in the region at risk whereas low intensity irradiation (0.90 W/cm2) induced late spontaneous reperfusion. The morphological appearance of apoptotic and necrotic cells was demonstrated by H&E, TUNEL and Hoechst stainings under light microscopy, immunohistochemistry and electron microscopy. This was further confirmed by gel electrophoresis showing DNA laddering that coexisted with DNA smear. Cell counts revealed that apoptotic cells appeared earlier (at 24 h) and remained as long as the necrotic cells, that is up to 72 hours after ischemic onset in regions with severe CBF reduction. After low intensity irradiation, we observed early and widespread increased expression of the anti-apoptotic protein bcl-w and a prolonged elevation of Bcl-2 with unchanged pro-apoptotic Bax in mitochondria. In contrast, decreased bcl-w and Bcl-2 with scattered Bax remained after high intensity irradiation. Correspondingly, the release of the pro-apoptotic factor Smac/DIABLO from the mitochondria to the cytosol was more persistent in high- compared with low-intensity irradiation.   

    Apoptotic and necrotic cell death coexisted in the same regions at the same time after photothrombotic ring stroke induced by low- or high-intensity irradiation, where spontaneous morphological recovery or pannecrosis were evident in the region at risk. The ratios between Bcl-w, Bcl-2 and Bax may direct the translocation of Smac/DIABLO from the mitochondria to the cytosol and thereby influence cell death or survival after focal cerebral ischemia.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2003. , p. 59
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 826
National Category
Medical and Health Sciences Neurosciences
Identifiers
URN: urn:nbn:se:umu:diva-133950ISBN: 91-7305-390-2 (print)OAI: oai:DiVA.org:umu-133950DiVA, id: diva2:1090139
Public defence
2003-02-28, Sal B, plan 9, By 1D, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2019-03-19 Created: 2017-04-22 Last updated: 2019-03-21Bibliographically approved
List of papers
1. A photothrombotic ring stroke model in rats with or without late spontaneous reperfusion in the region at risk
Open this publication in new window or tab >>A photothrombotic ring stroke model in rats with or without late spontaneous reperfusion in the region at risk
1999 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 849, no 1-2, p. 175-186Article in journal (Refereed) Published
Abstract [en]

This study aimed at developing a dual setup of the photothrombotic ring stroke model with or without late spontaneous reperfusion in the region at risk and to explore the morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser-irradiation beam (o.d. 5.0 mm, 0.35 mm thick) for 2 min simultaneously with intravenous erythrosin B (17 mg/kg) infusion. Transcardial carbon-black perfusion revealed that a laser intensity of 0.90 W/cm(2) resulted in late, that is, starting at 72 h, spontaneous reperfusion, whereas the lowest laser intensity that produced lack of reperfusion at 7 days post-irradiation was 1.84 W/cm(2). Laser-Doppler flowmetry showed prompt cortical cerebral blood flow (cCBF) reduction both in the ring lesion and region at risk (12% and 25% of control values) after high-intensity irradiation; these reduced flow values were more rapid and pronounced than in the low-intensity irradiation setup as previously shown. The high- compared with low-intensity irradiation setup produced more frequent occurrence of thrombi in the ring-lesion region and a larger ischemic cortical lesion with a more rapid pace of ischemic cellular changes in the ring-lesion region and the region at risk. The region at risk transformed into pannecrosis in the high-intensity, but recovered morphologically in the low-intensity irradiation setup. This dual photothrombotic setup with or without spontaneous reperfusion enables the study of events related to ischemic cell survival or death in an anatomically predefined region at risk.

National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-133949 (URN)10.1016/S0006-8993(99)02152-6 (DOI)000084486900019 ()10592300 (PubMedID)2-s2.0-0345008870 (Scopus ID)
Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2019-03-19Bibliographically approved
2. Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats
Open this publication in new window or tab >>Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats
Show others...
2001 (English)In: Brain Research Protocols, ISSN 1385-299X, E-ISSN 1872-809X, Vol. 7, no 1, p. 76-85Article in journal (Refereed) Published
Abstract [en]

Clinical stroke is mostly of thromboembolic origin, in which the magnitude of brain damage resulting from arterial occlusions depends on the degree and duration of the concomitant ischemia. To facilitate more controllable and reproducible study of stroke-related pathophysiological mechanisms, a photothrombotic ring stroke model was initially developed in adult rats. The ring interior zone comprises an anatomically well confined cortical region-at-risk which is gradually encroached by progressive hypoperfusion, thus mimicking the situation (albeit in inverse fashion) of an ischemic penumbra or stroke-in-evolution. Modification of this model using a thinner ring irradiation beam resulted in late spontaneous reperfusion in the cortical region-at-risk and a remarkable morphological tissue recovery in this ostensibly critically injured region. On the other hand, doubling the thin irradiating beam intensity facilitates a complementary situation in which lack of reperfusion in the region-at-risk after stroke induction leads to tissue pannecrosis. The dual photothrombotic ring stroke model, effectuated either with or without reperfusion and thereby tissue recovery or pannecrosis, may be well suited for the study of events related to postischemic survival or cell death in the penumbra region. To popularize the photothrombotic ring stroke model, we present a detailed protocol of how this model is induced in either version as well as protocols for transcardial carbon black perfusion and laser-Doppler flowmetry experiments.

Place, publisher, year, edition, pages
Elsevier, 2001
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-133948 (URN)10.1016/S1385-299X(01)00046-0 (DOI)000168088300010 ()11275527 (PubMedID)2-s2.0-0035080062 (Scopus ID)
Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2019-03-19Bibliographically approved
3. Long-lasting neuronal apoptotic cell death in regions with severe ischemia after photothrombotic ring stroke in rats
Open this publication in new window or tab >>Long-lasting neuronal apoptotic cell death in regions with severe ischemia after photothrombotic ring stroke in rats
Show others...
2002 (English)In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 104, no 5, p. 462-70Article in journal (Refereed) Published
Abstract [en]

Apoptotic and necrotic cell death may act in concert in focal cerebral ischemia. This study explored the temporal and spatial pattern of apoptosis and necrosis in a novel photothrombotic ring stroke model with or without spontaneous reperfusion. Adult male Wistar rats were subjected to a ring-shaped laser irradiation beam simultaneously with intravenous erythrosin B infusion. The presence and attributes of apoptosis and necrosis in the anatomically well-defined cortical region at risk and ring-lesion region were verified under light microscopy with TUNEL, Hoechst 33342, and hematoxylin and eosin staining. Cells exhibiting apoptotic morphology with chromatin condensation and apoptotic bodies and necrotic ghost appearance were observed. The occurrence of apoptosis and necrosis in the ischemic regions was confirmed by electron microscopy and gel electrophoresis, in which DNA isolated from the lesion area revealed both a ladder and a smear. Double staining with TUNEL and the cell markers NeuN, glial fibrillary acidic protein, and ED-1 revealed that the majority of apoptotic cells were of neuronal origin. Cells exhibiting pyknosis/eosinophilia, apoptosis, or ghost appearance were quantified by stereological means. In subregions with severe ischemia, the peak appearance of apoptotic cells started earlier, i.e., at 24 h, than the peak of necrotic cells, and the high concentration of the apoptotic cells remained as long as that of necrotic cells, i.e., until 72 h post-ischemia. The ratio of apoptotic to necrotic cells was approximately 1:2. Therefore, apoptosis may be an important contributor to neuronal cell death in brain regions with severely reduced blood flow after thrombo-embolic stroke.

Place, publisher, year, edition, pages
Springer, 2002
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-133946 (URN)10.1007/s00401-002-0579-8 (DOI)000178750700004 ()12410394 (PubMedID)
Available from: 2017-04-22 Created: 2017-04-22 Last updated: 2019-03-19Bibliographically approved
4. Dynamic changes of the anti- and pro-apoptotic proteins Bcl-w, Bcl-2, and Bax with Smac/Diablo mitochondrial release after photothrombotic ring stroke in rats
Open this publication in new window or tab >>Dynamic changes of the anti- and pro-apoptotic proteins Bcl-w, Bcl-2, and Bax with Smac/Diablo mitochondrial release after photothrombotic ring stroke in rats
Show others...
2004 (English)In: European Journal of Neuroscience, ISSN 0953-816X, E-ISSN 1460-9568, Vol. 20, no 5, p. 1177-1188Article in journal (Refereed) Published
Abstract [en]

The anti‐apoptotic proteins Bcl‐w and Bcl‐2 and the pro‐apoptotic protein Bax may mediate cell death or survival via regulation of the mitochondria including second mitochondria‐derived activator of caspase (Smac)/direct inhibitor of apoptosis protein (IAP)‐binding protein with low pI (DIABLO) release. This study aimed to explore alterations in Bcl‐w, Bcl‐2, and Bax and the relationship between these proteins and Smac/DIABLO by means of in situ hybridization, immunohistochemical (IHC) staining, and Western blots after low‐ and high‐intensity photothrombotic ring stroke. At 4 h after low‐intensity irradiation, we found widespread bcl‐w overexpression on both the mRNA and protein levels in the bilateral cortex except the ring lesion region and in subcortical regions. A prolonged elevation of Bcl‐2 with relatively unchanged Bax in the mitochondrial fraction was demonstrated from 4 to 72 h. These upregulated anti‐apoptotic proteins combined with little Smac/DIABLO release might be associated with increased cell survival and thereby remarkable morphological recovery after low‐intensity irradiation. After high‐intensity irradiation, we observed decreased bcl‐w and bcl‐2 mRNA with increased Bcl‐2 protein in the cytosolic fraction, whereas the Bax protein remained in scattered ischaemic cells in the ring lesion and the region at risk that corresponded with release of Smac/DIABLO from mitochondria to the cytosol at 1–24 h. These changes might be related to the massive cell death observed after high‐intensity irradiation. Taken together, the balance and the location of anti‐apoptotic proteins vs. pro‐apoptotic proteins could be associated with the translocation of Smac/DIABLO from the mitochondria to the cytosol and therefore closely related to cell death or survival after focal cerebral ischaemia.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2004
Keywords
Animals, Apoptosis/*physiology, Apoptosis Regulatory Proteins, Brain/metabolism, Carrier Proteins/analysis/*biosynthesis, Cell Survival/physiology, Cerebrovascular Accident/*metabolism, Intracranial Thrombosis/metabolism, Mitochondrial Proteins/analysis/*biosynthesis, Protein Biosynthesis, Proteins/analysis, Proto-Oncogene Proteins c-bcl-2/analysis/*biosynthesis, RNA; Messenger/analysis/biosynthesis, Rats, bcl-2-Associated X Protein
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-12979 (URN)10.1111/j.1460-9568.2004.03554.x (DOI)000224041300004 ()15341589 (PubMedID)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2019-03-19Bibliographically approved

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