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The role of microclot formation in an acute subarachnoid hemorrhage model in the rabbit.

Andereggen L, Neuschmelting V, von Gunten M, Widmer HR, Fandino J, Marbacher S - Biomed Res Int (2014)

Bottom Line: Our results showed significantly more TUNEL-positive cells (SAH: 115 ± 13; controls: 58 ± 10; P = 0.016) and fibrinogen-positive microthromboemboli (SAH: 9 ± 2; controls: 2 ± 1; P = 0.03) in the hippocampus after aneurysmal SAH.We found clear evidence of early microclot formation in a rabbit model of acute SAH.The extent of microthrombosis did not correlate with early apoptosis or CPP depletion after SAH; however, the total number of TUNEL positive cells in the cortex and the hippocampus significantly correlated with mean CPP reduction during the phase of maximum depletion after SAH induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Bern University Hospital, Inselspital Bern, 3012 Bern, Switzerland ; Laboratories for Neuroscience Research in Neurosurgery, Boston Children's Hospital, Boston, MA 02115, USA ; Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Microvascular dysfunction and microthrombi formation are believed to contribute to development of early brain injury (EBI) after aneurysmal subarachnoid hemorrhage (SAH).

Objective: This study aimed to determine (i) extent of microthrombus formation and neuronal apoptosis in the brain parenchyma using a blood shunt SAH model in rabbits; (ii) correlation of structural changes in microvessels with EBI characteristics.

Methods: Acute SAH was induced using a rabbit shunt cisterna magna model. Extent of microthrombosis was detected 24 h post-SAH (n = 8) by fibrinogen immunostaining, compared to controls (n = 4). We assessed apoptosis by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in cortex and hippocampus.

Results: Our results showed significantly more TUNEL-positive cells (SAH: 115 ± 13; controls: 58 ± 10; P = 0.016) and fibrinogen-positive microthromboemboli (SAH: 9 ± 2; controls: 2 ± 1; P = 0.03) in the hippocampus after aneurysmal SAH.

Conclusions: We found clear evidence of early microclot formation in a rabbit model of acute SAH. The extent of microthrombosis did not correlate with early apoptosis or CPP depletion after SAH; however, the total number of TUNEL positive cells in the cortex and the hippocampus significantly correlated with mean CPP reduction during the phase of maximum depletion after SAH induction. Both microthrombosis and neuronal apoptosis may contribute to EBI and subsequent DCI.

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Microclot formation analyzed in the hippocampus and cortex. (a) Schematic drawing of regions used for analysis of fibrinogen immunostained microclots in the hippocampus (red bar) and the cortex (blue bar). (b) Specific regions of interest (ROI) from the hippocampus and cortex used for dimensional analysis.
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fig1: Microclot formation analyzed in the hippocampus and cortex. (a) Schematic drawing of regions used for analysis of fibrinogen immunostained microclots in the hippocampus (red bar) and the cortex (blue bar). (b) Specific regions of interest (ROI) from the hippocampus and cortex used for dimensional analysis.

Mentions: Intracardiac perfusion-fixation was carried out 24 hours after SAH-induction at room temperature with 400 mL of 0.1 M phosphate-buffered solution (PBS) followed by 400 mL fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.3). Brains were removed from the skull and cut into four blocks between the forebrain (olfactory bulb) and cerebellum, embedded in paraffin, and cut into consecutive 7 μm sections. The cut surface of block one was placed through the cortical punch defect of the ICP and rCBF probes. The first section of blocks two to four was stained with hematoxylin and eosin, and the most representative fields containing the hippocampus and basal cortex were selected for additional cuts of ten consecutive sections used for immunohistochemical analysis in order to analyze the same subsection to eliminate bias. Apoptosis was detected using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL, Roche Diagnostics AG, Rotkreuz, Switzerland) as described above [7]. Quantitative analysis of apoptosis was performed within predefined regions of interest (ROI) of 300 μm × 300 μm on coronal sections for each hemisphere (Figure 1(b)). Thereby, 9 ROIs were used for analysis of apoptosis in the basal cortex (9 × 300 μm × 300 μm) and 3 ROIs along the hippocampal sectors CA1 and CA3 with (3 × 300 μm × 300 μm). Nuclei were counterstained with DAPI (Roche Diagnostics AG, Basel, Switzerland). Slides were visualized under a fluorescent microscope operating with a digital camera (Olympus BX 51, Olympus, Hamburg, Germany) using 2, 10, and 20x magnifications. Thereby, TUNEL red, FJB green, and DAPI blue were excited at 570–620 nm (maximum 580 nm), 450–490 nm (maximum 480 nm), and 340–380 nm (maximum 350 nm), respectively. The extent of microthrombosis was detected by fibrinogen immunohistochemistry using the Leica Bond III IHC staining system and analyzed in a blinded manner according to the schematic drawing depicted in Figure 1. For the fibrinogen immunohistochemistry, heat-induced epitope retrieval was carried out at 95°C for 20 minutes, followed by incubation with the primary antibody (polyclonal fibrinogen sheep anti-rabbit antibody; Acris, AP08879PU-N, 1:2′000, Herford, Germany) and secondary antibody (biotin-SP-conjugated AffiniPure donkey anti-sheep antibody; Jackson ImmunoResearch Laboratories, 713-065-003, 1:1′000, West Grove; USA), followed by incubation with a streptavidin-conjugated horseradish peroxidase reagent (Streptavidin-HRP, Leica Biosystems, RE7104).


The role of microclot formation in an acute subarachnoid hemorrhage model in the rabbit.

Andereggen L, Neuschmelting V, von Gunten M, Widmer HR, Fandino J, Marbacher S - Biomed Res Int (2014)

Microclot formation analyzed in the hippocampus and cortex. (a) Schematic drawing of regions used for analysis of fibrinogen immunostained microclots in the hippocampus (red bar) and the cortex (blue bar). (b) Specific regions of interest (ROI) from the hippocampus and cortex used for dimensional analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109416&req=5

fig1: Microclot formation analyzed in the hippocampus and cortex. (a) Schematic drawing of regions used for analysis of fibrinogen immunostained microclots in the hippocampus (red bar) and the cortex (blue bar). (b) Specific regions of interest (ROI) from the hippocampus and cortex used for dimensional analysis.
Mentions: Intracardiac perfusion-fixation was carried out 24 hours after SAH-induction at room temperature with 400 mL of 0.1 M phosphate-buffered solution (PBS) followed by 400 mL fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.3). Brains were removed from the skull and cut into four blocks between the forebrain (olfactory bulb) and cerebellum, embedded in paraffin, and cut into consecutive 7 μm sections. The cut surface of block one was placed through the cortical punch defect of the ICP and rCBF probes. The first section of blocks two to four was stained with hematoxylin and eosin, and the most representative fields containing the hippocampus and basal cortex were selected for additional cuts of ten consecutive sections used for immunohistochemical analysis in order to analyze the same subsection to eliminate bias. Apoptosis was detected using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL, Roche Diagnostics AG, Rotkreuz, Switzerland) as described above [7]. Quantitative analysis of apoptosis was performed within predefined regions of interest (ROI) of 300 μm × 300 μm on coronal sections for each hemisphere (Figure 1(b)). Thereby, 9 ROIs were used for analysis of apoptosis in the basal cortex (9 × 300 μm × 300 μm) and 3 ROIs along the hippocampal sectors CA1 and CA3 with (3 × 300 μm × 300 μm). Nuclei were counterstained with DAPI (Roche Diagnostics AG, Basel, Switzerland). Slides were visualized under a fluorescent microscope operating with a digital camera (Olympus BX 51, Olympus, Hamburg, Germany) using 2, 10, and 20x magnifications. Thereby, TUNEL red, FJB green, and DAPI blue were excited at 570–620 nm (maximum 580 nm), 450–490 nm (maximum 480 nm), and 340–380 nm (maximum 350 nm), respectively. The extent of microthrombosis was detected by fibrinogen immunohistochemistry using the Leica Bond III IHC staining system and analyzed in a blinded manner according to the schematic drawing depicted in Figure 1. For the fibrinogen immunohistochemistry, heat-induced epitope retrieval was carried out at 95°C for 20 minutes, followed by incubation with the primary antibody (polyclonal fibrinogen sheep anti-rabbit antibody; Acris, AP08879PU-N, 1:2′000, Herford, Germany) and secondary antibody (biotin-SP-conjugated AffiniPure donkey anti-sheep antibody; Jackson ImmunoResearch Laboratories, 713-065-003, 1:1′000, West Grove; USA), followed by incubation with a streptavidin-conjugated horseradish peroxidase reagent (Streptavidin-HRP, Leica Biosystems, RE7104).

Bottom Line: Our results showed significantly more TUNEL-positive cells (SAH: 115 ± 13; controls: 58 ± 10; P = 0.016) and fibrinogen-positive microthromboemboli (SAH: 9 ± 2; controls: 2 ± 1; P = 0.03) in the hippocampus after aneurysmal SAH.We found clear evidence of early microclot formation in a rabbit model of acute SAH.The extent of microthrombosis did not correlate with early apoptosis or CPP depletion after SAH; however, the total number of TUNEL positive cells in the cortex and the hippocampus significantly correlated with mean CPP reduction during the phase of maximum depletion after SAH induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Bern University Hospital, Inselspital Bern, 3012 Bern, Switzerland ; Laboratories for Neuroscience Research in Neurosurgery, Boston Children's Hospital, Boston, MA 02115, USA ; Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Microvascular dysfunction and microthrombi formation are believed to contribute to development of early brain injury (EBI) after aneurysmal subarachnoid hemorrhage (SAH).

Objective: This study aimed to determine (i) extent of microthrombus formation and neuronal apoptosis in the brain parenchyma using a blood shunt SAH model in rabbits; (ii) correlation of structural changes in microvessels with EBI characteristics.

Methods: Acute SAH was induced using a rabbit shunt cisterna magna model. Extent of microthrombosis was detected 24 h post-SAH (n = 8) by fibrinogen immunostaining, compared to controls (n = 4). We assessed apoptosis by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in cortex and hippocampus.

Results: Our results showed significantly more TUNEL-positive cells (SAH: 115 ± 13; controls: 58 ± 10; P = 0.016) and fibrinogen-positive microthromboemboli (SAH: 9 ± 2; controls: 2 ± 1; P = 0.03) in the hippocampus after aneurysmal SAH.

Conclusions: We found clear evidence of early microclot formation in a rabbit model of acute SAH. The extent of microthrombosis did not correlate with early apoptosis or CPP depletion after SAH; however, the total number of TUNEL positive cells in the cortex and the hippocampus significantly correlated with mean CPP reduction during the phase of maximum depletion after SAH induction. Both microthrombosis and neuronal apoptosis may contribute to EBI and subsequent DCI.

Show MeSH
Related in: MedlinePlus