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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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Evidence of microclot formation, neuronal apoptosis, and CPP depletion after experimental SAH. Quantification of TUNEL-positive cells and fibrinogen-positive stained microthrombi in cortex and hippocampus. (a) There were significant increases in the number of TUNEL-positive cells in both cerebral cortex and hippocampus as compared to controls (P = 0.016 and P = 0.017, resp., Student's t-test). (b) There was a nonstatistically significant trend towards an increase in fibrinogen-positive microvessels in the cerebral cortex (P = 0.06) and a statistically significant increase in fibrinogen-positive microvessels in the hippocampus (P = 0.03). No correlations of TUNEL-positivity with fibrinogen-positivity were observed in cortex (R2 = 0.094; (c)) or in hippocampus (R2 = 0.2; (d)). All data are expressed as mean ± SEM, n = 8 in SAH group, and n = 4 in control group. A P value of <0.05 was considered statistically significant. (e) Time course of the CPP depletion after induction of SAH compared to control animals.
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fig3: Evidence of microclot formation, neuronal apoptosis, and CPP depletion after experimental SAH. Quantification of TUNEL-positive cells and fibrinogen-positive stained microthrombi in cortex and hippocampus. (a) There were significant increases in the number of TUNEL-positive cells in both cerebral cortex and hippocampus as compared to controls (P = 0.016 and P = 0.017, resp., Student's t-test). (b) There was a nonstatistically significant trend towards an increase in fibrinogen-positive microvessels in the cerebral cortex (P = 0.06) and a statistically significant increase in fibrinogen-positive microvessels in the hippocampus (P = 0.03). No correlations of TUNEL-positivity with fibrinogen-positivity were observed in cortex (R2 = 0.094; (c)) or in hippocampus (R2 = 0.2; (d)). All data are expressed as mean ± SEM, n = 8 in SAH group, and n = 4 in control group. A P value of <0.05 was considered statistically significant. (e) Time course of the CPP depletion after induction of SAH compared to control animals.

Mentions: Fibrinogen staining showed distinct microclot formation in vessels of the hippocampus (Figures 2(a) and 2(c)) and cerebral cortex (Figures 2(b) and 2(d)) after SAH ((a), (b)) compared to control animals ((c), (d)). Immunohistochemistry analysis revealed a significant increase of the number of TUNEL-positive cells in both cerebral cortex and hippocampus (Figure 3(a)). Namely, there were 68 ± 8 TUNEL-positive cells in the cortex after SAH compared to 36 ± 2 cells in the control animals (differences between means 32 ± 11; P = 0.017). In the hippocampus, there were 115 ± 13 TUNEL-positive cells after SAH compared to 58 ± 10 positive cells in the control animals (differences between means 58 ± 20; P = 0.016). Taking into account the differences in the density of neurons in the hippocampus and the cerebral cortex, immunohistochemistry analyses showed in the hippocampus 115 ± 13 TUNEL-positive cells after SAH compared to 68 ± 8 positive cells in the cerebral cortex (differences between means 48 ± 15; P = 0.014). In control animals, there were 58 ± 10 TUNEL-positive cells in the hippocampus compared to 36 ± 2 in the cerebral cortex (differences between means 22 ± 9; P = 0.13).


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)

Evidence of microclot formation, neuronal apoptosis, and CPP depletion after experimental SAH. Quantification of TUNEL-positive cells and fibrinogen-positive stained microthrombi in cortex and hippocampus. (a) There were significant increases in the number of TUNEL-positive cells in both cerebral cortex and hippocampus as compared to controls (P = 0.016 and P = 0.017, resp., Student's t-test). (b) There was a nonstatistically significant trend towards an increase in fibrinogen-positive microvessels in the cerebral cortex (P = 0.06) and a statistically significant increase in fibrinogen-positive microvessels in the hippocampus (P = 0.03). No correlations of TUNEL-positivity with fibrinogen-positivity were observed in cortex (R2 = 0.094; (c)) or in hippocampus (R2 = 0.2; (d)). All data are expressed as mean ± SEM, n = 8 in SAH group, and n = 4 in control group. A P value of <0.05 was considered statistically significant. (e) Time course of the CPP depletion after induction of SAH compared to control animals.
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Related In: Results  -  Collection

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fig3: Evidence of microclot formation, neuronal apoptosis, and CPP depletion after experimental SAH. Quantification of TUNEL-positive cells and fibrinogen-positive stained microthrombi in cortex and hippocampus. (a) There were significant increases in the number of TUNEL-positive cells in both cerebral cortex and hippocampus as compared to controls (P = 0.016 and P = 0.017, resp., Student's t-test). (b) There was a nonstatistically significant trend towards an increase in fibrinogen-positive microvessels in the cerebral cortex (P = 0.06) and a statistically significant increase in fibrinogen-positive microvessels in the hippocampus (P = 0.03). No correlations of TUNEL-positivity with fibrinogen-positivity were observed in cortex (R2 = 0.094; (c)) or in hippocampus (R2 = 0.2; (d)). All data are expressed as mean ± SEM, n = 8 in SAH group, and n = 4 in control group. A P value of <0.05 was considered statistically significant. (e) Time course of the CPP depletion after induction of SAH compared to control animals.
Mentions: Fibrinogen staining showed distinct microclot formation in vessels of the hippocampus (Figures 2(a) and 2(c)) and cerebral cortex (Figures 2(b) and 2(d)) after SAH ((a), (b)) compared to control animals ((c), (d)). Immunohistochemistry analysis revealed a significant increase of the number of TUNEL-positive cells in both cerebral cortex and hippocampus (Figure 3(a)). Namely, there were 68 ± 8 TUNEL-positive cells in the cortex after SAH compared to 36 ± 2 cells in the control animals (differences between means 32 ± 11; P = 0.017). In the hippocampus, there were 115 ± 13 TUNEL-positive cells after SAH compared to 58 ± 10 positive cells in the control animals (differences between means 58 ± 20; P = 0.016). Taking into account the differences in the density of neurons in the hippocampus and the cerebral cortex, immunohistochemistry analyses showed in the hippocampus 115 ± 13 TUNEL-positive cells after SAH compared to 68 ± 8 positive cells in the cerebral cortex (differences between means 48 ± 15; P = 0.014). In control animals, there were 58 ± 10 TUNEL-positive cells in the hippocampus compared to 36 ± 2 in the cerebral cortex (differences between means 22 ± 9; P = 0.13).

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