Name: Brunelli Da Rós Peruch
Type: MSc dissertation
Publication date: 26/02/2015
Advisor:

Namesort descending Role
Breno Valentim Nogueira Advisor *

Examining board:

Namesort descending Role
Breno Valentim Nogueira Advisor *
Cristina Martins e Silva Internal Examiner *
Thiago de Melo Costa Pereira External Examiner *

Summary: Introduction. The stroke is classically characterized as a neurological deficit attributed to an acute focal lesion of the central nervous system (CNS), for vascular cause, and a major cause of disability and death worldwide. In all cases, 87% are ischemic. The main risk factors are age, race, atherosclerosis, hyperlipidemia, smoking, and others. To date drug therapy approved by the National Health Surveillance Agency (ANVISA) and the Food and Drug Administration (FDA) for the treatment of acute ischemic stroke is the use of the class of thrombolytic agents activators of fibrinolysis. Several cytokines appear to play a role in neurogenesis and /or neuronal regeneration. Recent studies have demonstrated that granulocyte colony stimulating factor (G-CSF) has neuroprotective effects in mice subjected to cerebral ischemia, but its effects opposite to increased oxidative stress and hypercholesterolemia/atherosclerosis, a major cause of stroke, although is unknown and was the object of this study. Methods. Were used apolipoprotein E knockout (ApoE-/-) and C57BL/6 mice, male, young adults, aged between 12-14 weeks and weighing on average 28 g divided into three groups: control ApoE-/-, treated G-CSF ApoE-/- and control C57BL/6. All animals underwent occlusion of common carotid arteries (temporary) and left (standing) then administration of glucose solution 5% (controls) and G-CSF 100μg/kg/day (treated) just after and 24 hours following surgery. On the third day after occlusion the animals were sacrificed and the wet weight was obtained from the spleen, blood sample collected and extracted the brain. Statistical comparisons were performed by test t Student's and ANOVA 1 way, followed by Fisher's post hoc. Results and discussion. The count of circulating leukocytes in the G-CSF ApoE-/- group was statistically higher (6661 ± 1059 cells/ mm3) compared to the control ApoE-/- group (4044 ± 610 cells/mm3) and the control C57BL/6 group (3965 ± 409 cells/mm3) highlighting the systemic effect of G-CSF in the recruitment and maturation of blood granulocytes. In the measurement of plasma total cholesterol G-CSF did not reverse the hypercholesterolemia in ApoE-/- animals. The macroscopic area of cerebral infarction was significantly lower in group ApoE-/- treated with G-CSF (0.03 ± 0.01 cm2) compared with the group ApoE-/- control (0.18 ± 0.03 cm2). There was also significant difference in the infarcted area of control C57BL/6 group (0.08 ± 0.02 cm2) with the control ApoE-/- group. Muscle strength, measured by the grip test, was significantly reduced in ApoE-/- control animals 48 hours after surgery compared to preoperative controls (97.9 ± 9.8g for 70.69 ± 10,62g), while the group G-CSF ApoE-/- force recorded before surgery was preserved after surgery. In the assay for oxidative stress plasma treatment with G-CSF did not change the group parameters in ApoE-/- (control = 23,76±3,36 vs G-CSF = 23,0±3,20 μMC-T/mg) which was statistically increased compared to the C57BL/6 control group (3.97 ± 1.22 μMC-T/mg). The test TUNEL (TdT-mediated dUTP Nick End Labeling), to show the tissue cell death was related to positive cells/negative less in the group treated ApoE-/- as compared to the control group ApoE-/-. The immunostaining of neurons and G-CSF receptor showed stimulation for neuronal survival and increased presence of receptors in the treated group compared to the control. Conclusion. Our findings are in agreement with the literature and contribute to highlight the neuroprotective effect of G-CSF, strongly linked to antiapoptotic property in global cerebral ischemia model even in the presence of the major risk factor for stroke, atherosclerosis/hypercholesterolemia, and of increased oxidative stress.

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