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FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia.

Liesz A, Sun L, Zhou W, Schwarting S, Mracsko E, Zorn M, Bauer H, Sommer C, Veltkamp R - PLoS ONE (2011)

Bottom Line: Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment.Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO.This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University Heidelberg, Heidelberg, Germany. Arthur.Liesz@med.uni-heidelberg.de

ABSTRACT

Background: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/principal findings: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/significance: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

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FTY720 does not alter infarct volume in alternative experimental protocols.(A) Representative images of brain sections stained by the silver-staining technique from coagulation-MCAO and transient 60 min filament-MCAO as used for determination of infarct volumes. (B) Brain ischemia was induced by 60 min reversible MCAO and infarct volumes were determined at 24 h after ischemia in animals receiving daily oral administration of FTY720 or PBS starting 48 h before stroke induction (n = 12, p = 0.42, 3 individual experiments). (C, D) FTY720 or PBS were administered by daily i.p. injection starting 48 h berfore MCAO. (C) MCAO was induced by transcranial permanent coagulation and infarct columes determined at 7d after MCAO (n = 10, p = 0.36, 2 individual experiments). (D) MCAO was induced by transient 60 min filament-occlusion of the MCA and infarct volume measured at 24 h after MCAO (n = 9, p = 0.86, 2 individual experiments).
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pone-0021312-g003: FTY720 does not alter infarct volume in alternative experimental protocols.(A) Representative images of brain sections stained by the silver-staining technique from coagulation-MCAO and transient 60 min filament-MCAO as used for determination of infarct volumes. (B) Brain ischemia was induced by 60 min reversible MCAO and infarct volumes were determined at 24 h after ischemia in animals receiving daily oral administration of FTY720 or PBS starting 48 h before stroke induction (n = 12, p = 0.42, 3 individual experiments). (C, D) FTY720 or PBS were administered by daily i.p. injection starting 48 h berfore MCAO. (C) MCAO was induced by transcranial permanent coagulation and infarct columes determined at 7d after MCAO (n = 10, p = 0.36, 2 individual experiments). (D) MCAO was induced by transient 60 min filament-occlusion of the MCA and infarct volume measured at 24 h after MCAO (n = 9, p = 0.86, 2 individual experiments).

Mentions: To test the hypothesis of a differential effect of per os FTY720 treatment in an infarct model of ischemia-reperfusion injury of the brain in contrast to permanent MCA occlusion, we analyzed infarct volumes 24 h after MCAO in 48 h-pretreated animals undergoing 60 min reversible occlusion of the MCA (Fig. 3B). We did not detect a significant effect of FTY720 treatment compared to PBS on the resulting infarct volume. Additionally, we tested whether intraperitoneal (i.p.) administration of FTY720 would have a neuroprotective effect in contrast to administration by oral gavage. Neither in at 7d after MCAO in the coagulation model (Fig. 3C) nor at 24 h after MCAO in the 60 min filament-occlusion model (Fig. 3D) a significant difference between FTY720 and PBS treated animals was detected. Furthermore we analyzed brain edema formation as a marker of blood-brain-barrier dysfunction in normal animals receiving FTY720 or PBS and in mice at 3d after MCAO in both treatment groups (Fig. 4A). We detected a significant increase in brain water content of the ischemic hemisphere at 3d after MCAO compared to brains 3d after Sham operation, but we did not measure a difference between edema in PBS treated and FTY720 treated animals (Fig. 4A). Additionally, blood-brain-barrier permeability was analyzed by measuring Evans blue fluorescence intensity in brain homogenates at 3d after permanent MCAO in FTY720 treated mice and controls. We did not measure a significant difference in the ratio of ischemic/non-ischemic hemispheres for the Evans blue fluorescence intensity (Fig. 4B).


FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia.

Liesz A, Sun L, Zhou W, Schwarting S, Mracsko E, Zorn M, Bauer H, Sommer C, Veltkamp R - PLoS ONE (2011)

FTY720 does not alter infarct volume in alternative experimental protocols.(A) Representative images of brain sections stained by the silver-staining technique from coagulation-MCAO and transient 60 min filament-MCAO as used for determination of infarct volumes. (B) Brain ischemia was induced by 60 min reversible MCAO and infarct volumes were determined at 24 h after ischemia in animals receiving daily oral administration of FTY720 or PBS starting 48 h before stroke induction (n = 12, p = 0.42, 3 individual experiments). (C, D) FTY720 or PBS were administered by daily i.p. injection starting 48 h berfore MCAO. (C) MCAO was induced by transcranial permanent coagulation and infarct columes determined at 7d after MCAO (n = 10, p = 0.36, 2 individual experiments). (D) MCAO was induced by transient 60 min filament-occlusion of the MCA and infarct volume measured at 24 h after MCAO (n = 9, p = 0.86, 2 individual experiments).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3119049&req=5

pone-0021312-g003: FTY720 does not alter infarct volume in alternative experimental protocols.(A) Representative images of brain sections stained by the silver-staining technique from coagulation-MCAO and transient 60 min filament-MCAO as used for determination of infarct volumes. (B) Brain ischemia was induced by 60 min reversible MCAO and infarct volumes were determined at 24 h after ischemia in animals receiving daily oral administration of FTY720 or PBS starting 48 h before stroke induction (n = 12, p = 0.42, 3 individual experiments). (C, D) FTY720 or PBS were administered by daily i.p. injection starting 48 h berfore MCAO. (C) MCAO was induced by transcranial permanent coagulation and infarct columes determined at 7d after MCAO (n = 10, p = 0.36, 2 individual experiments). (D) MCAO was induced by transient 60 min filament-occlusion of the MCA and infarct volume measured at 24 h after MCAO (n = 9, p = 0.86, 2 individual experiments).
Mentions: To test the hypothesis of a differential effect of per os FTY720 treatment in an infarct model of ischemia-reperfusion injury of the brain in contrast to permanent MCA occlusion, we analyzed infarct volumes 24 h after MCAO in 48 h-pretreated animals undergoing 60 min reversible occlusion of the MCA (Fig. 3B). We did not detect a significant effect of FTY720 treatment compared to PBS on the resulting infarct volume. Additionally, we tested whether intraperitoneal (i.p.) administration of FTY720 would have a neuroprotective effect in contrast to administration by oral gavage. Neither in at 7d after MCAO in the coagulation model (Fig. 3C) nor at 24 h after MCAO in the 60 min filament-occlusion model (Fig. 3D) a significant difference between FTY720 and PBS treated animals was detected. Furthermore we analyzed brain edema formation as a marker of blood-brain-barrier dysfunction in normal animals receiving FTY720 or PBS and in mice at 3d after MCAO in both treatment groups (Fig. 4A). We detected a significant increase in brain water content of the ischemic hemisphere at 3d after MCAO compared to brains 3d after Sham operation, but we did not measure a difference between edema in PBS treated and FTY720 treated animals (Fig. 4A). Additionally, blood-brain-barrier permeability was analyzed by measuring Evans blue fluorescence intensity in brain homogenates at 3d after permanent MCAO in FTY720 treated mice and controls. We did not measure a significant difference in the ratio of ischemic/non-ischemic hemispheres for the Evans blue fluorescence intensity (Fig. 4B).

Bottom Line: Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment.Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO.This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University Heidelberg, Heidelberg, Germany. Arthur.Liesz@med.uni-heidelberg.de

ABSTRACT

Background: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/principal findings: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/significance: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

Show MeSH
Related in: MedlinePlus