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Neuroprotection by the histone deacetylase inhibitor trichostatin A in a model of lipopolysaccharide-sensitised neonatal hypoxic-ischaemic brain injury.

Fleiss B, Nilsson MK, Blomgren K, Mallard C - J Neuroinflammation (2012)

Bottom Line: Also only in females, TSA reduced grey matter and white matter injury at 5 days post-LPS/HI.Treatment altered animal behaviour in the open field and improved learning in the fear-conditioning test in females compared with LPS/HI-only females at 25 days post-HI.TSA did not impair oligodendrocyte maturation, which increases the possible clinical relevance of this strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Perinatal Center, Department of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Box 432, Gothenburg, 405 30, Sweden. bobbi.fleiss@inserm.fr

ABSTRACT

Background: Perinatal brain injury is complex and often associated with both inflammation and hypoxia-ischaemia (HI). In adult inflammatory brain injury models, therapies to increase acetylation are efficacious in reducing inflammation and cerebral injury. Our aim in the present study was to examine the neuropathological and functional effects of the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) in a model of neonatal lipopolysaccharide (LPS)-sensitised HI. We hypothesised that, by decreasing inflammation, TSA would improve injury and behavioural outcome. Furthermore, TSA's effects on oligodendrocyte development, which is acetylation-dependent, were investigated.

Methods: On postnatal day 8 (P8), male and female mice were exposed to LPS together with or without TSA. On P9 (14 hours after LPS), mice were exposed to HI (50 minutes at 10% O2). Neuropathology was assessed at 24 hours, 5 days and 27 days post-LPS/HI via immunohistochemistry and/or Western blot analysis for markers of grey matter (microtubule-associated protein 2), white matter (myelin basic protein) and cell death (activated caspase-3). Effects of TSA on LPS or LPS/HI-induced inflammation (cytokines and microglia number) were assessed by Luminex assay and immunohistochemistry. Expression of acetylation-dependent oligodendrocyte maturational corepressors was assessed with quantitative PCR 6 hours after LPS and at 24 hours and 27 days post-LPS/HI. Animal behaviour was monitored with the open-field and trace fear-conditioning paradigms at 25 days post-LPS/HI to identify functional implications of changes in neuropathology associated with TSA treatment.

Results: TSA induced increased Ac-H4 in females only after LPS exposure. Also only in females, TSA reduced grey matter and white matter injury at 5 days post-LPS/HI. Treatment altered animal behaviour in the open field and improved learning in the fear-conditioning test in females compared with LPS/HI-only females at 25 days post-HI. None of the inflammatory mechanisms assessed that are known to mediate neuroprotection by HDACi in adults correlated with improved outcome in TSA-treated neonatal females. Oligodendrocyte maturation was not different between the LPS-only and LPS + TSA-treated mice before or after exposure to HI.

Conclusions: Hyperacetylation with TSA is neuroprotective in the female neonatal mouse following LPS/HI and correlates with improved learning long-term. TSA appears to exert neuroprotection via mechanisms unique to the neonate. Deciphering the effects of age, sex and inflammatory sensitisation in the cerebral response to HDACi is key to furthering the potential of hyperacetylation as a viable neuroprotectant. TSA did not impair oligodendrocyte maturation, which increases the possible clinical relevance of this strategy.

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Neonatal trichostatin A (TSA) treatment altered open-field behaviours and improved learning in young adulthood after lipopolysaccharide-sensitised hypoxia-ischaemia (LPS/HI).(A) Representative trace recordings from LPS/HI- and LPS + TSA/HI-treated mice. (B) Output from the multivariate partial least squares discriminant analysis (PLS-DA) of open-field data illustrating significant differences between LPS/HI- and LPS + TSA/HI-treated mice in young adulthood. Each point represents the cumulative value for all behavioural variables for one individual, and red circles represent female LPS/HI. Green squares represent female LPS + TSA/HI. The y-axis is for visualisation purposes only and should not be overinterpreted. The statistics are described further in the Materials and methods section. (C) Trace fear-conditioning data illustrating time spent immobile (frozen) pretone and after exposure to the fear-conditioned stimulus (light and tone) on day 2 (n = 15 to 19). Data are means ± SEM. *P<0.05 by post hoc Student’s t-test.
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Figure 7: Neonatal trichostatin A (TSA) treatment altered open-field behaviours and improved learning in young adulthood after lipopolysaccharide-sensitised hypoxia-ischaemia (LPS/HI).(A) Representative trace recordings from LPS/HI- and LPS + TSA/HI-treated mice. (B) Output from the multivariate partial least squares discriminant analysis (PLS-DA) of open-field data illustrating significant differences between LPS/HI- and LPS + TSA/HI-treated mice in young adulthood. Each point represents the cumulative value for all behavioural variables for one individual, and red circles represent female LPS/HI. Green squares represent female LPS + TSA/HI. The y-axis is for visualisation purposes only and should not be overinterpreted. The statistics are described further in the Materials and methods section. (C) Trace fear-conditioning data illustrating time spent immobile (frozen) pretone and after exposure to the fear-conditioned stimulus (light and tone) on day 2 (n = 15 to 19). Data are means ± SEM. *P<0.05 by post hoc Student’s t-test.

Mentions: Multivariate analysis was used to analyse the open-field data. A single extreme outlier was identified. As multivariate analyses are sensitive to extreme outliers and to prevent the analysis’s becoming focused on the difference between the one identified outlier and the rest of the group, it was excluded from the final analysis, although this animal’s data is illustrated in the output plot (green point far top left quadrant in Figure 7). The analysis yielded a one-component model, explaining 73% of the variance in the behavioural variables with a predicted validity of 42% (R2 × (cum) = 0.732; Q2 (cum) = 0.424)) (Figure 8A). A complementary loading plot for the analysis illustrated behavioural variables as having a variable importance value larger than 1. We created simple time curves and applied two-way ANOVA to these data ( Additional file 11: Figure S5). The simple time curves consolidated the multivariate analysis, and, compared with LPS/HI, only LPS + TSA/HI animals were characterised by spending more time in the centre zone (P = 0.016), having a higher occurrence of ambulation in total (P = 0.006) as well as in the centre zone (P = 0.002), showing longer distance moved in the centre (P = 0.006), and demonstrating more head stretches (P = 0.039) and tail moves in the centre (P = 0.006).


Neuroprotection by the histone deacetylase inhibitor trichostatin A in a model of lipopolysaccharide-sensitised neonatal hypoxic-ischaemic brain injury.

Fleiss B, Nilsson MK, Blomgren K, Mallard C - J Neuroinflammation (2012)

Neonatal trichostatin A (TSA) treatment altered open-field behaviours and improved learning in young adulthood after lipopolysaccharide-sensitised hypoxia-ischaemia (LPS/HI).(A) Representative trace recordings from LPS/HI- and LPS + TSA/HI-treated mice. (B) Output from the multivariate partial least squares discriminant analysis (PLS-DA) of open-field data illustrating significant differences between LPS/HI- and LPS + TSA/HI-treated mice in young adulthood. Each point represents the cumulative value for all behavioural variables for one individual, and red circles represent female LPS/HI. Green squares represent female LPS + TSA/HI. The y-axis is for visualisation purposes only and should not be overinterpreted. The statistics are described further in the Materials and methods section. (C) Trace fear-conditioning data illustrating time spent immobile (frozen) pretone and after exposure to the fear-conditioned stimulus (light and tone) on day 2 (n = 15 to 19). Data are means ± SEM. *P<0.05 by post hoc Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3420244&req=5

Figure 7: Neonatal trichostatin A (TSA) treatment altered open-field behaviours and improved learning in young adulthood after lipopolysaccharide-sensitised hypoxia-ischaemia (LPS/HI).(A) Representative trace recordings from LPS/HI- and LPS + TSA/HI-treated mice. (B) Output from the multivariate partial least squares discriminant analysis (PLS-DA) of open-field data illustrating significant differences between LPS/HI- and LPS + TSA/HI-treated mice in young adulthood. Each point represents the cumulative value for all behavioural variables for one individual, and red circles represent female LPS/HI. Green squares represent female LPS + TSA/HI. The y-axis is for visualisation purposes only and should not be overinterpreted. The statistics are described further in the Materials and methods section. (C) Trace fear-conditioning data illustrating time spent immobile (frozen) pretone and after exposure to the fear-conditioned stimulus (light and tone) on day 2 (n = 15 to 19). Data are means ± SEM. *P<0.05 by post hoc Student’s t-test.
Mentions: Multivariate analysis was used to analyse the open-field data. A single extreme outlier was identified. As multivariate analyses are sensitive to extreme outliers and to prevent the analysis’s becoming focused on the difference between the one identified outlier and the rest of the group, it was excluded from the final analysis, although this animal’s data is illustrated in the output plot (green point far top left quadrant in Figure 7). The analysis yielded a one-component model, explaining 73% of the variance in the behavioural variables with a predicted validity of 42% (R2 × (cum) = 0.732; Q2 (cum) = 0.424)) (Figure 8A). A complementary loading plot for the analysis illustrated behavioural variables as having a variable importance value larger than 1. We created simple time curves and applied two-way ANOVA to these data ( Additional file 11: Figure S5). The simple time curves consolidated the multivariate analysis, and, compared with LPS/HI, only LPS + TSA/HI animals were characterised by spending more time in the centre zone (P = 0.016), having a higher occurrence of ambulation in total (P = 0.006) as well as in the centre zone (P = 0.002), showing longer distance moved in the centre (P = 0.006), and demonstrating more head stretches (P = 0.039) and tail moves in the centre (P = 0.006).

Bottom Line: Also only in females, TSA reduced grey matter and white matter injury at 5 days post-LPS/HI.Treatment altered animal behaviour in the open field and improved learning in the fear-conditioning test in females compared with LPS/HI-only females at 25 days post-HI.TSA did not impair oligodendrocyte maturation, which increases the possible clinical relevance of this strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Perinatal Center, Department of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Box 432, Gothenburg, 405 30, Sweden. bobbi.fleiss@inserm.fr

ABSTRACT

Background: Perinatal brain injury is complex and often associated with both inflammation and hypoxia-ischaemia (HI). In adult inflammatory brain injury models, therapies to increase acetylation are efficacious in reducing inflammation and cerebral injury. Our aim in the present study was to examine the neuropathological and functional effects of the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) in a model of neonatal lipopolysaccharide (LPS)-sensitised HI. We hypothesised that, by decreasing inflammation, TSA would improve injury and behavioural outcome. Furthermore, TSA's effects on oligodendrocyte development, which is acetylation-dependent, were investigated.

Methods: On postnatal day 8 (P8), male and female mice were exposed to LPS together with or without TSA. On P9 (14 hours after LPS), mice were exposed to HI (50 minutes at 10% O2). Neuropathology was assessed at 24 hours, 5 days and 27 days post-LPS/HI via immunohistochemistry and/or Western blot analysis for markers of grey matter (microtubule-associated protein 2), white matter (myelin basic protein) and cell death (activated caspase-3). Effects of TSA on LPS or LPS/HI-induced inflammation (cytokines and microglia number) were assessed by Luminex assay and immunohistochemistry. Expression of acetylation-dependent oligodendrocyte maturational corepressors was assessed with quantitative PCR 6 hours after LPS and at 24 hours and 27 days post-LPS/HI. Animal behaviour was monitored with the open-field and trace fear-conditioning paradigms at 25 days post-LPS/HI to identify functional implications of changes in neuropathology associated with TSA treatment.

Results: TSA induced increased Ac-H4 in females only after LPS exposure. Also only in females, TSA reduced grey matter and white matter injury at 5 days post-LPS/HI. Treatment altered animal behaviour in the open field and improved learning in the fear-conditioning test in females compared with LPS/HI-only females at 25 days post-HI. None of the inflammatory mechanisms assessed that are known to mediate neuroprotection by HDACi in adults correlated with improved outcome in TSA-treated neonatal females. Oligodendrocyte maturation was not different between the LPS-only and LPS + TSA-treated mice before or after exposure to HI.

Conclusions: Hyperacetylation with TSA is neuroprotective in the female neonatal mouse following LPS/HI and correlates with improved learning long-term. TSA appears to exert neuroprotection via mechanisms unique to the neonate. Deciphering the effects of age, sex and inflammatory sensitisation in the cerebral response to HDACi is key to furthering the potential of hyperacetylation as a viable neuroprotectant. TSA did not impair oligodendrocyte maturation, which increases the possible clinical relevance of this strategy.

Show MeSH
Related in: MedlinePlus