Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat - PubMed (original) (raw)

Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

Qin Lu et al. J Neuroinflammation. 2014.

Abstract

Background: Mammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates many processes, including cell growth and the immune response. mTOR is also dysregulated in several neurological diseases, such as traumatic brain injury (TBI), stroke, and neurodegenerative disease. However, the role of mTOR in intracerebral hemorrhage (ICH) remains unexplored. The aims of our study were to determine whether inhibiting mTOR signaling could affect the outcome after ICH and to investigate the possible underlying mechanism.

Methods: A rat ICH model was induced by intracerebral injection of collagenase IV into the striatum, and mTOR activation was inhibited by administration of rapamycin. mTOR signaling activation was determined by western blotting. Neurobehavioral deficit after ICH was determined by a set of modified Neurological Severity Scores (mNSS). The levels of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and cytokines were examined using flow cytometry and ELISA, respectively.

Results: Our results demonstrated thatmTOR signaling was activated 30 minutes and returned to its basal level 1 day after ICH. Increased p-mTOR, which mean that mTOR signaling was activated, was predominantly located around the hematoma. Rapamycin treatment significantly improved the neurobehavioral deficit after ICH, increased the number of Tregs, increased levels of interleukin-10 and transforming growth factor-β and reduced interferon-γ both in peripheral blood and brain.

Conclusions: Our study suggests that mTOR improves ICH outcome and modulates immune response after ICH.

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Figures

Figure 1

Figure 1

Mammalian target of rapamycin (mTOR) was activated after intracerebral hemorrhage (ICH). (A) Protein from the ipsilateral hemisphere was analyzed by western blotting using anti-p-mTOR (Ser 2448)., which was was greatly increased at 30 minutes after ICH and lasted up to 14 days after ICH. The level of p-mTOR was normalized to the level of tubulin. *_P<_0.05.

Figure 2

Figure 2

Increased mammalian target of rapamycin (mTOR) activation in the ipsilateral striatum after intracerebral hemorrhage (ICH). (A) Protein from the striatum was analyzed by western blotting using anti-p-mTOR (Ser 2448). The level of p-mTOR was normalized to the level of tubulin. The p-mTOR significantly increased at 30 minutes after ICH, and returned to basal level at 1 day after ICH. (B) The change in total mTOR was not significant after being normalized to tubulin. *_P<_0.05; **_P<_0.001.

Figure 3

Figure 3

Mammalian target of rapamycin (mTOR) activation in the ipsilateral cortex after intracerebral hemorrhage (ICH). The levels of both (A) p-mTOR and (B) total mTOR did not show significant changes after ICH (after normalization to tubulin).

Figure 4

Figure 4

Increased p70S6 activation in the ipsilateral striatum after intracerebral hemorrhage (ICH). (A) Protein from the striatum was analyzed by western blotting using anti-p-p70S6 (Thr 389). The level of p-p70S6 was normalized to the level of tubulin. p-mTOR was significantly increased at 1 hour and returned to basal level at 1 day after ICH. (B) The change in total p70S6 was not significant after being normalized to tubulin. _*P<_0.05.

Figure 5

Figure 5

Rapamycin improves recovery of neurobehavioral function after intracerebral hemorrhage (ICH). The scores in both the ICH and rapamycin groups were similar before ICH. At 1 hour after ICH, rapamycin-treated groups were injected with rapamycin using different concentrations: 50, 150, 250, and 500 μg/kg. The behaviors were evaluated and compared at 1, 3, 7, and 14 days after ICH. We observed a significant functional recovery in rapamycin-treated groups compared with the ICH group. The mean modified Neurological Severity Score (mNSS) values ± SEM are depicted, *_P<_0.05; ** _P<_0.001 compared with ICH rats.

Figure 6

Figure 6

Rapamycin inhibited p-mammalian target of rapamycin (mTOR) in the striatum. (A) Compared with the intracerebral hemorrhage (ICH) group (4 h after ICH), significantly lower levels of p-mTOR were observed in the 150, 250, and 500 μg/kg treated groups while there was no significant change in the 50 μg/kg treated group. (B) The total mTOR level in rapamycin-treated groups was similar to that of the ICH group. Both p-mTOR and total mTOR were normalized to tubulin. *_P<_0.05; **_P<_0.001.

Figure 7

Figure 7

Rapamycin increased the level of regulatory T cells (Tregs) in the blood. Dot plots labeled with CD4 and Foxp3 show the blood lymphocytes derived from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significant differences between the control group and rapamycin-treated groups, and between the ICH and rapamycin-treated groups. _*P<_0.05.

Figure 8

Figure 8

Rapamycin increased the level of regulatory T cells (Tregs) in the ipsilateral hemisphere. Dot plots labeled with CD4 and Foxp3 show the brain lymphocytes from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significantly higher levels of Tregs in the rapamycin-treated group than the control and ICH groups. *_P<_0.001.

Figure 9

Figure 9

Levels of cytokines in the serum after rapamycin treatment. (A) The levels of interferon (IFN)-γ in each rapamycin-treated group were lower than the intracerebral hemorrhage (ICH) group. (B) Rapamycin-treated groups presented higher interleukin (IL)-10 expression than the ICH group. (C) The ratio of interleukin (IL)-10 to IFN-γ increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. There were no significant differences between the 150, 250, and 500 μg/kg rapamycin-treated groups. *_P<_0.05.

Figure 10

Figure 10

Levels of cytokines around the hematoma after rapamycin treatment. (A) Compared with the intracerebral hemorrhage (ICH) group, the levels of **interferon (IFN)-**γ were reduced in the rapamycin-treated groups except for the 50 μg/kg group. (B) Rapamycin-treated groups had higher levels of **interleukin (IL)**-10 than the ICH group, except for the 50 μg/kg group. (C) The ratio of IL-10 to IFN-γ was increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. *_P<_0.05.

Figure 11

Figure 11

Effect of rapamycin on the levels of interferon (IFN)-γ and interleukin (IL)-10 at an autologous blood-injection model of intracerebral hemorrhage (ICH). (A, B) Similar to the collagenase-injection model, the levels of IFN-γ was downregulated after treatment with rapamycin both in serum and around the hematoma in an autologous blood-injection model. (C, D) Rapamycin upregulated IL-10 both in serum and around the hematoma. AU, Autologous blood-injection model of ICH, CO, Collagenase-injection model of ICH, RA, Rapamycin. *_P<_0.05.

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