Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats - PubMed (original) (raw)
doi: 10.1186/s12974-017-0942-2.
Li Hu 1, Jiaxiang Wu 1, Qin Wu 2, Wenjing Cheng 2, Yuhong Guo 2, Ruijuan Guan 2, Yahui Wang 3, Xingxin Chen 2, Xanxia Yan 2, Danian Zhu 2, Jijiang Wang 2, Shutian Zhang 4, Yanfang Guo 5, Chunmei Xia 6
Affiliations
- PMID: 28835252
- PMCID: PMC5569471
- DOI: 10.1186/s12974-017-0942-2
Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats
Dongshu Du et al. J Neuroinflammation. 2017.
Abstract
Background: Neuroinflammation plays hypertensive roles in the uninjured autonomic nuclei of the central nervous system, while its mechanisms remain unclear. The present study is to investigate the effect of neuroinflammation on autophagy in the neurons of the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside.
Methods: Stress-induced hypertension (SIH) was induced by electric foot-shock stressors with noise interventions in rats. Systolic blood pressure (SBP) and the power density of the low frequency (LF) component of the SAP spectrum were measured to reflect sympathetic vasomotor activity. Microglia activation and pro-inflammatory cytokines (PICs (IL-1β, TNF-α)) expression in the RVLM were measured by immunoblotting and immunostaining. Autophagy and autophagic vacuoles (AVs) were examined by autophagic marker (LC3 and p62) expression and transmission electron microscopy (TEM) image, respectively. Autophagy flux was evaluated by RFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors transfected into the RVLM. Tissue levels of glutamate, gamma aminobutyric acid (GABA), and plasma levels of norepinephrine (NE) were measured by using high-performance liquid chromatography (HPLC) with electrochemical detection. The effects of the cisterna magna infused minocycline, a microglia activation inhibitor, on the abovementioned parameters were analyzed.
Results: SIH rats showed increased SBP, plasma NE accompanied by an increase in LF component of the SBP spectrum. Microglia activation and PICs expression was increased in SIH rats. TEM demonstrated that stress led to the accumulation of AVs in the RVLM of SIH rats. In addition to the Tf-LC3 assay, the concurrent increased level of LC3-II and p62 suggested the impairment of autophagic flux in SIH rats. To the contrary, minocycline facilitated autophagic flux and induced a hypotensive effect with attenuated microglia activation and decreased PICs in the RVLM of SIH rats. Furthermore, SIH rats showed higher levels of glutamate and lower level of GABA in the RVLM, while minocycline attenuated the decrease in GABA and the increase in glutamate of SIH rats.
Conclusions: Collectively, we concluded that the neuroinflammation might impair autophagic flux and induced neural excitotoxicity in the RVLM neurons following SIH, which is involved in the development of SIH.
Keywords: Autophagic flux; Hypertension; Microglia activation; Neuroinflammation; Pro-inflammatory cytokines; Stress.
Conflict of interest statement
Ethics approval and consent to participate
All experimental protocols and animal-handling procedures were performed according to protocols approved by the Animal Care and Use Committee (ACUC) of Fudan University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Figures
Fig. 1
Representative gels (inset) or densitometric analysis (a) of protein level of ionized calcium-binding adaptor molecule-1 (Iba-1) detected from RVLM of SIH rats or age-matched normotensive rats or IC infusion of minocycline for 1 week in SIH rats. Microglial activation expressed as a percent of total microglia (b) in RVLM or adjacent to it and total number of microglia (activated + non-activated) in the same region. Photomicrographs showed microglia (c) taken from the RVLM of rats in different groups. The outline of the RVLM is highlighted by the dashed lines. Values are mean ± SEM of quadruplicate analyses on samples pooled from five to eight animals in each group. *P < 0.05 vs. control rats, and #P < 0.05 vs. the SIH group in the post hoc Scheffé multiple range analysis. RVLM rostral ventrolateral medulla. Bar = 50 μm in the top and 200 μm at the bottom panel of c
Fig. 2
Effect of minocycline on interleukin (IL)-1β and tumor necrosis factor (TNF)-α expression in the RVLM. Bar graphs illustrate the expressions of IL-1β or TNF-α mRNAs (a) in the RVLM of control, SIH, minocycline only, and minocycline + SIH groups. Representative photomicrographs of Western blot (b) and densitometric analysis (c) for TNF-α in RVLM. Values are mean ± SEM of quadruplicate analyses on samples pooled from five to eight animals in each group. *P < 0.05 vs. control rats, and #P < 0.05 vs. the SIH group in the post hoc Scheffé multiple range analysis
Fig. 3
Temporal changes of systolic blood pressure (SAP; a) or the power density of the low frequency (LF) component of SBP spectrum (b) after intracisternal infusion of minocycline for 1 week in SIH. SIH rats showed higher level of NE (pg/ml) in plasma than that of control (c), which was attenuated by minocycline microinjection. SIH rats showed higher levels of and glutamate (d) and lower level of GABA (e) in the RVLM. One week of intracisternal infusion minocycline attenuated the decrease in RVLM GABA and the increase in glutamate in SIH rats. Values are mean ± SEM of 12 animals in each group. Statistical analysis was performed using one-way ANOVA. *P < 0.05 vs. the control group, #P < 0.05 vs. the SIH group, respectively. n = 12 (or 13) in the post hoc Scheffé multiple range analysis
Fig. 4
Transmission electronic microscopy photomicrographs of the RVLM neurons in the control and SIH groups. The control group showed a number of normal ribosomes, rough endoplasmic reticulum, mitochondrion, and a few autophagic bodies (a). The SIH group showed an increased double membrane structure containing the mitochondrion (b) in comparison with the control (c). AVs are marked with the yellow arrow and blue arrows indicate the mitochondrion. N nucleus. Scale bar, 2 μm. Values are mean ± SEM of four animals in each group. *P < 0.05 vs. control rats in the post hoc Scheffé multiple range analysis
Fig. 5
LC3 expression analysis. a Representative gels (inset) or densitometric analysis of ratio between light chain 3 (LC3)-II and LC3-I protein level in different groups. b Representative immunofluorescent photomicrographs for LC3B. c Qualitative analysis of the number of LC3B detected from RVLM neurons of SIH or normotensive rats. d Representative photomicrographs of LC3 dots in rats when the GFP-LC3 adeno-associated virus was transfected into the RVLM, implying the enhancement of autophagosomes in SIH. Values are mean ± SEM of quadruplicate analyses on samples pooled from five to six animals in each group. *P < 0.05 vs. control rats in the post hoc Scheffé multiple range analysis. RVLM rostral ventrolateral medulla. Scale bar, 20 μm in b and 40 μm in d
Fig. 6
The expression of p62 in RVLM neurons. a Representative double immunofluorescent staining for p62 and neurons marker NeuN. b Qualitative analysis of the number of p62 immuno-positivities detected from RVLM neurons of SIH or normotensive rats or intracisternal infusion minocycline for 1 week in SIH. c Representative gels (inset) or densitometric analysis of p62 protein with an image analyzer. Bar represents mean ± SEM from five to eight rats in each group. Statistical analysis was performed using one-way ANOVA. *P < 0.05 vs. the control group, #P < 0.05 vs. the SIH group. RVLM rostral ventrolateral medulla. Scale bar 20 μm
Fig. 7
LC3 dots were visualized under fluorescent confocal microscope (a) and quantified (b) following RFP-GFP-tandem fluorescent LC3 adeno-associated virus transfected to RVLM for 2 weeks. At least 20 cells per group were included for the counting of RFP- and GFP-LC3 puncta. Statistical analysis was performed using one-way ANOVA. *P < 0.05 vs. the control group, #P < 0.05 vs. the SIH group. RVLM rostral ventrolateral medulla. Scale bar, 50 μm
Fig. 8
Double immunofluorescent staining showed LC3 and lysosomal marker LAMP2 was co-localized in RVLM of control and SIH rats. a Representative double immunofluorescent staining for LC3 and LAMP2. b Qualitative analysis of the number of LC3 and LAMP2 immuno-positivities detected in RVLM. Bar represents mean ± SEM from five to eight rats in each group. Statistical analysis was performed using one-way ANOVA. *P < 0.05 vs. the control group. Scale bar, 50 μm. RVLM rostral ventrolateral medulla, LAMP2 lysosomal-associated membrane proteins 2
Fig. 9
Schematic diagrams illustrating the putative mechanisms that enhanced microglial PICs contributed to autophagic flux disruption and caused the neural excitotoxicity in the RVLM neurons
References
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