Multi-parametric MRI assessment of melatonin regulating the polarization of microglia in rats after cerebral ischemia/reperfusion injury.

OBJECTIVES: Multi-parametric magnetic resonance imaging (MRI) can provide comprehensive and valuable information for precise diagnosis and treatment evaluation of a number of diseases. In this study, the neuroprotective effects of melatonin (Mel) on a rat model of cerebral ischemia/reperfusion injury (CIRI) were assessed by multi-parametric MRI combined with histopathological techniques for longitudinal monitoring of the lesion microenvironment. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into three groups: the Sham, CIRI and CIRI + Mel groups. At multiple time points after ischemia, MRI scanning was performed on a 7.0 Tesla MRI scanner. Multi-parametric MRI includes T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and chemical exchange saturation transfer (CEST)-MRI. CEST effects were calculated by the Lorentzian difference method, 3.5 ppm indicates amide protons of mobile proteins/peptide (Amide-CEST) and 2.0 ppm indicates amine protons (Guan-CEST). Multiple histopathological techniques were used to examine the histopathological changes and explore the therapeutic effects of Mel. RESULTS: T2WI and DWI-MRI could localize the infarct foci and areas in CIRI rats, which was further validated by staining, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining. After Mel treatment, T2WI and DWI-MRI showed smaller infarct volume, and neurons displayed improved morphology with less apoptosis rates. Notably, Amide-CEST and Guan-CEST signal decreased as early as 2 h after CIRI (all P <0.001), reflecting the change of pH after ischemia. After Mel treatment, both Amide-CEST and Guan-CEST signal increased in ischemic cortex and striatum compared with control group (all P < 0.001). The immunofluorescence staining and western blotting analysis suggested the expression of M2 microglia increased after Mel treatment; While，after Mel treatment the inflammatory factor interleukin-1ß (IL-1ß) decreased compared with control CIRI rats. CONCLUSIONS: Multi-parametric MRI was shown to be an effective method to monitor the brain damage in a rat model of CIRI and assess the therapeutic effects of Mel treatment. Amide-CEST and Guan-CEST were especially sensitive to the changes in brain microenvironment during the early stage after CIRI. Furthermore, the neuroprotective effect of Mel treatment is associated with its promotion of the microglia polarized to M2 type in CIRI rats.

N-acetylserotonin alleviated the expression of interleukin-1ß in retinal ischemia-reperfusion rats via the TLR4/NF-κB/NLRP3 pathway.

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1ß (IL-1ß) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1ß in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4+ and cleaved Caspase-1+ with IL-1ß+ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4+, p-NF-κB+, NLRP3+, cleaved Caspase-1+, and IL-1ß+ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1ß in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1ß+ cells was significantly correlated with those of TLR4+ and cleaved Caspase-1+ cells in the RIRI + NAS group (r2 = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1ß by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

[Effects of different melatonin treatment regimens on the proliferation of endogenous neural stem cells in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effects of different melatonin treatment regimens on the proliferation of neural stem cells (NSCs) and long-term histopathology in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to identify better melatonin treatment regimens. METHODS: A total of 96 Sprague-Dawley rats aged 7 days were randomly divided into normal control, HIBD, single-dose immediate melatonin treatment (SDIT), and 7-day continuous melatonin treatment (7DCT) groups, with 24 rats in each group. The rat model of HIBD was prepared by isolation and electrocoagulation of the right common carotid artery as well as hypoxic treatment in a hypoxic chamber (oxygen concentration 8.00% ±â€…0.01%) for 2 hours. On day 7 after modeling, proliferating cell nuclear antigen/Nestin double-labeling immunofluorescence was used to measure the proliferation of endogenous NSCs in the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG) region in 8 rats in each group, and Western blot was used to measure the protein expression of Nestin in brain. On day 28 after modeling, hematoxylin-eosin (HE) staining and Nissl staining were used to observe the changes in the histopathology and the number of pyramidal cells in the hippocampal CA1 region in 8 rats in each group. RESULTS: Immunofluorescent staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of PCNA+Nestin+DAPI+ cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01). Western blot showed that the SDIT and 7DCT groups had significantly higher protein expression of Nestin than the HIBD group, and the 7DCT group had significantly higher expression than the SDIT group (P<0.05). HE staining showed that the SDIT and 7DCT groups had alleviated cell injury, and Nissl staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of pyramidal cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01). CONCLUSIONS: Both single-dose immediate melatonin treatment and 7-day continuous melatonin treatment can promote the proliferation of endogenous NSCs and alleviate long-term histological injury in the brain of neonatal rats with HIBD. A 7-day continuous melatonin treatment has a better effect than single-dose immediate melatonin treatment.