Bingyin Su

Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage

BackgroundInflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.MethodsIn this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.ResultsCompared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.ConclusionsOur findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

Alterations of steroid receptor coactivator-1 (SRC-1) immunoreactivities in specific brain regions of young and middle-aged female Sprague–Dawley rats

Previous studies have shown that steroid receptor coactivator-1 (SRC-1) is involved in the regulation of Purkinje cell development and motor learning, neural stem cell differentiation and reproductive-related plasticity. It is widely distributed in the adult brain, but the aging-related changes in the brain remain unclear. In this study age-related alterations of SRC-1 expression in female brain were examined. The results showed that striking age-related decreases of SRC-1 were noticed in those regions related to central regulation of motor (substantia nigra, pontine nuclei, lateral reticular nucleus and Purkinje cells, etc.), learning and memory (olfactory bulb, hippocampus, Purkinje cells, etc.), and neural stem cell (olfactory, dentate gyrus, cerebral cortex, etc.). Surprisingly, although SRC-1 immunopositive materials were predominantly detected in the cell nuclei, they were also detected in the extra-nuclear components predominantly in these motor-regulation sub-regions. The above results showing age-related decrease of SRC-1 in specific motor, learning and memory nuclei suggested its potential roles in neurodegenerative disorders, which may be one of the underlying mechanisms of the vulnerability of the aged brain.

Expression of Steroid Receptor Coactivator-1 Was Regulated by Postnatal Development but Not Ovariectomy in the Hippocampus of Rats

Female steroids such as estrogens and progestins, through their nuclear receptors, play important roles in regulation of the structure and function of the hippocampus. Steroid receptor coactivator-1 (SRC-1) has been detected in embryonic and/or adult hippocampus of rodents, and SRC-1 null mice showed significantly longer escape latency in the Morris maze test, indicating a role of this coactivator in the regulation of hippocampus function. Whether this is regulated by development and circulating ovary hormones remains unclear. In this study, postnatal development and ovariectomy for regulation of hippocampal SRC-1 in female rats were investigated by Western blot and immunohistochemistry. The results showed that SRC-1-immunopositive materials were predominantly detected in the CA1 pyramidal cell layer and dentate gyrus granular cell layer. Very low levels of SRC-1 were detected at postnatal day 0, but they increased with development. The highest levels of SRC-1 were detected at postnatal day 14, then they decreased to adult levels from postnatal day 30; significantly lower levels of SRC-1 were detected in the middle-aged (18-month-old) hippocampus when compared with that of the adult. Western blot and immunohistochemistry demonstrated that hippocampal SRC-1 expression was unchanged after ovariectomy, no significant differences were noticed from day 3 to 8 weeks postsurgery when compared with sham animals. The above results showed that hippocampal SRC-1 is regulated by postnatal development but not ovariectomy, and that the exact role of SRC-1 in the estradiol regulation of hippocampus needs further investigation.

Microglial TIR-domain-containing adapter-inducing interferon-β (TRIF) deficiency promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB.

BackgroundTIR-domain-containing adapter-inducing interferon-β (TRIF) is the sole downstream adaptor of Toll-like receptor (TLR)3, which is one of the major signaling pathways in immune cells leading to neuroinflammation in the central nervous system. Overexpression of TRIF may lead to activation of inflammatory responses, and contribute to pathophysiological progression in both acute and chronic neurodegenerative retinal diseases. In the present study, was aimed to elucidate the contributions of TRIF to optic nerve (ON) regeneration and retinal ganglion cell (RGC) survival following injury to the ON, a widely studied model of central nervous system injury and of degenerative diseases such as glaucoma.MethodsWe used retrograde labeling with a fluorochrome, hydroxystilbamidine (Fluorogold) to evaluate RGC survival, and immunostaining with growth-associated protein-43 to evaluate axon regeneration in an ON crush model. Changes in microglial cytokines following RGC injury was examined with ELISA and real-time PCR. In vivo studies were carried out in wild-type and trif-/- mice. A Transwell co-culture system and migration test were used to mimic the crosstalk between microglia and RGCs. TRIF-associated downstream adaptors were determined by western blotting.ResultsCompared with wild-type (WT) mice, TRIF knockout (KO) mice displayed a robust ability to regenerate axons 3 or 7 days after nerve injury. In addition, RGC survival was considerably higher in trif-/- than in WT mice. ON lesion induced less microglial activation in trif-/- than in WT mice. and more WT microglia distorted and migrated toward the foramen opticum. In the transwell system, few trif-/- microglia migrated through the membrane when stimulated by the performed lesion on RGC axons in a transwell system. Inactivation of microglial cells in trif-/- mice was associated with reduced production of inflammatory cytokines, as detected with real-time RT-PCR and ELISA. Furthermore western blot analysis showed that activation of known downstream effectors of TRIF, including TBK1, IKKε and NF-κB, were significantly inhibited by TRIF deficiency.ConclusionOur results indicate that TRIF deficiency promotes ON axon regeneration by attenuating microglial activation and consequently reducing the release of harmful cytokines via NF-κB inactivation.

Effects of C3 deficiency on inflammation and regeneration following spinal cord injury in mice

Inflammation can activate the complement system, which in turn enhances inflammation and aggravates secondary injury after spinal cord injury (SCI). As the three complement activation pathways converge at the cleavage of C3, we investigated whether inhibiting complement activation in C3-deficient mice would reduce secondary injury after SCI and improve axon regeneration. Weight-drop contusion injury (5g, 6cm) was created in wild-type or C3-deficient mice. Astrocytes (ASTs) activation, TNF-α expression, and axon regeneration were investigated in vivo. In other studies, dorsal root ganglia (DRGs) were co-cultured with mechanically injured ASTs in vitro to evaluate effects on neurite outgrowth. Our results show that, after injury, C3-deficient mice exhibit higher BBB scores than wild-type mice. In addition, ASTs activation was inhibited, TNF-α expression process was delayed in vivo and inhibited in vitro, and nerve fiber regeneration was improved in C3-deficient mice. DRGs co-cultured with mechanically injured ASTs from C3-deficient mice also showed improved neurite outgrowth. We conclude that C3 deficiency can inhibit inflammation through suppressing ASTs activation and TNF-α expression, thereby reducing secondary injury and improving neural regeneration and functional recovery after SCI. The above results suggest that complement inhibition may be a potential therapy to promote central nervous system regeneration by targeting C3.

Expression profiling of Rab GTPases reveals the involvement of Rab20 and Rab32 in acute brain inflammation in mice

Rab GTPases have emerged as central regulators of vesicle trafficking and are essential for cytokine production during the pathogenesis of neuroinflammation. To characterize the roles of different Rab proteins in brain inflammation, we used quantitative PCR (qPCR) to examine the expression profiles of all members of the Rab family in an experimental model of brain inflammation in mice. We found that Rab20 and Rab32 were substantially up-regulated during the acute phase of inflammation. The increased expression of Rab20 was also confirmed by immunostaining of inflamed brains at different timepoints. The concomitant overexpression of Rabs (Rab20 and Rab32) and early response proinflammatory cytokines (TNF-α and IL-1β) suggested that these Rabs may be important for subsequent inflammatory responses in brain. Furthermore, we found that the expression of certain Rabs was dramatically reduced in cultured primary microglia, which was not observed in the in vivo profiling. In N9, a microglial cell line, however, there was no increase in the expression of Rab20 or Rab32, but Rab3c was significantly overexpressed. These results collectively indicate that Rabs may participate in inflammatory response in microglia during brain inflammation. The differential regulation of individual Rabs in different experimental systems is a caveat for the analysis of Rab functions.

Delayed post-injury administration of C5a improves regeneration and functional recovery after spinal cord injury in mice

The activation of a complement system can aggravate the secondary injury after spinal cord injury (SCI). However, it was reported recently that the activation of a complement could have both a secondary injury and a neuroprotective effect, in which C5a is the most important factor, but there is no direct evidence for this dual effect of C5a after SCI. In order to investigate the potential neuroprotective effect of C5a after SCI, in this study ectogenic C5a was injected intraperitoneally before/after SCI in vivo, or administrated to mechanically injured neurones in vitro; following this, neurone apoptosis, neurite outgrowth, axonal regeneration and functional recovery were investigated. The in‐vivo experiments indicated that, following treatment with C5a 24 h before or immediately after injury, locomotor function was impaired significantly. However, when treatment with C5a took place 24 h after injury, locomotor function improved significantly. In‐vitro experiments indicated that a certain concentration of C5a (50–100 nM) could inhibit caspase‐3‐mediated neurone apoptosis by binding to its receptor CD88, and that it could even promote the neurite outgrowth of uninjured neurones. In conclusion, delayed post‐injury administration of C5a within a certain concentration could exert its neuroprotective effect through inhibiting caspase‐3‐mediated neurone apoptosis and promoting neurite outgrowth of uninjured neurones as well. These data suggest that C5a may have opposite functions in a time‐ and concentration‐dependent manner after SCI. The dual roles of C5a have to be taken into account when measures are taken to inhibit complement activation in order to promote regeneration after SCI.

Morphological changes of cholinergic nerve fibers in the urinary bladder after establishment of artificial somatic-autonomic reflex arc in rats

To establish an artificial somatic-autonomic reflex arc in rats and observe the following distributive changes of neural fibers in the bladder. Adult Sprague-Dawley rats were randomly divided into three groups: control group, spinal cord injury (SCI) group, and reinnervation group. DiI retrograde tracing was used to verify establishment of the model and to investigate the transport function of the regenerated efferent axons in the new reflex arc. Choline acetyltransferase (ChAT) in the DiI-labeled neurons was detected by immunohistochemistry. Distribution of neural fibers in the bladder was observed by acetylcholine esterase staining. DiI-labeled neurons distributed mainly in the left ventral horn from L3 to L5, and some of them were also ChAT-positive. The neural fibers in the bladder detrusor reduced remarkably in the SCI group compared with the control (P < 0.05). After establishment of the somatic-autonomic reflex arc in the reinnervation group, the number of ipsilateral fibers in the bladder increased markedly compared with the SCI group (P < 0.05), though still much less than that in the control (P < 0.05). The efferent branches of the somatic nerves may grow and replace the parasympathetic preganglionic axons through axonal regeneration. Acetylcholine is still the major neurotransmitter of the new reflex arc. The controllability of detrusor may be promoted when it is reinnervated by the pelvic ganglia efferent somatic motor fibers from the postganglionic axons. 观察体神经, 内脏神经人工反射弧建立后?大鼠膀胱肌间神经丛分布的改变以及神经肌肉接头处的变化。 Sprague-Dawley 大鼠随机分为三组: 对照组、 脊髓横断组和手术重建组。 手术重建组大鼠术后饲养 3 个月, 与脊髓横断组大鼠一起进行脊髓横断, 再继续饲养3 个月, 对照组不做任何处理。 DiI 进行逆行神经追踪; 免疫荧光的方法显示 DiI 阳性标记细胞中的胆碱乙酰转移酶(choline acetyltransferase, ChAT); 改良的 Karnovsky-Roots法 检测膀胱铺片中神经纤维的分布。 DiI 阳性标记细胞主要分布于脊髓 L3 尾部至 L5 头侧前角, ChAT 阳性细胞和 DiI 阳性标记细胞部分重叠。 手术重建组和对照组相比, 膀胱肌间神经纤维的数量较少, 染色浓度也较浅(P < 0.05); 而手术重建组神经纤维密度较脊髓横断组增大, 染色浓度增强(P < 0.05); 且出现明显的神经再分布。 人工体内脏神经反射弧建立后, 新的传出支为体神经, 可以长入副交感神经纤维, 传出神经元的递质仍为乙酰胆碱, 膀胱内胆碱能神经纤维再生和乙酰胆碱活性增强且出现神经再分布, 这可能在膀胱的控制性排尿中起作用。

Spatial and Temporal Distribution of Dopaminergic Neurons during Development in Zebrafish

As one of the model organisms of Parkinson’s disease (PD) research, the zebrafish has its advantages, such as the 87% homology with human genome and transparent embryos which make it possible to observe the development of dopaminergic neurons in real time. However, there is no midbrain dopaminergic system in zebrafish when compared with mammals, and the location and projection of the dopaminergic neurons are seldom reported. In this study, Vmat2:GFP transgenic zebrafish was used to observe the development and distribution of dopaminergic neurons in real time. We found that diencephalons (DC) 2 and DC4 neuronal populations were detected at 24 h post fertilization (hpf). All DC neuronal populations as well as those in locus coeruleus (LC), raphe nuclei (Ra) and telencephalon were detected at 48 hpf. Axons were detected at 72 hpf. At 96 hpf, all the neuronal populations were detected. For the first time we reported axons from the posterior tubercle (PT) of ventral DC projected to subpallium in vivo. However, when compared with results from whole mount tyrosine hydroxylase (TH) immunofluorescence staining in wild type (WT) zebrafish, we found that DC2 and DC4 neuronal populations were mainly dopaminergic, while DC1, DC3, DC5 and DC6 might not. Neurons in pretectum (Pr) and telencephalon were mainly dopaminergic, while neurons in LC and Ra might be noradrenergic. Our study makes some corrections and modifications on the development, localization and distribution of zebrafish dopaminergic neurons, and provides some experimental evidences for the construction of the zebrafish PD model.

Expressions of C5a and Its Receptor CD88 After Spinal Cord Injury in C3-deficient Mice

The activation of complement system can aggravate the secondary injury after spinal cord injury (SCI). Our previous study indicates that the interception of complement activation by C3 deficiency can reduce the secondary injury and improve the regeneration and functional recovery after SCI. However, recently, it was reported that C5a which was generated during the complement activation pathways also had a protective effect on neurons, but whether it has the similar effect after SCI is unknown. To investigate the possibility and mechanism of the protective effect of C5a on neurons, it is necessary to study the expression profiles of C5a and its receptor CD88 after SCI and the influence on their expression when C3 was knocked out. By immunohistochemistry and Western blot, we found that in wild‐type (WT) mice, both the expression of C5a and its receptor CD88 increased significantly, and there were two peaks during their expression after SCI. However, in C3‐deficient mice, the expression of C5a still increased after SCI, although it was lower than that in WT group at every time points after SCI, and the expression of CD88 remained stable. Our study suggests that the expressions of C5a and CD88 can be inhibited in different degrees after SCI when the activation of complement system is blocked through C3 deficiency, which can reduce the secondary injury caused by C5a after SCI on one hand but deprive neurons of the possible protective effect from C5a on the other hand.