莱菔子乙醇提取物对ApoE-/-小鼠血脂血糖及肝脂肪变性的影响*

目的:探讨莱菔子乙醇提取物(AERS)对ApoE^-/-小鼠血脂血糖及肝脂肪变性的影响及其机制。方法:随机将60只ApoE^-/-小鼠分为对照组(CG)、生理盐水组(NG)、罗格列酮组(RG)、AERS治疗组,后者再分成AERS低、中、高剂量组(AERS-L/M/H),每组10只。除CG常规饲料外,其他动物均以高脂高糖高盐饲料饲养9周,CG和NG分别每天以等容量NS灌胃1次,RG每天灌胃罗格列酮(1.33 mg·kg^-1,NS稀释成等容量溶液,每次0.2 ml·10 g^-1),AERS组动物每天分别予AERS灌胃(100、300和900 mg·kg^-1)9周后。肝病理学观察,检测FPG、FIns,计算胰岛素抵抗指数(IRI)和肝系数(LC)。检测血清ALT、AST、瘦素(LEP)、TNF-α及血脂水平(TC、FFA等)。Western blot检测肝脂质代谢相关蛋白表达(HMG-R、LDL-R、LEP-R)。结果:与CG相比,NG和RG肝脏外观(色泽、肿胀情况等)和病理学(脂肪变性、肝细胞坏死等)改变明显,而AERS-M/H与CG相似;与CG相比,血清FPG、Fins、IRI、ALT、AST、TNF-α、LC、TG、LDL-C、FFA、LEP等指标明显升高(P＜0.05);与NG相比,AERS呈现剂量依赖性降低(P＜0.05);与RG相比,AERS-H的FPG、Fins明显升高(P＜0.05),而IRI明显降低(P＜0.05);与NG和RG相比,AERS组的HMG-R和LEP-R蛋白表达呈剂量依赖性减少,而LDL-R蛋白表达呈剂量依赖性增加(P＜0.05)。结论:AERS能阻止高脂高糖饲料诱导ApoE^-/^-小鼠血脂血糖升高及肝脂肪变性,其机制与减少FFA和LEP的生成,抑制TNF-α、HMG-R、LEP-R蛋白表达和促进LDL-R的蛋白表达有关。     

基于转录组测序探究ATP7B基因缺陷小鼠铜累积诱导肝细胞自噬的相关机制*

目的：分析ATP7B基因缺陷(Wilson's disease,WD)小鼠肝脏组织中自噬相关基因的表达和自噬相关蛋白的相互作用方式,探讨铜累积诱导肝内自噬活化的可能机制。方法：对4周龄和12周龄WD小鼠肝组织进行铜含量检测和转录组测序,对差异基因进行GO和KEGG富集分析,筛选自噬相关差异基因做qRT-PCR和Western blot验证,采用GeneMANIA数据库构建自噬相关差异蛋白的互作网络(PPI)并进行功能注释分析,抑制自噬相关蛋白的表达分析其对自噬的影响。结果：与野生型小鼠相比,WD小鼠肝铜含量显著升高,铜累积导致基因表达模式改变;基于GO数据库统计自噬相关差异基因数目,4周龄和12周龄分别有8个、51个,基于KEGG数据库统计,4周龄和12周龄分别有5个、19个;筛选Ulk1、Ddit4、Plk3等9个基因进行qRT-PCR,定量结果与测序结果表达趋势基本一致;其编码的蛋白质通过共表达、共定位等方式互相作用;Western blot结果显示铜累积导致Ulk1、Plk3、Park2蛋白表达显著增加和细胞自噬发生,抑制Ulk1、Plk3、Park2的蛋白质表达可显著下调细胞自噬水平。结论：WD不同阶段的铜累积可调节肝脏多个自噬相关基因的表达,通过其编码的自噬相关蛋白的互相作用共同诱导肝脏自噬活化以缓解肝损伤。     

Portrait of visual cortical circuits for generating neural oscillation dynamics

The mouse primary visual cortex (V1) has emerged as a classical system to study neural circuit mechanisms underlying visual function and plasticity. A variety of efferent-afferent neuronal connections exists within the V1 and between the V1 and higher visual cortical areas or thalamic nuclei, indicating that the V1 system is more than a mere receiver in information processing. Sensory representations in the V1 are dynamically correlated with neural activity oscillations that are distributed across different cortical layers in an input-dependent manner. Circuits consisting of excitatory pyramidal cells (PCs) and inhibitory interneurons (INs) are the basis for generating neural oscillations. In general, INs are clustered with their adjacent PCs to form specific microcircuits that gate or filter the neural information. The interaction between these two cell populations has to be coordinated within a local circuit in order to preserve neural coding schemes and maintain excitation–inhibition (E–I) balance. Phasic alternations of the E–I balance can dynamically regulate temporal rhythms of neural oscillation. Accumulating experimental evidence suggests that the two major sub-types of INs, parvalbumin-expressing (PV⁺) cells and somatostatin-expressing (SOM⁺) INs, are active in controlling slow and fast oscillations, respectively, in the mouse V1. The review summarizes recent experimental findings on elucidating cellular or circuitry mechanisms for the generation of neural oscillations with distinct rhythms in either developing or matured mouse V1, mainly focusing on visual relaying circuits and distinct local inhibitory circuits.     

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUNDThe development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIMTo investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs).METHODSSeven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle.RESULTSFollow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones.CONCLUSIONOur data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Pre-induced adult human peripheral blood mononuclear cells migrate widely into the degenerative retinas of rd1 mice

Background aimsRecent advances in stem cell research have raised the possibility of stem cells repairing or replacing retinal photoreceptor cells that are either dysfunctional or lost in many retinal diseases. Various types of stem cells have been used to replace retinal photoreceptor cells. Recently, peripheral blood stem cells, a small proportion of pluripotent stem cells, have been reported to mainly exist in the peripheral blood mononuclear cells (PBMCs).MethodsIn this study, the effects of pre-induced adult human PBMCs (hPBMCs) on the degenerative retinas of rd1 mice were investigated. Freshly isolated adult hPBMCs were pre-induced with the use of the conditioned medium of rat retinas for 4 days and were then labeled with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI) and then transplanted into the subretinal space of the right eye of rd1 mice through a trans-scleral approach. The right eyes were collected 30 days after transplantation. The survival and migration of the transplanted cells in host retinas were investigated by whole-mount retinas, retinal frozen sections and immunofluorescent staining.ResultsAfter subretinal transplantation, pre-induced hPBMCs were able to survive and widely migrate into the retinas of rd1 mice. A few CM-DiI–labeled cells migrated into the inner nuclear layer and the retinal ganglion cell layer. Some transplanted cells in the subretinal space of rd1 host mice expressed the human photoreceptor–specific marker rhodopsin.ConclusionsThis study suggests that pre-induced hPBMCs may be a potential cell source of cell replacement therapy for retinal degenerative diseases.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

Expression of the murine transcription factor SOX3 during embryonic and adult neurogenesis

Previous studies have shown that Sox3 is expressed in nascent neuroprogenitor cells and is functionally required in mammals for development of the dorsal telencephalon and hypothalamus. However, Sox3 expression during embryonic and adult neurogenesis has not been examined in detail. Using a SOX3-specific antibody, we show that murine SOX3 expression is maintained throughout telencephalic neurogenesis and is restricted to progenitor cells with neuroepithelial and radial glial morphologies. We also demonstrate that SOX3 is expressed within the adult neurogenic regions and is coexpressed extensively with the neural stem cell marker SOX2 indicating that it is a lifelong marker of neuroprogenitor cells. In contrast to the telencephalon, Sox3 expression within the developing hypothalamus is upregulated in developing neurons and is maintained in a subset of differentiated hypothalamic cells through to adulthood. Together, these data show that Sox3 regulation is region-specific, consistent with it playing distinct biological roles in the dorsal telencephalon and hypothalamus.     

Epoxiconazole‐Induced Degeneration in Rat Placenta and the Effects of Estradiol Supplementation

Epoxiconazole (CAS‐No. 133855‐98‐8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7–18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole‐mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose‐dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 μg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter. Birth Defects Res (Part B) 98:208–221, 2013. © 2013 Wiley Periodicals, Inc.