Microinjection of a growth factor cocktail affects activated microglia in the neocortex of adult rats

Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.     

点突变Uchl1不影响小鼠卵母细胞成熟

目的 通过观察突变蛋白与野生蛋白过量对卵母细胞成熟过程的影响,分析Uchl1在小鼠卵母细胞离体成熟中发挥的作用及其作用方式。方法 通过原核重组蛋白技术制备点突变（C90S和I93M）和野生型Uchl1蛋白,通过蛋白微量注射技术将突变蛋白与对照蛋白注射到未成熟卵母细胞,或体外培养液中添加突变蛋白与野生型蛋白,分析野生型Uchl1过量,或突变Uchl1-I93M蛋白添加,或泛素水解酶活性缺失的Uchl1-C90S突变蛋白添加,对于卵母细胞体外成熟的影响。结果 显微注射UCHL1融合蛋白的各组之间以及与注射PBS之间, GVBD率差异没有达到统计学显著性;同时培养液中添加Uchl1的GST融合蛋白,相对于无注射对照组,也没有统计学显著差异（P>0.05）。注射GST-C90S融合蛋白组少量GV期卵母细胞体外发育为MII期卵母细胞后呈现极体偏大于对照组。I93M点突变小鼠与WT小鼠比较,GV期卵母细胞体外GVBD率无显著差异。结论 在小鼠卵母细胞中,添加外源性Uchl1,或者有毒性作用的I93M突变体,或者水解酶活性结构改变的C90S突变体,均不影响卵母细胞成熟的GVBD进程。即使构建的I93M点突变小鼠卵母细胞GVBD率无异常。但是,其水解酶活性位点突变影响极体的形成。     

卵子激活剂对无精子症患者冻融睾丸精子ICSI后妊娠结局的影响

目的研究卵子激活剂CultActive在临床中能否提高无精子症患者冻融睾丸精子行卵胞浆内单精子显微注射(ICSI)后的妊娠结局。方法选择2015年1月~2019年12月我院收治的188例行冻融睾丸精子ICSI助孕治疗的无精子症患者作为研究对象,按照患者ICSI后是否进行应用卵子激活剂CultActive随机分为冻融睾丸精子组(n=107)和卵子激活组(n=81),对照组为新鲜睾丸精子组(n=129),分别比较了三组患者在年龄、不孕年限、促性腺激素用量、促性腺激素使用天数等临床资料的差异以及获卵数、受精率、2PN率、分裂率、优胚率、可利用胚胎率、临床妊娠率等胚胎发育情况的差异。结果三组患者在年龄、不孕年限、促性腺激素用量、促性腺激素使用天数、获卵数上均无统计学差异(P>0.05)。B组在2PN率、优胚率、可利用胚胎率上均高于A、C组,但无统计学差异(P>0.05)。三组患者在分裂率上也无统计学差异(P>0.05)。B组在受精率上明显高于C组(P<0.05),且显著高于A组(p<0.01)。B组在临床妊娠率上显著高于A、C组(p<0.01)。结论卵子激活剂CultActive可以提高冻融睾丸精子ICSI效率,值得在临床中推广。     

Transgenesis and neuronal ablation in parasitic nematodes: revolutionary new tools to dissect host-parasite interactions

Ease of experimental gene transfer into viral and prokaryotic pathogens has made transgenesis a powerful tool for investigating the interactions of these pathogens with the host immune system. Recent advances have made this approach feasible for more complex protozoan parasites. By contrast, the lack of a system for heritable transgenesis in parasitic nematodes has hampered progress toward understanding the development of nematode-specific cellular responses. Recently, however, significant strides towards such a system have been made in several parasitic nematodes, and the possible applications of these in immunological research should now be contemplated. In addition, methods for targeted cell ablation have been successfully adapted from Caenorhabditis elegans methodology and applied to studies of neurobiology and behaviour in Strongyloides stercoralis. Together, these new technical developments offer exciting new tools to interrogate multiple aspects of the host-parasite interaction following nematode infection.     

Enhanced cognitive flexibility in reversal learning induced by removal of the extracellular matrix in auditory cortex

During brain maturation, the occurrence of the extracellular matrix (ECM) terminates juvenile plasticity by mediating structural stability. Interestingly, enzymatic removal of the ECM restores juvenile forms of plasticity, as for instance demonstrated by topographical reconnectivity in sensory pathways. However, to which degree the mature ECM is a compromise between stability and flexibility in the adult brain impacting synaptic plasticity as a fundamental basis for learning, lifelong memory formation, and higher cognitive functions is largely unknown. In this study, we removed the ECM in the auditory cortex of adult Mongolian gerbils during specific phases of cortex-dependent auditory relearning, which was induced by the contingency reversal of a frequency-modulated tone discrimination, a task requiring high behavioral flexibility. We found that ECM removal promoted a significant increase in relearning performance, without erasing already established—that is, learned—capacities when continuing discrimination training. The cognitive flexibility required for reversal learning of previously acquired behavioral habits, commonly understood to mainly rely on frontostriatal circuits, was enhanced by promoting synaptic plasticity via ECM removal within the sensory cortex. Our findings further suggest experimental modulation of the cortical ECM as a tool to open short-term windows of enhanced activity-dependent reorganization allowing for guided neuroplasticity.Structural remodeling and stabilization of synaptic networks are key mechanisms underlying learning in the adult brain. During early life, high structural and functional plasticity is required for the experience-shaped development of basic neuronal circuits (1). With brain maturation, juvenile plasticity of so-called critical or sensitive periods is decreased and is accompanied by the appearance of the brain’s extracellular matrix (ECM) and its specialized compact form named “perineuronal net” (PNN) enwrappping cell bodies and synaptic contacts (2, 3). Enzymatic degradation of the ECM in adult animals has been demonstrated to restore such forms of developmental (juvenile) plasticity with respect to topographical map plasticity in the visual cortex (4), fear-response–mediating circuits in the amygdala (5), spinal cord injuries (6, 7), and song learning circuits of zebra finches (8). In addition, enzymatic ECM removal altered several forms of synaptic plasticity in vitro and in vivo (9–12). However, even though structural stability of networks acquired during developmental phases is essential for neuronal efficiency, mechanisms allowing synaptic remodeling are key events during learning and memory formation throughout life (13). We recently demonstrated that endogenous proteases moderately digesting specific components of the ECM are regulated in an activity-dependent manner (2, 14) and ECM removal modulates synaptic short-term plasticity by synaptic exchange of postsynaptic glutamate receptors (10, 15). Further, ECM modulation enhances synaptic short-term plasticity by affecting voltage-dependent calcium channels (9). These findings challenge the view of the purely stabilizing role of ECM in the brain and indicate a potential regulatory switch for plastic network adaptations within the adult brain at the level of individual synapses by modulating the extracellular space (16). However, it remains open to what extent ECM modulations influence learning-related plasticity in the adult brain and its specific effects on behavior during a cognitive task.In the present study, we aimed at evaluating the potential role of experimental ECM removal within the auditory cortex (ACx) of Mongolian gerbils, which has been found to be particularly rich in ECM (17), during a cognitively demanding auditory go/no go shuttle-box task. We selected discrimination and reversal learning of frequency-modulated (FM) tones as a cognitive task, which necessarily requires ACx plasticity (18, 19). Injections of the ECM-degrading enzyme hyaluronidase (HYase) into the ACx after the first acquisition phase significantly enhanced subsequent reversal learning compared with sham-treated animals (injection of 0.9% saline). Particularly, after ECM degradation, animals abandoned the inappropriate discrimination strategy from the initial acquisition phase faster and thus promoted successful discrimination performance of the new contingency during reversal learning. ECM removal did not further influence the initial acquisition learning or interfere with already established—that is, learned—capacities in later learning stages, suggesting an enhanced activity-dependent neuroplastic reorganization of established synaptic networks in ACx during reversal learning.Our findings suggest that experimental degradation of the ECM in sensory cortex, although not affecting general sensory learning, does, however, enhance the cognitive flexibility that can build on the learned behaviors. Thereby, ECM degradation could be used as a tool for guided neuroplasticity, which might also bear therapeutic potential.     

YMDD突变株HBV转基因小鼠的制备及检测

目的建立YMDD耐药突变株HBV转基因小鼠,为乙肝防治研究提供转基因动物模型。方法采用受精卵显微注射法,将带有YMDD突变的对拉米夫定有耐药性的1.3拷贝HBV基因注入FVB/N单细胞受精卵的原核内,制备YMDD耐药突变株HBV转基因小鼠。采用PCR检测外源基因的整合和传代情况,采用ELISA和免疫组化等方法检测HBsAg在肝、肾中的复制和表达情况。结果注射受精卵3401枚,产269只F0代仔鼠,PCR阳性33只,外源基因的整合率12.3%。9只转基因鼠血清HBVDNA弱阳性,拷贝数低于103拷贝/ml;免疫组化结果显示肝组织和肾组织均有HBsAg表达,且肾组织中的表达强于肝组织。经传代产下47只F1代转基因鼠,目的基因PCR阳性率为27.6%,且肝组织和肾组织中HBsAg均有表达,分布特性与F0代一致。结论成功制备出体内有复制表达而且可以传代YMDD耐药突变株HBV的转基因小鼠。     

新型振荡注射仪在冰冻血小板中的应用研究

目的：研究手工法与某新型振荡注射仪冰冻血小板之异同。方法：通过手工法及与某厂家合作研制的振荡注射仪冰冻血小板,检测2组血小板冰冻前后的关键指标及临床应用指标,并比较2者之间的差异。结果：手工组和振荡注射仪组在加入DMSO后冰冻前与对照组比较,在血小板P选择蛋白和PF4方面差异有统计学意义（P〈0.01）,在加入DMSO冰冻后复溶与对照组比较,血小板黏附功能、血小板P选择蛋白、血小板第4因子（PF4）和血块收缩试验之间差异有统计学意义（P〈0.01）,但手工组与振荡注射仪组之间差异无统计学意义（P〉0.05）,振荡注射仪组与手工组在融化后报废率方面差异有统计学意义（P〈0.01）。结论：振荡注射仪组在融化后报废率方面优于手工组。     

Decreased levels of pNR1 S897 protein in the cortex of neonatal Sprague Dawley rats with hypoxic-ischemic or NMDA-induced brain damage

Our objective was to investigate the protein level of phosphorylated N-methyl-D-aspartate (NMDA) receptor-1 at serine 897 (pNR1 S897) in both NMDA-induced brain damage and hypoxic-ischemic brain damage (HIBD), and to obtain further evidence that HIBD in the cortex is related to NMDA toxicity due to a change of the pNR1 S897 protein level. At postnatal day 7, male and female Sprague-Dawley rats (13.12 ± 0.34 g) were randomly divided into normal control, phosphate-buffered saline (PBS) cerebral microinjection, HIBD, and NMDA cerebral microinjection groups. Immunofluorescence and Western blot (N = 10 rats per group) were used to examine the protein level of pNR1 S897. Immunofluorescence showed that control and PBS groups exhibited significant neuronal cytoplasmic staining for pNR1 S897 in the cortex. Both HIBD and NMDA-induced brain damage markedly decreased pNR1 S897 staining in the ipsilateral cortex, but not in the contralateral cortex. Western blot analysis showed that at 2 and 24 h after HIBD, the protein level of pNR1 S897 was not affected in the contralateral cortex (P > 0.05), whereas it was reduced in the ipsilateral cortex (P < 0.05). At 2 h after NMDA injection, the protein level of pNR1 S897 in the contralateral cortex was also not affected (P > 0.05). The levels in the ipsilateral cortex were decreased, but the change was not significant (P > 0.05). The similar reduction in the protein level of pNR1 S897 following both HIBD and NMDA-induced brain damage suggests that HIBD is to some extent related to NMDA toxicity possibly through NR1 phosphorylation of serine 897.     

Effect of catecholamines on the swallowing reflex after pressure microinjections into the lateral solitary complex of the medulla oblongata

The present study was carried out to elucidate the influence of catecholamines on swallowing, a polysynaptic reflex organized by an interneuronal network localized mainly within the lateral solitary complex (LSC) of the medulla oblongata. The effects of catecholaminergic agents were investigated in the rat, on rhythmic swallowing elicited by repetitive stimulation of the superior laryngeal nerve (SLN). Catecholaminergic agents were microinjected by pressure application, through multibarrelled glass micropipettes, into the LSC including the tractus solitarius, the swallowing region of the nucleus of the solitary tract and the adjacent reticular formation. Microinjections of noradrenaline (NA, 0.1–5 nmol, 50 nl) induced a significant decrease of the number and the amplitude of the rhythmic swallows elicited by stimulation of the ipsilateral SLN. This inhibitory effect was dose-related. Microinjections of clonidine (2.5 nmol, 50 nl), dopamine (0.25–2.5 nmol, 50 nl) and apomorphine (0.5 nmol, 50 nl), also inhibited swallowing. No significant modification of swallowing was induced by control injections of the vehicle (50 nl) within the active sites. Moreover the NA-induced inhibition of swallowing, was significantly antagonized by pretreatment with the -adrenergic blocker phentolamine applied locally in the LSC. Furthermore neither blood pressure, nor respiratory rhythm were consistently modified by the catecholaminergic microinjections, indicating that the catecholamine-induced inhibition of swallowing was not a secondary side effect originating from alteration of these functions. It can therefore be concluded that the present results demonstrate the existence within the LSC of a catecholaminergic inhibition of the swallowing reflex. This inhibitory effect likely arises from activation of specific catecholaminergic receptors and effects the swallowing structures localized within the LSC, i.e., the laryngeal swallowing afferents running in the solitary tract and/or the swallowing interneurons within the nucleus of the solitary tract.     

Norepinephrine applied in the paraventricular hypothalamic nucleus stimulates vasopressin release

The present experiments were designed to investigate the effect of norepinephrine (NE) applied directly in the area of the paraventricular nucleus (PVN) of the hypothalamus on arginine-vasopressin (AVP) release and blood pressure. A microinjection of 0.4 micrograms NE in the PNV produced a plasma AVP level of 26.3 +/- 5.3 pg/ml compared to 5.3 +/- 0.6 pg/ml in controls receiving dextrose (P less than 0.001). This rise was associated with blood pressure elevations varying between 10 and 13 mm Hg, lasting for about 5 min. Systemic injection of an antivasopressor AVP antagonist reversed or prevented the blood pressure rise induced by NE microinjection. The data suggest that locally applied NE in vasopressinergic neurons of the hypothalamus stimulates the release of AVP and induces an AVP-dependent rise in blood pressure.