Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo

Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47–57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47–57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.     

Penetration in 3D tumor spheroids and explants: Adding a further dimension to the structure-activity relationship of cell-penetrating peptides

Drug delivery into tumors and metastases is a major challenge in the eradication of cancers such as epithelial ovarian carcinoma. Cationic cell-penetrating peptides (CPPs) are a promising group of delivery vehicles to mediate cellular entry of molecules that otherwise poorly enter cells. However, little is known about their penetration behavior in tissues. Here, we investigated penetration of cationic CPPs in 3D ovarian cancer spheroids and patient-derived 3D tumor explants. Penetration kinetics and distribution after long-term incubation were imaged by confocal microscopy. In addition, spheroids and tumor explants were dissociated and cell-associated fluorescence determined by flow cytometry. CPPs with high uptake activity showed enhanced sequestration in the periphery of the spheroid, whereas less active CPPs were able to penetrate deeper into the tissue. CPPs consisting of d-amino acids were advantageous over l-amino acid CPPs as they showed less but long lasting cellular uptake activity, which benefitted penetration and retention over time. In primary tumor cultures, in contrast to nonaarginine, the amphipathic CPP penetratin was strongly sequestered by cell debris and matrix components pointing towards arginine-rich CPPs as a preferred choice. Overall, the data show that testing in 3D models leads to a different choice of the preferred peptide in comparison to a standard 2D cell culture.     

Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain--in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish.     

Thermally Targeted Delivery of a c-Myc Inhibitory Polypeptide Inhibits Tumor Progression and Extends Survival in a Rat Glioma Model

Treatment of glioblastoma is complicated by the tumors’ high resistance to chemotherapy, poor penetration of drugs across the blood brain barrier, and damaging effects of chemotherapy and radiation to normal neural tissue. To overcome these limitations, a thermally responsive polypeptide was developed for targeted delivery of therapeutic peptides to brain tumors using focused hyperthermia. The peptide carrier is based on elastin-like polypeptide (ELP), which is a thermally responsive biopolymer that forms aggregates above a characteristic transition temperature. ELP was modified with cell penetrating peptides (CPPs) to enhance delivery to brain tumors and mediate uptake across the tumor cells’ plasma membranes and with a peptide inhibitor of c-Myc (H1). In rats with intracerebral gliomas, brain tumor targeting of ELP following systemic administration was enhanced up to 5-fold by the use of CPPs. When the lead CPP-ELP-fused c-Myc inhibitor was combined with focused hyperthermia of the tumors, an additional 3 fold increase in tumor polypeptide levels was observed, and 80% reduction in tumor volume, delayed onset of tumor-associated neurological deficits, and at least doubled median survival time including complete regression in 80% of animals was achieved. This work demonstrates that a c-Myc inhibitory peptide can be effectively delivered to brain tumors.     

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R₆-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.     

细胞穿膜肽研究应用的新进展

细胞穿膜肽(Cell-penetrating peptides,CPPs)是一类能够穿过细胞膜或组织屏障的短肽。CPPs可通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。相比于其他非天然的化学分子,CPPs具有生物相容性佳、对细胞造成的毒性小、完成入胞转运后可降解、并能与生物活性蛋白直接融合重组表达等优点,因此成为以胞内分子为靶标的药物递送技术发展的重要工具,并在生物医学研究领域具有良好的应用前景。文中针对CPPs的分类特点、入胞转运机制及其治疗应用的新近研究进展进行综述和讨论。     

Mechanisms of Zero-Lag Synchronization in Cortical Motifs

Zero-lag synchronization between distant cortical areas has been observed in a diversity of experimental data sets and between many different regions of the brain. Several computational mechanisms have been proposed to account for such isochronous synchronization in the presence of long conduction delays: Of these, the phenomenon of “dynamical relaying” – a mechanism that relies on a specific network motif – has proven to be the most robust with respect to parameter mismatch and system noise. Surprisingly, despite a contrary belief in the community, the common driving motif is an unreliable means of establishing zero-lag synchrony. Although dynamical relaying has been validated in empirical and computational studies, the deeper dynamical mechanisms and comparison to dynamics on other motifs is lacking. By systematically comparing synchronization on a variety of small motifs, we establish that the presence of a single reciprocally connected pair – a “resonance pair” – plays a crucial role in disambiguating those motifs that foster zero-lag synchrony in the presence of conduction delays (such as dynamical relaying) from those that do not (such as the common driving triad). Remarkably, minor structural changes to the common driving motif that incorporate a reciprocal pair recover robust zero-lag synchrony. The findings are observed in computational models of spiking neurons, populations of spiking neurons and neural mass models, and arise whether the oscillatory systems are periodic, chaotic, noise-free or driven by stochastic inputs. The influence of the resonance pair is also robust to parameter mismatch and asymmetrical time delays amongst the elements of the motif. We call this manner of facilitating zero-lag synchrony resonance-induced synchronization, outline the conditions for its occurrence, and propose that it may be a general mechanism to promote zero-lag synchrony in the brain.     

Neutralization of BDNF Attenuates the in vitro Protective Effects of Olfactory Ensheathing Cell-Conditioned Medium on Scratch-Insulted Retinal Ganglion Cells

Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.     

The Response of Cerebral Cortex to Haemorrhagic Damage: Experimental Evidence from a Penetrating Injury Model

Understanding the response of the brain to haemorrhagic damage is important in haemorrhagic stroke and increasingly in the understanding the cerebral degeneration and dementia that follow head trauma and head-impact sports. In addition, there is growing evidence that haemorrhage from small cerebral vessels is important in the pathogenesis of age-related dementia (Alzheimer’s disease). In a penetration injury model of rat cerebral cortex, we have examined the neuropathology induced by a needlestick injury, with emphasis on features prominent in the ageing and dementing human brain, particularly plaque-like depositions and the expression of related proteins. Needlestick lesions were made in neo- and hippocampal cortex in Sprague Dawley rats aged 3–5 months. Brains were examined after 1–30 d survival, for haemorrhage, for the expression of hyperphosphorylated tau, Aβ, amyloid precursor protein (APP), for gliosis and for neuronal death. Temporal cortex from humans diagnosed with Alzheimer’s disease was examined with the same techniques. Needlestick injury induced long-lasting changes–haem deposition, cell death, plaque-like deposits and glial invasion–along the needle track. Around the track, the lesion induced more transient changes, particularly upregulation of Aβ, APP and hyperphosporylated tau in neurons and astrocytes. Reactions were similar in hippocampus and neocortex, except that neuronal death was more widespread in the hippocampus. In summary, experimental haemorrhagic injury to rat cerebral cortex induced both permanent and transient changes. The more permanent changes reproduced features of human senile plaques, including the formation of extracellular deposits in which haem and Aβ-related proteins co-localised, neuronal loss and gliosis. The transient changes, observed in tissue around the direct lesion, included the upregulation of Aβ, APP and hyperphosphorylated tau, not associated with cell death. The findings support the possibility that haemorrhagic damage to the brain can lead to plaque-like pathology.     

Unit Operation Optimization for the Manufacturing of Botanical Injections Using a Design Space Approach: A Case Study of Water Precipitation

Quality by design (QbD) concept is a paradigm for the improvement of botanical injection quality control. In this work, water precipitation process for the manufacturing of Xueshuantong injection, a botanical injection made from Notoginseng Radix et Rhizoma, was optimized using a design space approach as a sample. Saponin recovery and total saponin purity (TSP) in supernatant were identified as the critical quality attributes (CQAs) of water precipitation using a risk assessment for all the processes of Xueshuantong injection. An Ishikawa diagram and experiments of fractional factorial design were applied to determine critical process parameters (CPPs). Dry matter content of concentrated extract (DMCC), amount of water added (AWA), and stirring speed (SS) were identified as CPPs. Box-Behnken designed experiments were carried out to develop models between CPPs and process CQAs. Determination coefficients were higher than 0.86 for all the models. High TSP in supernatant can be obtained when DMCC is low and SS is high. Saponin recoveries decreased as DMCC increased. Incomplete collection of supernatant was the main reason for the loss of saponins. Design space was calculated using a Monte-Carlo simulation method with acceptable probability of 0.90. Recommended normal operation region are located in DMCC of 0.38–0.41 g/g, AWA of 3.7–4.9 g/g, and SS of 280–350 rpm, with a probability more than 0.919 to attain CQA criteria. Verification experiment results showed that operating DMCC, SS, and AWA within design space can attain CQA criteria with high probability.     

SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity

In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)–specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.     

Depth Sensitivity and Source-Detector Separations for Near Infrared Spectroscopy Based on the Colin27 Brain Template

Understanding the spatial and depth sensitivity of non-invasive near-infrared spectroscopy (NIRS) measurements to brain tissue–i.e., near-infrared neuromonitoring (NIN) – is essential for designing experiments as well as interpreting research findings. However, a thorough characterization of such sensitivity in realistic head models has remained unavailable. In this study, we conducted 3,555 Monte Carlo (MC) simulations to densely cover the scalp of a well-characterized, adult male template brain (Colin27). We sought to evaluate: (i) the spatial sensitivity profile of NIRS to brain tissue as a function of source-detector separation, (ii) the NIRS sensitivity to brain tissue as a function of depth in this realistic and complex head model, and (iii) the effect of NIRS instrument sensitivity on detecting brain activation. We found that increasing the source-detector (SD) separation from 20 to 65 mm provides monotonic increases in sensitivity to brain tissue. For every 10 mm increase in SD separation (up to ∼45 mm), sensitivity to gray matter increased an additional 4%. Our analyses also demonstrate that sensitivity in depth (S) decreases exponentially, with a “rule-of-thumb” formula S = 0.75*0.85^depth. Thus, while the depth sensitivity of NIRS is not strictly limited, NIN signals in adult humans are strongly biased towards the outermost 10–15 mm of intracranial space. These general results, along with the detailed quantitation of sensitivity estimates around the head, can provide detailed guidance for interpreting the likely sources of NIRS signals, as well as help NIRS investigators design and plan better NIRS experiments, head probes and instruments.     

Robust Short-Term Memory without Synaptic Learning

Short-term memory in the brain cannot in general be explained the way long-term memory can – as a gradual modification of synaptic weights – since it takes place too quickly. Theories based on some form of cellular bistability, however, do not seem able to account for the fact that noisy neurons can collectively store information in a robust manner. We show how a sufficiently clustered network of simple model neurons can be instantly induced into metastable states capable of retaining information for a short time (a few seconds). The mechanism is robust to different network topologies and kinds of neural model. This could constitute a viable means available to the brain for sensory and/or short-term memory with no need of synaptic learning. Relevant phenomena described by neurobiology and psychology, such as local synchronization of synaptic inputs and power-law statistics of forgetting avalanches, emerge naturally from this mechanism, and we suggest possible experiments to test its viability in more biological settings.     

Model for Concomitant Microdialysis Sampling of the Pons and Cerebral Cortex in Rhesus Macaques (Macaca mulatta)

Pediatric diffuse intrinsic pontine gliomas are aggressive brainstem tumors that fail to respond to treatment. We hypothesize that the protective features of the pons may hinder chemotherapeutic agents from entering pontine tissue compared with cortical brain tissue. To test this hypothesis, we developed a unique nonhuman primate model using microdialysis, a continuous in vivo extracellular sampling technique, to compare drug exposure concurrently in pontine tissue, cortical tissue, CSF, and plasma after intravenous administration of chemotherapeutic agents. The surgical coordinates and approach for microdialysis cannula–probe placement were determined in 5 adult male rhesus monkeys (Macaca mulatta) by using MRI. Microdialysis cannulas–probes were implanted stereotactically in the brain, retrodialysis was performed to measure relative recovery, and a 1-h intravenous infusion of temozolomide was administered. Continuous microdialysis samples were collected from the pons and cortex over 4 h with concurrent serial plasma and CSF samples. Postsurgical verification of microdialysis cannula–probe placement was obtained via MRI in 3 macaques and by gross pathology in all 5 animals. The MRI-determined coordinates and surgical methodologies resulted in accurate microdialysis probe placement in the pons and cortex in 4 of the 5 macaques. Histologic examination from these 4 animals revealed negligible tissue damage to the pontine and cortical tissue from microdialysis. One macaque was maintained for 8 wk and had no deficits attributed to the procedure. This animal model allows for the determination of differences in CNS penetration of chemotherapeutic agents in the pons, cortex, and CSF after systemic drug administration.Abbreviations: DIPG, diffuse intrinsic pontine glioma; ECF, extracellular fluidPediatric diffuse intrinsic pontine gliomas (DIPG) are aggressive tumors that cannot be surgically resected due to their location, and are resistant to chemotherapeutic and radiation therapies. As a result, children with DIPG have a dismal prognosis with median survival less than one year from diagnosis. One hypothesis for the poor efficacy of treatment is that innate CNS protective features, such as the blood–brain barrier and the blood–CSF barrier, shield the brainstem to a higher degree given its critical functions, and isolate pontine gliomas from treatment. To test this hypothesis, we developed a nonhuman primate model in rhesus monkeys to evaluate pontine tissue pharmacokinetics by using microdialysis, a continuous in vivo extracellular sampling technique based on diffusion. Microdialysis is the ‘gold standard’ for in vivo sampling methodologies in the CNS, enabling the collection of extracellular tissue fluid via passive diffusion by using a semipermeable membrane probe.A nonhuman primate model demonstrating the feasibility of microdialysis sampling from cortical brain tissue with concurrent pharmacokinetic sampling during chemotherapeutic drug administration has previously been established,^3-5,7 but there are no current animal models that measure drug penetration into the pons. The location of the pons deep within the brain, as well as the vital brainstem functions associated with the pons, present additional obstacles to accurate microdialysis probe placement and sample collection. The objectives of the current study were to develop imaging and surgical procedures for the accurate placement of a microdialysis probe within the pons of rhesus monkeys for sample collection, to establish a method to perform microdialysis simultaneously in multiple CNS regions, and to develop a mechanism to perform repeated microdialysis in the same areas with a single invasive surgical procedure. This model allows for the pharmacokinetic comparison of drug penetration into pontine tissue, in conjunction with cortical tissue, plasma, and CSF, after intravenous administration.     

A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.     

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca²⁺. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca²⁺ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca²⁺ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca²⁺ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca²⁺ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.     

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.     

Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.