Electrical and Neurotrophin Enhancement of Neurite Outgrowth Within a 3D Collagen Scaffold

Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 h in culture media containing NGF (10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8–10 V/m DC, then incubated for 24 h with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment.     

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.     

P53和P21在PC12细胞分化中的作用

目的探讨P53及其下游P21蛋白在PC12细胞分化中的可能作用机制。方法用反转录病毒质粒pBabe-P53/m175转染未分化PC12细胞,经筛选后建立野生型P53蛋白功能丧失的细胞系PC12(P53/m175);利用倒置相差显微镜、流式细胞术及Western blot等方法,比较两种细胞在神经生长因子(NGF)作用后,细胞分化表型、细胞周期及相关蛋白表达的改变。结果NGF作用于正常PC12细胞组,P53/P21蛋白表达持续升高,细胞G1期阻滞出现的时间与蛋白表达开始增加的时间相一致;而在NGF作用的PC12(P53/m175)细胞组,细胞不能表达P21蛋白,且细胞周期G1期阻滞的程度也出现显著下降。NGF作用下,两组细胞都能观察到神经突起的生长,表现出明显的分化表型。结论PC12细胞经NGF作用后出现P53及P21蛋白持续表达增加,主要介导了细胞分化过程中细胞周期G1期阻滞这一特征的出现,但并不是导致神经突起进行性生长这一特征的必需条件。     

A laminin and nerve growth factor-laden three-dimensional scaffold for enhanced neurite extension.

Agarose hydrogel scaffolds were engineered to stimulate and guide neuronal process extension in three dimensions in vitro. The extracellular matrix (ECM) protein laminin (LN) was covalently coupled to agarose hydrogel using the bifunctional cross-linking reagent 1,19- carbonyldiimidazole (CDI). Compared to unmodified agarose gels, LN-modified agarose gels significantly enhanced neurite extension from three-dimensionally (3D) cultured embryonic day 9 (E9) chick dorsal root ganglia (DRGs), and PC 12 cells. After incubation of DRGs or PC 12 cells with YIGSR peptide or integrin beta1 antibody respectively, the neurite outgrowth promoting effects in LN-modified agarose gels were significantly decreased or abolished. These results indicate that DRG/PC 12 cell neurite outgrowth promoting effect of LN-modified agarose gels involves receptors for YIGSR/integrin beta1 subunits respectively. 1,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC)-based lipid microcylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels. The resulting trophic factor gradients stimulated directional neurite extension from DRGs in agarose hydrogels. A PC 12 cell-based bioassay demonstrated that NGF-loaded lipid microcylinders can release physiologically relevant amounts of NGF for at least 7 days in vitro. Agarose hydrogel scaffolds may find application as biosynthetic 3D bridges that promote regeneration across severed nerve gaps.     

Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.     

Neural mechanisms of subliminal priming for traumatic episodic memory: an ERP study

Nerve regeneration and functional recovery have been a major issue following injury of nerve tissues. Electrospun nanofibers are known to be suitable scaffolds for neural tissue engineering applications. In addition, modified substrates often provide better environments for neurite outgrowth. This study was conducted to determine if multi-walled carbon nanotubes (MWCNTs)-coated electrospun poly (l-lactic acid-co-caprolactone) (PLCL) nanofibers improved the neurite outgrowth of rat dorsal root ganglia (DRG) neurons and focal adhesion kinase (FAK) expression of PC-12 cells. To accomplish this, the DRG neurons in either uncoated PLCL scaffolds (PLCL group) or MWCNTs-coated PLCL scaffolds (PLCL/CNT group) were cultured for nine days. MWCNTs-coated PLCL scaffolds showed improved neurite outgrowth of DRG neurons. Moreover, FAK expression was up-regulated in the PLCL/CNT group when compared to the PLCL group in a non-time-dependent manner. These findings suggest that MWCNTs-coated nanofibrous scaffolds may be alternative materials for nerve regeneration and functional recovery in neural tissue engineering.     

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration

Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325 + 383 nm, large diameter fibers; 759 + 179 nm, intermediate diameter fibers; and 293 + 65 nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5 days and then the explants were stained against neurofilament and S100. DAPI stain was used to assess SC migration. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.     

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3β signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

Adhesive proteins linked with focal adhesion kinase regulate neurite outgrowth of PC12 cells

Adhesive proteins existing in the extracellular matrix (ECM) play important roles in the regulation of neuronal cell behavior, including cell adhesion, motility and neurite outgrowth. Herein we show the effects of a series of adhesive proteins on the neurite outgrowth of PC12 cells and elucidate that this is closely related to the activation of focal adhesion kinase (FAK). For this we prepared culture substrates by coating tissue culture plastic with either collagen (Col), fibronectin (FN) or laminin (LN) and investigated the neurite outgrowth behavior. The results demonstrated that neurite outgrowth was highly dependent on the particular type of adhesive protein. While neurite number was comparable on all the coated surfaces, the length of neurites was greater on the FN- and LN-coated ones (greatest on the LN-coated one). In particular, FAK expression was highly up-regulated in the FN- and LN-coated surfaces, as revealed by Western blot analysis. A knock-down experiment further supported the idea that neurite outgrowth was largely suppressed in cells transfected with a FAK knock-down gene. Taken together, the neurite outgrowth of PC12 cells was greatly affected by adhesive proteins of the ECM, particularly FN and LN, and this is considered to be closely related to FAK intracellular signaling. This study may be useful in the consideration and design of nerve guidance and three-dimensional scaffolds which are appropriate to promote neuronal growth and nerve tissue regeneration.     

Nerve growth factor expression by PLG-mediated lipofection

Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.     

Hypoxia induces neurite outgrowth in PC12 cells that is mediated through adenosine A2A receptors

Development of the nervous system is a complex process, involving coordinated regulation of diverse cellular processes including proliferation, differentiation and synaptogenesis. Disturbances to brain development such as pre- and perinatal hypoxia have been linked to behavioural and late onset of neurological disorders. This study examines the effect of hypoxia on neurite outgrowth in PC12 cells. Hypoxia not only caused a rapid induction of neurite outgrowth, but also synergistically enhanced nerve growth factor (NGF)-induced neurite outgrowth up to 24 h. Transactivation of TrkA receptors was ruled out since the TrkA inhibitor K252a did not block hypoxia-induced neurite outgrowth. Adenosine deaminase prevented hypoxia-induced neurite outgrowth indicating that the effect is mediated by adenosine. Use of the specific adenosine A2A receptor agonist CGS21680 and antagonist 8-3(chlorostyryl)caffeine demonstrated that activation of this receptor is critical for hypoxia-induced neurite outgrowth. Hypoxia-induced neurite outgrowth was blocked by the adenylate cyclase inhibitor, MDL-12,330A, indicating a role for activation of this enzyme in the pathway. Hypoxia was further shown to cause a decrease in growth-associated protein (GAP)-43 levels and a lack of induction of betaIII tubulin, in contrast to NGF treatment which resulted in increased cellular levels of both of these proteins. These findings suggest that hypoxia induces neurite outgrowth in PC12 cells via a pathway distinct from that activated by NGF. Thus, exposure to hypoxia at critical stages of development may contribute to aberrant neurite outgrowth and could be a factor in the pathogenesis of certain delayed developmental neurological disorders.     

An aligned 3D neuronal-glial co-culture model for peripheral nerve studies

In this study we report on the development of a 3D in vitro peripheral nerve model using aligned electrospun polycaprolactone fibre scaffolds manufactured under tightly controlled and reproducible conditions with uniform diameters of 1 μm, 5 μm and 8 μm. Fibres were characterized by SEM for diameter, density and alignment properties and formed in to scaffolds for 3D in vitro culture. Three different approaches were adopted using: i) neuronal or primary Schwann cell cultures alone; ii) neuronal and primary Schwann cells in co-culture and iii) isolated dorsal root ganglion cultures, containing both neuronal and Schwann cells, with immunohistochemical and 3D confocal microscopy analysis. Neurite guidance was evident on all fibres diameters with the longest neurites detected on 8 μm fibres when cultured alone. However, co-culture with primary Schwann cells was found to enable neurite formation on all scaffolds. Dorsal root ganglion explants when grown on scaffolds showed both organised aligned neurite guidance and notably the co-localization of Schwann cells with neurites. Neurite lengths of up to 2.50 mm were routinely observed using 1 μm diameter fibres after 10 days and all cultures demonstrated a migrating Schwann cell 'front' of up to 2.70 mm (1 μm diameter fibres). Thus, a direct relationship was found between fibre diameter, neurite outgrowth and Schwann cell morphology. This work therefore supports the use of aligned electrospun PCL microfibres for the development of 3D peripheral nerve models in vitro. We envisage such models having future value in a number of areas including developmental biology, disease studies and the design of devices and scaffolds for peripheral nerve repair.     

A synthetic analog of verbenachalcone potentiates NGF-induced neurite outgrowth and enhances cell survival in neuronal cell models

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2 ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2 μM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.     

The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.     

The precision structural regulation of PLLA porous scaffold and its influence on the proliferation and differentiation of MC3T3-E1 cells

A thermal-induced phase separation combined sugar template method was used to fabricate the Poly (L-lactide) acid (PLLA) scaffolds with precisely regulated porous structure. The effect of tuned porous structure of scaffolds on osteoblasts proliferation and differentiation was investigated. The results showed that the pore diameters (200–300, 300–400, 400–500 μm), porosity and interconnectivity of PLLA scaffolds can be accurately controlled indicated by scanning electron microscope. The results of cell experiments showed that the porous structure including the pore size and interconnectivity of scaffolds dramatically influence the cell proliferation and differentiation. The scaffold with pore diameter of 400–500 μm exhibited the highest cell viability and alkaline phosphatase activity among all the scaffolds for the MC3T3-E1 cells. The higher cell proliferation and biocompatibility observed in the 400–500 μm scaffold indicated the high selectivity for MC3T3-E1cells on the pore size of scaffold in tissue engineering. The precise control of the porous structure of scaffold may better guide the cell–matrix interaction in the future research.     

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.     

ERK在NGF诱导PC12 细胞分化中的作用

目的探讨细胞外信号调节激酶(ERK)在NGF。诱导的PC12细胞分化中的作用机制。方法以NGF处理PC12细胞建立分化模型,运用免疫印迹检测不同浓度不同作用时间时NGF对ERK1／2蛋白和磷酸化ERK1／2蛋白水平的影响,并观察MAPK／ERK激酶(MEK)抑制剂U0126对NGF诱导的细胞形态学改变的影响。结果ERK1／2蛋白的磷酸化呈现NGF剂量和时间依赖性。NGF作用细胞5min即可观察到明显的ERK1／2蛋白磷酸化,持续1h左右,2h时降低到初始水平,而细胞形态的改变出现在NCF作用12h以后。倒置相差显微镜观察可见PC12细胞分化的程度与ERK1／2活化持续的时间正相关,U0126可完全即时抑制ERK1／2的活化,而ERK1／2活化的抑制可完全阻断。NGF诱导的PC12细胞分化。结论ERK1／2的活化是PC12细胞发生分化的必需事件,其活化时间的长短对分化具有决定作用。     

ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth

Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.