Neural tissue engineering: a self-organizing collagen guidance conduit

We report a novel implantable device that will deliver a tethered aligned collagen guidance conduit containing Schwann cells into a peripheral nerve injury site. Cells (Schwann cells and fibroblasts) incorporated into tethered rectangular collagen gels contracted and resulted in uniaxial alignment. This tissue-engineered construct was tested in three-dimensional culture and demonstrated the ability to guide neurite extension from dissociated dorsal root ganglia. A silicone tube was adapted to provide tethering sites for an implantable construct such that uniaxial cell-generated tension resulted in the formation of a bridge of aligned collagen fibrils, with a resident Schwann cell population. The potential of this device for surgical nerve regeneration was assessed in a 5-mm defect in a rat sciatic nerve model. Neural regeneration through this device was significantly greater than in controls, demonstrating that this system has potential both as a simple robust clinical implant and as a three-dimensional engineered tissue model.     

Polystyrene replicas of neuronal basal lamina act as excellent guides for regenerating neurites

Various scaffolds, natural or artificial, have been used for neural repair, including basal lamina scaffolds obtained through extraction of nerves. Here we tested whether plastic casts of such preparations could be used for neurite guidance. To this end, longitudinal micron thick sections of rat sciatic nerve were extracted with detergents and treated with Dnase, yielding an acellular basal lamina master. From the basal lamina master a polydimethylsiloxane (PDMS) mold was made. Then a polystyrene replica was made using the PDMS mold as the master. The polystyrene replica showed high similarity to the master within nanometer resolution as revealed by scanning electron microscopy. Organ cultured mouse dorsal root ganglia grown on the polystyrene replica and the master preparation exhibited guided outgrowth of neurites as assayed by two-dimensional fast Fourier transform analysis on preparations, where the neurites had been visualized by β-III-tubulin staining. The neurites aligned longitudally in the direction of the original basal lamina tubes. Thus, using inexpensive methods it is possible to make replicas of basal lamina which can be used for neurite guidance. This opens a new avenue for nerve reconstruction.     

Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend

Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.     

Spatial confinement of neurite regrowth from dorsal root ganglia within nonporous microconduits

Tissue engineering is founded on the concept of controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. These interdependent signals include cytokine delivery, extracellular matrix interactions, and cell-cell communication. Here, we report on the effect of spatial guidance as a signal for nerve tissue regeneration, using a simple in vitro model. We observe the acceleration of neurite extension from rat dorsal root ganglia within micron-scale tubes. Within these hydrogel-filled conduits, neurites were observed to extend more rapidly than when cultured within the hydrogel alone. The spatial cue also induced a change in tissue architecture, with the cabling of cells within the microconduit. The acceleration of neurite extension was found to be independent of conduit diameter within the range of 200 to 635 microm. Finally, our in vitro model enabled quantification of the effect of combining spatial control and localized nerve growth factor delivery.     

Redirection of Neurite Outgrowth by Coupling Chondroitin Sulfate Proteoglycans to Polymer Membranes

Upon nerve injury, the body creates an environment consisting of permissive and non-permissive cues that instruct the function of cells involved in nerve repair. Among other roles, the developing extracellular matrix (ECM) acts as an underlying substrate to guide the union of neurites extending from the proximal stump for bridging the nerve gap. Chondroitin sulfate proteoglycans (CSPGs) are present in the nerve ECM and inhibit axon growth, potentially providing molecular cues to prevent aberrant growth and direct regeneration. In this study, we examined the potential of CSPGs to guide dorsal root ganglia (DRG) neurite outgrowth when freely available in the media or presented from a polymeric membrane. Soluble CSPGs added to the media of DRG explant cultures inhibited neurite outgrowth without spatial bias, caused retraction of axons, and decreased neurite extension in a dose-dependent manner. Poly-l-lactic acid membranes were chemically treated to enhance adsorption of CSPGs to the surface. CSPGs bound to 1,6-hexanediamine-treated membranes directed the orientation of neurite outgrowth, as neurites avoided bound CSPGs and a higher number and percentage grew on treated membranes lacking CSPGs. DRG explants cultured on CSPG-coated membranes without 1,6-hexanediamine-treatment had a smaller number of neurites and decreased neurite outgrowth, suggesting CSPGs were not retained on the membrane and were released into the culture medium. Taken together, these data demonstrate the potential of CSPG presentation to guide axonal growth. This approach offers a strategy to improve upon existing nerve guidance conduits by incorporating axon guidance molecules to direct nerve regeneration.     

Neurite outgrowth in fibrin gels is regulated by substrate stiffness

Fibrin is a promising matrix for use in promoting nerve repair given its natural occurrence in peripheral nerve injuries, and the biophysical properties of this matrix can be regulated to modulate tissue regeneration. In this study, we examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia (DRG) neurite extension. Increases in fibrinogen concentration increased the number of fibrin strands, resulting in decreased pore size and increased stiffness. Neurite extension was reduced when DRG explants were cultured within fibrin gels of increasing fibrinogen concentrations (from 9.5 to 141 mg/mL). The addition of NaCl also increased the number of fibrin strands, reducing fiber diameter and porosity, while increasing mechanical strength, and reductions in neurite extension correlated with increases in NaCl content. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and matrix metalloproteinases by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. Taken together, the results of this study provide new insight into the effect of fibrin gel biophysical properties on neurite extension and suggest new opportunities to improve the efficacy of these materials when used as nerve guidance conduits.     

Experience with examination of the spinal cord and peripheral nervous system (PNS) in mice: A brief overview

The representative areas for examination of the mouse peripheral nervous system are the spinal cord, containing central components of the peripheral nervous system that needs to be examined at least at cervical and lumbar level, the sciatic and the tibial nerve. Skeletal muscle samples should include the soleus muscle and the quadriceps femoris or long digital extensor, as well as the medial gastrocnemius. Examination can be extended to the thoracic spinal cord, lumbar dorsal root ganglia and spinal nerve roots, as well as the plantar nerve, and other areas of interest. Perfusion fixation is considered optimal for the nervous system; however, immersion fixation allows producing microscopic sections of excellent quality as well. Paraffin-embedded, hematoxylin and eosin-stained sections can be made from all areas, save for small nerves such as the tibial or plantar nerve, which are examined with advantage in hard plastic sections. It is possible to produce hard plastic sections also of the vertebral column, including the spinal cord, dorsal root ganglia and nerve roots. For special investigations, mice can be fixed in toto, decalcified, embedded and sectioned to reveal the areas of interest. In the mouse peripheral nerves, myelination progresses until the adult age. In aging peripheral nerves there is axonal atrophy, degeneration, nerve fiber loss, increase of collagen and sporadic demyelination, especially radiculoneuropathy. The dorsal root ganglia of untreated control animals show frequent cytoplasmic vacuolation. Axonal degeneration is distally, primary demyelination proximally accentuated. Mouse is not very sensitive to peripheral neurotoxicity: to induce toxic peripheral neuropathy mostly parenteral administration and/or newborn animals are used. Naturally occurring infection affecting the spinal cord and peripheral nerves is Theiler's encephalomyelitis virus inducing acute poliomyelitis or chronic demyelination. Any experimental results are to be assessed taking into account spontaneous, age-related, background changes.     

Bioactive hydrogel-filament scaffolds for nerve repair and regeneration

The design of novel biomaterials is crucial for the advancement of tissue engineering in nerve regeneration. In this study we developed and evaluated novel biosynthetic scaffolds comprising collagen crosslinked with a terpolymer of poly(N-isopropylacrylamide) (PNiPAAm) as conduits for nerve growth. These collagen-terpolymer (collagen-TERP) scaffolds grafted with the laminin pentapeptide YIGSR were previously used as corneal substitutes in pigs and demonstrated enhanced nerve regeneration compared to allografts. The purpose of this project was to enhance neuronal growth on the collagen-TERP scaffolds through the incorporation of supporting fibers. Neuronal growth on these matrices was assessed in vitro using isolated dorsal root ganglia as a nerve source. Statistical significance was assessed using a one-way ANOVA. The incorporation of fibers into the collagen-TERP scaffolds produced a significant increase in neurite extension (p<0.05). The growth habit of the nerves varied with the type of fiber and included directional growth of the neurites along the surface of certain fiber types. Furthermore, the presence of fibers in the collagen-TERP scaffolds appeared to influence neurite morphology and function; neurites grown on fibers-incorporated collagen-TERP scaffolds expressed higher levels of Na channels compared to the scaffolds without fiber. Overall, our results suggest that incorporation of supporting fibers enhanced neurite outgrowth and that surface properties of the scaffold play an important role in promoting and guiding nerve regeneration. More importantly, this study demonstrates the potential value of tissue engineered collagen-TERP hybrid scaffolds as conduits in peripheral nerve repair.     

Salicylic acid-derived poly(anhydride-ester) electrospun fibers designed for regenerating the peripheral nervous system

Continuous biomaterial advances and the regenerating potential of the adult human peripheral nervous system offer great promise for restoring full function to innervated tissue following traumatic injury via synthetic nerve guidance conduits (NGCs). To most effectively facilitate nerve regeneration, a tissue engineering scaffold within a conduit must be similar to the linear microenvironment of the healthy nerve. To mimic the native nerve structure, aligned poly(lactic-co-glycolic acid)/bioactive polyanhydride fibrous substrates were fabricated through optimized electrospinning parameters with diameters of 600 ± 200 nm. Scanning electron microscopy images show fibers with a high degree of alignment. Schwann cells and dissociated rat dorsal root ganglia demonstrated elongated and healthy proliferation in a direction parallel to orientated electrospun fibers with significantly longer Schwann cell process length and neurite outgrowth when compared to randomly orientated fibers. Results suggest that an aligned polyanhydride fiber mat holds tremendous promise as a supplement scaffold for the interior of a degradable polymer NGC. Bioactive salicylic acid-based polyanhydride fibers are not limited to nerve regeneration and offer exciting promise for a wide variety of biomedical applications.     

Responses of macrophages in rat dorsal root ganglia following peripheral nerve injury

Summary Immunohistochemical studies with monoclonal antibodies to macrophage antigens were performed on sections of rat lumbar dorsal root ganglia. In confirmation of previous observations, cells with macrophage antigenicity were detected in normal ganglia. Many of these presumptive macrophages were perineuronal in contact with the neuron/satellite cell complex, a few were perivascular, and others were in interstitial position not in apparent contact with either blood vessels or neurons. The number of macrophages in lumbar dorsal root ganglia started to increase 2–4 days after sciatic nerve transection and remained elevated for four weeks. Perineuronal macrophages resembled satellite glial cells in light microscope appearance but were distinguished from glial cells by their lack of S-100 immunoreactivity. Following this sciatic nerve injury, macrophage counts were modestly increased in contralateral lumbar dorsal root ganglia but not in cervical dorsal root ganglia. Thus peripheral nerve injury induces a recruitment and/or proliferation of macrophages in the corresponding dorsal root ganglion. Although the functions of these macrophages are unclear, those in perineuronal position could contribute to the survival or regeneration of axotomized neurons.     

狗肾脏感觉神经来源

目的探讨狗肾脏感觉神经来源。方法应用辣根过氧化物酶(HRP)逆行追踪技术,将CB-HRP注入一侧狗肾脏内,动物存活2~3 d,TMB法成色反应,光镜下观察。结果狗肾脏感觉神经来源于双侧背根节T5~L3节段,主要集中在T10~L2节段,占背根节内标记细胞总数90.56%,符合既集中(T10~L2)又分散(T5~L3)的分布特点,无左右侧差异;背根节内标记细胞以卵圆形、圆形的细胞为主,少见细胞突起,细胞分布弥散,多分布于节的边缘部位。在4例注射侧迷走神经结状节内也见有少量标记细胞,体积大,突起不明显。结论狗肾脏感觉神经来源于双侧背根节T5~L3段和同侧迷走神经结状节细胞。     

Cannabinoid receptors undergo axonal flow in sensory nerves.

Cannabinoids modulate nociceptive processing through central and peripheral mechanisms. The present study was conducted to evaluate axonal flow of cannabinoid receptors from the dorsal root ganglion to the periphery and to identify the putative involvement of CB1 and/or CB2 receptor subtypes. The sciatic nerve was tightly ligated to dam the flow of cannabinoid receptors to the periphery. The densities of cannabinoid receptors proximal and distal to one or two tightly constrictive ligatures was evaluated using in vitro receptor binding and high-resolution emulsion autoradiography. In both models, [3H]CP55,940 binding accumulated proximal as opposed to distal to the ligature. These data indicate that axonal transport of cannabinoid receptors to the periphery was occluded by tight constriction of the sciatic nerve. In situ hybridization histochemistry revealed that dorsal root ganglia cells synthesize CB1 but not CB2 receptor messenger RNA. By contrast, CB2 messenger RNA was highly expressed in sections of rat spleen that were processed together with the dorsal root ganglia, as previously described. These data demonstrate that neuronal cannabinoid CB1 receptors are synthesized in cells of the dorsal root ganglia and inserted on terminals in the periphery.     

胶原神经导管对外周神经缺损修复的实验研究

目的探讨组织工程化胶原神经导管的构建及对外周神经缺损的修复。方法分别取10g胶原蛋白利用自制模具分别制备含0.25g川芎嗪和无川芎嗪的胶原神经导管(2%京尼平交联);对交联前后的胶原神经导管进行拉伸实验并评价其力学特性;用扫描电子显微镜观察胶原神经导管交联前后的空间结构;将大鼠骨髓间充质干细胞(MSC)与细胞外基质(ECM)混合后接种于导管内腔,用扫描电子显微镜观察细胞与材料的复合情况;质量浓度10g/L的川芎嗪诱导MSC7d后,荧光免疫化学方法鉴定MSC分化为神经元细胞;用8只Wister大鼠复制坐骨神经10mm缺损模型,复合MSC的神经导管连接缺损神经,其中4只为无中药导管作为对照,90d后处死大鼠观察再生神经形态,免疫组织化学鉴定其功能。结果京尼平交联前后胶原纤维结构发生明显改变,交联后胶原纤维排列致密,胶原纤维之间形成不规则孔隙且韧性增强;力学实验结果表明:交联前后的胶原神经导管的最大载荷和断裂载荷分别为(0.23±0.09)、(0.20±0.12)N和(0.76±0.15)、(0.69±0.17)N,两者差异具有显著统计学意义(P﹤0.01);川芎嗪能促进MSC表达神经元特异性烯醇酶并向神经细胞分化;神经导管与MSC复合培养两者具有良好的组织相容性;复合川芎嗪的胶原神经导管能促进缺损神经的修复。结论构建的组织工程化胶原神经导管能有效修复外周神经缺损。     

Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth

Nerve injury, a significant cause of disability, may be treated more effectively using nerve guidance channels containing longitudinally aligned fibers. Aligned, electrospun nanofibers direct the neurite growth of immortalized neural stem cells, demonstrating potential for directing regenerating neurites. However, no study of neurite guidance on these fibers has yet been performed with primary neurons. Here, we examined neurites from dorsal root ganglia explants on electrospun poly-L-lactate nanofibers of high, intermediate, and random alignment. On aligned fibers, neurites grew radially outward from the ganglia and turned to follow the fibers upon contact. Neurite guidance was robust, with neurites never leaving the fibers to grow on the surrounding cover slip. To compare the alignment of neurites to that of the nanofiber substrates, Fourier methods were used to quantify the alignment. Neurite alignment, however striking, was inferior to fiber alignment on all but the randomly aligned fibers. Neurites on highly aligned substrates were 20 and 16% longer than neurites on random and intermediate fibers, respectively. Schwann cells on fibers assumed a very narrow morphology compared to those on the surrounding coverslip. The robust neurite guidance demonstrated here is a significant step toward the use of aligned, electrospun nanofibers for nerve regeneration. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.     

In vitro and in vivo modulation of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and calcitonin-gene related peptide-like immunoreactivities in adult rat sensory neurons.

In a previous work we have shown that culturing adult rat dorsal root ganglia neurons modifies their neurotransmitter phenotype in such a way that cultured neurons synthesize transmitters that are not found in situ, while several other transmitters are expressed in a much higher percentage of neurons in culture than in situ [Schoenen J. et al. (1989) J. Neurosci. Res. 22, 473-487]. The aim of the present study was to investigate the origin and the nature of the relevant environmental signals that allow this plasticity to be expressed, focusing on three neurotransmitters: 5-hydroxytryptamine, thyrotropin-releasing hormone and calcitonin-gene related peptide. The main results can be summarized as follows: (1) culturing cells in fetal calf serum or on feeder layers of astrocytes, Schwann cells or fibroblasts partially inhibits the serotoninergic phenotype of dorsal root ganglia neurons; (2) in vivo disconnection of dorsal root ganglia from their spinal targets but not from their peripheral or supraspinal targets induces a significant increase of the percentage of 5-hydroxytryptamine- and thyrotropin-releasing hormone-positive neurons in disconnected ganglia; (3) growth factors such as ciliary neuronotrophic factor or basic fibroblast growth factor but not nerve growth factor repress 5-hydroxytryptamine and calcitonin gene-related peptide immunoreactivity in cultured sensory neurons. In conclusion, neurotransmitter gene expression of adult dorsal root ganglia neurons is controlled by complex influences. Our data suggest that thyrotropin-releasing hormone and 5-hydroxytryptamine gene expression are tonically repressed in vivo by factors originating from the spinal segmental level and that growth factors such as ciliary neurotrophic factor or basic fibroblast growth factor could be potential vectors of this repressing effect.     

Directed axonal growth towards axolotl limb blastemas in vitro

Limb amputation in urodele amphibia is followed by formation of a blastema, which subsequently develops into a complete limb with normal pattern of innervation. In this study, we investigated the effects of axolotl limb blastemas on axonal growth in gels of collagen and extracellular matrix (matrigel). When peripheral nerves with attached dorsal root ganglia were cultured in collagen gels together with blastemas, axonal outgrowth was markedly increased compared with control preparations. Blastemas contain fibroblast growth factors, and may also contain neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4, glial cell line-derived neurotrophic factor and hepatocyte growth factor/scatter factor, since these factors are expressed in developing limbs in other vertebrates. In collagen gels the neurotrophins and glial cell line-derived neurotrophic factor stimulated axonal growth, but outgrowing axons were shorter than in co-cultures with blastemas. The tyrosine kinase inhibitor K252a blocked the stimulatory effects of the neurotrophins on axonal growth but had relatively little effect on axonal growth in co-cultures with blastemas. In experiments in which peripheral nerves, with attached dorsal root ganglia, were cultured in matrigel, axons grew towards blastemas over distances of about 1mm. Directed axonal growth even occurred in these co-cultures after addition of high concentrations of all the above neurotrophic factors, suggesting that blastemas may release a different factor which stimulates axonal growth.The results indicate that during early stages of limb regeneration in amphibia, factor(s) are released which are capable of attracting the growth of peripheral nerves and may play an important role in the development of innervation of regenerated limbs. The identity of the factor(s) remains to be determined.     

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.     

Micropatterns of positive guidance cues anchored to polypyrrole doped with polyglutamic acid: a new platform for characterizing neurite extension in complex environments

This paper describes a method for preparing substrates with micropatterns of positive guidance cues for the purpose of stimulating the growth of neurons. This method uses an oxidizing potential, applied to a micropattern of indium tin oxide in the presence of pyrrole and polyglutamic acid, to electrodeposit a matrix consisting of polypyrrole doped with polyglutamic acid. The resulting matrix subsequently can be modified with positive guidance cues via standard amide coupling reactions. Cells adhered to the micropatterned substrates can be stimulated electrically by the underlying electrodeposited matrix while they are in contact with positive guidance cues. This method can be extended to include both positive and negative guidance cues in a variety of combinations. To demonstrate the suitability of this method in the context of nerve guidance, dorsal root ganglia were grown in the presence of a micropatterned substrate whose surface was modified with molecules such as polylysine, laminin, or both. Cell adhesion and neurite extension were found to occur almost exclusively in areas where positive guidance cues were attached. This method is easy to execute and is of general utility for fundamental studies on the behavior of neurons in the presence of complex combinations of guidance cues as well as advanced bioelectronic devices such as neuronal networks.     

Hierarchically structured nerve guidance channels based on poly-3-hydroxybutyrate enhance oriented axonal outgrowth

Traumatic peripheral nerve lesions can cause local anesthesia, paralysis and loss of autonomic control. Reconstruction using engineered nerve guidance conduits (NGCs) is rarely successful due to the sub-optimal characteristics of the conduits. To address the demands of clinical practice, we developed a hierarchically structured NGC from slowly resorbing poly(3-hydroxybutyric acid) (P3HB). The NGC consists of a permeable single-lumen tube and melt-spun fibrillar lumen fillers. Permeable tubes were constructed from P3HB/poly(ɛ-caprolactone) (PCL) blends or poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) (P(3HB-co-4HB)). Polyvinylpyrrolidone was used as a porogen in solvent-free thermoplastic processing, followed by selective polymer leaching. All tested material compositions showed hydrolytic degradation after 16 weeks in phosphate buffered saline, whereas P3HB/PCL tubes maintained mechanical strength compared to (P(3HB-co-4HB)). The porous scaffolds allowed diffusion of large molecules (∼70 kDa). In vitro studies demonstrated that mouse fibroblasts survived and proliferated inside closed porous tubes. An in vitro model of axonal regeneration using dorsal root ganglia and sympathetic cervical ganglia demonstrated that the NGCs successfully supported neuron survival and neurite outgrowth. The introduction of fibrillar lumen fillers promoted oriented neurite growth and coating with extracellular matrix proteins further increased ganglia attachment and cell migration. In this study we show that P3HB-based NGCs scaffolds have potential in long gap peripheral nerve repair strategies.     

Rescue and regeneration of injured peripheral nerve axons by intrathecal insulin

Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both alpha and beta insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor alpha subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.