The Regulatory Effects Of Neurotrophic Factors And Skeletal Muscle Cells On DRG Neuronal Outgrowth

Skeletal muscle (SKM) is controlled by motor and sensory neurons. Muscle spindle is a sensor for a feeling of proprioception. Dorsal root ganglion (DRG) is consisted of primary sensory neuron cell body sending central and peripheral process and established sensory contact between nerve endings and spinal cord. In vitro co-culture of mammalian neurons and muscle cells is a popular model system to study formation, function, and maintenance of neuromuscular junction (NMJ), as well as nerve-muscle disorders, signal computation at the nerve-muscle interface and other applications ranging from drug screening to biorobotics and tissue engineering. Previous studies suggest that sensory neurons may simply follow motoneurons to their target tissues. Sensory axons grow out together with, but slightly later than, motor axons. But some findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.Neurotrophins (NTs) are the most profound known regulators of survival in the developing nervous system. Nerve growth factor (NGF), brain-derived ncurotrophic factor (BDNF), and neurotrophin3(NT-3) are three members of the neurotrophic factor family. Exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration. Exchange of NTs and other molecules is likely to be an important source of nerve-muscle communication. Release and reception of various factors both neurons and target tissues must be considered as means of mutual influences. Both NTs and target SKM cells are essential for the maintenance of neuronal function and nerve-muscle communication.Once neurons recognize their appropriate targets, specific neuron-target contacts involving modulation of neurite growth dynamics and formation of functional synaptic connections are established. In the present study, neuromuscular cocultures of DRG neurons and SKM cells were established. Using these culture systems, the neuromuscular connection between DRG neurons and target SKM was observed and the regulatory effects of NTs and target SKM on DRG neuronal growth was determined.Part Ⅰ The effects of target skeletal muscle cells on neuronal outgrowth and migration from organotypically cultured DRG explantsThe interdependent relation exists between neurons and target tissue. During development, neurons extend axons to their targets, and become dependent for their survival on trophic substances secreted by their target cells. Of course, the neurons themselves have the survival, development, and metabolism of its own, but the effect of the target tissue on neuronal survival is beyond doubt. In the embryonic stage of development, because of loss of nutrition of the limb bud, sensory neurons will be dominant disorder, even lose the ability to survive and to programmed cell death (PCD). Extracellular application of myosin Ⅱ or skeletal muscle extracts promoted axon formation and branching or neuronal survival. Peripheral nerve recovery after crush injury was suppressed by chronic inflammation in peripheral target tissue. Growth factors are derived from target tissues and cells promoted neuronal survival and neurite outgrowth.Growth associated protein43(GAP-43), as a kind of neuron specific calcium binding protein and actin binding protein, is highly expressed in the presynaptic membrane. GAP-43is often used as markers of development, nerve regeneration and synaptic growth after nerve injury. The expression of sensory neuropeptides in DRG neurons represents the function of DRG neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide derived from the alternative splicing of the calcitonin gene. It is expressed in DRG neurons, and thus possesses diverse biological effects, including the modulation of many sensations and SKM motility, vasodilation, and inflammatory responses. Neurofilament heavy chain (NF-H), such as neurofilament-200(NF-200), plays an important role in healthy neurons. With the maturation of neurons, the appearance of NF-H represents a critical event about axonal stabilization. The neuropeptide-immunoreactive (NP-IR) and neurofilament-immunoreactive (NF-IR) neurons are two major phenotypical classes in DRG neurons. NP-IR neurons, such as CGRP-IR neurons, are considered to be with unmyelinated or thinly myelinated nociceptive afferent fiber distributed skin and viscera. NF-IR neurons, such as NF-200-IR neurons, typically send myelinated axons distributed muscle spindle.It remains unknown whether target SKM influences growth of DRG neurons and neuronal phenotype. In the present study, using neuromuscular cocultures of organotypic DRG and SKM cells, the expression of GAP-43, CGRP and NF-200, neurites outgrowth, neuronal migration of DRG explants were observed.Under aseptic conditions and using the newborn rat (within24h after birth). SKM was removed from the hind limb of each animal. After digestion, centrifugation, filtration, dissociated SKM cells were plated in24-well clusters which precoated with poly-L-lysine. Upon the fusion of SKM cells at3days post-culture, embryonic rats at embryonic day15(E15) were used for organotypic DRG culture. Under aseptic conditions, the bilateral dorsal root ganglia (DRGs) were removed from each embryo. The membranous tissue of DRGs was removed. Each DRG explant was plated at the bottom of each well of24-well clusters which precoated with poly-L-lysine or which would contain a single layer of SKM cells prepared as above. According to the experimental design, the cultures were divided into two groups.(1) Coculture group: Organotypic DRG explant and dissociate SKM cells were cocultured for6days.(2) DRG culture:Organotypic DRG explants were cultured for6days. At6days of culture age, DRG explant culture and neuromuscular coculture of DRG explant and SKM cells were processed for scanning electron microscope (SEM) observation, immunofluorescent labeling, Western blot assay and real time-PCR analysis. The results are as follows.(1) At6days of culture age, nerve fiber bundles extended from DRG explants from the edge of a quarter of each DRG explants were counted. The number of nerve fiber bundles extended from DRG explant in the neuromuscular coculture is more than that in DRG explant culture alone.(2) The nerve fiber bundles extended from DRG explant in the neuromuscular coculture are much more stronger and extend far more distance from the DRG explant as compared with that in DRG explant culture alone observed under SEM. And also, much more migrating neurons from DRG explant were observed under SEM in the neuromuscular coculture as compared with those in DRG explant culture alone. The migrating distance (some up to0.5mm) of the neurons in the neuromuscular coculture is larger than that in DRG explant culture alone. Neurites of DRG explants cross over multiple myotubes, or eventually enlarge and terminate on the surfaces of myotubes. Single neuron migrated from DRG explants scattered between myotubes and sends projections joining or terminating on the myotube surface.(3) Quantitative analysis of the migrating neurons showed that the number of neurons migrated from DRG explant in the neuromuscular coculture is higher than that in DRG explant culture alone after immunofluorescent with microtubule-associated protein2(MAP-2).(4) The percentage of migrating GAP-43-IR and NF-200-IR, but not CGRP-IR, neurons is higher in the neuromuscular coculture than that in DRG explant culture alone.(5) The protein and mRNA levels of GAP-43and NF-200in DRG are higher in the neuromuscular coculture than that in DRG explant culture alone.These results show that, nerve-muscle connection is formed between fiber bundles of DRG explants or neurites of migrating neurons from DRG explants and myotubes in neuromuscular cocultures of organotypic DRG and dissociated SKM cells. In neuromuscular cocultures, target SKM cells not only promote the growth and migration of DRG neurons, but also increase the expression of NF-IR, but not NP-IR, neuronal phenotype. The results imply that the anatomical nerve-muscle connection between DRG neurons and SKM cells has been established. Target SKM cells play a specific role in regulating distinct neuronal phenotype. These data provide new clues for a better understanding of the functional association of target SKM with DRG neurons in present study.Part Ⅱ The effects of different neurotrophins on growth-associated protein43expression in cocultures of dissociated DRG neurons and skeletal muscle cellsTargets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin (NT)-responsive neurons. Sensory nerve cross-anastomosis (sensory protection) provides a modified trophic environment by modulating neurotrophic factor synthesis in muscle. Target tissues contribute to the phenotype and function of sensory neurons. In the process of axonal regeneration, GAP-43is selectively distributed to the axonal domain in developing neurons. GAP-43expression increased in the injured nerve and in the corresponding DRG after peripheral nerve injury. The expression of GAP-43in cultured DRG neurons was affected by different NTs, such as NGF and BDNF. GAP-43plays an important role in growth cone formation and neurite outgrowth of cultured DRG neurons. DRG neurons contacting with target tissue, compared with no target tissue, plastic changes in their growth process may be different. It remains unknown whether NTs influence GAP-43expression of DRG neurons contacting with target tissue. In the present study, using neuromuscular cocultures of dissociate DRG neurons and SKM cells, the levels of GAP-43expression were determined after administration of NGF, BDNF, and NT-3.SKM cell culture preparations were utilized newborn Wistar rats. SKM was removed from the hind limb of each animal. After digestion, centrifugation and filtration, dissociated SKM cells were plated at a density of2×105cells/ml in24-well clusters which precoated with poly-L-lysine and then incubated at37℃in a5%CO2incubator. After3days, DRG cell cultures were prepared by using embryonic rats at E15, DRG were removed bilaterally using a sharp pair of forceps from each embryo. Dissociated DRG cells were plated at a density of2×105cells/ml in clusters which would contain a single layer of SKM cells prepared as above. All the cocultures were divided into4groups:(1) NGF group:The neuromuscular cocultures were treated with NGF (10ng/ml) during the6days of coculture.(2) BDNF group:The neuromuscular cocultures were treated with BDNF (10ng/ml) during the6days of coculture.(3) NT-3group:The neuromuscular cocultures were treated with NT-3(10ng/ml) during the6days of coculture.(4) Control group:The neuromuscular cocultures were continuously exposed to growth media as control. At6days of culture age, All cultures were processed for SEM observation, immunofluorescent labeling, Western blot assay and real time-PCR assay. The following results were obtained:(1) Compared with control, DRG neurons in neuromuscular coculture treated with NTs sent more axons, ranging from1to5, which from fibrous network on the surface of dissociated SKM cells. Some axons would widen into a varicosity, some would become smaller in caliber, and many appeared no different from the immediate proximal configuration. The endings enlarged and terminated on the surface of SKM cells.(2) By analysis of immunofluorescent labeling, Western blot assay and real time-PCR analysis, the percentage of GAP-43-IR neurons, GAP-43protein and mRNA levels increased in NGF treated cocultures, BDNF treated cocultures, and NT-3treated cocultures as compared with that in cocultures absence of NT.These results revealed that in neuromuscular cocultures of dissociate DRG neurons and SKM cells, exogenous NGF, BDNF, and NT-3promoted neurite growth and GAP-43expression. These results indicated that specific NTs may improve regeneration microenvironment of DRG neurons and specific effect of different NTs for different subsets of DRG neuron needed further discussion.Part III The effects of NGF or NT-3on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2in cocultures of dissociated DRG neurons and skeletal muscle cellsSpecific NTs promoted growth of DRG neurons, may also affect various neurotransmitters synthesis and protein expression. Both substance P (SP) and CGRP, as peptide neurotransmitters involving in vascular relaxation and sensory transmission, are expressed in DRG neurons. NF-200which belongs to NF-H, plays an important role in healthy neurons. MAP-2proteins, involving in regulation of neuronal growth and cell polarity, are known to interact with microtubules, NFs and actin, and contribute to maintenance of neuronal cytoarchitecture within the neuronal cell. In order to further detect the influence of specific NTs on these neurotransmitters and protein in DRG neurons, Using neuromuscular cocultures of dissociated DRG neurons and SKM cells systems, the levels of mRNAs for preprotachykinin (PPT) encoding for SP, CGRP, NF-200, and MAP-2was determined after administration of NGF or NT-3.The preparations of coculture are the same as the second part. All the cocultures were divided into3groups.(1) NGF group:The neuromuscular cocultures were treated with NGF (10ng/ml) during the6days of coculture.(2) NT-3group:The neuromuscular cocultures were treated with NT-3(10ng/ml) during the6days of coculture.(3) Control group:The neuromuscular cocultures were continuously exposed to growth media as control. The results revealed that PPT mRNA, CGRP mRNA, NF-200mRNA, but not MAP-2mRNA, levels increased in NGF or NT-3treated cocultures as compared with that in control group. The effects of NGF and NT-3on promoting expression of PPT and CGRP mRNA indicated that specific NTs can promote development and maturation of peptide neurotransmitter in DRG neurons. The effects of NGF and NT-3on promoting expression of NF-200mRNA indicated that both NGF and NT-3were involved in regulation of the later stage of DRG development. No effect of NGF and NT-3on expression of MAP-2mRNA indicated that its expression is sufficiently by the presence of target SKM cells or DRG neurons itself, not depending on the presence of specific NTs. The effects of NGF and NT-3on promoting mRNA expression of various neurotransmitters and protein reflected the reaction of DRG sensory neurons to specific NTs. These results offer new evidence that specific NTs may affect the plasticity of DRG sensory neurons in different conditions or status in further research.