Neurons - Docenti.unina.it
Transcription
Neurons - Docenti.unina.it
Rigenerazione del tessuto nervoso Ingegneria dei Tessuti 13 – Nov 2012 The Nervous system has three major functions: Sensory – monitors internal & external environment through presence of receptors Integration – interpretation of sensory information (information processing); complex (higher order) functions Motor – response to information processed through stimulation of effectors muscle contraction glandular secretion General Organization of the nervous system • Two Anatomical Divisions Central nervous system (CNS) – • • Brain Spinal cord Peripheral nervous system (PNS) – • • • All the neural tissue outside CNS Afferent division (sensory input) Efferent division (motor output) – – Somatic nervous system Autonomic nervous system General Organization of the nervous system Brain & spinal cord Histology of neural tissue Two types of neural cells in the nervous system: Neurons - For processing, transfer, and storage of information Neuroglia – For support, regulation & protection of neurons Neuroglia (glial cells) CNS neuroglia: • astrocytes • oligodendrocytes • microglia • ependymal cells PNS neuroglia: • Schwann cells (neurolemmocytes) • satellite cells Astrocytes • create supportive framework for neurons • create “blood-brain barrier” • monitor & regulate interstitial fluid surrounding neurons • secrete chemicals for embryological neuron formation • stimulate the formation of scar tissue secondary to CNS injury Oligodendrocytes • create myelin sheath around axons of neurons in the CNS. Myelinated axons transmit impulses faster than unmyelinated axons Microglia • “brain macrophages” • phagocytize cellular wastes & pathogens Ependymal cells • line ventricles of brain & central canal of spinal cord • produce, monitor & help circulate CSF (cerebrospinal fluid) Schwann cells • surround all axons of neurons in the PNS creating a neurilemma around them. Neurilemma allows for potential regeneration of damaged axons • creates myelin sheath around most axons of PNS Satellite cells • support groups of cell bodies of neurons within ganglia of the PNS Neuron structure of Ranvier •Most axons of the nervous system are surrounded by a myelin sheath (myelinated axons) •The presence of myelin speeds up the transmission of action potentials along the axon •Myelin will get laid down in segments (internodes) along the axon, leaving unmyelinated gaps known as “nodes of Ranvier” •Regions of the nervous system containing groupings of myelinated axons make up the “white matter” •“gray matter” is mainly comprised of groups of neuron cell bodies, dendrites & synapses (connections between neurons) Classification of neurons Structural classification based on number of processes coming off of the cell body: Anaxonic neurons • no anatomical clues to determine axons from dendrites • functions unknown Multipolar neuron • multiple dendrites & single axon • most common type Bipolar neuron • two processes coming off cell body – one dendrite & one axon • only found in eye, ear & nose Unipolar (pseudounipolar) neuron • single process coming off cell body, giving rise to dendrites (at one end) & axon (making up rest of process) Classification of neurons Functional classification based on type of information & direction of information transmission: • Sensory (afferent) neurons – • transmit sensory information from receptors of PNS towards the CNS • most sensory neurons are unipolar, a few are bipolar • Motor (efferent) neurons – • transmit motor information from the CNS to effectors (muscles/glands/adipose tissue) in the periphery of the body • all are multipolar • Association (interneurons) – • transmit information between neurons within the CNS; analyze inputs, coordinate outputs • are the most common type of neuron (20 billion) • are all multipolar Conduction across synapses In order for neural control to occur, “information” must not only be conducted along nerve cells, but must also be transferred from one nerve cell to another across a synapse Most synapses within the nervous system are chemical synapses, & involve the release of a neurotransmitter The Structure of a Typical Synapse Neuronal Pools Anatomical organization of neurons Neurons of the nervous system tend to group together into organized bundles The axons of neurons are bundled together to form nerves in the PNS & tracts/pathways in the CNS. Most axons are myelinated so these structures will be part of “white matter” The cell bodies of neurons are clustered together into ganglia in the PNS & nuclei/centers in the CNS. These are unmyelinated structures and will be part of “gray matter” Neural Tissue Organization Origins of peripheral nervous system axons. The junction between the spinal cord and the PNS, with schematically drawn cell bodies of sensory neurons (green) and motor neurons (red), and their corresponding neurites. For the length of the peripheral nerve (PN), only axons are present in the tissue. The motor neurons are typically in the brain; however, other intermediate neurons located within the grey matter can play a role in transferring the electrical impulse. The righthand side text and arrows indicate the direction of the signal for a motor command – a sensory signal is not shown. Repairing Peripheral Nerve Injuries - Myelin associated glycoproteins are released after injury. Macrophages must remove these proteins in order to allow axon regrowth - Regrowth starts at the proximal end and is directed towards distal end. Here Schwann cell release secrete factors stimulating axon growth Repairing Peripheral Nerve Injuries End-to-end suturing of peripheral nerves. Alignment of the fascicles is critical in successful regeneration and microsurgical techniques have been developed to optimize this surgery Repairing Peripheral Nerve Injuries Autologous Nerve Grafts Repairing Peripheral Nerve Injuries Nerve Guides Sequence of events in empty silicone nerve guides. a. fluid and cytokines fill up the nerve guide b. formation of a fibrin matrix inside the lumen which initially supports invading fibroblasts (blue) c. infiltration of Schwann cells (green) and axons (red), which eventually penetrate to the distal stump. d. regenerated nerve (yellow) Repairing Peripheral Nerve Injuries Critical gap length Nerve regeneration depends on the extent of the lesion. The chance of successful reinnervation with nerve guides is dramatically reduced once an injury gap reaches a certain value. This length, termed the critical gap length, LC, is where successful regeneration occurs 50% of the time. Repairing Peripheral Nerve Injuries – Scoring the devices Examples of the critical gap length gap, Lc, for the rat sciatic nerve. a. empty silicone (PDMS) nerve guides have a Lc of 9.7 mm b. PDMS tube filled with collagen–GAG matrix increases the Lc to at least 14.8 mm with a L of 5.1 mm. c. PDMS tube filled oriented fibrin matrices results in a L of at least 4.7 mm when compared to the same, nonoriented matrices. d. degradable collagen nerve guides have L of at least 5.4 mm compared to silicone tubes. e. Tube with polyamide or collagen fibers the L is at least 7.4 mm compared to empty nerve guides f. Tube containing Schwann cells results in a similar L value of 7.4 mm, compared to nerve guides filled with phosphate-buffered saline. Repairing Peripheral Nerve Injuries SEM micrographs of three types of nerve guides approved for clinical use: (A) NeuraGenTM made from collagen, (B) Neurolac made from polylactide/caprolactone, (C) Neurotube made from polyglycolide Repairing Peripheral Nerve Injuries – Aligned Scaffolds Directing axon ingrowth through contact guidance a. magnetically aligned fibres of fibringel b. close-up of fibres c. aligned pores d. porous morphology of uniaxial freezing of PEG hydrogel (scale 100 micron) Repairing Peripheral Nerve Injuries Effect of fibre diameter on directed neuron growth Normalized distributions of neurite outgrowth for all filament sizes and model fit to data set. Confocal images of DRG neurons with fluorescently labelled neurofilament on 35-μmdiameter (a) and 500μmdiameter (b) filaments. The angle distributions of length segments collected for all filament sizes are shown as the blue histograms (c). The fit of the Boltzmann model is shown as the black curve overlying the distributions Repairing Peripheral Nerve Injuries Conductive polymers Repairing Peripheral Nerve Injuries Tube-like porosity (similar to solvent casting particulate leaching) PCL template in pHEMA matrix Dissolution of the fibres with acetone Repairing Peripheral Nerve Injuries Tube-like porosity (injection molding – solvent casting) Laminin pore wall coating and Schwann cells seding Natural graft PLGA+acetic acid Repairing Peripheral Nerve Injuries – Factor delivery approaches for delivering growth factors into peripheral nerve guide Growth factors can be incorporated into the nerve guide (a) or introduced closer to the lumen with a rod (b) or as growth factor containing microspheres (c). The growth factors may be incorporated into the matrix as non bound (d) or as a Non covalent controlled release system (e). Development and Growth • Axon Stretch Growth – Mechanical tension in nerves is natural during development in most species Background • Nerve Guidance Conduits for Treating Nerve Injury – Hollow tubes used in support of nerve regeneration 2D Axon Stretch-Growth • Building a Nerve Tissue Transplant: “Axon Stretch-Growth” 2-D Stretch-Growth 2D Axon Stretch-Growth 2D Axon Stretch-Growth 2D Axon Stretch-Growth In vivo transplant: throughout the transplanted region, there was an intertwining plexus of host and graft axons, suggesting that the transplanted axons mediated host axonal regeneration across the lesion 3D Axon Stretch-Growth • Building a Nerve Tissue Transplant: “Axon Stretch-Growth” 3-D Stretch-Growth 3D Axon Stretch-Growth • Design Considerations – Sustaining stretch-grown axons in a gel 3D Axon Stretch-Growth • Axons crossing through mesh! 3D Axon Stretch-Growth • Axons also grow along the fibers of the nylon mesh!!! Axon Outgrowth on 2D and 3D Collagen Substrates • Evaluation of Candidate Materials for Nerve Tissue Construct – Rat Tail Collagen Type I – Bovine Tendon Collagen Type I – Study Axon Growth Behavior • 2D coating on dishes (Collagen) • 3D Hydrogels (Collagen) Axon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen Neurite Outgrowth on 2D Rat Tail and Bovine Tendon Type I Collagen 2500 2000 2D Rat Tail Collagen 1500 307.37µm/day 2D Bovine Tendon Collagen 1000 228.03µm/day 500 0 0 2 4 6 Days 8 10 Axon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen • Morphology Differences a) DRG on Bovine Tendon Collagen Type I b) DRG on Rat Tail Collagen Type I 2D vs. 3D Axonal Outgrowth DRG Neurite Outgrowth on Collagen Substrates 3500 3000 2500 Neurite Outgrowth in 2D 246.32um/day Neurite Outgrowth in 3D 153.63um/day 2000 1500 1000 500 0 0 2 4 6 Days 8 10 Central Nervous System Injuries A. Sagittal section of the spinal cord illustrating astrocyte hypertrophy (red) and chondroitin sulphate proteoglycan (CSPG) upregulation (blue, denoted by dashed lines). Note the longitudinal, thickened bands of reactive astrocytes forming an extremely dense wall of cells. B. High magnification of the banded reactive astrocytes, further demonstrating the extreme hypertrophy of astroglia at this late time point after the lesion. C. High magnification of the injury region clearly illustrates the presence of CSPG still remaining, along with the fibrous banding of reactive astrocytes Central Nervous System Injuries – Damage Models Contusion the formation of an astrocytic scar (often termed glial scar) presents a barrier to the axons which lines a fluidfilled cyst. The reactive astrocytes (yellow) lining the cyst are a formidable barrier to regenerating axons. Hemisection Full-transection In hemisections, the space can be filled with a matrix/scaffold (dark blue), either injected to fill the lesion or preformed and implanted In full-transections, the cord is severed and a scaffold is positioned between or the stumps are inserted into a nerve guide (green and sectioned to expose lumen) filled with a therapeutic agent (light blue). In some species, the spinal cord will retract after full transection, creating a gap to fill with cells/matrix/scaffold. Central Nervous System Injuries – Damage Models Contusion Hemisection Difficult to assess axon ingrowth (what neurons are infiltrating?) Full-transection Reliable model. Problems with lesion stability Central Nervous System Repair – axons ingrowth Illustration of the effect of poly(acrylonitrileco -vinylchloride) [P(AN-VC)] nerve guides implanted within hemisections. When Schwann cells are not included within the nerve guides (Matrigel™ only) there is no penetration of axons (red) into the graft. While Schwann cell inclusion with the Matrigel™ demonstrates axonal penetration, the axons can be guided out of the nerve guide via the delivery of BDNF and/or NT-3 into the caudal end of the distal nerve a few millimeters from the implant. Without elevated levels of neurotrophic factor, then axons do not exit the implant. Central Nervous System Repair – matrices with bound peptides Fibrin gel modified with laminin derived peptides Effect of soluble / bound factors 24h 48h (A, B) empty (C, D) filled with unmodified fibrin (E, F) filled with fibrin modified with the four laminin peptides, RGD, IKVAV, RNIAEIIKDI, and YIGSR, at a final density of 1.7 mol peptide/mol fibrinogen each Central Nervous System Repair – matrices with soluble factors Biomimetic approach: delivery of growth factors through Heparin binding sites Neurotrophins are a family of growth factors that play an important role in the survival of neurons and axonal elongation during embryonic development. Brain-derived neurotrophic factor (BDNF) and NT-3, also support survival and regeneration when delivered to the site of a spinal cord injury. Neural fiber staining at the lesion site at 9 days following treatment with (A) unmodified fibrin gels or (B) fibrin gels containing the HBDS and NT-3 (100 ng/ml). A. white line shows the boundaries of the lesion. B. HBDS-treated lesions showed more neural fiber sprouting and greater integration of the fibrin gel with rostral and caudal spinal cord segments. Central Nervous System Repair – self assembling matrices Central Nervous System Repair – self assembling matrices A. IKVAV-containing peptide amphiphile molecule and its self-assembly into nanofibers B. Scanning electron micrograph of an IKVAV nanofiber network formed by adding cell media (DMEM) to a peptide amphiphile aqueous solution. C and D. Micrographs of the gel formed by adding to IKVAV peptide amphiphile solutions (C) cell culture media and (D) cerebral spinal fluid. E. Micrograph of an IKVAV nanofiber gel surgically extracted from an enucleated rat eye after intraocular injection of the peptide amphiphile solution. Central Nervous System Repair – self assembling matrices (A and B) The same field of view in two different planes of focus showing immunocytochemistry of NPCs encapsulated in IKVAVPA gels at 1 day. Differentiated neurons were labeled for - tubulin (in green), and differentiated astrocytes (glial cells) were labeled for GFAP (in orange). (C) Immunocytochemistry of an NPC neurosphere encapsulated in an IKVAV-PA nanofiber network at 7 days. (D) NPCs cultured on laminin-coated cover slips at 1 day. (E) NPCs cultured on laminin-coated cover slips at 7 days. The prevalence of astrocytes is apparent. (F) Percentage of total cells that differentiated into neurons (-tubulin). (G) Percentage of total cells that differentiated into astrocytes (GFAP). (H) Percentage of total cells that differentiated into neurons after 1 day in nanofiber networks containing different amounts of IKVAV-PA and EQS-PA (solid line) and in EQS-PA nanofiber networks to which different amounts of soluble IKVAV peptide were added (dashed line). Central Nervous System Repair – scaffolds with oriented pores Freeze drying technique - PLA in dimethylcarbonate supplemented with BDNF PLA foam contained pores oriented along the cooling direction (a) Horizontal section: presence of small cavities in the spinal cord near the interface with the foam (F) post 2 wk. (b) Same as (a) but showing a larger cavity near the cord– foam interface, post 4 wk. (c) GFAP-positive cells and processes in the spinal cord, but not in the foam (F), post 2 wk. (d) GFAP-positive processes (arrows) in control foam (F) at 4 wks (e) Laminin-positive profiles in BDNF foam (F) at 8 wks (f) Same area as in (e) but demonstrating CSPGimmunoreactive profiles in BDNF foam (F). Central Nervous System Injuries – Damage Models Retina and optic nerve Central Nervous System Injuries – Damage Models Retina and optic nerve