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