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OLD MACKINNON - 2018 ASSH - Nerve Research

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Andrew Yee

on 21 August 2018

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Transcript of OLD MACKINNON - 2018 ASSH - Nerve Research

+4 MILLION VIDEO PLAYS
75% denervation
to see muscle deficit
Myelinated Axon with Schwann Cell
Schwann Cell "Senescence"
Dispersed Chromatin
HYPOXIA
MACROPHAGE
VASCULARITY
SCHWANN CELL
MIGRATION
INFILTRATION
NERVE
REGENERATION
MYELINATED AXONS
FUNCTIONAL FORCE
p16
p53
SAHFs - Senescence-associated Herterochromatin Foci
SASP - Senescence-associated Secretory Profile
sa-B-galactosidase
Normal
3cm ANA
6cm ANA
MYELINATED AXONS
p16
GFP INTENSITY
Crush Injury
Transection Injury
Isograft
Allograft
FK-506
enhances
nerve regeneration in a large animal model.
Enhancement of Nerve Regeneration
FK-506
enhances
nerve regeneration and
accelerates
functional recovery in rodent models following...
Swine Model
Allograft
Autograft
Untreated
+ FK-506
15%
Proximal
Distal
Length of Growth
Rate of Growth
3 Days - Preload
3 Days - After
Nerve Injury
3 day preload
enhances
FK-506 regenerative effect
Placebo
Immediate FK-506
3 Day Preload
*
*
FK-506 effect
diminishes
after administered
more then 3 days after injury
No FK-506
Delay 0
Delay 3
Delay 5
Low dose

FK-506
+
anti-CD40 ligand (CSB)

enhances
nerve regeneration.
FK-506
+
anti-CD40 ligand (CSB)

abrogates
neuroregenerative effects of FK-506.
Isograft
Isograft +
FK-506
Allograft +
Low Dose FK-506
Allograft +

Low Dose FK-506
+
CD40
Allograft +
FK-506
Allograft +
CD40
Allograft +
CsA
Allograft +
CD40
+
FK-506
Allograft +
CD40
+
CsA
Total Number of Fibers
Immunosuppressive Dose FK-506 - 2.0 mg/kg
Low Dose FK-506 - 0.5 mg/kg
Isograft
Isograft +
FK-506
Allograft +
Low Dose FK-506
Allograft +

Low Dose FK-506
+
CD40
Allograft +
FK-506
Allograft +
CD40
Allograft +
CsA
Allograft +
CD40
+
FK-506
Allograft +
CD40
+
CsA
Total Number of Fibers
Allograft
Untreated
Allograft
Untreated
FK-506 + anti-CD40 ligand (CSB)
abrogates

neuroregenerative effects of FK-506.
Low dose FK-506
+
anti-CD40 ligand (CSB)
enhances
nerve regeneration.
Day 10
Acute Rejection
Nerve Injury
Allograft Repair
Short window to rescue from rejection.
FK-506 could rescue nerve allograft within 10 days.
Isograft
Allograft
Allograft +
CD40
+
FK506
Allograft +
CD40
+
CTLA4
Isograft +
Preload FK-506
Total Number of Fibers
~15%
increase
in regeneration.
Isograft
Allograft
Allograft +
CD40
+
FK506
Allograft +
CD40
+
CTLA4
Isograft +
Preload FK-506
Total Number of Fibers
Abrogation
of regeneration with
FK-506
and
CSB.
Similar findings in VCA model...
- FK-506
enhancing
nerve regeneration
.
-
Abrogation
of regeneration with FK-506 and CSB.
No FK-506 treatment
Continuous FK-506 treatment
FK-506 treatment until specific time point
Crush Injury
Transection Injury
Isograft
Return of Functional Recovery
FK-506
FK506
SUMMARY
FK-506
Immunosuppressive
Functional Recovery
Increases
Regeneration
enhances
abrogates
15%
<
<
<
-
CONDUITS
AUTOGRAFTS
ACELLULARIZED
ALLOGRAFTS
ALLOGRAFTS
NATIVE HOST
ALLOGRAFT DONOR
SCHWANN CELLS
SCHWANN CELLS
IMMUNOSUPPRESSION
CONDUCTION BLOCK
Early Immunosupression Withdrawl
Allograft Retrieval
ABO Blood-type Match
Cold Preservation
(4°C) x 7 Days
Preload FK-506
3 Days Prior to Operation
Azathioprine
FK-506 (Tacrolimus)
HOW MUCH
~
~
ADMINISTRATION
DURATION
DOSAGE / CSB
TIMING OF
CLINICAL REGIMEN
Immunosuppressive dose
enhances nerve regeneration by
~15%

Pre-loading
FK-506 with
continuous
dosage.

FK-506 within short window
(10 days) can rescue allograft
.

Similar findings in VCA model
"GOLD STANDARD"
"BRONZE STANDARD"
Schwann Cell Senescence
"Cellular Growth Arrest in Response to Over-Proliferation or Stress"
PROXIMAL
6cm ANA NERVE GRAFT
DISTAL
Control
Proximal Graft
(Regeneration)
Distal Graft
Distal End
Dispersed Chromatin
Dispersed Chromatin
SCHWANN CELL MORPHOLOGY
SENESCENCE MARKERS
-B-Galactosidase
p16
-B-GALACTOSIDASE AND P16
ISOGRAFT
ANA
I
I
I
I
Fluorescence Markers for Senescence and SCs
Colocalization
-B-GALACTOSIDASE AND P16
sa-B-galactosidase
NEGATIVE EFFECTS OF SENESCENCE AND RESCUE
AUTOGRAFT RESCUE
UNCOVERING THE ROLE OF SENESCENCE SCHWANN CELLS
Schwann Cell
Schwann Cell
Stressed
Aphidicolin (DNA Polymerase Inhibitor)
Stressed Schwann Cell
Schwann Cell
B-galactosidase
Adult DRG Dissociated Neurons
(Thy1-GFP Rats)
EXPERIMENTAL DESIGN
5mm Conduit
Schwann Cell
Schwann Cell
Stressed
Control
Schwann Cell
Stressed
Schwann Cell
Isograft
Acellularized Allograft
2cm
4cm
2cm
4cm
6cm
6cm
14mm
28mm
60mm
40mm
Assessment of Acellularized Nerve Allografts
Acellularized Nerve Allografts in Long Nerve Allograft Model
Cold Preservation
Avance Graft
Long NERVE ALLOGRAFT MODEL
Schwann Cell Senescence
"Cellular Growth Arrest in Response to Over-Proliferation or Stress"
WHEN AXONS ARREST
0 Weeks
4 Weeks
sa
sa
VASCULARIZATION IN LONG NERVE GRAFTS
SUSAN E. MACKINNON, MD
ANDREW YEE
SURGICALEDUCATION
.WUSTL.EDU
NERVESURGERY.WUSTL.EDU
EDUCATION WEBSITE
EDUCATION VIDEOS
CREDIT
WASHINGTON UNIVERSITY
PLASTIC & RECONSTRUCTIVE SURGERY
SCHOOL OF MEDICINE
NERVE
RESEARCH
sa
5 DAYS
10 DAYS
20 DAYS
3 DAYS
MECHANISM OF STRESSED SCHWANN CELLS
DLL4
CS56
SEMA3D
TO PROMOTE NERVE REGENERATION
STEPPING STONE
...UNCOVERING the Role for Senescence and Stress
DISCOVERING ABNORMAL SCHWANN CELLS...
MYELINATED AXONS
LONG NERVE GRAFT MODEL
5,000
MATTHEW WOOD, PhD
ISOGRAFT
Growth Factors
GDNF
Nerve
Injury
Upregulation

Distal Nerve
Muscle
...
promotes nerve regeneration
, especially in motorneurons.

...upregulation is
transient
and

can be insuffienct.
Exogenous
"short window"
GDNF
Distal Nerve
...to
extend
the "short window" of enhanced nerve regeneration.
Lentiviral Vector

GDNF
Normal
+GDNF
...GDNF causes
axonal trapping
at delivery site.
+GDNF
Axonal Trapping
...allograft +GDNF
reduces distal regeneration
.
Increased
fiber count.
Decreased
fiber count.
Schwann Cells + GDNF
Delivery System
Developing a...
Conduit with Affinity Fibrin Matrix
...development of fibrin-GDNF conduit delivery.
Fibrin-GDNF Conduit = Isograft
Fibrin-GDNF
Isograft
Distal Nerve
Distal Nerve
5mm Gap
5mm Gap
16mm Gap
...
controlling
overexpression of +GDNF
#1 - Tetracylcine
induction
...
#2 -
Production
of Cre...
#3 -
Excision
of GDNF by laxP...
Neurotrophic Factor
Neurotrophic Factors
- survial factor for motor neurons
- less sensative and negative dosing issues
- large number of neuronal receptors for it

NGF / BDNF
Mechanism of Axonal Elongation
HDAC5 - Axonal Elongation
Site of Injury
Cell Body
HDAC5 tubulin deacetylase
promotes axonal growth...
HDAC5 nuclear export
activates a
pro-regenerative transcriptional program.
Control
HDAC5
Knock-out
HDAC5
Over-expression
Overexpression Schwann Cells
Moderate Exercise
Excessive Exercise
Depression
reduces levels of neurotrophic factors...
Exercise
can help promote nerve regeneration...
Clinical Impact on Patients
MMD - Major Depression Disorder
Remission
GDNF
NT-3
Inactivity
Detrimental
Improve...
Detrimental
BDNF role in activity-dependent therapies after injury.
Pain
may influence nerve regeneration...
PO-GABA-B1 Knock-out Mice
Hyperalgesic and Allodynic
increases unmyelinated fibers and Remak bundles
increases nociceptive C-fibers
Neuregulin-1
GABA-B Receptor Knock-out
Type-1
Type-3
Exercise
Pain
Depression
upregulation of growth factors
decreases allodynia pain
downregulation of growth factors
altered levels of Neuregulin-1
Electrical Stimulation
Purdue Pegboard Test
Semmes Weinstein
no functional improvement
IMMUNOSUPPRESSION
SCAFFOLDS AND TISSUE ENGINEERING
NERVE GAP
Biological Strategies for Nerve Regeneration
Where are we now, where are we going?
Time and Regenerative ANA Distance
6 Weeks
22 Weeks
14mm Graft
28mm Graft
1511 ± 3490
302 ± 799
5590 ± 2533
5786 ± 5488
13803 ± 3977
13271 ± 3616
Isograft
Acellularized Allograft
Conduit
Fiber Count
Schwann Cells
6 Weeks
NON-CRITICAL SENSORY RECONSTRUCTION
Ulnar Nerve with
Collagen Conduits (2x 6mm x 2cm)
Good Results
with Nerve Conduits
Failed Results
with Nerve Conduits
226 Digital Nerve Cases
18 Median / Ulnar Nerve Cases
Median Nerve with
Collagen Conduit

(7mm x 2cm)
Failed Case #1
Failed Case #2
Ulnar Nerve with
PGA Conduit (4mm x 2cm)
Proximal
Distal
Failed Case #3
Ulnar Nerve with
Collagen Nerve Wrap (7mm x 2cm)
Failed Case #4
Conduit Volume
Nerve Conduit Marketing
FDA/CE Approved Nerve Conduits
Conduit
Allograft
Conduit
Acellularized Allograft
Autograft
Acellularized Allograft
Schwann Cells
Conduit
Conduit
Matrix
Schwann Cells and Matrix
Bronze Standard
Clinical
Clinical
Research
Research
Research
Clinical
Experimental Progress...
Acellularized
+/-
+/-
+/-
+/-
+/-
+/-
ANIMAL CUT/REPAIR MODEL
PROXIMAL INTRAOP STIM
CONTROL (Repair Only)
ELECTRICAL STIM
0.2mA

&
0.5mA
FK506
=
DEVICE SETTING
STIMULATION DURATION
CONTROL (No Stim)
ELECTRICAL STIM
1-60 min
Modified
Stimulator
Lead

ACCELERATES REINNERVATION
CARPAL TUNNEL SYNDROME
IMPROVES CONDUCTION SPEED
histomorphometry
VIDEO-BASED LEARNING AND EDUCATION
dissemination & implementation
SURGICALEDUCATION
.WUSTl.EDU

MECHANISM
UPREGULATION OF REGENERATION-associated GENE (RAG)
Full transcript