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S11 Open Communication: SCI
S11-01
THE CGRP8-37 RECOMBINANT PEPTIDE CONSTRUCT
TO REDUCE CHRONIC PAIN FROM RAT SPINAL CORD
INJURY
Chenxu Han
1
, Pingping Chen
2
, Chelsea Cosner
2
, Stanislava Jergova
2
,
Shyam Gajavelli
2
, Jacqueline Sagen
2
1
Florida International University, Biomedical Engineering, Miami,
USA
2
University of Miami, Neurosurgery, Miami, USA
Chronic pain following spinal cord injury (SCI) is challenging clinical
problem with few effective treatments. It is necessary to identify new
therapeutic targets and approaches. Calcitonin gene related peptide
(CGRP) is produced by neurons in the dorsal root ganglia and thought
to play a key role in nociceptive neurotransmission in the spinal dorsal
horn. Hypersensitivity to CGRP and/or sprouting in response to injury
may contribute to allodynia and hyperalgesia in persistent neuropathic
pain. A truncated CGRP peptide, CGRP
8-37
, can reverse symptoms of
neuropathic and inflammatory pain in animal models. This study aims
to test the analgesic potential of the neuropathic pain gene therapy
candidate CGRP
8-37
. The CGRP
8-37
fragment from human CGRP
cDNA was cloned to the peptidylglycine-amidating monooxygenase
(ssPAM/pGEMT) signal peptide to allow CGRP
8-37
to be amidated
and secreted, and subcloned into AAV- and Lenti-EGFP plasmids.
Immunocytochemical colocalization of anti-CGRP and Golgi marker
anti-Giantin antibody confirmed secretable CGRP
8-37
peptide. For
initial screening, CGRP
8-37
supernatant transfected HEK cells was
intrathecally injected into rats with chronic constriction injury (CCI)
and formalin-evoked inflammatory pain model. Results showed that
reduction of mechanical, tactile, cold allodynia and formalin-evoked
pain responses treated with AAV-CGRP
8-37
supernatant was compa-
rable with the effect of 10 nM CGRP
8-37
peptide, but not in controls.
Spinal cord clip compression injury was induced pain-related behavior
in rats. At 4 weeks post-injury when pain-related behavior was clearly
established, animals were injected with lenti-ssPAM-CGRP
8-37
-EGFP
or control virus intraspinally into lumbar dorsal horn. Attenuation of
tactile, mechanical and cold allodynia was observed by 2 weeks post
injection with gradual improvement of behavioral outcomes towards
pre-injury levels by 12 weeks post-SCI. In contrast, allodynia per-
sisted in rats receiving control virus. Our findings suggest that en-
gineered analgesic CGRP
8-37
peptide have the potential to alleviate
SCI-induced pain.
Supported by the Sheila and David Fuente Neuropathic Pain Pro-
gram, University of Miami Research Support Award, and Buoniconti
Fund to Cure Paralysis
Keywords: CGRP8-37, spinal cord injury, gene therapy, chronic
pain
S11-02
LONGITUDINAL OPTOGENETIC MAPPING OF THE COR-
TICOSPINAL TRACT AS A NOVEL APPROACH FOR FUNC-
TIONAL EVALUATION OF SPINAL CORD INJURY
Xiaoming Jin
1,2
, Xingjie Ping
1
, Wei Wu
2
, Tyler Nguyen
1
, Wenhui
Xiong
1
, Xiao-Ming Xu
1,2
1
Indiana University School of Medicine, Anatomy and Cell Biology,
Indianapolis, USA
2
Indiana University School of Medicine, Neurological Surgery, In-
dianapolis, USA
Spinal cord injury (SCI) causes immediate disruption of ascending
and descending pathways, which are commonly followed by plasticity
and reorganization of these pathways at various levels of the central
nervous system. For the motor system, evaluating longitudinal chan-
ges in the integrity and function of the corticospinal tract (CST) fol-
lowing SCI is important for understanding injury mechanisms and
assessing the efficacy of therapeutic interventions. However, no
techniques are currently available for this purpose. Our goal was to
use
in vivo
transcranial optogenetic mapping of the motor cortex for
longitudinally assessing the function of the CST after SCI. In trans-
genic mice that expressed channelrhodopsin-2 in cortical layer V
pyramidal neurons, we used a blue laser to scan the region of motor
cortex through intact skull. Optogenetically evoked limb movements
were precisely detected by motion detectors or by recording electro-
myogram (EMG). In uninjured mice, motor maps of the forelimb area
made at different times were generally stable and reproducible. To
determine whether optogenetic mapping would reflect CST function
at different stages after SCI, we simultaneously assessed changes in
motor maps and motor behavior of the forelimb before and at different
times following unilateral pyramidotomy or cervical spinal hemi-
contusion. Unilateral pyramidotomy caused immediate loss of the
forelimb motor area, which was followed by partial recovery of motor
map and behavior in 3–4 weeks after SCI. In contrast, spinal hemi-
contusion at the cervical level (C5) resulted in an acute expansion of
the motor map, which was followed by progressive loss of map area
and impairment of motor behavior at 3–4 weeks after injury. Further
analyses indicated positive correlations between map size and motor
function. We conclude that optogenetic mapping of cortical motor
area may be an efficient and minimally invasive technique for lon-
gitudinal functional evaluation of the CST following SCI.
Keywords: Optogenetic, Motor cortex, Spinal cord injury, Corti-
cospinal tract
S11-03
3D IMAGING OF AXONS IN TRANSPARENT SPINAL CORDS
FROM RODENTS AND NON-HUMAN PRIMATES
Pantelis Tsoulfas
, Cynthia Soderblom, Do Hun Lee, Abdul Dawood,
Vance Lemmon, Jae Lee
University of Miami School of Medicine, Neurosurgery and The Mi-
ami Project to Cure Paralysis, Miami, USA
Failure of axons to regenerate is the primary reason for paralysis after
spinal cord injury (SCI). Thus, discovering mechanisms to promote axon
regeneration has been an intense area of research. A technical challenge
has been visualizing axon trajectory in the injured spinal cord to provide
clear origin-target information. Recent advances in tissue clearing
methods have made it possible to overcome this hurdle, but previous
studies have been performed with transgenic mice in which the axons
were pre-labeled with green fluorescent protein (GFP). Thus, while these
studies have provided a proof-of-concept, a more practical approach to
investigating axon regeneration requires axon tracing. Using mouse and
rat models of SCI, we labeled different axon tracts using several types of
adeno-associated viruses and performed tissue clearing to image axons
using light sheet (LSFM) and confocal microscopy. AAV8-pUBC-eGFP
and tdTomato viral axon labeling combined with a tetrahydrofuran
(THF)/benzyl alcohol-benzyl benzoate (BABB) tissue clearing method is
effective in visualizing different axons in the intact and injured mouse
and rat spinal cord. While a LSFM can image the spinal cord with
exceptional speed, fine axonal projections such as corticospinal axons are
better suited for confocal microscopy imaging.
Keywords: Clearing, LSFM, Spinal cord injury, Light sheet fluo-
rescence microscopy, 3Disco, CST and RST
A-12