Biology Reference
In-Depth Information
Disorders and Stroke (NINDS) started a “Facilities in Research
Excellence—Spinal cord injury replication project” (FORE-SCI), which
were contracted to three different centers with a mandate to repeat key ex-
periments in SCI research (
Steward et al., 2012
). The result of the effort is
that of 12 contracted studies, only 2 could be fully repeated. There are many
reasons for the failures resulting from technical differences (
Steward et al.,
2012
), but two aspects deserve more attention: (1) Rigorous quality control
of the reagent being tested. Endotoxin is a frequent contaminant of labora-
tory solutions (
Weinstein, Swarts, Bishop, Hanisch, & M¨ ller, 2008
) and
may be an especially serious contaminant when recombinant proteins are
used in preclinical studies. Endotoxin, which is a lipopolysaccharide from
bacterial membranes, is highly inflammatory and should be monitored with
available kits, which would provide assurance that biological effects are due
to the product, and not inflammation from endotoxin. (2) The Basso,
Beattie, and Bresnahan (BBB) locomotor rating scale developed as a rela-
tively easy scoring system to measure locomotion in rats after thoracic
SCI (
Basso, Beattie, & Bresnahan, 1995
) has never been validated to be
robust and predictive, and most SCI studies do not carry out functional
analysis in a proper double-blind experiment (
Steward et al., 2012
). The
FORE-SCI replication studies highlight the weakness of the BBB assay,
which has been used by many, including by the authors of this chapter.
Inactivation of Rho signaling as a strategy to promote functional recov-
ery was not tested by the FORE-SCI contract studies, but this signaling
pathway has been well documented by many independent labs using differ-
ent outcome measures. A study of ROCKII
/
mice showed that neurons
placed in culture were less sensitive to growth inhibitory proteins than con-
trols, and mice with SCI showed some spontaneous axon regeneration
(
Duffy et al., 2009
). Moreover, ROCKII
/
mice showed improved use
of forelimbs after cervical dorsal root crush compared to ROCK
þ
/
þ
mice.
More recently, siRNA knockdown of RhoA after administration by lumbar
puncture improved motor recovery after SCI (
Otsuka et al., 2011
).
Inhibition of Rho signaling with C3 transferase or cell-permeable vari-
ants of C3 transferase provides strong evidence for the role of Rho in axon
regeneration after CNS injury. In the mouse, inactivation of Rho with C3
improves functional recovery, as detected by the BBB test (
Dergham et al.,
2002
). In these studies, the animals were treated with C3 delivered directly
to the injury site in a fibrin gel injected into the site of an overhemisection
lesion. The Mueller lab showed that cell-permeable C3 made with a Tat
construct also promotes robust function recovery in rats, as measured by
