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REVIEW
SA JOURNAL OF DIABETES & VASCULAR DISEASE
14
VOLUME 12 NUMBER 1 • JULY 2015
recombinant proteins injected into cells via polyarginine anchors,
which has coined the nomenclature ‘piPSCs’ – protein-induced
pluripotent stem cells;
16
and small-compound mimicking, which
raises reprogramming efficiency
.17
iPS cells offer the advantage of being: easily and non-invasively
harvested; useful tools for drug development; models for disease
processes
in vitro
; and a source of autologous cells for transplantation
due to a lower risk of immunorejection. Disadvantages include a
propensity towards tumorigenesis and a lack of long-term data on
stability and safety.
18
Peripheral and central injury models
Progress on the utility of cell-based therapies for neuropathic pain
research is dependent upon the application of appropriate experi-
mental animal models of peripheral and central nerve lesions.
There are two broad groups of experimental animal models of
neuropathic pain: those that localise the lesion e.g. dorsal root
ganglion lesion, peripheral nerve lesion, spinal cord lesion, and
dorsal and ventral root lesion; and those that describe the type
of lesion e.g. transection, tumour cell invasion or laser radiation,
cryoneurolysis, crush, stimulation of perineuronal inflammation,
and tight or loose ligature.
19
However, no single animal model
entirely recaptures the full range of neuropathic pain mechanisms.
Inanimalmodelstheassessmentandquantificationofneuropathic
pain by direct evaluation is not feasible. Rather subjectively, most
data obtained using animal models have relied on the use of evoked
pain-related behaviours such as withdrawal responses as surrogate
markers for neuropathic pain.
20
Leading on from this, complexities
regarding assessing neuropathic pain in animal models are further
exemplified by attempts to extrapolate and identify relevant markers
for spontaneous pain. This is particularly problematic for patients
with neuropathic pain but rather difficult to measure in rodents.
Surrogate indicators include: changes in general innate behaviours
such as locomotion, burrowing, digging, excessive grooming
and nesting;
21
and more complicated paradigms using Pavlovian
conditioning methods such as conditioned place preference and
aversion.
22
Notwithstanding these methods, laboratory tools for
objectively assessing neuropathic pain in animal models are available
and utilise two surrogate markers, namely: thermal hyperalgesia
using the acetone test; and mechano-allodynia using the von Frey
test.
19
Evidence on the utility of ES cells for the treatment of
neuropathic pain
ES cells have been used to treat neuropathic pain by regenerating
GABAergic interneurons with restoration of the inhibitory tone in
the dorsal horn of the spinal cord, the lack of which would other-
wise contribute to the hyperexcitability that underlies allodynia
and hyperalgesia. Evidence in support of this used mouse ES
cell-derived MGE cortical inhibitory precursor cells, which were
transplanted into a mouse model of peripheral sciatic nerve injury.
23
Using ES cell-derived MGE cells that expressed GFP under the
control of the Gad1 promoter, it was demonstrated that the ES
cell-derived MGE grafts adapted and thrived in the novel spinal
cord environment and migrated throughout the ipsilateral dorsal
and ventral horns. Within two weeks of transplantation the grafted
cells showed immunocytochemical evidence of differentiation
towards a neuronal phenotype (NeuN+) and likewise demonstrated
immunoreactivity for markers of cortical GABAergic interneurons,
namely GABA, neuropeptide Y, parvalbumin and somatostatin.
Furthermore, the transplants structurally integrated into host spinal
cord circuitry as evidenced by neurite outgrowth, sprouting, path-
finding andsynapse formation with host primary afferent and post-
synaptic neurons.
The grafts targeted and influenced a range of spinal cord
neurons including projection neurons of lamina I that normally
receive nociceptive stimuli. There was a close temporal relationship
between the improvement in mechanical allodynia and integration
of the grafted cells, which indicated the latter was instrumental
for recovery. There was no correlation between the number of
transplanted cells and observed anti-nociceptive effects, which
suggests there may be a threshold above which the number of
grafted cells is of less significance in attaining analgesia. There was
a differential effect observed for ES cell-derived MGE transplants
with efficacy demonstrated specifically for neuropathic pain, which
was not matched in a model of inflammatory pain in response to
formalin-induced tissue injury. This suggests MGE grafts are disease
rather than symptom modifying.
The strength of Braz
et al.
’s study is based on the novel observation
that ES cell-derived MGE grafts restored anti-nociceptive inhibitory
GABAergic neurotransmission by structural integration into host
spinal cord neuronal circuits. This contrasts with previous simplistic
mechanisms of achieving the same by studies that merely focused
on releasing GABA using adenoviral and HSV vectors expressing
the GABA synthesising enzyme GAD65 in neuropathic models of
trigeminal neuralgia
24
and spinal nerve ligation.
25
Leading on from
this, the conclusion of Braz
et al.
’s study has been corroborated by
other groups, which increases its reliability.
26
Braz
et al.
’s study failed to account for the anti-nociceptive
summative effects of other co-existent endogenous inhibitory
pathways, which may have confounded the cause and effect
relationship between the independent and dependent variables. It
was presumed the anti-nociceptive effects observed post ES cell-
derived MGE transplantation was GABA mediated, as evidenced
by normalisation of GAD65 mRNA levels in the peripheral nerve
injury model, which is normally associated with low GAD65 mRNA
levels. However, the inhibitory neurotransmitters glycine and
serotonin, which co-exist in some spinal cord GABAergic neurons,
may have accounted for the inhibition and anti-allodynic effects
recorded.
27
Additionally, evidence suggests that following nerve
injury, activation of microglia results in a BDNF-mediated shift in the
chloride gradient of projection neurons in lamina I and deep in the
dorsal horn, which leads to GABAergic inputs becoming somewhat
paradoxically excitatory and pro-nociceptive.
28,29
The results from
Mackie, De Koninck and Price’s studies do not corroborate the
functional integrative mechanism postulated by Braz
et al.
’s study.
The findings of Braz
et al.
’s study are further limited in their
long-term application; they only provide data on the utility of ES
cell-derived MGE grafts for the treatment of neuropathic pain for
28 days post-transplantation. Accordingly the study was unable
to determine whether: the anti-nociceptive effects observed were
sustained; if tolerance developed; and if the experimental models
showed delayed anti-allodynic effects or developed an analgesic
phenotype characterised by mechanical thresholds greater than the
baseline.
A separate study used a modified retinoic acid protocol to induce
differentiation of mouse ES cells into neural and glial precursors.
These were used for transplantation into a mouse model of
central neuropathic pain induced secondary to excitotoxic spinal