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Review Article
Open Access
Rodent Models of Painful Diabetic Neuropathy: What Can We Learn from
Them?
Anne-Sophie Wattiez1,2#, David André Barrière1,2*#, Amandine Dupuis1,2 and Christine Courteix1,2
1Clermont Université, Université d’Auvergne, Pharmacologie Fondamentale et Clinique de la Douleur, France
2Inserm, U 1107, Neuro-Dol, F-63001 Clermont-Ferrand, France
#Equal Contribution
*Corresponding author:
David André Barrière
Clermont Université, Université
d’Auvergne
Pharmacologie Fondamentale et Clinique de la Douleur
BP 10448,
F-63000 Clermont-Ferrand, France Tel: 0033 473 278 230 Fax: 0033 473 274
621 E-mail: david.barriere@u-clermont1.fr
Received April 02, 2012; Accepted May 26, 2012; Published May 29, 2012
Citation: Wattiez AS, Barrière DA, Dupuis A, Courteix C (2012) Rodent Models of
Painful Diabetic Neuropathy: What Can We Learn from Them? J Diabetes Metab
S5:008. doi:10.4172/2155-6156.S5-008
Diabetic peripheral neuropathy (DPN) is the most common clinical complication of diabetes mellitus, and can be
related to type 1 as well as type 2. To date, this highly invalidating neurological impairment is insufficiently known,
understood and the treatments proposed by physicians are still empirical and poorly efficient.
Animal rodent modeling of clinical DPN offers a powerful tool in order to understand diabetes-mediated peripheral
nerve injury. The majority of studies which have investigated DPN in rodent used the streptozotocin-induced rat
model which reproduces metabolic lesional mechanisms of Type 1 Diabetes Mellitus (T1DM) and usual symptoms
of evoked pain. Although the clinical relevance of this model is challenged due to 1) a high prevalence of type 2-
compared to type 1-diabetes in the adult population, 2) the important alteration of the general clinical state of the
animals and 3) the lack of morphological changes in peripheral nerves, many studies have contributed to a better
pathophysiological and pharmacological understanding of the DPN.
In this review we investigated rodent models of T1DM and T2DM, their contributions for a better understanding
of DPN, molecular targets and pharmacological strategies, which could be used for the enhancement of clinical care.
Finally, we proposed possible ways to improve animal modeling.
Introduction
Neuropathic pain has recently been redefined by the Neuropathic
Pain Special Interest Group (NeuPSIG) to correspond to “pain initiated
or caused by a primary lesion or dysfunction in the nervous system” [1].
Its prevalence in general population, all causes combined, is estimated
at 1.5 [2] and 6.9% [3]. The development of experimental models of
neuropathic pain secondary to lesions of traumatic origin (constriction,
partial section, infraorbital nerve ligation, spinal nerve ligation [4-6],
etc), metabolic (diabetes, [7]) or toxic (anticancer agent [8] or retroviral
[9]) has contributed to a better understanding of their pathophysiology.
These models try to stand as close as possible to the symptomatology
and / or clinical etiopathogeny of neuropathic pain and are currently
used to evaluate new therapeutic drugs.
The need for modeling diabetic neuropathic pain comes from a
clinical reality: diabetes is one of the largest providers of neuropathy
in the world. Indeed, of 246 million diabetic patients, between 20 and
30 million are affected by symptomatic diabetic neuropathy [10]. Neuropathy
occurs for both type 1 diabetes mellitus (T1DM) and type 2
diabetes mellitus (T2DM), suggesting that hyperglycemia is the primary
etiologic factor [11]. The most frequent clinical form is by far
the diabetic distal sensory or sensorimotor polyneuropathy, affecting
30% of community-based people with diabetes [12]. Sensory polyneuropathy
presents a typical distribution “in stocking and glove”, and can
sometimes be asymptomatic but usually causes abnormal sensations
(paresthesia and dysesthesia) and/or pain. Here, the longest fibers are
first affected, which explains the distal distribution. Continuous or intermittent,
spontaneous or evoked, pain and abnormal sensations precede
or accompany the neuropathy. Estimated prevalence of painful
polyneuropathy varies between 8 and 65% [13-16] according to studies
and the diagnostic tools used. Indeed, using the DN4 pain questionnaire,
an overall prevalence of painful diabetic peripheral neuropathy of
14% [17] or 65.3% [14] was found. By using the Michigan Neuropathy Screening Instrument (MNSI) and questions from the Brief Pain Inventory
(BPI), the prevalence rate of painful diabetic peripheral neuropathy
was 8% (MNSI score of 7 or higher and a 24 h average pain rating
BPI greater than 0) [16]. Just like neuropathic pain of other etiology,
diabetic neuropathic pain responds poorly to classical analgesics (acetaminophen,
NSAIDs) and the reference treatments are only partially
effective. Three molecules have specific authorization in this indication:
gabapentin and pregabalin, calcium channels α2δ subunit ligands, and
duloxetine, a serotonin and norepinephrine reuptake inhibitor (SNRI).
From these clinical settings, experimental rodent models of spontaneous
diabetes were developed (Type 1 diabetic insulinopenic BB /
Worchester Rats, type 2 diabetic hyperinsulinemic BBZDR / Worchester
Rats, NOD Mice, LETL Rats, Akita Mice spontaneous type 1 diabetes
[B6Ins2(Akita)]) or obtained by dietary manipulations (overeating,
fasting, shift from a high fat diet to a high carbohydrate diet) (High-fat
diet-fed Mice), genetic manipulations (Zucker diabetic fatty rat, Obese
leptin-deficient (ob / ob) Mice, Leptin receptor deficient (db / db) Mice,
nonobese diabetic Mice) or chemo-induced pancreatic toxicity (streptozotocin
(STZ), alloxan (ALX)) [18,19].
Experimental Models of T1DM Induced by Chemical
Pancreatectomy in Rats
Two agents can be used to induce chemical pancreatectomy, both
are glucose analogs: ALX, a pyrimidine derivative (synthesized in 1938)
and STZ, an alkylating and antimicrobial agent. Chemical properties
of these compounds are crucial for their ability to induce diabetes [20].
Both are hydrophilic and cannot cross plasma membrane. They use the
glucose transporter GLUT2, which is expressed by the pancreatic betacells.
Cytotoxic effects of ALX are due to its reduced reaction product,
dialuric acid, and to the production of reactive oxygen species (ROS)
(superoxide radicals O2°-, hydrogen peroxide H2O2 and hydroxyl OH°,
[21]).
STZ exerts its toxicity through DNA alkylation [21]. Protein glycosylation
is an additional deleterious factor. STZ induces ADP polymerase
over-stimulation leading to a decrease in NAD+ as well as in
ATP concentration and leads to the activation of apoptotic program
that destroys beta-cells and all the cells expressing the GLUT2 transporter
(cells from the kidney and liver). By performing a bibliography
research using the database MEDLINE (PUBMED) and the following
keywords: “diabetes” and “alloxan” or “streptozocin” and “neuropathy”
during the last 30 years (i.e. 1982 to 2012), 298 studies used the antimicrobial
agent STZ and only 48 used ALX to induce diabetes. During the
last 10 years (i.e. 2002 to 2012) the ratio ALX: STZ was 9: 139 probably
due to the poor specificity of alloxan compared to STZ against pancreatic
beta-cells. Indeed STZ generally produces greater cytotoxicity due
to its conversion to anionic radicals.
STZ is more commonly used because of its greater stability and relative
lack of extrapancreatic toxicity [22]. Thus, we focused our review
on STZ-induced diabetic neuropathy in rodents.
Clinical signs of STZ-induced diabetes in rats
After STZ administration, hyperglycemia and hypoinsulinemia appear
in the first days and persist, attesting to an irreversible toxicity. A
halt in weight growth and sometimes even a weight loss are also observed
[7]. Hyperglycemia is concomitant with polydipsia (water intake
10-times higher), polyuria and polyphagia [23]. While most morphological,
histological and electrophysiological studies show that diabetic
neuropathy is accompanied by nerve structural changes (segmental
demyelination and axonal degeneration) and functional changes (assessed
by nerve conduction velocity) in diabetic patients [10], structural
changes are rarely reported in STZ-induced diabetic rats or appear
slowly and later. Walker et al. [24] using tibial nerve biopsies from
diabetic rats reported the lack of abnormal nerve tissue regarding the
distribution of unmyelinated axons, diameter of myelinated axons, fascicular
area, absence of Wallerian degeneration. However, abnormalities
in the structure of endoneural capillaries presented increased luminal
surface and decreased endothelial cells size, related to impairment
in vaso nervorum. Therefore, in this experimental model, there is no
structural support for the functional abnormalities, and changes in pain
sensitivity.
In the absence of segmental demyelination and axonal degeneration
that characterizes human diabetic neuropathy, the diabetic rat model of
STZ could be considered as a short duration model of hyperglycemia
in which functional abnormalities reflect early stages of diabetic neuropathy.
Etiopathogenic factors of STZ-induced neuropathy
The involvement of chronic hyperglycemia in the development and
aggravation of T1DM complications in humans has been confirmed by
a North American multicentric study (Diabetes Control and Complications
Trial Research Group, 1993) performed on 1441 patients followed
during 6.5 years. In this study, the prophylactic importance of
glycemic control on the progression of retinopathy (34-76% reduction),
microalbuminuria (50%) and neuropathy (60% reduction) was further
supported. Etiopathogenic factors of sensory neuropathy are still unclear
but hypothesis coming from experimental work on diabetic rats
were made. Several mechanisms underlying glucotoxicity on peripheral
nerve fibers have been proposed [25]: an enzymatic mechanism involving
the polyol pathway, proteins glycation and expression of advanced
glycation end products receptors, as well as oxidative stress.
Glucose uptake by Schwann cells of nerves is independent of insulin:
glucose enters and accumulates in neurons initiating the aldose
reductase pathway. This metabolic pathway leads to the accumulation
of sorbitol and fructose, to the depletion of myo-inositol and compromises
glutathion cycle and ATPase Na+/K+ activity. Inhibitors of aldose
reductase, that are very efficient on functional impairment due to diabetes
in rats, are much less efficient in diabetic patients, a difference
probably related to the importance of the polyol pathway in rodents
compared to humans.
Hyperglycemia also induces a non-enzymatic glycation of proteins,
glycation end products in turn activate the transcription factor NFκB
responsible for the modification of many genes expression.
Finally, excess of glucose in neuron is responsible for the increase of
oxidative stress by combining free radical genesis and inefficient antioxidant
protection systems. Most ROS (O2°-, OH°, H2O2) are produced
by the mitochondrial respiratory chain; NADPH oxidase and xanthine
reductase, as well as reactive species of nitrogen (nitric oxide NO, peroxynitrite,
ONOO-) produced by the NO synthesis enzyme NO-synthase,
have been shown to be involved in the development of diabetic
peripheral neuropathy in STZ-treated rat. In STZ rats, it was shown that
free radicals exerted their deleterious effects on Schwann cells. Chain
reaction neutralization generated by ROS is assessed by superoxydismutase
(SOD), catalase and glutathione peroxidase. Thus treatment of
STZ rats with antioxidants not only prevents or suppresses functional
impairment [26], but also the pain-related behaviors [27]. Conversely,
treatment of healthy rats with a pro-oxidant agent (premaquine) induces
functional changes similar to those observed after induction of
diabetes [28].
Some signaling pathways involving the MAPKinases are activated
in sensory neurons exposed to increased glucose in vitro and in vivo in
rats and humans with diabetes [29].
Hypersensitivity in STZ-induced diabetic rat
Behavioral studies assessed in STZ-diabetic rats often focus on their
response to nociceptive or non-nociceptive stimuli, because of the absence
of quantifiable signs of spontaneous pain. These tests consist in
measuring time latency or withdrawal thresholds of an animal whose
paw or tail is exposed to a thermal, mechanical or tactile stimulation.
The place preference test, where the animal can chose between two temperatures
[30] presents the advantage of getting rid of animal handling,
therefore allowing the assessment of spontaneous behavior towards a
range of thermal stimuli, leaving the animal free to stand on one of the two plates of different temperatures. Using this test has allowed to reveal
a thermal hypersensibility (for a temperature of 45°C) in STZ rats [31].
However, thermal hyperalgesia towards hot temperatures is not a common
painful symptom in diabetic patients, which makes difficult the
extrapolation of these results toward clinic [32]. Some authors, using
the thermal ramp test that consists in placing the animal on a surface
which temperature increases of 1°C/sec from 30°C to 50°C, have also
observed hypersensitivity during the first few weeks of diabetes but that
transforms into a hypoalgesia 2 to 3 month later, signing an evolution of
the painful neuropathy toward an insensitive neuropathy towards nociceptive
hot stimuli, that can be found in humans [33]. Moreover, a loss
in thermal nociceptors was reported in diabetic patients [34].
The perception of tactile stimuli (light touch) and mechanical (pressure)
is also affected by diabetes. In STZ rats, the application of a von
Frey filament, producing a light static touch, causes a paw withdrawal
induced by the inappropriate activation of Aδ and C fibers, signing a
tactile allodynia. More recently, cotton swabs or brushes have been used
to measure dynamic tactile allodynia by caressing the plantar surface of
the hind paw of the animal, which evokes a paw withdrawal if Aβ fibers
are impaired. The comparison of the two different symptoms reveals
that dynamic allodynia has a later onset than static allodynia and both
painful symptoms worsen over time in STZ rats [35]. This observation
suggests that the presence of dynamic allodynia results in more severe
nerve damage than when static tactile allodynia is only symptom. The
STZ-induced diabetic rat model makes it possible to test the dynamic
tactile allodynia, which, unlike the static allodynia and thermal hypersensitivity
to hot temperatures, is a common symptom of neuropathic
pain in humans. The use of von Frey filaments can also be exploited for
the exploration of mechanical hypersensitivity: the application of a von
Frey filament (# 4.93) exerting a very static pressure point, results in a
two fold increase of the intensity of the response in diabetic animals
[36].
The search for a chemical sensitivity in diabetic rats, that could, at
best, mimic human inflammatory hypersensitivity observed in clinic,
revealed an increase in the tonic response while the phasic response is
not altered by the chemical agent [37].
Pathophysiology of STZ-induced neuropathic pain
Pain associated with nerve damage from diabetes initially involves
peripheral mechanisms causing sensory fibers hypersensitivity, which
secondarily leads to central rearrangements responsible for central nociceptive
system hyperexcitability. In this section, we discuss the main
peripheral and central mechanisms of diabetic neuropathic pain proposed
by the work using the STZ-rat model.
Peripheral changes:Involvement of voltage-dependent calcium
channels Cav: The T-type Cav channels (“LVA” low voltage activated)
or Cav 3.1, 3.2 and 3.3 are localized in cell bodies and dendrites of
primary afferent fibers, and play an important role in modulating the
neuronal excitability [38]. Their involvement in the pathophysiology of
neuropathic pain has also been demonstrated, particularly in models
of diabetes and traumatic neuropathies by sciatic nerve ligation, where
current density of type T is greatly increased [36]. The “knockdown”
strategy by Cav3.2 isoform antisense but not the Cav3.1 or Cav3.3 isoforms,
suppresses thermal (Hargreaves test) and mechanical hypersensitivity
(applying a von Frey filament # 4.93) in STZ-diabetic rats.
Electrophysiological recording from small cells (C fibers) of dorsal root
ganglia (DRG) and spinal cord (whole cell voltage-clamp) shows that
the same strategy inhibits the “up-regulation” of T-type currents induced
by diabetes [39].
Finally, over expression of the α2δ subunit of L-type calcium channels
belonging to the family of “HVA” (high activation threshold) in the
DRG of diabetic rats is contemporary with the development of tactile
allodynia appreciated by the test of von Frey filaments [40]. This α2δ
subunit is also the pharmacological target of certain antiepileptic drugs
such as gabapentin and pregabalin.
Involvement of voltage-dependent sodium channels Nav: Peripheral
nerve injury can alter the expression and function of NaV channels
α subunits which results in a change in neuronal excitability [41]. Hong
et al. [42] have shown that four weeks after induction of diabetes by STZ
in rats, the NaV currents sensitive (S) and resistant (R) to tetrodotoxin
(TTX) increased in small diameter DRG. Quantification by Western
blotting of different types of sodium channels showed an increased
expression of NaV1.3 and NaV1.7 (TTX-S) channels and a decreased
expression of NaV1.6 (TTX-S) and NaV1.8 (TTX-R) channels in DRG
of diabetic rats (four weeks post-STZ). These authors also reported that
phosphorylation of Thr / Ser residues of NaV1.8 and NaV1.6 channels,
and Tyr residues of NaV1.7 and NaV1.3 channels is increased by diabetes.
This fact is not unambiguous: while an increase and a decrease in
NaV1.3 and NaV1.8 channels expression (mRNA and protein) respectively,
have already been found, the expression of NaV1.6 (mRNA and
protein) has been shown to increase [43]. Sensitive or resistant TTX NaV channels play an important role in the pathophysiology of neuropathic
pain of all etiologies, including diabetic, by changing the electrical
properties of the membrane, thus contributing to the genesis of ectopic
discharges. These channels are also the target of different molecules
(tricyclic antidepressants, anticonvulsants, local anesthetics ...) which
therapeutic efficacy in the treatment of neuropathic pain is established.
Involvement of Transient Receptor Potential (TRP) channels:
Thermal sensitivity observed in STZ-treated animals [31] is probably
due to the sensitization of cutaneous nociceptors associated with Aδ
and C fibers. TRPV1 channel (Transient Receptor Potential Vanilloid
type 1), is a major actor in thermal sensitivity, predominantly present
in C fibers and, to a less extent, in Aδ fibers [44-46]. TRPV1 is a non-selective
calcium/sodium-permeable channel activated by temperatures
up to 43°C, capsaicin (extracted from red pepper), protons (pH < 5.9),
metabolites of arachidonic acid ... ; TRPV1 can be sensitized by phosphorylation,
by prostaglandins, bradykinin, glutamate, histamine, serotonin,
ATP or NGF. Any change in TRPV1 expression, associated with
changes in intracellular signal transduction, may lead to spontaneous
neuronal activity induced by normal body temperature; this is the case
if the response threshold of TRPV1 is lowered below 38°C [47]. Pabbidi
et al. [45], reported an increase in the amplitude of TRPV1 currents
induced by capsaicin in STZ-induced hyperalgesic mice compared to
STZ-induced hypoalgesic mice or normoglycemic control mice. The
expression of TRPV1 channels in Aδ and C fibers of STZ-treated mice
was increased in those presenting hyperalgesia, and reduced in hypoalgesic
mice. The same team also showed that thermal hypersensitivity
developed by diabetic wild-type mice is abolished when the gene
coding for TRPV1 channel is disabled (TRPV1-/- mouse). Finally, treatment
with anti-vanilloid VR1 receptor antiserum abolishes thermal
hyperalgesia in STZ-treated mice [44]. In physiological conditions, it
was shown that insulin positively modulates the activity and expression
of TRPV1 channels via protein kinase C (PKC) [48]. It is therefore
possible that the sudden decrease in insulin levels induced by STZ is
indirectly responsible for a decreased in TRPV1 activity, which would
lead to a compensatory increasing of the expression of these channels.
This could explain the thermal hypersensitivity appearing in the third week of diabetes in our study [31]. Another hypothesis brought by Pabbidi
et al. [48] suggests a direct action of STZ onto sensory neurons,
involving the ROS-p38 MAPkinase pathway, thereby altering expression
and function of TRPV1. However, a direct effect of STZ on the
expression and / or functionality of TRPV1 can be excluded because we
have shown that animals which failed to develop hyperglycemia after
STZ injection did not present thermal sensitivity disorders (unpublished
results).
On the other hand, a second TRP channel, TRPA1 (Transient Receptor
Potential Ankyrin type 1) seems to be involved in DPN, since
some studies showed that TRPA1 antagonists changed mechanical
thresholds in STZ-treated rat [49,50]. Moreover, the TRPA1 channel
can be activated in sensory neurons by ROS, alkenyl aldehydes and
15-deoxy-prostaglandin J2, which are generated during oxidative stress
leading to intracellular calcium rise [51,52]. Hence, TRPA1 receptor
through indirect activation by metabolites from oxidative stress seems
to be an important molecular protagonist in mechanical hypersensitivity
of DPN.
Involvement of HCN channels: Described for the first time in
pacemaker cells of the heart sinus node [53], HCN (hyperpolarizationactivated
cyclic nucleotide-gated cation) channels were discovered
in neurons and responsible for Ih currents [53]. HCN channels open
when the membrane is hyperpolarized (-60 to -50 mV, i.e. to the rest
potential) and generate a mixed Na+, K+ cationic current. Four genes
coding for HCN channels have been identified (HCN1-4). The most
abundant in neurons of DRG are the HCN1/2 type. The Ih current generated
is of greater amplitude, faster and more frequent in neurons of
large and medium diameter (type A) than in small diameter neurons
(type C). The administration of a HCN channel blocker, the ZD7288,
suppresses tactile allodynia in STZ-induced diabetic rats (three weeks
post-STZ) and reduces mechanical hypersensitivity as well (personal
results), whereas ivabradine, a more selective blocker of HCN channels,
suppresses cold allodynia in a model of toxic neuropathy induced by
oxaliplatin [54].
Together, these data obtained in diabetic rats underline the important
role played by ion channels in the balance of the neuronal membrane
and the importance of any change in expression levels or thresholds
of activation of these channels on the excitability of sensory fibers
and their deleterious effects on nociception.
Central changes
Involvement of N-Methyl-D aspartate (NMDA) receptors:In vivo analgesic effects of dizocilpine, memantine or D-CPP, NMDA receptor
non-competitive and competitive antagonists respectively, on mechanical
hypersensitivity in STZ rats [55-57] can also be obtained in human
clinical studies with ketamine, but they unfortunately induce debilitating
side effects [58], which compromise their clinical use. NMDA receptor
phosphorylation would be involved in the development of tactile
allodynia, mechanical and thermal hypersensitivity [23]. Our team has
also shown the importance of the specific activation of certain isoforms
of MAPKinases in painful hypersensitivity in STZ animals, as well as
the need for NMDA receptor activation for the phosphorylation of
these kinases [59], opening new prospects for a more targeted drug
therapy, thus better tolerated for diabetic neuropathic pain.
Alteration of descending systems: One of the pathophysiological
mechanisms involved in the pathogenesis of chronic pain including
neuropathic pain, is a loss of the inhibitory role of serotonin on persistent
pain, as evidenced by (i) the nearly ineffectiveness of selective serotonin
reuptake inhibitors (SSRIs) in neuropathic pain patients [60] and, (ii) results obtained in STZ-induced diabetic rats showing an alteration
of spinal 5-HT2A receptor-mediated analgesic effect, usually involved in
the analgesic effect of serotonin [31]. These receptors have the particularity
to be associated with specific multiprotein complexes, consisting
in part of proteins containing PDZ domains, which can modulate signal
transduction of receptors to which they are associated [61]. In STZ rats,
the administration of a cell-penetrating peptidyl mimetic of the 5-HT2A
receptor C-terminus ending, which disrupts their interaction with PDZ
proteins, induces antihyperalgesic effect per se and enhanced the analgesic
effect of fluoxetine, an SSRI [31].
Most of peripheral and central abnormalities in the transmission
and modulation of nociception that have been described in STZ rats
and were also found in other neuropathic pain models, especially traumatic
peripheral nerve injury (CCI or SNL) showing the lack of specificity
of the model. It would be simplistic to want to associate a pathophysiological
mechanism to an etiology because the same mechanism
can be found in neuropathies of different etiologies [62], and a given
injury may involve several mechanisms.
Activity of reference drugs
CaV channels α2δ subunit ligands: The antiallodynic and analgesic
efficacy of pregabalin [63] and gabapentin [55], whose action depends
on binding to the α2δ-1 subunit of the CaV2.X, has been shown
in many neuropathic pain models, including STZ-induced diabetic rat.
The increased expression of mRNAs encoding subunits α2δ of neurons
of small (C fibers) and medium (Aδ) caliber in diabetic rats has been
known for a decade [40] and would play a major role in the development
of pain hypersensitivity.
N-type CaV channel (CaV2.2) blockers: Leconotide and ziconotide,
synthetic versions of ω-conotoxins MVIIA and CVID produced by
marine mollusks, showed dose-dependent analgesic activity after intravenous
administration in the diabetic neuropathic pain model ,
on thermal hyperalgesia [64]. However, only the intrathecal route of
administration of ziconotide is effective in patients suffering from severe
chronic pain, emphasizing the difficulties and precautions needed
when extrapolating data obtained from animal experiments to human disease.
NaV channel blockers: NaV channels are the target of many analgesics.
Topical lidocaine (patch form), prescribed for the treatment of
postherpetic neuropathic pain, is one of the most used sodium channel
modulator in human therapeutics. Its analgesic activity when systemically
administrated had been reported in the model of diabetic neuropathy
[37,65]. Having a similar structure, mexiletine showed anti-allodynic
activity during the early stages of experimental diabetes (three
weeks post-STZ) [63], suggesting a reorganization of sodium channels
along diabetes.
Antidepressants: Literature which report the analgesic effect of antidepressants
in animal models are numerous and heterogeneous. We
have recently stressed out the importance of using protocols of administration
similar to those used clinically (repeated administration every
half-life time) for assessing the effects of antidepressants [66]. Using
chronic treatment, we have highlighted a differential profile of activity
of milnacipran (a SNRI) depending in the etiology of neuropathy,
and proposed selection criteria to use dual monoaminergic antidepressants
based on their opioidergic mechanism: such mechanism would
be predictive of modest efficacy, regardless of the neuropathy etiology
[66]. Other experimental works in animal models, based on comparative studies of the mechanism of action of antidepressants according to
the etiology and symptomatology of neuropathic pain, are necessary
before initiating clinical trials; those clinical trials will be useful to test
whether semiologic profiles of responders or nonresponders to a particular
antidepressant may be considered as selection criteria, source of
personalized treatment and thus improved efficiency.
Despite the relative lack of pathophysiological specificity of the diabetic
model, pharmacological data showed particular activity profiles
of analgesics that are considered to be efficient in this painful context,
and made possible recommendations to improve the clinical relevance
of the model.
When one considers that the predictability of a model refers to its
symptoms and its sensitivity to pain treatments recognized as effective
in a painful context, the experimental model of diabetic neuropathy
induced by STZ can be considered as predictive, bearing in mind that
the spontaneous pain has never been quantified in this model, probably
because of the lack of appropriate behavioral tools. Improvement of its
clinical relevance is still necessary and may come from a standardization
of procedures regarding the selection of the animal (strain, age,
sex, breeding), the chronicity of the disorder, the pain measurement
(operative and non operative testing) [67], or on how to use pharmacological
tools.
Type 2 Diabetes Model in Rodents
Type 2 diabetes (T2DM) is the most representative form of diabetes
mellitus in adult diabetic population. T2DM has affected 285 million
people worldwide in 2010 [68], and will probably affect more than 366
million in 2030 [69]. T2DM is characterized by an impairment of insulin
actions caused by insulin secretory defects and/or peripheral insulin
resistance. Peripheral insulin resistance is compensated by increasing
insulin secretion which leads to reduced pancreatic beta-cell (insulinopenia)
functions through local inflammatory processes which drive to
increase again glycaemia [70]. The prevalence of DPN is higher in type
2 (50.8%) than in type 1 (25.6%) diabetic patients; the prevalence of
painful DPN is 14% which, again, is higher in type 2 (17.9%) than in
type 1 (5.8%) patients [17]. Nevertheless, only few studies focused their
efforts to develop correct rodent model for investigating of T2DMinduced
neuropathy and develop new strategies against peripheral
neuropathy. A PUBMED search with « type 1 diabetes neuropathy »
finds 2349 matches which have been published between 2012 and 1964
whereas a search with « type 2 diabetes neuropathy rat » or « type 2
diabetes neuropathy mice » finds 32 and 41 matches respectively which
have been published between 2012 and 1988. In this sense, we have
selected for this review 29 articles, which explored pain behaviors, pain
thresholds and/or nerve conduction velocity in T2DM-induced neuropathy
(Table 1 and 2).
Table 1:Rat models used in T2D-induced neuropathy.
Table 2:Mice models used in T2D-induced neuropathy.
Obese models of T2DM
In rat almost 50 % of articles worked in the Zuker Diabetic Fatty
model (ZDF), a useful and well-known model of leptin receptor gene
deficiency, which displays hyperphagia, fat overstorage, glucose intolerance,
hyperglycemia, glucosuria, and polyuria. Authors using this
model reported tactile allodynia, mechanical and thermal hyperalgesia
and a decrease in nerve conduction velocity [71-75]. In ZDF rats, the
number of sural axons is preserved, but atrophy and a loss of largecaliber
dermal and small-caliber epidermal axons are observed [71].
Otto et al. [73] recently showed a temporal loss of opioid sensitivity in
these animals and a marked morphine hyposensitivity was evident at six months. Romanovsky et al. [74] also showed that the compensation
of hyperinsulinemia might not restore compromised nerve function.
On the other hand, Li et al. [72] showed that a 2% taurine diet reverses
mechanical hypersensitivity and neurovascular deficits. Eventually,
Sugimoto et al. [75] showed that ZDF animals also exhibited progression
from thermal hyperalgesia to hypoalgesia, which occurred more
rapidly and coincided with a rapid decline in pancreatic insulin secretion.
The same model of obesity and T2DM is available in mice since
ob/ob and db/db models, which display leptin and leptin receptor deficiency
respectively, have been developed. These models are often used
for the assessment of T2DM-induced neuropathy in mice. The most
obvious characteristic of leptin-deficient ob/ob mice is that they are
grossly overweight and have higher food consumption. They are also
hyperglycemic, hyperlipidemic, hyperinsulinemic and display lowered
physical activity [76]. Db/db mouse is the most widely used model for
the study of T2DM neuropathy in mice. First described in 1966, the
db gene encodes a G-to-T point mutation to the leptin receptor, which
is transmitted in an autosomal recessive fashion. This defect leads to
the development of hyperphagia, obesity, hyperlipidemia, hyperinsulinemia,
insulin resistance, and diabetes [77].
Ob/ob mice [78-80] and db/db mice [81-83] develop thermal hypoalgesia,
tactile allodynia and a decrease in nerve conduction velocity.
Ob/ob mice developed manifest sciatic motor nerve conduction velocity
(MNCV) and hind-limb- digital sensory nerve conduction velocity
(SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable
loss of intraepidermal nerve fibers [80]. In this mice, administration
of fidarestat, an aldose reductase inhibitor, was associated with
preservation of normal MNCV and SNCV, alleviation of thermal hypoalgesia
and decreasing of intraepidermal nerve fiber loss, but not tactile
allodynia [78]. Sciatic nerves of wild type C57BL6, ob/ob, and db/db mice were investigated by electronic microscopy, which revealed injuries
in myelin sheaths in small (< 5 μm), medium-sized (5-10 μm), and
large axons (>10 μm) of db/db mice compared with wild type mice. In
ob/ob mice, only large fibers showed a decrease in myelin sheath thickness.
Moreover, the basement membranes of endoneural microvessels
were thickened in both obese groups. The authors also explored laminin
expression by western blot and showed a decrease in db/db group
but not in ob/ob. Hence, changes in nerve fibers and in endoneural
microvessels are present in sciatic nerve of both mouse models [79].
Gene expression changes in db/db mice are consistent with structural
changes of axonal degeneration and interestingly Nerves Growth Factor
(NGF), Substance P (SP), and calcitonin gene-related peptide (CGRP)
are up-regulated in dorsal root ganglion (DRG) of db/db mice before or
during the development of mechanical allodynia [84]. Interestingly, upregulation
of NGF coincided with enhanced tyrosine kinase A (TrkA)
receptor phosphorylation in DRG. Further study aimed to identify the
detailed mechanism of astrocyte-induced allodynia in db/db mice. Results
showed that spinal activated astrocytes dramatically increased interleukin
1β expression which may induce the phosphorylation of NR1
subunit of NMDA on the serine residue 896 [81].
All these results show that T2DM neuropathy in obese rat and mice
models could be sustained by direct injuries onto the peripheral nervous
system, which involved classical molecular actors found in pain
sciences. Nevertheless, these models of leptin-deficient or leptin receptors
deficient obesity cannot represent a clinical reality since leptin mutation
in human population still rare and, typically, people risking to
develop T2DM or obesity, which could lead to T2DM have a complex association of inherited variations at many genetic sites and are exposed
to environmental stressors [85]. In this sense, few non-obese but more
pertinent models of T2DM were developed but, unfortunately, they are
seldom used for the study of T2DM-induced peripheral neuropathy.
Non-obese model of T2DM
The best described rat model of non-obese diabetes which does
not result of single point mutation is the congenic strain Goto Kakizaki (GK). GK is a moderately diabetic rat strain that was developed by Masaei
Kakizaki and Yoshio Goto by repeated inbreeding of glucose-intolerant
Wistar rats over several generations. In contrast to many other rodent
models of non-insulin-dependent diabetes GK rat does not exhibit
hyperlipidemia nor obesity [86].
Murakawa et al. [87] showed an impairment in the blood glucose
tolerance tests in GK rats, a decrease of 76 % of normal MNCV, a loss
of small myelinated fibers and an atrophy/loose of unmyelinated axons.
On the other hand, the levels of NGF in the sciatic nerve were significantly reduced, and concomitantly, TrkA and NGFp75 receptor
expression was decreased in DRG. These changes were accompanied
by significantly reduced immunoreactivity for SP and CGRP in DRG
neurons and sciatic nerve. Unfortunately, this interesting paper does
not correlate painful thermal and mechanical thresholds with peripheral
damages and impaired expression of molecular protagonists [87].
Most studies have highlighted the beneficial role of the GK model
in pharmacology by testing new drugs. Ueta et al. [88] reported that
GK rat presented thermal hypoalgesia and explored the anti-hypoalgic
effect of T-1095, an orally active inhibitor of Na+-glucose co-transporter
(SGLT). Throughout the study, T-1095 treatment significantly
decreased both blood glucose and hemoglobin A(1C) levels in the GK
rat and a concomitantly reduced the thermal impairment in tail-flick
test [88]. In the same manner, Kitahara et al. [89] examined the effect
of long-term suppression of postprandial hyperglycemia and glycemic
fluctuation in GK by nateglinide, an antidiabetic drug which stimulates
the release of insulin from pancreatic beta cells. Nateglinide treatment suppressed postprandial hyperglycemia by 50% and normalized delayed
motor nerve conduction but once again, authors do not correlate
these results with pain thresholds evaluation [89]. To finish, Liepinsh et
al. [90] showed that mildronate, an anti-ischaemic drug, significantly
decreased both the fed- and fasted-state blood glucose and the thermal
hyposensitivity [90].
The GK model is the one for which pharmacological studies have
been done to study T2DM-mediated peripheral neuropathy. However
it appears clearly that antidepressants, anticonvulsants, as well as α2δ
ligands, which display clinical efficiency, should be investigated in this
model in order to validate its clinical pertinence for the development of new analgesic compounds.
The last model used for studying T2DM induced-neuropathy is the
diet-induced diabetes model. Very few article explored pain sensitivity
in this model, which, nevertheless, displays neuropathic changes when
animals are fed with high fat diet (HFD). This model of T2DM-induced
neuropathy is exclusively caused by the dietary regimen, the most important
factor associated with idiopathic neuropathy in non-diabetic
human subjects [91]. In mice, two studies explored pain thresholds
in HFD which led to the conclusion that the development of thermal
hypoalgesia was identical in both females [92,93] and males [94]. In
females, tactile allodynia was also reported, but mechanical hypoalgesia was only reported in males [92,93,95]. These studies showed the role of
nitrosative stress in peripheral nerves and demonstrated the role 4-hydroxynonenal
adduct, nitrotyrosine, poly (ADP-ribose) accumulation
and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal
root ganglion neurons. Authors proposed that oxidative stress is a good
target for the treatment of diabetic peripheral neuropathy.
Other models
T2DM-induced neuropathy was also studied in another models but
their using still is marginal.
1) The Otsuka Long Evans Tokushima Fatty (OLETF) rat is a Cholecystokinine
1 receptor (CCK1) knockout model which allows studying
the multiple CCK functions. OLETF rats are grossly hyperphagic
probably due to the loss of a feedback satiety signal in the central nervous
system [96]. Administration of sucrose to OLETF rats caused significant
body weight increase and marked hyperglycemia. Sucrose-fed
OLETF rats demonstrated significantly delayed MNCV and their thermal
nociceptive thresholds is significantly decreased [97].
2) The inbred Bio-Breeding Zucker diabetic rat (BBZDR)/Wor, is a
relatively emerging model of T2DM. Diabetic male BBZDR/Wor rat
are homozygous for a leptin receptor gene mutation and shares genetic
background of original BB strain. BBZDR/Wor rats are hyperlipidemic
and hyperleptinemic, become insulin resistant, and ultimately develop
hyperglycemia as well as thermal hyperalgesia [98].
3) Tsumura Suzuki Obese Diabetes (TSOD) mice, were also obtained
by selective breeding of obese male mice of the ddY strain and using
indices of the heavy body weight and appearance of urinary glucose
[99]. Iizuka et al. [100] reported that TSOD mice develop mechanical
hyperalgesia between six to twelve months old.
4) A very interesting model is the stress-induced T2DM mice model
developed by Loizzo et al. [101], A post-natal psychological stress
produced a series of dysmetabolic signs highly similar to mild human
T2DM. Adult mice, receiving post-natal stress, display increased body
weight, fasting glycaemia and increased plasma level of corticosterone
and adrenocorticotropic hormone (ACTH). Mice present thermal hyperalgesia
in tail-flick test and administration of naloxone prevented
body overweight and abdominal overweight suggesting an involvement
of the opioid system and of the hypothalamus-pituitary axis. This model
of stress should be useful to study idiopathic diabetes mellitus and
neuropathy induced in these conditions [101].
5) STZ-induced T2DM model was developed by Srinivasan et al.
[102] in order to replicate the natural history and the metabolic characteristics
of human T2DM for suitable pharmacological screening.
Authors used male Sprague-Dawley rats, which were fed with HFD
(58% calories as fat), for a period of two weeks. HFD-fed rats exhibited
significant increase in body weight, basal glycaemia, and insulinemia
and also presented dyslipidemia. Then, rats received an intraperitoneal
injection of a low dose of streptozotocin (35 mg/kg), which produces a
decline of insulin secretion and transforms prediabetes status, induced
by high fat feeding, in diabetes. Hence, rats present hyperglycemia,
insulinopenia, insulin resistance, and dyslipidemia as patient with an
advanced T2DM [102]. From this model Xiu-ying Yang et al. [103]
showed that rats which received standard chow diet supplemented with
10% sucrose, 10% lard, 2% cholesterol and 0.2% cholic acid during one
month followed by intraperitoneal injection of STZ (30 mg/kg) present
thermal hypoalgesia and a decrease NCV which could be relieved by
salvianolic acid A, an antioxidant [103].
All these models display painful thresholds changes; nevertheless,
they are still marginal and most investigations will be necessary to improve
their predictability in pain research.
Nowadays, T2DM rat or mice models are not systematically used
for the study of DPN, probably because none of them is yet fully characterized
but also because housing, maintain and using of knockout
mice or congenic strains is more problematic than the use of the T1DM
model induced by STZ which, contributed to most of our knowledge in
DPN in the last thirty years.
Conclusion
Rodent models of T1DM and T2DM have vastly improved the understanding
of pathophysiology of diabetic neuropathic pain and the
development of new therapeutics. These models do not pretend to reproduce
diabetic neuropathy, as it develops in humans, but to approach
it, according to the “principle of similarity” defined by Bennett [104].
The conclusions from observations obtained in these models should be
drawn with care and validated in more than one model or condition
because diabetic patients with painful neuropathy come from a heterogenous
population in terms of etiopathogenesis, clinical course of diabetes
and, for some of them, co-morbidities. Cancer is one example of
co-morbidity and diabetes may negatively impact both cancer risk and
outcomes of treatment. Indeed several chemotherapeutic agents like
cisplatin, paclitaxel, and vincristine might cause or exacerbate neuropathy
[105]. The deleterious effect of paclitaxel chemotherapy on thermal
nociception was observed in STZ diabetic hyperglycemic rats [106] and
further support the need for development of animal models closer to
clinical reality.
This literature review reaffirms the need for collaboration between
clinical and preclinical research to increase the benefit of pharmacological
advances, and the relevance of the work on animal models.
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Fields HL, Basbaum AI (1999) Central nervous system mechanisms of pain modulation. In: MSBaK M, editor. Text book of Pain, 5th Edition. Churchill Livingston, Edinburgh: Elsevier Limited: 309-329.
Rice AS (2010) Predicting analgesic efficacy from animal models of peripheral neuropathy and nerve injury: a critical view from the clinic. In: J Mogil, editor. Pain 2010 an updated review. Seattle: IASP Press: 415-426.
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