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Neuroscience 250 (2013) 786–797
ROLE OF HYDROGEN SULFIDE IN THE PAIN PROCESSINGOF NON-DIABETIC AND DIABETIC RATS
M. E. VELASCO-XOLALPA, a P. BARRAGAN-IGLESIAS, b
J. E. ROA-CORIA, a B. GODINEZ-CHAPARRO, b
F. J. FLORES-MURRIETA, a,c J. E. TORRES-LOPEZ, d
C. I. ARAIZA-SALDANA, d A. NAVARRETE e ANDH. I. ROCHA-GONZALEZ a*
aSeccion de Estudios de Posgrado e Investigacion, Escuela Superior
de Medicina, Instituto Politecnico Nacional, Mexico D.F., Mexico
bDepartamento de Farmacobiologıa del Cinvestav, Sede Sur,
Mexico D.F., Mexico
cUnidad de Investigacion en Farmacologıa, Insituto Nacional de
Enfermedades Respiratorias ‘‘Ismael Cosio Villegas’’, Mexico
D.F., Mexico
dCentro de Investigacion y Posgrado, Division Academica de
Ciencias de la Salud, Universidad Juarez Autonoma de Tabasco,
Villahermosa, Tabasco, Mexico
eFacultad de Quımica, Departamento de Farmacia, Universidad
Nacional Autonoma de Mexico, Mexico D.F., Mexico
Abstract—Hydrogen sulfide (H2S) is a gasotransmitter
endogenously generated from the metabolism of L-cysteine
by action of two main enzymes called cystathionine b-syn-thase (CBS) and cystathionine c-lyase (CSE). This gas has
been involved in the pain processing and insulin resistance
produced during diabetes development. However, there is
no evidence about its participation in the peripheral neurop-
athy induced by this metabolic disorder. Experimental dia-
betes was induced by streptozotocin (50 mg/kg, i.p.) in
female Wistar rats. Streptozotocin injection increased for-
malin-evoked flinching in diabetic rats as compared to
non-diabetic rats after 2 weeks. Peripheral administration
of NaHS (an exogenous donor of H2S) and L-cysteine (an
endogenous donor of H2S) dose-dependently increased
flinching behavior in diabetic and non-diabetic rats. Con-
trariwise, hydroxylamine (HA, a CBS inhibitor) and DL-prop-
argylglycine (PPG, a CSE inhibitor) decreased formalin-
induced nociceptive behavior in both experimental groups.
In addition, an ineffective dose of HA and PPG partially pre-
vented the L-cysteine-induced hyperalgesia in diabetic and
non-diabetic rats. Interestingly, HA and PPG were three
order of magnitude more potent in diabetic rats respect to
non-diabetic rats, whereas NaHS was ten times more potent
in the streptozotocin-diabetic group. Nine to 11 weeks after
0306-4522/13 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.06.053
*Corresponding author. Address: Seccion de Estudios de Posgrado eInvestigacion, Escuela Superior de Medicina, Instituto PolitecnicoNacional, Plan de San Luis y Dıaz Miron s/n, Col. Casco de SantoTomas, Miguel Hidalgo, 11340, Mexico D.F., Mexico. Tel: +52-55-54-87-17-00x5600/5126; fax: +52-55-56-65-46-23.
E-mail address: hector.isaac@gmail.com (H. I. Rocha-Gonzalez).Abbreviations: ANOVA, analysis of variance; AUC, area under curve;CBS, cystathionine b-synthase; CSE, cystathionine c-lyase; ED30,effective dose 30; H2S, hydrogen sulfide; HA, hydroxylamine; NMDA,N-methyl-D-aspartate; PPG, DL-propargylglycine; TRP, transientreceptor potential.
786
diabetes induction, tactile allodynia was observed in the
streptozotocin-injected rats. On this condition, subcutane-
ous administration of PPG or HA reduced tactile allodynia
in diabetic rats. Paradoxically, H2S levels were decreased
in nerve sciatic, dorsal root ganglion and spinal cord, but
not paw nor blood plasma, during diabetes-associated
peripheral neuropathy development. Collectively, results
suggest that H2S synthesized by CBS and CSE participate
in formalin-induced nociception in diabetic and non-diabetic
rats, as well as; in tactile allodynia in streptozotocin-injected
rats. In addition, data seems to indicate that diabetic rats are
more sensible to H2S-induced hyperalgesia than normogly-
cemic rats. � 2013 IBRO. Published by Elsevier Ltd. All
rights reserved.
Key words: formalin test, hydrogen sulfide, inflammatory
pain, neuropathy, streptozotocin-induced diabetes, tactile
allodynia.
INTRODUCTION
Diabetes mellitus is one of the most serious problems in
developing as well as developed countries (Sharma
et al., 2009). Peripheral neuropathy is the most common
and debilitating complication of diabetes and results in
spontaneous pain, hyperalgesia and allodynia (Jolivalt
et al., 2008; Obrosova, 2009). The prevalence of
neuropathy is estimated to be about 8% in newly
diagnosed diabetic patients and greater than 50% in
patients with long-standing disease (Boulton et al.,
2005). In recent years, considerable progress has been
made toward understanding the mechanisms leading to
diabetic neuropathy; however, the etiology of this
condition is not fully understood yet (Edwards et al.,
2008). Actually, accepted medical approaches to treat
diabetic neuropathy are frequently unsuccessful and
have serious side effects (Tesfaye and Selvarajah,
2012). Therefore, a better knowledge of diabetes-
associated peripheral neuropathy pathogenesis is
necessary to relief this condition.
In the last decade, hydrogen sulfide (H2S) has been
studied as a gasotransmitter. This gas is endogenously
generated from the metabolism of L-cysteine by action
of two main enzymes called cystathionine b-synthase(CBS) and cystathionine c-lyase (CSE). Traditionally, it
is accepted that endothelium-independent and -
dependent vasodilatation is modulates by H2S
generated mostly from CSE, whereas H2S synthesized
by CBS has a main physiological role in the nervous
system. Notwithstanding, both enzymes are expressed
d.
M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797 787
in the nervous system and they could contribute to pain
processing (Smith, 2009).
Most of published literature points out a
pronociceptive role of H2S. Lee et al. (2008) showed
that H2S concentrations are increased locally after
intraplantar formalin injection at 5%, but not at 1.25%, in
homogenates prepared from hind paws. In addition,
formalin-induced flinching behavior was attenuated by
systemic administration of DL-propargylglycine (PPG). In
this study, authors suggested that the effect of H2S in
the pathogenesis of inflammatory pain depends on the
nociceptive stimulus intensity. Consequently, a growing
body of evidence proposed that H2S stimulates
capsaicin-sensitive nociceptive fibers (Patacchini et al.,
2005) inducing the release of neuropeptides as
neurokinin A, substance P and calcitonin gene-related
peptide (Patacchini et al., 2004, 2005; Trevisani et al.,
2005).
Kawabata et al. (2007) showed that intraplantar
injection of NaHS or L-cysteine produce mechanical
hyperalgesia through activation of T-type Ca2+
channels. Matsunami et al. (2009) confirmed this
mechanism in a visceral pain model, concluding that
hyperalgesia and allodynia observed in this model are
due to the stimulation of T-type Ca2+ channel currents,
but not L-type Ca2+ channel currents. Maeda et al.
(2009) and Okubo et al. (2011) reported similar results
in the paw pressure method and paclitaxel-evoked
hyperalgesia, respectively. Furthermore, H2S seems to
up-regulate CaV3.2 T-type calcium channels in some
neuropathic pain models (Takahashi et al., 2010).
Further CaV3.2 T-type calcium channels, recent studies
have shown that H2S increases excitability through
suppression of sustained Kv1.1 and Kv1.4 potassium
channel currents of trigeminal ganglion neurons (Feng
et al., 2013) and by sensitization and up-regulation of
voltage-gated NaV1.7 and NaV1.8 channels in dorsal
root ganglion neurons (Qu et al., 2013). Interestingly,
similar mechanisms have been associated to
nociceptive sensitization process in diabetic rats (Hong
et al., 2004; Sun et al., 2012; Khomula et al., 2013).
Besides, described mechanisms above, H2S could
produce its pronociceptive effect by activation of N-methyl-D-aspartate (NMDA) channels due this gas
selectively causes a cyclic AMP-dependent activation of
NMDA receptors and enhances hippocampal long-term
potentiation (Kimura, 2000; Eto et al., 2002). Moreover,
it has been proposed that several members of the
family of transient receptor potential (TRP) as TRPV1
and TRPA1 could be pharmacological targets of H2S,
since these channels are cysteine-rich proteins able to
react with thiols and H2S (Macpherson et al., 2007).
Notwithstanding, there is some evidence suggesting an
antinociceptive effect of this gas through production of
nitric oxide and activation of ATP-sensitive potassium
(KATP) channels (Distrutti et al., 2006).
Although there is some evidence that suggests a
pronociceptive effect for H2S in different pain paradigms.
There are no studies about the importance of H2S or
the two main endogenous H2S synthase enzymes in the
diabetes-associated peripheral neuropathy. Based on
the above considerations, this work was undertaken to
compare the H2S nociceptive effect and its producing
enzymes in normoglycemic and hyperglycemic rats. In
addition, H2S concentration was measured along
nociceptive pathway during diabetes development.
EXPERIMENTAL PROCEDURES
Animals
Experiments were performed on adult female Wistar rats
with a body weight between 200 and 220 g. Female rats
were used based on the fact that previous experiments
in our conditions have not shown significant differences
between males and females (Sanchez-Ramırez et al.,
2006). Animals were obtained from our own breeding
facilities and had free access to food and drinking water
before experiments. All experiments are in compliance
with the requirements published by SAGARPA in the
Technical Specifications for the Production, Care and
Use of Laboratory Animals (NOM-062-ZOO-1999),
Guidelines National Institute of Health Guide for the
Care and Use of Laboratory Animals (NIH Publications
No. 80-23) revised 1996, and Guidelines on Ethical
Standards for Investigation of Experimental Pain in
Animals (Zimmermann, 1983). In addition, the
experiments were approved by our local committee.
Every effort was done to minimize pain and suffering in
animals and the number of rats used was the minimal
required to obtain significant statistical power.
Formalin test
To determine the role of H2S in the processing of
inflammatory pain, the formalin test was performed.
Normoglycemic rats were placed in open observation
chambers for 30 min to allow them to acclimate to their
surroundings; then they were removed and gently
restrained while the dorsum of the hind paw was
injected with 50 lL of diluted formalin (0.5% for NaHS
and L-cysteine or 1% for inhibitors of CBS and CSE)
using a 30-gauge needle. The animals were returned to
the chambers and the nociceptive behavior was
observed immediately after formalin injection. Mirrors
were placed in each chamber to enable unhindered
observation. Nociceptive behavior was quantified as the
numbers of flinches of the injected paw during 1-min
periods every 5 min, up to 60 min after formalin injection
(Wheeler-Aceto et al., 1990). Flinching was readily
discriminated and was characterized as rapid and brief
withdrawal, or as flexing of the injected paw. At the end
of the experiments, rats were sacrificed in a CO2
chamber.
Induction of experimental diabetes by streptozotocin
Fasted rats were injected intraperitoneally with 50 mg/kg
of streptozotocin (Sigma, St. Louis, MO, USA) to
produce experimental diabetes (Sanchez-Ramırez et al.,
2006). Control animals (weight-matched) received saline
0.9%. Diabetes was confirmed 1 week after injection by
measurement of tail vein blood glucose levels with a
788 M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797
glucometer Glucolab (HMD Biomedical, Titusville, USA).
Two or 9–11 weeks after streptozotocin injection,
glycemia was again determined and only animals with
the final blood glucose level P400 mg/dL were included
in the study.
Measurement of chemical hyperalgesia
To evaluate the participation of H2S in the diabetic
neuropathy, the hyperalgesia and allodynia in
hyperglycemic rats of 2 and 9–11 weeks, respectively,
was determined. Hyperalgesia was assessed 2 weeks
after of intraperitoneal streptozotocin injection in
hyperglycemic rats by 0.5% formalin test as was
described above (Araiza-Saldana et al., 2005; Torres-
Lopez et al., 2007).
Measurement of tactile allodynia
Tactile allodynia was tested in rats with 9–11 weeks of
streptozotocin-induced experimental diabetes as
previously described (Araiza-Saldana et al., 2010).
Briefly, rats were transferred to a clear plastic, wire
mesh-bottomed cages and allowed to acclimatize for
30 min. von Frey filaments (Stoelting, WoodDale, IL,
USA) were used to determine the 50% paw withdrawal
threshold using the up–down method described by
Dixon (1980). A series of filaments, starting with one
that had a buckling weight of 2 g, were applied in
consecutive sequence to the plantar surface of the right
hindpaw with a pressure causing the filament to buckle
during 5 s. Lifting of the paw before of 5 s indicated a
positive response and prompted the use of the next
weaker filament whereas that absence of a paw
withdrawal after 5 s indicated a negative response and
prompted the use of the next filament of increasing
weight. This paradigm continued until four more
measurements had been made after the initial change
of the behavioral response or until five consecutive
negative (assigned a score of 15 g) or four consecutive
positive (assigned a score of 0.25 g) responses had
occurred. The resulting scores were used to calculate
the 50% of response threshold by using the formula:
50% g threshold ¼ 10ðXfþjdÞ
10000
where Xf = the value (in log units) of the final von Frey
filament used, j = the value (from table in Chaplan
et al., 1994) for the pattern of positive and negative
responses, and d = the mean difference (in log units)
between stimuli. 50% threshold withdrawal was
assessed before drug administration and every 30 min
during 8 h after drug administration. Allodynia was
considered to be present when paw withdrawal
threshold was below 4 g as was described above
(Chaplan et al., 1994; Araiza-Saldana et al., 2010).
Diabetic rats with a basal threshold withdrawal above
4 g were not included for the experiments.
Drugs
DL-propargylglycine (PPG), hydroxylamine hydrochloride
(HA), L-cysteine, H2S and streptozotocin were
purchased from Sigma (St. Louis, MO, USA).
Formaldehyde was purchased from Merck (Darmstadt,
Germany). All drugs, except streptozotocin, were
dissolved in isotonic saline solution. Streptozotocin was
dissolved in distilled and deionized water. PPG, HA and
H2S were injected in a subcutaneous manner in the
right hindpaw 10 min before formalin injection. L-cysteine
was administered 30 min before in the same way. All
solutions were used freshly prepared.
Measurement of endogenous levels of H2S
To determine the concentration of H2S through
nociceptive pathway during the experimental diabetes
development, we performed the technique described by
Chavez-Pina et al. (2010) with some modifications.
Briefly, normoglycemic (control) and hyperglycemic rats
with 2 and 9–11 weeks after streptozotocin injection
were sacrificed by decapitation. Then, blood samples
were taken and sciatic nerve, dorsal root ganglia from
L4 to L6, lumbar spinal cord and dorsal region of hind
paws were carefully excised. Tissues were weighted
and homogenized (500 mg) in an ice-cold 50 mM
phosphate buffer at pH 7.4 and 1% zinc acetate
(500 lL), whereas blood serum samples were diluted
with the same buffer in a ratio 1:4. The samples were
incubated for 10 min at room temperature and then
were centrifuged at 14000g during 10 min at 4 �C.Supernatants (200 lL) were mixed with 10%
trichloroacetic acid (160 lL) to stop the reaction. Again,
supernatants were centrifuged at 14000g during 10 min
at 4 �C and the new supernatants were collected and
mixed with 20 mM N,N-dimethyl-p-phenylenediamine
sulfate in 7.2 M HCl (70 lL) and 30 mM FeCl3 in 1.2 M
HCl (70 lL). After 20 min absorbances were measured
at 670 nm. The calibration curve of absorbance versus
H2S concentration was obtained using NaHS solution of
varying concentration. When NaHS is dissolved in
water, HS� is released and forms H2S with H+. H2S
concentration was taken as 30% of the NaHS
concentration in the calculation. The calibration curve
was linear from 6 to 96 lM H2S (r2 = 0.993).
Study design
Independent groups of animals (n= 6 or 7) were used for
each experimental condition. In order to determine the
role of H2S in the processing of inflammatory pain and
diabetes-induced peripheral hyperalgesia, were
constructed dose–response curves for NaHS and L-
cysteine in normoglycemic and hyperglycemic (2 weeks)
rats using the formalin test. Dose response curves were
carried out giving vehicle or increasing doses of NaHS
(0.1–300 lg/paw) and L-cysteine (10–100 lg/paw) 10
and 30 min before formalin injection, respectively. In an
attempt to determine the possible participation of CBS
and CSE in the NaHS-induced nociceptive response,
increasing doses of the inhibitors HA (0.001–100 lg/paw) and PPG (0.03–300 lg/paw) were administered
10 min before formalin injection. In non-diabetic rats,
0.5% formalin was used in experiments where the test
drug was anticipated to augment the response, whereas
1% formalin was administered when antinociceptive
effect was anticipated, as previously reported (Doak and
M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797 789
Sawynok, 1997; Rocha-Gonzalez et al., 2005;
Castaneda-Corral et al., 2009). On the other hand, in
diabetic rats, 0.5% formalin was used for H2S donor
drugs and H2S-producing enzyme inhibitors due this
formalin concentration let us determine in this metabolic
condition pro- and antinociceptive effects, respectively,
as previously validated (Torres-Lopez et al., 2007). To
determine whether drugs acted locally, the greatest
tested dose of drugs was administered individually to
the left paw (contralateral) whereas formalin was
injected into the right paw (ipsilateral), and the flinching
behavior was assessed. To establish whether L-
cysteine-induced pronociception was mediated by CBS
and CSE activation in normoglycemic and
hyperglycemic (2 weeks) rats, the highest dose tested of
L-cysteine (100 lg/paw, �30 min) was administered with
an ineffective dose of either the CBS inhibitor
hydroxylamine (�10 min) or CSE inhibitor PPG
(�10 min). For the study of allodynia, diabetic rats (9–
11 weeks) received the subcutaneous administration of
vehicle (isotonic saline solution), PPG (30 lg/paw) or
HA (0.1 lg/paw), and mechanical withdrawal threshold
was measured every 30 min during 8 h. Inhibitors were
chosen by their affinity on the studied enzymes whereas
L-cysteine and NaHS are the endogenous and
exogenous precursors of H2S, respectively (Alexander
et al., 2009). All doses of those drugs and NaHS were
selected from pilot experiments performed in our
laboratory. Rats in all groups were observed regarding
behavioral or motor function changes induced by the
treatments. This was assessed, but not quantified, by
testing the ability of animals to stand and walk in a
normal posture.
Fig. 1. (A) Time courses of the flinching behavior induced by
subcutaneous (s.c.) injection of 0.5% formalin to normoglycemic
(white circles) and hyperglycemic (black circles) rats. (B, C) Bar plots
show formalin-induced hyperalgesic effect during phase 2, but not
during phase 1, in hyperglycemic rats administered with 50 mg/kg
Data analysis and statistics
All results are presented as means ± standard error of
the mean (S.E.M.) for at least six animals per group.
Temporal courses were constructed plotting the number
of flinches as a function of time. Dose–response curves
and bar graphs were made from the area under the
number of flinches against time curves obtained by the
trapezoidal rule. In addition, data were transformed to %
nociception to determine effective dose 30 (ED30), the
area under the curve obtained with 0.5% or 1% formalin
was considered as 100% of nociception. ED30 and 95%
confidence intervals were calculated as is described by
Tallarida (2000). On the other hand, endogenous levels
of H2S obtained in the different tissues are presented as
means ± S.E.M. for three animals per group. In all
experiments, one-way analysis of variance (ANOVA)
followed by the Dunnett’s test was used to compare all
treatments with respect to the control group. Differences
were considered statistically significant when P< 0.05.
(i.p.) of streptozotocin 2 weeks before. Data are expressed as themean ± S.E.M. of six or seven animals per group. ⁄Significantlydifferent (P< 0.05) from normoglycemic group, as determined by
Student’s test. AUC: area under the curve, i.p.: intraperitoneal.
RESULTSFormalin-evoked flinching behavior in non-diabeticand diabetic rats
Streptozotocin, but not distilled water, injection caused
hyperglycemia. The blood glucose level measured in
these rats was 70.7 ± 4.1 and 65 ± 1.9 mg/dL before
distilled water or streptozotocin injection, and 76.3 ± 4.4
790 M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797
and 570.5 ± 7.1 mg/dL 2 weeks after distilled water or
streptozotocin injection, respectively. Furthermore, these
rats had an increase in food and water intake, showed
polyuria and did not gain body weight (data not shown).
Both the non-diabetic and diabetic (2 weeks) groups
exposed to 1% or 0.5% formalin, respectively, exhibited
a typical biphasic pattern of flinching. Phase 1 of the
nociceptive response began immediately after formalin
administration and then declined gradually in
approximately 10 min. Phase 2 began about 15 min
after formalin administration and lasted about 1 h, as
previously was reported (Wheeler-Aceto et al., 1990).
Formalin-evoked flinching was increased in
hyperglycemic rats respect to normoglycemic animals
(Fig. 1A). The overall analysis of formalin-evoked
nociceptive behavior as area under curve (AUC),
showed that increment in the flinching frequency of
hyperglycemic rats was in the second phase (P< 0.05),
but not during first phase, of the test (Fig. 1B, C). Since
streptozotocin mainly increased nociception during
second phase of the formalin test, in the following
studies only second phase was further analyzed.
Fig. 2. Pronociceptive effect induced by local peripheral administration of Na
rats submitted to the 0.5% formalin test (0.5% F). Data are expressed as t⁄Significantly different from the control group (P< 0.05), as determined b
administration of the highest tested dose for each drug, 0.5% F: control grou
Peripheral effect of NaHS and L-cysteine on formalin-induced pain in streptozotocin-diabetic and non-diabetic rats
The role of H2S in the behavioral response to
subcutaneous injection of 0.5% formalin was
investigated by the injection of NaHS (H2S exogenous
donor) and L-cysteine (H2S endogenous donor).
Peripheral injection of the NaHS significantly increased
in a dose-dependent manner (P< 0.05) the nociceptive
behavior induced by formalin during phase 2 in
normoglycemic (10, 100 and 300 lg/paw) and
hyperglycemic (0.1, 1 and 10 lg/paw) rats (Fig. 2A, C).
Moreover, NaHS induced-pronociceptive effect was local
since the greatest dose administered (300 lg/paw)ipsilateral, but not contralateral, augmented significantly
the flinching behavior. Considering that approximately
30% of NaHS is endogenously formed to H2S, the ED30
(30% pronociception) of this gas in normoglycemic and
hyperglycemic rats was 8.5 ± 2.2 and 0.98 ± 3.6 lg/paw, respectively. In other words, H2S was almost ten
times more potent in diabetic rats compared to non-
diabetic rats.
HS and L-cysteine in normoglycemic (A, B) and hyperglycemic (C, D)
he mean ± S.E.M. of six or seven animals per experimental group.
y analysis of variance followed by Dunnett’s test. CL: contralateral
p and s.c.: subcutaneous administration.
Fig. 3. Effect of local peripheral administration of DL-propargylglycine (PPG, a CSE inhibitor) and hydroxylamine (HA, a CBS inhibitor) in
normoglycemic (A, B) and hyperglycemic (C, D) rats submitted to the 1% or 0.5% formalin test (1% F or 0.5% F), respectively. Data are expressed
as the mean ± S.E.M. of six or seven animals per experimental group. ⁄Significantly different from the control group (P < 0.05), as determined by
analysis of variance followed by Dunnett’s test. CL: contralateral administration of the highest tested dose for each drug, 0.5% F or 1% F: control
group and s.c.: subcutaneous administration.
M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797 791
On the other hand, the ipsilateral, but not contralateral,
injection of exogenous L-cysteine (10, 30 and 100 lg/paw,�30 min) augmented in a dose-dependent manner
(P<0.05) the nociceptive behavior induced by 0.5%
formalin during phase 2 of the test in non-diabetic and
streptozotocin-diabetic rats (Fig. 2B, D). In both groups
the ED30 was similar, 12.6 ± 2.3 lg/paw for normogly-
cemic rats and 15.6 ± 3.5 lg/paw for hyperglycemic rats.
It is fair to say that the vehicle without formalin gave
areas under the curve of 12.9 ± 2.5 and 30.3 ± 2.2 for
the phases 1 and 2, respectively. So the differences in the
area under the curve obtained with the different
treatments for the phase 2 were due to pharmacological
treatment but not to the used vehicle.
Peripheral effect of PPG and HA on formalin-inducedflinching behavior in normoglycemic andhyperglycemic rats
To determine the participation of CBS and CSE on
formalin-induced pain in non-diabetic and diabetic rats,
inhibitors of CSE and CBS, HA and PPG, respectively,
were administered 10 min before formalin injection.
Comparison of H2S-producing enzyme inhibitors was
made using 1% formalin in non-diabetic rats and 0.5%
formalin in diabetic rats due pain intensity and duration
in 0.5% formalin-treated diabetic rats is similar to that
observed in non-diabetic rats injected with 1% formalin.
In non-diabetic rats, local peripheral ipsilateral, but not
contralateral, injection of HA (1, 10 and 100 lg/paw)and PPG (30, 100 and 300 lg/paw), reduced
significantly (P< 0.05) 1% formalin-induced flinching
during phase 2 (Fig. 3A, B). In these groups, ED30 (30%
antinociception) for HA and PPG was 7.1 ± 3.6 and
163.4 ± 37.1 lg/paw, respectively.Conversely, pre-treatment of HA (1, 10 and 100 ng/
paw) and PPG in hyperglycemic rats (0.03, 0.3 and
30 lg/paw, �10 min) injected in the dorsum of the right
hind paw reduced significantly 0.5% formalin-induced
nociceptive behavior during phase 2 (Fig. 3C, D). In
these animals, HA gave an ED30 = 5.6 ± 2.3 ng/paw
whereas PPG had an ED30 = 77.1 ± 80 ng/paw. Both
HA and PPG were around three order of magnitude
more potent in the diabetic group compared to non-
diabetic group.
792 M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797
PPG and HA reverses L-cysteine-inducedhyperalgesia in the formalin test
To assess the local participation of either CBS or CSE
enzymes on the pronociceptive activity of L-cysteine in
the 0.5% formalin test was performed the following
protocol in non-diabetic and diabetic groups. 100 lg/paw of L-cysteine were injected in the ipsilateral hind
paw 30 min before 0.5% formalin administration, 20 min
after L-cysteine injection, an ineffective dose of PPG or
HA was administered at the same site of L-cysteine
injection. Local peripheral pronociceptive effect of
exogenous L-cysteine (100 lg/paw) was significantly
reduced by local peripheral injection of the CBS inhibitor
HA and CSE inhibitor PPG in the formalin test
(Fig. 4A–D).
Antiallodynic effect of DL-propargylglycine andhydroxylamine in streptozotocin-diabetic rats
In order to confirm the antineuropathic effect of PPG and
HA, streptozotocin-diabetic rats were evaluated with von
Fig. 4. Effect of the inhibitors DL-propargylgycine (PPG, a CSE inhibitor
pronociception during phase 2 of 0.5% formalin test in normoglycemic (A, B)
effect by themselves at the tested dose for its administration with L-cysteine. D
of six or seven animals per group. ⁄Significantly different (P < 0.05) from c
cysteine group (100 Cist), as determined by one-way ANOVA followed by th
Frey filaments. Hyperglycemic rats developed tactile
allodynia between 9 and 11 weeks after streptozotocin
injection. In marked contrast, non-diabetic animals
remained with a normal threshold during the 11 weeks
(Fig. 5). No autotomy behavior was ever observed
during the experiment. On this condition, subcutaneous
administration of PPG (30 lg/paw) or HA (0.1 lg/paw),but not vehicle, increased the withdrawal threshold in
diabetic rats. The maximal antiallodynic effect was
reached at 2 h and it lasted for 8 h for PPG and 6 h for
HA at the dose tested.
Effect of streptozotocin injection on H2Sconcentration along nociceptive pathway
Based on the results obtained with donors and enzymatic
inhibitors of H2S in normoglycemic and hyperglycemic
animals, we considered of interest to determine if the
H2S concentration was altered during steptozotocin-
induced diabetes development along nociceptive
pathway. Measurements were conducted in naıve and
) and hydroxylamine (HA, a CBS inhibitor) on L-cysteine-induced
and hyperglycemic (C, D) rats. Inhibitors did not show any nociceptive
ata are expressed in bars as average of the % nociception ± S.E.M.
ontrol group (0.5% F) and #significantly different (P< 0.05) from L-
e Dunnett’s test. 100 Cist: 100 lg/paw of L-cysteine.
Fig. 5. Time course of the antiallodynic effect observed after acute
subcutaneous administration of 30 lg/paw DL-propargylglycine
(PPG) or 0.1 lg/paw hydroxylamine (HA) in rats administered with
streptozotocin (50 mg/kg, i.p.) 9–11 weeks before experiment (white
and black triangles, respectively). The non-diabetic group (gray
circles) is placed as a reference of the maximum possible effect,
whereas vehicle group shows that antiallodynic effect is induced by
PPG or HA, but not by vehicle (white circles). Data are presented as
mean ± S.E.M. for six animals. PPG group was significantly different
(P< 0.05) from control group (diabetic) during 8 h, whereas HA
group was significantly different (P< 0.05) from control group from
0.5 to 6 h by two-way ANOVA followed by Bonferroni’s test. Asterisks
indicating significant differences are omitted for the sake of clarity.
M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797 793
hyperglycemic rats with 2 or 9–11 weeks after
streptozotocin injection. H2S concentration decreased in
Fig. 6. H2S concentration obtained from plasma (A), dorsal root ganglion (L
during hyperalgesia (After 2 weeks) and allodynia (After 9–11 weeks) develo
11 weeks before, respectively. Data of H2S concentration are presented as th
tissue (B–E) obtained of at least three independent experiments where ea
(P< 0.05) from control group (naive), as was determined by one-way ANO
a significant manner (P< 0.05) as compared to non-
diabetic animals in dorsal root ganglia (L4–L6), sciatic
nerve and spinal cord, but not in paw or blood plasma,
after streptozotocin injection. The maximum reduction
was reached after 2 weeks in sciatic nerve and spinal
cord and was time-dependant in dorsal root ganglia.
Furthermore, H2S concentration had a tendency to
decrease with neuropathy development in the paw, but
was not statistically significant (Fig. 6).
DISCUSSION
In the current study, we have shown that NaHS, a H2S
exogenous donor, was able to increase formalin-induced
nociceptive behavior either normoglycemic or
streptozotocin-diabetic rats. According to our data, it has
been reported that local peripheral administration of
NaHS produces a pronociceptive effect in paw pressure
(Kawabata et al., 2007; Maeda et al., 2009), formalin
test (Lee et al., 2008), neuropathy induced by L5 spinal
nerve cutting (Takahashi et al., 2010) and visceral
hyperalgesia pain models (Nishimura et al., 2009; Xu
et al., 2009). However, to our knowledge, this is the first
report about the hyperalgesic effect of NaHS in
peripheral neuropathy associated to diabetes. The
pronociceptive effect of NaHS has been widely
explained through activation and sensitization of T-type
Ca2+ channels in several pain models (Maeda et al.,
2009; Matsunami et al., 2009; Nishimura et al., 2009;
Takahashi et al., 2010; Okubo et al., 2011) and
4–L6) (B), sciatic nerve (C), dorsal spinal cord (D) and hindpaw (E)
pment induced by administration of 50 mg/kg streptozotocin 2 and 9–
e mean ± S.E.M. of nmol of H2S/mL of plasma (A) or nmol H2S/ mg of
ch experiment is a tissue mix of three rats. ⁄Significantly different
VA followed by the Dunnett’s test.
794 M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797
inhibition of sustained potassium channel currents (Feng
et al., 2013). In addition, it has been also reported that
NaHS induces phosphorylation of extracellular signal-
regulated kinase (Fukushima et al., 2010) and increases
synthesis of pro-inflammatory cytokines through this
pathway (Zhi et al., 2007). Likewise, several reports
have suggested that H2S might stimulate the release of
neuropeptides from capsaicin-sensitive nociceptive
fibers and activates other ionic channels as NMDA,
TRPA1 and TRPV1 (Kimura, 2000; Eto et al., 2002;
Patacchini et al., 2004, 2005; Trevisani et al., 2005;
Macpherson et al., 2007).
Interestingly, in the current study, dose–response
curves of NaHS indicate that hyperglycemic rats are
more sensitive to H2S than normoglycemic rats since
NaHS had a pronociceptive ED30 10 times lower in
hyperglycemic rats. These results agree with a
preliminary study that proposed that H2S has a stimulus
intensity-dependent pronociceptive effect (Lee et al.,
2008). Consequently, dorsal administration in the right
hind paw of L-cysteine also increased flinching behavior
in normoglycemic and hyperglycemic rats. This result is
in line with the pronociceptive effect observed in the
paw pressure model in rats after intraplantar
administration of L-cysteine (Kawabata et al., 2007),
although this drug seems to have an antinociceptive
effect in visceral pain (Distrutti et al., 2006). Differences
between pronociceptive and antinociceptive effect of L-
cysteine may be attributed to pain model (inflammatory
versus visceral) or administration route (local versus
systemic), but further studies are necessary to clarify its
role in the processing of nociception.
In biological systems, it is well-known that H2S is
mainly synthesized from L-cysteine by CBS and CSE. In
the current study, PPG and HA, inhibitors of CSE and
CBS, respectively, abolished the chemical hyperalgesia
and allodynia tactile induced by hyperglycemia in
streptozotocin-induced diabetic rats, as well as
inflammatory pain in non-diabetic rats, suggesting the
participation of these two H2S-producing enzymes in the
diabetes-induced peripheral neuropathy and formalin-
induced acute pain in non-diabetic rats, but it should
keep in mind that HA not only inhibits CBS enzyme, but
also has other non-specific mechanisms, which could
explain antinociceptive effect observed, such as release
of nitric oxide (Antoine et al., 1996; Hervera et al., 2011)
or activation of A1 receptors (Fowler et al., 1999; Gong
et al., 2010). In order to exclude other possible
mechanisms, these two enzymatic inhibitors were given
in an ineffective dose with the endogenous donor, L-
cystein; in these experiments, both inhibitors were able
to prevent the hyperalgesic effect of L-cysteine in
streptozotocin-induced diabetes and non-diabetic rats,
confirming the probable participation of CBS and CSE in
inflammatory pain and hyperglycemia-induced peripheral
neuropathy. In the literature, the participation of CSE
and CBS has been documented in different pain
paradigms. CSE is the most studied H2S-producing
enzyme and its inhibition by PPG or b-cyanoalaninesuppressed hyperalgesia and allodynia in inflammatory
and neuropathic pain (Kawabata et al., 2007; Lee et al.,
2008; Nishimura et al., 2009; Takahashi et al., 2010;
Okubo et al., 2011). On the other hand, CBS is
expressed in approximately 85% of nociceptive neurons
innervating colon and 77.3% of neurons from trigeminal
ganglion (Xu et al., 2009; Feng et al., 2013).
Furthermore, its nociceptive participation has been
evaluated in visceral pain models (Xu et al., 2009;
Wang et al., 2012; Qu et al., 2013) with similar results
to those obtained in the current study.
As with H2S, both HA and PPG reached
antinociceptive ED30 three order of magnitude lower in
diabetic compared to non-diabetic rats. These data
suggest the participation of two main H2S-producing
enzymes in the chemical and mechanical
hypersensitivity observed in diabetes by a possible up-
regulation or an increased activity of both enzymes
during peripheral neuropathy development. These
findings are consequent with a study performed in
diabetic rats submitted to gastric balloon distension,
where is showed that up-regulation of CBS by NF-jbcontributes to gastric hypersensitivity (Zhang et al.,
2013). In addition, up-regulation of CBS and CSE has
been reported after administration of caerulein into the
pancreatic duct or acetic acid in the colon, respectively
(Nishimura et al., 2009; Xu et al., 2009). Regarding to
their activity, it has been published that streptozotocin-
induced diabetes is associated with enhanced tissue
hydrogen sulfide biosynthesis (Yusuf et al., 2005). In
that study, authors reported that H2S formation in
pancreas and liver was increased in diabetic rats and
both CSE and CBS mRNAs were increased in liver of
these animals. Similar results were found with CBS
mRNA in pancreas, although the activity of these
enzymes was not measured in the nervous system.
A growing body of evidence suggests that
hypersensitivity observed with H2S could be due to that
this gasotransmitter significantly enhanced currents of
depolarizing cation channels and inhibits currents of
hyperpolarizing channels. In this regard, some studies
point out that H2S significantly enhances currents
related T-type Ca2+ channels in dorsal root ganglion
neurons (Matsunami et al., 2009) and it up-regulates
CaV3.2 T-type Ca2+ channels in neuropathic pain
(Takahashi et al., 2010). Remarkably, T-type calcium
channels facilitate activity- and calcium-dependent long-
term potentiation at synapses from nociceptive nerve
fibers and inhibit nociceptive behavior of both phases of
formalin test (Ikeda et al., 2003; Cheng et al., 2007).
Furthermore, recent evidence in visceral inflammation
models point out that sensitization and up-regulation of
voltage-gated NaV1.7 and NaV1.8 channels in colon-
specific dorsal root ganglion neurons is dependent of
activity and expression of CBS (Wang et al., 2012; Qu
et al., 2013). Consequently, H2S also suppresses
sustained Kv1.1 and Kv1.4 potassium channel currents
in trigeminal ganglion neurons (Feng et al., 2013). In
this way, we think that H2S induces a hypersensitivity
state in primary afferent fibers and spinal nociceptive
neurons during peripheral neuropathy development
through sensitization of depolarizing channels and
inhibition of hyperpolarizing channels. Notwithstanding,
M. E. Velasco-Xolalpa et al. / Neuroscience 250 (2013) 786–797 795
future experiments are necessary to confirm if H2S-
induced sensitization in neurons during diabetic
neuropathy development is due to an increment in the
expression or activity of CSE and CBS enzymes. At this
moment, in the laboratory are performing preliminary
experiments to determine activity and expression of
both enzyme in diabetic rats.
Contrary to the expected result, H2S concentrations
were decreased in a time-dependent manner during
diabetic peripheral neuropathy development in sciatic
nerve, dorsal root ganglia and spinal cord, but not
plasma. Moreover, H2S concentration in the paw had a
tendency to decrease during peripheral neuropathy
development. These data suggest that under
physiological conditions H2S is present in sites related
to nociceptive processing and that nerve injury
development induced by diabetes leads a negative
feedback on the concentration of this gas along
nociceptive pathway. Actually, our results are supported
by a study performed in guinea pigs with allergic rhinitis,
where authors demonstrated that H2S concentration is
negatively correlated with the process of inflammation
and positively correlated with expression of CSE
(Shaoqing et al., 2009). In this sense, the experiments
performed in the current study, in which an ineffective
dose of PPG or HA is able to reduce the L-cistein-
induced hyperalgesic effect and the left shift in the
dose–response curve of both inhibitors in diabetic rats,
suggest that both enzymes are present in the diabetes-
associated peripheral neuropathy with a low activity
level. Furthermore, we believe that it is not necessary a
high concentration of H2S in diabetic rats to generate
neuropathic pain since hypersensitivity state originated
by this gas along nociceptive pathway might allow that
little changes in the concentration of H2S may be able to
generate greater nociceptive responses.
In a similar way, the decrement of H2S in the spinal
cord in the current study could also help to explain the
hypersensitive state in diabetic rats. In this regard, a
previous study showed that the H2S amount in the
spinal cord also decreased in a negative manner of
noxious stimuli given in the hindpaw (Lee et al., 2008),
suggesting that H2S concentration decrease with a
greater stimulus in the spinal cord. Here, a lot of
evidence supports a prominent role of glial cells in the
maintenance of neuropathic pain states, and it has been
reported that H2S inhibits the activation of microglia and
astrocytes in the central nervous system (Hu et al.,
2007). Hereof, the reduction of H2S in the spinal cord
during diabetes development may facility microglia
activation, leading a hypersensitivity state in neuropathic
rats.
In summary, our findings show a pronociceptive effect
of H2S and the participation of its two main producing
enzymes in the streptozotocin-induced peripheral
neuropathy. In addition, data suggest that hypergly-
cemic rats are more sensitive to H2S-induced
hyperalgesia than normoglycemic rats. Taken together
our results suggest that inhibition of CBS or CSE may
be an important target for the treatment of peripheral
neuropathy associated to diabetes.
AUTHOR’S CONTRIBUTION
All authors have read and approved the final manuscript.
H.I.R.-G. designed, performed, and supervised the
experiments, analyzed the data, prepared the figures
and wrote the manuscript. M.E.V.-X., P.B.-I., E.R.-C.,
B.G.-C. and C.I.A.-S. performed the experiments.
F.J.F.-M., J.E.T.-L. and A.-N. coordinated the project,
helped to interpreted the data and edited the manuscript.
Acknowledgments—Mario Emmanuel Velasco-Xolalpa, Paulino
Barragan-Iglesias and Beatriz Godınez-Chaparro are Conacyt
fellows. This work is part of the PhD dissertation of Eduardo
Roa-Coria and BSc. dissertation of Mario Emmanuel Velasco-
Xolalpa. This work was partially supported by Conacyt 154880
(H.I.R.-G.) and SIP 20113892 (H.I.R.-G.) grants.
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GLOSSARY
H2S: hydrogen sulfide
CBS: cystathionine b-synthaseCSE: cystathionine c-lyaseHA: hydroxylamine
PPG: DL-propargylglycine
(Accepted 23 June 2013)(Available online 2 July 2013)
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