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 the

mean ± 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.

RESULTS

Formalin-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)