ORIGINAL ARTICLE

Interleukin-1 receptor antagonist decreases cerebrospinal fluidnitric oxide levels and increases vasopressin secretion in the latephase of sepsis in rats

Fazal Wahab • Lucas F. Tazinafo • Evelin C. Carnio •

Fabio A. Aguila • Marcelo E. Batalhao •

Maria Jose A. Rocha

Received: 23 August 2014 / Accepted: 9 October 2014

� Springer Science+Business Media New York 2014

Abstract The aim of this study was to analyze the effect

of IL-1ra (an Interleukin-1 receptor antagonist) on sepsis-

induced alterations in vasopressin (AVP) and nitric oxide

(NO) levels. In addition, IL-1ra effect on the hypothalamic

nitric oxide synthase (NOS) activities and survival rate was

also analyzed. After Wistar rats were intracerebroventric-

ular injected with IL-1ra (9 pmol) or vehicle (PBS

0.01 M), sepsis was induced by cecal-ligation and puncture

(CLP). Blood, CSF, and hypothalamic samples were col-

lected from different groups of rats (n = 8/group) after 4,

6, and 24 h. AVP and NO levels were greatly increased in

CLP. Both total NOS and inducible NOS (iNOS) activities

were also greatly increased in CLP rats. These changes in

AVP, NO, and NOS were not observed in sham-operated

control rats. IL-1ra administration did not alter plasma

AVP levels after 4 and 6 h as compared to vehicle in CLP

animals but after 24 h were significantly (P \ 0.01) higher

in IL-1ra-treated animals. IL-1ra administration signifi-

cantly (P \ 0.01) decreased NO concentration in CSF but

not in plasma. Both total NOS and iNOS activities were

also significantly decreased by IL-1ra at 24 h in CLP ani-

mals. Moreover, the 24 h survival rate of IL-1ra-treated

rats increased by 38 % in comparison to vehicle adminis-

tered animals. The central administration of IL-1ra

increased AVP secretion in the late phase of sepsis which

was beneficial for survival. We believe that one of the

mechanisms for this effect of IL-1ra is through reduction of

NO concentration in CSF and hence lower hypothalamic

iNOS activities in the septic rats.

Keywords IL-1b � iNOS activity � Survival rate �Hypothalamus � Cecal puncture

Introduction

Sepsis is a fatal pathophysiological condition that develops

when the body respond with systemic inflammatory

response to the invading infectious agent (bacteria, fungi,

parasites, or viruses), damaging its own tissues and organs

[1–3]. This response can leads to multiple organ failure,

septic shock, and ultimately death, especially if not rec-

ognized in early stage. In the initial phase of sepsis, occurs

excessive production and release of inflammatory media-

tors, which may directly or indirectly activate the central

nervous system, affecting autonomic and neuroendocrine

functions [1, 4–8]. A biphasic response in the systemic

secretion of arginine vasopressin (AVP) is a major neuro-

endocrine change reported during sepsis [9–11].

In the initial phase of sepsis, there is an increase in the

plasma AVP concentration in an attempt to maintain blood

pressure and tissue perfusion, but in the late phase, release

of this hormone is basal, despite of persistent hypotension,

which ultimately contributes to vasodilatation and organ

dysfunction [9–14]. The causes of this drop in AVP,

despite of hypotension, are not fully clear.

The experimental data, documented until now, suggest

participation of various factors in inducing alterations in

AVP secretion during sepsis. Many studies report a role of

the interleukin-1 (IL-1), a pro-inflammatory cytokine, in

inducing sepsis-associated pathophysiological alterations

F. Wahab � L. F. Tazinafo � F. A. Aguila � M. J. A. Rocha (&)

Department of Morphology, Physiology and Basic Pathology,

School of Dentistry of Ribeirao Preto, Avenida do Cafe s/n CEP,

Ribeirao Preto, SP 14040-904, Brazil

e-mail: mjrocha@forp.usp.br

E. C. Carnio � M. E. Batalhao

Department of Specialized Nursing, Nursing School of Ribeirao

Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil

123

Endocrine

DOI 10.1007/s12020-014-0452-2

in neuroendocrine secretion. IL-1 concentration, peripher-

ally as well as centrally, is greatly increased in both acute

and chronic phase of sepsis [15–17]. In first instance after

induction of sepsis, IL-1 is synthesized and released

peripherally when the entry of microorganisms leads to the

activation of the immune system [17]. This pro-inflam-

matory cytokine reaches the components of the neuroen-

docrine system via various routes [16, 17]. Moreover, IL-1

production can be endogenously induced in the hypothal-

amus by the binding of peripheral IL-1 or other pro-

inflammatory cytokines with their receptors on neuronal

projections in the median eminence [16, 17]. In hypothal-

amus, IL-1 leads to activation of inducible nitric oxide

synthase (NOS) which in turn causes excessive production

of NO and neuronal bioenergetic changes inhibiting vaso-

pressin expression [18]. This inhibition of AVP expression

in the late phase of sepsis, apparently results in reduced

neurohypophyseal stocks and plasma hormone concentra-

tion contributing to hypotension and septic shock [10, 19].

Due to the prominent role of AVP in the vasopressor

response, the elucidation of mechanisms for deficient

hormone synthesis may contribute to therapy for septic

shock, a major cause of death in critical care patients.

Therefore, the specific objective of this study was to

investigate the involvement of IL-1b in the vasopressin

secretion and central production of nitric oxide in animals

with experimental polymicrobial sepsis.

Materials and methods

Animals

Adult Wistar rats, weighing 280 ± 30 g, were used in this

study. These animals were obtained from the Central

Animal Facility at the University of Sao Paulo Campus

Ribeirao Preto and placed in the animal room of the School

of Dentistry of Ribeirao Preto, USP. These animals were

kept in a photoperiod of 12:12 h in a thermostatically

temperature (25 ± 2 �C) control room, with free access to

water and commercial balanced diet. The experimental

protocol of this study was approved by the Ethics Com-

mittee on Animal Use (CEUA), University of Sao Paulo-

Campus de Ribeirao Preto (CEUA protocol number:

12.1.1205.53.0).

Cecal-ligation and puncture surgery

The cecal-ligation and puncture (CLP) of the experimental

rats was carried out for induction of severe sepsis as pre-

viously reported [13]. Briefly, under TBE (tribromoetha-

nol; 2.5 %, 250 mg/kg i.p.; Acros Organics) anesthesia, a

midline laparotomy of rats was done in sterile condition.

The cecum was exposed, ligated below the ileocecal valve

and then punctured 10 times with a sterile 16-gage needle.

After CLP, the abdominal cavity was closed with suture,

followed by a subcutaneous injection of normal saline

(5 mL/250 g; body weight). As a control, some rats were

sham-operated. In these rats, the cecum was manipulated

but neither ligated nor punctured.

Working solution of IL-1ra

Recombinant Interleukin-1 receptor antagonist (IL-1ra)

was purchased from the local agent of R&D systems

(Minneapolis, MN, USA). Stock (54 pmol) and working

(9 pmol) solutions of IL-1ra were prepared in sterile

phosphate buffered saline (PBS; 0.01 M).

Intracerebroventricular cannulation

Under anesthesia induced by a mixture of ketamine and

xylazine, a permanent 22-gage stainless steel guide cannula

(0.7 mm OD, 10 mm long) was stereotaxically implanted into

the right lateral ventricle. Coordinates for the guide cannulae

were 1.6 mm lateral to the midline, 1.5 mm posterior to

bregma, and 2.5 mm below the brain surface. The cannula

was fixed to the skull with screws and dental acrylic. All

animals were given an antibiotic treatment after the surgery. A

5-day recovery period was allowed before experimentation.

Animals showing misplaced, blocked cannula, or abnormal

patterns of weight gain were excluded from the study.

Experimental protocol

Experimental rats were given an intracerebroventricular

(icv) injection of IL-1 receptor antagonist (IL-1ra; 9 pmol

in total volume of 2 lL) or PBS (0.01 M; 2 lL) as vehicle

control. After 15 min of IL-1ra/PBS injection, they were

anesthetized with TBE and then sham-operated or sub-

jected to CLP. After 4, 6, or 24 h of surgery, the cere-

brospinal fluid (CSF) was harvested and subsequently the

rats were decapitated for blood collection and brain

removal. The CSF was used for NO analysis while the

blood was used for determination of IL-1 b, NO, and AVP

concentrations. The hypothalamus was dissected from the

brain and then stored at -70 �C until used for measure-

ment of NOS activities.

Harvesting of cerebrospinal fluid (CSF)

and hypothalamus

A single CSF sample was collected as described by

Consiglio and Lucion [20]. Samples were maintained in the

dark under ice until centrifugation at 1,3009g for 15 min;

thereafter, the supernatant were stored at -80 �C until

Endocrine

123

analysis. When contaminated with blood, samples were

discarded. Immediately after CSF collection, the animals

were killed by decapitation and their brains rapidly

removed. The whole hypothalamus was dissected from

brain using the following limits: the anterior border of optic

chiasm, the anterior border of the mammillary bodies, and

the lateral hypothalamic sulcus, with a depth of 2 mm. The

total dissection time was less than 2 min from decapitation

and then the hypothalamus was stored at -70 �C until

analysis.

Plasma AVP and IL-1 b levels

Plasma AVP and IL-1b levels were evaluated by enzyme-

linked immunosorbent assay (ELISA; Enzo Life Sciences

Inc. Farmingdale NY, USA). The assays were performed

according to the manufacturer’s instructions. For AVP, the

plasma samples (100 lL) were extracted using acetone and

petroleum ether and lyophilized [21] and then reconstituted

with assay buffer. The sensitives of the assays were 3.39

and 5.0 pg/mL for AVP and IL-1b, respectively. The intra-

and inter-assays coefficients of variance were less than

10 %.

Plasma and CSF nitrate determination

The determination of plasma and CSF nitrate was per-

formed by the technique of chemiluminescence NO/ozone.

The procedure was performed using 5 lL of plasma or CSF

deproteinized by incubation with 95 % ethanol at 4 �C for

30 min and then centrifuged for 5 min at 5,0009g. The

samples were injected into a reaction vessel containing a

reducing agent (0.8 % vanadium chloride in HCl 1 N at

95 �C) that converted nitrate to NO in equimolar amounts.

NO was drawn, using helium gas into the chamber of the

Sievers chemiluminescence NO Analyzer. The NO mea-

surement was determined from calculation of its reaction

with ozone, emitting red light. The emitted photons were

detected and converted into an electrical signal. The cur-

rent generated was converted by an analog–digital con-

verter and analyzed by computer. The area under the curve

generated by the electric current was the concentration of

nitrate in the sample. The concentration is calculated by

comparison with a standard curve using known concen-

trations (0, 5, 10, 15, 30, and 60 mmol) of sodium nitrate.

Hypothalamic NOS activity determination

Determination of NOS activity was performed by modi-

fying the citrulline method described by Bredt and Synder

[22]. The hypothalami were removed from brains of

experimental animals. They were sonicated in 200 lL of

homogenization buffer [50 mM Tris, pH 7.4, 3.2 mM

sucrose, 1 mM EDTA, 10 mg/mL Leupeptina, 1 mM

DTT, 2 g/mL aprotinin, 1 mM PMSF, and 10 mg/mL of

protease inhibitor (Roche C11836145001)]. The homog-

enates were centrifuged (10 min, 10,0009g, 4 �C) and

aliquots of supernatant (40 lL) were added to 100 lL of

buffer assay (20 mM HEPES, 1.25 mM CaCl2, 1 mM

DDT, 100 mM tetrahydrobiopterin (BH4), 1 mM

NADPH, and 80.000 cpm [14 C]-L-arginine) and incu-

bated for 1 h at room temperature. The reaction was

stopped with the addition of 1 mL of ion exchange resin

Dowex 50 W, 4 �C, previously activated with NaOH and

diluted in 100 mM HEPES, 10 mM EDTA, pH 5.5. The

resin was removed by centrifugation (10,0009g, 10 min),

400 lL of supernatant containing L-citrulline was added to

3 mL of liquid scintillation (Ecolite/ICN-882,475) and the

radioactivity was determined by beta scintillation counter.

The supernatant was used to determine protein concen-

tration by the method of Brad Ford-Coomassie plus Pro-

tein Assay Reagent (Thermo Scientific-1856210). The

NOS activity was expressed as picomoles [14 C]—L-ci-

truline/mg protein/min.

Statistical analysis

All results are expressed as mean ± SEM. For statistical

analysis of results, we used two ways ANOVA followed by

Bonferroni post hoc tests for each analysis. P values\0.05

is considered statistically significant different in all cases.

4 6 240

100

200

300

400V-ShamIL-1ra-ShamV-CLPIL-1ra-CLP

******

*

**

#**

Time (h)

Mea

n Pl

asm

a IL

-1β

(pg/

mL)

Fig. 1 IL-1b concentration significantly (P \ 0.05–0.005) increased

in CLP animals at all time points. Vehicle injection did not alter

concentrations of IL-1b in both CLP and sham animals. IL-1ra

injection significantly (#P \ 0.05) decreased plasma level of IL-1b at

24 h in CLP rats as compared to vehicle-treated CLP rats

Endocrine

123

Results

Plasma concentration of IL-1b in control and septic

animals after IL-1ra and vehicle injection

Plasma concentration of IL-1b is shown in Fig. 1. There

were no significant differences in plasma IL-1b concen-

tration between vehicle and IL-1ra treatments in sham-

operated animals. IL-1b concentration significantly

(P \ 0.005) increased in vehicle-treated CLP animals at all

time points as compared to control animals. In IL-1ra-

treated CLP animals, plasma level of IL-1b was signifi-

cantly increased at 4 and 6 h as compared to control ani-

mals while significantly (P \ 0.05) lowered at 24 h as

compared to vehicle-treated CLP rats.

Survival rate of septic rats after IL-1ra and vehicle

injection

The survival rate of septic rats treated with IL-1ra was

higher as compared to vehicle-treated CLP rats. The 24 h

survival rate of CLP rats treated with PBS was 48 % while

that of CLP rats treated with 9 pmol of IL-1ra was 82 %

(Fig. 2).

48

82

0

20

40

60

80

100

V-CLP IL-1ra-CLPExperimental Groups

Surv

ival

Rat

e

Fig. 2 IL-1ra administration greatly enhanced 24 h survival rate of

CLP rats in comparison to vehicle-treated CLP rats

4 6 240

10

20

30V-ShamIL-1ra-ShamV-CLPIL-1ra-CLP

*** ***

******

#

***

Time (h)

Mea

n Pl

asm

a A

VP (p

g/m

L)

Fig. 3 Administration of IL-1ra significantly (P \ 0.005) enhanced

24 h plasma AVP levels in CLP animals as compared to vehicle

treatment as well as sham-operated animals. There was no visible

significant effect of IL-1ra and vehicle treatments on the AVP

concentration at post-CLP 4, 6 h and in the sham-operated animals.

***P \ 0.005 increase versus vehicle-sham and IL-1ra-sham while#P \ 0.05–0.005 decrease versus post-CLP (vehicle and IL-1ra) 4 and

6 h and IL-1ra-CLP 24 h. Both vehicle and IL-1ra administration did

not alter plasma AVP after 4 and 6 h as compared to vehicle injection

in CLP animals

4 240

1

2

3

4 Sham-V

V-CLP

* **

#

IL-1ra-CLP

A

Time (h)

iNO

S A

ctiv

ity (p

mol

/mg/

min

)

4 240

1

2

3

V-ShamV-CLPIL-1ra-CLP

**

*#

C

Time (h)

Tota

l NO

S A

ctiv

ity4 24

0.0

0.1

0.2

0.3

0.4

0.5V-ShamV-CLPIL-1ra-CLP

B

Time (h)

Con

stitu

tive

NO

S A

ctiv

ity

(pm

ol/m

g/m

in)

(pm

ol/m

g/m

in)

Fig. 4 Vehicle and IL-1ra administration did not alter NOS activities

in sham-operated animals. In vehicle-treated CLP rats, total NOS and

iNOS activities were significantly (*P \ 0.05) increased at both 4 and

24 h. In IL-1ra-treated CLP rats, both total NOS and iNOS activities

were significantly (#P \ 0.05) lowered at 24 h as compared to vehicle

treatment in CLP rats at same time point. There was no effect of

IL-1ra on constitutive NOS activity

Endocrine

123

Plasma AVP concentration after IL-1ra and vehicle

injection in septic and control animals

Plasma concentration of AVP is shown in Fig. 3. IL-1ra

administration did not alter plasma AVP after 4 and 6 h as

compared to vehicle injection in CLP animals. In contrast,

after 24 h plasma AVP levels were significantly (P \ 0.01)

higher in IL-1ra-treated CLP animals as compared to 24 h

plasma AVP levels of vehicle-treated CLP animals as well

as sham-operated control animals. There were no signifi-

cant differences in plasma AVP concentrations of vehicle-

and IL-1ra-treated sham-operated animals.

Comparison of hypothalamic NOS activity in septic

and control animals after IL-1ra and vehicle

administration

Total, inducible, and constitutive NOS activities are shown

in Fig. 4. After 4 and 24 h of CLP surgery, there was an

increase of total and inducible (iNOS) activities but no

alteration in the constitutive NOS as compared to sham

animals. IL-1ra injections decreased total and inducible

NOS activities but only 24 h following sepsis induction

when compared with the vehicle group that alone had no

effect in CLP rats.

Plasma and CSF nitric oxide concentration in septic

and control animals after IL-1ra and vehicle injection

Plasma and CSF NO concentrations are shown in Fig. 5.

After 6 and 24 h of CLP surgery, both plasma and CSF NO

levels were significantly (P \ 0.01–0.005) increased when

compared to sham-operated animals. IL-1ra administration

did not significantly alter plasma NO levels but at 24 h

CSF NO levels were significantly (P \ 0.02) lowered in

CLP rats as compared to the vehicle group. The injection of

the vehicle alone did not alter CSF or plasma NO levels in

both CLP and sham-operated groups.

Discussion

Many studies have reported alterations in IL-1, NO, and

AVP during sepsis. In good relation to the findings of

previous scientific investigations [10, 13, 16, 18, 23–25], in

the present study, we also observed that plasma AVP

concentration highly increased in first phase of sepsis while

returned to basal in late phase of sepsis. Plasma NO con-

centration was also higher in septic rats at all time points.

Of note for the first time, in this study, we reported gradual

increase of CSF NO concentration, in good relation to

plasma. Moreover, total NOS and iNOS activities were

significantly increased in CLP rats. Plasma concentrations

of IL-1b were also greatly elevated in septic rats as com-

pared to control animals.

Most importantly, our results suggest that the IL-1 sig-

naling is a key pathway for linking inflammatory status

with the hypothalamic-neurohypophyseal system emer-

gency response. Our findings demonstrated that blocking

the IL-1 receptor by central administration of IL-1ra can

revert many of the pathophysiological responses during

sepsis among these is the impaired vasopressin secretion

during late phase of sepsis. In the late phase of sepsis,

4 6 240

50

100

150V-ShamIL-1ra-ShamV-CLPIL-1ra-CLP ***

***

***A

***

Time (h)

Mea

n Pl

asm

a N

O ( μ

M)

4 6 240

10

20

30

40V-ShamIL-1ra-ShamV-CLPIL-1ra-CLP #

***

*

B

Time (h)

Mea

n C

SF N

O ( μ

M)

Fig. 5 Plasma and CSF NO levels were significantly

(P \ 0.01–0.005) increased at post-CLP 6 and 24 h as compared to

sham-operated animals. In CLP rats, IL-1ra administration did not

significantly alter CSF NO concentration at 4 and 6 h as compared to

vehicle treatment. In contrast, at 24 h CSF NO levels were

significantly (P \ 0.02) lowered in IL-1ra-treated CLP animals as

compared to vehicle-treatment in CLP animals at same time point.

There was no statistically significant effect of IL-1ra on plasma NO

levels in both CLP and sham-operated rats

Endocrine

123

despite of severe hypotension, plasma AVP concentrations

drop to basal levels. This drop in AVP concentration is

responsible for poor survival rate of the animals during

severe septic condition. The blockade of the IL-1 signaling

pathway augments plasma AVP secretion in the late phase

of sepsis. This augmentation in plasma AVP is associated

with increased 24 h survival rate of the septic rats as

compared to vehicle-treated rats.

Our investigations give clue to the mechanism of the

IL-1ra effect on the augmentation of plasma AVP and

higher 24 h survival rate of the septic rats. Previous studies

have implicated excessive local hypothalamic production

of IL-1 and NO for many of the neuroendocrine alterations

during sepsis [6, 13, 26–28]. In our results, we also noted

very high levels of NO in plasma and CSF, and the

hypothalamic iNOS activity in CLP septic rats. The

administration of IL-1 receptor antagonist decreases CSF

NO concentration and hypothalamic iNOS activity in the

septic rats.

The mutual communication between the immune system

and the brain take place at the circumventricular organs

(CVOs), which are highly vascularized structures lacking a

blood–brain barrier [16, 24, 29, 30]. The vascular walls of

the CVOs and perivascular glia constitutively express

receptor for IL-1, which, when activated, induces local

production of IL-1b expression [16, 28]. IL-1b [31] and

other pro-apoptotic stimuli like TNFa [32] have been

reported to induce NO synthesis. Furthermore, mitochon-

drial membranes have been noted to generate NO when

treated with various apoptotic stimuli along with mito-

chondrial respiratory dysfunction as an early event of

apoptosis [33]. Indeed, elevated level of NO is one of the

major causes of metabolic hypoxia in tissues. It has been

reported that NO competes with oxygen for binding to

mitochondrial cytochrome C oxidase enzyme. NO inhibits

the cytochrome C oxidase even at physiological concen-

trations [34–36]. High concentrations of NO during sepsis

prevent use of available oxygen thus leading to inhibition

of mitochondrial respiration [28, 35, 36]. Inhibition of

mitochondrial respiration also favors the generation of

superoxide anions [35] and consequently of peroxynitrite

and hydrogen peroxide (H2O2) [37] which may further

stimulate the expression of iNOS [36] and NO production.

The state of hypoxia generated by the increase of NO can

also induce expression of a subunit of the hypoxia-induced

factor 1 (HIF1a) in neurons. Hypoxia inhibits prolyl

hydrolase preventing the hydroxylation of HIF1 a and

decreasing its degradation by the ubiquitin–proteasome

system. The stabilization of HIF1a favors the rapid buildup

and when in the phosphorylated form dimerize with HIF1ß

building the HIF1 [38]. The HIF1 regulates the expression

of several genes related to energy metabolism, but also

induces the expression of caspase 3 and pro-apoptotic

members of BCL2 family [38, 39]. Moreover, NO also

promotes cell death indirectly by the regulation of anti-

apoptotic Bcl-2 family members via the activation of the

ASK1–JNK1 pathway, which leads to BAX/BAK-depen-

dent apoptosis of cell.

Accordingly, we saw recently an increased expression

of receptor of IL-1 (IL-1R1) and IL-1b in the hypo-

thalamus of septic rats that was accompanied by a pro-

gressive increased expression of the iNOS encoding gene

in the SON. These changes were parallel with an

increased expression of HIF1a protein and cytochrome

C, suggesting cellular changes in vasopressinergic mag-

nocellular neurons [18]. Occurrence of apoptosis was

also detected by increased caspase 3 and annexin-V

affinity assay [40].

We then observed in this study that antagonist of IL-1

receptor was able to block the IL-1 pathway by decreasing

IL-1b levels, iNOS activity, and the central production of

NO levels preventing the mitochondrial bioenergetics

changes and/or the apoptosis caused by the gas in the late

phase of sepsis. This recovered the vasopressin secretion

and possibly the blood pressure, increasing the survival of

the septic rats.

Conclusions

Our results have demonstrated that blocking IL-1 signaling

by central administration of receptor antagonist, IL-1ra,

increases AVP secretion in the late phase of sepsis which

may be beneficial for survival. We believe that the mech-

anism for this effect of IL-1ra is through reduction in

hypothalamic iNOS activity and hence diminished NO

production in the hypothalamus of CLP rats.

Acknowledgments The authors thank Nadir Martins Fernandes and

Milene Mantovani for the technical assistant. Fernando Queiroz

Cunha and Jose Antunes Rodrigues provided the infrastructure for the

NOS activity analysis. Financial support from Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP) is gratefully

acknowledged.

Disclosures The authors have nothing to disclose.

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