Peptides 61 (2014) 56–60

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Peptides
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Short communication

Exploring LPS-induced sepsis in rats and mice as a model to study

potential protective effects of the nociceptin/orphanin FQ system
Roisin C. Thomas a , Michael F. Bath a , Cordula M. Stover b , David G. Lambert a,1 ,
Jonathan P. Thompson a,∗,1
a
Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Robert Kilpatrick Clinical
Sciences Building, Leicester Royal Infirmary, Leicester LE1 5WW, United Kingdom
b
Department of Infection, Inflammation and Immunity, University of Leicester, Medical Sciences Building, University Road, Leicester LE1 9HN, United
Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert
Received 2 July 2014 a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental
Received in revised form 14 August 2014 lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6
Accepted 14 August 2014
mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a noci-
Available online 23 August 2014
ceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced
no septic response, whereas 1.2 mg/kg produced a profound response within 5 h. In BALB/c mice, LPS
Keywords:
4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24 h. In Wistar
Nociceptin
Nociceptin/Orphanin FQ rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25 mg/kg resulted in marked
Nociceptin receptor lethargy before 24 h. Splenic interleukin-1␤ mRNA in BALB/c mice, and serum TNF-␣ concentrations in
Sepsis Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control
UFP-101 animals, indicating that LPS had stimulated an inflammatory reaction. IL-1␤ and TNF-␣ concentrations
SB-612111 in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antago-
nists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a
variety of tissues. In these animal models, the dose–response curve for LPS was too steep to allow use
in survival studies and no changes in the N/OFQ system occurred within 24 h. We conclude that LPS-
inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in
sepsis.
© 2014 Elsevier Inc. All rights reserved.

Introduction administration of exogenous N/OFQ increased mortality whilst NOP

receptor blockade significantly reduced mortality [6]. We want
The nociceptin system comprises the nociceptin receptor (NOP) to establish an animal model of sepsis that is suitable to evalu-
and its 17 amino acid peptide ligand nociceptin/orphanin FQ ate both gene expression changes in NOP and ppN/OFQ mRNA,
(N/OFQ), which is cleaved from a precursor protein pre-pro noci- and the effects of nociceptin system modulation on survival. The
ceptin/orphanin FQ (ppN/OFQ) [10]. This system has been linked CLP model has been criticized because of its variability within and
to many aspects of inflammation and sepsis [15] and has been between studies [4,7]. Hence we hypothesized that LPS-induced
shown to modulate the cellular and functional responses to inflam- sepsis would be a suitable and reproducible model in which to
matory stimuli in animals and humans [2,3,11,18]; in a rat caecal study these potential changes, because by using the same dose and
ligation and puncture (CLP) experimental model of sepsis, the serotype of LPS for each experiment, the inherent variability should
be reduced. The aim of these studies was to explore the responses
to different doses of LPS, produce a LPS dose–response curve, and
to examine the effects of NOP antagonists on these responses. We
∗ Corresponding author at: Tel.: +44 01162523132; fax: +44 0116 2523125.
used a peptide antagonist ([Nphe1 , Arg14 , Lys15 ] N/OFQ-NH2 : UFP-
E-mail addresses: [email protected] (R.C. Thomas), [email protected]
101) [5] as it is widely considered to be a reference antagonist for
(M.F. Bath), [email protected] (C.M. Stover), [email protected] (D.G. Lambert),
[email protected] (J.P. Thompson). NOP, though as a peptide there are stability issues. Hence a non-
1
Both the authors are joint senior authors. peptide NOP antagonist, SB-612111, [20] that is pharmacologically

http://dx.doi.org/10.1016/j.peptides.2014.08.009
0196-9781/© 2014 Elsevier Inc. All rights reserved.
 R.C. Thomas et al. / Peptides 61 (2014) 56–60 57

active in a range of behavioral assays was also included in some culled by cervical dislocation after obtaining a blood sample by
experiments [14,17]. cardiac puncture.

Materials and methods Welfare assessment

All experiments were performed in accordance with UK Home Animals were monitored for responses to LPS every 30 min to
Office regulations (project license number 60/4327). Animals were 2 h. All assessments were carried out blinded, by the same assessor.
obtained from Charles River, UK and held in specific pathogen-free An in-house welfare assessment scoring system was used to score
conditions in the University of Leicester’s Central Research Facil- common signs of illness/disease in rodents, including piloerection,
ity for at least one week prior to commencing studies. C57BL/6 orbital tightening and lethargy. Assessments were carried out over
and BALB/c Mice and Wistar rats were fed (ad libitum) on PMI Lab a period of 24 h, at the end of which animals who had not shown
Diet (5FL2) (BCM IPS LTD London) and allowed UV treated water signs of illness in response to LPS were culled, whilst those showing
at all times. Environmental enrichment was provided to all ani- signs of illness were culled upon reaching the severity limit stipu-
mals based on the requirements of the particular mouse/rat strain. lated in the project license. This was determined by the presence
Temperature was maintained at 21 ◦ C and animals were exposed of a high degree of lethargy.
to a 12 h light, 12 h dark cycle with a 15 min ramp up and ramp
down to simulate dusk and dawn. Mice were housed in groups of Measurement of NOP and ppN/OFQ mRNA
five per cage and rats in groups of 3. All animals were randomly
allocated to experimental groups using the online Graphpad® ran- Total RNA extraction and processing
domizer tool [8]. All injections were administered intraperitoneally At the end of each experiment, organs were harvested and stored
or intravenously in a volume of 100 ␮l (mice) or 1 ml (rats) between at −80 ◦ C. The tissues taken from mice were cerebral cortex, cere-
9.30 and 10.30 am. bellum, brainstem, spleen, lung, heart and thymus and the tissues
taken from the rats were cerebral cortex, spleen and heart. Total
RNA was extracted using Tri-reagent® (Sigma–Aldrich, Dorset, UK)
Drugs/solutions
according to the manufacturers’ instructions following homoge-
nization with a TissueRuptor® (QIAGEN). The concentration and
The peptide NOP antagonist UFP-101 was synthesized by the
purity of the mRNA was determined using a NanoDrop ND2000
group of Guerrini and Calò, (University of Ferrara, Italy), as previ-
spectrophotometer. Equal concentrations of mRNA from each sam-
ously described [9] and suspended in phosphate buffered saline
ple were treated to remove any genomic DNA contamination with
(PBS). UFP-101was used in similar doses to previous studies in
a Turbo DNA-free® kit (Life Technologies, UK).
a mg/kg conversion dose for mice [3,6]. The non-peptide NOP
antagonist SB-612111 (Tocris, UK) was dissolved in ethanol and
Reverse transcription
diluted with PBS (final concentration of ethanol in 1 ml injection
Complimentary DNA (cDNA) synthesis was performed using a
∼2.5%). Lipopolysaccharide (LPS) serotype O127:B7 was purchased
High Capacity cDNA Reverse Transcription Kit (Life Technologies,
from Sigma–Aldrich, UK (rat studies) and LPS Serotype O111:B4
UK) according to the manufacturer’s instructions.
was purchased from Enzo Life Sciences (mouse studies). UFP-101
was administered via the intraperitoneal route so that it could be
PCR
included together with the LPS as a single injection. SB-612111
Gene expression of NOP, ppN/OFQ, the inflammatory marker IL-
1 mg/kg was administered intravenously via the tail vein 15 min
1␤ and reference genes were assessed using TaqMan® probes (Life
before the LPS inoculation. The dose and route of injection for SB-
Technologies, UK). Assays were performed on the StepOne real time
612111 has been shown to be effective in a range of behavioral
PCR system. Expression levels of the target genes were normalized
assays in mice [14,20]. Phosphate buffered saline (137 mM NaCl,
to the endogenous reference genes ␤-actin (all mouse tissues), ELF1
2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ) was used in con-
and POP4 (rat spleen), Ywhaz and HPRT1 (rat cortex), and ELF1 and
trols (administered intraperitoneally where UFP-101 was used and
HPRT (rat heart). The expression stability of the reference genes
intravenously where SB-612111 was used).
used in the rat PCR assays was assessed by analyzing the reference
gene data with two algorithms: GeNorm [19] and Normfinder [1].
Animal studies The geometric mean value of the two most stable genes in each
tissue was used for normalization.
C57BL/6 mice
In a series of small pilot experiments the dose response to mg/kg ELISA
LPS 0.004–4 ± UFP-101 2 ng (∼80 ng/kg) was assessed in male and
female C57BL/6 mice (∼25 g). At the end of the experiment mice To measure the extent of the inflammatory response in rats,
were culled by cervical dislocation (n = 19). TNF-␣ was measured in serum obtained from the blood of LPS-
treated rats (15 or 25 mg/kg LPS). Analysis was performed using a
BALB/c mice DuoSet® ELISA kit (R&D Systems, Abingdon, UK) according to the
In a second series of experiments the dose response to LPS 3, 7 or manufacturers’ instructions.
10 mg/kg ± UFP-101 0.03 or 0.3 mg/kg was assessed in male BALB/c
mice (∼25 g) (n = 36). Mice were allocated to three experimental Results
groups: (A) LPS, (B) LPS + UFP-101, and (C) PBS (control). At the end
of the experiment mice were culled by cervical dislocation. Effects of LPS on survival

Wistar rats C57BL/6 mice

To assess the dose response to LPS in rats, male Wistar rats LPS at a dose of 0.85 mg/kg did not produce a septic response,
(∼250 g) were administered with either LPS 15 mg/kg ± SB-612111 whereas an increase in dose to 1.2 mg/kg resulted in all animals
1 mg/kg or LPS 25 mg/kg ± UFP-101 0.03 mg (n = 30). At the end of reaching the welfare severity limit before 5 h (Fig. 1A). This dose
the experiment rats were anaesthetized with isoflurane (3%) and response was too steep to allow use in survival studies and there
58 R.C. Thomas et al. / Peptides 61 (2014) 56–60

Fig. 1. Effects of LPS on survival and interleukin-1␤ in mice and rats. (A) Survival of C57BL/6 mice following administration of 0.004–4 mg/kg LPS ± 2 ng (∼80 ng/kg) UFP-101
(UFP-101 treatment made no difference to survival so is not represented as a separate group). (B) Survival of BALB/c mice following administration of 4–10 mg/kg LPS and
0.03–0.3 mg/kg UFP-101. (C) Levels of IL-1␤ in the spleen of LPS treated BALB/c mice (7 mg/kg (1) and (2) refer to two separate experiments). Median and range shown (n = 3).
(D) Survival of Wistar rats following administration of 15 ± 1 mg/kg SB-612111 (first three columns) and 25 mg/kg LPS ± 0.03 mg/kg UFP-101. Data expressed as median
(range).

was a suggestion of strain differences [16] so we changed to use there were median fold changes of 1.05 after LPS 4 mg/kg, 1.90–2.12
BALB/c mice. after 7 mg/kg, (in the in the two studies where this dose was used)
and 25.83 after LPS10 mg/kg. (Fig. 1C)
BALB/c mice
LPS 4 mg/kg (±UFP-101) did not produce a septic response.
Doses of 7 and 10 mg/kg (±UFP-101; 0.03 or 0.3 mg/kg) produced TNF-˛
severe responses such that the severity limit was reached before There was a significant increase in TNF-␣ concentrations com-
24 h. No differences were noted in survival times between LPS- pared to controls after injection of LPS 25 mg/kg. The low number
treated and LPS + UFP-101-treated groups (Fig. 1B). Therefore we of rats that responded to LPS at a dose of 15 mg/kg meant that
abandoned using mouse as a species and moved to use rats. although an increase in TNF-␣ concentration was observed in rats
that reached the severity limit, the difference was not statisti-
Wistar rats cally significant compared to controls. No significant differences
Administration of 15 mg/kg LPS did not produce a septic occurred between groups that had received LPS alone and groups
response in three out of five treated rats and in three out that had received LPS + a NOP antagonist at either dose of LPS
of five LPS + SB-612111 treated rats. LPS at 25 mg/kg ± UFP-101 (Table 1).
0.03 mg/kg resulted in 10/10 rats reaching the severity limit by 12 h.
There was no difference in the survival times between groups that
had received LPS alone and groups that had received LPS + a NOP PCR
antagonist (Fig. 1D)
NOP and ppN/OFQ mRNA was detected in the cerebral cortex of
Effects of LPS on inflammatory cytokine levels Wistar rats. ppN/OFQ was detected in the heart and spleen but NOP
was undetected in these tissues (Table 1). NOP and ppN/OFQ mRNA
IL-1ˇ was detected in the cerebral cortex, brain stem and cerebellum of all
To assess the extent of the inflammatory response to LPS, IL-1␤ mice, whereas the remaining tissues showed very low/no expres-
mRNA concentrations were measured in the spleen of BALB/c mice sion (data not shown). No significant differences in expression of
by qPCR. In the LPS-treated groups (relative to control animals), NOP or ppN/OFQ mRNA between groups were observed.
 R.C. Thomas et al. / Peptides 61 (2014) 56–60 59

Table 1
ELISA and qPCR results. (A) TNF-␣ concentrations in serum obtained from rats treated with either 15 mg/kg or 25 mg/kg LPS ± a NOP antagonist (controls received saline)
n = 5 per group. *There was a significant increase in TNF-␣ concentration in the LPS-treated group relative to the control group, p < 0.05. Data expressed as median (range). (B)
qPCR of NOP and ppN/OFQ in cortex, spleen and heart of Wistar rats. Ct values for two reference genes, (geometric mean calculated), and the genes of interest were detected.
Median Ct values (and range) for each group are shown. ND, not determined.

Control (saline) LPS LPS + UFP-101 LPS + SB-612111

(A) TNF− ␣ (pg/ml)

15 mg/kg LPS 0 (0, 0) 0 (0–441.7) ND ND 0 (0–131.2)
25 mg/kg LPS 0 (0, 0) *223.2 (0–3978.0) 350.1 (41.1–3397.0) ND

(B) PCR (Ct )

Cortex (15 mg/kg LPS) NOP 6.77 (6.54–6.92) 6.61 (5.87–6.66) ND 6.21 (6.08–7.00)
ppN/OFQ 7.89 (7.55–8.33) 7.75 (7.52–8.12) ND 7.70 (6.96–8.13)

Cortex (25 mg/kg LPS) NOP 9.14 (8.83–9.33) 8.60 (8.00–9.25) 8.54 (8.44–8.84) ND
ppN/OFQ 7.89 (7.55–8.33) 7.75 (7.52–8.12) 7.70 (6.96–8.13) ND

Spleen (15 mg/kg LPS) NOP 12.98 (12.62–14.09) 14.94 (14.66–16.30) ND 14.64 (13.95–16.71)
ppN/OFQ 10.64 (8.41–11.47) 10.11 (8.92–10.35) ND 9.61 (7.62–10.10)

Spleen (25 mg/kg LPS) NOP Undetected Undetected Undetected ND

ppN/OFQ 10.52 (10.25–11.36) 9.90 (9.17–12.47) 10.42 (9.78–11.24) ND

Heart (15 mg/kg LPS) NOP Undetected Undetected ND Undetected

ppN/OFQ 7.22 (6.61–9.13) 6.73 (6.48–8.91) ND 8.46 (7.15–9.09)

Heart (25 mg/kg LPS) NOP Undetected Undetected Undetected ND

ppN/OFQ 9.07 (7.85–9.55) 8.83 (7.48–10.43) 7.61 (5.85–9.17) ND

Discussion to identify genes suited for normalization, applied to bladder and colon cancer
data sets. Cancer Res 2004;64:5245–50.
[2] Brookes ZL, Stedman EN, Brown NJ, Hebbes CP, Guerrini R, Calò G,
In these studies, the administration of LPS in C57BL/6 mice, et al. The nociceptin/orphanin FQ receptor antagonist UFP-101 reduces
BALB/c mice and Wistar rats produced a steep dose-response curve microvascular inflammation to lipopolysaccharide in vivo. PLOS ONE 2013;8:
so that at lower doses animals showed little or no signs of ill- e74943.
[3] Brookes ZL, Stedman EN, Guerrini R, Lawton BK, Calo G, Lambert DG. Proinflam-
ness 24 h after treatment, but at higher doses the animals reached matory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric
the severity limit determined by our project license before 24 h microcirculation are mediated by histamine. Am J Physiol Heart Circ Physiol
had passed. Increases in pro-inflammatory cytokine concentrations 2007;293:H2977–85.
[4] Buras JA, Holzmann B, Sitkovsky M. Animal models of sepsis: setting the stage.
confirm that an inflammatory response was produced in LPS-
Nat Rev Drug Discov 2005;4:854–65.
treated animals; however administration of LPS had no effect on [5] Calo G, Rizzi A, Rizzi D, Bigoni R, Guerrini R, Marzola G, et al.
the gene expression of NOP and ppN/OFQ. One reason for this could [Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antag-
onist of the nociceptin/orphanin FQ receptor. Br J Pharmacol 2002;136:
be that the changes that occur in the nociceptin system are occur-
303–11.
ring at a time after the animals in our experiments have reached the [6] Carvalho D, Petronilho F, Vuolo F, Machado RA, Constantino L, Guerrini R,
permitted limit of illness severity. In vitro LPS-treated splenocytes et al. The nociceptin/orphanin FQ-NOP receptor antagonist effects on an animal
show a significant increase in N/OFQ secretion compared to con- model of sepsis. Intensive Care Med 2008;34:2284–90.
[7] Gonnert FA, Recknagel P, Seidel M, Jbeily N, Dahlke K, Bockmeyer CL, et al.
trols at 24 h after stimulation [12] and unpublished in vitro work Characteristics of clinical sepsis reflected in a reliable and reproducible rodent
from our lab found upregulation of ppN/OFQ mRNA in a precursor sepsis model. J Surg Res 2011;170:e123–34.
eosinophilic cell line did not occur until 48 h post LPS-treatment. A [8] GraphPad QuickCalcs. http://www.graphpad.com/quickcalcs/randMenu/
[9] Guerrini R, Calo G, Rizzi A, Bianchi C, Lazarus LH, Salvadori S, et al. Address and
recent study using a CLP model of sepsis in rats found increased pp message sequences for the nociceptin receptor: a structure-activity study of
N/OFQ mRNA at 72 h, and the authors suggested that a model of 5–7 nociceptin-(1-13)-peptide amide. J Med Chem 1997;40:1789–93.
days might be required [11]. Another reason for a lack of response [10] Lambert DG. The nociceptin/orphanin FQ receptor: a target with broad thera-
peutic potential. Nat Rev Drug Discov 2008;7:694–710.
in the nociceptin system could be that LPS-induced sepsis does not [11] Laufenberg LJ, Weller GE, Lang CH, Ruiz-Velasco V. Nociceptin receptor
activate the relevant pathways or transcription factors involved in signaling in sympathetic neurons from septic rats. J Surg Res 2013;184:
upregulation of NOP and ppN/OFQ transcripts. Furthermore, the 973–80.
[12] Miller TR, Fulford AJ. Regulation of nociceptin/orphaninFQ secretion
use of LPS as a model has been criticized for failing to reproduce
by immune cells and functional modulation of interleukin-2. Peptides
physiological events that occur in human sepsis [13] and is there- 2007;28:2243–52.
fore not considered as clinically relevant as other models. For these [13] Nemzek JA, Hugunin KM, Opp MR. Modeling sepsis in the laboratory: merging
sound science with animal well-being. Comp Med 2008;58:120.
reasons, we recommend that in vivo research into the nociceptin
[14] Rizzi A, Gavioli EC, Marzola G, Spagnolo B, Zucchini S, Ciccocioppo R, et al.
system during sepsis employs the use of a longer term polymicro- Pharmacological characterization of the nociceptin/orphanin FQ receptor
bial model of sepsis. Our future plan is to evaluate a model which antagonist SB-612111 [(−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-
induces sepsis via intraperitoneal injection of a fecal slurry as this 1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vivo studies. J
Pharmacol Exp Ther 2007;321:968–74.
has been proposed as a more reliable and reproducible model than [15] Serrano-Gomez A, Thompson J, Lambert D. Nociceptin/orphanin FQ in inflam-
CLP [7]. mation and sepsis. Br J Anaesth 2011;106:6–12.
[16] Shen DF, Buggage RR, Eng HC, Chan C-C. Cytokine gene expression in different
Acknowledgment strains of mice with endotoxin-induced uveitis (EIU). Ocul Immunol Inflamm
2000;8:221–5.
[17] Spagnolo B, Carrà G, Fantin M, Fischetti C, Hebbes C, McDonald J, et al.
This work was funded entirely from within our Department, Pharmacological characterization of the nociceptin/orphanin FQ receptor
with no external funding. antagonist SB-612111 [(−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-
1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vitro studies.
J Pharmacol Exp Ther 2007;321:961–7.
References [18] Thompson JP, Serrano-Gomez A, McDonald J, Ladak N, Bowrey S, Lambert
DG. The nociceptin/orphanin FQ system is modulated in patients admitted
[1] Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative to ICU with sepsis and after cardiopulmonary bypass. PLOS ONE 2013;8:
reverse transcription-PCR data: a model-based variance estimation approach e76682.
60 R.C. Thomas et al. / Peptides 61 (2014) 56–60

[19] Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Glossary

et al. Accurate normalization of real-time quantitative RT-PCR data by geo-
metric averaging of multiple internal control genes. Genome Biol 2002;3,
NOP: The nociceptin/orphanin FQ receptor. G-protein coupled receptor, structurally
research0034.
similar to opioid receptors.
[20] Zaratin PF, Petrone G, Sbacchi M, Garnier M, Fossati C, Petrillo
N/OFQ: A 17 amino acid peptide nociceptin/orphanin FQ (N/OFQ). Endogenous ligand
P, et al. Modification of nociception and morphine tolerance
by the selective opiate receptor-like orphan receptor antagonist for the nociceptin receptor.
(−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9- ppN/OFQ: Precursor protein from which N/OFQ is cleaved.
tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J Pharmacol Exp Ther LPS: Lipopolysaccharide component of gram negative bacterial cell wall which leads
2004;308:454–61. to activation of immune cells.