Involvement of cellular prion protein in the nociceptive response in mice

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Involvement of cellular prion protein in the nociceptive response in mice Flavia Carla Meotti c,1 , Cristiane Lima Carqueja b,1 , Vinícius de Maria Gadotti a , Carla I. Tasca b , Roger Walz d,e , Adair R.S. Santos a,⁎ a

Departamento de Ciências Fisiológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brasil Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brasil c Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, 97110-000, Brasil d Departamento de Clínica Médica, Hospital Universitário, Universidade Federal, de Santa Catarina – UFSC, Florianópolis, SC, 88.040-970, Brasil e Centro de Cirurgia de Epilepsia, Hospital Governador Celso Ramos, Florianópolis, SC, 88.015-270, Brasil b

A R T I C LE I N FO

AB S T R A C T

Article history:

The role of the cellular prion protein (PrPc) in neuronal functioning includes neuronal

Accepted 8 March 2007

excitability, cellular adhesion, neurite outgrowth and maintenance. Here we investigated

Available online 13 March 2007

the putative involvement of the PrPc function on the nociceptive response using PrPc null (Prnp0/0) and wild-type (Prnp+/+) mice submitted to thermal and chemical models of

Keywords:

nociception. PrPc null mice were more resistant than wild-type mice to thermal

Cellular prion protein

nociception of the tail-flick test. However, no significant difference was found on the hot

Thermal and chemical nociception

plate test. In the acetic acid-induced visceral nociception, PrPc null mice showed an

Inflammation

enhanced response when compared to wild-type mice. However, there was no difference between Prnp0/0 and wild-type mice on glutamate- and formalin-induced licking behaviour and Freund's Complete Adjuvant (FCA)-induced mechanical allodynia. PrPc null mice developed significantly lower paw edema than wild-type mice. In addition, the visceral conditioning stimuli produced by a previous injection of acetic acid (20 days before testing) significantly reduced early and late phases of formalin-induced nociception in wild-type mice. In contrast, the same pre-treatment did not alter the formalin response in PrPc null mice. These results indicate a role of PrPc in the nociceptive transmission, including the thermal tail-flick test and visceral inflammatory nociception (acetic acid-induced abdominal constriction). Our findings show that PrPc is involved with a response mediated by inflammation (paw edema) and by visceral conditioning stimuli. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Cellular prion protein (PrPc) is a glycosyl-phosphatidylinositol anchored cell surface glycoprotein that is mainly expressed in

neurons and, to a lesser extent, in other types of cells. An alteration in the PrPc secondary structure, with a much higher proportion of beta-sheet conformational domains, leads to an accumulation of the abnormal protein prion scrapie. This

⁎ Corresponding author. Fax: +55 48 37219672. E-mail address: [email protected] (A.R.S. Santos). 1 Both authors have contributed equally to this study. 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.024

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results in spongiform encephalopathy, in humans and animals (Prusiner, 1998). Recent studies have investigated the physiological function of PrPc (Martins et al., 2002; Walz et al., 2002). Animals in which the PrPc gene (Prnp) was constitutively ablated presented an enhance in the neuronal excitability in vitro, either in constitutively or post-natal PrPc null mice (Walz et al., 2002; Colling et al., 1996; Collinge et al., 1994; Maglio et al., 2004; Mallucci et al., 2002). These animals also showed a higher sensitivity to seizures in vivo (Walz et al., 1999). The clearance of extracellular glutamate through astrocytic uptake was decreased in PrPc null mice (Brown and Mohn, 1999). However, PrPc is not directly related to neuronal glutamate uptake or release (Thais et al., 2006). PrPc is also implicated in the protection against oxidative stress (Brown, 2001; Klamt et al., 2001), modulation of neuronal apoptosis (Chiarini et al., 2002; Lopes et al., 2005; Zanata et al., 2002), cellular adhesion, neurite outgrowth (Lopes et al., 2005; Graner et al., 2000a) and maintenance (Walz et al., 2002; Graner et al., 2000b). Although most findings concerning PrPc physiology are related to the nervous system, studies have found a role for PrPc in antioxidant protection not only in the brain, but also in the liver, muscle and heart (Klamt et al., 2001). It has recently been shown that PrPc modulates phagocytosis and the inflammatory response in vitro and in vivo (Almeida et al., 2005). Related cases of spongiform encephalopathy in humans reported that patients presented with a progressive increase in pain (Lundberg, 1998; Sugai et al., 2000). Although a higher pain sensibility was attributed to nerve degeneration (Sugai et al., 2000), how the alteration in PrPc structure affects responsiveness to pain is still unclear. In an attempt to approach the PrPc role in pain transmission, the present study investigated the nociceptive response in PrPc null and wild-type mice that were submitted to thermal and chemical models of nociception.

Fig. 1 – Response to thermal noxious stimuli in tail-flick (A) and hot-plate tests (B). The black columns (Prnp+/+) and white columns (Prnp0/0) represent the mean ± SE of seven animals. A one-way ANOVA was performed in A and a two-way ANOVA (considering genotype and temperature) was performed in B. The asterisk denotes significant difference from Prnp+/+ mice by Student's t-test (*P < 0.05).

[F(1,10) = 20.9, P < 0.01], 10 min [F(1,10) = 8.4, P < 0.05] and 15 min [F(1,10) = 28.7, P < 0.001] (Fig. 2). The total amount of abdominal constrictions in 20 min of observation is shown in the inset of Fig. 2 [F(1,10) = 225.5, P < 0.001].

2.3. Mice nociceptive response induced by intraplantar injection of glutamate and formalin The direct activation of peripheral sensorial fibers C by intraplantar injection of glutamate caused a licking behaviour that was similar between PrPc null and wild-type mice [F(1,14) = 0.21, P > 0.05; Fig. 3A]. The intraplantar treatment with formalin exhibited the same profile between both genotype [F(1,14) = 0.47, P > 0.05] in the early phase and [F(1,14) = 0.02, P > 0.05] in the late phase (Fig. 3B).

2.4.

2.

Results

2.1. Thermal nociceptive response to hot-plate and tail-flick test Acute nociceptive response evoked by a noxious heat stimulus in the tail-flick assay displayed different responses between PrPc null and wild-type mice. Fig. 1A illustrates that PrPc null mice were more resistant than wild-type mice to the thermal stimulus [F(1,11) = 7.1, P < 0.05]. No significant differences were found between PrPc null and wild-type mice on the hot-plate nociceptive heat stimulus (50.5, 55.0 and 58.0 °C) [F(1,36) = 3.73, P > 0.05]. Two-way analysis of variance (ANOVA) revealed no significant interaction between temperature and genotype [F(2,36) = 0.48, P > 0.05] (Fig 1B).

2.2.

Abdominal constriction induced by acetic acid

The response to acetic acid-induced visceral nociception was increased in PrPc null mice in the first 20 min after acetic acid administration. The number of abdominal constrictions was significantly different among the groups at 5 min

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Paw edema and allodynia induced by FCA

The intraplantar injection of FCA produced a profound and long-lasting mechanical allodynia in the injected paw of PrPc null and wild-type mice. The allodynia was initiated 2 h after FCA administration and it was maintained for 8 days [F(1,196) = 52.5, P < 0.05, from baseline]. A three-way analysis of variance revealed no interaction between genotype, presence of FCA and time after FCA injection [F(6,196) = 0.04, P > 0.05]. No difference was found between PrPc null and wild-type mice in mechanical allodynia (Fig. 4A). The FCA injection enhanced paw volume (edema) in PrPc null and wild-type mice from 2 h after FCA injection and it was maintained for 8 days [F(1,224) = 492.05, P < 0.05, from baseline]. A three-way analysis of variance revealed no interaction between genotype, presence of FCA and time after FCA injection [F(7,224) = 0.626, P > 0.05]. Edema development was significantly lower in PrPc null than wild-type mice at 12, 48 and 96 h after FCA administration (Fig. 4B).

2.5.

Visceral conditioning stimuli

The treatment of animals with acetic acid (AA 0.6%, 10 ml/kg, i.p.) produces a visceral conditioning stimulus, which reduces

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the late phase. Thus, the response of animals submitted to conditioning stimulus depends on the presence or absence of PrPc.

3.

Fig. 2 – Number of abdominal constriction induced by i.p. injection of acetic acid in wild-type (closed circle) and PrPc null (open circle) mice. Inset: total number of abdominal constrictions in 20 min of observation in wild-type (black column) and PrPc null (white column) mice. The results represent the mean ± SE of six animals. Statistical analysis was performed by one-way repeated-measures ANOVA. The asterisks denote significant difference (**P < 0.01 or ***P < 0.001) from Prnp+/+ mice by Student–Newman–Keuls test.

phase 2 formalin licking behaviour in mice C57BL/6J (Kurihara et al., 2003). Here we observed that either Prnp0/0 or Prnp+/+ mice pre-treated with saline (control group), 20 days before formalin intraplantar injection, presented an ipsilateral licking behaviour. This can be observed over two distinct phases, an early phase (0–5 min) and a late phase (15–30 min) (Figs. 5A and B). The results on Figs. 5A and B demonstrate that pretreatment with acetic acid significantly reduced the formalin response at both, early and late phases in wild-type mice. In contrast, the same pre-treatment did not alter the formalin response in PrPc null mice (Figs. 5A and B). A two-way ANOVA indicated a significant interaction between acetic acid pretreatment (conditioning stimulus) and Prnp gene disruption [F (1,21) = 6.38, P < 0.05] for the early and [F(1,21) = 5.32, P < 0.05] for

Discussion

The function of PrPc is not well established. However, previous studies have demonstrated a correlation between PrPc dysfunction and enhancement of neuronal excitability, higher sensitivity to seizures, oxidative damage and cellular growth disorders (Walz et al., 1999; Graner et al., 2000a; Brown, 2001; Klamt et al., 2001; Lopes et al., 2005). It has been assumed that the PrPc-dependent signaling may contribute to cell homeostasis and participate in the fine-tuning of neuronal neurotransmitter-associated functions (Mouillet-Richard et al., 2005). Therefore, the involvement of PrPc on the nociceptionintegrated system is biologically plausible. Nociceptive transmission comprises a network of neuronal arrangement distributed between peripheral, spinal and supra-spinal levels. This occurs through the action of neurotransmitters, neuromodulators and intracellular messengers (Julius and Basbaum, 2001; Ji and Stricharstz, 2004). In this study, we have demonstrated that PrPc is needed in the transmission of thermal and chemical nociception in mice. PrPc null mice showed significant analgesia when compared to wildtype controls in the tail-flick test. However, the absence of PrPc protein did not alter latency to response in the hot-plate test. This different may occur because behavioural responses (paw licking and jumping) produced by the hot-plate test are considered to be supraspinally integrated reactions, while the tail-flick test corresponds to a reflex action from the spinal medulla (Chapman et al., 1985; Sinclair et al., 1988; Le Bars et al., 2001). Conversely, in the acetic acid-induced visceral nociception, PrPc null mice were more sensitive to nociception than wildtype controls. Acetic acid administration activates resident macrophages and mast cells in the abdominal cavity, causing release of endogenous inflammatory mediators, including bradykinin, substance P, prostanoids and cytokines (TNF-α, IL1β, IL-8) (Collier et al., 1968; Ribeiro et al., 2000). Therefore, the

Fig. 3 – Nociceptive response to intraplantar injection of 20 μl of glutamate (10 μmol/paw) (A) or formalin (2.5%, 0.92% formaldehyde) (B). Nociceptive response was estimated as total time mice spent licking hindpaw in 15 min after glutamate; 0–5 min (early phase) and 15–30 min (late phase) after formalin administration. Each column represents the mean ± SE of eight mice. Statistical analysis were performed by one-way ANOVA followed by post hoc Student's t-test.

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Fig. 4 – Allodynia (A) and paw edema (B) induced by i.pl. injection of 20 μl FCA in wild-type (closed circle) and PrPc null (open circle) mice. The results represent the mean ± SE of eight animals. Statistical analysis was performed by three-way repeated-measures ANOVA (considering genotype, FCA and time after FCA injection). The asterisk denotes significant difference from Prnp+/+ by Student–Newman–Keuls test (*P < 0.05).

nociception induced by acetic acid is greatly dependent on macrophages and mast cells. It has been postulated that mice lacking the PrPc had an augmentation in macrophage-dependent phagocytosis. In these mice, an intraperitoneal administration of chemicals caused an increased migration of macrophages (Almeida et al., 2005). Our data agree with these studies since the absence of PrPc caused an increase in the number of abdominal constrictions, possibly associated with greater activation of macrophages in PrPc null mice. In contrast to the effect of PrPc upon macrophage activation, this protein was reported to exert a positive effect upon polymorphonuclear cell (neutrophils and eosinophils) recruitment (Almeida et al., 2005). Since edema induced by FCA is mainly due to polymorphonuclear cells infiltration (Meotti et al., 2006), our results at FCA-induced paw edema corroborate with those found by Almeida et al. (2005). They demonstrated that PrPc null mice had a lower infiltration of polymorphonuclear cells than wild-type mice. Although FCA administration increased the sensitivity in response to an innocuous stimulus (mechanical allodynia), there was no significant difference between wild-type and PrPc null mice. This was an unexpected because inflamma-

tion (estimated by edema) was more pronounced in wildtype mice. Similarly to mechanical allodynia, nociceptive response caused by direct sensitization of C fibres, through an intraplantar injection of glutamate or formalin (early phase), showed the same profile in wild-type and PrPc null mice. Interestingly, the absence of PrPc did not affect the late phase of nociception triggered by formalin. Similarly to acetic acid model, the late phase of the formalin test is also an inflammatory-mediated response. In acetic acid and formalin nociception excitatory neurotransmitter are released into the dorsal horn and activating intracellular kinases (Malmberg and Yaksh, 1995; Feng et al., 2003; Galan et al., 2003; Svensson et al., 2006). The peripheral inflammatory response caused by acetic acid is mediated by macrophages and mast cells, while that caused by formalin is mainly mediated by mast cells (Ribeiro et al., 2000; Parada et al., 2001). Therefore, a differential role of PrPc in controlling activation of macrophages should be considered. Indeed, Almeida et al. (2005) demonstrated a different pattern of leucocytes activation in PrPc null mice. However, investigations of PrPc effects on mast cell activation are necessary to test this hypothesis.

Fig. 5 – Effect of i.pl. injection of formalin in wild-type and PrPc null mice submitted to acetic acid conditioned stimulus in the first (A) and second (B) phases of formalin. The columns represent mean ± SE of eight animals pre-treated with saline i.p. (white columns) or acetic acid i.p. (gray columns). Statistical analysis was performed by two-way ANOVA (considering genotype and acetic acid treatment). The asterisk denotes significant difference (*P < 0.05) from non-conditioned mice and hash symbol denotes significant difference (#P < 0.05) from Prnp0/0 conditioned mice by Student–Newman–Keuls test.

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The visceral conditioning stimuli data provides evidence that PrPc is required by nociceptive transmission. We have observed that acetic acid administration, 20 days before formalin injection caused a reduction in the formalin-induced nociceptive response (somatic stimulus) in wild-type mice. This result corroborates previous findings that injection with acetic acid results in a long-lasting (2 to 3 weeks) inhibition of somatic inflammatory pain in C57BL/6J mice (Kurihara et al., 2003). Conversely, PrPc null mice did not display such effect. It has been postulated that antinociception provoked by visceral conditioning stimuli are dependent on the serotonergic system in the spinal cord (Kurihara et al., 2003). Hence, activation of 5-HT2 receptors in the spinal cord produce antinociception through depolarization of the inhibitory interneurons, such as GABAergic and/or glycinergic neurons (Sugiyama and Huang, 1995; Millan, 1997; Khasabov et al., 1999). Thus, an involvement of the cellular prion protein in the serotonergic system may be speculated. Previous studies carried out in cell cultures have demonstrated that PrPc modulates the coupling and cross-talk of serotonergic receptors (Mouillet-Richard et al., 2005). Therefore, mice devoid of PrPc present with a disturbance in serotonergic system activation might have an alteration in the control of pain. However, further studies are required to clarify this point. In conclusion, the results of the present study have demonstrated the role of PrPc in the nociceptive transmission mechanisms. This approach allowed the in vivo observation of an interaction of PrPc and visceral conditioning stimuli. The mechanisms of macrophage-dependent acute inflammatory pain, inflammatory peripheral edema, and reflex response from spinal medulla to thermal nociception are closely related to PrPc function.

4.

Experimental procedure

4.1.

Animals

A total of 52 adult male knockout mice (3 months old, weighing 20–30 g) homozygous for disrupted PrPc gene, PrnP (designated Prnp0/0 mice) produced as previously described (Bueler et al., 1992) were used and 52 male wild-type (Prnp+/+) mice of the same age and weight were used as controls. The Prnp0/0 mice were descendants of Zrch I animals (Bueler et al., 1992). Wild-type controls were generated by crossing F1 descendants from a 129/Sv × C57BL/6J maintained for more than 10 years at the Ludwig Institute for Cancer Research (São Paulo Branch). To confirm the genotype of the animals we used PCR procedures with DNA extracted from the tail. The reactions were performed with an annealing temperature of 60 °C in 35 cycles using the following primers: forward (5′-ATCAGTCATCATGGCGAAC-3′) and reverse (5′-AGAGAATTCTCAGCTGGATCTTCTCCCGTC-3′). A band of 693 bp corresponds to the Prnp sequence in the wild-type animals, while a band of 1635 bp represents the neomycin cassette which replaced the Prnp. Thus identifying the Prnp0/0 mice. Animals were housed five to a cage (30 × 20 × 15 cm) with food and water ad libitum. They were kept in 12 h light/dark cycles (lights on at 7:00 a.m.) at the temperature of 23 ± 1 °C. The experiments reported were carried out in accordance with the current guidelines for the

care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals as specified by Zimmermann (1983). The number of animals and the intensity of noxious stimuli used were the minimum necessary to demonstrate consistent effects of the nociceptive response.

4.2.

Materials

Acetic acid and formaldehyde were obtained from Merck (Darmstadt, Germany). Sodium chloride (NaCl), glutamate and Freund's Complete Adjuvant (FCA) were obtained from Sigma (St. Louis, USA). Drugs were dissolved in a sterile solution of 0.9% of NaCl.

4.3.

Hot-plate and tail-flick tests

Thermal sensitivity of Prnp0/0 mice was evaluated employing two different heat stimuli, hot plate and tail flick tests. The response latencies on the hot-plate test were measured according to the method described by Woolfe and MacDonald (1944), with minor modifications. The apparatus (Ugo Basile, model–DS 37) was maintained at 50.5, 55.0 or 58.0 °C. Animals were placed into a glass cylinder of 24 cm diameter on the heated surface, and the time between placement and jumping, shaking or licking of paws was recorded as the index of response latency. Each animal was submitted to two trials with an interval of more than 30 min between the first and second trials. The average of these trials was used to quantify latency. An automatic 30-s cut-off was used to prevent tissue damage. In the tail-flick test a radiant heat analgesiometer was used to measure response latencies according to the method described previously by D'Amour and Smith (1941), with minor modifications. Animals responded to a focused heat stimulus (90 W) by flicking or removing their tail, exposing a photocell in the apparatus immediately below it. An automatic 30-s cutoff was used to minimize tissue damage. Three measurements were done (3 trials) at each time and the mean was used to quantify latency.

4.4.

Acetic acid-induced abdominal constriction

The abdominal constrictions were induced according to procedures described previously (Collier et al., 1968). It results in the contraction of the abdominal muscle together with a stretching of the hind limbs in response to an intraperitoneal (i.p.) injection of acetic acid (0.6 %, 0.45 ml/mouse). After i.p. acetic acid injection, mice were individually placed into glass cylinders of 20 cm diameter and the abdominal constrictions were recorded cumulatively every 5 min, during a 60-min period.

4.5.

Peripheral excitatory amino acid-induced paw licking

The licking behaviour was examined immediately after the glutamate hind paw injection. The procedure used was similar to that described previously (Beirith et al., 2002). A volume of 20 μl of glutamate (10 μmol/paw), prepared in saline, was injected intraplantarly into the right hind paw. Animals were

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observed individually for 15 min following the glutamate injection. The amount of time spent licking the injected paw was timed and considered as indicative of nociception.

4.6.

Formalin-induced nociception

The procedure used was essentially similar to that described previously (Hunskaar and Hole, 1987). Animals were injected intraplantarly with 20 μl of 2.5% formalin solution (0.92% of formaldehyde), made up in saline solution (137 mM NaCl). Mice were immediately placed in a glass cylinder 20 cm in diameter and observed from 0 to 30 min following formalin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The first phase of the nociceptive response normally peaked 5 min after the formalin injection and the second phase 15 to 30 min after the formalin injection, representing the neurogenic and inflammatory nociceptive responses, respectively (Hunskaar and Hole, 1987).

4.7.

FCA-induced paw edema and mechanical allodynia

Mice received 20 μl of Freund's Complete Adjuvant (FCA, 1 mg/ml of heat killed Mycobacterium tuberculosis in 85% paraffin oil and 15% mannide monoleate), subcutaneously in the intraplantar (i.pl.) surface of the right hind paw. Effects were evaluated against the paw edema and mechanical allodynia. Paw edema was measured by use of a plethysmometer (Ugo Basile) at several time-points (baseline, 2, 4, 8, 12, 24, 48, 96 and 192 h). The paw volume was expressed in μl and indicated the degree of inflammation. The mechanical allodynia was measured as 50% paw withdrawal threshold testing, in response to 6 applications of Von Frey Hair (VFH, Stoelting, Chicago, USA) as described before (Chaplan et al., 1994). The frequency of withdrawal was determined before (baseline) FCA injection, in order to obtain data purely derived from the treatments of FCA allodynia. The mechanical allodynia was recorded at 2, 4, 8, 12, 24, 48 and 192 h after FCA injection.

4.8.

Visceral conditioning stimuli

PrPc null and wild-type mice were treated with acetic acid (0.6%, 0.45 ml), by i.p. route for inducing a visceral conditioning stimulus (Kurihara et al., 2003). Three weeks after acetic acid or saline (control group) injection, we examined the somatic noxious stimulus induced by intraplantar injection of 20 μl of formalin 2.5% (formaldehyde 0.92%) in the right hind paw. The observation chamber was a glass cylinder of 20 cm diameter and each animal was placed in the chamber for 5 min before formalin injection. Immediately after formalin administration, the time mice spent licking the injected paw was considered indicative of nociception. The nociceptive response (licking time) was recorded at 0–5 min (early or acute phase) and 15–30 min (late or tonic phase) after formalin injection (Hunskaar and Hole, 1987).

4.9.

Statistical analyses

The data are expressed as mean ± SE from six to eight animals. A one-way analysis of variance (ANOVA) was performed for

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tail-flick, formalin and glutamate tests. A one-way analysis of variance with repeated measures was carried out for acetic acid-induced abdominal constriction. A three-way repeatedmeasure analysis of variance was performed for paw edema and allodynia induced by FCA (considering genotype, presence of FCA and time after FCA administration). A two-way analysis of variance was performed for hot plate (considering genotype and temperature) and for the visceral conditioning stimulus test (considering genotype and acetic acid treatment). Analyses were followed by post-hoc Student–Newman–Keuls test where appropriate. Differences between groups were considered significant when P < 0.05.

Acknowledgments This work was supported by grants from FAPESP, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa de Apoio aos Núcleos de Excelência (PRONEX) and Fundação de Apoio à Pesquisa Científica e Tecnológica do Estado de Santa Catarina (FAPESC), Brazil. R. Walz is supported by CNPq (301379/2005-0) and FAPESP (03-13189-2). F.C. Meotti and V.M. Gadotti, Ph.D. students in Biochemical Toxicology and Neuroscience Programs, thank CAPES for fellowship support. The authors thank V.R. Martins, A.M. Hampton and M. Hampton for critical reading of the manuscript.

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