Neonatal arthritis disturbs sleep and behaviour of adult rat offspring and their dams

European Journal of Pain 14 (2010) 985–991

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Neonatal arthritis disturbs sleep and behaviour of adult rat offspring and their dams Suely Roizenblatt a,1, Monica L. Andersen a,*,1, Magda Bignotto a, Vania D’Almeida b, Paulo J.F. Martins a, Sergio Tufik a a b

Department of Psychobiology, Universidade Federal de São Paulo (UNIFESP), Brazil Department of Biosciences, Universidade Federal de São Paulo (UNIFESP), Brazil

a r t i c l e

i n f o

Article history: Received 14 October 2009 Received in revised form 10 February 2010 Accepted 16 March 2010 Available online 18 April 2010 Keywords: Maternal behaviour Pain Sleep Prolactin Corticosterone EEG Anxiety Hot plate Serotonin Arthritis

a b s t r a c t This study aims to evaluate the impact of neonatal arthritis on adult pain threshold, sleep and general behaviours in rats and their lactating dams. Male pups were injected in the hind paw with complete Freund’s adjuvant or saline on postnatal day (PN) 1. After weaning, dams were tested for anxiety, sleep recording or hormone profiling (ACTH, corticosterone and prolactin) and brain sampling (pineal melatonin and hippocampus serotonin). At adulthood (PN90), distinct subgroups of neonatal arthritic (AR) and control rats (CR) were also assessed for anxiety and pain thresholds, sleep recording, and blood/brain sampling. Compared to their respective controls at PN12, dams of arthritic rats (DAR) showed a longer latency in expressing pup retrieval and dam–pup interaction. DAR and AR showed a lower pain threshold, anxiety-like behaviour, and sleep fragmentation. Compared to controls, DAR displayed longer sleep latency, reduced paradoxical sleep latency and sleep efficiency, a decrease in prolactin and serotonin levels and increased melatonin levels. This model of unilateral hindpaw inflammation has a wide range of long-term effects in both lactating dams and their adult offspring. Ó 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

1. Introduction As recently pointed out by Butkevich et al. (2009), the infant stage of rat development is very important for the correction of adverse consequences produced by negative prenatal events. Pain experience during this period is a potent stressor that elicits physiological and behavioural responses (Stevens et al., 2000) with long-term consequences on the ability of the adult offspring to cope with stress (Levine, 2002; Walker et al., 2003). Neural plasticity during the neonatal period was reported by inducing neonatal arthritis by injection of complete Freund’s adjuvant (CFA) into the hindpaw of neonatal rats (Ruda et al., 2000; Tachibana et al., 2001). Enhanced maternal care has been implicated for long-term hyporeactivity to stress following short-term local inflammatory insult in neonatal rats (Lidow et al., 2001; Wang et al., 2004). Indeed, maintenance of maternal behaviour depends on the responses of the litter to a variety of stimuli from the dam (Wei et al., 2010 and reference therein). Although stress responses are

* Corresponding author. Address: Department of Psychobiology, Universidade Federal de São Paulo, Rua Napoleão de Barros, 925, Vila Clementino. SP-04024-002, São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (M.L. Andersen). 1 The first two authors contributed equally to this study.

noticeably reduced during lactation, developing rodent pups place substantial demand on their dams particularly during the postpartum period. The ability to move toward the maternal ventrum and nest bedding, and vocalisation in the ultrasonic range instigate the dam to search, retrieve, give care and provide access to her ventrum to the pups. Maternal behaviour is a dynamic interactive process for the neonate with its mother and the environment, and dams expend considerable energy in retrieving pups with disabled locomotion for nest building and other maternal behaviours. Our group has investigated the bidirectional relationship between disordered sleep and pain in animal models and clinical conditions (Andersen and Tufik, 2003, Andersen et al., 2006, Andersen et al., 2009; Roizenblatt et al., 2001; Schütz et al., 2003; Nascimento et al., 2007; Silva et al., 2008). For example, it has been demonstrated that arthritis induced by Freund’s adjuvant injected into rat hind paws causes reduced sleep efficiency, slow wave sleep and paradoxical sleep, as well as increased latency to the first sleep episode and arousal events (Andersen and Tufik, 2000). The importance of the maternal-pup interaction lies in the prevention of early insults during the neonatal period. Such detrimental experiences can lead to long-term abnormal behaviours, particularly in terms of reactivity to pain and stress (Ren et al., 2004). However, satisfactory animal models for studying the impact of impaired maternal care-giving practices on the physiology and behaviour of the mother are limited. In the present study, we

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hypothesised that exposure of the dam to disabled offspring would yield impaired maternal behaviour and behavioural alterations in both dams and adult rats. To address the consequences of dysfunctional, fragmented maternal behaviour in the dam, we applied the paradigm of neonatal arthritis to its offspring. The endpoint of this prospective, controlled study was to compare maternal anxiety, pain threshold and sleep patterns between dams of arthritic versus saline injected offspring. The secondary outcomes were to evaluate the same parameters in both groups of offspring and to analyse the hormonal profile of the dams. 2. Methods 2.1. Animals All procedures used in the present study complied with the Guidelines for the Care and Use of Laboratory Animals. We obtained 58 nulliparous and pregnant Wistar rats (90-day old) from our animal breeding facility and housed them individually in plastic cages covered with soft sawdust. They were provided with chow and water ad libitum. The entire study was conducted under a controlled 12 h light–dark cycle (lights on at 7 a.m.) and room temperature (22 ± 1 °C) in the animal facility of the Department of Psychobiology at the Universidade Federal de São Paulo. 2.2. Study design This study was conducted as a prospective, controlled analysis. Dams were assigned to one of the following groups: dams of arthritic rats (DAR), in which pups were injected with complete Freund’s adjuvant (CFA) in the left hind paw; and control dams (CD), in which the offspring of were injected with saline using the same procedure (control rats – CR). The original female parent was always included (six newborn male rats per dam were allowed). Each group was comprised of 29 dams: a set of 10 dams was subjected to the maternal behaviour test on PN12 and pain threshold evaluation on PN30; another set of 10 dams was used for 48-h sleep recording; and the remaining nine dams were used for blood and brain sampling at PN30 (Fig. 1). Post-partum day 0 corresponded to the day on which newborn litters were found. On postnatal day 1 (PN1), litters were culled, when necessary, to six male pups within 12 h of birth and the pups were allowed to stay with each dam during the 21-day nursing period. At PN1, the littermates were assigned to two study groups: the arthritic pup rats (AR) injected with CFA in the left hind paw; and control rats (CR), in which saline was similarly injected in the left hind paw. The sample size ranged from 9 to 10 animals per group. After reaching adulthood (PN90), the rats (n = 31/group) were distributed into a series of experiments. In the first series, anxiety and pain thresholds were measured using the elevated plus maze and hot-plate test (also performed at PN30, n = 10). In a second series, consisting of 10 rats each, we measured sleep patterns. In the third series of experiments, nine rats from each group were euthanised for blood and brain sampling. Additionally, two pups from each group were euthanised (PN30) to allow histological analysis of the inflammatory process in the injected paw. 2.3. Arthritis induction in the offspring On PN1, conscious pups were gently restrained by the experimenter and injected in the left hind paw with 10 ll of sterile saline or CFA containing 6 mg/ml of denatured Mycobacterium butyricum suspended in mineral oil (Sigma, USA). The intraplantar subcutaneous injection was performed between 9–11 a.m. with a 30-gauge needle.

2.4. Behavioural tests 2.4.1. Maternal behaviour On PN12, at 9 a.m., pups were removed from their home cages and placed in a similar clean cage in an adjacent room. After a 10-min period, the pups were returned to the dam’s cage, and distributed throughout the cage. Then, dam–litter interactions were continuously monitored for 60 min by the same experimenter, who was blinded to the group assignment and located approximately 1 m away from the cage. Latency to the following maternal behaviours was calculated as the time elapsed from a stimulus to engagement in maternal behaviour for an uninterrupted period of 5 s: (a) retrieval behaviour; (b) other dam-pup interactions. The duration of dam-pup interactions was also assessed, and if the dam did not attract the pups during the 60 min period, the behaviour was categorised as ‘‘ignoring’’ the litter.

2.4.2. Elevated plus maze test The plus maze consisted of two open arms (28.5  7.0 cm) and two closed arms (50  10  40 cm) arranged perpendicularly. The maze was elevated 50 cm above the floor. Each rat was placed in the centre of the apparatus and the number of entries and time spent in open and closed arms were recorded for 5 min. Percent time spent in the open arms was calculated by the formula: [time in open arms/(time in open arms + time in closed arms)]  100. Anxiety-like behaviour of dams at PN30 and adult offspring at PN90 was measured by the plus maze. Reduced exploration of open arms indicated increased anxiety-related behaviour.

2.5. Pain threshold evaluation Pain sensitivity tests were carried out in dams (hot-plate and laser tests) at PN30, and adult offspring at PN30 (hot-plate test) and PN90 (hot-plate and laser tests).

2.5.1. Hot-plate test For evaluation of pain sensitivity, individual rats were placed on a hot plate (Ugo Basile, Biological Research Apparatus Company, Comerio, Italy) maintained at 50 ± 1 °C. Hot plate latency was defined in adult rats as lifting, licking or flinching of the hindpaw or forepaw, or jumping off the plate to avoid the thermal stimulus. For PN30 pups, latency to paw-lift was considered. The animal was removed from the hot plate immediately after measurement. A latency period of 90 s was defined as complete analgesia and used as the cut-off time for rats that did not respond. Tests were always conducted between 8 a.m. and 10 a.m.

2.5.2. Laser test Cutaneous heat stimuli (0.5–2.0 W, 50 ms) were delivered using a 810 nm near-infrared diode laser (Opto-FTC/TTT-2000) to a skin area on the external part of the thigh proximal to the left hind paw. The target skin area was blackened with a thin layer of India ink. To provide non-contact activation of heat receptors on the skin surface, the fibreoptic cable was coupled to a collimator to yield a beam diameter of 0.1 mm, and the beam was focused at a point 1 mm beyond the exit point of the collimator. Singlestimulus pulses lasting 50 m were delivered arrhythmically at intervals of 8–15 s by the fibreoptic cable, which was moved slightly after each stimulus to minimise nociceptor fatigue or central habituation. The potency of laser stimuli was increased gradually until the rat displayed a perceptual response, such as withdrawal reflex movements of the hindpaw or jumping behaviour (Fan et al., 1995).

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CD (N=29) DAR (N=29)

Maternal behavior

Weaning

(n=10/group)

Anxiety Pain threshold (n=10/group)

Sleep recording (n=10/group)

Blood and brain sampling (n=9/group)

Weaning

CR (N=31)

Histology (n=2/group)

AR (N=31)

Pain threshold (n=10/group)

Anxiety Pain threshold (n=10/group)

Sleep recording (n=10/group)

Blood and brain sampling (n=9/group)

PN1

PN12

PN21

PN30

PN90

Fig. 1. Schematic representation of the experimental design. A timeline illustration of the sequence of events throughout the experiments showing animals designated for behavioural tests and for blood and tissue collection, as well as the animals subjected to sleep recovery recording. CD = control dams; DAR = dams of arthritic rats; CR = control rats; AR = arthritic rats, PN = post natal day.

2.6. Implantation of electrodes and sleep recording evaluation To perform sleep recordings, electrodes were surgically implanted in a second set of lactating dams (DAR and CD) and in adult rats (AR and CR). Anaesthesia was induced by i.p. administration of ketamine–xylazine. The recordings were performed on a Nihon Koden Co. (Tokyo, Japan) model QP-223A apparatus using three channels for each animal: two pairs of electrodes (steel screws) were implanted in the fronto-parietal medial derivation (one pair on each side of the skull) for ECoG recording, and one additional pair of nickel-chrome electrodes was also implanted in the dorsal muscle of the rat’s neck for EMG recording. The electrodes were soldered to a connector, which was fixed to the cranium with acrylic dental cement. Rats were allowed to recover from surgery for two weeks. ECoG signals were amplified and filtered with a low pass filter at 0.5 Hz and a higher cut-off filter at 35 Hz as the EEG frequency band between 0.5 and 30 Hz is the most relevant during a conscious state. EMG activity was filtered with a low pass filter at 5.3 Hz as this allowed the recording of the fast range of frequencies. Sleep recordings were monitored during light and dark periods lasting 12 h each (food and water were available ad libitum) and were evaluated at baseline (prior to administration) as well as on days 1, 10, 15, 20 and 28 after the injection of saline or iodoacetate, or assignment to SHAM or CTRL groups. During the experimental period, rats were kept in their own cages placed inside a Faraday chamber. The ECoG traces were visually and manually scored blindly for 30 s periods. All recordings were scored by

only one researcher, blinded to the group assignment, thus assuring consistency of the data. The following sleep parameters were assessed: sleep efficiency (the total sleep time percentage during the recording time); slow wave sleep (SWS, the deep sleep time percentage throughout recording); paradoxical sleep (PS, the PS time percentage throughout the recording); and PS episodes and microarousals (events at least 15 s long with abrupt modification of baseline ECoG frequency accompanied by high amplitude EMG activity followed by SWS). PS episodes and microarousal values were expressed as absolute numbers. 2.7. Assays Dams were euthanised by decapitation at PN30, and adult rats at PN90. Blood samples were collected in ice-chilled glass tubes containing 10% EDTA solution and kept on melting ice until centrifugation (3500 rpm, 10 min, 4 °C). Separated plasma was stored in 200 ll aliquots at 80 °C for ACTH and corticosterone analysis. The brain was placed rapidly in an ice-chilled petri dish and bathed in saline (0.9%); the hippocampus and pineal gland were dissected. The hippocampus (20%w/v) and pineal gland (1 ml/pineal) were homogenised by politron in 0.1 M tricine buffer (0.9% NaCl, 0.1% gelatin) and centrifuged at 30,000 rpm for 30 min at 4 °C. The supernatants were frozen at 80 °C until serotonin and melatonin determination. Radioimmunoassay was used for hippocampal serotonin (Alpco, NH), pineal melatonin (Bühlmann Laboratories

S. Roizenblatt et al. / European Journal of Pain 14 (2010) 985–991

2.8. Statistical analyses A sample size of 20 rats per group was calculated by estimating a 30% change between control and treatment groups with an error of 0.025 and a power of 0.8. The normality of the distribution was tested using the Shapiro–Wilk test and intervalar values were expressed as the mean ± standard deviation. The differences between DAR or AR and respective controls were evaluated by Student’s t-test for independent samples, whereas for sleep parameters, treatment condition (saline or CFA) and animals (dam or adult offspring) were included in a repeated measures analysis of variance (ANOVA), followed by the Tukey post hoc test. The twotailed Fisher’s exact probability test was used for nominal data. Significance was defined as p < 0.05.

3. Results 3.1. Maternal/adult rat behaviour Table 1 summarises the maternal behaviour on PN12. DAR were less often in contact with their litter and exhibited a delay in the onset of maternal behaviour as compared to CD. The latency to the first retrieval and to dam–pup interactions were significantly increased in DAR compared to CD (p < 0.001). Nursing behaviour was reduced in DAR compared to CD (p < 0.05). Furthermore, 20% of the DAR ignored their pup in contrast to none in the CD group (p < 0.05). Pups with neonatal-induced arthritis weighed less than controls until PN30 (data not shown). Histopathological signs of inflammation or tissue destruction were observed between 24 h and day 21 after CFA injection. At PN30, no significant differences were observed between the groups (data not shown).

60

Num f r entere ed mberr of floo

Switzerland) and plasma corticosterone (Diagnostic Division, ICN Biomedicals, USA). Plasma ACTH was determined by a sequential immunometric assay using an IMMULITE analyser (Diagnostic Products Corporation, USA). Serum prolactin concentration was determined in dams using a double antibody radioimmunoassay using specific kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, USA). The antiserum for prolactin was anti-rat PRL-S9 and the reference preparations were PRL-RP3. The assays were performed in duplicate.

CR/CD

50

AR/DAR

*

40

* 30

20

10

0 Adults

Dams

Groups Fig. 2. Mean values of the number of total locomotion events displayed by control dams (CD) and dams of arthritic rats (DAR) and adult (AR and CR) rats in the elevated plus maze test. Significantly different from respective control group. n = 10/group.

3.3. Pain threshold evaluation Fig. 3 shows the differences in pain thresholds between groups of dams assessed by the hot-plate and laser tests. On PN90 but not on PN30, AR showed a lower pain threshold than the respective control rats, as assessed by the latency to lick a paw in contact with the 50 °C surface of the hot plate (p < 0.01, Fig. 3A). Similarly, DAR also exhibited a lower pain threshold on the hot-plate test. In comparison to controls, both DAR and AR showed significantly higher sensitivity to laser stimulation at PN90 (p < 0.01, Fig. 3B).

A

90 CR/CD

Hot platte la cies (sec atenc c)

988

80

AR/DAR

70 60 50

*

40 30

*

20 10

3.2. Elevated plus maze test

0 PN30

Table 1 Reduced maternal behaviour displayed by dams of arthritic pups on postnatal day 12. N = 10/group. Behaviour

CD

DAR

Latency to first retrieval (s) Latency to other dam-pup interactions Duration of nursing behaviour (s) Ignored the pups (% of dams)

182.5 ± 10.2 68.1 ± 19.4 99.1 ± 32.2 0

394.3 ± 29.0** 159.9 ± 30.5** 65.4 ± 28.8* 20%#

Maternal behaviours of dams of control dams (CD) and dams of arthritic rats (DAR) during a 60-min observation on PN12 (n = 10/group) after dam-pup separation for 15 min. The data (in seconds) obtained within an observation period of 60 min are presented as mean ± sd. * p < 0.05, compared to controls (unpaired two-tailed Student t-test). ** p < 0.001, compared to controls (unpaired two-tailed Student t-test). # p < 0.05 (two-tailed exact probability test).

PN90

PN30 Dams

Adults

B ensiity (m att) La aserr inte mWa

In comparison to respective controls, both DAR and AR had fewer entries into the open arms (p < 0.01 for adults rats, and p < 0.05 for dams). These results are depicted in Fig. 2.

2500

2000

1500

* 1000

*

500

0 Adults

Dams

Groups p Fig. 3. Pain threshold evaluation as the latency of withdrawal displayed by control dams (CD) and dams of arthritic rats (DAR) and adult rats in the hot-plate test (seconds) and laser potency required to elicit aversive behaviour. Significantly different from respective control group. n = 10/group.

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3.4. Sleep parameters

3.5. Assays

Table 2 depicts the alterations in sleep patterns in all groups studied. Compared to respective controls, both DAR and their adult littermates displayed the following changes in sleep architecture during four 12-h-light/dark periods: increase in waking after sleep onset, number of arousals, paradoxical sleep (except for the light period in DAR), sleep latency (except for the dark period in DAR). Significant decreases in sleep efficiency, latency to PS, and SWS (except for p12 in DAR) were also observed.

Biochemical assays were performed on blood and brain samples in each group (DAR and CD) at PN30, and on PN90 for AR and CR. The results are shown in Table 3. No significant differences in stress hormones, such as ACTH and corticosterone were observed between the groups of dams or adults (CD and AR) relative to controls. Moreover, DAR showed lower plasma prolactin (p < 0.03), hippocampal serotonin (p < 0.03), and higher pineal melatonin (p < 0.04) levels in comparison to CD. In contrast, AR did not show any significant differences from CR in biochemical assays. 4. Discussion

Table 2 Sleep alterations induced by injection of the hind paw with complete Freund’s adjuvant on postnatal day (PN) 1. Sleep parameters were assessed over a 48-h period: first day light (p12), first day dark (p24), second day light (p36) and second day dark (p48) period. Results were compared between control dams (CD) and dams of arthritic rats (DAR) on PN30, and between control rats (CP) and arthritic rats (AR) on PN90. n = 10/group. CD (PN30) Wake12 Wake24 Wake36 Wake48 SWS12 SWS24 SWS36 SWS48 PS12 PS24 PS36 PS48 LAT12 LAT24 LAT36 LAT48 LATPS12 LATPS24 LATPS36 LATPS48 Arousal12 Arousal24 Arousal36 Arousal48 Efficiency12 Efficiency24 Efficiency36 Efficiency48

34.5 ± 5.2 66.1 ± 9.0 31.1 ± 4.0 64.4 ± 10.2 83.8 ± 4.8 91.0 ± 2.2 83.2 ± 4.4 88.8 ± 3.5 16.1 ± 5.0 16.8 ± 4.4 16.7 ± 4.4 11.2 ± 3.5 54.6 ± 6.7 118.0 ± 86.0 35.8 ± 14.8 76.0 ± 20.9 61.3 ± 13.8 123.2 ± 116.8 53.8 ± 13.1 100.6 ± 87.5 14.4 ± 5.4 10.0 ± 2.9 12.3 ± 2.8 8.3 ± 4.1 65.5 ± 5.2 33.9 ± 9.0 68.9 ± 4.0 35.6 ± 10.2

DAR (PN30) **

59.9 ± 10.6 78.6 ± 7.8* 60.4 ± 5.1** 80.3 ± 2.6** 78.9 ± 6.3 84.8 ± 5.0* 80.6 ± 2.7* 80.7 ± 6.1* 21.1 ± 6.3 20.9 ± 4.0* 20.8 ± 4.0 19.7 ± 6.0* 90.7 ± 12.8* 86.2 ± 53.3 94.3 ± 14.0* 77.1 ± 41.4 13.9 ± 6.7* 14.6 ± 5.1* 15.2 ± 9.2* 76.0 ± 20.9* 25.3 ± 5.0* 16.1 ± 2.3* 24.3 ± 5.9** 12.1 ± 2.3* 40.1 ± 10.6** 21.4 ± 7.8* 39.5 ± 5.1** 19.7 ± 2.6**

CR (PN90)

AR (PN90)

34.3 ± 2.6 62.7 ± 4.4 31.7 ± 3.9 62.8 ± 4.6 87.2 ± 3.1 93.3 ± 2.0 85.1 ± 3.1 91.4 ± 3.1 14.5 ± 1.5 7.7 ± 1.4 15.5 ± 4.2 8.7 ± 3.8 30.7 ± 9.0 40.0 ± 10.3 21.8 ± 7.4 41.3 ± 11.5 20.0 ± 4.0 26.5 ± 6.5 20.2 ± 7.8 24.7 ± 9.1 16.1 ± 2.7 14.7 ± 3.6 17.7 ± 3.4 14.9 ± 4.6 65.6 ± 2.6 36.7 ± 4.1 66.3 ± 3.1 36.7 ± 4.1

49.3 ± 3.2** 73.7 ± 5.6** 51.2 ± 4.9** 72.6 ± 3.1** 77.3 ± 6.0** 83.1 ± 5.2** 75.5 ± 4.4** 81.84 ± 5.1** 22.6 ± 6.0** 16.8 ± 5.2** 22.5 ± 2.7** 15.9 ± 3.5** 57.5 ± 15.1** 66.4 ± 7.8** 62.5 ± 12.1** 64.4 ± 4.9** 20.8 ± 8.1 23.1 ± 8.2 21.8 ± 11.4 24.9 ± 8.2 34.0 ± 3.6** 26.6 ± 4.4** 34.8 ± 4.4** 29.1 ± 5.8** 49.0 ± 2.7** 26.2 ± 5.6** 48.0 ± 4.4** 26.2 ± 5.6**

Sleep parameters assessed in 48-h period. First day light (p12); first day dark (p24); second day light (p36) and second day dark (p48) period. Control dams (CD) vs. dams of arthritic rats (DAR) on PN30 (n = 10/group), and control rats (CR) vs. arthritic rats (AR) on PN90. (n = 10/group). Wake = time spent awake; SWS = slow wave sleep; PS = paradoxical sleep; efficiency = sleep efficiency. Except for arousal, all sleep parameters are expressed in percentage. Values are expressed as mean ± sd. * p < 0.05 compared to controls (unpaired two-tailed Student t-test). ** p < 0.01 compared to controls (unpaired two-tailed Student t-test).

Our results show that neonatal arthritis is associated with anxiety and sleep impairment as well as reduced pain thresholds in both dams and offspring during the nursing period and in adulthood for arthritic offspring. Decreased maternal behaviour and lower locomotor activity were observed in the pups that were injected with Freund’s adjuvant as opposed to saline. AR also exhibited persistent histological evidence of inflammation in the injected paw, and anxiety-like behaviours and lower pain threshold. Because ventral and perioral somatosensory contact is considered the sensory-motor response from the litter for maternal behaviour (Sibolboro-Mezzacappa et al., 2003; Moore, 2007), the lower weight gain observed in arthritic pups may reflect reduced nursing behaviour in dams of disabled pups, as observed previously (Guerra and Nunes, 2001). Hence, the impaired gait of the arthritic pups during the nursing period may result from the increased latency of the dams to retrieve arthritic pups, establish a nest and express maternal behaviours in addition to other pupdirected activities. The rapidity in retrieving pups is related to the physical state of the pups (Deviterne et al., 1990), and dampup interactions require that the pups stimulate nursing (Stern and Lonstein, 2001). Oxytocin and prolactin inhibit HPA axis responses in addition to acting as anxiolytics (Torner et al., 2002). The down-regulation of stress responses and inhibition of brain CRH activity in lactation may also be related to changes in limbic regions that affect the emotional evaluation of an external stimulus (Wartella et al., 2003), resulting in altered behavioural responses (Gammie et al., 2004), including reduced levels of arousal (Glynn et al., 2004). Therefore, it is likely that stress in arthritic offspring is, at least, partly blunted by these adaptive mechanisms. To what extent litter well-being can elicit a maternal stress response remains controversial (Deschamps et al., 2003). In the present study, neither dams nor offspring showed significant alterations in plasma levels of HPA-associated stress markers. Increased anxiety among DAR is supported by findings from other studies that demonstrated that anxiety expression in lactating dams is modulated by changes in cues from the pups

Table 3 Neonatal arthritis alters biochemical and neurochemical responses to the injection of complete Freund’s adjuvant on postnatal day (PN) 1. Results were compared between control dams (CD) and dams of arthritic pups (DAP), and between control rats (CP) and arthritic rats (AR) on PN90. n = 9/group.

Corticosterone (ng/ml) ACTH (ng/ml) Prolactin (ng/ml) Serotonin (hippocampus, ng/mg) Melatonin (pineal, pg/gland)

CD (PN30)

DAR (PN30)

CR (PN90)

AR (PN90)

10.2 ± 173.2 16.0 ± 13.0 12.7 ± 8.9 0.07 ± 0.02 4.1 ± 1.6

62.1 ± 123.8 10.4 ± 4.4 6.8 ± 4.7* 0.05 ± 0.03* 5.5 ± 2.3*

83.2 ± 55.3 13.8 ± 5.2 – 1.47 ± 0.03 5.2 ± 2.1

106.0 ± 41.7 11.6 ± 3.1 – 1.44 ± 0.05 4.6 ± 2.4

Dosages carried out in control dams (CD) vs. dams of arthritic rats (DAR) on PN30 (n = 9/group), and in control (CR) vs. arthritic rats (AR) on PN90, (n = 9/group). The data are presented as mean ± sd. * p < 0.05 compared to controls (unpaired two-tailed Student t-test).

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(Fernández-Guasti et al., 2001). Reduced prolactin levels in DAR are consistent with higher anxiety and lower maternal behaviour (Wartella et al., 2003). In light of the controversy surrounding the long-term effects of neonatal arthritis on anxiety-related behaviour, it is tempting to speculate that persistent histologic alterations and locomotor disability of the pups may impair the dam-pup interaction and contribute to long-term expression of anxiety in the offspring, as observed in the present study. Using different methodologies, other studies investigating the long-term effects of neonatal pain stimulation have yielded conflicting results. According to Anseloni et al. (2005), rats receiving short-term early local inflammatory insults produced by a single injection of 0.25% carrageenan into the hind paw grow into adults with low anxiety, a trait associated with reduced emotional responsiveness to stress. This reduction on reactivity to pain and stress may be modulated by maternal attention to the pups (Ren et al., 2004), however, if pain stimulation persists during the first week of life, the amount of care falls below control levels, and the initial inflammation-induced increase in care gives way to maternal neglect toward the impaired pups (Anseloni et al., 2005). Changes in the injected paw and abnormal locomotion persisted for 21 and 30 days, respectively, following induction of neonatal arthritis. These findings confirm that if the insult is applied within a restricted neonatal ‘window of vulnerability’, maternal care toward pups is reduced and anxiety-related behaviour among the offspring increases over the long term. These results are not related to stress, as neither dams nor offspring showed significant changes in plasma levels of HPA-associated stress markers, as determined at baseline and after the immobilisation test. Our data are in agreement with those of Anand et al. (1999), who used repeated daily needle sticks during the first week of life as a neonatal pain stimulus and observed enhanced long-term emotionality unaccompanied by abnormal plasma levels of ACTH. Blunted neuroendocrine responses to stress have been reported in lactating females after exposure to various stressors (Walker et al., 1991), and the ability of the litter to elicit the stress response in the dam remains controversial (Deschamps et al., 2003). The presence and type of long-term outcomes depend on the type of neonatal pain-inducing insult (Lidow, 2002), such as repeated daily hotplate exposures during the neonatal period (Bernardi et al., 1986). Laser stimuli are currently used to explore pain and heat sensation pathways. By spreading the heating over the surface of the skin, the thermal effects of the diode laser (Tzabazis et al., 2005) induces an indirect activation of nociceptors as a result of heat conduction through the skin (Arendt-Nielsen and Bjerring, 1988), rather than as a direct effect of the diode laser on C or Ad thermonociceptors (Tzabazis et al., 2005). Responses to such brief laser stimulation reflect a less integrated level of cortical processing, which is a more reliable neurophysiological correlate of noxious input because it is not affected by the cognition and temporal summation of responses elicited by long-lasting thermal stimulation. Dams of arthritic rats and their offspring, even upon reaching adulthood showed lower pain thresholds than their respective controls. The observed avoidance of the closed arms of the plus maze and jumping behaviour on the hot plate are suggestive of anxietylike states. In a state of heightened anxiety, both humans and animals express elevated pain sensitivity, and high-anxiety rat strains show increased responsiveness to acute pain. Nevertheless, laser experiments confirmed that arthritic rats (PN90) and their dams have a lower pain threshold than controls, strengthening the hypothesis of an interaction between maternal behaviour, pain threshold, anxiety and sleep patterns. Decreased 5-HT levels in dams and arthritic offspring may also be implicated in aversive behaviours displayed by dams and their arthritic offspring, as serotonergic mechanisms participate in vari-

ous active and passive coping mechanisms and behaviours related to anxiety and/or depression (Ho et al., 2002). Regarding the dams of arthritic offspring, a lower pain threshold may be related to serotonin levels that are lower than those in controls. The decreased levels of prolactin that we observed in DAR may indicate the long-term effect that pain in neonatal offspring has on the central nervous system of dams. Indeed, mean serum concentrations of prolactin in fibromyalgia patients are significantly lower than in control patients during the early sleep period (Landis et al., 2001). The influence of PS periods on prolactin secretion is supported by sleep recording studies that reveal a reduction in PS among dams of arthritic pups. There is a clear-cut relationship between hippocampal 5-HT levels and sleep-wake behaviours in the rat. Stress has been shown to cause a greater increase in 5-HT levels in female rats in multiple brain areas (Peters, 1988). In particular, low sleep quality interferes with the maternal-offspring relationship. Indeed, as reviewed recently by Marcus (2009), during the neonatal period, there is a correlation between the entrainment of infant sleep patterns and maternal depressive symptoms. For instance, women with depressive symptoms have infants who experience longer sleep latency (time to sleep), less sleep efficiency and total sleep time compared to infants whose mothers who are not experiencing depressive symptoms (Heringhausen et al., 2008). These patterns occur from 2 weeks post-partum through 30 weeks following delivery. Regarding our results, we also observed sleep impairment in both dams (approximately four weeks after delivery) and adult rats, and these included increased sleep latency and awake time, lower sleep efficiency, among other alterations. Thus, sleep alterations in both dams and offspring may constitute one of the mechanisms of anxiety and hyperalgesia in adult rats that were subjected as neonates to local inflammatory insults during the first month of life. Melatonin levels during waking hours provide a good index of nocturnal secretion. We observed higher levels of melatonin in DAR and in AR, suggesting a circadian rhythm sleep disorder with a delayed sleep period as well as melatonin secretion. The relationship of such a condition with depression in humans has been proposed, but it remains controversial. The reduction in PS latency is consistent with this finding; poor sleep causes more naps, which alters the phases of melatonin secretion and increases daytime secretion. In studies of depressive disorders related to the reproductive cycle, melatonin levels were higher or showed delayed offset (Wehr et al., 2001). Although controversial, the inhibitory effect of melatonin on the secretion of prolactin in the rat (Chu et al., 2001) is supported by decreased prolactin levels in DAR in the present study. Melatonin may be present in animals younger than 10 days of age by transfer from the mother (Navarová et al., 2004; Vázquez et al., 2007). Possible differences in melatonin levels in offspring are unlikely to affect the results in the present study, as no significant differences were detected between AR and CD, suggesting that the pregnancy conditions of the dams were the same. We acknowledge that a limitation of this study was the failure to assess HPA function using several measurements of stress responses. However, the collection of such data was precluded by the study design, as indoleamine concentrations would have been influenced by previous stress exposure. Of note, our study included a long-term investigation with several behavioural and electrophysiological evaluations, in addition to various biomarkers assays. Based on the current findings, our animal model provides intriguing findings regarding the impact of early pain on future somatosensory processing, and demonstrates that tissue injury or exposure to noxious stimuli at infancy leads to changes in adult sensory behaviour and sleep. Additional and multifaceted studies are warranted to provide a comprehensive understanding of the overall long-lasting consequences induced by neonatal injury challenge on the maternal-offspring relationship.

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