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Early neonatal inflammation affects adult pain reactivity and anxiety related traits in mice: genetic background counts
Int. J. Devl Neuroscience 27 (2009) 661–668
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Early neonatal inflammation affects adult pain reactivity and anxiety related traits in mice: genetic background counts Cristina Benatti a, Silvia Alboni a, Giacomo Capone a, Daniela Corsini a, Federica Caggia a, Nicoletta Brunello a, Fabio Tascedda a, Joan M.C. Blom a,b,* a b
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy Department of Paediatrics, University Hospital of Modena, Via del Pozzo 71, 41100 Modena, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2009 Received in revised form 3 July 2009 Accepted 30 July 2009
Protracted or recurrent pain and inflammation in the early neonatal period may cause long-lasting changes in central neural function. However, more research is necessary to better characterize the longterm behavioral sequelae of such exposure in the neonatal period. Objectives: (1) to study whether timing of postnatal exposure to persistent inflammation alters responsiveness to thermal pain in the adult animal; (2) to assess whether animals experiencing early postnatal chronic inflammation display altered anxiety related behavior; (3) to study the importance of genetic background. Newborn mice (outbred strain, CD1 and F1 hybrid strain, B6C3F1) received an injection of Complete Freund’s Adjuvant (CFA) or saline on either postnatal day 1 or 14 (PND1; PND14) into the left hind paw. Pain to radiant heat and anxiety were examined in 12-week-old adult animals. Adult baseline PWL was significantly decreased in CD1 mice exposed to CFA on PND 1 and 14 as compared to their saline treated counterparts. B6C3F1 mice exposed to CFA on PND14 showed markedly reduced baseline PWL compared to the PND14 saline group. Persistent inflammation experienced by B6C3F1 mice on PND1 failed to affect baseline adult thermal responsiveness. Adult mice, CD1 and B6C3F1, displayed low anxiety traits only if they had been exposed to persistent inflammation on PND1 and not on PND14. Our research suggests a role for genetic background in modulating long-term behavioral consequences of neonatal persistent inflammation: the data support the hypothesis that pain experienced very early in life differentially affects adult behavioral and emotional responsiveness in outbred (CD1) and hybrid mice (B6C3F1). ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Pain Development Long-term consequences Strain differences Anxiety traits CFA
The early neonatal period is characterized by great plasticity and reorganization. Premature infants undergo numerous invasive and painful procedures necessary to assure their survival while in the neonatal intensive care unit (Simons and Tibboel, 2006). Recurrent painful stimulation during the neonatal period produces long-term behavioral sequelae that last well into adulthood, affecting behavioral responsiveness to pain or stress later in life (Bhutta and Anand, 2002; Fitzgerald, 2005; Garg et al., 2003; Grunau et al., 2006). Neonatal pain and inflammation cause aberrant activation of central nervous circuits, inducing developmental consequences that depend on the type and intensity of the pain-inducing insult (Anand et al., 1999; Lidow, 2002; Peng et al., 2003). Further research is needed to characterize more specifically the behavioral sequelae, such as
* Corresponding author at: Department of Paediatrics, University Hospital of Modena, Via del Pozzo 71, 41100 Modena, Italy. Tel.: +39 059 2055162; fax: +39 059 2055625. E-mail address: [email protected] (Joan M.C. Blom). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.07.009
anxiety related behavior, of a strong noxious stimulation in the neonatal period. Rodents, and more specifically rats (Ruda et al., 2000; Lin and Al-Chaer, 2003; Ren et al., 2004; Boisse et al., 2005; Hohmann et al., 2005), are widely used as a model system to study the impact of early life pain and stress on future responsivity and represent a valuable resource to enhance our understanding concerning the long-term consequences of neonatal pain. However, a limited number of studies have been conducted using mice (Blom et al., 2006). At birth, the neurological maturity of pups is similar to that of human preterm neonates at 24 weeks of gestation, while the nociceptive system of a 1-year-old child closely resembles that of 2-week-old mice (Anand et al., 1999). Our previous findings demonstrated that CD1 adult mice exposed to a robust long-lasting local inflammatory insult (i.e. Complete Freund’s Adjuvant, CFA) early during development, are characterized by heightened sensitivity to pain (Blom et al., 2006). However, each rodent strain is characterized by a unique pattern of behavior and their genetic inheritance influences behavioral traits such as sensitivity to pain and stressors as well as anxiety and depression related behaviors (Bolivar et al., 2000; Bouwknecht and
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Paylor, 2002; Jacobson and Cryan, 2007; Mogil et al., 1999a,b, 2006; Mogil and McCarson, 2000). To further investigate long-term behavioral sequelae following a robust neonatal inflammation, we assessed whether genetic background could affect long-term behavioral consequences of neonatal pain in the adult life of mice in two different strains: a randomly outbred strain (CD1) and a F1 hybrid strain (B6C3F1). These strains, routinely used in our laboratory (Blom et al., 2002), are characterized by their own distinctive expression pattern of specific neuronal plasticity linked markers as well as by their display of behavioral differences, B6C3F1 being proner to show anxiety related behavior (data not published). In this study we set out to test whether timing of postnatal exposure (postnatal day 1 (PND1) or PND14) to a persistent inflammatory insult (CFA) could impact adult responsiveness to thermal pain as well as to a mild emotional stressor caused by placing adult animals on an elevated plus maze (EPM) using these two strains. To our knowledge this is the first study testing the effects of neonatal robust inflammation on adult mice comparing two different strains. Little is known on how genes interact with neonatal environmental cues and how this may impact on shaping individual behavioral differences, even more so, when the developmental equilibrium is shaken by a traumatic event.
natural aversion of rodents to enter open places/spaces. Consequently, high anxiety is thought to correlate with higher aversion to enter open spaces, while low anxiety corresponds to the willingness of the animal to explore. The EPM consisted of two open (5 cm wide, 30 cm long) and two closed arms (5 cm wide, 30 cm long, enclosed by a wall of 15 cm height) arranged in a plus configuration, joined by a central square (5 cm 5 cm) (Lister, 1990; Wall and Messier, 2000). The apparatus was made of opaque Plexiglas and kept on a wooden base 40 cm above the floor. The floor and walls of the maze were wiped between trials with a 70% alcohol:distilled water solution to prevent individual mice from following the scent of a previously tested animal. The illumination at the level of the maze was 100 lx. All animals were subjected to tests of a standard 5-min duration at 11 weeks of age; at the beginning of each test the mouse was placed individually in the center of the maze, its head facing an open arm (the same for all mice). All testing was conducted during the morning phase of the light:dark cycle between 08:00 and 14:00 h and recorded using a video camera. The parameters measured, selected among the battery of the EPM anxiety indices of Wall and Messier (2000), included four negatively correlated anxiety indices: open arm entries, percent of time spent – duration – in the open arm, number of scannings (dipping of the head/shoulders over the sides of the open arms) and scanning duration and four positively anxiety correlated indices: closed arm entries, percent of time spent – duration – in the closed arm, number of risk assessments (head/shoulder dipping over the closed arm) and risk assessment duration. An entry in a given arm was counted only when the mouse had completely entered the arm. The duration was expressed as percentage of the entire 5 min of testing; otherwise, the data were expressed as the number of occurrences during testing.
1. Experimental procedures
The behavioral response to heat nociception of 12-week-old male mice was determined using a stainless steel hot plate (maintained at 48 0.1 8C) (UGO BASILE, Hot Plate DS37). The mouse’s right hind limb, both forelimbs, and trunk were gently supported by the experimenter, allowing the left hind limb of the animal to rest lightly but stably on the heated surface (Blass and Blom, 1996). The timing circuit was activated when the limb was brought into contact with the hot plate surface and stopped upon unambiguous hind limb withdrawal; this defined the latency for heat withdrawal. Paw withdrawal latency (PWL) was tested for five times at intervals of 5 min and was calculated as the mean, excluding the first familiarization trial (Ruda et al., 2000). Data were collected by an experimenter who was uninformed about the experimental condition. A 40 s cut-off was applied to prevent tissue damage. At 12 weeks of age, neonatal CFA- and neonatal saline treated animals received an unilateral injection of 100 mL of CFA (1:1) in the left hind paw. All adult animals irrespective of neonatal treatment, displayed pain related behaviors after CFA injection. Baseline paw withdrawal latencies of the left hind paw to a radiant heat source were determined in all the experimental groups before receiving the inflammatory injection. Withdrawal latency was tested again 24 h after injection with CFA into the adult left hind paw. All testing was conducted during the morning phase of the light:dark cycle between 08:00 and 14:00 h
1.1. Animals Male offspring from multiparous CD1 and B6C3F1 female mice (Charles River, Lecco, Italy) served as subjects. Pregnant females were individually housed in plastic tubs (28 cm 17 cm 12 cm) with stainless steel wire lids. The floor of each cage was covered with bedding of wood shavings. Tap water and food were available ad libitum. Lights were on from 08.00 to 20.00 h. Colony room temperature ranged from 20 to 24 8C, with humidity uncontrolled. Newborn litters discovered before 11.00 h were considered born that day and designated PND0 (postnatal day 0). Sexing of the pups was conducted at birth by evaluation of the ano-genital distance. Pups were treated between 12.00 and 18.00 h. Only male mice were used in the experiments. 1.2. Experimental procedure Outbred CD1 and hybrid B6C3F1 pups were injected with Complete Freund’s Adjuvant (CFA) in their left hind paw at postnatal day 1 (PND1) or PND14. Neonatal mice pups received a single injection of CFA (CFA, Sigma Aldrich1 F5881, 0.5 mg/mL heat killed Mycobacterium tuberculosis suspended in an stock emulsion of oil:saline (1:1)) or saline alone on PND1 or PND14. At PND1, pups were injected with 20 mL of the CFA emulsion previously prepared, diluted with saline (2:1) and PND14 pups were injected with 40 mL of this suspension. Thus, the amount of CFA injected was approximately 5 mg for PND1 treated animals and 10 mg for PND14 treated animals. In this way we adjusted dose to weight and maturation of the mice, procedure previously used by us (Blom et al., 2006) as well as by others (Ruda et al., 2000). The animals were then left undisturbed to mature. The pups were checked by the experimenter for distinct pain related behaviors at the time of CFA injection (immediate shaking and licking). Mice injected with CFA at PND1 or PND14, responded by immediate shaking and licking of the paw that persisted for at least 10 min. Saline injected neonatal animals did not show persistent pain related licking or shaking. For the next 14 days post-injection pups were observed closely with respect to the formation of edema and erythema. Within 7 days swelling of the injected hind paw was reduced to baseline (matching the contra-lateral untreated paw) except for the group treated with CFA at PND14 where swelling persisted for 10 days following injection (data not shown). No significant differences in body weight were found in saline or CFA treated animals during the entire duration of the experiment. After weaning at PND21, pups were housed three per cage until the end of the experimental procedure. Animals were checked for signs of discomfort as indicated by animal care and use guidelines (National Academy of Sciences. Guide for the care and use of laboratory animals, 1998, ‘‘Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research’’ (National Research Council 2003)). EC guidelines (EEC Council Directive 86/609 1987), local (University of Modena) and Italian legislation on animal experimentation (Decreto Legislativo 116/92) and the International Association for the Study of Pain ethical guidelines were applied to the experiments. 1.3. Elevated plus maze anxiety-like behavior test Anxiety traits of each animal were measured using an elevated plus maze (EPM), a behavioral model of anxiety based on the conflict between exploration and the
1.4. Thermal nociception
1.5. Data analysis Behavioral data were analyzed with a two-factor (age treatment) analysis of variance (ANOVA). Multiple comparisons were conducted with one-way’s ANOVA. At no time more than four groups were compared among each other. All mean differences were considered statistically significant if p < 0.05.
2. Results 2.1. Baseline paw withdrawal latency in neonatally treated CD1 mice As previously observed in our laboratory (Blom et al., 2006), CD1 mice injected at PND1 or PND14 with CFA, displayed reduced latency in lifting their left hind paw from the hot plate with respect to their saline exposed counterparts as adults. We tested the hypothesis that a strong noxious inflammation experienced by CD1 pups either on PND1 or PND14 enhances adult responsivity to a radiant heat source. CD1 baseline values differed significantly among each other [ANOVA univariate; F (3,71) = 16.630; p < 0.001; Fig. 1A and B]. A two-factor ANOVA (neonatal treatment age of injection) revealed an overall main effect of type of neonatal treatment [p < 0.0001; Fig. 1A and B] and of age of injection [p = 0.024]. No interaction was observed between the two terms (p > 0.05). Pairwise comparisons showed no difference between PND1 and PND14 saline injected mice (p > 0.05). Similarly, PND1CFA treated mice behaved as mice injected with CFA on PND14 (p > 0.05).
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Fig. 1. Paw withdrawal latency (PWL) of CD1 adult mice treated with saline or CFA on postnatal day 1 (PND1) (A), and on postnatal day 14 (PND14) (B). Adult baseline PWL of mice injected with CFA on either PND1 or PND14 was lower than saline injected animals (*p < 0.05). 24 h after baseline measurement, each animal received an injection with CFA in the left hind paw. Adult treatment with CFA reduced PWL in all experimental groups irrespective of neonatal age of injection (§p < 0.05; ruled box). PWL was measured at 12 weeks of age at baseline and 24 h after exposure to CFA as adults. Data are represented as means (SEM); *p < 0.05 is significant difference between baseline values of saline treated animals compared to baseline values of CFA treated mice; §p < 0.05 is significant difference between baseline and mice treated with CFA at 12 weeks of age.
Fig. 2. Paw withdrawal latency (PWL) of B6C3F1 adult mice treated with saline or CFA on postnatal day 1 (PND1) (A), and on postnatal day 14 (PND14) (B). CFA injected mice displayed lower baseline PWL than saline injected mice (*p < 0.05), only if exposed to the inflammatory agent on PND14. 24 h after baseline measurement, each animal received an injection with CFA in the left hind paw. Adult treatment with CFA reduced PWL in all experimental groups irrespective of neonatal age of injection (§p < 0.05; ruled box). PWL was measured at 12 weeks of age at baseline and 24 h after exposure to CFA as adults. Data are represented as means (SEM); *p < 0.05 is significant difference between baseline values of saline treated animals compared to baseline values of CFA treated mice; §p < 0.05 is significant difference between baseline and mice treated with CFA at 12 weeks of age.
2.2. Paw withdrawal latency after adult exposure to CFA in neonatally treated CD1 mice
The behavior observed with respect to heat nociception of adult B6C3F1 is strongly affected by the type of neonatal insult as well as by the timing of the insult.
24 h after measuring basal PWL, CD1 adult animals received an unilateral injection of CFA into the left hind paw that produced a reduction in PWL (Fig. 1A and B). To assess whether the type of treatment and the developmental stage at which treatment was received were able to influence thermal nociception even following an inflammatory challenge as an adult, a two-factor ANOVA (neonatal treatment age of injection) was performed. A main effect was observed for the type of treatment the mice received as neonates [F (3,71) = 5.458; p = 0.022], but not for the age at which they were first treated (p > 0.05). 2.3. Baseline paw withdrawal latency in neonatally treated B6C3F1 mice Similar to CD1 outbred animals, the hypothesis was tested that a strong noxious inflammation experienced by B6C3F1 hybrid pups either on PND1 or PND14 increases adult responsivity to a radiant heat source. We observed that adult thermal nociception of hybrid B6C3F1 mice treated neonatally with CFA or saline followed a different pattern with respect to that observed in outbred CD1 mice. B6C3F1 baseline values differed significantly among each other [F (3,32) = 6.948; p = 0.001; Fig. 2A and B]. However, an injection of CFA at PND1 failed to increase adult thermal nociception, since no difference was observed in baseline PWL among these animals and in mice receiving saline 1 day after birth (p > 0.05). On the other hand, animals injected at PND14 with CFA showed reduced PWL measured at 12 weeks of age with respect to their saline exposed counterparts (p < 0.05). Pairwise comparisons revealed no difference between PND1 and PND14 saline injected mice (p > 0.05), while animals exposed to CFA on PND1 were hypoalgesic with respect to the group exposed to the same inflammatory agent 2 weeks after birth (p < 0.05). A two-factor ANOVA (neonatal treatment age of injection) showed an overall main effect of both neonatal treatment [F (3,32) = 6.331, p = 0.017] and of age of injection [F (3,32) = 15.764; p < 0.0001]. No interaction was observed between the two terms.
2.4. Paw withdrawal latency after adult exposure to CFA in neonatally treated B6C3F1 mice Re-exposure to CFA, 24 h after measuring their basal PWL, caused a decrease in PWL in adult B6C3F1 mice exposed to either saline or CFA at PND1 or PND14 (Fig. 2A and B). B6C3F1 PND1 saline injected animals displayed a significant reduction in PWL [down by approximately 69%] after being exposed to CFA at 12 weeks of age. Even so, mice exposed to CFA at PND1 exhibited a decrease [down by approximately 49%] in PWL when re-exposed to CFA at 12 weeks of age. A similar pattern was observed in hybrid mice injected at PND14: adult exposure to CFA reduced PWL by 51% in mice treated with saline while those treated with CFA at PND14 showed a less marked reduction [down by approximately 40%]. To assess whether the type of treatment and the developmental stage at which treatment was received were able to influence thermal nociception even following an inflammatory challenge as an adult, a two-factor ANOVA (neonatal treatment age of injection) was performed. A main effect was found for age of injection [F (3,32) = 5.837; p = 0.022] but not for neonatal treatment [F (3,32) = 0.129; p > 0.05]. Furthermore, an interaction was observed between the two terms [F (3,32) = 4.452; p = 0.043], suggesting that the developmental stage in which the animals experienced a noxious stimulus is a critical factor influencing their behavior as an adult. Pairwise comparisons revealed a significant difference between PWL of animals exposed to CFA on PND1 as compared to PND14, in the presence of an ongoing inflammation (p < 0.05). When comparing adult baseline thermal sensitivity of CD1 and B6C3F1 mice treated neonatally with saline (Figs. 1 and 2), their PWLs differed significantly [ANOVA univariate; F (3,52) = 3.983; p = 0.013; Figs. 1 and 2]. However, further analyses revealed that only CD1 and B6C3F1 mice treated at PND14 differed among each other (p < 0.05). A two-factor ANOVA (strain age of injection)
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2.5. Paw withdrawal latency difference between baseline and adult CFA exposure in neonatally treated CD1 and B6C3F1 mice
Fig. 3. Paw withdrawal latency difference between baseline and adult CFA exposure. Adult reactivity to thermal pain with or without an ongoing inflammation is influenced by genotype, by the developmental maturity of animals when they received the insult and by the type of treatment they received as pups. *p < 0.05 is significant difference between neonatal saline and CFA treated mice. §p < 0.05 is significant difference between PND1 and PND14 saline treated mice.
revealed that neither strain nor age of injection produce a main effect, while a significant interaction was observed between the two terms [F (3,32) = 11.096, p = 0.002]. CD1 or B6C3F1 mice receiving an injection with saline at either PND1 or PND14, when re-exposed to CFA as adults, were statistically undistinguishable [ANOVA univariate; F (3,52) = 0.490; p > 0.05; Figs. 1 and 2].
A difference score was obtained by calculating the difference between PWL after adult CFA exposure and their respective baseline values (Fig. 3; Blom et al., 2006). Adult CFA administration reduced the time spent on the hot plate in all experimental groups, but behavioral reactivity to thermal nociception in the presence of an ongoing inflammation was differentially affected by type and age of injection. A two-factor ANOVA (neonatal treatment age of injection) revealed an overall main effect of both neonatal treatment [F (3,32) = 8.390; p = 0.07] and age of injection [F (3,32) = 11.969; p = 0.03]. No interaction was observed between the two terms (p > 0.05). This suggests that adult reactivity to thermal pain with or without an ongoing inflammation is strongly influenced by the developmental maturity of animals receiving the insult, as well as by the type of treatment they received as pups. Re-exposure to CFA at 12 weeks of age reduced the time spent on the hot plate, as observed in our laboratory also in CD1 mice (Fig. 3; Blom et al., 2006), pairwise comparisons though revealed a significant difference between PND1 (saline) and PND14 (saline) groups [F (1,17) = 6.197; p = 0.023], as well as between mice exposed to CFA on postnatal day 1 and 14 [F (1,15) = 6.400; p = 0.023]. The two types of neonatal treatment differentially affected adult behavioral reactivity only in mice treated on PND14 [F (1,14) = 10.996; p = 0.005], while exposure to either CFA or saline at PND1 did not affect the difference in PWL to a radiant heat
Fig. 4. Anxious behavior of 11-week-old CD1 mice treated with saline or CFA on PND1 (A and B), and on PND14 (C and D). Positively correlated anxiety related behaviors (closed arm entries, closed arm duration, number of risk assessment (head/shoulder dipping over the closed arm) and duration) and negatively correlated anxiety related behaviors (open arm entries, open arm duration, number of scanning (dipping of the head/shoulders over the sides of the open arms) and duration) were evaluated on an elevated plus maze. PND1-CFA treated animals displayed less anxious behavior with respect to their saline injected-counterparts; animals exposed to CFA on PND14 did not differ from saline treated mice. Data are expressed as number of occurrences and % of total testing time, each column represents a mean (SEM); *p < 0.05 is significant difference with baseline values of saline treated animals.
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source before and after adult exposure to CFA (p > 0.05). This suggest that in B6C3F1 mouse strain, exposure to a painful procedure in a period of great plasticity may cause alterations in the behavioral reactivity of animals that may induce a heightened sensitivity when re-exposed to a robust inflammatory insult. 2.6. Behavior of CD1 adult animals experiencing a robust neonatal inflammatory insult in the elevated plus maze Anxious behavior of CD1 outbred mice exposed to CFA at PND1 or PND14 was assessed on an elevated plus maze (EPM) with respect to their saline exposed counterparts. We tested the hypothesis that a strong noxious inflammation experienced either on PND1 or PND14 affects adult anxious behavior. Exposure to CFA on postnatal day 1 strongly affected anxiety behavior with respect to mice exposed to saline at the same postnatal age. Univariate ANOVA of animals receiving either saline or CFA at the same postnatal age revealed that PND1-CFA treated animals displayed a less anxious phenotype (i.e. low anxiety traits), as behaviors positively correlated with anxiety were diminished while behavioral indices correlating negatively with anxiety were increased when compared to saline PND1-exposed animals (Fig. 4A and B). Furthermore, a reduction was observed in the time spent in the closed arms [F (1,21) = 10.048; p = 0.005; down by approximately 20%] and time spent in risk assessment [F (1,21) = 4.512; p = 0.046; down by approximately 20%]. Also, exposure to CFA on PND1 caused a significant increase in closed arms entries when
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compared to exposure to saline [F (1,21) = 6.967; p = 0.015; up by approximately 40%], while the number of risk assessments performed during the test was not affected by the type of treatment received on PND1 (p > 0.05). CD1 animals exposed to CFA on PND1 showed a robust increase in negatively anxiety correlated behavioral indices such as time spent in the open arms of the maze [F (1,21) = 9.203; p = 0.007; up by approximately 240%], number of entries in these arms [F (1,21) = 8.642; p = 0.008; up by approximately 225%], time spent in scanning [F (1,21) = 8.458; p = 0.009; up by approximately 110%] and number of scannings [F (1,21) = 16.530; p = 0.001; up by approximately 120%]. On the other hand, a strong noxious stimuli experienced on PND 14 failed to affect any of the behavioral parameters considered (p > 0.05) (Fig. 4C and D). These results suggest that adult CD1 animals displayed low anxiety traits only after receiving a CFA injection on PND1 and not on PND14. 2.7. Behavior of B6C3F1 adult animals experiencing a robust neonatal inflammatory insult in the elevated plus maze B6C3F1 mice treated with saline or CFA on postnatal day 1 or 14, were tested on an elevated plus maze as were their CD1 counterparts. B6C3F1 hybrid mice were less affected by a persistent inflammation than the outbred strain. PND1-CFA treated B6C3F1 were undistinguishable from their saline exposed counterparts in all the behaviors that positively
Fig. 5. Anxious behavior of 11-week-old B6C3F1 mice treated with saline or CFA on PND1 (A and B), and on PND14 (C and D). Positively correlated anxious behaviors (closed arm entries, closed arm duration, number of risk assessment (head/shoulder dipping over the closed arm) and duration) and negatively correlated anxious behaviors (open arm entries, open arm duration, number of scanning (dipping of the head/shoulders over the sides of the open arms) and duration) were evaluated on an elevated plus maze. PND1-CFA treated animals displayed less anxious behavior with respect to their saline injected-counterparts; animals exposed to CFA on PND14 did not differ from saline treated mice. Data are expressed as number of occurrences and % of total testing time, each column represents a mean (SEM); *p < 0.05 is significant difference with baseline values of saline treated animals.
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correlated with anxiety, with the exception of time spent in the closed arms [F (1,18) = 5.642; p = 0.029; down by approximately 20%]. Among the negatively anxiety correlated behavioral indices only time spent in scanning [F (1,18) = 6.912; p = 0.018; up by approximately 120%] and the number of scannings [F (1,18) = 5.791; p = 0.028; up by approximately 90%] were enhanced following a robust early inflammation when compared to saline PND1-exposed animals (Fig. 5A and B). Time spent in the open arms of the maze and number of entries in these arms were not influenced by the type of neonatal treatment that animals had received (p > 0.05). A strong noxious stimuli experienced on PND 14 failed to affect any of the behavioral parameters considered (p > 0.05) (Fig. 5C and D). B6C3F1 mice treated on PND1 with CFA displayed low anxiety with respect to their saline exposed counterparts, and to a lesser extent than CD1 animals receiving the same type of neonatal treatment. On the other hand, a robust inflammation experienced on PND14 failed to affect adult anxious behavior in both strains considered. 2.8. Spontaneous litter size CD1 litters were significantly larger (11 pups for each dam) than B6C3F1 (8 pups for each dam) [F (1,17) = 10.891; p < 0.01], with approximately 50% males for each strain. Moreover, CD1 pups were bigger and more mature than their B6C3F1 counterparts (visual observation collected by the experimenter). 3. Discussion In this study, we investigated for the first time the long-term consequences of a robust neonatal inflammatory insult on the response to pain and subsequent anxious behavior in adult animals of two different mouse strains: randomly outbred CD1 mice and the hybrid strain B6C3F1. The latter is a first-generation (F1) hybrid strain produced by crossing C57BL/6 females and C3H males (Kalueff et al., 2007; Roy et al., 2007). In summary, our results show that: (1) CD1 outbred mice, exposed to a robust long-lasting local inflammatory insult early during development, grow into adults that display enhanced responsiveness to thermal pain. Also, adult animals display low anxiety traits only after receiving a CFA injection on PND1 and not on PND14. (2) B6C3F1 hybrid adult mice exposed to a robust long-lasting local inflammatory insult on PND14 are characterized by hyperalgesia, while treatment with CFA on PND1 fails to affect adult reactivity to thermal nociception. Moreover, adult animals display low anxiety traits only if they received a neonatal persistent inflammation on PND1 and not on PND14. Each mouse strain exhibits its own specific phenotype; behavioral differences between strains range from anxiety to locomotor activity, to influencing depressive-like behavior or the response to drugs (Bolivar et al., 2000; Bouwknecht and Paylor, 2002; Ducottet and Belzung, 2005; Griebel et al., 2000; Wahlsten et al., 2006). Also, distinctive patterns of responsiveness to various nociceptive tests were described for different strains (Lariviere et al., 2002; Mogil et al., 1999a,b). To our knowledge, this is the first study to investigate the effects of a strong persistent noxious stimulus on the behavior of two different mouse strains. CD1 mice, as already reported by our laboratory (Blom et al., 2006), receiving CFA as neonates grow into hyperalgesic adults, which is consistent with previous findings obtained in rats exposed to several types of inflammatory insults (Boisse et al., 2005; Hohmann et al., 2005; Ren et al., 2004; Ruda et al., 2000). CD1 mice receiving CFA on PND1 are characterized by a lower baseline PWL with respect to saline treated animals while
B6C3F1 PND1-CFA treated mice were indistinguishable from their saline exposed counterparts. CD1 mice handled and treated at PND1, an extremely sensitive period in development, are characterized as adults by a blunted ability to process noxious stimuli. In fact in adults, an ongoing inflammation with a strong impact on nociceptive circuit formation, like the one caused by CFA, resulted in a more marked reduction in paw withdrawal latencies in mice exposed during development (PND14), at an higher degree of maturity (Blom et al., 2006). B6C3F1 hybrids on the other hand, are more reactive in the presence of an ongoing inflammation if handled and treated very early during development, while persistent inflammation experienced on PND14 caused a less robust reactivity when exposed to CFA as adults. Neonatal treatment differentially affected adult behavioral reactivity only in mice treated on PND14, while in adult mice exposed to either CFA or saline on PND1, pain related behavioral response did not differ. This suggests that neonatal experience at this young age is not of influence in the B6C3F1 strain. Thermal sensitivity was shown to depend on the severity of the neonatal insult: a robust inflammation caused by CFA resulted in basal hyperalgesia (Hohmann et al., 2005; Ruda et al., 2000) while a milder insult such as a local action that lasted for only 48 h resulted in basal hypoalgesia (Anseloni et al., 2005; Ren et al., 2004). CFA injection is a very strong and painful stimulus and produces interstitial infiltration of lymphocytes that lasts until 8 weeks after the neonatal injection (Walker et al., 2003). Since our mice were tested at 12 weeks of age, we cannot exclude that the hyperalgesic condition, observed in these 2 strains after an injection with CFA on PND14, may be mediated, at least in part, by a state of chronic inflammation caused by the massive effect of CFA on the immune system. This, then, would suggest that the model adopted by us and proposed by Hohmann et al. (2005) before, may represent a useful behavioral tool to better understand a condition such as juvenile rheumatoid arthritis, which is characterized by persistent inflammation and pain with an early onset during development. Furthermore, neonatal exposure to an early inflammatory insult affected behavioral reactivity in a commonly used paradigm testing anxiety. The inter-relationship between emotional and nociceptive outcomes is an issue still under investigation (Craig, 1999; Wilson et al., 2007) Compared to randomly outbred CD1 mice, exposure to CFA on PND1 affected adult anxiety behavior to a lesser extent in hybrid B6C3F1 mice. Interestingly, no significant differences were found in any of the behaviors examined in the EPM when considering mice treated at PND14 either with saline or with CFA, irrespective of strain. It seems that the ‘‘critical window’’ of vulnerability for the effects of neonatal inflammatory pain on anxiety lies within the first week of age (Spencer et al., 2006). The hypoanxious traits we observed in CD1 mice and, to a lesser extent, in B6C3F1 animals are similar to those reported previously by Anseloni et al. (2005) in rats. It is remarkable that inflammatory insults, like CFA and carrageenan with a different physical impact on pups, produce similar anxiety related behaviors, but affect basal sensitivity to thermal stimulation in an opposite manner. Furthermore, aberrant activation of the immune system in animals at PND1 may induce prolonged changes in the developing hypothalamic–pituitary– adrenal axis (LaPrairie and Murphy, 2007), which in turn may alter anxiety related behavior later in life. The effects of a neonatal inflammatory insult on adult anxious behavior seem to depend more on the timing of the insult rather than on its severity. Thus, behavioral sequelae of neonatal pain are part of a complex and highly coordinated response and depend on the intensity and duration of the inflammatory insult (Lim et al., 2009), on the genetic background of each strain (Mogil, 1999), but also on nongenetic mechanisms that are known to influence the behavior of neonatal animals.
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In fact, variations in maternal behavior can influence genetic programming of the offspring and represent a non-genomic transmission of stress reactivity by influencing hypothalamic neuroendocrine functioning of their offspring and ultimately altering neurotransmitter systems (Meaney, 2001; Weaver et al., 2002, 2005, 2006; Champagne et al., 2003; Szyf et al., 2007). Furthermore, litter size has been recently proposed to affect adult anxious behavior in rodents: rats reared in big litters were less anxious than animals growing up in smaller litters, probably because of a complex interaction between prenatal environment, maternal behavior and interaction with other pups (Dimitsantos et al., 2007). We observed that B6C3F1 animals are reared in a significantly smaller litter with respect to CD1 mice. Moreover, B6C3F1 mice grow less fast and remain smaller compared to pups of the B6 parental strain (Turturro et al., 1999). Maternal behavior in both our mice strains is an issue that needs to be investigated as mice strains exhibit differences in maternal phenotype (Champagne et al., 2007). On the other hand, various studies have reported that neonatal stress influences maternal behavior (Anseloni et al., 2005; Walker et al., 2008) as well. During a short lasting inflammation and throughout the time the hind paw of the pup was inflamed, the mother spent more time nursing the pup while this period was followed by a more-alone phase (Anseloni et al., 2005). Given that inflammation caused by CFA has a deep and lasting impact on the health of pups, further studies are necessary to explore the role of maternal behavior in the behavioral outcomes we observed in mice that experienced such a pervasive type of inflammation. Naturally occurring variations in maternal care may shape adult pain sensitivity irrespective of a mild neonatal inflammation (Walker et al., 2008). On the whole, individual differences, caused by epigenetic programming, combined with genetic influences, the type of insult that perturbs the nest, and the degree of maturation of the pup may all account for the blunted reactivity on an elevated plus maze we observed after an injection with CFA on PND1 in CD1 adult mice. In the same way a different combination or interaction among these factors, may dampen emotional reactivity in B6C3F1 to a lesser extent. 4. Conclusions Although the interaction between the neonatal animal and its immediate environment is of great importance, our results clearly indicate that the choice of which mouse strain should be used in an experiment is an issue of extreme importance, and that further efforts must be directed to better characterize different mouse strains. Future research will focus on enhancing our understanding of the molecular impact of a robust neonatal insult in B6C3F1 parental inbred strains (C57BL6 and C3H) since these two strains possess different reactivity to inflammatory pain (Mogil, 1999). Quantitative trait locus (QLT) mapping studies could be performed on B6C3F1 mice to fully elucidate the role of genetic background in shaping behavioral outcome of an adverse neonatal experience. If we really want to learn something useful from animal models or paradigms, especially when it comes to understanding the consequences of neonatal pain, we must abide by their interstrain differences and try to find a way to learn from them. Only in this way, animal models will be useful and will help to get a better grip on the behavioral outcome of complex gene environment interaction. References Anand, K.J.S., Coskun, V., Thrivikraman, K.V., Nemeroff, C.B., Plotsky, P.M., 1999. Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol. Behav. 66, 627–637. Anseloni, V.C.Z., He, F., Novikova, S.I., Turnbach, Robbins, M., Lidow, I.A., Ennis, M., Lidow, M.S., 2005. Alteration in stress-associated behaviours and neurochem-
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Early neonatal inflammation affects adult pain reactivity and anxiety related traits in mice: genetic background counts Cristina Benatti a, Silvia Alboni a, Giacomo Capone a, Daniela Corsini a, Federica Caggia a, Nicoletta Brunello a, Fabio Tascedda a, Joan M.C. Blom a,b,* a b
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy Department of Paediatrics, University Hospital of Modena, Via del Pozzo 71, 41100 Modena, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2009 Received in revised form 3 July 2009 Accepted 30 July 2009
Protracted or recurrent pain and inflammation in the early neonatal period may cause long-lasting changes in central neural function. However, more research is necessary to better characterize the longterm behavioral sequelae of such exposure in the neonatal period. Objectives: (1) to study whether timing of postnatal exposure to persistent inflammation alters responsiveness to thermal pain in the adult animal; (2) to assess whether animals experiencing early postnatal chronic inflammation display altered anxiety related behavior; (3) to study the importance of genetic background. Newborn mice (outbred strain, CD1 and F1 hybrid strain, B6C3F1) received an injection of Complete Freund’s Adjuvant (CFA) or saline on either postnatal day 1 or 14 (PND1; PND14) into the left hind paw. Pain to radiant heat and anxiety were examined in 12-week-old adult animals. Adult baseline PWL was significantly decreased in CD1 mice exposed to CFA on PND 1 and 14 as compared to their saline treated counterparts. B6C3F1 mice exposed to CFA on PND14 showed markedly reduced baseline PWL compared to the PND14 saline group. Persistent inflammation experienced by B6C3F1 mice on PND1 failed to affect baseline adult thermal responsiveness. Adult mice, CD1 and B6C3F1, displayed low anxiety traits only if they had been exposed to persistent inflammation on PND1 and not on PND14. Our research suggests a role for genetic background in modulating long-term behavioral consequences of neonatal persistent inflammation: the data support the hypothesis that pain experienced very early in life differentially affects adult behavioral and emotional responsiveness in outbred (CD1) and hybrid mice (B6C3F1). ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Pain Development Long-term consequences Strain differences Anxiety traits CFA
The early neonatal period is characterized by great plasticity and reorganization. Premature infants undergo numerous invasive and painful procedures necessary to assure their survival while in the neonatal intensive care unit (Simons and Tibboel, 2006). Recurrent painful stimulation during the neonatal period produces long-term behavioral sequelae that last well into adulthood, affecting behavioral responsiveness to pain or stress later in life (Bhutta and Anand, 2002; Fitzgerald, 2005; Garg et al., 2003; Grunau et al., 2006). Neonatal pain and inflammation cause aberrant activation of central nervous circuits, inducing developmental consequences that depend on the type and intensity of the pain-inducing insult (Anand et al., 1999; Lidow, 2002; Peng et al., 2003). Further research is needed to characterize more specifically the behavioral sequelae, such as
* Corresponding author at: Department of Paediatrics, University Hospital of Modena, Via del Pozzo 71, 41100 Modena, Italy. Tel.: +39 059 2055162; fax: +39 059 2055625. E-mail address: [email protected] (Joan M.C. Blom). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.07.009
anxiety related behavior, of a strong noxious stimulation in the neonatal period. Rodents, and more specifically rats (Ruda et al., 2000; Lin and Al-Chaer, 2003; Ren et al., 2004; Boisse et al., 2005; Hohmann et al., 2005), are widely used as a model system to study the impact of early life pain and stress on future responsivity and represent a valuable resource to enhance our understanding concerning the long-term consequences of neonatal pain. However, a limited number of studies have been conducted using mice (Blom et al., 2006). At birth, the neurological maturity of pups is similar to that of human preterm neonates at 24 weeks of gestation, while the nociceptive system of a 1-year-old child closely resembles that of 2-week-old mice (Anand et al., 1999). Our previous findings demonstrated that CD1 adult mice exposed to a robust long-lasting local inflammatory insult (i.e. Complete Freund’s Adjuvant, CFA) early during development, are characterized by heightened sensitivity to pain (Blom et al., 2006). However, each rodent strain is characterized by a unique pattern of behavior and their genetic inheritance influences behavioral traits such as sensitivity to pain and stressors as well as anxiety and depression related behaviors (Bolivar et al., 2000; Bouwknecht and
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Paylor, 2002; Jacobson and Cryan, 2007; Mogil et al., 1999a,b, 2006; Mogil and McCarson, 2000). To further investigate long-term behavioral sequelae following a robust neonatal inflammation, we assessed whether genetic background could affect long-term behavioral consequences of neonatal pain in the adult life of mice in two different strains: a randomly outbred strain (CD1) and a F1 hybrid strain (B6C3F1). These strains, routinely used in our laboratory (Blom et al., 2002), are characterized by their own distinctive expression pattern of specific neuronal plasticity linked markers as well as by their display of behavioral differences, B6C3F1 being proner to show anxiety related behavior (data not published). In this study we set out to test whether timing of postnatal exposure (postnatal day 1 (PND1) or PND14) to a persistent inflammatory insult (CFA) could impact adult responsiveness to thermal pain as well as to a mild emotional stressor caused by placing adult animals on an elevated plus maze (EPM) using these two strains. To our knowledge this is the first study testing the effects of neonatal robust inflammation on adult mice comparing two different strains. Little is known on how genes interact with neonatal environmental cues and how this may impact on shaping individual behavioral differences, even more so, when the developmental equilibrium is shaken by a traumatic event.
natural aversion of rodents to enter open places/spaces. Consequently, high anxiety is thought to correlate with higher aversion to enter open spaces, while low anxiety corresponds to the willingness of the animal to explore. The EPM consisted of two open (5 cm wide, 30 cm long) and two closed arms (5 cm wide, 30 cm long, enclosed by a wall of 15 cm height) arranged in a plus configuration, joined by a central square (5 cm 5 cm) (Lister, 1990; Wall and Messier, 2000). The apparatus was made of opaque Plexiglas and kept on a wooden base 40 cm above the floor. The floor and walls of the maze were wiped between trials with a 70% alcohol:distilled water solution to prevent individual mice from following the scent of a previously tested animal. The illumination at the level of the maze was 100 lx. All animals were subjected to tests of a standard 5-min duration at 11 weeks of age; at the beginning of each test the mouse was placed individually in the center of the maze, its head facing an open arm (the same for all mice). All testing was conducted during the morning phase of the light:dark cycle between 08:00 and 14:00 h and recorded using a video camera. The parameters measured, selected among the battery of the EPM anxiety indices of Wall and Messier (2000), included four negatively correlated anxiety indices: open arm entries, percent of time spent – duration – in the open arm, number of scannings (dipping of the head/shoulders over the sides of the open arms) and scanning duration and four positively anxiety correlated indices: closed arm entries, percent of time spent – duration – in the closed arm, number of risk assessments (head/shoulder dipping over the closed arm) and risk assessment duration. An entry in a given arm was counted only when the mouse had completely entered the arm. The duration was expressed as percentage of the entire 5 min of testing; otherwise, the data were expressed as the number of occurrences during testing.
1. Experimental procedures
The behavioral response to heat nociception of 12-week-old male mice was determined using a stainless steel hot plate (maintained at 48 0.1 8C) (UGO BASILE, Hot Plate DS37). The mouse’s right hind limb, both forelimbs, and trunk were gently supported by the experimenter, allowing the left hind limb of the animal to rest lightly but stably on the heated surface (Blass and Blom, 1996). The timing circuit was activated when the limb was brought into contact with the hot plate surface and stopped upon unambiguous hind limb withdrawal; this defined the latency for heat withdrawal. Paw withdrawal latency (PWL) was tested for five times at intervals of 5 min and was calculated as the mean, excluding the first familiarization trial (Ruda et al., 2000). Data were collected by an experimenter who was uninformed about the experimental condition. A 40 s cut-off was applied to prevent tissue damage. At 12 weeks of age, neonatal CFA- and neonatal saline treated animals received an unilateral injection of 100 mL of CFA (1:1) in the left hind paw. All adult animals irrespective of neonatal treatment, displayed pain related behaviors after CFA injection. Baseline paw withdrawal latencies of the left hind paw to a radiant heat source were determined in all the experimental groups before receiving the inflammatory injection. Withdrawal latency was tested again 24 h after injection with CFA into the adult left hind paw. All testing was conducted during the morning phase of the light:dark cycle between 08:00 and 14:00 h
1.1. Animals Male offspring from multiparous CD1 and B6C3F1 female mice (Charles River, Lecco, Italy) served as subjects. Pregnant females were individually housed in plastic tubs (28 cm 17 cm 12 cm) with stainless steel wire lids. The floor of each cage was covered with bedding of wood shavings. Tap water and food were available ad libitum. Lights were on from 08.00 to 20.00 h. Colony room temperature ranged from 20 to 24 8C, with humidity uncontrolled. Newborn litters discovered before 11.00 h were considered born that day and designated PND0 (postnatal day 0). Sexing of the pups was conducted at birth by evaluation of the ano-genital distance. Pups were treated between 12.00 and 18.00 h. Only male mice were used in the experiments. 1.2. Experimental procedure Outbred CD1 and hybrid B6C3F1 pups were injected with Complete Freund’s Adjuvant (CFA) in their left hind paw at postnatal day 1 (PND1) or PND14. Neonatal mice pups received a single injection of CFA (CFA, Sigma Aldrich1 F5881, 0.5 mg/mL heat killed Mycobacterium tuberculosis suspended in an stock emulsion of oil:saline (1:1)) or saline alone on PND1 or PND14. At PND1, pups were injected with 20 mL of the CFA emulsion previously prepared, diluted with saline (2:1) and PND14 pups were injected with 40 mL of this suspension. Thus, the amount of CFA injected was approximately 5 mg for PND1 treated animals and 10 mg for PND14 treated animals. In this way we adjusted dose to weight and maturation of the mice, procedure previously used by us (Blom et al., 2006) as well as by others (Ruda et al., 2000). The animals were then left undisturbed to mature. The pups were checked by the experimenter for distinct pain related behaviors at the time of CFA injection (immediate shaking and licking). Mice injected with CFA at PND1 or PND14, responded by immediate shaking and licking of the paw that persisted for at least 10 min. Saline injected neonatal animals did not show persistent pain related licking or shaking. For the next 14 days post-injection pups were observed closely with respect to the formation of edema and erythema. Within 7 days swelling of the injected hind paw was reduced to baseline (matching the contra-lateral untreated paw) except for the group treated with CFA at PND14 where swelling persisted for 10 days following injection (data not shown). No significant differences in body weight were found in saline or CFA treated animals during the entire duration of the experiment. After weaning at PND21, pups were housed three per cage until the end of the experimental procedure. Animals were checked for signs of discomfort as indicated by animal care and use guidelines (National Academy of Sciences. Guide for the care and use of laboratory animals, 1998, ‘‘Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research’’ (National Research Council 2003)). EC guidelines (EEC Council Directive 86/609 1987), local (University of Modena) and Italian legislation on animal experimentation (Decreto Legislativo 116/92) and the International Association for the Study of Pain ethical guidelines were applied to the experiments. 1.3. Elevated plus maze anxiety-like behavior test Anxiety traits of each animal were measured using an elevated plus maze (EPM), a behavioral model of anxiety based on the conflict between exploration and the
1.4. Thermal nociception
1.5. Data analysis Behavioral data were analyzed with a two-factor (age treatment) analysis of variance (ANOVA). Multiple comparisons were conducted with one-way’s ANOVA. At no time more than four groups were compared among each other. All mean differences were considered statistically significant if p < 0.05.
2. Results 2.1. Baseline paw withdrawal latency in neonatally treated CD1 mice As previously observed in our laboratory (Blom et al., 2006), CD1 mice injected at PND1 or PND14 with CFA, displayed reduced latency in lifting their left hind paw from the hot plate with respect to their saline exposed counterparts as adults. We tested the hypothesis that a strong noxious inflammation experienced by CD1 pups either on PND1 or PND14 enhances adult responsivity to a radiant heat source. CD1 baseline values differed significantly among each other [ANOVA univariate; F (3,71) = 16.630; p < 0.001; Fig. 1A and B]. A two-factor ANOVA (neonatal treatment age of injection) revealed an overall main effect of type of neonatal treatment [p < 0.0001; Fig. 1A and B] and of age of injection [p = 0.024]. No interaction was observed between the two terms (p > 0.05). Pairwise comparisons showed no difference between PND1 and PND14 saline injected mice (p > 0.05). Similarly, PND1CFA treated mice behaved as mice injected with CFA on PND14 (p > 0.05).
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Fig. 1. Paw withdrawal latency (PWL) of CD1 adult mice treated with saline or CFA on postnatal day 1 (PND1) (A), and on postnatal day 14 (PND14) (B). Adult baseline PWL of mice injected with CFA on either PND1 or PND14 was lower than saline injected animals (*p < 0.05). 24 h after baseline measurement, each animal received an injection with CFA in the left hind paw. Adult treatment with CFA reduced PWL in all experimental groups irrespective of neonatal age of injection (§p < 0.05; ruled box). PWL was measured at 12 weeks of age at baseline and 24 h after exposure to CFA as adults. Data are represented as means (SEM); *p < 0.05 is significant difference between baseline values of saline treated animals compared to baseline values of CFA treated mice; §p < 0.05 is significant difference between baseline and mice treated with CFA at 12 weeks of age.
Fig. 2. Paw withdrawal latency (PWL) of B6C3F1 adult mice treated with saline or CFA on postnatal day 1 (PND1) (A), and on postnatal day 14 (PND14) (B). CFA injected mice displayed lower baseline PWL than saline injected mice (*p < 0.05), only if exposed to the inflammatory agent on PND14. 24 h after baseline measurement, each animal received an injection with CFA in the left hind paw. Adult treatment with CFA reduced PWL in all experimental groups irrespective of neonatal age of injection (§p < 0.05; ruled box). PWL was measured at 12 weeks of age at baseline and 24 h after exposure to CFA as adults. Data are represented as means (SEM); *p < 0.05 is significant difference between baseline values of saline treated animals compared to baseline values of CFA treated mice; §p < 0.05 is significant difference between baseline and mice treated with CFA at 12 weeks of age.
2.2. Paw withdrawal latency after adult exposure to CFA in neonatally treated CD1 mice
The behavior observed with respect to heat nociception of adult B6C3F1 is strongly affected by the type of neonatal insult as well as by the timing of the insult.
24 h after measuring basal PWL, CD1 adult animals received an unilateral injection of CFA into the left hind paw that produced a reduction in PWL (Fig. 1A and B). To assess whether the type of treatment and the developmental stage at which treatment was received were able to influence thermal nociception even following an inflammatory challenge as an adult, a two-factor ANOVA (neonatal treatment age of injection) was performed. A main effect was observed for the type of treatment the mice received as neonates [F (3,71) = 5.458; p = 0.022], but not for the age at which they were first treated (p > 0.05). 2.3. Baseline paw withdrawal latency in neonatally treated B6C3F1 mice Similar to CD1 outbred animals, the hypothesis was tested that a strong noxious inflammation experienced by B6C3F1 hybrid pups either on PND1 or PND14 increases adult responsivity to a radiant heat source. We observed that adult thermal nociception of hybrid B6C3F1 mice treated neonatally with CFA or saline followed a different pattern with respect to that observed in outbred CD1 mice. B6C3F1 baseline values differed significantly among each other [F (3,32) = 6.948; p = 0.001; Fig. 2A and B]. However, an injection of CFA at PND1 failed to increase adult thermal nociception, since no difference was observed in baseline PWL among these animals and in mice receiving saline 1 day after birth (p > 0.05). On the other hand, animals injected at PND14 with CFA showed reduced PWL measured at 12 weeks of age with respect to their saline exposed counterparts (p < 0.05). Pairwise comparisons revealed no difference between PND1 and PND14 saline injected mice (p > 0.05), while animals exposed to CFA on PND1 were hypoalgesic with respect to the group exposed to the same inflammatory agent 2 weeks after birth (p < 0.05). A two-factor ANOVA (neonatal treatment age of injection) showed an overall main effect of both neonatal treatment [F (3,32) = 6.331, p = 0.017] and of age of injection [F (3,32) = 15.764; p < 0.0001]. No interaction was observed between the two terms.
2.4. Paw withdrawal latency after adult exposure to CFA in neonatally treated B6C3F1 mice Re-exposure to CFA, 24 h after measuring their basal PWL, caused a decrease in PWL in adult B6C3F1 mice exposed to either saline or CFA at PND1 or PND14 (Fig. 2A and B). B6C3F1 PND1 saline injected animals displayed a significant reduction in PWL [down by approximately 69%] after being exposed to CFA at 12 weeks of age. Even so, mice exposed to CFA at PND1 exhibited a decrease [down by approximately 49%] in PWL when re-exposed to CFA at 12 weeks of age. A similar pattern was observed in hybrid mice injected at PND14: adult exposure to CFA reduced PWL by 51% in mice treated with saline while those treated with CFA at PND14 showed a less marked reduction [down by approximately 40%]. To assess whether the type of treatment and the developmental stage at which treatment was received were able to influence thermal nociception even following an inflammatory challenge as an adult, a two-factor ANOVA (neonatal treatment age of injection) was performed. A main effect was found for age of injection [F (3,32) = 5.837; p = 0.022] but not for neonatal treatment [F (3,32) = 0.129; p > 0.05]. Furthermore, an interaction was observed between the two terms [F (3,32) = 4.452; p = 0.043], suggesting that the developmental stage in which the animals experienced a noxious stimulus is a critical factor influencing their behavior as an adult. Pairwise comparisons revealed a significant difference between PWL of animals exposed to CFA on PND1 as compared to PND14, in the presence of an ongoing inflammation (p < 0.05). When comparing adult baseline thermal sensitivity of CD1 and B6C3F1 mice treated neonatally with saline (Figs. 1 and 2), their PWLs differed significantly [ANOVA univariate; F (3,52) = 3.983; p = 0.013; Figs. 1 and 2]. However, further analyses revealed that only CD1 and B6C3F1 mice treated at PND14 differed among each other (p < 0.05). A two-factor ANOVA (strain age of injection)
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2.5. Paw withdrawal latency difference between baseline and adult CFA exposure in neonatally treated CD1 and B6C3F1 mice
Fig. 3. Paw withdrawal latency difference between baseline and adult CFA exposure. Adult reactivity to thermal pain with or without an ongoing inflammation is influenced by genotype, by the developmental maturity of animals when they received the insult and by the type of treatment they received as pups. *p < 0.05 is significant difference between neonatal saline and CFA treated mice. §p < 0.05 is significant difference between PND1 and PND14 saline treated mice.
revealed that neither strain nor age of injection produce a main effect, while a significant interaction was observed between the two terms [F (3,32) = 11.096, p = 0.002]. CD1 or B6C3F1 mice receiving an injection with saline at either PND1 or PND14, when re-exposed to CFA as adults, were statistically undistinguishable [ANOVA univariate; F (3,52) = 0.490; p > 0.05; Figs. 1 and 2].
A difference score was obtained by calculating the difference between PWL after adult CFA exposure and their respective baseline values (Fig. 3; Blom et al., 2006). Adult CFA administration reduced the time spent on the hot plate in all experimental groups, but behavioral reactivity to thermal nociception in the presence of an ongoing inflammation was differentially affected by type and age of injection. A two-factor ANOVA (neonatal treatment age of injection) revealed an overall main effect of both neonatal treatment [F (3,32) = 8.390; p = 0.07] and age of injection [F (3,32) = 11.969; p = 0.03]. No interaction was observed between the two terms (p > 0.05). This suggests that adult reactivity to thermal pain with or without an ongoing inflammation is strongly influenced by the developmental maturity of animals receiving the insult, as well as by the type of treatment they received as pups. Re-exposure to CFA at 12 weeks of age reduced the time spent on the hot plate, as observed in our laboratory also in CD1 mice (Fig. 3; Blom et al., 2006), pairwise comparisons though revealed a significant difference between PND1 (saline) and PND14 (saline) groups [F (1,17) = 6.197; p = 0.023], as well as between mice exposed to CFA on postnatal day 1 and 14 [F (1,15) = 6.400; p = 0.023]. The two types of neonatal treatment differentially affected adult behavioral reactivity only in mice treated on PND14 [F (1,14) = 10.996; p = 0.005], while exposure to either CFA or saline at PND1 did not affect the difference in PWL to a radiant heat
Fig. 4. Anxious behavior of 11-week-old CD1 mice treated with saline or CFA on PND1 (A and B), and on PND14 (C and D). Positively correlated anxiety related behaviors (closed arm entries, closed arm duration, number of risk assessment (head/shoulder dipping over the closed arm) and duration) and negatively correlated anxiety related behaviors (open arm entries, open arm duration, number of scanning (dipping of the head/shoulders over the sides of the open arms) and duration) were evaluated on an elevated plus maze. PND1-CFA treated animals displayed less anxious behavior with respect to their saline injected-counterparts; animals exposed to CFA on PND14 did not differ from saline treated mice. Data are expressed as number of occurrences and % of total testing time, each column represents a mean (SEM); *p < 0.05 is significant difference with baseline values of saline treated animals.
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source before and after adult exposure to CFA (p > 0.05). This suggest that in B6C3F1 mouse strain, exposure to a painful procedure in a period of great plasticity may cause alterations in the behavioral reactivity of animals that may induce a heightened sensitivity when re-exposed to a robust inflammatory insult. 2.6. Behavior of CD1 adult animals experiencing a robust neonatal inflammatory insult in the elevated plus maze Anxious behavior of CD1 outbred mice exposed to CFA at PND1 or PND14 was assessed on an elevated plus maze (EPM) with respect to their saline exposed counterparts. We tested the hypothesis that a strong noxious inflammation experienced either on PND1 or PND14 affects adult anxious behavior. Exposure to CFA on postnatal day 1 strongly affected anxiety behavior with respect to mice exposed to saline at the same postnatal age. Univariate ANOVA of animals receiving either saline or CFA at the same postnatal age revealed that PND1-CFA treated animals displayed a less anxious phenotype (i.e. low anxiety traits), as behaviors positively correlated with anxiety were diminished while behavioral indices correlating negatively with anxiety were increased when compared to saline PND1-exposed animals (Fig. 4A and B). Furthermore, a reduction was observed in the time spent in the closed arms [F (1,21) = 10.048; p = 0.005; down by approximately 20%] and time spent in risk assessment [F (1,21) = 4.512; p = 0.046; down by approximately 20%]. Also, exposure to CFA on PND1 caused a significant increase in closed arms entries when
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compared to exposure to saline [F (1,21) = 6.967; p = 0.015; up by approximately 40%], while the number of risk assessments performed during the test was not affected by the type of treatment received on PND1 (p > 0.05). CD1 animals exposed to CFA on PND1 showed a robust increase in negatively anxiety correlated behavioral indices such as time spent in the open arms of the maze [F (1,21) = 9.203; p = 0.007; up by approximately 240%], number of entries in these arms [F (1,21) = 8.642; p = 0.008; up by approximately 225%], time spent in scanning [F (1,21) = 8.458; p = 0.009; up by approximately 110%] and number of scannings [F (1,21) = 16.530; p = 0.001; up by approximately 120%]. On the other hand, a strong noxious stimuli experienced on PND 14 failed to affect any of the behavioral parameters considered (p > 0.05) (Fig. 4C and D). These results suggest that adult CD1 animals displayed low anxiety traits only after receiving a CFA injection on PND1 and not on PND14. 2.7. Behavior of B6C3F1 adult animals experiencing a robust neonatal inflammatory insult in the elevated plus maze B6C3F1 mice treated with saline or CFA on postnatal day 1 or 14, were tested on an elevated plus maze as were their CD1 counterparts. B6C3F1 hybrid mice were less affected by a persistent inflammation than the outbred strain. PND1-CFA treated B6C3F1 were undistinguishable from their saline exposed counterparts in all the behaviors that positively
Fig. 5. Anxious behavior of 11-week-old B6C3F1 mice treated with saline or CFA on PND1 (A and B), and on PND14 (C and D). Positively correlated anxious behaviors (closed arm entries, closed arm duration, number of risk assessment (head/shoulder dipping over the closed arm) and duration) and negatively correlated anxious behaviors (open arm entries, open arm duration, number of scanning (dipping of the head/shoulders over the sides of the open arms) and duration) were evaluated on an elevated plus maze. PND1-CFA treated animals displayed less anxious behavior with respect to their saline injected-counterparts; animals exposed to CFA on PND14 did not differ from saline treated mice. Data are expressed as number of occurrences and % of total testing time, each column represents a mean (SEM); *p < 0.05 is significant difference with baseline values of saline treated animals.
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correlated with anxiety, with the exception of time spent in the closed arms [F (1,18) = 5.642; p = 0.029; down by approximately 20%]. Among the negatively anxiety correlated behavioral indices only time spent in scanning [F (1,18) = 6.912; p = 0.018; up by approximately 120%] and the number of scannings [F (1,18) = 5.791; p = 0.028; up by approximately 90%] were enhanced following a robust early inflammation when compared to saline PND1-exposed animals (Fig. 5A and B). Time spent in the open arms of the maze and number of entries in these arms were not influenced by the type of neonatal treatment that animals had received (p > 0.05). A strong noxious stimuli experienced on PND 14 failed to affect any of the behavioral parameters considered (p > 0.05) (Fig. 5C and D). B6C3F1 mice treated on PND1 with CFA displayed low anxiety with respect to their saline exposed counterparts, and to a lesser extent than CD1 animals receiving the same type of neonatal treatment. On the other hand, a robust inflammation experienced on PND14 failed to affect adult anxious behavior in both strains considered. 2.8. Spontaneous litter size CD1 litters were significantly larger (11 pups for each dam) than B6C3F1 (8 pups for each dam) [F (1,17) = 10.891; p < 0.01], with approximately 50% males for each strain. Moreover, CD1 pups were bigger and more mature than their B6C3F1 counterparts (visual observation collected by the experimenter). 3. Discussion In this study, we investigated for the first time the long-term consequences of a robust neonatal inflammatory insult on the response to pain and subsequent anxious behavior in adult animals of two different mouse strains: randomly outbred CD1 mice and the hybrid strain B6C3F1. The latter is a first-generation (F1) hybrid strain produced by crossing C57BL/6 females and C3H males (Kalueff et al., 2007; Roy et al., 2007). In summary, our results show that: (1) CD1 outbred mice, exposed to a robust long-lasting local inflammatory insult early during development, grow into adults that display enhanced responsiveness to thermal pain. Also, adult animals display low anxiety traits only after receiving a CFA injection on PND1 and not on PND14. (2) B6C3F1 hybrid adult mice exposed to a robust long-lasting local inflammatory insult on PND14 are characterized by hyperalgesia, while treatment with CFA on PND1 fails to affect adult reactivity to thermal nociception. Moreover, adult animals display low anxiety traits only if they received a neonatal persistent inflammation on PND1 and not on PND14. Each mouse strain exhibits its own specific phenotype; behavioral differences between strains range from anxiety to locomotor activity, to influencing depressive-like behavior or the response to drugs (Bolivar et al., 2000; Bouwknecht and Paylor, 2002; Ducottet and Belzung, 2005; Griebel et al., 2000; Wahlsten et al., 2006). Also, distinctive patterns of responsiveness to various nociceptive tests were described for different strains (Lariviere et al., 2002; Mogil et al., 1999a,b). To our knowledge, this is the first study to investigate the effects of a strong persistent noxious stimulus on the behavior of two different mouse strains. CD1 mice, as already reported by our laboratory (Blom et al., 2006), receiving CFA as neonates grow into hyperalgesic adults, which is consistent with previous findings obtained in rats exposed to several types of inflammatory insults (Boisse et al., 2005; Hohmann et al., 2005; Ren et al., 2004; Ruda et al., 2000). CD1 mice receiving CFA on PND1 are characterized by a lower baseline PWL with respect to saline treated animals while
B6C3F1 PND1-CFA treated mice were indistinguishable from their saline exposed counterparts. CD1 mice handled and treated at PND1, an extremely sensitive period in development, are characterized as adults by a blunted ability to process noxious stimuli. In fact in adults, an ongoing inflammation with a strong impact on nociceptive circuit formation, like the one caused by CFA, resulted in a more marked reduction in paw withdrawal latencies in mice exposed during development (PND14), at an higher degree of maturity (Blom et al., 2006). B6C3F1 hybrids on the other hand, are more reactive in the presence of an ongoing inflammation if handled and treated very early during development, while persistent inflammation experienced on PND14 caused a less robust reactivity when exposed to CFA as adults. Neonatal treatment differentially affected adult behavioral reactivity only in mice treated on PND14, while in adult mice exposed to either CFA or saline on PND1, pain related behavioral response did not differ. This suggests that neonatal experience at this young age is not of influence in the B6C3F1 strain. Thermal sensitivity was shown to depend on the severity of the neonatal insult: a robust inflammation caused by CFA resulted in basal hyperalgesia (Hohmann et al., 2005; Ruda et al., 2000) while a milder insult such as a local action that lasted for only 48 h resulted in basal hypoalgesia (Anseloni et al., 2005; Ren et al., 2004). CFA injection is a very strong and painful stimulus and produces interstitial infiltration of lymphocytes that lasts until 8 weeks after the neonatal injection (Walker et al., 2003). Since our mice were tested at 12 weeks of age, we cannot exclude that the hyperalgesic condition, observed in these 2 strains after an injection with CFA on PND14, may be mediated, at least in part, by a state of chronic inflammation caused by the massive effect of CFA on the immune system. This, then, would suggest that the model adopted by us and proposed by Hohmann et al. (2005) before, may represent a useful behavioral tool to better understand a condition such as juvenile rheumatoid arthritis, which is characterized by persistent inflammation and pain with an early onset during development. Furthermore, neonatal exposure to an early inflammatory insult affected behavioral reactivity in a commonly used paradigm testing anxiety. The inter-relationship between emotional and nociceptive outcomes is an issue still under investigation (Craig, 1999; Wilson et al., 2007) Compared to randomly outbred CD1 mice, exposure to CFA on PND1 affected adult anxiety behavior to a lesser extent in hybrid B6C3F1 mice. Interestingly, no significant differences were found in any of the behaviors examined in the EPM when considering mice treated at PND14 either with saline or with CFA, irrespective of strain. It seems that the ‘‘critical window’’ of vulnerability for the effects of neonatal inflammatory pain on anxiety lies within the first week of age (Spencer et al., 2006). The hypoanxious traits we observed in CD1 mice and, to a lesser extent, in B6C3F1 animals are similar to those reported previously by Anseloni et al. (2005) in rats. It is remarkable that inflammatory insults, like CFA and carrageenan with a different physical impact on pups, produce similar anxiety related behaviors, but affect basal sensitivity to thermal stimulation in an opposite manner. Furthermore, aberrant activation of the immune system in animals at PND1 may induce prolonged changes in the developing hypothalamic–pituitary– adrenal axis (LaPrairie and Murphy, 2007), which in turn may alter anxiety related behavior later in life. The effects of a neonatal inflammatory insult on adult anxious behavior seem to depend more on the timing of the insult rather than on its severity. Thus, behavioral sequelae of neonatal pain are part of a complex and highly coordinated response and depend on the intensity and duration of the inflammatory insult (Lim et al., 2009), on the genetic background of each strain (Mogil, 1999), but also on nongenetic mechanisms that are known to influence the behavior of neonatal animals.
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In fact, variations in maternal behavior can influence genetic programming of the offspring and represent a non-genomic transmission of stress reactivity by influencing hypothalamic neuroendocrine functioning of their offspring and ultimately altering neurotransmitter systems (Meaney, 2001; Weaver et al., 2002, 2005, 2006; Champagne et al., 2003; Szyf et al., 2007). Furthermore, litter size has been recently proposed to affect adult anxious behavior in rodents: rats reared in big litters were less anxious than animals growing up in smaller litters, probably because of a complex interaction between prenatal environment, maternal behavior and interaction with other pups (Dimitsantos et al., 2007). We observed that B6C3F1 animals are reared in a significantly smaller litter with respect to CD1 mice. Moreover, B6C3F1 mice grow less fast and remain smaller compared to pups of the B6 parental strain (Turturro et al., 1999). Maternal behavior in both our mice strains is an issue that needs to be investigated as mice strains exhibit differences in maternal phenotype (Champagne et al., 2007). On the other hand, various studies have reported that neonatal stress influences maternal behavior (Anseloni et al., 2005; Walker et al., 2008) as well. During a short lasting inflammation and throughout the time the hind paw of the pup was inflamed, the mother spent more time nursing the pup while this period was followed by a more-alone phase (Anseloni et al., 2005). Given that inflammation caused by CFA has a deep and lasting impact on the health of pups, further studies are necessary to explore the role of maternal behavior in the behavioral outcomes we observed in mice that experienced such a pervasive type of inflammation. Naturally occurring variations in maternal care may shape adult pain sensitivity irrespective of a mild neonatal inflammation (Walker et al., 2008). On the whole, individual differences, caused by epigenetic programming, combined with genetic influences, the type of insult that perturbs the nest, and the degree of maturation of the pup may all account for the blunted reactivity on an elevated plus maze we observed after an injection with CFA on PND1 in CD1 adult mice. In the same way a different combination or interaction among these factors, may dampen emotional reactivity in B6C3F1 to a lesser extent. 4. Conclusions Although the interaction between the neonatal animal and its immediate environment is of great importance, our results clearly indicate that the choice of which mouse strain should be used in an experiment is an issue of extreme importance, and that further efforts must be directed to better characterize different mouse strains. Future research will focus on enhancing our understanding of the molecular impact of a robust neonatal insult in B6C3F1 parental inbred strains (C57BL6 and C3H) since these two strains possess different reactivity to inflammatory pain (Mogil, 1999). Quantitative trait locus (QLT) mapping studies could be performed on B6C3F1 mice to fully elucidate the role of genetic background in shaping behavioral outcome of an adverse neonatal experience. If we really want to learn something useful from animal models or paradigms, especially when it comes to understanding the consequences of neonatal pain, we must abide by their interstrain differences and try to find a way to learn from them. Only in this way, animal models will be useful and will help to get a better grip on the behavioral outcome of complex gene environment interaction. References Anand, K.J.S., Coskun, V., Thrivikraman, K.V., Nemeroff, C.B., Plotsky, P.M., 1999. Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol. Behav. 66, 627–637. Anseloni, V.C.Z., He, F., Novikova, S.I., Turnbach, Robbins, M., Lidow, I.A., Ennis, M., Lidow, M.S., 2005. Alteration in stress-associated behaviours and neurochem-
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