All procedures performed in this study were approved by the Institutional Ethics Committee for Animal Research (CETEA - Comite de Etica em Experimentacao Animal, protocol number 044/2010) from the School of Medicine of Ribeirao Preto, University of Sao Paulo, and were carried out according to the guidelines of the International Association for the Study of Pain (IASP) and National Institutes of Health (NIH).

Female Wistar rats were accompanied throughout pregnancy. All animals were maintained on 12 hour light/dark cycle, controlled temperature and relative air humidity and noise, with food and water ad libitum. Immediately after birth, each litter was randomly assigned in pain groups or control groups. Pain groups received noxious stimuli that consisted of rapid insertions of a 30-gauge needle into the plantar foot pad and the lateral surface of the right hind paw (15 insertions), twice per day for 15 days, starting at birth. Control groups received tactile stimuli with a cotton-swab into the plantar and the lateral surface of the right paw twice per day for 15 days starting at birth. This protocol was adapted from Anand et al.35 and used in previous studies from our laboratory33,34,36.

A total of 157 neonate Wistar rats (Rattus norvegicus) were divided into 16 groups, being 8 control groups and 8 pain groups. Control or pain animals were studied at 15, 30, 90 and 180 days of life and subdivided by gender, as shown on Table 1.

The behavioral test was performed using the Elevated Plus-Maze (EPM). This apparatus consist of two open arms (50 x 10 cm) and two enclosed arms (50 x 10 x 40 cm) with an open roof, connected by a central platform (10 x 10 cm) and elevated 50 cm from the floor7,37.

Before evaluation, the animals were weighed and then remained in the experiment room in their cages for at least 15 minutes so they could get adapted to the environment. Afterwards, they were placed for 5 minutes in the EPM and recorded with a video camera. Each animal was exposed to the EPM only once so they would not get used to the apparatus.

At the beginning of the test the animals were placed individually on the central platform of the maze, with the head turned to one of the open arms. Each entry in one of the arms, the time spent in the open or the closed arm as well the time spent on the central platform (time to make a decision) was recorded, counted and analyzed by the software X-Plot Rat. An entry in one of the arms was computed when the animal placed all four feet within the specific arm.

The Sigma Stat software, version 3.01 (Jandel Scientific) was used for the analysis. The values were expressed as mean ± SEM.

Statistical analysis was performed by unpaired t-test for comparisons between two groups (males x females, same age and control x pain groups, same age). Different ages were compared by the one-way ANOVA, followed by Holm-Sidak post-test. The interaction between ages and treatments was investigated by the repeated measure two-way ANOVA, followed by Duncan s test.

Statistical significance was considered if p values were equal or smaller than 0.05.

Body weight data from all groups is shown in Table 2. Body weight increased significantly with age in all groups studied independently of gender or treatment (male control and pain, and female control and pain). Also, males were heavier than females at 90 and 180 days of age, in control and pain groups.

The comparison between pain and control groups showed that pain group animals presented smaller body weight at ages of 90 days for females (p = 0.028) and 180 days for males (p = 0.001), with a non-significant (p = 0.068) smaller weight in females with this age.

The number of entries in the open arms of the EPM for all experimental groups is shown in Figure 1, upper panels.

The comparison between ages on control animals showed an increase in the number of entries from 15 to 30 days of age followed by a decrease of this number at older ages. For males, significant differences were detected between 15 and 30 days (P = 0.009) and between 30 and 180 days (p = 0.009). For females, differences were detected between 15 and 30, 30 and 90 and 30 and 180 days (p = 0.001). For the pain groups, males showed a similar pattern described for controls but the increased number of entries was detected at 90 days of age, with difference between 15 and 90, 15 and 180 and 90 and 180 days (p = 0.002). For the females of the pain group, no significant differences were observed with aging (p = 0.160).

The comparison between genders on control groups showed no significant difference in all ages studied. On pain groups, gender differences were detected only at 15 days of age, being the number of entries in the open arm in females larger than males (p = 0.006).

The comparison between treatments (control and pain) showed, for males, a tendency for a reduced number of entries in the open arms in the pain group at 15 (p = 0.078) and 30 (p = 0.092) days and the opposite situation at 180 days (p = 0.05). No differences were found between pain and control groups in females.

The percentage of the time that the animals spent in the open arms of the EPM from all experimental groups is shown in Figure 1, lower panels. As expected, results are in line with those for the number of entries in the open arms. The most important difference was that males from pain group at 180 days of age spent more time in the open arms of the EPM compared to all other ages. They also spent more time in the open arms compared to controls of the same age. For the females, 30 days old animals spent more time in the open arms of the EPM that animals with 15, 90 and 180 days on the control group while no differences were observed for the pain group.

The number of entries in the closed arms of the EPM from all experimental groups is shown in Figure 2, upper panels.

The comparison between ages on control animals showed an increase in the number of entries from 15 to 30 days of age followed by a decrease of this number in older ages. For males, significant difference was detected between 15 and 30 days. For females, differences appeared between 15 and 30 and 30 and 90 days. For the pain groups, males showed a similar pattern described for controls but the increased number of entries was detected at 90 days of age, with differences between 15 and 30, 15 and 90 and 15 and 180 days. For the females in the pain group, a difference was present only between 15 and 90 days, also with the 90 days animals showing the larger values compared to the other ages.

The comparison between genders on control groups showed no significant difference in all ages studied. On pain groups, gender differences were detected only at 15 days of age, being the number of entries in the closed arm in females larger than males.

The comparison between treatments (control and pain) showed, for males, a reduced number of entries in the closed arms in the pain group at 15 days (p = 0.015) with no differences for the other ages. No differences were found between pain and control groups in females.

The percentage of the time that the animals spent in the closed arms of the EPM from all experimental groups is shown in Figure 2, lower panels. Interestingly, the more time the animals entered in the closed arms, the less time they spent in these arms, in all groups and ages studied. This is the opposite behavior of the animals in the open arms as the larger number of entries in those arms, the larger the time they spent in there.

The comparison between ages on control animals showed an increased time spent in the closed arms at 180 days compared to 30 days of age. For females, animals at 90 days and at 180 days spent more time in the closed arms compared to 30 days old animals. For the pain groups, male animals at 90 days and at 180 days spent less time in the closed arms compared to 15 days old animals while no differences were observed between female pain groups.

The comparison between genders on control groups showed no significant difference in all ages studied. On pain groups, gender differences were detected only at 15 days of age, being the time spent in the closed arm in females smaller than males.

The comparison between treatments (control and pain) showed, for males, an increase in the time spent in the closed arms in the pain group at 15 days (p = 0.005) with no differences for the other ages. For the females, differences were detected on ages 30 and 90 days, with opposite behaviors. While 30 days old controls spent less time in the closed arms compared to the pain animals, 90 days old controls spent more time in the closed arms compared to the pain animals.

Male and female control animals reduced the time spent in the central platform with aging in a very similar way. The large difference observed was between ages 30 and 90 days, in both genders. No differences between genders were observed between the controls.

Females from the pain groups showed a similar pattern of reduction of the time spent in the central platform (difference found between 15 and 180 days old animals), but with higher values compared to female controls in all ages. Statistical significance between control and pain groups was attained only at 15 days, while for the other ages only a trend towards higher values was observed.

Male pain animals showed an erratic behavior when analyzing the time spend in the central platform. Younger animals (15 days old) spent significantly less time in the central platform compared to controls but increased this time up to 90 days of age, then decreased again at 180 days. No differences between ages were detected.

There are neural circuits and connections involved both in pain and anxiety and innate fear. It is known that dorsal periaqueductal gray matter play a role in descendent modulation of pain but it also receive projections from the amygdala, being an important structure on defensive behavior38,39. Thus, we hypothesized that painful stimulus could affect behavioral circuits producing, perhaps, an anxiety-like behavior. The elevated plus-maze (EPM) is an apparatus largely used to detect anxiety-related behaviors in rats and it is described that a lower number of entries and time spent in the open arms suggest an anxiety-like behavior37.