A Painful Beginning: Early Life Surgery Produces Long-Term Behavioral Disruption in the Rat

Abstract

Early life surgery produces peripheral nociceptive activation, inflammation, and stress. Early life nociceptive input and inflammation have been shown to produce long-term processing changes that are not restricted to the dermatome of injury. Additionally stress has shown long-term effects on anxiety, depression, learning, and maladaptive behaviors including substance abuse disorder and we hypothesized that early life surgery would have long-term effects on theses complex behaviors in later life. In this study surgery in the rat hindpaw was performed to determine if there are long-term effects on anxiety, depression, audiovisual attention, and opioid reward behaviors. Male animals received paw incision surgery and anesthesia or anesthesia alone (sham) at postnatal day 6. At 10 weeks after surgery, open field center zone entries were decreased, a measure of anxiety (n = 20) (P = 0.03) (effect size, Cohen's d = 0.80). No difference was found in the tail suspension test as a measure of depression. At 16-20 weeks, attentional performance in an operant task was similar between groups at baseline and decreased with audiovisual distraction in both groups (P < 0.001) (effect size, η² = 0.25), but distraction revealed a persistent impairment in performance in the surgery group (n = 8) (P = 0.04) (effect size, η² = 0.13). Opioid reward was measured using heroin self-administration at 16-24 weeks. Heroin intake increased over time in both groups during 24-h free access (P < 0.001), but was greater in the surgery group (P = 0.045), with a significant interaction between time and treatment (P < 0.001) (effect size, Cohen f ² = 0.36). These results demonstrate long-term disruptions in complex behaviors from surgical incision under anesthesia. Future studies to explore sex differences in early life surgery and the attendant peripheral neuronal input, stress, and inflammation will be valuable to understand emerging learning deficits, anxiety, attentional dysfunction, and opioid reward and their mechanisms. This will be valuable to develop optimal approaches to mitigate the long-term effects of surgery in early life.

Keywords: anxiety; attention; incision; neonate; opioid; pain; reward; surgery.

FIGURE 1

Experimental groups and behavior outcomes timeline. Animals used in the study were male Sprague Dawley pups. Random assignment was made of 80 male pups from 8 dams to either have treatment at postnatal day 6 (P6) of anesthesia and surgery or anesthesia alone with half of the pups in each group from each dam. Sutures were removed at P10 and animals weaned at P21. Random assignment of animals into behavioral outcome measurement was made with equal numbers of surgery and anesthesia and sham anesthesia from each dam; 16 animals were placed in the five choice serial reaction time titration variant (5CTV) group for measurement of attention and distraction, 12 animals were placed into the heroin self-administration (SA) group, and 40 animals were placed into the open field discrimination (OFT)/Tail Suspension Test (TST). Twelve additional animals were not included as no animal was excluded from loss or failure to train and all animals initially entered into the experiment randomly were used for the experiment. As a result a total of 68 animals were studied as only 16 could be studied in the 5CTV and 12 in the self-administration paradigm.

FIGURE 2

Five choice titration variant to measure attentional performance. A light and sound attenuated activity chamber is used with a bank of 5 lights on one wall and a food trough with a light on the opposite wall. Animals are trained in stages to: (1) poke head in trough for food when lighted, (2) poke head in center cue light and get food reward in trough, (3) poke head in any cue randomly lighted and get food reward in trough, and (4) poke head in any cue randomly lighted of varied duration (the titration) of illumination based on performance. Median cue duration (MCD) was calculated from trials 25–75 and used for assessment of attention on day 1 for baseline and an audio and visual distraction was performed from trials 25–75 on days 2, 3, 4 and no distraction on day 5. Premature responses are nose pokes any time during the inter-trial interval and only reset the inter-trial interval and are not considered a trial and do not contribute to the MCD.

FIGURE 3

Anxiety measured with open field exploration. Open field testing was performed at 10 weeks after incision and anesthesia (N = 20) or anesthesia alone (sham) (N = 20). (A) Ambulation in the center zone is reduced in the incision group while there is no difference in ambulation in the corners between the two groups representing reduced exploration of the center zone once entered. (B) Center zone entries are significantly reduced in the incision group which suggests increased anxiety and reduced exploration in the open zone. *Statistically significant difference between groups (unpaired t-test).

FIGURE 4

Depression or despair measured in the tail suspension test. The tail suspension test was conducted at 12 weeks after in the initial incision. There was no difference found between the anesthesia and incision (N = 20) and the anesthesia alone (sham) groups (N = 20) with respect to the time to first stop moving and the total time moving during the test (unpaired t-test).

FIGURE 5

Attention and Distraction after Early Life Surgery. Attentional performance was assessed using the median cue duration (MCD) from trials 25–75 at baseline and for 3 days of audiovisual distraction and then no distraction on Day 4 (N = 8 anesthesia alone (sham) and N = 8 incision and anesthesia animals). (A) No difference in baseline MCD between sham and incision was present (unpaired t-test). Distraction over days impaired performance in animals as measured by an increase in the MCD in both groups of animals (two way repeated measures ANOVA). In animals after early surgery, the five choice serial titration variant showed a persistent disruption seen as elevated MCD during days 1 to 4 (D1–D4) while exposed to distracting light and noise during task compared to control anesthesia sham. The incision group MCD was increased compared to baseline at all time points and remained elevated from baseline on Day 4 with no distraction (Holms-Sidak pairwise correction for comparisons). The bar graph shows a significant difference between group means. (B) Premature responses in the five choice serial titration variant were determined reflecting decreased inhibitory control when increased and either more inhibitory control or increased anxiety when decreased. Baseline responses were no different (N = 8 control anesthesia alone and N = 8 incision and sham anesthesia animals) (unpaired t-test). There was an effect of distraction and a treatment effect with only the animals in the surgery group with reduced premature responding from baseline with distraction and not the sham group. The bar graph shows a significant difference between group means. Data are presented as means and standard deviation and bar graphs are group means and standard error of the mean. *p < 0.05 between groups.

FIGURE 6

Correct, Incorrect and Omission Responses during the attention trials. Responses are shown for sham (N = 8) and incision and anesthesia animals (N = 8). (A) Correct responses are shown at the top of the graph (circles). No difference in baseline correct response between sham and incision was present (unpaired t-test). Distraction decreased correct responses in both incision and sham with no difference between groups (two way repeated measures ANOVA). The bar graph shows no significant difference between correct group means. Incorrect responses are shown at the bottom of the graph (triangles). No difference in baseline incorrect response between sham and incision was present (unpaired t-test). Distraction decreased incorrect responses in both incision and sham with a significant difference between groups (two way repeated measures ANOVA). The bar graph shows a significant difference between incorrect group means. (B) Response omissions are shown with no difference in baseline omission responses between sham and incision (unpaired t-test). Distraction increased omissions in both incision and sham with an overall difference between groups (two way repeated measures ANOVA). The bar graph shows a significant difference between omission group means. Data are presented as means and standard deviation and bar graphs are group means and standard error of the mean. *p < 0.05 between groups.

FIGURE 7

Heroin Self Administration after Early Life Surgery. Heroin self-administration was initiated at 12–16 weeks after surgery and anesthesia (N = 6) or the anesthesia alone (sham) (N = 6). (A) Acquisition was reduced in the incision and anesthesia group compared to sham. No difference was seen at baseline or after 10 days at steady state between the 2 groups. The bar graph shows a significant difference between group means. (B) After steady-state heroin self-administration within session was achieved, a dose response was performed over 3 sessions which revealed a dose-dependent decrease in heroin consumption in both groups, but no difference between groups. The bar graph shows no significant difference between group means. (C) Animals were given 24-h access to heroin. No difference between baseline heroin consumption was present. There was escalation of heroin intake in both groups compared to baseline over time. Animals after early life surgery self-administered/escalated heroin more than sham (mixed model with random effects). The bar graph shows a significant difference between group means. Data are presented as means and standard deviation and bar graphs are group means and standard error of the mean. *Statistically significant difference between groups.

FIGURE 8

Early Life Surgery Behavioral Effects. Effects of surgery in early life result in long-term behavioral changes in anxiety, learning, attention, and altered opioid reward behavior. Early life surgery and anesthesia likely produce these effects through a systemic response or neural pathway from the stress, inflammation or noxious nociceptive activation. Transient or permanent changes in the brain during development are likely responsible for long-term changes that result in altered or maladaptive behaviors in later life.