Long-term behavioral effects of repetitive pain in neonatal rat pups

Abstract

Human preterm neonates are subjected to repetitive pain during neonatal intensive care. We hypothesized that exposure to repetitive neonatal pain may cause permanent or long-term changes because of the developmental plasticity of the immature brain. Neonatal rat pups were stimulated one, two, or four times each day from P0 to P7 with either needle prick (noxious groups N1, N2, N4) or cotton tip rub (tactile groups T1, T2, T4). In groups N2, N4, T2, T4 stimuli were applied to separate paws at hourly intervals;each paw was stimulated only once a day. Identical rearing occurred from P7 to P22 days. Pain thresholds were measured on P16, P22, and P65 (hot-plate test), and testing for defensive withdrawal, alcohol preference, air-puff startle, and social discrimination tests occurred during adulthood. Adult rats were exposed to a hot plate at 62 degrees C for 20 s, then sacrificed and perfused at 0 and 30 min after exposure. Fos expression in the somatosensory cortex was measured by immunocytochemistry. Weight gain in the N2 group was greater than the T2 group on P16 (p < 0.05) and P22 (p < 0.005); no differences occurred in the other groups. Decreased pain latencies were noted in the N4 group [5.0 +/- 1.0 s vs. 6.2 +/- 1.4 s on P16 (p < 0.05); 3.9 +/- 0.5 s vs. 5.5 +/- 1.6 s on P22 (p < 0.005)], indicating effects of repetitive neonatal pain on subsequent development of the pain system. As adults, N4 group rats showed an increased preference for alcohol (55 +/- 18% vs. 32 +/- 21%; p = 0.004); increased latency in exploratory and defensive withdrawal behavior (p < 0.05); and a prolonged chemosensory memory in the social discrimination test (p < 0.05). No significant differences occurred in corticosterone and ACTH levels following air-puff startle or in pain thresholds at P65 between N4 and T4 groups. Fos expression at 30 min after hot-plate exposure was significantly greater in all areas of the somatosensory cortex in the T4 group compared with the N4 group (p < 0.05), whereas no differences occurred just after exposure. These data suggest that repetitive pain in neonatal rat pups may lead to an altered development of the pain system associated with decreased pain thresholds during development. Increased plasticity of the neonatal brain may allow these and other changes in brain development to increase their vulnerability to stress disorders and anxiety-mediated adult behavior. Similar behavioral changes have been observed during the later childhood of expreterm neonates who were exposed to prolonged periods of neonatal intensive care.

FIG. 1

Alcohol preference was tested by the proportion of total fluid intake by the adult rat given a choice between: 2% sucrose or 5% alcohol in 2% sucrose. Each rat was exposed to three testing periods (each period = 24 h) and average intake was calculated for each rat. Data were compared between noxious and tactile stimulation groups using the Mann–Whitney U-test.

FIG. 2

Defensive withdrawal behavior was tested by placing the rat in a dark PVC tube that was located in a corner of the open field. Behavior was monitored using a video camera (Model SS3-M370; Sony Corp., Tokyo, Japan) and (a) number of entries into the open field, (b) latency for the first open-field entry, and (c) total time spent by the rats in the PVC tube were compared between N₄ and T₄ groups using the Mann–Whitney U-test. Significantly greater latency and total time spent in the tube by rats in the N₄ group suggest increased anxiety following noxious stimulation in the neonatal period.

FIG. 3

Social discrimination tested the juvenile recognition and discrimination abilities of adult rats (see text for details of the test procedure). In the T₄ group, significantly greater (p = 0.008) investigation of the novel juvenile after an IET (interexposure time) of 30 min implies a chemosensory memory for the previously exposed juvenile, and this memory was lost following an IET of 120 min (no difference between novel and same juvenile, p = 0.421). In the N₄ group, chemosensory memory for the previously exposed juvenile was retained after an IET of 30 min (p = 0.013) as well as 120 min (p = 0.021), implying greater vigilance for intruders following noxious stimulation during the neonatal period.

FIG. 4

Photomicrographs of matched coronal sections from the (A) N₄ and (B) T₄ groups located in the rostral part of the left somatosensory cortex (Paxinos Rat Brain Atlas: corresponding to Fig. #16; stereotaxic parameters: bregma 0.48 mm, interaural 9.48 mm). Sections were stained with a 1:2000 dilution of Fos antibody, and Fos-like immunoreactivity was visualized by the avidin–biotin complex method using Nickel peroxidase intensification. The darkly stained nuclei represent the nuclear expression of Fos-like immunoreactivity at 30 min following exposure to a hot plate shown at 5× magnification (scale = 400 μm). Greater cortical expression of Fos-like immunoreactivity occurred at 30 min after hot plate exposure in the T₄ group (B) compared with the N₄ group (A).

FIG. 5

Photomicrographs of matched coronal sections from the (A) N₄ and (B) T₄ groups located in the rostral part of the left somatosensory cortex shown at 20× magnification (scale = 100 μm). Greater cortical expression of Fos-like immunoreactivity is evident in the T₄ group (B) compared with the N₄ group (A).

FIG. 6

Photomicrographs of matched coronal sections from the (A) N₄ and (B) T₄ groups located in the caudal part of the right somatosensory cortex (Paxinos Rat Brain Atlas: corresponding to Fig. #33; stereotaxic locations: bregma 3.80 mm, interaural 5.20 mm). Sections were stained with a 1:2000 dilution of Fos antibody, and Fos-like immunoreactivity was visualized by the avidin–biotin complex method using Nickel peroxidase intensification. The darkly stained nuclei represent the nuclear expression of Fos-like immunoreactivity shown at 5× magnification (scale = 400 μm). The cortical expression of Fos-like immunoreactivity at 30 min following exposure to a hot plate is much greater in the T₄ group (B) compared with the N₄ group (A).

FIG. 7

Photomicrographs of matched coronal sections from the (A) N₄ and (B) T₄ groups located in the caudal part of the right somatosensory cortex shown at 20× magnification (scale = 100 μm) (Paxinos Rat Brain Atlas: corresponding to Fig. #33; stereotaxic locations: bregma 3.80 mm, interaural 5.20 mm). The cortical expression of Fos-like immunoreactivity at 30 min following exposure to a hot plate is much greater in the T₄ group (B) compared with the N₄ group (A).

FIG. 8

The number of Fos-immunoreactive cells were counted in the somatosensory cortex according to procedures detailed in the text. Rats from the N₄ and T₄ groups were paired based on gender and body weight. From each pair (one N₄ rat and one T₄ rat), six repre- sentative sections from the somatosensory cortex were matched for the anatomical level and stained in the same immunocytochemistry assay. Anatomically matched sections included in the same ICC assay from matched N₄ and T₄ rats were used for cell counting. (a) This shows data from rats killed immediately after exposure to the hot plate. No significant differences occurred in the number of Fos-positive cells between N₄ and T₄ rats. (b) This shows data from rats killed at 30 min after exposure to the hot plate. Significantly greater numbers of Fos-positive cells were counted in the somatosensory cortex of rats from the T₄ group compared to the N₄ group (p < 0.0001, repeated measures ANOVA). These data indicate that the constitutive level of Fos expression was similar at baseline for both groups, and that the noxious stimulation activated a significantly greater number of neurons in the somatosensory cortex of the T₄ group compared to the N₄ group.