Increased spinal prodynorphin gene expression in reinflammation-associated hyperalgesia after neonatal inflammatory insult

Abstract

Background: Neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. We investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behaviour at the physiological and molecular levels after peripheral reinflammation in adulthood.

Results: A decrease in paw withdrawal latency (PWL) after a heat stimulus was documented in rats that received inflammatory injections in their left hind paws on postnatal day one (P1) and a reinflammation stimulus at postnatal 6-8 weeks of age, compared with normal rats. An increase in the expression of the prodynorphin (proDYN) gene was noted after reinflammation in the spinal cord ipsilateral to the afferents of the neonatally treated hind paw. The involvement of the activation of extracellular signal-regulated kinases (ERK) in peripheral inflammatory pain hypersensitivity was evidenced evident by the increase in phospho-ERK (pERK) activity after reinflammation.

Conclusions: Our results indicate that peripheral inflammation in neonates can permanently alter the pain processing pathway during the subsequent sensory stimulation of the region. Elucidation of the mechanism underlying the developing pain circuitry will provide new insights into the understanding of the early pain behaviours and the subsequent adaptation to pain.

Figure 1

Behavioural analysis using thermal tests after reinflammation. The PWL to a noxious thermal stimulus at baseline and at 24 h after CFA reinflammation was compared between the neonatal CFA group and the neonatal saline group. The results revealed an absence of differences in the baseline PWL to a noxious thermal stimulus between the neonatal CFA group and the neonatal saline group. However, 24 h after reinflammation via CFA injection into the left hind paw, the PWL in the neonatal CFA group was significantly decreased compared with that observed in the neonatal saline group (* P < 0.05).

Figure 2

Determination of proDYN mRNA expression levels in the spinal cord after reinflammation using real-time RT-PCR. A significant increase in proDYN mRNA expression was found in the neonatal CFA group compared with the neonatal non-treated and neonatal saline groups 24 h after reinflammation with CFA (* P < 0.05). No significant differences in proDYN expression levels was noted between the neonatal non-treated group and the neonatal saline group. Little or no proDYN expression was noted in the naïve group, which did not receive any CFA injection during the neonatal and the adult time.

Figure 3

In situ hybridization study showing an upregulation of the proDYN mRNA in the dorsal horns after reinflammation. A low-magnification image shows neurons expressing spinal proDYN mRNA in the superficial and deeper laminae of the dorsal horn 24 h after reinflammation in the CFA group (A). A high-magnification image of Figure 3A shows a comparable increase in proDYN mRNA-positive neurons in laminae I-II of the dorsal horn ipsilateral (B) side that experienced neonatal peripheral inflammation compared with the contralateral side, which did not receive neonatal peripheral CFA insults (C). Scale bars: a, 200 μm; b and c, 100 μm

Figure 4

Quantification of proDYN mRNA-positive neurons using in situ hybridization in laminae I-II and laminae III-V of the dorsal horn 24 h after reinflammation with CFA injection into bilateral hind paws at postnatal time 6-8 weeks in the neonatal CFA group. A significant increase in the number of proDYN mRNA-positive neurons was found in laminae I-II of the left side that received a neonatal peripheral CFA insults compared to the right dorsal horn, which did not receive neonatal CFA insults (* P < 0.05). There was also an increase in the number of proDYN mRNA-positive neurons in laminae III-V of the neonatal CFA-treated side compared with the contralateral side; however, this difference was not significant.

Figure 5

Western blot analysis of pERK activation in spinal cords subjected to CFA-induced peripheral reinflammation. Western blots of spinal cord tissues from animals in the neonatal CFA group, neonatal saline group and the naive group revealed varied immunoreactivity against different isomers of pERK 10 min after reinflammation (A). The intensity of the total ERK was the same in the three groups. A significant increase in pERK activity was found in the spinal cord tissues of animals in the neonatal CFA group compared with the neonatal saline group and the naive group (* P < 0.05) (B).