Altered formalin-induced pain and Fos induction in the periaqueductal grey of preadolescent rats following neonatal LPS exposure

Abstract

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this process.

Figure 1. Neonatal LPS exposure enhances formalin-induced nociception in preadolescent rats.

Time course of flinching (A) and licking (B) responses following an injection of 1.1% formalin (mean ± SEM). AUC: the Area Under the Curve.

Figure 2. Fos immunoreactivity in the midbrain periaqueductal grey (PAG) following a neonatal immune challenge and subsequent inflammatory pain.

Representative examples illustrate the distribution of Fos-positive nuclei in the dorsal and ventrolateral PAG (DPAG, VLPAG, respectively) at the rostral (A & B) and caudal (C & D) axes following formalin injection in preadolescent rats. (E) Quantification of Fos-positive nuclei in DPAG between neonatal saline and LPS-treated rats. (F) Quantification of Fos-positive nuclei in lateral PAG (LPAG). (G) Quantification of Fos-positive nuclei in VLPAG. Data are presented as mean ± SEM. *p<.05; **p<.01. Scale bar = 100 µm.

Figure 3. Fos immunoreactivity in the habenula following a neonatal immune challenge and subsequent inflammatory pain.

(A) Representative examples illustrate the distribution of Fos-positive nuclei in the lateral and medial habenula (LHb & MHb, respectively). (B) Quantification of Fos-positive nuclei in LHb and MHb in neonatal saline (white bar) and LPS-treated rats (grey bar) after formalin injection in preadolescent rats. 3V: third ventricle. ***p<.001. Scale bar = 100 µm for the 10x microscopic objective and 200 µm for 20x microscopic objective.