The emergence of adolescent onset pain hypersensitivity following neonatal nerve injury

David Vega-Avelaira, Rebecca McKelvey, Gareth Hathway, Maria Fitzgerald, David Vega-Avelaira, Rebecca McKelvey, Gareth Hathway, Maria Fitzgerald

Abstract

Background: Peripheral nerve injuries can trigger neuropathic pain in adults but cause little or no pain when they are sustained in infancy or early childhood. This is confirmed in rodent models where neonatal nerve injury causes no pain behaviour. However, delayed pain can arise in man some considerable time after nerve damage and to examine this following early life nerve injury we have carried out a longer term follow up of rat pain behaviour into adolescence and adulthood.

Results: Spared nerve injury (SNI) or sham surgery was performed on 10 day old (P10) rat pups and mechanical nociceptive reflex thresholds were analysed 3, 7, 14, 21, 28, 38 and 44 days post surgery. While mechanical thresholds on the ipsilateral side are not significantly different from controls for the first 2-3 weeks post P10 surgery, after that time period, beginning at 21 days post surgery (P31), the SNI group developed following early life nerve injury significant hypersensitivity compared to the other groups. Ipsilateral mechanical nociceptive threshold was 2-fold below that of the contralateral and sham thresholds at 21 days post surgery (SNI-ipsilateral 28 (± 5) g control groups 69 (± 9) g, p < 0.001, 3-way ANOVA, n = 6 per group). Importantly, no effect was observed on thermal thresholds. This hypersensitivity was accompanied by macrophage, microglial and astrocyte activation in the DRG and dorsal horn, but no significant change in dorsal horn p38 or JNK expression. Preemptive minocycline (daily 40 mg/kg, s.c) did not prevent the effect. Ketamine (20 mg/kg, s.c), on the other hand, produced a dose-dependent reversal of mechanical nociceptive thresholds ipsilateral to the nerve injury such that thresholds return to control levels at the highest doses of 20 mg/Kg.

Conclusions: We report a novel consequence of early life nerve injury whereby mechanical hypersensitivity only emerges later in life. This delayed adolescent onset in mechanical pain thresholds is accompanied by neuroimmune activation and NMDA dependent central sensitization of spinal nociceptive circuits. This delayed onset in mechanical pain sensitivity may provide clues to understand the long term effects of early injury such as late onset phantom pain and the emergence of complex adolescent chronic pain syndromes.

Figures

Figure 1
Figure 1
Nerve injury at the age of 10 days has no effect over the first 2–3 weeks but gradually ipsilateral mechanical hypersensitivity emerges in adolescence (3-way ANOVA, p B) Thermal pain thresholds show a general developmental reduction in latency in all experimental groups with no significant differences in the SNI group when compared to sham group.
Figure 2
Figure 2
Shows sections through the L4 dorsal horn at various times after sham (top) and SNI (bottom) surgery at P10 (left and middle) and adult (right). Sections are immunostained with the microglia marker IBA-1 (red) and the non peptidergic C-fibre marker isolectin IB-4 (green). SNI surgery at P10 produces non significant microglia activation after 7 days compared to sham in (P10) rats. However, there is a late onset of microglia activation at 21 days post surgery which is significantly greater than sham and similar to that seen in adult rats 7 days post surgery. The gap in isolectin IB-4 is a marker of the terminal region of the nerves damaged by SNI surgery. B) Quantification of microglia activation in the three experimental groups illustrated in A. IBA-1 stained microglial cells were counted and the results are expressed as fold difference of the contralateral side. *: (n = 3 animals per experimental group, 3-way ANOVA, p < 0.001, Tukey, p < 0.05).
Figure 3
Figure 3
Shows sections through the L4 dorsal horn at various times after sham (top) and SNI (bottom) surgery at P10 (left and middle) and adult (right). Sections are immunostained with the astrocytic marker GFAP (red) and the non peptidergic C-fibre marker isolectin IB-4 (green). SNI surgery at P10 produces non significant astrocyte activation after 7 days compared to sham in (P10) rats. However, there is a late onset of astrocyte activation at 21 days post surgery which is significantly greater than sham and similar to that seen in adult rats 7 days post surgery. The gap in isolectin IB-4 is a marker of the terminal region of the nerves damaged by SNI surgery. B) Quantification of astrocyte activation in the three experimental groups illustrated in A. GFAP stained astrocytes with signs of gliosis were counted and the results are expressed as fold difference of the contralateral side. *: (n = 3 animals per experimental group, 3-way ANOVA, p < 0.001, Tukey, p < 0.05).
Figure 4
Figure 4
Shows sections through the L4 dorsal root ganglion at various times after sham (top) and SNI (bottom) surgery at P10 (left and middle) and adult (right). Sections are immunostained with the macrophage marker IBA-1 (red) and the large A cell neuronal population marker NF200 (green). Macrophage activation is observed as macrophages clustering around large neuronal cell bodies in characteristic ‘ring-like’ structures with processes extending from their enlarged cell bodies towards large neurons. SNI surgery at P10 produces non significant macrophage activation after 7 days compared to sham in (P10) rats. However, there is a late onset of macrophage activation at 21 days post surgery which is significantly greater than sham and similar to that seen in adult rats 7 days post surgery. B) Quantification of these activated macrophage ‘ring-like’ structures in the three experimental groups illustrated in A and expressed as a percentage accordingly to large sensory neurons*: (n = 3 animals per experimental group, 3-way ANOVA, p < 0.001, Tukey, p < 0.05).
Figure 5
Figure 5
Mechanical pain thresholds in animals with early-life (P10) nerve injury (S) or sham surgery H) tested in later life (P31). Mechanical pain thresholds were measured in minocycline (M) (40 mg/Kg daily ip injections) after SNI nerve injury compared to SNI saline (S) group and controls (H). n = 6 per experimental group; *: ANOVA, p B) Representative images of western-blot for phospho-p38 and phospho-JNK (pJNK) and the housekeeping gene Gapdh. Quantification was done by densitometry of p38 (C) and pJNK(D). *: n = 4 per experimental group; *: ANOVA, p < 0.001, Tukey, p < 0.05.
Figure 6
Figure 6
Mechanical pain thresholds in animals with early-life (P10) nerve injury (SNI-ipsi) or sham surgery (sham-ipsi) tested in later life (P31), before and after i.p ketamine. SNI surgery animals have lower baseline mechanical pain thresholds but the NMDA antagonist ketamine increases mechanical pain thresholds to normal levels in a dose dependant manner. The clearance of ketamine (72 h post injection) results in the mechanical pain thresholds falling back to pre-ketamine levels. B) The same dose dependent reversal of mechanical hypersensitivity by ketamine is observed when measuring foot paw withdrawal to a von Frey hair. *: (n = 6 per experimental group, 3-way ANOVA, p < 0.001, Tukey, p < 0.05), SNI: spared nerve injury, Ipsi: ipsilateral, Contra: contralateral, ip: intraperitoneal, FPW: frequency of paw withdrawal. Ketamine had no effect upon contralateral (SNI-contra) thresholds or upon sham operated animals.

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