Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain

Abstract

Background: Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing.

Results: We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

Figure 1

Mechanical allodynia in different models of neuropathic pain. A severe and persistent hypersensitivity developed following SNL, SNI and CCI surgery. Calibrated von Frey filaments were used to determine the withdrawal threshold for punctate mechanical stimulation. Data were expressed as means of ± SEM of threshold of mechanical sensitivity. ***P < 0.001 for all three models in comparison to the sham group. SNL n = 6, SNI n = 8, CCI n = 5.

Figure 2

Expression of autophagic markers in the spinal dorsal horn in three different models of peripheral nerve injury (SNL, SNI and CCI). The representative Western blots show levels of the autophagic markers LC3-I, LC3-II, Beclin 1 and p62 and voltage-gated calcium channel subunit α₂δ-1 in the spinal dorsal horn ipsilateral (I) and contralateral (C) to the side of injury.

Figure 3

Densitometric analysis of autophagic markers changes detected by Western blot in the spinal dorsal horn following three different models of peripheral nerve injury (SNL, SNI and CCI). The SNL model (A) was characterised by increased LC3-II and p62, whereas in the SNI (B) and CCI (C) only LC3-II and Beclin 1 increase were detected, respectively. A statistically significant upregulation of the voltage-gated calcium channel subunit α₂δ-1 was observed in the injured side of the SNL and the SNI model. Signals from each band were normalised towards the corresponding GAPDH signal. Values were expressed as mean ± SEM. *P < 0.05, SNL n = 8, SNI n = 5, CCI n = 5.

Figure 4

Distribution and cellular localisation of p62 immunoreactivity in the spinal dorsal horn following SNL. (A) Although ubiquitously expressed (see positive cell bodies in the ventral spinal cord), high levels of p62 were present in the superficial layers of the dorsal horn, particularly on the side ipsilateral to injury (A, insert). Spots of p62 immunoreactivity were detected within glial processes as shown by some p62 immunolabelling within GFAP-positive structures (B and C arrowheads). However, high p62 levels were particularly evident in some neuronal cell, as shown by the strong signal within NeuN-positive cell bodies (D and E arrows). Each image is a single focal plane from spinal cord sections of mice undergone SNL; B, C, D, E show ipsilateral side. Scale bars: A 250 μm; B, D 100 μm; C 20 μm; E, 50 μm.

Figure 5

Effect of chloroquine intratechal injection on Beclin 1, LC3 and p62 in the spinal dorsal horn. (A) Representative Western blots of Beclin 1, LC3 and p62 protein levels after chloroquine (300pmoles/3 μl, i.t.) according to the treatment protocol in Figure 6A. (B) Densitometric analysis of Beclin 1, LC3-II and p62 expression. While Beclin 1 expression was not affected, LC3-I, LC3-II and p62 levels were significantly increased in the chloroquine group compared to the vehicle-injected group. The appearance of a second band has been previously suggested as a p62 splicing variant or a partially cleaved product [42]. Signals of each band were normalised to the respective GAPDH. Data were expressed as mean ± SEM. *P < 0.05. Vehicle n = 3, choloroquine (CQ) n = 6 (n = 5 for Beclin 1).

Figure 6

Effect of chloroquine intratechal injection on mechanical sensitivity. (A) Baseline mechanical thresholds were assessed in naïve mice for 2 consecutive days before the first injection. Chloroquine (300pmoles/3 μl) or vehicle were then injected intrathecally once daily. Two hours after treatment, mechanical sensitivity was measured by Von Frey’s test. The protocol was repeated for three consecutive days. (B) Localised spinal block of autophagy significantly reduced the threshold of mechanical sensitivity, in comparison to vehicle-treated mice. Data were expressed as mean ± SEM of the threshold of mechanical sensitivity. **P < 0.01. Vehicle n = 3, chloroquine (CQ) n = 6.