GFP Adeno-associated virus(AAV Serotype 5)

Toll-like receptor 7 (TLR7) is expressed in neurons of the dorsal root ganglion (DRG), but whether it contributes to neuropathic pain is unknown. Here, researchers found that peripheral nerve injury caused by ligation of the fourth lumbar (L₄) spinal nerve (SNL) or chronic constriction injury of the sciatic nerve resulted in a significant increase in the expression of TLR7 at the mRNA and protein levels in the injured DRG of mice. Blocking this increase by microinjection of TLR7 shRNA-expressing adeno-associated virus (AAV) 5 into the ipsilateral L₄ DRG attenuated SNL-induced mechanical, thermal, and cold pain hypersensitivity in male and female mice. In contrast, mimicking this increase by microinjection of AAV5 expressing full-length TLR7 into the unilateral L_3/4 DRG resulted in increased amounts of p-ERK1/2 and GFAP in the dorsal horn, augmented responses to mechanical, thermal and cold stimuli, and induced the spontaneous pain on the ipsilateral side in the absence of SNL. These findings suggest that DRG TLR7 contributes to neuropathic pain through activation of NF-κB in primary sensory neurons.

To determine whether increased DRG TLR7 is sufficient to cause neuropathic pain, the researchers microinjected AAV5 expressing full-length TLR7 protein (AAV5-TLR7) into the unilateral L3 and L4 DRG of naive adult male mice. AAV5 expressing green fluorescent protein (AAV5-GFP) was used as a control. A sharp increase in the amount of TLR7 and TRPA1 was detected in the injected DRG 8 weeks after microinjection of AAV5-TLR7 compared with microinjection of AAV5-GFP (Figure 1a). Mice microinjected with AAV5-TLR7 but not AAV5-GFP showed a significant increase in paw withdrawal frequencies in response to 0.07 g and 0.4 g von Frey filament stimuli (Figure 1b, c) and marked decreases in paw withdrawal latencies in response to thermal and cold stimuli (Figure 1d, e) on the ipsilateral side. These changes occurred 4 weeks after microinjection and persisted for at least 8 weeks (Figure 1a-e), consistent with the 3–4 week lag phase of AAV5 expression. Neither virus affected motor function or basal contralateral paw withdrawal responses (Figure 1b-e). Furthermore, mice microinjected with AAV5-TLR7 (but not AAV5-GFP) remained longer in the lidocaine-paired chamber (Figure 1f-g), indicating stimulus-independent spontaneous pain. These findings suggest that increased TLR7 in the DRG produces spontaneous and evoked pain hypersensitivity, a common clinical symptom of neuropathic pain.

Figure 1. Effect of DRG TLR7 overexpression on nociceptive thresholds and dorsal horn central sensitization in naïve mice. (He L, et al., 2020)