Histamine upregulates Nav1.8 expression in primary afferent neurons via H2 receptors: involvement in neuropathic pain

Abstract

Introduction: The upregulation of Nav1.8 in primary afferents plays a critical role in the development and persistence of neuropathic pain. The mechanisms underlying the upregulation are not fully understood.

Aims: The present study aims to investigate the regulatory effect of histamine on the expression of Nav1.8 in primary afferent neurons and its involvement in neuropathic pain.

Results: Histamine at 10(-8) M increased the expression of Nav1.8 in cultured DRG neurons. This effect could be blocked by H2 receptor antagonist cimetidine or famotidine, but not by H1 receptor antagonist pyrilamine or dual H3 /H4 antagonist thioperamide. Peri-sciatic administration of histamine increased Nav1.8 expression in the sciatic nerve and L4/L5 DRG neurons in a dose-dependent manner, accompanied with remarkable mechanical allodynia and heat hyperalgesia in the ipsilateral hindpaw. Famotidine but not pyrilamine or thioperamide inhibited Nav1.8 upregulation and pain hypersensitivity. In addition, famotidine (40 mg/kg, i.p.) not only suppressed autotomy behavior in the rat neuroma model of neuropathic pain but also attenuated mechanical allodynia and thermal hyperalgesia following partial sciatic nerve ligation. Moreover, famotidine inhibited Nav1.8 upregulation in the neuroma and ligated sciatic nerve.

Conclusions: Our findings indicate that histamine increases Nav1.8 expression in primary afferent neurons via H2 receptor-mediated pathway and thereby contributes to neuropathic pain. H2 receptor antagonists may potentially be used as analgesics for patients with neuropathic pain.

Keywords: H2 antagonists; Histamine; Nav1.8; Neuropathic Pain; Primary afferents.

Figure 1

Effect of histamine on Nav1.8 expression in cultured DRG neurons. (A) Representative microphotographs showing the expression of Nav1.8 in cultured DRG neurons incubated with histamine for 24 h at indicated concentrations. (B) The normalized fluorescent intensity of Nav1.8 immunostaining in immunocytochemistry (n = 5/group). The fluorescent intensity was normalized to that in the control group. (C) Nav1.8 expression in cultured DRG neurons treated with histamine for 24 h was measured by western blot (upper panels) and semiquantitative analysis of immunostaining intensity is shown in the histogram (n = 4/group). (D) Nav1.8 expression in cultured DRG neurons treated with H₁ or H₂ receptor antagonists followed by histamine for 24 h was measured by western blot (upper panels) and semiquantitative analysis of immunostaining intensity is shown in the histogram (n = 4/group). * P < 0.05 versus the control group. # and ## P < 0.05 and 0.01, respectively, versus the histamine 10⁻⁸ M group. Scale bar, 100 μm.

Figure 2

Peri‐sciatic administration of histamine for 3 days induced the upregulation of Nav1.8 expression in the sciatic nerve and L4/L5 DRG and hindpaw hypersensitivity. (A) Representative microphotographs showing the expression of Nav1.8 in the sciatic nerve and DRG neurons. (B and C) Histograms showing the normalized fluorescence intensity of Nav1.8 immunostaining in the sciatic nerves (B) and L4/L5 DRG (C) (n = 6/group). The fluorescent intensity was normalized to that in the saline group. ** P < 0.01 versus the saline group; # P < 0.05 versus each other. (D and E) The hind paw withdrawal threshold to mechanical (D) and thermal stimuli (E) (n = 6–8/group). * and ** P < 0.05 and 0.01, respectively, versus the corresponding baseline. Scale bar, 100 μm.

Figure 3

Famotidine but not pyrilamine attenuated the hindpaw hypersensitivity and reduced the upregulation of Nav1.8 expression in the sciatic nerve and L4/L5 DRGs induced by peri‐sciatic administration of histamine for 3 days. (A and B) The hind paw withdrawal threshold to mechanical (A) and thermal stimuli (B) (n = 6/group) in rats. (C) Representative microphotographs showing the expression of Nav1.8 in the sciatic nerve and DRG neurons in vehicle‐, pyrilamine‐ or famotidine‐treated rats. (D and E) Histograms showing the normalized fluorescence intensity of Nav1.8 immunostaining in the sciatic nerve (B) and L4/L5 DRG (C) (n = 6/group). The fluorescent intensity was normalized to that in the saline group. * and ** P < 0.05 and 0.01, respectively. $ P = 0.051, # P = 0.06 versus the histamine 2 nM +Saline group. Scale bar, 100 μm.

Figure 4

Famotidine suppressed the progression and severity of autotomy in the rat neuroma model of neuropathic pain. Famotidine (40 mg/kg, i.p., n = 8) given immediately after the surgery for 30 days significantly suppressed the progression of autotomy (A), delayed the onset of autotomy (B), and reduced the area under the curve (AUC) during the periods of postoperative days 0–30 (C) and 31–50 (D). Famotidine (40 mg/kg, i.p., n = 6) given when the autotomy score reached 3 or 4 points suppressed the autotomy progression (E) and the AUC during the period of drug treatment (0–30 days) (F). *, ** and *** P < 0.05, 0.01 and 0.001, respectively, versus the saline group (n = 6).

Figure 5

Famotidine but not loratadine attenuated the mechanical allodynia and thermal hyperalgesia in the rat PSL model of neuropathic pain. Famotidine (40 mg/kg, i.p., n = 11) given immediately after the surgery attenuated the ipsilateral (A) and contralateral (B) mechanical allodynia, ipsilateral (C), but not contralateral (D) thermal hyperalgesia. * and ** P < 0.05 and 0.01, respectively, # P = 0.054 versus the saline group (n = 8) at the same time points.

Figure 6

Famotidine reduced Nav1.8 expression in the sciatic nerve in the rat neuroma model and PSL model. (A and C) Representative microphotographs showing the expression of Nav1.8 in the sciatic nerve after 30 days of famotidine (40 mg/kg, i.p.) in the neuroma model (A) and after 7 days of famotidine (40 mg/kg, i.p.) in the PSL model (C). (B and D) Histograms showing the normalized fluorescence intensity of Nav1.8 immunostaining in the sciatic nerve in the neuroma model (B) and PSL model (D). The fluorescence intensity was normalized to that in the sham group. * and ** P < 0.05 and 0.01, respectively, versus the sham group. ## P < 0.01 versus each other. n = 6/group. Scale bar, 100 μm.