3.1. Sensitized DRG neurons enhanced response to chemical of stimuli after CCD
To explore the behavioral effects of TRPV1, Histamine, 5-HT and MrgprA3 receptor after CCD, we used the calf model that allows the differentiation of side-directed itch- and pain-like behaviors in response to pruritic and algesic chemical stimuli [41]. Mice with high dose of capsaicin injection displayed less site-directed spontaneous biting behaviors than mice with low dose of capsaicin injection (P<0.05, 1 μg/10 μL vs 0.1 μg/10 μL; P<0.01, 10 μg/10 μL vs 0.1 μg/10 μL) [Fig. 1A]. Mice with high dose of capsaicin injection displayed less site-directed spontaneous licking behaviors than mice with low dose of capsaicin injection (P<0.05, 1 μg/10 μL vs 0.1 μg/10 μL; P<0.01, 10 μg/10 μL vs 0.1 μg/10 μL) [Fig. 1E]. Intradermal (i.d.) injection of capsaicin (1 μg/10 μL) into the calf of CCD mice significantly increased the number of licking bouts but not the number of biting as compared to mice before CCD (Fig.1E). In contrast, injection of capsaicin to CCD mice did not significantly change the number of licking and biting as compared to control mice (Fig.1A). These results indicate that capsaicin elicits more pain than itch within receptive field of DRG neurons after CCD. These findings suggest that capsaicin acts on TRPV1 to trigger pain behaviors in vivo under the CCD conditions.
At 5 days after CCD surgery, mice with histamine injection displayed more site-directed spontaneous biting behaviors than control mice [Fig. 1B]. Mice with high dose of histamine injection displayed more site-directed spontaneous biting behaviors than mice with low dose of histamine injection (P<0.01, 20 μg/10 μL and 50 μg/10 μL vs 10 μg/10 μL) [Fig. 1B]. Intradermal (i.d.) injection of histamine (20 μg/10 μL) into the calf of CCD mice significantly increased the number of biting bouts but not the number of licking as compared to control mice (Fig.1B, D). These results suggest that histamine elicits more itch than pain within receptive field of DRG neurons after CCD. These findings indicate that histamine acts on histamine receptor to trigger itch behaviors in vivo under the CCD conditions.
We then tested histamine-independent pruritogen, BAM8-22 on evoking skin itch. At 5 days after CCD surgery, mice with BAM injection displayed more site-directed spontaneous biting behaviors than control mice [Fig. 1C]. Intradermal (i.d.) injection of BAM (1 μg/10 μL) into the calf of CCD mice significantly increased the number of biting bouts but not the number of licking as compared to control mice (Fig.1C, G). Intradermal (i.d.) injection of BAM (10 μg/10 μL) into the calf of control mice significantly increased the number of licking as compared to low dose BAM injection to control mice [Fig.1G]. These results suggest that BAM elicits more itch than pain within receptive field of DRG neurons after CCD.
Mice with 5-HT (0.003 μg/10 μL) injection displayed no more site-directed spontaneous biting and licking behaviors between CCD mice and control mice [Fig. 1D, H]. At 1 day after CCD surgery, intradermal (i.d.) injection of 5-HT (0.3 μg/10 μL) into the calf of CCD mice significantly increased the number of biting bouts and licking behaviors as compared to control mice (Fig.1D, H). These results suggest that high dose of 5-HT elicits more itch and pain within receptive field of DRG neurons between control mice and CCD mice.
To compare DRG neurons respond to chemical stimuli between control and CCD, we found the most suitable concentration of chemical. In low and high concentration, there are no significantly different at behavior results between control and CCD. Compared with control group, the calf licking of CCD group increased significantly by using medium concentration chemical (Capsaicin 1 μg/10 μL, Histamine 20 μg/10 μL and BAM 1 μg/10 μL) (Fig.2A). 0.3 μg/10 μL of 5-HT injection increased the calf licking time in CCD group compared with control group (Fig. 2A). Histamine and BAM increased the calf biting time in CCD group compared with control group (Fig.2B).
3.2. Confocal imaging of DRG
To evaluate neuronal activity in DRG somata, we used Pirt-GCaMP3 mice to image Ca2+ response in L4 DRG.
The percentage of neurons that responded to capsaicin (1 µg/10 µL) was significantly increased in CCD (169/382, n=6) compared with the control DRG (75/398, n=4), P<0.01, as shown Fig 3A-D, M. The percentage of neurons that responded to histamine (20 µg/10 µL) was significantly increased in CCD (149/385, n=4) compared with the control DRG (92/440, n=5), P<0.01, as shown Fig 3E-H, N.
The percentage of neurons that responded to BAM8-22 (1 µg/10 µL) was significantly increased in CCD (79/352, n=3) compared with the control DRG (60/362, n=3), P<0.01, as shown Fig 3I-L, O.
In the present study, a model of CCD was used to mimic a chronic neuropathic state. Bilateral L4 DRG of mice were kept in ACSF at 37°C on 5 days after CCD. Using confocal image, we investigated MrgprA3+ neuronal activity in L4 DRG somata in vitro. The total number of MrgprA3+ neurons was significantly increased in CCD (230, 38.33±3.07, n=6) compared with the control DRG (133, 18.83±1.7, n=6), P<0.01, as shown Fig 4A-B.
3.3 H1R and TRPV1 of DRG neurons immunoreactivity after CCD surgery
Histamine receptor 1 immunoreactivity was detected in 10.60% (42/396, n=6) of DRG sensory neurons in control. However, Histamine receptor 1 immunoreactivity was detected in 16.15% (62/384, n=6) in CCD, as shown in Fig 5A. H1R+ DRG sensory neurons were of small diameter (average 30 µm), positive for marker TRPV1+. TRPV1 immunoreactivity was detected in 12.82% (155/1209, n=10) of DRG sensory neurons in control. However, TRPV1 immunoreactivity was detected in 18.55% (218/1175, n=6) in CCD, as shown in Fig 5B. TRPV1+ DRG sensory neurons were of small diameter (average 30 µm), positive for marker CGRP. Immunofluorescent staining revealed very few H1- and TRPV1-immunopositive DRG neurons in control mice (Fig. 6A-C). In contrast, the mean percentage of H1- and TRPV1-immunopositive DRG neurons was significantly greater in CCD mice (Fig. 6E-G). In addition, some H1-immunopositive DRG neurons in CCD mice were also significantly increased immunopositive for TRPV1 (detected in 44.14% of neurons with H1-immunopositive, Fig. 6H) compared with control mice (detected in 26.31% of neurons with H1-immunopositive, Fig. 6D).
Immunofluorescent staining revealed very few TRPV1- and CGRP-immunopositive DRG neurons in control mice (Fig. 7A- C). In contrast, the mean percentage of H1- and TRPV1-immunopositive DRG neurons was significantly greater in CCD mice (Fig. 7D-F). In addition, some TRPV1-immunopositive DRG neurons in CCD mice were also significantly increased immunopositive for TRPV1 (detected in 40% of neurons with CGRP-immunopositive, Fig. 7H) compared with control mice (detected in 21.88% of neurons with CGRP-immunopositive, Fig. 7G).
3.4 Expression of TRPV1/Histamine Receptor on mouse DRG
TRPV1 is expressed in primary sensory neurons in the DRG and is distributed in small-diameter, nociceptive neurons. Because TRPV1 may contribute to production of itch sensation at the primary sensory neurons similar to the function of histamine receptor, we investigated the potential interaction of the two receptors. In our immunofluorescence staining study, immunoactivity for TRPV1 and that for H1R in DRG neurons of CCD mice increased compared with control mice.
It is possible that the sensitization of DRG we detected after CCD is mediated by upregulated and spontaneously released Histamine via activation of HR1A on DRG.
At the protein level, immunofluorescence results revealed that a significantly larger percentage e of DRG neurons of CCD mice stained TRPV1 compared with controls (Fig.3A, E), indicating an increased number of cutaneous sensory neurons expressing TRPV1 after the development of CCD (12.82%:155/1209 vs 18.55%:218/1175).
CGRP receptors are expressed on the nervous system. Both peripheral and central neurons released CGRP that play role in inducing central sensitisation to tactile stimuli.
We further determined the expression pattern of H1R in DRG after CCD. The percentage of DRG neurons stained with H1R from CCD mice (16.15%, 62/384) was significantly greater as compared with that from control mice (12.17%, 50/411).
3.5 CCD elevated mRNA expression of TRPV1 and MrgprA3 in CCD DRG
As shown in Fig.8, mean mRNA expression levels of TRPV1, Histamine receptor 1 and MrgprA3 measured in the DRG, were significantly elevated in the CCD model group (P<0.05) compared to control.