The Role of Pruriceptors in Enhanced Sensitivities to Pruritogens in a Murine Model of Chronic Compression of Dorsal Root Ganglion

Background : Chronic pruritus is a symptom that commonly observed in neurological diseases. It has been hypothesized that the chronic pruritus may result from sensitization of itch-signaling pathways but the mechanisms remain obscure. Methods: In this study, we established a mouse model of chronic compression of dorsal root ganglion (CCD) and injected various pruritogenic and algogenic agents intradermally to the calf skin ipsilateral to the compressed DRG. We additionally investigated if pruritogen-evoked activities of dorsal root ganglion (DRG) neurons is enhanced in this model. The expression of TRPV1, CGRP and H1R was detected with immunoflorescent staining. DRG neurons response to four agents using in vivo calcium imaging. Results: Compared to the naïve mice, a significant increase in itch-related behaviors was observed in the CCD mice after the injection of pruritogens including histamine and BAM8-22, but not after the injection of algogenic agents including capsaicine and 5-HT, although all the above agents evoked enhanced pain-related behaviors toward the injected site. In vivo calcium imaging revealed that compressed DRG neurons exhibited significantly enhanced responses to histamine and BAM8-22. Immunoflorescent staining also showed that the histamine receptor H1 and the capsaicin receptor TRPV1 were significantly upregulated in DRG neurons. Conclusions: Our findings indicated that sensitization of primary pruriceptive neurons may underlie the enhanced itch sensation after chronic compression of DRG neurons in mice, and may play a role in chronic pruritus in neurological diseases.


Background
Itching (pruritus) has been defined as an "unpleasant skin sensation that elicits the desire or reflex to scratch'' [1]. Primary sensory neurons in dorsal root ganglia (DRG) play an important role in generating itch by detecting pruritogenic stimuli via their peripheral axons in the skin and sending the signals to the spinal cord through their central axons [2]. Although both somatosensory sensations activating sensory nerves, itch and pain can be differentiated by psychophysiological and molecular characteristics [3]. Compression of the cervical spinal cord or the spinal ganglia at C5/C6 occurs in brachioradial pruritus (BRP), causing unilateral or bilateral pruritus in the forearms [4].
Chronic compression of the dorsal root ganglion (CCD) is an animal model of lumbar intraforaminal stenosis and radicular pain [5,6]. CCD increased the incidence of paw-shaking to a normally suprathreshold nociceptive force ("hyperalgesia") [7,8]. Hypersensitivity of mechanical behavior may arise in the rat after CCD or after the local application of either proinflammatory tissue to the lumbar DRG [9][10][11]. The cell bodies of sensory neurons of the compressed DRG become hyperexcitable as evidenced by the presence of spontaneous activity originating in the DRG [12,13], raised responses to electrical, thermal and chemical nociceptive stimuli [14]. Although the pathophysiology of low back pain is well studied, the neural mechanisms accompanying itch are not largely explored.
Histamine is released from mast cells when tissues are inflamed or stimulated by allergens, and once released, histamine induces itch is triggered by the excitation of a subset of unmyelinated C-fibers [15].
BAM8-22 was agonist which can bind to and activate hMrgX1, mMrgC11 and rMrgC receptors with nanomolar affinities [16][17][18]. The study of BAM8-22 is of particular interest not only because of its important role in pain transmission and modulation, but also for its highest metabolic stability and longest duration of action compared to the other Mrg neuropeptides agonists [18].
The present study was designed to explore whether there might be an enhanced behavioral responses to pruritogens in the CCD mice and the potential role of primary pruriceptive neurons in mediating the itch-related behaviors. were divided into a control group and CCD model group.

Surgical treatment
Under 3% isoflurane anesthesia, a midline incision was made along the back and the intervertebral foramina of L3 and L4, exposed after separating the paraspinal muscles from the mammillary process and the transverse process [4]. CCD was produced by the insertion of an L-shaped stainless steel rod (0.3 mm diameter, each arm, 2 mm in length), into each foramen to compress DRGs [7].
The incision was closed in layers and topically treated with ointment containing an antibiotic (TriTop), which is a local anesthetic and an anti-inflammatory agent. A systemic antibacterial was also administered (Baytril, 10 mg/mL, i.m.).
After completion of all behavioral testing, mice were euthanized, and DRGs receiving CCD were microscopically examined to confirm rod placement and, after removal of the epineurium and flushing with saline.

Behavioral test
In this test three groups of mice were given subcutaneous injection of capsaicin (0.1, 1, 10 mg/10 mL), histamine (10, 20, 50 mg/10 mL) and BAM8-22(0.1, 1, 10 mg/10 mL) into the calf of hind leg respectively and sequent behavior was videotaped using a high definition camera for 30 min on pre-CCD 1d and post-CCD 1, 3, 5, 7d. According to present studies, the injection of capsaicin tends to bring out nociceptive (painful) sensations which lead to licking toward injection site in calf models while pruritic stimulus generally aroused biting behaviors. Hence cumulative durations of licking and biting the injection site were counted through video, taking as the assessment of chemical-induced pain and itch.
The chamber was specially made from a cylindrical glass container (20cm, diameter) with two small mirrors attached to plastic bricks and placed as right angle inside, offering a wide view on every act of the animal. There were 10 min of habituation before each test and recording started immediately after the injection.

DRG exposure surgery for in vivo imaging of the whole L4 DRG
For all imaging experiments, mice 8 weeks or older were anesthetized by injection of sodium 5 pentobarbital (40-50 mg/kg, i.p.). After deep anesthesia was reached, the animal's back was shaved and aseptically prepared, and ophthalmic ointment was applied to the eyes to prevent drying. During the surgery, mice were kept on a heating pad (DC temperature controller, FHC) to maintain body temperature at 37±0.5 °C as monitored by a rectal probe.
Dorsal laminectomy in DRG was performed usually at spinal level L5 to L3below the lumbar enlargement but without removing the dura. A 1.5 cm long midline incision was made around the lower part of the lumbar enlargement area; these were dissected away to expose the lower lumbar part which surrounds (L3-L5) vertebra bones. The L4 DRG transverse processes were exposed and cleaned. Using small rongeurs, the surface aspect of the L4 DRG transverse process near the vertebra was removed (only the L4 DRG transverse process was removed but the bone over the spinal cord was intact) to expose the underlying DRG without damaging the DRG and spinal cord. Bleeding from the bone was stopped using styptic cotton.

In vivo L4 DRG calcium imaging
In vivo imaging of whole L4 DRG in live mice was performed for 5 days after CCD surgery. After surgery mice were laid down in the abdomen down position on a designed microscope stage. The spinal column was stabilized using clamps to minimize movements caused by breathing and heart beats. The mice were maintained under continuous anesthesia for the duration of the imaging experiment with 1-2% isoflurane gas using a gas vaporizer. Pure oxygen air was used to deliver the gas to the mouse.
The microscope stage was fixed under a laser-scanning confocal microscope (Nikon C2 microscope system), which was equipped with macro based large objective and fast EM-CCD camera. Live images were acquired at typically 8-10 frames with 600 Hz in frame-scan mode per 6-7 s, using a 5* 0.5 N.A. macro dry objective, at typically 512*512 pixel resolution with solid diode lasers (Nikon) tuned at 448 wavelength, and emission at 500-550 nm for green fluorescence, respectively. For analysis, raw image stacks (512*512 pixels in the x-y plane; typically 8 optical sections) were imported into Nikon Instrument system-element for further analysis. DRG neurons were at the focal plane and imaging was monitored during the activation of DRG neuron cell bodies by peripheral chemical stimuli. The 6 imaging parameters were chosen to allow repeated imaging of the same cell over many stimuli, without causing damage to the imaged cells or to surrounding tissue.

Immunofluenscence
Five days after CCD surgery, the L3 and L4 DRGs of five mice were removed after transcardial perfusion, first with PBS and second, 4% paraformaldehyde, and post-fixed in the same fixative for 4 h, and cryoprotected in 30% sucrose overnight. Tissue was frozen and sectioned at 12 μm thickness by a cryostat and processed for immunofluorescence labeling [19]. The sections on slides were dried at 37°C for 1 hr, and fixed with 4% paraformaldehyde at room temperature for 10 min. The slides were preincubated in blocking solution (10% normal horse serum (vol/vol), 0.2% Triton X-100 (vol/vol) in PBS, pH 7.4) for 1hr at room temperature, then incubated overnight at 4°C with primary antibodies.
Secondary antibody incubation was performed at room temperature for 1 hr.

Quantitative Realtime-RT-PCR
The mRNA levels of TRPV1, Histamine receptor 1, Histamine receptor 4, MrgprA3 receptor in the DRG were measured by real-time PCR (RT-qPCR). Total RNA was extracted by using Trizol reagent according to the manufacturer's instructions. The cDNA was synthesized from 1µg of total RNA by PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time). Each cDNA sample was amplified for the gene of interest and GAPDH in a 25 µL reaction volume using SYBR1 Premix Ex TaqTM II (Tli RNaseH Plus). All primers used are listed in Table 1. The realtime RT-PCR conditions were 94 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s and 72 °C for 60 s. The mRNA levels of all 7 genes were normalized to GAPDH.

Statistical analyses
For in vivo experiments, the animals were distributed into various treated groups randomly. All of the results are given as means ± SEM. Data distribution was assumed to be normal but this was not formally tested. The data were statistically analyzed with two-tailed, paired/unpaired Student's t test and a one-way or two-way ANOVA. When ANOVA showed significant difference, pairwise comparisons between means were tested by the post hoc Tukey method (SigmaStat, San Jose, CA).

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 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 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).

Confocal imaging of DRG
To evaluate neuronal activity in DRG somata, we used Pirt-GCaMP3 mice to image Ca 2+ response in L4 DRG.

H1R and TRPV1 of DRG neurons immunoreactivity after CCD surgery
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).

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 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).

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.

Discussion
In this study, we first found that CCD upregulated H1R and TRPV1 coexpression in primary sensory neurons of dorsal root ganglion. The injection of low dose of histamine and BAM into the calf of mice evoked itch-like behaviors after CCD. There were a significantly greater percentage of primary sensory neurons expressing not only H1R but also TRPV1 after CCD.
Researcher found that both H1R and H4R are expressed on C-afferent fiber terminals, and also that these antagonists can directly inhibit the transmission of itching responses from the peripheral to central nervous system [20]. Using single-cell calcium imaging, Rossbach et al. found that histamine induced an increase in calcium levels in a subset of skin-specific sensory neurons in mice by activating H1R and H4R as well as inhibiting H3R [21]., Histamine is a well-known mediator of acute inflammatory and immediate hypersensitivity responses.
Though the physiological role of histamine was well studied, much is known of signaling pathway that leads to the excitation of the sensory neurons, which induces the adapt neural signals for itching. Our study provides in vivo and in vitro evidence that histamine requires the activation of TRPV1 to excite primary sensory neurons of DRG after CCD. H1R can activate phospholipase C, and increases intracellular Ca 2+ level. Pruritus is elicited by the activation of H1R. The strong relationship between histamine and TRPV1 in primary sensory neurons of DRG has been showed in our study. Coexpression of TRPV1 and histamine receptor is in a subset of sensory neurons [26,27], and primary afferent C-fibers that respond to histamine are also sensitive to capsaicin [26,28]. Moreover, repetitive application of capsaicin is known to desensitize TRPV1 or sensory nerves and was found to alleviate the pruritus induced by histamine [29]. In a word, these results further strengthen the notion that TRPV1 mediates histamine-induced itching. There are some reports suggest that histamine H1 and H4 receptors are co-invovled in the pathway to transmit the itch signal to the central system [30,31]. In the present study, we showed that CCD increased histamine H1 and TRPV1 receptor agonist-induced itching behaviors.
After the histamine H1 receptor was activated, the Gαq proteins coupled with the histamine H1 receptor downstream signal pathway induced TRPV1 to open and excited the neurons to transmit the itch signal [24, 25].
BAM8-22 was fragment from the proenkephalin A gene, which was identified as ligand capable of potently activating rat MrgC11 and MrgA3. After CCD, the expression of MrgA3 protein in DRG elevated compared to control mice.
In this study, mRNA of DRG tissue was detected, single cell PCR should be applied to detect mRNA expression of histamine receptor and TRPV1 positive neuron in next study. Undoubtedly, more work is needed to understand how these pruriceptors enhances pruriceptic behaviors in mouse.

Conclusions
In summary, the present study shows that lower concentration of histamine and BAM excite sensory neurons to induce itching behavior in CCD mice, not in control mice.
The responses of compressed DRG neurons to histamine and BAM8-22 were significantly enhanced, and its H1R and TRPV1 were markedly upregulated. Moreover, histamine is an important cause of itching in dermatitis patient, the present study provides clues concerning the treatment of evokeditching and inflammatory pain.

Availability of data and materials:
There is no data, software, databases, and application/tool available apart from the reported in the present study. All data is provided in manuscript and supplementary data.

Author contributions:
JT collected the behavioral data and performed DRG calcium imaging, TW performed animal surgery,  Table   Table 1 The sequence of the primers used in the experiment.       Upregulation of the expression of H1R and TRPV1 in the DRG in CCD mice. A and B.
Immunofluorescence staining showed a significantly higher percentage of H1R-and TRPV1immunopositive DRG neurons in CCD mice compared with that in control mice (n=6 mice/group). *P<0.05, CCD vs Control, Student's t-test.
26 Figure 5 Upregulation of the expression of H1R and TRPV1 in the DRG in CCD mice. A and B.
Immunofluorescence staining showed a significantly higher percentage of H1R-and TRPV1immunopositive DRG neurons in CCD mice compared with that in control mice (n=6 mice/group). *P<0.05, CCD vs Control, Student's t-test.