Developmental analysis of reconstructed embryos of second-generation cloned transgenic goats.

To improve the efficiency of the production of transgenic cloned goats by somatic cell nuclear transfer (SCNT), the development of reconstructed embryos of first-generation (G1) and second-generation (G2) cloned transgenic goats was compared and analysed. Primary transgenic foetal fibroblasts were used as donor cells for G1 somatic cell nuclear transfer (SCNT). When the G1 transgenic embryos developed to 35 days in the recipient goats, transgenic foetal fibroblasts were isolated from them and used as donor cells for the G2 clone. In the G1 clones, the average fusion rate of reconstructed embryos was 73.62 ± 2.9%, the average development rate (2-4 cells) was 33.96 ± 2.36%, and the pregnancy rate of transplant recipients was 31.91%. In the G2 clones, the average fusion rate of the reconstructed embryos was 90.29 ± 2.03%, the average development rate was 66.46 ± 3.30%, and the pregnancy rate was 58.14%. These results indicate that in the G2 clones, the fusion rate of eggs, the development rate of reconstructed embryos and the pregnancy rate of transplant recipients were significantly higher than those of G1 clones. We believe these results will lay a solid foundation for the efficient production of transgenic cloned animals in the future.

Facilitation of MrgprD by TRP-A1 promotes neuropathic pain.

Neuropathic pain remains a therapeutic challenge because of its complicated mechanisms. Mas-related GPCR D (MrgprD) is specifically expressed in small-diameter, nociceptive neurons of dorsal root ganglia (DRGs) and is implicated in pain modulation. However, the underlying mechanism of MrgprD involved in neuropathic pain remains elusive. In this study, we used behavioral experiments and physiologic examination methods to investigate the role of MrgprD in chronic constriction injury (CCI)-induced neuropathic pain. We found that MrgprD is necessary for the initiation of mechanical hypersensitivity and cold allodynia, but not for heat allodynia. Moreover, we demonstrated that transient receptor potential cation channel (TRP)-A1 was the ion channel downstream of MrgprD, and the ß-alanine-induced calcium signal was attributed mostly to TRP-A1 function. We further showed that PKA serves as a downstream mediator of ß-alanine-activated MrgprD signaling to activate TRP-A1 in DRG neurons and in human embryonic kidney 293 cells, to coexpress MrgprD and TRP-A1 plasmids. Finally, we found that the ß-alanine-induced pain behavior was increased, whereas the itching behavior was unchanged in CCI models compared with sham-injured animals. Knockout of TRPA1 also attenuated the ß-alanine-induced pain behavior in CCI models. In conclusion, MrgprD is essential in cold allodynia in CCI-induced neuropathic pain through the PKA-TRP-A1 pathway. TRP-A1 facilitates MrgprD to development of neuropathic pain. Our findings reveal a novel mechanism of neuropathic pain formation and highlight MrgprD as a promising drug target for the treatment of neuropathic pain.-Wang, C., Gu, L., Ruan, Y., Geng, X., Xu, M., Yang, N., Yu, L., Jiang, Y., Zhu, C., Yang, Y., Zhou, Y., Guan, X., Luo, W., Liu, Q., Dong, X., Yu, G., Lan, L., Tang, Z. Facilitation of MrgprD by TRP-A1 promotes neuropathic pain.

Pirt Together with TRPV1 Is Involved in the Regulation of Neuropathic Pain.

Neuropathic pain is a chronic pain and reduces the life quality of patients substantially. Transient receptor potential vanilloid channel 1 (TRPV1), a nonselective cation channel, has been shown to play a crucial role in neuropathic pain. Although TRPV1 plays an important role in neuropathic pain, the mechanism of how TRPV1 was regulated in neuropathic pain remains unclear. Pirt is a membrane protein and binds to TRPV1 to enhance its activity. It was suggested that Pirt should also be involved in neuropathic pain. In this study, we investigated the role of Pirt in neuropathic pain (CCI model); the results show that mechanical allodynia and thermal hyperalgesia were alleviated in Pirt-/- mice in CCI models. TRPV1 expression was increased by immunofluorescence and real-time PCR experiments. The increase in TRPV1 expression was less in Pirt knockout mice in CCI models. Moreover, the number of capsaicin-responding neurons and the magnitude of evoked calcium response were attenuated in DRG neurons from Pirt-/- mice in CCI models. Finally, we found that the pain behavior attenuated in dysfunction of both Pirt and TRPV1 was much stronger than in dysfunction of Pirt or TRPV1 only in a CCI model in vitro study. Taken together, Pirt together with TRPV1 is involved in CCI-induced neuropathic pain.

A Combined Water Extract of Frankincense and Myrrh Alleviates Neuropathic Pain in Mice via Modulation of TRPV1.

Frankincense and myrrh are widely used in clinics as a pair of herbs to obtain a synergistic effect for relieving pain. To illuminate the analgesia mechanism of frankincense and myrrh, we assessed its effect in a neuropathic pain mouse model. Transient receptor potential vanilloid 1 (TRPV1) plays a crucial role in neuropathic pain and influences the plasticity of neuronal connectivity. We hypothesized that the water extraction of frankincense and myrrh (WFM) exerted its analgesia effect by modulating the neuronal function of TRPV1. In our study, WFM was verified by UHPLC-TQ/MS assay. In vivo study showed that nociceptive response in mouse by heat and capsaicin induced were relieved by WFM treatment. Furthermore, thermal hypersensitivity and mechanical allodynia were also alleviated by WFM treatment in a chronic constriction injury (CCI) mouse model. CCI resulted in increased TRPV1 expression at both the mRNA and protein levels in predominantly small-to-medium neurons. However, after WFM treatment, TRPV1 expression was reverted in real-time PCR, Western blot, and immunofluorescence experiments. Calcium response to capsaicin was also decreased in cultured DRG neurons from CCI model mouse after WFM treatment. In conclusion, WFM alleviated CCI-induced mechanical allodynia and thermal hypersensitivity via modulating TRPV1.

An effective and concise device for detecting cold allodynia in mice.

Detection of cold allodynia is a very important aspect in the study of pain behavior. An effective and concise device for detecting cold pain has always been the hope of many researchers. Here, an easily produced and operated cold plate device is presented for the assessment of cold allodynia in mice. The device used to detect cold allodynia has two components: a chamber consists of a cylinder for animal experiment and a cube box around the chamber for holding ice to keep temperature stable. In the testing chamber, a mouse was placed on the circular plexiglass plate steady at 4 °C above ice for five minutes. The tested mouse will lift its paw when exposed to the cold plate. The number of lifts will present animal's response to the degree of cold stimulation. To evaluate this approach, three commonly used pain models of mice were tested: formalin test, bone cancer pain (BCP), and chronic constriction injury (CCI). As is reported in other literatures, these three pain mice models showed increased sensitivity to cold stimulation. The new device is indeed suitable for detecting cold allodynia behavior in mice. Comparisons with existing devices of detecting cold allodynia, such as the cold plate in the market (UGO, Panlab, Columbus, etc.), the new device has the advantages of low cost, simple operation and easy popularization and can detect cold allodynia behavior of mice very well. This is a very practical and economical device to detect cold allodynia behavior.

Matrine inhibits itching by lowering the activity of calcium channel.

Sophorae Flavescentis Radix (SFR) is a medicinal herb with many functions that are involved in anti-inflammation, antinociception, and anticancer. SFR is also used to treat a variety of itching diseases. Matrine (MT) is one of the main constituents in SFR and also has the effect of relieving itching, but the antipruritic mechanism is still unclear. Here, we investigated the effect of MT on anti-pruritus. In acute and chronic itch models, MT significantly inhibited the scratching behavior not only in acute itching induced by histamine (His), chloroquine (CQ) and compound 48/80 with a dose-depended manner, but also in the chronic pruritus models of atopic dermatitis (AD) and acetone-ether-water (AEW) in mice. Furthermore, MT could be detected in the blood after intraperitoneal injection (i.p.) and subcutaneous injection (s.c.). Finally, electrophysiological and calcium imaging results showed that MT inhibited the excitatory synaptic transmission from dorsal root ganglion (DRG) to the dorsal horn of the spinal cord by suppressing the presynaptic N-type calcium channel. Taken together, we believe that MT is a novel drug candidate in treating pruritus diseases, especially for histamine-independent and chronic pruritus, which might be attributed to inhibition of the presynaptic N-type calcium channel.

MrgprA3 shows sensitization to chloroquine in an acetone-ether-water mice model.

Chronic itch, a distressing symptom of many cutaneous and systemic diseases, significantly impairs quality of life. However, its underlying molecular mechanism is still unclear. Mas-related G protein-coupled receptor A3 (MrgprA3) is considered an itch-specific receptor. MrgprA3 neurons are identified as a class of itch-specific neurons, but the role of MrgprA3 in chronic itch remains elusive. An acetone-ether-water (AEW) model as a histamine-independent itch model is often used in the study of chronic pruritus. In this study, behavioral tests, immunostaining, cell culture, calcium imaging, and other experiments were carried out to examine the expression of MrgprA3. The results showed that the scratching bouts induced by chloroquine increased significantly under the AEW condition; the density of MrgprA3 sensory fibers in the AEW-treated skin area and the number of MrgprA3 neurons in dorsal root ganglia from the AEW model mice also increased significantly. Further analysis showed that the MrgprA3 in mRNA level was also increased after AEW treatment. These results indicated that MrgprA3 played a crucial role in chronic pruritus in the AEW model.