Unveiling adcyap1 as a protective factor linking pain and nerve regeneration through single-cell RNA sequencing of rat dorsal root ganglion neurons.

BACKGROUND: Severe peripheral nerve injury (PNI) often leads to significant movement disorders and intractable pain. Therefore, promoting nerve regeneration while avoiding neuropathic pain is crucial for the clinical treatment of PNI patients. However, established animal models for peripheral neuropathy fail to accurately recapitulate the clinical features of PNI. Additionally, researchers usually investigate neuropathic pain and axonal regeneration separately, leaving the intrinsic relationship between the development of neuropathic pain and nerve regeneration after PNI unclear. To explore the underlying connections between pain and regeneration after PNI and provide potential molecular targets, we performed single-cell RNA sequencing and functional verification in an established rat model, allowing simultaneous study of the neuropathic pain and axonal regeneration after PNI. RESULTS: First, a novel rat model named spared nerve crush (SNC) was created. In this model, two branches of the sciatic nerve were crushed, but the epineurium remained unsevered. This model successfully recapitulated both neuropathic pain and axonal regeneration after PNI, allowing for the study of the intrinsic link between these two crucial biological processes. Dorsal root ganglions (DRGs) from SNC and naïve rats at various time points after SNC were collected for single-cell RNA sequencing (scRNA-seq). After matching all scRNA-seq data to the 7 known DRG types, we discovered that the PEP1 and PEP3 DRG neuron subtypes increased in crushed and uncrushed DRG separately after SNC. Using experimental design scRNA-seq processing (EDSSP), we identified Adcyap1 as a potential gene contributing to both pain and nerve regeneration. Indeed, repeated intrathecal administration of PACAP38 mitigated pain and facilitated axonal regeneration, while Adcyap1 siRNA or PACAP6-38, an antagonist of PAC1R (a receptor of PACAP38) led to both mechanical hyperalgesia and delayed DRG axon regeneration in SNC rats. Moreover, these effects can be reversed by repeated intrathecal administration of PACAP38 in the acute phase but not the late phase after PNI, resulting in alleviated pain and promoted axonal regeneration. CONCLUSIONS: Our study reveals that Adcyap1 is an intrinsic protective factor linking neuropathic pain and axonal regeneration following PNI. This finding provides new potential targets and strategies for early therapeutic intervention of PNI.

Cephalic inferior vena cava non-clamping technique versus standard procedure for robot-assisted laparoscopic level II-III thrombectomy: a prospective cohort study.

BACKGROUND: Renal tumour can invade the venous system and ~4-10% patients with renal tumour had venous thrombus. Although the feasibility of robot-assisted laparoscopic inferior vena cava thrombectomy (RAL-IVCT) in patients with inferior vena cava (IVC) thrombus has been validated, the wide application is still a challenge due to the complexity of IVC control. The objective was to describe our novel cephalic IVC non-clamping technique and to compare the outcomes versus standard RAL-IVCT. MATERIALS AND METHODS: A prospective single-centre cohort containing 30 patients with level II-III IVC thrombus was established since August 2020. Fifteen patients underwent cephalic IVC non-clamping approach and 15 patients received standard RAL-IVCT. The authors decided the surgical technique according to the echocardiographic assessment of the right heart and IVC. RESULTS: The non-clamping group had less operative time (median 148 versus 185 min, P =0.04), and lower Clavien-grade II complication rate (26.7% versus 80.0%, P =0.003). The median intraoperative blood loss were 400 ml [interquartile range (IQR) 275-615 mL] and 800 ml (IQR 350-1300 ml), respectively ( P =0.05). The most common complication in standard RAL-IVCT group was liver dysfunction. No gas embolism, hypercapnia or tumour thrombus dislodgment occurred in non-clamping group. After a median follow-up of 17.0 months (IQR 13.5-18.5 months) and 15.5 months (IQR 13.0-17.0 months), two patients (16.7%) in the non-clamping group and 3 patients (20.0%) in the standard RAL-IVCT group died (hazard ratio 0.59, 95% CI 0.10-3.54, P =0.55). CONCLUSIONS: The cephalic IVC non-clamping technique can be performed safely with acceptable surgical outcomes and short-term oncologic outcomes in patients with level II-III IVC thrombus. Compared with standard procedure, it had less operative time and lower complication rate.

Role of ß-1,3-galactosyltransferase 2 in trigeminal neuronal sensitization induced by peripheral inflammation.

Glycosyltransferases are enzymes that catalyze the formation of a variety of glycoconjugates. Glycoconjugates play important roles in the nervous system. ß-1,3-Galactosyltransferase 2 (B3galt2) belongs to the family of ß-1,3-galactosyltransferase, which is one of the major types of glycosyltransferases. Dental pulp inflammation may cause neurophysiological alterations in the trigeminal ganglion (TG), and serve as a good model for investigating the peripheral inflammation and trigeminal neuronal sensitization. In the present study, we investigated the expression of B3galt2 in neuroinflammation using the dental pulp inflammatory model induced by lipopolysaccharide in rat. The expression of B3galt2 gene and protein were determined by reverse transcription PCR, immunohistochemistry and western blot analysis. ELISA assays were used to measure the levels of cytokines in the TG neurons. Toll-like receptor 4 (TLR4) and nuclear factor-κB (NFκB) were evaluated by immunohistochemistry and western blotting. Our results demonstrated that B3galt2 was expressed in the TG, and dental pulp inflammation up-regulated B3galt2 expression in the TG. B3galt2 gene knockdown reduced the secretion of TNFα and IL-6 in the TG neurons. The expression of TLR4 and NFκB in the TG was activated during the inflammation, but B3galt2 gene knockdown inhibited the expression of TLR4 and NFκB. These observations indicated that dental pulp inflammation could induce B3galt2 expression in TG, and that B3galt2 might play a regulatory role in TG neuronal sensitization. These findings suggest that B3galt2 may play an important role in trigeminal neuronal sensitization induced by peripheral inflammation via mediating TLR4/NFκB signaling pathway.

Dynamic Regulation of Delta-Opioid Receptor in Rat Trigeminal Ganglion Neurons by Lipopolysaccharide-induced Acute Pulpitis.

INTRODUCTION: Delta-opioid receptor (DOR) and its endogenous ligands distribute in trigeminal system and play a very important role in modulating peripheral inflammatory pain. DOR activation can trigger p44/42 mitogen-activated protein kinase (ERK1/2) and Akt signaling pathways, which participate in anti-inflammatory and neuroprotective effects. In this study, our purpose was to determine the dynamic changes of DOR in trigeminal ganglion (TG) neurons during the process of acute dental pulp inflammation and elucidate its possible mechanism. METHODS: Forty rats were used to generate lipopolysaccharide-induced acute pulpitis animal models at 6, 12, and 24 hours and sham-operated groups. Acute pulpitis was confirmed by hematoxylin-eosin staining, and TG neuron activation was determined by anti-c-Fos immunohistochemistry. DOR protein and gene expression in TG was investigated by immunohistochemistry, Western blotting, and real-time polymerase chain reaction, and DOR expression in trigeminal nerves and dental pulp was also determined by immunohistochemistry. To further investigate the mechanism of DOR modulating acute inflammation, the change of pErk1/2 and pAkt in TG was examined by immunohistochemistry. RESULTS: Lipopolysaccharide could successfully induce acute pulpitis and activated TG neurons. Acute pulpitis could dynamically increase DOR protein and gene expression at 6, 12, and 24 hours in TG, and DOR dimerization was significantly increased at 12 and 24 hours. Acute pulpitis also induced the dynamic change of DOR protein in trigeminal nerve and dental pulp. Furthermore, ERK1/2 and Akt signaling pathways were inhibited in TG after acute pulpitis. CONCLUSIONS: Increased DOR expression and dimerization may play important roles in peripheral acute inflammatory pain.

Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats.

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression significantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibited, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.