Chemogenetic silencing of NaV1.8 positive sensory neurons reverses chronic neuropathic and bone cancer pain in FLEx PSAM4-GlyR mice

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically-activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging upon activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.Significance StatementChronic pain is a massive problem. Peripheral nerve block is effective in many chronic pain conditions, demonstrating the importance of peripheral drive in chronic pain. We used chemogenetic tools based on the modified ligand-gated chloride channel PSAM4-GlyR to silence dorsal root ganglion neurons in vitro and in vivo This approach reduces pain-like behavior in acute and chronic pain models, including resistant pain conditions like neuropathic pain or cancer-induced bone pain. We generated a mouse line that expresses PSAM4-GlyR in a Cre-dependent manner, providing a useful research tool to address not only the role of nociceptive sensory neurons in pain states but also the function of genetically defined sets of neurons throughout the nervous system in normal and pathological conditions

Exploring potential analgesic targets and tools for chronic pain treatment

This thesis investigated two main topics. The first was the preclinical evaluation of various analgesic modalities for cancer-induced bone pain (CIBP). The second involved the potential application of chemogenetic tools for pain research. I optimised and used an in vivo model of CIBP involving the injection of Lewis Lung Carcinoma cells into the intramedullary space of the femur of C57BL/6 mice or transgenic mice with C57BL/6 background. In this model, mice gradually reduce the use of the affected limb resulting in altered weight bearing. Symptoms of secondary cutaneous cold, heat, and mechanical sensitivity also manifest. Three potential analgesic modalities were assessed, which can be divided into three categories; targeting the NaV1.7 voltage-gated Na+ channel, targeting neuronal subsets (namely the µ-opioid receptor-expressing neurons and the sensory neurons that express NaV1.8 channels), and finally, the dual targeting of two of the tumour-derived products (nerve growth factor (NGF) and tumour necrosis factor (TNF)). Results from these experiments indicated the congenital deletion or chemogenetic-based silencing of the NaV1.8 expressing neurons reduced pain-like behaviour associated with CIBP. Moreover, dual inhibition of NGF and TNF resulted in an impressive reduction in CIBP-driven weight-bearing and prevented the development of secondary cutaneous heat hyperalgesia. The second half of this work focused on modified ligand-gated ion channels, namely PSAM4-GlyR and PSAM4-5HT3. This work showed that expressing PSAM4-GlyR in dorsal root ganglia (DRG) neurons and agonism with varenicline silences DRG neurons and elevates the withdrawal thresholds of mice in various sensory tests. Additionally, PSAM4-GlyR activation in the NaV1.8+ neurons reversed signs of mechanical, thermal, and cold sensitivity associated with neuropathic pain. Moreover, chemogenetic-based activation of specific neurons in the central amygdala was shown to increase pain thresholds. These techniques will be useful for studies investigating the effects of manipulating neuronal subsets

Efficacy and Safety of Warfarin Therapy: Comparison Between Specialized INR Clinic and General Medical Clinic

Abstract Background: Although warfarin is known as effective oral anticoagulant to prevent thromboembolic events, its' narrow therapeutic index requires ambient and good follow-up to reduce its therapeutic complications. There is a continuous debate whether the best practice to accomplish this goal is in a specialized international normalized ratio clinic (INR-C) or in a general medical clinic (General-C). Few, if any, studies have been done in Sudan to compare the safety and efficacy of anticoagulant therapy in those clinics. Thus, the objective of this study was to compare the efficacy and safety of anticoagulant therapy in INR-C and in General-C. Methods: This is a prospective hospital-based study where 200 patients were divided into two groups (group A and B) of 100 patients. Group A were in the INR-C at Ahmed Gasim specialized hospital and group B in the General-Cat AL-Shaab teaching hospital. The study was conducted from September 2019 to April 2020. All patients were on warfarin treatment and regular follow-ups were conducted. Demographic and clinical data were collected and analyzed statistically using SPSS version 20. Ethical approval was obtained from the ethical committee of the Sudanese Medical Specialization Board (SMSB). Results: Of the 200 patients, 118/59% were females and 82/41% were males. Target international normalized ratio (INR) for group (A) was achieved in 56% of the patients in the first visit, increased to 63% in the second visit, and 75% in the third follow-up, compared with 24% of the patients from group (B) in the initial and second follow-up visit, to 43% in the third visit (P value=0.05). Knowledge about drug and food interaction of coagulation agents was higher (91%) among patients in group (A) compared with group (B) (56%). Drug interaction awareness was found in 89% of the patients in group (A) compared with only 40% in group (B) (P value=0.05). Major bleeding was reported in 2% and 14% of the patients of group (A) and (B) respectively, whereas minor bleeding was seen in 4% of group (A) and 11% of group (B). Conclusion: The study showed that INR-C is more efficient and safer for patients on regular warfarin therapy compared with the General-C