Model Of Painful Diabetic Neuropathy Research Articles

Liquiritin ameliorates painful diabetic neuropathy in SD rats by inhibiting NLRP3-MMP-9-mediated reversal of aquaporin-4 polarity in the glymphatic system.

Despite advancements in diabetes treatment, the management of Painful Diabetic Neuropathy (PDN) remains challenging. Our previous research indicated a significant correlation between the expression and distribution of Aquaporin-4 (AQP4) in the spinal glymphatic system and PDN. However, the potential role and mechanism of liquiritin in PDN treatment remain uncertain. This study established a rat model of PDN using a combination of low-dose Streptozotocin (STZ) and a high-fat, high-sugar diet. Rats were treated with liquiritin and MCC950 (an NLRP3 inhibitor). We monitored fasting blood glucose, body weight, and mechanical allodynia periodically. The glymphatic system's clearance function was evaluated using Magnetic Resonance Imaging (MRI), and changes in proteins including NLRP3, MMP-9, and AQP4 were detected through immunofluorescence and Western blot techniques. The rats with painful diabetic neuropathy (PDN) demonstrated several physiological changes, including heightened mechanical allodynia, compromised clearance function within the spinal glymphatic system, altered distribution of AQP4, increased count of activated astrocytes, elevated expression levels of NLRP3 and MMP-9, and decreased expression of AQP4. However, following treatment with liquiritin and MCC950, these rats exhibited notable improvements. Liquiritin may promote the restoration of AQP4 polarity by inhibiting NLRP3 and MMP-9, thereby enhancing the clearance functions of the spinal cord glymphatic system in PDN rats, alleviating the progression of PDN.

Keratinocyte-Derived Exosomes in Painful Diabetic Neuropathy.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.

10 kHz spinal cord stimulation improves metrics of spinal sensory processing in a male STZ rat model of diabetes

To explore why clinical 10 kHz spinal cord stimulation (10 kHz SCS) might improve neurological function in a model of painful diabetic neuropathy (PDN), the short-term behavioral, electrophysiological, and histological effects of 10 kHz SCS were studied using adult male streptozotocin (STZ)-induced diabetic Sprague-Dawley rats. Four testing groups were established: Naïve controls (N=8), STZ controls (N=7), STZ+Sham SCS (N=9), and STZ+10 kHz SCS (N=11). After intraperitoneal injection (60 mg/kg) of STZ caused the rats to become hyperglycemic, SCS electrodes were implanted in the dorsal epidural space over the L5-L6 spinal segments in the STZ+Sham SCS and STZ+10 kHz SCS groups and were stimulated for 14 days. The von Frey filament paw withdrawal threshold was measured weekly. At termination, animals were anesthetized and the electrophysiologic response of dorsal horn neurons (receptive field size, vibration, radiant warmth) of the ipsilateral foot was measured. Tissue from the plantar paw surface was obtained post-euthanization for intraepidermal nerve fiber density measurements. In comparison to other control groups, while no significant effect of 10 kHz SCS on peripheral intraepidermal nerve fiber density was observed, 10 kHz SCS ‘normalized’ the central neural response to vibration, receptive field, and paw withdrawal threshold, and elevated the neural response to tissue recovery from warm stimuli. These results suggest that short-term, low intensity 10 kHz SCS operates in the spinal cord to ameliorate compromised sensory processing, and may compensate for reduced peripheral sensory functionality from chronic hyperglycemia, thereby treating a broader spectrum of the sensory symptoms in diabetic neuropathy.

Effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy

Previous studies have explored the antinociceptive effects of riboflavin (vitamin B2) across various experimental models. However, there remains a gap in the literature regarding its potential to alleviate neuropathic pain in diabetes. This study aims to investigate the effects of riboflavin on hyperalgesia and serum glutamine-to-glutamate ratio in rats with painful diabetic neuropathy. In fasted rats, a model of painful diabetic neuropathy was induced through intraperitoneal injection of streptozotocin. In the fifth week post-injection, diabetic rats experiencing neuropathic pain were administered daily doses of riboflavin (25 or 50 mg), dissolved in their drinking water, for a duration of two weeks. Results demonstrate that riboflavin significantly reduced mechanical and cold-induced hyperalgesia in diabetic rats compared to controls. Formalin-induced hyperalgesia was alleviated by riboflavin in the second phase. Additionally, riboflavin supplementation increased the serum glutamine-to-glutamate ratio in these animals. These findings highlight the therapeutic potential of riboflavin in managing neuropathic pain associated with diabetes.

J-2156, a small molecule somatostatin type 4 receptor agonist, alleviated hindpaw hypersensitivity in the streptozotocin-induced rat model of painful diabetic neuropathy but with a 2-fold decrease in potency at an advanced stage in the model, mimicking morphine.

There is a large unmet need for novel pain-killers to improve relief of painful diabetic neuropathy (PDN). Herein, we assessed the efficacy of the somatostatin type 4 (SST4) receptor agonist, J-2156, for relief of PDN in rats. Diabetes was induced with streptozotocin (STZ; 70mg/kg) and bilateral hindpaw hypersensitivity was fully developed by 8-week post-STZ. In the intervals, 8-12-weeks (morphine-sensitive phase; Phase 1) and 16-18-weeks (morphine-hyposensitive phase; Phase 2) post-STZ, rats received a single dose of intraperitoneal (i.p.) J-2156 (10, 20, 30mg/kg), gabapentin (100mg/kg i.p.), subcutaneous morphine (1mg/kg) or vehicle. Hindpaw withdrawal thresholds (PWTs) were assessed using von Frey filaments pre-dose and at regular intervals over 3-h post-dose. In Phase 1, J-2156at 30mg/kg evoked significant anti-allodynia in the hindpaws with maximal effect at 1.5h compared with 1h for gabapentin and morphine. The durations of action for all three compounds were greater than 3h. The corresponding mean (±SEM) extent and duration of anti-allodynia (ΔPWT AUC) for gabapentin did not differ significantly from that for J-2156 (30mg/kg) or morphine. However, in Phase 2, the ΔPWT AUC for morphine was reduced to approximately 25% of that in Phase 1, mirroring our previous work. Similarly, the mean (±SEM) ΔPWT AUC for J-2156 (30mg/kg) in Phase 2 was approximately 45% of that for Phase 1 whereas for gabapentin the mean (±SEM) ΔPWT AUCs did not differ significantly (p > 0.05) between the two phases. Our findings further describe the preclinical pain relief profile of J-2156 and complement previous work in rat models of inflammatory pain, neuropathic pain and low back pain. SST4 receptor agonists hold promise as novel therapeutics for the relief of PDN, a type of peripheral neuropathic pain that is often intractable to relief with clinically used drug treatment options.

The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and changes in cutaneous innervation. However, the complete molecular profile underlying the hyperexcitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches that are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing of DRGs, we demonstrated an increased expression of the Mas-related G protein-coupled receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established high-fat diet (HFD) mouse model of PDN. Importantly, limiting Mrgprd signaling reversed mechanical allodynia in the HFD mouse model of PDN. Furthermore, in vivo calcium imaging allowed us to demonstrate that activation of Mrgprd-positive cutaneous afferents that persist in diabetic mice skin resulted in an increased intracellular calcium influx into DRG nociceptors that we assess in vivo as a readout of nociceptors hyperexcitability. Taken together, our data highlight a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising and accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN.

BTD: A TRPC5 activator ameliorates mechanical allodynia in diabetic peripheral neuropathic rats by modulating TRPC5-CAMKII-ERK pathway

Mechanical allodynia is a serious complication of painful diabetic neuropathy (PDN) with limited treatment options. The transient receptor potential canonical 5 (TRPC5) channel is a promising target in pain; however, its role in painful diabetic neuropathy has not yet been elucidated. In this study, we have investigated the role of TRPC5 channels using BTD [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ6,2,4-benzothiadiazin-3-yl)-propanamide)],a potent TRPC5 activator and HC070, as TRPC5 channel inhibitor in rat model of PDN. In this study, streptozotocin was used to induce diabetes in male Sprague-Dawley rats. The alterations in mechanical and thermal pain thresholds, nerve functional deficits in diabetic animals were assessed by various behavioral and functional parameters.TRPC5 involvement was investigated by treating neuropathic rats with BTD, TRPC5 channel activator (1 and 3 mg/kg, i.p. for 14 days) and HC070, a TRPC5 channel inhibitor (1 and 3 mg/kg). BTD and HC070 effects in pain reduction were assessed by western blotting, estimating oxidative stress and inflammatory markers in the lumbar spinal cord. BTD treatment (3 mg/kg, i.p.) once daily for 14 days ameliorated mechanical allodynia but not thermal hyposensation or nerve functional deficit in diabetic neuropathic rats. BTD treatment down-regulated TRPC5 expression by increasing the activity of protein kinase C. It also subsequently down-regulated the downstream pain markers (CAMKII, ERK) in the spinal cord. Additionally, a decrease in inflammatory cytokines (TNF-α, IL-6) also demonstrated BTD's potent anti-inflammatory properties in reducing mechanical allodynia. On the other hand, HC070 did not exert any beneficial effects on behavioural and nerve functional parameters. The study concludes that BTD ameliorated mechanical allodynia in a rat model of painful diabetic neuropathy not only through modulation of the TRPC5-CAMKII-ERK pathway but also through its anti-inflammatory and anti-apoptotic properties. Overall, BTD is a promising therapeutic molecule in the treatment of mechanical allodynia in painful diabetic neuropathy.

Swimming exercise attenuates mechanical hypersensitivity and mitigates peripheral nerve degeneration in rats with painful diabetic neuropathy (PDN)

BackgroundThis study aimed to assess the effectiveness of swimming exercise in alleviating mechanical hypersensitivity and peripheral nerve degeneration associated with a pre-clinical model of painful diabetic neuropathy (PDN). MethodsThis study is a pre-clinical study conducted using the streptozocin (STZ)-induced PDN rat model. Rats were randomly allocated to three groups: a vehicle group of non-diabetic rats (Vehicle, n = 9), a group of rats with PDN (PDN, n = 8), and a group of rats with PDN that performed a swimming exercise program (PDN-SW, n = 10). The swimming exercise program included daily 30-minute swimming exercise, 5 days per week for 4 weeks. Von Frey testing was used to monitor hindpaw mechanical sensitivity over 4 weeks. Assessment of cutaneous peripheral nerve fiber integrity was performed after the 4-week study period via immunohistochemistry for protein gene product 9.5-positive (PGP9.5+) intra-epidermal nerve fiber density (IENFD) in hind-paw skin biopsies by a blinded investigator. ResultsThe results showed that swimming exercise mitigated but did not fully reverse mechanical hypersensitivity in rats with PDN. Immunohistochemical testing revealed that the rats in the PDN-SW group retained higher PGP9.5+ IENFD compared to the PDN group but did not reach normal levels of the Vehicle group. ConclusionsThe results of this study indicate that swimming exercise can mitigate mechanical hypersensitivity and degeneration of peripheral nerve fibers in rats with experimental PDN.

481-P: 10kHz SCS Restores Mechanical and Thermal Neural Processing in a Rodent Model of Painful Diabetic Neuropathy

A recent randomized controlled trial of 10 kHz spinal cord stimulation (SCS) in patients with painful diabetic neuropathy (PDN) has demonstrated significant and durable improvements in neurologic status, particularly sensory restoration of the foot (Petersen et al 2021, Argoff et al 2023). In order to gain further mechanistic insight into the improvements in sensory processing by 10kHz SCS, we analyzed the receptive field (RF) size and responses of spinal dorsal horn (DH) neurons to mechanical and thermal stimuli of the paw in a PDN rat model. For PDN-model, Rats were given a single effective dose of streptozotocin (STZ), while Naïve animals were injected with 0.9% saline. Four groups were studied: Naïve, STZ, STZ+ShamSCS, and STZ+10kHzSCS. Rats were anesthetized with urethane and a laminectomy was performed for DH neurons recording. We analyzed RF size and the response of DH neurons to vibrotactile stimulation and thermal sensation. Both the Naïve and STZ+10kHzSCS groups had significantly smaller RFs than the STZ Control and STZ+ShamSCS groups. In response to a 1 Hz vibration frequency, Naïve and STZ+10kHzSCS groups had higher peak power spectral density (PSD) of DH firing than STZ+ShamSCS, and PSD for the STZ+10kHzSCS group was statistically higher than for the STZ Control group. Response of DH neurons showed no significant differences between groups during the thermal stimulation, but the post-stimulus firing rates for the STZ+10kHzSCS group were significantly higher compared to STZ Control and STZ+ShamSCS. These data suggest that 10 kHz SCS amplified the DH response to large fiber (vibration) and small fiber (warm) signals and normalized the receptive field of the paw (acuity) in PDN rats. In diabetes, where peripheral sensory compromise from chronic hyperglycemia can lead to loss of protective sensation, such ‘tuned amplification’ as provided by 10 kHz SCS in the central nervous system may compensate for the reduced functionality seen in the peripheral sensory systems. Disclosure K.Lee: Employee; Nevro Corp. D.Lee: Employee; Nevro Corp. D.Wang: Employee; Nevro Corp. Z.B.Kagan: Employee; Nevro Corp. K.Bradley: Employee; Nevro Corp.

482-P: Behavioral Assessment of 10kHz Spinal Cord Stimulation–Mediated Pain Relief in a Rat Model of Painful Diabetic Neuropathy

Painful diabetic neuropathy (PDN) is a serious complication of diabetes and occurs in approximately 20% of diabetic patients. In a recent RCT, 10kHz spinal cord stimulation (10kHz SCS) was shown to be a promising therapy for PDN, in both pain reduction and possible neurological improvements (Petersen et al 2021). In this study, we aimed to observe the behavioral effects of 10kHz SCS on PDN using streptozotocin (STZ)-induced diabetic rats. We established four testing groups: Naïve controls, STZ controls, STZ+Sham SCS, and STZ+10kHz SCS. A single effective dose (60 mg/kg) of the cytotoxic agent STZ caused the rats to become hyperglycemic within 72 hours and lasted for several weeks. These STZ-injected rats also showed a significant and continuous reduction in body weight in comparison to Naïve control rats. Using the von Frey test for mechanical allodynia, we observed that 14 days of 24h/day low-intensity (30% of motor threshold) 10kHz SCS significantly elevated the mechanical pain withdrawal thresholds compared to STZ controls or STZ+Sham SCS animals, reaching statistical equivalence to Naïve controls. In a thermal preference assay, all animals exhibited a training effect towards less activity and more preference to the room temperature side vs the non-painful heat side of a two-temperature chamber over the several-week protocol. We found that STZ-injected animals demonstrated significantly less exploration activity in the 10min testing periods compared to Naïve controls but neither the thermal preference nor the total distance they traveled were restored after 14 days of 10kHz SCS. Our results demonstrate that 10 kHz SCS resulted in behavioral outcomes reflective of the mechanical pain reduction in the PDN rodent model. These findings provide supporting evidence for the recent RCT data and underscore the conclusion that 10kHz SCS can effectively treat patients with refractory PDN. Disclosure D.Wang: Employee; Nevro Corp. D.Lee: Employee; Nevro Corp. K.Lee: Employee; Nevro Corp. Z.B.Kagan: Employee; Nevro Corp. K.Bradley: Employee; Nevro Corp.

Spinal Cord Stimulation Alleviates Pain Hypersensitivity by Attenuating Neuroinflammation in a Model of Painful Diabetic Neuropathy.

Painful diabetic neuropathy (PDN) is an intractable chronic pain condition affecting a growing number of adults in China. Spinal cord stimulation (SCS) has been employed in the treatment of PDN for several decades. However, the efficacy and underlying mechanisms of SCS are still inconclusive. In this study, we adopted an implantable pulse generator to deliver electrical stimulation (50 Hz, 200 us pulse width, 12 hours/day in 5 weeks) via a quadripolar electrode in the lumbar epidural space to treat pain hypersensitivity in the rat model of PDN. Electronic von Frey and Hargreaves tests were used to measure the responses to mechanical and heat stimuli, respectively. Quantitative PCR, western blotting, and enzyme-linked immunosorbent assay (ELISA) were adopted to explore the changes in neuroinflammation after SCS. SCS alleviated mechanical allodynia and heat hyperalgesia over a period of 3 weeks in diabetic rats. SCS completely suppressed neuropathy-induced Tlr4 and NFκB p65 elevation, resulting in the reduction of pain-promoting Il1β, Il6, and Tnfα proteins in the spinal cord dorsal horn. SCS may alleviate diabetic neuropathy-induced pain hypersensitivity via attenuating neuroinflammation in the spinal cord dorsal horn.

Synergism between metformin and analgesics/vitamin B12 in a model of painful diabetic neuropathy

Metformin (a widely used antidiabetic drug) has demonstrated efficacy in models of painful diabetic neuropathy (PDN), as well as certain clinical efficacy in relieving/preventing PDN. This study aimed to determine the type of interaction between metformin and duloxetine/oxycodone/eslicarbazepine acetate [ESL]/vitamin B12 in relieving diabetic pain hypersensitivity. Antihyperalgesic efficacy was determined using a Von Frey apparatus in mice with streptozotocin-induced PDN. We examined metformin’s efficacy following oral (acute and prolonged 7-day treatment) and local (spinal and peripheral) application. The examined analgesics were administered in a single oral dose, whereas vitamin B12 was intraperitoneally administered for 7 days. In combination experiments, metformin (prolonged treatment) and analgesics/vitamin B12 were co-administered in fixed-dose fractions of their ED50 values and the type of interaction was determined using isobolographic analysis. Metformin produced dose-dependent antihyperalgesic effects in diabetic mice after oral (acute and prolonged 7-day treatment) and local spinal/peripheral application. Two-drug metformin combinations with analgesics/vitamin B12 also dose-dependently reduced mechanical hyperalgesia. The isobolographic analysis revealed that metformin synergises with analgesics/vitamin B12, with a 6–7 fold dose reduction of both drugs in the examined combinations. In conclusion, metformin reduces hyperalgesia in diabetic animals, most likely by acting at the spinal and peripheral level. Additionally, it synergizes with duloxetine/oxycodone/ESL/vitamin B12 in reducing hyperalgesia. Metformin co-treatment may increase analgesic efficacy and enable the use of lower (and potentially safer) analgesic doses for treating PDN. Combined metformin-vitamin B12 use may provide more effective pain relief and mitigate metformin-induced vitamin B12 deficiency.

Low-Intensity 10 kHz Spinal Cord Stimulation Reduces Behavioral and Neural Hypersensitivity in a Rat Model of Painful Diabetic Neuropathy.

BackgroundLow-intensity 10 kHz spinal cord stimulation (SCS) has been shown to provide pain relief in patients with chronic pain resulting from diabetic peripheral neuropathy (DPN). However to date, there have been no studies of 10 kHz SCS in animal models of diabetes. We aimed to establish correlative data of the effects of this therapy on behavioral and electrophysiological measures in a DPN model.MethodsTwenty-five adult male Sprague-Dawley rats were injected once intraperitoneally with 60 mg/kg streptozotocin (STZ) to induce diabetes over a subsequent 4 w period, while 4 naïve control animals were not injected. After approximately 21 d, 12 of STZ-injected rats had mini epidural SCS leads implanted: 8 received continuous low intensity (~30% motor threshold) 10 kHz SCS, and 4 received sham SCS (0 mA) over 7 d. Behavioral assays (von Frey filament probe of hindpaw) were measured in 18 animals and in vivo dorsal horn electrophysiological studies (receptive field; response to afferent brush, von Frey probe, pinch) were performed in 17 animals.ResultsAcross behavioral assays of mechanical allodynia and electrophysiological assays of receptive field size and mechanosensitivity, diabetic animals stimulated with 10 kHz SCS showed statistically significant improvements compared to sham SCS.ConclusionLow-intensity 10 kHz SCS produced several measures associated with a reduction of pain in diabetic rodent models that may help explain the clinical benefits of 10 kHz SCS in patients with painful diabetic neuropathy.

Neuron-Keratinocyte Communication in the Epidermis in Painful Diabetic Neuropathy

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration, which can progress to complete loss of cutaneous innervation and is accompanied by neuropathic pain. Uncovering the mechanisms underlying axonal degeneration in PDN remains a major challenge to finding effective and disease-modifying therapies. Sensory nerve afferents normally extend into the epidermis in close juxtaposition to keratinocytes but degenerate in diabetic skin. Our aim is to identify the changes in gene expression profiles and the interactions between dorsal root ganglion (DRG) neurons and keratinocytes to explore the mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts axonal degeneration underlying neuropathic pain in PDN. We used a mouse model of PDN where mice were fed a regular diet (RD, 11% fat) or a high-fat diet (HFD, 42% fat) for 10 weeks during which these mice develop glucose intolerance, mechanical allodynia, small fiber neuropathy. Using a single-cell RNA (scRNA-seq) sequencing approach we captured DRG and keratinocytes gene expression profiles and generated interactome maps. scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes between RD and HFD from the DRG. We were able to identify several clusters of immune cells and keratinocytes at different stages of differentiation. scRNA-seq results were validated using RNAscope on DRG and skin frozen sections. Moreover, we generated interactome maps between DRG neurons and the peripheral cells to highlight ligand-receptor interactions and we looked to identify genes that were differentially expressed in these interactions. Taken together our data highlights the importance of studying neurons in conjunction with the cells in the tissues with which they interact to identify ligand-receptor interactions that may lead to the identification of neuron signaling in a chronic pain state such as PDN. Grant support from 1R01AR77691-01. Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration, which can progress to complete loss of cutaneous innervation and is accompanied by neuropathic pain. Uncovering the mechanisms underlying axonal degeneration in PDN remains a major challenge to finding effective and disease-modifying therapies. Sensory nerve afferents normally extend into the epidermis in close juxtaposition to keratinocytes but degenerate in diabetic skin. Our aim is to identify the changes in gene expression profiles and the interactions between dorsal root ganglion (DRG) neurons and keratinocytes to explore the mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts axonal degeneration underlying neuropathic pain in PDN. We used a mouse model of PDN where mice were fed a regular diet (RD, 11% fat) or a high-fat diet (HFD, 42% fat) for 10 weeks during which these mice develop glucose intolerance, mechanical allodynia, small fiber neuropathy. Using a single-cell RNA (scRNA-seq) sequencing approach we captured DRG and keratinocytes gene expression profiles and generated interactome maps. scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes between RD and HFD from the DRG. We were able to identify several clusters of immune cells and keratinocytes at different stages of differentiation. scRNA-seq results were validated using RNAscope on DRG and skin frozen sections. Moreover, we generated interactome maps between DRG neurons and the peripheral cells to highlight ligand-receptor interactions and we looked to identify genes that were differentially expressed in these interactions. Taken together our data highlights the importance of studying neurons in conjunction with the cells in the tissues with which they interact to identify ligand-receptor interactions that may lead to the identification of neuron signaling in a chronic pain state such as PDN. Grant support from 1R01AR77691-01.

Antinociceptive and Antiallodynic Activity of Some 3-(3-Methylthiophen-2-yl)pyrrolidine-2,5-dione Derivatives in Mouse Models of Tonic and Neuropathic Pain

Antiseizure drugs (ASDs) are commonly used to treat a wide range of nonepileptic conditions, including pain. In this context, the analgesic effect of four pyrrolidine-2,5-dione derivatives (compounds 3, 4, 6, and 9), with previously confirmed anticonvulsant and preliminary antinociceptive activity, was assessed in established pain models. Consequently, antinociceptive activity was examined in a mouse model of tonic pain (the formalin test). In turn, antiallodynic and antihyperalgesic activity were examined in the oxaliplatin-induced model of peripheral neuropathy as well as in the streptozotocin-induced model of painful diabetic neuropathy in mice. In order to assess potential sedative properties (drug safety evaluation), the influence on locomotor activity was also investigated. As a result, three compounds, namely 3, 6, and 9, demonstrated a significant antinociceptive effect in the formalin-induced model of tonic pain. Furthermore, these substances also revealed antiallodynic properties in the model of oxaliplatin-induced peripheral neuropathy, while compound 3 attenuated tactile allodynia in the model of diabetic streptozotocin-induced peripheral neuropathy. Apart from favorable analgesic properties, the most active compound 3 did not induce any sedative effects at the active dose of 30 mg/kg after intraperitoneal (i.p.) injection.

Poly (ADP-ribose) polymerase 1-mediated defective mitophagy contributes to painful diabetic neuropathy in the db/db model.

Studies have shown that poly (ADP-ribose) polymerase 1 (PARP1) is involved in the pathological process of diabetes. Mitophagy is widely acknowledged to be a key regulatory process in maintaining reactive oxygen species homeostasis via lysosome degradation of damaged mitochondria. However, the regulatory role of PARP1 in mitophagy-related mitochondrial oxidative injury and progression of painful diabetic neuropathy (PDN) is unclear. In this study, we studied the in vitro and in vivo mechanisms of PARP1-mediated mitophagy blockade in a leptin gene-mutation (db/db) mouse model of PDN. Db/db mice models of PDN were established by assessing the sciatic nerve conduction velocity (SNCV), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). The results showed that PARP1 activity and mitochondrial injury of dorsal root ganglion (DRG) neurons were increased, and mitophagy was impaired in PDN mice. PARP1 was found to mediate the impairment of mitophagy in DRG neurons isolated from PDN mice. PARP1 inhibitors (PJ34 or AG14361) attenuated diabetes-induced peripheral nerve hyperalgesia, restored DRG neuron mitophagy function and decreased mitochondrial oxidative injury. Mitophagy impairment induced by lysosome deacidificant (DC661) aggravated diabetes-induced DRG neuron mitochondrial oxidative stress and injury. Taken together, our data revealed that PARP1-induced defective mitophagy of DRG neurons is a key mechanism in diabetes-induced peripheral neuropathic injury. Inhibition of PARP1 and restoration of mitophagy function are potential therapeutic targets for PDN.

Neuroprotective role of Tinospora cordifolia extract in streptozotocin induced neuropathic pain

Diabetic Neuropathy (DN) is one of the prevailing micro vascular complications of diabetes which can be characterized by neuropathic pain. Streptozotocin (STZ) induced diabetes in the rat has been increasingly used as a model of painful diabetic neuropathy. STZ injection leads to neurotoxicity of peripheral nerves that leads to development of Peripheral Diabetic Neuropathy in rat model. The present study was aimed at exploring the protective role of Tinospora cordifolia extract in STZ induced neurotoxicity and evaluating mechanisms responsible for attenuating neuropathic pain. Neuropathic pain markers like hyperalgesia, allodynia and motor deficits were assessed before STZ injection and after the treatment with 250 mg/kg and 500 mg/kg dose of Tinospora cordifolia. Oxidative stress markers, NGF expression in sciatic nerve were observed after seven weeks treatment. Our results demonstrated that seven weeks treatment with Tinospora cordifolia leaf extract significantly relieved thermal hyperalgesia and allodynia by increasing the antioxidant enzyme levels, decreasing the lipid peroxidation and by increasing the Nerve growth factor (NGF) expression in diabetic rat sciatic nerves. Our findings highlighted the beneficial effects of oral administration of Tinospora cordifolia extract in attenuating diabetic neuropathic pain, possibly through a strong antioxidant activity and by inducing NGF m RNA in sciatic nerves.

Upregulation of TLR9 may contribute to activation of microglia and painful diabetic neuropathy via the p38 MAPK pathway in rats.

Painful diabetic neuropathy is a common chronic complication of diabetes, and the underlying mechanism remains largely elusive. A rat model of painful diabetic neuropathy was established via streptozotocin (STZ) injection and assessed as increased heat and mechanical hypersensitivity. An upregulation of TLR9 was observed in the spinal cords of rats injected with STZ and rat microglia (primary microglia and immortalized microglia HAPI) treated with high glucose. To investigate the role of TLR9 in high glucose-induced microglia activation, short hairpin RNAs targeting TLR9 were used in vitro to knock down TLR9 in HAPI cells. TLR9 interference suppressed the high glucose-induced expression and secretion of inflammatory cytokines (TNF-α, IL-1β, and IL-6), IBA-1 expression and the chemotaxis of HAPI microglia. Similar results were obtained when HAPI microglia were incubated with a p38 inhibitor (SB203580). P38 and ERK were downstream of TLR9 because TLR9 ablation markedly inhibited the phosphorylation of p38 and ERK. TLR9 was also knocked down in vivo via the injection of shTLR9 lentiviral vector into the rat spinal cord. Relief of STZ-induced heat and mechanical hypersensitivity was observed in rats with TLR9 interference, and TLR9 knockdown prevented STZ-induced inflammatory cytokine secretion and microglial and MAPK signaling activation. Our study revealed the participation of TLR9 in microglial activation and diabetes-induced hyperalgesia likely via the MAPK pathway. The targeting of TLR9 may be an effective strategy for the treatment of painful diabetic neuropathy.

Loganin Ameliorates Painful Diabetic Neuropathy by Modulating Oxidative Stress, Inflammation and Insulin Sensitivity in Streptozotocin-Nicotinamide-Induced Diabetic Rats.

Loganin is an iridoid glycoside with antioxidant, anti-inflammatory, glucose-lowering activities which may address the pathological mechanisms of painful diabetic neuropathy (PDN) related to inflammation, oxidative stress, and hyperglycemia. This study investigated the underlying mechanisms of action of loganin on PDN. The in vivo model of PDN was established by streptozotocin-nicotinamide (STZ-NA) induction in Sprague Dawley (SD) rats. Subsequently, loganin (5 mg/kg) was administered by daily intraperitoneal injection. High-glucose stimulated human SH-SY5Y cells co-incubated with loganin were used to mimic the in vitro model of PDN. Loganin improved PDN rats’ associated pain behaviors (allodynia and hyperalgesia), insulin resistance index (HOMA-IR), and serum levels of superoxide dismutase (SOD), catalase and glutathione. Loganin also reduced pain-associated channel protein CaV3.2 and calcitonin gene-related peptide (CGRP) in the surficial spinal dorsal horn of PDN rats. Loganin inhibited oxidative stress and NF-κB activation and decreased the levels of mRNA and protein of proinflammatory factors IL-1β and TNF-α. Moreover, loganin attenuated insulin resistance by modulating the JNK-IRS-1 (insulin receptor substrate-1)-Akt-GSK3β signaling pathway in PDN rats. These results suggested that loganin improved PDN-mediated pain behaviors by inhibiting oxidative stress-provoked inflammation in the spinal cord, resulting in improved neuropathic pain.

Mitochondrial calcium uniporter deletion prevents painful diabetic neuropathy by restoring mitochondrial morphology and dynamics.

Painful diabetic neuropathy (PDN) is an intractable complication affecting 25% of diabetic patients. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, resulting in calcium overload, axonal degeneration, and loss of cutaneous innervation. The molecular pathways underlying these effects are unknown. Using high-throughput and deep-proteome profiling, we found that mitochondrial fission proteins were elevated in DRG neurons from mice with PDN induced by a high-fat diet (HFD). In vivo calcium imaging revealed increased calcium signaling in DRG nociceptors from mice with PDN. Furthermore, using electron microscopy, we showed that mitochondria in DRG nociceptors had fragmented morphology as early as 2 weeks after starting HFD, preceding the onset of mechanical allodynia and small-fiber degeneration. Moreover, preventing calcium entry into the mitochondria, by selectively deleting the mitochondrial calcium uniporter from these neurons, restored normal mitochondrial morphology, prevented axonal degeneration, and reversed mechanical allodynia in the HFD mouse model of PDN. These studies suggest a molecular cascade linking neuropathic pain to axonal degeneration in PDN. In particular, nociceptor hyperexcitability and the associated increased intracellular calcium concentrations could lead to excessive calcium entry into mitochondria mediated by the mitochondrial calcium uniporter, resulting in increased calcium-dependent mitochondrial fission and ultimately contributing to small-fiber degeneration and neuropathic pain in PDN. Hence, we propose that targeting calcium entry into nociceptor mitochondria may represent a promising effective and disease-modifying therapeutic approach for this currently intractable and widespread affliction. Moreover, these results are likely to inform studies of other neurodegenerative disease involving similar underlying events.