Dorsal Root Ganglion Explants Research Articles

Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervation.

Low back pain, knee osteoarthritis, and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Axonal dieback induced by capsaicin (Caps) denervation has been FDA-approved to treat painful neuropathies and knee osteoarthritis but with short-term efficacy and discomfort. Herein, we propose to evaluate pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as axonal dieback compounds for denervation with potential to alleviate pain. Previous literature suggests Pyr, Vcr, and Imy can cause undesired axonal degeneration, but no previous work has evaluated axonal dieback and cytotoxicity on adult rat dorsal root ganglia (DRG) explants. Thus, we performed axonal dieback screening using adult rat DRG explants in vitro with Caps as a positive control and assessed cytotoxicity. Imy inhibited axonal outgrowth and slowed axonal dieback, while Pyr and Vcr at high concentrations produced significant reduction in axon length and robust axonal dieback within three days. DRGs treated with Caps, Vcr, or Imy had increased DRG cytotoxicity compared to matched controls, but overall cytotoxicity was minimal and at least 88% lower compared to lysed DRGs. Pyr did not lead to any DRG cytotoxicity. Further, neither Pyr nor Vcr triggered intervertebral disc cell death or affected cellular metabolic activity after three days of incubation in vitro. Overall, our findings suggest Pyr and Vcr are not toxic to DRGs and intervertebral disc cells, and there is potential for repurposing these compounds for axonal dieback compounds to cause local denervation and alleviate pain.

Strain-induced bands of Büngner formation promotes axon growth in 3D tissue-engineered constructs

Treatment of peripheral nerve lesions remains a major challenge due to poor functional recovery; hence, ongoing research efforts strive to enhance peripheral nerve repair. In this study, we aimed to establish three-dimensional tissue-engineered bands of Büngner constructs by subjecting Schwann cells (SCs) embedded in fibrin hydrogels to mechanical stimulation. We show for the first time that the application of strain induces (i) longitudinal alignment of SCs resembling bands of Büngner, and (ii) the expression of a pronounced repair SC phenotype as evidenced by upregulation of BDNF, NGF, and p75NTR. Furthermore, we show that mechanically aligned SCs provide physical guidance for migrating axons over several millimeters in vitro in a co-culture model with rat dorsal root ganglion explants. Consequently, these constructs hold great therapeutic potential for transplantation into patients and might also provide a physiologically relevant in vitro peripheral nerve model for drug screening or investigation of pathologic or regenerative processes.

Preliminary Insights into JWH182: a Synthetic Cannabinoid’s Neuroprotective Role Against Paclitaxel-Induced Neuronal Toxicity

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN), a frequently encountered consequence of neurotoxic chemotherapy, affects approximately 30 to 40% of patients. Taxanes are associated with an exceptionally high incidence of peripheral neuropathy, with Paclitaxel (PTX) accounting for approximately 70,8% of CIPN cases. Patient’s symptoms are severe, compelling oncologists to consider dosage reduction or even complete abandonment of the treatment plan. Several recent studies have shown the potential efficacy of either synthetic, endogenous or phytocannabinoids in alleviating CIPN symptoms. In case of in vitro studies, neural protection can be assessed by measuring the axon length of the cultured neurons. Therefore, our study aimed to investigate whether the synthetic cannabinoid JWH182 could emerge as a promising new candidate for managing Paclitaxel-induced peripheral neuropathy, in case of an in vitro neural model. METHODS: Primary neuronal cultures were obtained from mouse-derived dorsal root ganglia (DRG) explants. The harvested ganglia were subjected to a series of enzymatic and mechanical dissociating processes, followed by a density-gradient centrifugation to isolate neurons, which were then seeded in Poly-D-Lysine coated 6-well plates and incubated for 24h. Thereafter, cells were exposed to an equal parts solution of 20 uM JWH182 and 1uM PTX. As a means of comparison, the neurons from the positive control group were exclusively exposed to a 1uM PTX solution, whereas the negative control group was left untreated. Photographs of the neurons were taken before the treatment and subsequently at 6, 24, 48 and 72 hours, with a particular focus on observing changes in axon length and cell viability. RESULTS: Unlike our positive control group, which displayed noticeable adverse effects on axon length, the sample treated with both PTX and JWH182 presented more promising outcomes. To be precise, there was a less important reduction in axon length at all time points following drug administration. In the meantime, the negative control exhibited no changes, maintaining a typical morphology and rate of axonal growth. CONCLUSION: These findings suggest that the synthetic cannabinoid JWH182 confers a protective effect on DRG neurons treated with Paclitaxel. As a result, this compound holds the potential to emerge as a novel treatment option for managing Paclitaxel-induced peripheral neuropathy. This could lead to the alleviation of symptoms in oncological patients, thereby enhancing their quality of life and their overall disease prognosis. Ultimately, these initial results lay the foundation for subsequent in vitro and in vivo experiments, aimed at validating our hypothesis.

One-Pot Assembly of Drug-Eluting Silk Coatings with Applications for Nerve Regeneration.

Clinical use of polymeric scaffolds for tissue engineering often suffers from their inability to promote strong cellular interactions. Functionalization with biomolecules may improve outcomes; however, current functionalization approaches using covalent chemistry or physical adsorption can lead to loss of biomolecule bioactivity. Here, we demonstrate a novel bottom-up approach for enhancing the bioactivity of poly(l-lactic acid) electrospun scaffolds though interfacial coassembly of protein payloads with silk fibroin into nanothin coatings. In our approach, protein payloads are first added into an aqueous solution with Bombyx mori-derived silk fibroin. Phosphate anions are then added to trigger coassembly of the payload and silk fibroin, as well as noncovalent formation of a payload-silk fibroin coating at poly(l-lactic) acid fiber surfaces. Importantly, the coassembly process results in homogeneous distribution of protein payloads, with the loading quantity depending on payload concentration in solution and coating time. This coassembly process yields greater loading capacity than physical adsorption methods, and the payloads can be released over time in physiologically relevant conditions. We also demonstrate that the coating coassembly process can incorporate nerve growth factor and that coassembled coatings lead to significantly more neurite extension than loading via adsorption in a rat dorsal root ganglia explant culture model.

Type I collagen concentration affects neurite outgrowth of adult rat DRG explants by altering mechanical properties of hydrogels

Type I collagen is a predominant fibrous protein that makes up the extracellular matrix. Collagen enhances cell attachment and is commonly used in three-dimensional culture systems, to mimic the native extracellular environment, for primary sensory neurons such as dorsal root ganglia (DRG). However, the effects of collagen concentration on adult rat DRG neurite growth have not been assessed in a physiologically relevant, three-dimensional culture. This study focuses on the effects of type I collagen used in a methacrylated hyaluronic acid (MAHA)-laminin-collagen gel (triple gel) on primary adult rat DRG explants in vitro. DRGs were cultured in triple gels, and the neurite lengths and number of support cells were quantified. Increased collagen concentration significantly reduced neurite length but did not affect support cell counts. Mechanical properties, fiber diameter, diffusivity, and mesh size of the triple gels with varying collagen concentration were characterized to further understand the effects of type I collagen on hydrogel property that may affect adult rat DRG explants. Gel stiffness significantly increased as collagen concentration increased and is correlated to DRG neurite length. Collagen concentration also significantly impacted fiber diameter but there was no correlation with DRG neurite length. Increasing collagen concentration had no significant effect on mesh size and diffusivity of the hydrogel. These data suggest that increasing type I collagen minimizes adult rat DRG explant growth in vitro while raising gel stiffness. This knowledge can help develop more robust 3D culture platforms to study sensory neuron growth and design biomaterials for nerve regeneration applications.

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell-neuron cocultures.

Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1β, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

The Impact of the Molecular Weight of Degradation Products with Silicon from Porous Chitosan-Siloxane Hybrids on Neuronal Cell Behavior.

Silicon (Si) is an essential trace element in the human body and it exists in connective tissue as aqueous orthosilicic acid. Porous chitosan-3-glycidoxypropyltrimethoxysilane (GPTMS) hybrids can regenerate nerve tissue and recover sensor and motor functions. However, the structures and roles of the degradation products with Si extracted from the hybrids in nerve regeneration are not clear. In this study, we prepared porous chitosan-GPTMS hybrids with different amounts of GPTMS to amino groups of chitosan (chitosan:GPTMS = 1:0.5 and 1:1 molar ratios). The structures of the degradation products with Si from the hybrids were examined using time-of-flight mass spectrometry, and biological assessments were conducted in order to evaluate their potential use in the preparation of devices for nerve repair. Glial and motor cell lines and ex vivo explants of dorsal root ganglia were used in this study for evaluating their behavior in the presence of the different degradation products with Si. The structure of the degradation products with Si depended on the starting composition. The results showed that glial cell proliferation was lower in the medium with the higher-molecular-weight degradation products with Si. Moreover, motor cell line differentiation and the neurite outgrowth of dorsal root ganglion explants were improved with the lower-molecular-weight degradation products with Si. The results obtained could be useful for designing a new nerve regeneration scaffold including silicon components.

Aminosilane Functionalized Aligned Fiber PCL Scaffolds for Peripheral Nerve Repair.

Silane modification is a simple and cost-effective tool to modify existing biomaterials for tissue engineering applications. Aminosilane layer deposition has previously been shown to control NG108-15 neuronal cell and primary Schwann cell adhesion and differentiation by controlling deposition of ─NH2 groups at the submicron scale across the entirety of a surface by varying silane chain length. This is the first study toreport depositing 11-aminoundecyltriethoxysilane (CL11) onto aligned Polycaprolactone (PCL) scaffolds for peripheral nerve regeneration. Fibers are manufactured via electrospinning and characterized using water contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Confirmed modified fibers are investigated using in vitro cell culture of NG108-15 neuronal cells and primary Schwann cells to determine cell viability, cell differentiation, and phenotype. CL11-modified fibers significantly support NG108-15 neuronal cell and Schwann cell viability. NG108-15 neuronal cell differentiation maintains Schwann cell phenotype compared to unmodified PCL fiber scaffolds. 3D ex vivo culture of Dorsal root ganglion explants (DRGs) confirms further Schwann cell migration and longer neurite outgrowth from DRG explants cultured on CL11 fiber scaffolds compared to unmodified scaffolds. Thus, a reproducible and cost-effective tool is reported to modify biomaterials with functional amine groups that can significantly improve nerve guidance devices and enhance nerve regeneration.

In vitro myelination using explant culture of dorsal root ganglia: An efficient tool for analyzing peripheral nerve differentiation and disease modeling.

Peripheral nerves conducting motor and somatosensory signals in vertebrate consist of myelinated and unmyelinated axons. In vitro myelination culture, generated by co-culturing Schwann cells (SCs) and dorsal root ganglion (DRG) neurons, is an indispensable tool for modeling physiological and pathological conditions of the peripheral nervous system (PNS). This technique allows researchers to overexpress or downregulate molecules investigated in neurons or SCs to evaluate the effect of such molecules on myelination. In vitro myelination experiments are usually time-consuming and labor-intensive to perform. Here we report an optimized protocol for in vitro myelination using DRG explant culture. We found that our in vitro myelination using DRG explant (IVMDE) culture not only achieves myelination with higher efficiency than conventional in vitro myelination methods, but also can be used to observe Remak bundle and non-myelinating SCs, which were unrecognizable in conventional methods. Because of these characteristics, IVMDE may be useful in modeling PNS diseases, including Charcot Marie Tooth disease (CMT), in vitro. These results suggest that IVMDE may achieve a condition more similar to peripheral nerve myelination observed during physiological development.

Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons.

The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

Multimodular Bio-Inspired Organized Structures Guiding Long-Distance Axonal Regeneration.

Axonal bundles or axonal tracts have an aligned and unidirectional architecture present in many neural structures with different lengths. When peripheral nerve injury (PNI), spinal cord injury (SCI), traumatic brain injury (TBI), or neurodegenerative disease occur, the intricate architecture undergoes alterations leading to growth inhibition and loss of guidance through large distance. In order to overcome the limitations of long-distance axonal regeneration, here we combine a poly-L-lactide acid (PLA) fiber bundle in the common lumen of a sequence of hyaluronic acid (HA) conduits or modules and pre-cultured Schwann cells (SC) as cells supportive of axon extension. This multimodular preseeded conduit is then used to induce axon growth from a dorsal root ganglion (DRG) explant placed at one of its ends and left for 21 days to follow axon outgrowth. The multimodular conduit proved effective in promoting directed axon growth, and the results may thus be of interest for the regeneration of long tissue defects in the nervous system. Furthermore, the hybrid structure grown within the HA modules consisting in the PLA fibers and the SC can be extracted from the conduit and cultured independently. This “neural cord” proved to be viable outside its scaffold and opens the door to the generation of ex vivo living nerve in vitro for transplantation.

Magnetothermal Modulation of Calcium‐Dependent Nerve Growth

AbstractNerve injuries are common, and the available treatments including invasive surgeries do not guarantee complete regeneration of the injured nerves and restoration of function. Despite the ability of peripheral nerves to regenerate, the slow rate of axonal growth hampers the functional recovery. Development of new approaches to discover underlying mechanisms that may accelerate axonal growth is needed to overcome these limitations and augment available treatments of nerve injury. In addition to chemical factors, recent studies suggested the use of optogenetics and electrical stimulation to promote axonal growth. The underlying mechanisms of these approaches, however, require further investigation. Furthermore, their application relies on invasive hardware, which may not be compatible with injured nerves under significant mechanical deformation. Here, it is shown that thermal activation of a heat sensitive ion channel TRPV1 promotes axonal growth in a calcium‐dependent manner. By leveraging heat dissipation of magnetic nanoparticles in alternating magnetic fields, the calcium influx through TRPV1 channels endogenously expressed in dorsal root ganglion explants is triggered remotely. The accelerated axonal growth through elongation of neurofilaments and increased Schwann cell migration following magnetothermal stimulation is observed. These findings suggest future applications of magnetothermal modulation of axonal growth as a minimally invasive approach to accelerate nerve regeneration.

Advantages of Adult Mouse Dorsal Root Ganglia Explant Culture in Investigating Myelination in an Inherited Neuropathic Mice Model.

A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease.

Amine Functionalized Trimetallic Nitride Endohedral Fullerenes: A Class of Nanoparticle to Tackle Low Back/Leg Pain.

Low back pain is the most common health problem with a prevalence of over 80% worldwide and an estimated annual cost of $100 billion in the United States. Intervertebral disc degeneration accounts for a major cause of low back pain. However, there is still a lack of safe and effective treatment to tackle this devastating condition. In this study, we synthesized four functionalized trimetallic nitride endohedral metallofullerenes (carboxyl-f-Sc3N@C80, carboxyl-f-Gd3N@C80, amino-f-Sc3N@C80, and amino-f-Gd3N@C80) and characterized them with X-ray photoelectron spectroscopy, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and UV-vis. Via electron paramagnetic resonance, all four metallofullerene derivatives possessed dose-dependent radical scavenging capabilities (hydroxyl radicals and superoxide anions), with the most promising radical scavenging properties shown in the amine functionalized C80 metallofullerenes. Both amino-f-Sc3N@C80 and amino-f-Gd3N@C80 at 1 μM significantly reduced lipopolysaccharide induced reactive oxygen species production and mRNA expressions of pro-inflammatory mediators (inos, tnf-α, il-1, and cox-2) in macrophages without apparent cytotoxicity through regulating activity of p38 MAPK, p65, and nuclear translocation of NF-κB. Furthermore, in an established mouse model of lumbar radiculopathy, amino-f-Sc3N@C80 and amino-f-Gd3N@C80 effectively alleviated ipsilateral mechanical hyperalgesia for up to 2 weeks. In dorsal root ganglia explant culture, we also showed that amino-f-Sc3N@C80 and amino-f-Gd3N@C80 ameliorated TNF-α elicited neuroinflammation. In summary, we presented results for a potent radical scavenging, anti-inflammatory and analgesic nanoparticle, amino-functionalized eighty-carbon metallofullerenes in vitro and in vivo. Our study provides important assets for developing pleiotropic treatment strategies to tackle the inflammation, a significant pathological hallmark in the intervertebral disc degeneration and associated pain.

Nonwoven spidroin materials as scaffolds for ex vivo cultivation of aortic fragments and dorsal root ganglia

Recombinant spidroins (RS; the analogues of silk proteins of spider’s web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

The Integrated Stress Response and Eukaryotic Initiation Factor 2a Regulate Methylglyoxal-induced Pain Hypersensitivity

Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB. Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB.

March PC-FACS

March 2, 2022 Combined Chemotherapy and Anti-PD-1 Effect of Spiritual Care for Cancer Patients Mechanical Evaluation at EOL Effect of Identified Support on Older Populations Prescribing Antibiotics in Hospice Care Understanding Suicidal Self-Directed Violence Scopolamine Butylbromide to Prevent Death Rattle Rave Reviews Effects of Combined Treatment on Peripheral Neuropathy and Neuroinflammation Background: Chemotherapy-induced peripheral neuropathy (CIPN) is common and can affect patients’ long-term quality of life posttreatment.1–4 How does a program cell death protein-1 (PD-1) blockade impact CIPN development? Design and Participants: This study investigated the effects of monotherapy or co-therapy with the chemotherapeutic drug paclitaxel (associated with neurotoxicity, neuroinflammation, and CIPN) and immunotherapeutic drug anti-PD-1 (enhances the immune response against cancer cells) on pain hypersensitivity, neurite outgrowth, and immune cells in mice. Mice were randomized into control, paclitaxel, anti-PD-1, and paclitaxel+anti-PD-1 groups. Mice were injected intraperitoneally (days 0, 2, 4, 7, 9, and 11), using 5 mg/kg for paclitaxel (or vehicle control) and 12.5 mg/kg for anti-PD-1 (or IgG control). Behavioral (mechanical paw withdrawal thresholds [days -1, 20, and 27]) and tissue (collected days 28–30) analyses were performed. ANOVA and Fisher's exact test were used. Results: Mice (n = 6/group) treated with paclitaxel or co-therapy, but not with anti-PD-1 alone, exhibited increased hind paw mechanical sensitivity. Anti-PD-1 did not cause mechanical hypersensitivity or exacerbate paclitaxel-induced allodynia. However, both paclitaxel and anti-PD-1 reduced ex vivo neurite outgrowth of dorsal root ganglion (DRG) explants derived from treated mice, whereas only paclitaxel reduced the neurite outgrowth after direct in vitro treatment. Anti-PD-1 and co-therapy, but not paclitaxel, changed the T-cell profile in lymphoid organs and caused increased T-cell infiltration into the DRG. Paclitaxel and co-therapy increased DRG macrophage levels, and all treatments induced altered microglia marker expression in the dorsal horn of the spinal cord. Thus, both paclitaxel and anti-PD-1 induced neuroinflammatory and axonal degenerative effects; however, these were not cumulative in co-therapy. All P < 0.05. Commentary: Chemotherapy-induced peripheral neuropathy, observed in up to 80% of patients, can limit adherence to treatment and decrease quality of life.5,6 Although some treatments can be used for neuropathic pain, such as opioids or duloxetine, they are only partially effective. Immunotherapy is increasingly used for cancer treatment as monotherapy or in conjunction with chemotherapy. PD-1 and PD-L1 are expressed in the nervous system, including trigeminal ganglion, spinal dorsal horn, thalamic and cortical neurons,7 and blocking the PD-1 or PD-L1 pathway may unleash existing pain syndromes, induce allodynia,8–10 and suppress GABAergic neurotransmission in the spinal neurons.7 A recent systematic review found that combining chemotherapy and immune checkpoint inhibitors may increase the incidence of peripheral neuropathy in patients with solid tumors;11 however, the current study conducted in mice does not support that combination therapy increases CIPN. Bottom Line: Combination treatment with paclitaxel-anti-PD-1 did increase the severity of neuroinflammation and was associated with changes in spinal cord immunity with unclear significance. Reviewer: Regina M. Mackey, MD, Center for Palliative Medicine, Department of Internal Medicine, Mayo Clinic, Rochester, MN.

BET protein inhibition in macrophages enhances dorsal root ganglion neurite outgrowth in female mice.

Peripheral nerve regeneration is limited after injury, especially in humans, due to the large distance the axons have to grow in the limbs. This process is highly dependent on the expression of neuroinflammatory factors produced by macrophages and glial cells. Given the importance of the epigenetic BET proteins on inflammation, we aimed to ascertain if BET inhibition may have an effect on axonal outgrowth. For this purpose, we treated female mice with JQ1 or vehicle after sciatic nerve crush injury and analyzed target reinnervation. We also used dorsal root ganglion (DRG) culture explants to analyze the effects of direct BET inhibition or treatment with conditioned medium from BET‐inhibited macrophages. We observed that although JQ1 produced an enhancement of IL‐4, IL‐13, and GAP43 expression, it did not have an effect on sensory or motor reinnervation after crush injury in vivo. In contrast, JQ1 reduced neurite growth when interacting directly with DRG neurons ex vivo, whereas conditioned medium from JQ1‐treated macrophages promoted neurite outgrowth. Therefore, BET‐inhibited macrophages secrete pro‐regenerative factors that induce neurite outgrowth, and that may counteract the direct inhibition of BET proteins in neurons in vivo. Finally, we observed an activation of the STAT6 pathway in DRG explants treated with conditioned medium from JQ1‐treated macrophages. In conclusion, this study demonstrates that BET protein inhibition in macrophages provides a mechanism to enhance axonal outgrowth. However, specific targeting of BET proteins to macrophages will be needed to efficiently enhance functional recovery after nerve injury.