Nerve injury–induced neuropathic pain is difficult to treat. In this study, we used exosomes derived from human umbilical cord mesenchymal stem cell (UCMSC) as a cell-free therapy for nerve injury–induced pain in rats. Isolated UCMSC exosomes range in size from 30 to 160 nm and contain CD63, HSP60, and CD81 exosome markers. After L5/6 spinal nerve ligation surgery, single intrathecal injection of exosomes reversed nerve ligation–induced mechanical and thermal hypersensitivities of right hindpaw of rats at initial and well-developed pain stages. Moreover, continuous intrathecal infusion of exosomes achieved excellent preventive and reversal effects for nerve ligation–induced pain. In immunofluorescent study, lots of Exo-green-labelled exosomes could be found majorly in the ipsilateral L5 spinal dorsal horn, dorsal root ganglion, and peripheral axons, suggesting the homing ability of UCMSC exosomes. They also appeared in the central terminals or cell bodies of IB4⁺, CGRP⁺, and NF200⁺ sensory neurons. In addition, exosome treatment suppressed nerve ligation–induced upregulation of c-Fos, CNPase, GFAP, and Iba1. All these data suggest that the analgesic effects of exosomes may involve their actions on neuron and glial cells. Exosomes also inhibited the level of TNF-α and IL-1β, while enhanced the level of IL-10, brain-derived neurotrophic factor, and glial cell line–derived neurotrophic factor in the ipsilateral L5/6 dorsal root ganglion of nerve-ligated rats, indicating anti-inflammatory and proneurotrophic abilities. Protein analysis revealed the content of vascular endothelial growth factor C, angiopoietin-2, and fibroblast growth factor-2 in the exosomes. In summary, intrathecal infusion of exosomes from UCMSCs may be considered as a novel therapeutic approach for nerve injury–induced pain.

Central and peripheral nerve injuries can lead to permanent paralysis and organ dysfunction. In recent years, many cell and exosome implantation techniques have been developed in an attempt to restore function after nerve injury with promising but generally unsatisfactory clinical results. Clinical outcome may be enhanced by bio-scaffolds specifically fabricated to provide the appropriate three-dimensional (3D) conduit, growth-permissive substrate, and trophic factor support required for cell survival and regeneration. In rodents, these scaffolds have been shown to promote axonal regrowth and restore limb motor function following experimental spinal cord or sciatic nerve injury. Combining the appropriate cell/exosome and scaffold type may thus achieve tissue repair and regeneration with safety and efficacy sufficient for routine clinical application. In this review, we describe the efficacies of bio-scaffolds composed of various natural polysaccharides (alginate, chitin, chitosan, and hyaluronic acid), protein polymers (gelatin, collagen, silk fibroin, fibrin, and keratin), and self-assembling peptides for repair of nerve injury. In addition, we review the capacities of these constructs for supporting in vitro cell-adhesion, mechano-transduction, proliferation, and differentiation as well as the in vivo properties critical for a successful clinical outcome, including controlled degradation and re-absorption. Finally, we describe recent advances in 3D bio-printing for nerve regeneration.

Background: Systemic administration of gabapentin was shown previously to attenuate mechanical allodynia in a rat model of postoperative pain. Because intrathecal administration of gabapentin is effective in other hypersensitivity states, the authors tested its effect in the postoperative model, its interaction with another antiallodynic agent (clonidine), and a possible mechanism of gabapentin action (entry into sites of action via an L-amino acid transporter). Methods: Male Sprague-Dawley rats were anesthetized with halothane, and an incision of the plantaris muscle of right hind paw induced punctate mechanical allodynia. Withdrawal threshold to von Frey filament application near the incision site was determined before and 2 h after surgery. Then, an intrathecal injection was performed and thresholds were determined every 30 min for 3 h thereafter. Results: Paw incision induced a mechanical hypersensitivity (mechanical threshold > 25 g before incision and < 5 g after). Intrathecal gabapentin dose-dependently (10-100 microg) reduced mechanical allodynia. Intrathecal injection of an inhibitor of L-amino acid transporters or a competitor for this transporter, L-leucine, did not reverse the intrathecal effect of gabapentin. The ED50 of intrathecal gabapentin, clonidine, and their combination were 51, 31, and 9 microg, respectively, and isobolographic analysis showed synergy between gabapentin and clonidine. Conclusions: Intrathecal gabapentin is effective against tactile allodynia that occurs after paw incision, and interacts synergistically with clonidine. Unlike results in vitro, gabapentin does not obligatorily need to enter cells via the L-amino acid transporter mechanism to achieve its effects in vivo.

Background: Spinal cord injury (SCI) is a debilitating illness in humans that causes permanent loss of movement or sensation. To treat SCI, exosomes, with their unique benefits, can circumvent limitations through direct stem cell transplantation. Therefore, we utilized Gelfoam encapsulated with exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-EX) in a rat SCI model. Methods: SCI model was established through hemisection surgery in T9 spinal cord of female Sprague-Dawley rats. Exosome-loaded Gelfoam was implanted into the lesion site. An in vivo uptake assay using labeled exosomes was conducted on day 3 post-implantation. Locomotor functions and gait analyses were assessed using Basso-Beattie-Bresnahan (BBB) locomotor rating scale and DigiGait Imaging System from weeks 1 to 8. Nociceptive responses were evaluated through von Frey filament and noxious radiant heat tests. The therapeutic effects and potential mechanisms were analyzed using Western blotting and immunofluorescence staining at week 8 post-SCI. Results: For the in vivo exosome uptake assay, we observed the uptake of labeled exosomes by NeuN⁺, Iba1⁺, GFAP⁺, and OLIG2⁺ cells around the injured area. Exosome treatment consistently increased the BBB score from 1 to 8 weeks compared with the Gelfoam-saline and SCI control groups. Additionally, exosome treatment significantly improved gait abnormalities including right-to-left hind paw contact area ratio, stance/stride, stride length, stride frequency, and swing duration, validating motor function recovery. Immunostaining and Western blotting revealed high expression of NF200, MBP, GAP43, synaptophysin, and PSD95 in exosome treatment group, indicating the promotion of nerve regeneration, remyelination, and synapse formation. Interestingly, exosome treatment reduced SCI-induced upregulation of GFAP and CSPG. Furthermore, levels of Bax, p75NTR, Iba1, and iNOS were reduced around the injured area, suggesting anti-inflammatory and anti-apoptotic effects. Moreover, exosome treatment alleviated SCI-induced pain behaviors and reduced pain-associated proteins (BDNF, TRPV1, and Cav3.2). Exosomal miRNA analysis revealed several promising therapeutic miRNAs. The cell culture study also confirmed the neurotrophic effect of HucMSCs-EX. Conclusion: Implantation of HucMSCs-EX-encapsulated Gelfoam improves SCI-induced motor dysfunction and neuropathic pain, possibly through its capabilities in nerve regeneration, remyelination, anti-inflammation, and anti-apoptosis. Overall, exosomes could serve as a promising therapeutic alternative for SCI treatment.

Purpose: Nerve injury-induced pain is difficult to treat. In this study, we developed an alginate scaffold with human umbilical cord mesenchymal stem cell exosomes (EX-SC) to treat nerve injury-induced pain. Materials and Methods: The scaffold was prepared and characterized for its physical traits and biocompatibility. In vitro studies of PC12 and HEK293 cells were used to evaluate the neuroprotective and neurotrophic effects of exosomes. Right L5/6 spinal nerve ligation (SNL) was performed in Sprague-Dawley rats to induce mechanical allodynia and thermal hyperalgesia, evaluated by von Frey hair and radiant heat tests. The EX-SC was wrapped around ligated L5/6 spinal nerves for treatment. Western blotting and immunofluorescence staining were used to evaluate neuron/glial activation, cytokines and neurotrophic factor of affected dorsal root ganglion (DRG). Results: In cell culture assay, the exosomes induce neurite outgrowth of PC12 cells and protect PC12 and HEK293 cells against formaldehyde acid treatment. On post-ligation day 21, rats receiving EX-SC had significantly higher median (interquartile range) withdrawal threshold and latency [14.1 (13.7– 15.5) g, 14.2 (13.7– 15.3) s] than saline-SC-treated rats [2.1 (1.7– 3.0) g, 2.0 (1.8– 2.4) s, P=0.02 and 0.002]. The EX-SC also attenuated SNL-induced up-regulation of c-Fos, GFAP, Iba1, TNF-α and IL-1β, while enhancing the level of IL-10 and GDNF, in the ipsilateral L5/6 DRG. After implantation for 21 days, the EX-SC enhanced the expression of myelin basic protein and IL-10 in injured L5/6 axons. Conclusion: We demonstrate the EX-SC possesses antinociceptive, anti-inflammation and pro-neurotrophic effects in the SNL pain model. It could be a promising therapeutic alternative for nerve injury-induced pain.

The central amygdala (CeA) nucleus, a subcortical structure composed of mostly GABA-releasing (GABAergic) neurons, controls fear expression via projections to downstream targets in the hypothalamus and brainstem. The CeA consists of the lateral (CeL) and medial (CeM) subdivisions. The CeL strongly gates information transfer to the CeM, the main output station of the amygdala, but little is known about the functional organization of local circuits in this region. Using cluster analysis, we identified two major electrophysiologically distinct CeL neuron classes in mouse amygdala slices, the early-spiking (ES) and late-spiking (LS) neurons. These two classes displayed distinct autaptic transmission. Compared with LS neurons, ES neurons had strong and depressing autapses, which enhanced spike-timing precision. With multiple patch-clamp recordings, we found that CeL neurons made chemical, but not electrical, synapses. Analysis of individual connections revealed cannabinoid type 1 receptor-mediated suppression of the ES, but not of the LS cell output synapse. More interestingly, the efficacy of the ES→LS or LS→ES synapse was ∼2-fold greater than that of the LS→LS or ES→ES synapse. When tested at 20 Hz, synapses between different neurons, but not within the same class, were markedly depressing and were more powerful to sculpt activity of postsynaptic neurons. Moreover, neurons of different classes also form synapses with higher degree of connectivity. We demonstrate that ES and LS neurons represent two functionally distinct cell classes in the CeL and interactions between presynaptic and postsynaptic neurons dictate synaptic properties between neurons.SIGNIFICANCE STATEMENTThe central lateral amygdala (CeL) is a key node in fear circuits, but the functional organization of local circuits in this region is largely unknown. The CeL consists of mostly GABAergic inhibitory neurons with different functional and molecular features. Here, we report that the presynaptic cell class determines functional properties of autapses and cannabinoid-mediated modulation of synaptic transmission between neurons, whereas presynaptic versus postsynaptic cell classes dictate the connectivity, efficacy, and dynamics of GABAergic synapses between any two neurons. The wiring specificity and synaptic diversity have a great impact on neuronal output in amygdala inhibitory networks. Such synaptic organizing principles advance our understanding of the significance of physiologically defined neuronal phenotypes in amygdala inhibitory networks.