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Objectives: Muscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles., Materials and Methods: We collected sensory neurons from dorsal root ganglions of 14-day-old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups., Results: Our results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group., Conclusions: Cell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Background: Cell-based therapy may hold promise for treatment of chronic pain. Mesenchymal stem cells (MSCs) are readily available and robust, and their secretion of therapeutic peptides can be enhanced by genetically engineering. We explored the analgesic potential of transplanting bone marrow-derived MSCs that have been transduced with lentivectors. To optimize efficacy and safety, primary sensory neurons were targeted by MSC injection into the dorsal root ganglia (DRGs)., Results: MSCs were transduced using lentivectors to express enhanced green fluorescent protein (EGFP) or to co-express the analgesic peptide glial cell line-derived neurotrophic factor (GDNF) and EGFP by a viral 2A bicistronic transgene cassette. Engineered MSCs were injected into the 4(th) lumbar (L4) and L5 DRGs of adult allogeneic rats to evaluate survival in the DRGs. MSCs were detected by immunofluorescence staining up to 2-3 weeks after injection, distributed in the extracellular matrix space without disrupting satellite glial cell apposition to sensory neurons, suggesting well-tolerated integration of engrafted MSCs into DRG tissue. To examine their potential for inhibiting development of neuropathic pain, MSCs were injected into the L4 and L5 DRGs ipsilateral to a spinal nerve ligation injury. Animals injected with GDNF-engineered MSCs showed moderate but significant reduction in mechanical allodynia and hyperalgesia compared to controls implanted with MSCs expressing EGFP alone. We also observed diminished long-term survival of allografted MSCs at 3 weeks, and the development of a highly-proliferating population of MSCs in 12% of DRGs after transplantation., Conclusions: These data indicate that genetically modified MSCs secreting analgesic peptides could potentially be developed as a novel DRG-targeted cell therapy for treating neuropathic pain. However, further work is needed to address the challenges of MSC survival and excess proliferation, possibly with trials of autologous MSCs, evaluation of clonally selected populations of MSCs, and investigation of regulation of MSC proliferation.

Tissue engineered nerve grafts are considered as a promising alternative to autologous nerve grafts used for peripheral nerve repair. The differences between these two types of nerve grafts are mainly in the regenerative microenvironment established by them. To construct ideal tissue engineered nerve grafts, it is therefore required to develop a better way to introduce biochemical cues into a neural scaffold, as compared to single or combined use of support cells and growth factors. Here, we used a co-culture system of dorsal root ganglia and Schwann cells to create an in vitro formed nerve equivalent, which was introduced into a silk fibroin-based scaffold to furnish a tissue engineered nerve graft (TENG). At 4- and 12- weeks after the TENG was implanted to bridge a 10-mm-long sciatic nerve defect in rats, histological and functional assessments as well as Western blot analysis were performed to evaluate the influences of the TENG on peripheral nerve regeneration. We found that at an early stage of nerve regeneration, the TENG significantly accelerated axonal growth, and up-regulated expressions of N-cadherin and PMP22. Twelve weeks after nerve grafting, the TENG produced a further improved outcome of nerve regeneration and functional recovery, which was more close to that of the autologous nerve graft than that of the silk fibroin-based scaffold. The introduction of an in vitro cultured nerve equivalent into a scaffold might contribute to establishing a native-like microenvironment for nerve regeneration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Varicella-zoster virus (VZV) is a medically important human alphaherpesvirus. Investigating pathogenic mechanisms that contribute to VZV neurovirulence are made difficult by a marked host restriction. Our approach to investigating VZV neurotropism and neurovirulence has been to develop a mouse-human xenograft model in which human dorsal root ganglia (DRG) are maintained in severe compromised immunodeficient (SCID) mice. In this review, we will describe our key findings using this model in which we have demonstrated that VZV infection of SCID DRG xenograft results in rapid and efficient spread, enabled by satellite cell infection and polykaryon formation, which facilitates robust viral replication and release of infectious virus. In neurons that persist following this acute replicative phase, VZV genomes are present at low frequency with limited gene transcription and no protein synthesis, a state that resembles VZV latency in the natural human host. VZV glycoprotein I and interaction between glycoprotein I and glycoprotein E are critical for neurovirulence. Our work demonstrates that the DRG model can reveal characteristics about VZV replication and long-term persistence of latent VZV genomes in human neuronal tissues, in vivo, in an experimental system that may contribute to our knowledge of VZV neuropathogenesis.

β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid-β peptides that are present in plaques of Alzheimer's disease. BACE1 has been an attractive target for drug development. In cultured embryonic neurons, BACE1-cleaved N-terminal APP is further processed to generate a fragment that can trigger axonal degeneration, suggesting a vital role for BACE1 in axonal health. In addition, BACE1 cleaves neuregulin 1 type III, a protein critical for myelination of peripheral axons by Schwann cells during development. Here, we asked whether axonal degeneration or axonal regeneration in adult nerves might be affected by inhibition or elimination of BACE1. We report that BACE1 knock-out and wild-type nerves degenerated at a similar rate after axotomy and to a similar extent in the experimental neuropathies produced by administration of paclitaxel and acrylamide. These data indicate N-APP is not the sole culprit in axonal degeneration in adult nerves. Unexpectedly, however, we observed that BACE1 knock-out mice had markedly enhanced clearance of axonal and myelin debris from degenerated fibers, accelerated axonal regeneration, and earlier reinnervation of neuromuscular junctions, compared with littermate controls. These observations were reproduced in part by pharmacological inhibition of BACE1. These data suggest BACE1 inhibition as a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage.

Secondary lymphoid organs are dominant sites of T cell activation, although many T cells are subsequently retained within peripheral tissues. Currently, these nonlymphoid compartments are viewed as sites only of effector T cell function, without the involvement of renewed induction of immunity via the interactions with professional antigen-presenting cells. We describe a method of reactivation of herpes simplex virus to examine the stimulation of tissue-resident T cells during secondary challenge. The results revealed that memory CD8+ T cell responses can be initiated within peripheral tissues through a tripartite interaction that includes CD4+ T cells and recruited dendritic cells. These findings lend evidence for the existence of a sophisticated T cell response mechanism in extra-lymphoid tissues that can act to control localized infection.

Varicella-zoster virus (VZV) causes varicella, establishes latency in sensory ganglia, and reactivates as herpes zoster. Human dorsal root ganglia (DRGs) xenografts in immunodeficient mice provide a model for evaluating VZV neuropathogenesis. Our investigation of the role of glycoprotein I (gI), which is dispensable in vitro, examines the functions of a VZV gene product during infection of human neural cells in vivo. Whereas intact recombinant Oka (rOka) initiated a short replicative phase followed by persistence in DRGs, the gI deletion mutant, rOkaDeltagI, showed prolonged replication with no transition to persistence up to 70 days after infection. Only a few varicella-zoster nucleocapsids and cytoplasmic virions were observed in neurons, and the major VZV glycoprotein, gE, was retained in the rough endoplasmic reticulum in the absence of gI. VZV neurotropism was not disrupted when DRG xenografts were infected with rOka mutants lacking gI promoter elements that bind cellular transactivators, specificity factor 1 (Sp1) and upstream stimulatory factor (USF). Because gI is essential and Sp1 and USF contribute to VZV pathogenesis in skin and T cells in vivo, these DRG experiments indicate that the genetic requirements for VZV infection are less stringent in neural cells in vivo. The observations demonstrate that gI is important for VZV neurotropism and suggest that a strategy to reduce neurovirulence by deleting gI could prolong active infection in human DRGs.

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory ganglia. VZV reactivation results in herpes zoster. We developed a model using human dorsal root ganglion (DRG) xenografts in severe combined immunodeficient (SCID) mice to investigate VZV infection of differentiated neurons and satellite cells in vivo. DRG engrafted under the kidney capsule and contained neurons and satellite cells within a typical DRG architecture. VZV clinical isolates infected the neurons within DRG. At 14 days postinfection, VZ virions were detected by electron microscopy in neuronal cell nuclei and cytoplasm but not in satellite cells. The VZV genome copy number was 7.1 x 10(7) to 8.0 x 10(8) copies per 10(5) cells, and infectious virus was recovered. This initial phase of viral replication was followed within 4-8 weeks by a transition to VZV latency, characterized by the absence of infectious virus release, the cessation of virion assembly, and a reduction in VZV genome copies to 3.7 x 10(5) to 4.7 x 10(6) per 10(5) cells. VZV persistence in DRG was achieved without any requirement for VZV-specific adaptive immunity and was associated with continued transcription of the ORF63 regulatory gene. The live attenuated varicella vaccine virus exhibited the same pattern of short-term replication, persistence of viral DNA, and prominent ORF63 transcription as the clinical isolates. VZV-infected T cells transferred virus from the circulation into DRG, suggesting that VZV lymphotropism facilitates its neurotropism. DRG xenografts may be useful for investigating neuropathogenic mechanisms of other human viruses.

Progress in techniques and strategies for tissue engineering has increased our interest in allografting and xenografting in various organ systems. Previous work has shown that peripherally implanted fetal dorsal root ganglion neurons (DRGs) can grow axons across the boundary between the central and peripheral nervous system in the dorsal root and make functional connections within the spinal cord. We have extended this experimental paradigm to the auditory system and successfully implanted fetal DRG neurons into the normal and deafened cochlea, adjacent to deafferented auditory spiral ganglion neurons. These findings demonstrate the feasibility of using fetal sensory cells in a new strategy to repair or replace the auditory nerve. Further studies will show whether the surviving DRGs can restore a functional conduit from the cochlea to the brainstem. If so, implanting neuronal tissue close to the auditory nerve could be used to regain auditory function in e.g. profoundly deaf patients.

CNS lesions induce production of ECM molecules that inhibit axon regeneration. One major inhibitory family is the chondroitin sulfate proteoglycans (CSPGs). Reduction of their glycosaminoglycan (GAG) chains with chondroitinase ABC leads to increased axon regeneration that does not extend well past the lesion. Chondroitinase ABC, however, is unable to completely digest the GAG chains from the protein core, leaving an inhibitory "stub" carbohydrate behind. We used a newly designed DNA enzyme, which targets the mRNA of a critical enzyme that initiates glycosylation of the protein backbone of PGs, xylosyltransferase-1. DNA enzyme administration to TGF-beta-stimulated astrocytes in culture reduced specific GAG chains. The same DNA enzyme applied to the injured spinal cord led to a strong reduction of the GAG chains in the lesion penumbra and allowed axons to regenerate around the core of the lesion. Our experiments demonstrate the critical role of PGs, and particularly those in the penumbra, in causing regeneration failure in the adult spinal cord.

Previous studies suggest that sensory innervation of muscles is patterned by motor innervation. Muscle afferent projections mirror motor projections after various experimental manipulations and muscle afferents fail to project to muscle in the absence of motoneurons. It is not known, however, whether muscle afferents are specified with respect to the corresponding motoneurons or target muscles. To test this possibility we rotated three to four segments of neural crest in St. 15-17 chick embryos, leaving motoneurons intact, to reverse the rostrocaudal order of dorsal root ganglia (DRGs) T7/LS1-LS3. This caused sensory neurons derived from one segmental level to grow into the limb with motor axons from a different level. The resulting innervation patterns were assessed at St. 28-37 by injecting DiI and DiA into the sartorius and femorotibialis muscles or into the spinal cord and DRG. DiI labeling of crest prior to rotation showed that DRGs in the operated region were derived primarily from rotated cells. Muscle afferents from rotated DRGs grew to muscles in accord with their new rostrocaudal position, together with "inappropriate" motor axons from the same segmental level. The segmental distribution of sensory neurons innervating each muscle was more widespread in embryos operated at older than at younger stages. In contrast, sensory axons projected to the appropriate muscles in accord with their embryonic origin when segments of the whole neural tube, including motoneurons, were rotated, as reported previously. Thus, sensory neurons do not appear to be selectively matched with motoneurons or target muscles at stages when the corresponding motoneurons have clear identities.

Sensory ganglia latently infected with herpes simplex virus (HSV) were transplanted beneath the renal capsule of syngeneic recipients, and the latent infection remaining was investigated. HSV latency-associated transcript (LAT) expression and reactivation of HSV after explant of transplanted dorsal root ganglia were monitored as markers of latency. Two to four weeks after transplantation, both indicated evidence of HSV latency in transplants. At those times, infectious virus was not detected in direct ganglion homogenates. In addition, viral antigen and infected cell polypeptide 4 RNA were not detected. Taken together, the results suggested that HSV latent infection rather than persistent infection was present in transplants. From these results, two explanations seemed possible: latency was maintained in transplanted neurons, or alternatively, latency developed after transplantation, in neurons not previously latently infected. The latter was considered putative secondary latency and was investigated in three ways. First, evidence of reactivation which might serve as a source for secondary latency was evaluated. Reactivation of HSV in transplants was evident from HSV antigen expression (52% of transplants) and the presence of cell-free virus (38% of transplants) 3 to 5 days after transplantation. Second, putative secondary latency was investigated in recipients immunized with HSV prior to receiving latently infected ganglia. Reactivation was not detected 3 to 5 days after transplantation in immunized recipients, and LAT expression was rare in these recipients after 3 to 4 weeks. Lastly, the possibility of secondary latency was investigated by comparing results obtained with standard HSV and with reactivation-defective thymidine kinase-negative (TK-) HSV. Defective reactivation of TK- HSV was demonstrated by immunohistochemistry and by the inability to isolate infectious virus. Donor dorsal root ganglia latently infected with TK+ HSV showed many LAT-positive neurons 2 or more weeks after transplantation (average, 26 per transplant). However, LAT expression was undetectable or minimal > 2 weeks after transplantation in donor ganglia latently infected with TK- HSV (average, 0.2 per transplant). Impaired reactivation of TK- HSV-infected donor ganglia after transplantation, therefore, was correlated with subsequent limited LAT expression. From these results, the occurrence of secondary latency was concluded for ganglia latently infected with TK+ HSV and transplanted beneath the kidney capsule. In vivo reactivation in this transplant model may provide a more useful means to investigate HSV reactivation than in usual in vitro explant models and may complement other in vivo reactivation models. The occurrence of secondary latency was unique. The inhibition of secondary latency by the immune system may provide an avenue to evaluate immunological control of HSV latency.

Amphibian forelimb regeneration is a nerve-dependent process; nerves presumably release one or more neurotrophic factors that stimulate blastema cell division. To date several candidate molecules/factors have been shown to stimulate macromolecular synthesis and/or mitosis but sustained cell cycle activity and blastema development have not been achieved. Because dorsal root ganglia (DRG) implants are capable of promoting regeneration of denervated adult newt limbs (Kamrin & Singer, 1959), we have evaluated the DRG stimulation of regeneration in denervated limbs of adult newts and larval axolotls; two alternative timing strategies were tested as a step toward defining bioassay parameters that best reflect neurotrophic activity. The frequency of regeneration in denervated adult newt limbs was compared after providing DRG before or at the time of denervation (to maintain neurotrophic and cell cycle activity) versus DRG implantation at various postdenervation times (to resupply neurotrophic activity and restimulate suppressed cell cycle activity). The results show that denervated adult newt limbs regenerated most frequently using the maintenance strategy, but as the denervation interval was extended in the restimulation strategy, the frequency of regeneration declined. Larval axolotl limbs responded positively in both maintenance and restimulation DRG-grafting protocols. These results suggest that the efficacy of DRG stimulation of regeneration in adult newts was related to the relative number of blastema cells present at the time of denervation and the proliferative status of the blastema cells; bioassays with denervated adult newt limbs should be designed with these constraints in mind. Because such constraints are not as problematic with the larval axolotl, this species may provide the best opportunity for further defining bioassay parameters related to the neurotrophic stimulation of regeneration.

The cell types present in quail dorsal root ganglia during early development were identified using markers for neurons, glial cells, and fibroblasts (Rohrer et al., 1985). Using the quail-chick transplantation technique, the potential of quail dorsal root ganglion cells to differentiate to adrenergic chromaffin cells, as identified by tyrosine hydroxylase immunostaining, was analyzed. A population of undifferentiated cells, which is present in quail dorsal root ganglia at embryonic day 5, was separated from neurons and glial cells. We show that this population contains cells that differentiate to adrenergic chromaffin cells after back-transplantation into 2-d-old chick embryos. A large proportion of these undifferentiated cells also differentiates to neurons in vitro. Precursors for adrenal chromaffin cells and for neurons are present in dorsal root ganglia in significant numbers only during early development.

Thoracic dorsal root ganglia in bullfrogs contain sensory neurons that innervate the skin of the trunk and have synaptic connections in the dorsal horn of the spinal cord. The ganglion that innervates the forelimb contains, in addition to cutaneous afferents, many muscle afferents that project more ventrally in the spinal cord and make monosynaptic connections with motoneurons. In the present study, we have transplanted thoracic sensory neurons to the brachial level in tadpoles to discover whether they can innervate forelimb muscles and, if so, whether they form central connections characteristic of forelimb muscle afferents. The ganglion that normally supplies the forelimb was removed from tadpoles and replaced with 2 thoracic ganglia. After the tadpoles completed metamorphosis, the peripheral and central connections of the transplanted thoracic sensory neurons were examined with anatomical and electrophysiological techniques. When the ganglia were transplanted at stage XIV or earlier, transplanted sensory neurons innervated the forelimb and projected into the brachial spinal cord. Electrical stimulation of forelimb muscle nerves evoked impulses in the dorsal root, indicating that some centrally projecting sensory neurons were muscle afferents. Furthermore, muscle afferents were also activated by stretching muscles which suggest that they terminated on spindles. HRP labeling of the central projections revealed that transplanted sensory neurons terminated at sites characteristic of both cutaneous and muscle afferents. The pattern of synaptic connections was assessed by recording intracellularly from motoneurons. Stimulation of muscle afferents produced monosynaptic EPSPs in motoneurons. As in normal frogs, triceps muscle afferents projected more strongly to triceps motoneurons than to subscapularis and pectoralis motoneurons, while subscapularis afferents projected to all 3 types of motoneurons. Thus, the transplanted sensory neurons formed central connections appropriate to their novel peripheral targets. These observations suggest that interactions between sensory neurons and their targets may be important in determining their central connections.