Innervation of Meissner's corpuscles and Merkel -cells by transplantation of embryonic dorsal root ganglion cells after peripheral nerve section in rats.

Transplantation of embryonic motor neurons has been shown to improve motor neuron survival and innervation of neuromuscular junctions in peripheral nerves. However, there have been no reports regarding transplantation of sensory neurons and innervation of sensory receptors. Therefore, we hypothesized that the transplantation of embryonic sensory neurons may improve sensory neurons in the skin and innervate Merkel cells and Meissner's corpuscles. We obtained sensory neurons from dorsal root ganglia of 14-day rat embryos. We generated a rat model of Wallerian-degeneration by performing sciatic nerve transection and waiting for one week after. Six months after cell transplantation, we performed histological and electrophysiological examinations in naïve control, surgical control, and cell transplantation groups. The number of nerve fibers in the papillary dermis and epidermal-dermal interface was significantly greater in the cell transplantation than in the surgical control group. The percent of Merkel cells with nerve terminals, as well as the average number of Meissner corpuscles with nerve terminals, were higher in the cell transplantation than in the surgical control group, but differences were not significant between the two groups. Moreover, the amplitude and latency of sensory conduction velocity were evoked in rats of the cell transplantation group. We demonstrated that the transplantation of embryonic dorsal root ganglion cells improved sensory nerve fiber number and innervation of Merkel cells and Meissner's corpuscles in peripheral nerves.

Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats.

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.

Analgesia for neuropathic pain by dorsal root ganglion transplantation of genetically engineered mesenchymal stem cells: initial results.

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.

Allotransplanted DRG neurons or Schwann cells affect functional recovery in a rodent model of sciatic nerve injury.

OBJECTIVE: In this study, the functional recoveries of Sprague-Dawley rats following repair of a complete sciatic nerve transection using allotransplanted dorsal root ganglion (DRG) neurons or Schwann cells were examined using a number of outcome measures. METHODS: Four groups were compared: (1) repair with a nerve guide conduit seeded with allotransplanted Schwann cells harvested from Wistar rats, (2) repair with a nerve guide conduit seeded with DRG neurons, (3) repair with solely a nerve guide conduit, and (4) sham-surgery animals where the sciatic nerve was left intact. The results corroborated our previous reported histology findings and measures of immunogenicity. RESULTS: The Wistar-DRG-treated group achieved the best recovery, significantly outperforming both the Wistar-Schwann group and the nerve guide conduit group in the Von Frey assay of touch response (P < 0.05). Additionally, Wistar-DRG and Wistar-Schwann seeded repairs showed lower frequency and severity in an autotomy measure of the self-mutilation of the injured leg because of neuralgia. CONCLUSION: These results suggest that in complete peripheral nerve transections, surgical repair using nerve guide conduits with allotransplanted DRG and Schwann cells may improve recovery, especially DRG neurons, which elicit less of an immune response.

Bridging peripheral nerve defects with a tissue engineered nerve graft composed of an in vitro cultured nerve equivalent and a silk fibroin-based scaffold.

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.

Investigation of varicella-zoster virus neurotropism and neurovirulence using SCID mouse-human DRG xenografts.

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.

Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured peripheral nervous system.

ß-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid-ß 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.

Functional evaluation of a cell replacement therapy in the inner ear.

HYPOTHESIS: Cell replacement therapy in the inner ear will contribute to the functional recovery of hearing loss. BACKGROUND: Cell replacement therapy is a potentially powerful approach to replace degenerated or severely damaged spiral ganglion neurons. This study aimed at stimulating the neurite outgrowth of the implanted neurons and enhancing the potential therapeutic of inner ear cell implants. METHODS: Chronic electrical stimulation (CES) and exogenous neurotrophic growth factor (NGF) were applied to 46 guinea pigs transplanted with embryonic dorsal root ganglion (DRG) neurons 4 days postdeafening. The animals were evaluated with the electrically evoked auditory brainstem responses (EABRs) at experimental Days 7, 11, 17, 24, and 31. The animals were euthanized at Day 31, and the inner ears were dissected for immunohistochemistry investigation. RESULTS: Implanted DRG cells, identified by enhanced green fluorescent protein fluorescence and a neuronal marker, were found close to Rosenthal canal in the adult inner ear for up to 4 weeks after transplantation. Extensive neurite projections clearly, greater than in nontreated animals, were observed to penetrate the bony modiolus and reach the spiral ganglion region in animals supplied with CES and/or NGF. There was, however, no significant difference in the thresholds of EABRs between DRG-transplanted animals supplied with CES and/or NGF and DRG-transplanted animals without CES or NGF supplement. CONCLUSION: The results suggest that CES and/or NGF can stimulate neurite outgrowth from implanted neurons, although based on EABR measurement, these interventions did not induce functional connections to the central auditory pathway. Additional time or novel approaches may enhance functional responsiveness of implanted cells in the adult cochlea.

Axon growth across a lesion site along a preformed guidance pathway in the brain.

Our previous studies showed that axonal outgrowth from dorsal root ganglia (DRG) transplants in the adult rat brain could be directed toward a specific target location using a preformed growth-supportive pathway. This pathway induced axon growth within the corpus callosum across the midline to the opposite hemisphere. In this study, we examined whether such pathways would also support axon growth either through or around a lesion of the corpus callosum. Pathways expressing GFP, NGF, or FGF2/NGF were set up by multiple injections of adenovirus along the corpus callosum. Each pathway included the transplantation site in the left corpus callosum, 2.8 mm away from the midline, and a target site in the right corpus callosum, 2.5 mm from the midline. At the same time, a 1 mm lesion was made through the corpus callosum at the midline in an anteroposterior direction. A group of control animals received lesions and Ad-NGF injections only at the transplant and target sites, without a bridging pathway. DRG cell suspensions from postnatal day 1 or 2 rats were injected at the transplantation site three to four days later. Two weeks after transplantation, brain sections were stained using an anti-CGRP antibody. The CGRP+ axons were counted at 0.5 mm and 1.5 mm from the lesion site in both hemispheres. Few axons grew past the lesion in animals with control pathways, but there was robust axon growth across the lesion site in the FGF2/NGF and NGF-expressing pathways. This study indicated that preformed NGF and combination guidance pathways support more axon growth past a lesion in the adult mammalian brain.

Dendritic cell-induced memory T cell activation in nonlymphoid tissues.

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.

In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix.

The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.

Implanted embryonic sensory neurons project axons toward adult auditory brainstem neurons in roller drum and Stoppini co-cultures.

Previously we have shown in vivo the survival, migration and integration of embryonic dorsal root ganglion (DRG) neurons that were grafted into the inner ear and peripheral auditory nervous system. In order to evaluate relevant factors determining integration of sensory neurons further into the central auditory nervous system, complementary in vitro techniques are necessary. The advantages of in vitro systems are that a large number of factors including various grafts and different conditions can be efficiently examined for. Hence, we co-cultured 300 microm thick postnatal rat brainstem slices containing the cochlear nucleus including the central part of the 8th cranial nerve with mouse embryonic DRG neurons. The organotypic co-cultures were either grown on coverslips using the roller drum method described by Gähwiler or on membranes according to the interface method described by Stoppini. Neurons in the cochlear nucleus were labeled with DiI. The results demonstrate that (1) brainstem slices survive for up to 5 weeks in culture, and that (2) co-cultures of embryonic sensory neurons and brainstem show a high degree of neuronal survival, and that (3) survival and axonal outgrowth from the implanted embryonic neurons are dependent on the presence of the brainstem slice rather than on exogenous NGF and that (4) implanted embryonic neurons send axons toward neurons in the cochlear nucleus.

Aberrant infection and persistence of varicella-zoster virus in human dorsal root ganglia in vivo in the absence of glycoprotein I.

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.

Central nervous system stem/progenitor cells form neurons and peripheral glia after transplantation to the dorsal root ganglion.

We asked whether neural stem/progenitor cells from the cerebral cortex of E14.5 enhanced green fluorescent protein transgenic mice are able to survive grafting and differentiate in the adult rat dorsal root ganglion. Neurospheres were placed in lumbar dorsal root ganglion cavities after removal of the dorsal root ganglia. Alternatively, dissociated neurospheres were injected into intact dorsal root ganglia. Enhanced green fluorescent protein-positive cells in the dorsal root ganglion cavity were located in clusters and expressed beta-III-tubulin or glial fibrillary acidic protein after 1 month, whereas after 3 months, surviving grafted cells expressed only glial fibrillary acidic protein. In the intact adult DRG, transplanted neural stem/progenitor cells surrounded dorsal root ganglion cells and fibers, and expressed glial but not neuronal markers. These findings show that central nervous system stem/progenitor cells can survive and differentiate into neurons and peripheral glia after xenotransplantation to the adult dorsal root ganglion.

Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions.

While most approaches to repair spinal cord injury (SCI) rely on promoting axon outgrowth, the extensive distance that axons would have to grow to bridge SCI lesions remains an enormous challenge. In this study, we used a new tissue-engineering technique to create long nervous tissue constructs spanned by living axon tracts to repair long SCI lesions. Exploiting the newfound process of extreme axon stretch growth, integrated axon tracts from dorsal root ganglia (DRG) neurons were mechanically elongated in vitro to 10 mm over 7 days and encased in a collagen hydrogel to form a nervous tissue construct. In addition, a modified lateral hemisection SCI model in the rat was developed to create a 1 cm long cavity in the spinal cord. Ten days following SCI, constructs were transplanted into the lesion and the animals were euthanized 4 weeks post-transplantation for histological analyses. Through cell tracking methods and immunohistochemistry, the transplanted elongated cultures were consistently found to survive 4 weeks in the injured spinal cord. In addition, DRG axons were observed extending out of the transplanted construct into the host spinal cord tissue. These results demonstrate the promise of nervous tissue constructs consisting of stretch-grown axons to bridge even extensive spinal cord lesions.

Varicella-zoster virus infection of human dorsal root ganglia in vivo.

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.

The astrocytic barrier to axonal regeneration at the dorsal root entry zone is induced by rhizotomy.

After dorsal rhizotomy, sensory axons fail to regenerate beyond the astrocytic glia limitans at the dorsal root entry zone (DREZ) but this inhibition can be overcome with the delivery of exogenous neurotrophin-3. We investigated whether axonal inhibition at the DREZ is constitutive or induced after dorsal rhizotomy. Primary afferent neurones from enhanced green fluorescent protein-expressing mice were transplanted into adult rat dorsal root ganglia in the presence or absence of dorsal rhizotomy. In the absence of dorsal rhizotomy mouse axons freely extended into the rat central nervous system. After host dorsal rhizotomy, mouse axons were unable to cross the DREZ. However, in rats that received a dorsal rhizotomy concomitant with intrathecal neurotrophin-3, the mouse axons were able to cross the DREZ. These results indicate that, under normal circumstances, the adult DREZ is permissive to the regeneration of adult sensory axons and that it only becomes inhibitory once dorsal root axons have been injured and astrocytes at the DREZ have become reactive.

NGF stimulates extensive neurite outgrowth from implanted dorsal root ganglion neurons following transplantation into the adult rat inner ear.

Neuronal tissue transplantation is a potential way to replace degenerated spiral ganglion neurons (SGNs) since these cells cannot regenerate in adult mammals. To investigate whether nerve growth factor (NGF) can stimulate neurite outgrowth from implanted neurons, mouse embryonic dorsal root ganglion (DRG) cells expressing enhanced green fluorescent protein (EGFP) were transplanted into the scala tympani of adult rats with a supplement of NGF or artificial perilymph. DRG neurons were observed in the cochlea for up to 6 weeks postoperatively. A significant difference was identified in the number of DRG neurons between the NGF and non-NGF groups. In the NGF group, extensive neurite projections from DRGs were found penetrating the osseous modiolus towards the spiral ganglion. These results suggest the possibility that embryonic neuronal implants may become integrated within the adult auditory nervous system. In combination with a cochlear prosthesis, a neuronal implantation strategy may provide a possibility for further treatment of profoundly deaf patients.

[Treatment of severe hearing loss. Hopeful results with cochlear implants and nerve cell implantations]. / Bot vid svår hörselnedsättning. Hoppfulla resultat av kokleaimplantat och nervcellsimplantationer.

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.