Peripheral nerve injury (PNI) is common and despite modern microsurgical techniques of repair, functional restoration is always incomplete. This results in impaired sensation and reduced motor function alongside pain and cold intolerance. Traumatic PNI are often associated with loss of nerve tissue, creating a gap, and direct repair of the two damaged nerve stumps is not possible. These types of injuries are reconstructed using autologous nerve grafts but this is far from ideal since it necessitates the sacrifice of a functional nerve from elsewhere in the body. Chronic muscle atrophy because of the prolonged delay in nerve regeneration across gaps is a significant impediment to an optimal functional recovery.   Tissue engineering and regenerative medicine approaches to nerve repair might one day replace the need for autologous nerve grafts. This thesis investigates the effects of adipose derived stem cells (ASC) on nerve regeneration and muscle recovery by using the stem cells for intramuscular injection and combined with a biomaterial, poly-3-hydroxybutyrate (PHB), to create a bioengineered artificial nerve repair construct.  The mechanisms of interaction between the stem cells and neuromuscular system cells were investigated and with a view to translating this work into clinical practice, an optimal source of cells was investigated from human donors.   It was hypothesized that injecting regenerative cells into muscle would reduce nerve injury induced muscle atrophy. A rat sciatic nerve lesion was performed and three different types of cells were injected into the denervated gastrocnemius muscle; either (1) undifferentiated ASC, (2) ASC induced to a ‘Schwann cell-like’ phenotype (dASC) or (3) primary Schwann cells. Nerves were either repaired by direct end-end suture or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. The Schwann cells and dASC groups showed significantly better scores on functional tests when compared with control injections of growth medium alone. Muscle weight and histology were also significantly improved in the cell groups in comparison with the control group.   PHB strips seeded with either primary Schwann cells or dASC suspended in a fibrin glue matrix were used to bridge a 10mm rat sciatic nerve gap. After 12 weeks, functional and morphological analysis (walking track test, electromyography, muscle weight and muscle and nerve histology) was performed. The results showed significantly better functional results for the PHB strips seeded with cells versus the control group with fibrin matrix only. This correlated with less muscle atrophy and greater distal axon myelination in the cell groups.   To further optimize the nerve regeneration and muscle recovery, the nerve gap lesion was repaired by treatment with the bioengineered constructs seeded with dASC or nerve autograft in combination with stem cell injection in the muscle. After 6 weeks, the best results were obtained in the nerve graft group combined with intramuscular dASC injection which showed significantly less atrophy than the other groups. The results also showed that using the stem cells in a matrix on a PHB strip in combination with intramuscular injections could significantly reduce muscle atrophy.   In vitro experiments showed that dASC expressed a wide range of neurotrophic and myogenic factors including BDNF, VEGF-A, IGF-1 and HGF. Stem cell conditioned medium enhanced the proliferation of myoblast cell lines and primary Schwann cells. Various signaling pathways (PKA, MAP kinase) were involved in these effects dependent on the cell type investigated. Furthermore, in direct co-culture with myoblast cells, a small population of the cells fused together to form myotube-like structures and expressed myogenic markers.   Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Superficial layer ASC induced significantly enhanced neurite outgrowth from neuronal cell lines when compared with the deep layer cells.  However, RT-PCR and ELISA analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF and GDNF.   In summary, these results show that stem cell therapy at both levels (the nerve lesion site and in the target denervated muscle) offers a promising approach for clinical application for treatment of peripheral nerve lesions. The bioengineered artificial nerve construct, combining PHB strip with cells, also provides a beneficial environment for nerve regeneration. Many of the benefits of the ASC are likely to be mediated through their secretome, a rich source of neurotrophic and myogenic factors. Thus adipose tissue contains a pool of regenerative stem cells which have significant potential application to tissue engineering and regenerative medicine for nerve repair.

Substantial evidence shows that activation of glial cells in the spinal cord may promote central sensitization and enhancement of pain. Descending facilitation from the rostroventromedial medulla (RVM) is also recognized as a critical component in the maintenance of chronic pain states, although the precise mechanisms driving this activity are unclear. Here, we investigated the possibility that glial activation in the RVM could promote descending facilitation from the RVM in states of enhanced pain. Peripheral inflammation was induced with carrageenan injected into the plantar aspect of the hindpaw of male Sprague-Dawley rats and behavioral responses to noxious thermal and light tactile stimuli were determined. Microinjection of the glial inhibitors minocycline or fluorocitrate, or of SB 203580, a p38 MAPK inhibitor, produced a significant and time-related reversal of behavioral hypersensitivity resulting from hindpaw inflammation. Moreover, carrageenan-induced inflammation appeared to produce an increase in immunolabeling for activated microglia and astrocytes in the RVM, as well as for phosphorylated p38 MAPK; the latter was localized to both microglia and neurons of the RVM. Microinjection of the glial inhibitors into the RVM appeared to diminish immunofluorescent labeling for activated RVM microglia and astrocytes. Carrageenan-induced inflammation also increased RVM protein levels of Iba1 and GFAP and administration of minocycline or fluorocitrate into the RVM attenuated this effect. To examine a possible mechanism of glial activation, α, β-methylene-ATP was microinjected into the RVM, inducing thermal hyperalgesia, and pre-treatment with the P2X antagonists, PPADS and TNP-ATP, delayed the initiation of ATP-induced hyperalgesia. Post-treatment with the antagonists had no effect on established ATP-induced or carrageenan-induced hypersensitivity. The activation of P2X receptors initiates a signaling cascade leading to the production and release of nociceptive mediators, including BDNF. The RVM microinjection of an anti- BDNF antibody reversed carrageenan-induced thermal hyperalgesia. A model of morphine-induced paradoxical pain was also used to examine the role of glial activation in the RVM. Sustained morphine administration induced tactile allodynia and RVM microinjection of minocycline, but not fluorocitrate, attenuated the behavioral hypersensitivity. Sustained morphine also induced morphological changes in microglia of the RVM, suggesting microglial activation. A third model of enhanced pain used to study medullary glial activation was the spinal nerve ligation (SNL) model of neuropathic pain. The SNL injury induced astrocyte activation within the RVM and microinjection of the astrocyte inhibitor fluorocitrate attenuated the nerve injury-induced tactile allodynia. Minocycline administered to the RVM did not attenuate the behavioral hypersensitivity, suggesting a role for astrocytes, not microglia, in nerve injury-induced enhanced pain. The data show that inflammatory, opioid-induced and neuropathic pain is associated with glial activation in the RVM which likely participates in driving descending pain facilitation via glial-neuronal communication. These findings reveal a novel site of glial modulation of pain.

Anandamide (AEA), a well characterized endocannabinoid that has actions at multiple targets in the eye, may have potential as a novel therapeutic in the treatment of retinal disease. However, AEA is rapidly degraded by fatty acid amide hydrolase (FAAH). Therefore this study examined the drug URB597, that inhibits FAAH degradation of AEA, to assess AEA effects in experimental models of retinal damage. The objectives were to: 1) evaluate changes present in the aging retina, 2) determine whether the aging retina is more susceptible to tissue damage, and 3) investigate whether increasing AEA can provide retinal neurovascular protection in young and aged retina following damage. The results from this study showed that URB597 had protective effects on retinal ganglion cells and retinal capillaries and inhibited phagocytotic MG in models of retinal damage in young, but not the aged retina.

Severe injury may result in disconnection between the peripheral and central nervous system. Regeneration of the central portion of sensory neurons into the spinal cord is notoriously poor in adult mammals, with low regenerative drive and an unpermissive central environment, most likely resulting in persistent loss of sensory function. A variety of strategies have been addressedto augment regeneration, including application of growth promoting factors, counteraction of inhibitory molecules, and provision of growth permissive substrates. Stem cells have been investigated in these contexts, as well as for the possibility of providing new neurons to act as a relay between the periphery and spinal cord. Here we have investigated different sources of neural stem cells for their ability to form neurons and glia after transplantation to the periphery; to project axons into the spinal cord; and to assist regeneration of surviving sensory neurons. These have been performed at two locations: the "dorsal root ganglion cavity", and the transitional zone following dorsal root avulsion. Neurons and glia were generated form mouse boundary cap neural crest stem cells and embryonic stem cell derived ventral spinal cord progenitors, and in addition to this, regeneration of sensory fibers was observed after transplantation of human fetal spinal cord derived progenitors and human embryonic stem cell derived ventral spinal cord progenitors. Further, delivery of neurotrophic factor mimetics via mesoporous silica nanoparticles proved a valuable tool for stem cell survival and differentiation. While technological advances make in vivo differentiation a realistic goal, our findings indicate that so far assisting regeneration of host sensory fibers to reconnect with the spinal cord by transplantation of stem cells is a more reliable strategy.

Indiana University-Purdue University Indianapolis (IUPUI) / The adaptive arm of the immune system is necessary for facial motoneuron (FMN) survival after facial nerve axotomy (FNA). CD4+ T cells mediate FMN survival after FNA in an interleukin-10 (IL-10) dependent manner, but are not themselves the cellular source of neuroprotective IL-10. The aims of this study are to elucidate the neuroprotective capacity of cell-specific IL-10 expression, and to investigate the manner in which CD4+ T cells participate in IL-10 signaling after FNA. Immunohistochemistry revealed that FMN themselves were constitutive producers of IL-10, and astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 is critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 was necessary for FMN survival. Gene expression analysis of wild-type, immunodeficient, and immune cell-reconstituted animals was performed to determine the role of the immune system in modulating the central IL-10 signaling cascade. This revealed that CD4+ T cells were necessary for full upregulation of central IL-10 receptor (IL-10R) expression after FNA, regardless of their own IL-10R beta (IL-10RB) expression or IL-10R signaling capability. Surprisingly, the ability of CD4+ T cells to respond to IL-10 was critical for their ability to mediate neuroprotection. Adoptive transfer of IL-10RB-deficient T cells resulted in increased central expression of genes associated with microglial activation, antigen presentation, T cell co-stimulation, and complement deposition in response to injury. These data suggest that IL-10RB functions on the T cell to prevent non-neuroprotective immune activation after axotomy. The conclusions drawn from this study support a revised hypothesis for the mechanisms of IL-10-mediated neuroprotection, in which IL-10 serves both trophic and immune-modulating roles after axotomy. This research has implications for the development of immune-modifying therapies for peripheral nerve injury and motoneuron diseases. / 2 years (2021-05-24)

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Currently, changes to endogenous opioid circuits in various pain modalities, including surgical and neuropathic pain, remain unclear. Dynorphin, which is released by prodynorphin-expressing neurons (Pdyn+ neurons), is the endogenous opioid ligand to kappa opioid receptors (KOR). Moreover, a recent study has shown an increase in prodynorphin (Pdyn) mRNA expression in the prelimbic cortex (PL) in a mouse model of chronic pain. However, alterations in the activity of PL Pdyn-expressing neurons (PLPdyn+ neurons) in postoperative and chronic pain have never been explored. Firstly, I found that the population of PLPdyn+ neurons consists of both pyramidal and inhibitory subtypes. Secondly, I found that one day after surgical incision of the mouse hind paw, the excitability of pyramidal PLPdyn+ neurons was increased in both male and female mice, while the excitability of inhibitory PLPdyn+ neurons was unchanged. However, when postoperative pain behavior subsided, inhibitory PLPdyn+ neurons were hyperexcitable in male mice, while pyramidal PLPdyn+ neurons were hypoexcitable in female mice. Lastly, I dissected electrophysiological changes to PLPdyn+ neurons in the spared nerve injury (SNI) model of chronic neuropathic pain. At both early and late stages of SNI pain development, increased excitability of pyramidal PLPdyn+ neurons was detected in both male and female mice. However, in both male and female mice, the excitability of inhibitory PLPdyn+ neurons decreased 3 days after SNI but was conversely increased when measured 14 days after SNI. My findings suggest that different subtypes of PLPdyn+ neurons manifest distinct alterations in the development of different pain modalities in a sex-specific manner.

No description available.

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

Despite surgical innovation, the sensory and motor outcome after peripheral nerve injury is incomplete. In this thesis, the biological pathways potentially responsible for the poor functional recoveries were investigated in both the distal nerve stump/target organ, spinal motoneurons and dorsal root ganglia (DRG). The effect of delayed nerve repair was determined in a rat sciatic nerve transection model. There was a dramatic decline in the number of regenerating motoneurons and myelinated axons found in the distal nerve stumps of animals undergoing nerve repair after a delay of 3 and 6 months. RT-PCR of the distal nerve stumps showed a decline in expression of Schwann cells (SC) markers, with a progressive increase in fibrotic and proteoglycan scar markers, with increased delayed repair time. Furthermore, the yield of SC which could be isolated from the distal nerve segments progressively fell with increased delay in repair time. Consistent with the impaired distal nerve stumps the target medial gastrocnemius (MG) muscles at 3- and 6-months delayed repair were atrophied with significant declines in wet weights (61% and 27% compared with contralateral sides). The role of myogenic transcription factors, muscle specific microRNAs and musclespecific E3 ubiquitin ligases in the muscle atrophy was investigated in both gastrocnemius and soleus muscles following either crush or nerve transection injury. In the crush injury model, the soleus muscle showed significantly increased recovery in wet weight at days 14 and 28 (compared with day 7) which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. Morphological and molecular changes in spinal motoneurons were compared after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA). Neuronal degeneration was indicated by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Immunostaining for ED1-reactive microglia and GFAPpositive astrocytes was significantly elevated in all experimental groups. qRT-PCR analysis and Western blotting of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of apoptotic cell death mediators including caspases-3 and -8 and a range of related death receptors following VRA. In contrast, following PNA, only caspase-8 was moderately upregulated. The mechanisms of primary sensory neuron degeneration were also investigated in the DRG following peripheral nerve axotomy, where several apoptotic pathways including those involving the endoplasmic reticulum were shown to be upregulated. In summary, these results show that the critical time point after which the outcome of regeneration becomes too poor appears to be 3-months. Both proximal and distal injury affect spinal motoneurons morphologically, but VRA induces motoneuron degeneration mediated through both intrinsic and extrinsic apoptotic pathways. Primary sensory neuron degeneration involves several different apoptotic pathways, including the endoplasmic reticulum.