Nerve terminals in the tumor microenvironment as targets for local infiltration analgesia.

Nerve terminals within the tumor microenvironment as potential pain-mitigating targets for local infiltration analgesia is relatively less explored. In this study, we examine the role of key analgesics administered as local infiltration analgesia in a model of cancer-induced bone pain (CIBP). CIBP was induced by administration of allogenic MRMT1 breast cancer cells in the proximal tibia of rats, and tumor mass characterized using radiogram, micro-CT, and histological analysis. In vitro responsiveness to key analgesics δ-opioid receptor agonist (DOPr), Ca2+ channel and TRPV1 antagonists was assessed using ratiometric Ca2+ imaging in sensory neurons innervating the tumor site. Effectiveness of locally infiltrated analgesics administered independently or in combination was assessed by quantifying evoked limb withdrawal thresholds at two distinct sites for up to 14 days. CIBP animals demonstrated DOPr, N-, and L-type and TRPV1 expression in lumbar dorsal root ganglion neurons (DRG), comparable to controls. Evoked Ca2+ transients in DRG neurons from CIBP animals were significantly reduced in response to treatment with compounds targeting DOPr, N-, L-type Ca2+ channels and TRPV1 proteins. Behaviourally, evoked hyperalgesia at the tumor site was strongly mitigated by peritumoral injection of the DOPr agonist and T-type calcium antagonist, via its activity on bone afferents. Results from this study suggest that nerve terminals at tumor site could be utilized as targets for specific analgesics, using local infiltration analgesia.

Secretome mediated interactions between sensory neurons and breast cancer cells.

The role of the nervous system in aiding cancer progression and metastasis is an important aspect of cancer pathogenesis. Interaction between cancer cells and neurons in an in vitro platform is a simple and robust method to further understand this phenomenon. In our study, we aimed to examine in vitro reciprocal effect between breast cancer cells and cancer-sensitized peripheral primary sensory neurons. Secretome obtained from either cultured DRG neurons from tumor-burdened rats, or MRMT1 breast cancer cells were used to study neuronal and cancer cell reciprocity. We utilized neurite analysis, modified cell migration assay and cell signaling pathway inhibitors to determine neurite growth patterns and cell migration in PC12/DRG neurons and MRMT1 cells, respectively. MRMT1 secretome was found to induce significant neurite outgrowth in PC12 and primary sensory neurons. Secretome-induced neurite growth in PC12 cells was partly mediated by PI3K and ERK pathways, but not by adenylyl cyclase. Conversely, secretome from tumor-sensitized sensory neuron cultures induced increased rate of migration in cultured MRMT1 cells. Results from our study provide additional support to the hypothesis that both breast cancer cells and nerve terminals secrete signaling messengers that have a reciprocal effect on each other.

A Brief Review of In Vitro Models for Injury and Regeneration in the Peripheral Nervous System.

Nerve axonal injury and associated cellular mechanisms leading to peripheral nerve damage are important topics of research necessary for reducing disability and enhancing quality of life. Model systems that mimic the biological changes that occur during human nerve injury are crucial for the identification of cellular responses, screening of novel therapeutic molecules, and design of neural regeneration strategies. In addition to in vivo and mathematical models, in vitro axonal injury models provide a simple, robust, and reductionist platform to partially understand nerve injury pathogenesis and regeneration. In recent years, there have been several advances related to in vitro techniques that focus on the utilization of custom-fabricated cell culture chambers, microfluidic chamber systems, and injury techniques such as laser ablation and axonal stretching. These developments seem to reflect a gradual and natural progression towards understanding molecular and signaling events at an individual axon and neuronal-soma level. In this review, we attempt to categorize and discuss various in vitro models of injury relevant to the peripheral nervous system and highlight their strengths, weaknesses, and opportunities. Such models will help to recreate the post-injury microenvironment and aid in the development of therapeutic strategies that can accelerate nerve repair.

Effect of gold nanoparticle treated dorsal root ganglion cells on peripheral neurite differentiation.

The use of gold nanoparticles (AuNps) in applications connected to the peripheral nervous system (PNS) holds much promise in terms of therapeutic and diagnostic strategies. Despite their extensive use, a clear understanding of their effects on neurons and glia in the PNS is lacking. In this study, we set out to examine the effects of AuNps on dorsal root ganglion (DRG) cells, and how such AuNp-exposed cells could in-turn affect neurite differentiation. DRG cultures were exposed to mono-dispersed spherical-shaped AuNps of diameter 24.3 ± 2.3, 109.2 ± 14.7 or 175 ± 19.2 nm at varying concentrations. Cellular uptake and viability were quantified using flow-cytometry. Neurite differentiation was quantified using neurite tracing analysis in PC-12 and DRG neurons exposed to conditioned media derived from AuNp-treated DRG cells. Both neurons and glia were found to internalize AuNps. DRG cell viability was significantly reduced upon treatment with higher concentration of 175 nm sized AuNps, while 24 nm and 109 nm sized AuNps had no effect. Further, conditioned media from AuNp-treated DRG cells produced comparable neurite outgrowth and neurite branching measurement as controls in PC-12 and DRG neurons. DRG cells were quite resilient to AuNp exposure in mild-moderate concentration. AuNp-exposed DRG cells, irrespective of size and concentration range tested, did not affect neuronal differentiation.

Neuronal delivery of nanoparticles via nerve fibres in the skin.

Accessing the peripheral nervous system (PNS) by topically applied nanoparticles is a simple and novel approach with clinical applications in several PNS disorders. Skin is richly innervated by long peripheral axons that arise from cell bodies located distally within ganglia. In this study we attempt to target dorsal root ganglia (DRG) neurons, via their axons by topical application of lectin-functionalized gold nanoparticles (IB4-AuNP). In vitro, 140.2 ± 1.9 nm IB4-AuNP were found to bind both axons and cell bodies of DRG neurons, and AuNP applied at the axonal terminals were found to translocate to the cell bodies. Topical application of IB4-AuNP on rat hind-paw resulted in accumulation of three to fourfold higher AuNP in lumbar DRG than in contralateral control DRGs. Results from this study clearly suggest that topically applied nanoparticles with neurotropic targeting ligands can be utilized for delivering nanoparticles to neuronal cell bodies via axonal transport mechanisms.

Electrical stimulation of co-woven nerve conduit for peripheral neurite differentiation.

Electrically stimulable nerve conduits are implants that could potentially be utilized in patients with nerve injury for restoring function and limb mobility. Such conduits need to be developed from specialized scaffolds that are both electrically conductive and allow neuronal attachment and differentiation. In this study, we investigate neural cell attachment and axonal differentiation on scaffolds co-woven with poly-(L-lactic acid) (PLLA) yarns and conducting threads. Yarns obtained from electrospun PLLA were co-woven with polypyrrole (PPy)-coated PLLA yarns or ultrathin wires of copper or platinum using a custom built low-resistance semi-automated weaving machine. The conducting threads were first electrically characterized and tested for stability in cell growth media. Suitability of the conducting threads was further assessed via cell viability studies using PC12 cells. Neurite growth was then quantified after electrically stimulating rat dorsal root ganglion (DRG) sensory neurons cultured on the woven scaffolds. Electrical conductivity tests and cellular viability studies demonstrated better bio-tolerability of platinum wires over PPy-coated PLLA yarns and copper wires. Electrically stimulated DRG neurons cultured on platinum-PLLA co-woven scaffolds showed enhanced neurite outgrowth and length. We demonstrate that a woven scaffold design could be utilized to incorporate conducting materials into cell-tolerable polymer yarns for developing electrically stimulable nerve conduits.

Effect of Peritumoral Bupivacaine on Primary and Distal Hyperalgesia in Cancer-Induced Bone Pain.

BACKGROUND: Cancer-induced bone pain (CIBP) is a debilitating chronic pain condition caused by injury to bone nerve terminals due to primary or metastasized bone tumors. Pain manifests as enhanced sensitivity, not only over the affected bone site but also at distal areas that share common nerve innervation with the tumor. In this study, we aim to understand how tumor-induced primary and distal pain sensitivities are affected by bupivacaine-induced block of bone nerve endings in a rat model of CIBP. METHODS: MRMT-1 breast cancer cells were injected into the proximal segment of tibia in female Sprague-Dawley rats. Radiograms and micro-CT images were obtained to confirm tumor growth. Bupivacaine was injected peritumorally at day 7 or day 14 post-tumor induction, and withdrawal thresholds in response to pressure and punctate mechanical stimulus were recorded from the knee and hind-paw, respectively. Immunohistochemical studies for the determination of ATF3 and GFAP expression in DRG and spinal cord sections were performed. RESULTS: Rats developed primary and distal hyperalgesia after MRMT-1 administration that was sustained for 2 weeks. Peritumoral administration of bupivacaine in 7-day post-tumor-induced (PTI) rats resulted in a reversal of both primary and distal hyperalgesia for 20-30 mins. However, bupivacaine failed to reverse distal hyperalgesia in 14 day-PTI rats. ATF3 and GFAP expression were much enhanced in 14 day-PTI animals, compared to 7 day-PTI group. CONCLUSION: Results from this study strongly suggest that distal hyperalgesia of late-stage CIBP demonstrates differential characteristics consistent with neuropathic pain as compared to early stage, which appears more inflammatory in nature.

Hydrogel systems and their role in neural tissue engineering.

Neural tissue engineering (NTE) is a rapidly progressing field that promises to address several serious neurological conditions that are currently difficult to treat. Selecting the right scaffolding material to promote neural and non-neural cell differentiation as well as axonal growth is essential for the overall design strategy for NTE. Among the varieties of scaffolds, hydrogels have proved to be excellent candidates for culturing and differentiating cells of neural origin. Considering the intrinsic resistance of the nervous system against regeneration, hydrogels have been abundantly used in applications that involve the release of neurotrophic factors, antagonists of neural growth inhibitors and other neural growth-promoting agents. Recent developments in the field include the utilization of encapsulating hydrogels in neural cell therapy for providing localized trophic support and shielding neural cells from immune activity. In this review, we categorize and discuss the various hydrogel-based strategies that have been examined for neural-specific applications and also highlight their strengths and weaknesses. We also discuss future prospects and challenges ahead for the utilization of hydrogels in NTE.

Neuroglia as targets for drug delivery systems: A review.

Targeted drug delivery within the nervous system is an emerging topic of research that involves designing and developing vehicular delivery systems that have the ability to target specific neuronal and non-neuronal cell types in the central and peripheral nervous system. Drugs, genetic material, or any other payloads can be loaded onto such delivery systems and could be used to treat, prevent or manage various neurological disorders. Currently, majority of studies in this field have been concentrated around targeted delivery to neurons. However, the non-neuronal cells within the nervous system, collectively called neuroglia, have been largely ignored, though it is well known that they play a significant role in the pathophysiology of almost all neurological disorders. In this review, we present current developments in the specific area of neuroglia targeted delivery systems and highlight the use of polymeric, metallic, liposomal and other delivery systems used for this purpose.