harvesting-embedding-culturing-dorsal-root-ganglia-multi-compartment

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

University of Nebraska-Lincoln

Locomotive changes are often associated with disease or injury, and these changes can be quantified through gait analysis. Gait analysis has been applied to preclinical studies, providing quantitative behavioural assessment with a reasonable clinical analogue. However, available gait analysis technology for small animals is somewhat limited. Furthermore, technological and analytical challenges can limit the effectiveness of preclinical gait analysis. The Gait Analysis Instrumentation and Technology Optimized for Rodents (GAITOR) Suite is designed to increase the accessibility of preclinical gait analysis to researchers, facilitating hardware and software customization for broad applications. Here, the GAITOR Suite's utility is demonstrated in 4 models: a monoiodoacetate (MIA) injection model of joint pain, a sciatic nerve injury model, an elbow joint contracture model, and a spinal cord injury model. The GAITOR Suite identified unique compensatory gait patterns in each model, demonstrating the software's utility for detecting gait changes in rodent models of highly disparate injuries and diseases. Robust gait analysis may improve preclinical model selection, disease sequelae assessment, and evaluation of potential therapeutics. Our group has provided the GAITOR Suite as an open resource to the research community at www.GAITOR.org , aiming to promote and improve the implementation of gait analysis in preclinical rodent models.

The Open Source GAITOR Suite for Rodent Gait Analysis.

Preservation of tissue structure is often a primary goal when optimizing tissue and organ decellularization methods. Many current protocols nonetheless rely on detergents that aid extraction of cellular components but also damage tissue architecture. It may be more beneficial to leverage an innate cellular process such as apoptosis and promote cell removal without the use of damaging reagents. During apoptosis, a cell detaches from the extracellular matrix, degrades its internal components, and fragments its contents for easier clearance. We have developed a method that leverages this process to achieve tissue decellularization using only mild wash buffers. We have demonstrated that treating peripheral nerve tissue with camptothecin induced both an early marker of apoptosis, cleaved caspase-3 expression, as well as a late stage marker, TUNEL DNA fragmentation. Clearance of the cellular components was then achieved in an apoptosis-dependent manner using a gentle wash in hypertonic phosphate buffered saline followed by DNase treatment. This wash paradigm did not significantly affect collagen or glycosaminoglycan content, but it was sufficient to remove any trace of the cytotoxic compound based on conditioned media experiments. The resulting acellular tissue graft was immunogenically tolerated in vivo and exhibited an intact basal lamina microarchitecture mimicking that of native, unprocessed nerve. Hence, ex vivo induction of apoptosis is a promising method to decellularize tissue without the use of harsh reagents while better preserving the benefits of native tissue such as tissue-specific composition and microarchitecture.

Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure.

Decellularized peripheral nerve has been proven to be an effective clinical intervention for peripheral nerve repair and a preclinical cell carrier after spinal cord injury. However, there are currently a lack of decellularization methods for peripheral nerve that remove cells and maintain matrix similar to the previously established, clinically translated technique (the Hudson method) that relies on the discontinued Triton X-200 detergent. Therefore, the aim of this study was to optimize a novel chemical decellularization method for peripheral nerves based on the currently available anionic detergent sodium deoxycholate. Sprague Dawley rat sciatic nerves were isolated, frozen in buffered solution, and then subject to sequential washes in water, salt buffer, zwitterionic detergents sulfobetaines -10 and -16, and varying concentrations of sodium deoxycholate (SDC). To optimize DNA removal after SDC decellularization, nerves were subjected to deoxyribonuclease (DNase) incubation and salt buffer washes. Immunohistochemical results demonstrated that utilization of 3% SDC in the decellularization process preserved extracellular matrix (ECM) components and structure while facilitating significantly better removal of Schwann cells, axons, and myelin compared with the Hudson method. The addition of a 3-h DNase incubation to the 3% SDC decellularization process significantly removed cellular debris compared with the Hudson method. Proteomic analysis demonstrated that our novel decellularization method based on 3% SDC +3-h DNase used in conjunction with zwitterionic detergents, and salt buffers (new decellularization method using 3% SDC + 3-h DNase, zwitterionic detergents, and salt buffers [SDD method]) produced a similar proteomic profile compared with the Hudson method and had significantly fewer counts of cellular proteins. Finally, cytotoxicity analysis demonstrated that the SDD decellularized scaffolds do not contain significant cytotoxic residuals as eluted media supported metabolically active Schwann cells . Overall, this study demonstrates that SDD decellularization represents a novel alternative utilizing currently commercially available chemical reagents. Impact Statement Decellularized nerves are clinically relevant materials that can be used for a variety of regenerative applications such as peripheral nerve and spinal cord injury repair. However, discontinuation of key detergents used in a proven chemical decellularization process necessitates the optimization of an equivalent or better method. This research presents the field with a novel chemical decellularization method to replace the previous validated standard. Scaffolds generated from this method provide an extracellular matrix-rich material that can be used in a variety of  applications to understand cellular behavior and  applications to facilitate regeneration after neural injury.

Novel Sodium Deoxycholate-Based Chemical Decellularization Method for Peripheral Nerve.

Natural and synthetic hydrogels have been widely investigated as biomaterial scaffolds to promote tissue repair and regeneration. Nevertheless, the scaffold alone is often insufficient to drive new tissue growth, instead requiring continuous delivery of therapeutics, such as proteins or other biomolecules that work in concert with structural support provided by the scaffold. However, because of the high-water content, hydrogels tend to be permeable and cause rapid release of the encapsulated drug, which could lead to serious complications from local overdose and may result in the significant waste of encapsulated therapeutic(s). To this end, we designed an oligonucleotide-functionalized hydrogel that can provide sustained and controlled delivery of therapeutics for up to 4 weeks. To prove this concept, we successfully achieved sustained release (for over 28 days) of model anti-Nogo receptor (anti-NgR) RNA aptamer from oligonucleotide-functionalized hyaluronic acid-based hydrogel by changing the complementarity between the short antisense sequences and the aptamer. Furthermore, the released aptamer successfully blocked neuro-inhibitory effects of myelin-derived inhibitors and promoted neurite outgrowth from rat dorsal root ganglia in vitro. Because antisense sequences can be designed to bind to proteins, peptides, and aptamer, our oligonucleotide-functionalized hydrogel offers a promising therapeutic delivery system to obtain controlled release (both bolus and sustained) of various therapeutics for the treatment of complex diseases and injury models, such as spinal cord injury. STATEMENT OF SIGNIFICANCE: Producing a therapeutic effect often requires the administration of multiple injections with high dosages. This regimen causes discomfort to the patient and raises cost of treatment. Additionally, systemic delivery of therapeutics often results in adverse effects; therefore, local delivery at the site of injury is desirable. Therefore, in this study, we designed an oligonucleotide-functionalized biomaterial platform using ssDNA oligonucleotides (immobile species) as antisense sequences to increase residence time and fine-tune the release of anti-nogo receptor aptamer (mobile species) for spinal cord injury application. Because antisense sequences can be designed to bind proteins, peptides, and aptamer, our hydrogel offers a promising delivery system to obtain controlled release of various therapeutics for the treatment of complex diseases and injury models.

Oligonucleotide-functionalized hydrogels for sustained release of small molecule (aptamer) therapeutics.

Pain originating from an intervertebral disc (discogenic pain) is a major source of chronic low back pain. Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain, so treatment with biomaterials that have the ability to inhibit neurite growth will greatly benefit novel disc therapeutics. Currently, disc therapeutic biomaterials are rarely screened for their ability to modulate nerve growth, mainly due to a lack of models to screen neuromodulation. To address this deficit, our lab has engineered a three dimensional in vitro disc innervation model that mimics the interface between primary sensory nerves and the intervertebral disc. Further, herein we have demonstrated the utility of this model to screen the efficacy of chondroitin sulfate biomaterials to inhibit nerve fiber invasion into the model disc. Biomaterials containing chondroitin-4-sulfate (CS-A) decrease neurite growth in a uniform gel and at an interface between a growth-permissive and a growth-inhibitory gel, while chondroitin-6-sulfate (CS-C) is less neuroinhibitory. This in vitro model holds great potential for screening inhibitors of nerve fiber growth to further improve intervertebral disc replacements and therapeutics. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1016-1026, 2020.

Development of an in vitro intervertebral disc innervation model to screen neuroinhibitory biomaterials.

Congenital heart disease (CHD) affects almost one percent of all live births. Despite diagnostic and surgical reparative advances, the causes and mechanisms of CHD are still primarily unknown. The extracellular matrix plays a large role in cell communication, function, and differentiation, and therefore likely plays a role in disease development and pathophysiology. Cell adhesion and gap junction proteins, such as integrins and connexins, are also essential to cellular communication and behavior, and could interact directly (integrins) or indirectly (connexins) with the extracellular matrix. In this work, we explore disparities in the expression and spatial patterning of extracellular matrix, adhesion, and gap junction proteins between wild type and   mutant mice. Decellularization and proteomic analysis, Western blotting, histology, immunostaining, and mechanical assessment of embryonic and neonatal wild type and  mutant mouse hearts were performed. An increased abundance of collagen IV, fibronectin, and integrin β-1 was found in  mutant neonatal mouse hearts, as well as increased expression of connexin 43 in embryonic mutant hearts. Furthermore, a ventricular noncompaction phenotype was observed in both embryonic and neonatal mutant hearts, as well as spatial disorganization of ECM proteins collagen IV and laminin in mutant hearts. Characterizing such properties in a mutant mouse model provides valuable information that can be applied to better understanding the mechanisms of congenital heart disease.

Extracellular Matrix Disparities in an  Mutant Mouse Model of Congenital Heart Disease.

Over the past 50 years, several different types of extracellular vesicles have been discovered including exosomes, microvesicles, and matrix vesicles. These vesicles are secreted by cells for specific purposes and contain cargo such as microRNA, cytokines, and lipids. A novel extracellular vesicle, the matrix-bound nanovesicle (MBV), has been recently discovered. The MBV is similar to the microvesicle, however, it is attached to the extracellular matrix, instead of being secreted. This review compares MBVs to other types of extracellular vesicles to try and better understand their origin and function. Further, this review will explain various extracellular vesicle isolation methods and how these can be used for MBVs and summarize characterization of MBV cargo such as microRNA, proteins, and lipids. Lastly, we will summarize the effects of MBVs on cells. MBVs are a novel class of extracellular vesicles that hold great promise as a platform for delivery of targeted gene and drug therapeutics.

Matrix-Bound Nanovesicles: What Are They and What Do They Do?

Chronic low back pain (LBP) is a leading cause of disability, but treatments for LBP are limited. Degeneration of the intervertebral disc due to loss of neuroinhibitory sulfated glycosaminoglycans (sGAGs) allows nerves from dorsal root ganglia to grow into the core of the disc. Treatment with a decellularized tissue hydrogel that contains sGAGs may inhibit nerve growth and prevent disc-associated LBP.

Injectable decellularized nucleus pulposus tissue exhibits neuroinhibitory properties.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Chronic low back pain is a global socioeconomic crisis and treatments are lacking in part due to inadequate models. Etiological research suggests that the predominant pathology associated with chronic low back pain is intervertebral disc degeneration. Various research teams have created rat models of disc degeneration, but the clinical translatability of these models has been limited by an absence of robust chronic pain-like behavior. To address this deficit, disc degeneration was induced via an artificial annular tear in female Sprague Dawley rats. The subsequent degeneration, which was allowed to progress for 18-weeks, caused a drastic reduction in disc volume. Furthermore, from week 10 till study conclusion, injured animals exhibited significant axial hypersensitivity. At study end, intervertebral discs were assessed for important characteristics of human degenerated discs: extracellular matrix breakdown, hypocellularity, inflammation, and nerve sprouting. All these aspects were significantly increased in injured animals compared to sham controls. Also of note, 20 significant correlations were detected between selected outcomes including a moderate and highly significant correlation ( = 0.59,  < 0.0004) between axial hypersensitivity and disc nerve sprouting. These data support this model as a rigorous platform to explore the pathobiology of disc-associated low back pain and to screen treatments.

Axial hypersensitivity is associated with aberrant nerve sprouting in a novel model of disc degeneration in female Sprague Dawley rats.

Oxidative stress due to excess reactive oxygen species (ROS) is related to many chronic illnesses including degenerative disc disease and osteoarthritis. MnTnBuOE-2-PyP (BuOE), a manganese porphyrin analog, is a synthetic superoxide dismutase mimetic that scavenges ROS and has established good treatment efficacy at preventing radiation-induced oxidative damage in healthy cells. BuOE has not been studied in degenerative disc disease applications and only few studies have loaded BuOE into drug delivery systems. The goal of this work is to engineer BuOE microparticles (MPs) as an injectable therapeutic for long-term ROS scavenging.

Encapsulation of Manganese Porphyrin in Chondroitin Sulfate-A Microparticles for Long Term Reactive Oxygen Species Scavenging.

Despite the significant global prevalence of chronic pain, current methods to identify pain therapeutics often fail translation to the clinic. Phenotypic screening platforms rely on modeling and assessing key pathologies relevant to chronic pain, improving predictive capability. Patients with chronic pain often present with sensitization of primary sensory neurons (that extend from dorsal root ganglia [DRG]). During neuronal sensitization, painful nociceptors display lowered stimulation thresholds. To model neuronal excitability, it is necessary to maintain three key anatomical features of DRGs to have a physiologically relevant platform: (1) isolation between DRG cell bodies and neurons, (2) 3D platform to preserve cell-cell and cell-matrix interactions, and (3) presence of native non-neuronal support cells, including Schwann cells and satellite glial cells. Currently, no culture platforms maintain the three anatomical features of DRGs. Herein, we demonstrate an engineered 3D multicompartment device that isolates DRG cell bodies and neurites and maintains native support cells. We observed neurite growth into isolated compartments from the DRG using two formulations of collagen, hyaluronic acid, and laminin-based hydrogels. Further, we characterized the rheological, gelation and diffusivity properties of the two hydrogel formulations and found the mechanical properties mimic native neuronal tissue. Importantly, we successfully limited fluidic diffusion between the DRG and neurite compartment for up to 72 h, suggesting physiological relevance. Lastly, we developed a platform with the capability of phenotypic assessment of neuronal excitability using calcium imaging. Ultimately, our culture platform can screen neuronal excitability, providing a more translational and predictive system to identify novel pain therapeutics to treat chronic pain.

Engineering a multicompartment in vitro model for dorsal root ganglia phenotypic assessment.

Low back pain, knee osteoarthritis, and cancer patients suffer from chronic pain. Aberrant nerve growth into intervertebral disc, knee, and tumors, are common pathologies that lead to these chronic pain conditions. Axonal dieback induced by capsaicin (Caps) denervation has been FDA-approved to treat painful neuropathies and knee osteoarthritis but with short-term efficacy and discomfort. Herein, we propose to evaluate pyridoxine (Pyr), vincristine sulfate (Vcr) and ionomycin (Imy) as axonal dieback compounds for denervation with potential to alleviate pain. Previous literature suggests Pyr, Vcr, and Imy can cause undesired axonal degeneration, but no previous work has evaluated axonal dieback and cytotoxicity on adult rat dorsal root ganglia (DRG) explants. Thus, we performed axonal dieback screening using adult rat DRG explants in vitro with Caps as a positive control and assessed cytotoxicity. Imy inhibited axonal outgrowth and slowed axonal dieback, while Pyr and Vcr at high concentrations produced significant reduction in axon length and robust axonal dieback within three days. DRGs treated with Caps, Vcr, or Imy had increased DRG cytotoxicity compared to matched controls, but overall cytotoxicity was minimal and at least 88% lower compared to lysed DRGs. Pyr did not lead to any DRG cytotoxicity. Further, neither Pyr nor Vcr triggered intervertebral disc cell death or affected cellular metabolic activity after three days of incubation in vitro. Overall, our findings suggest Pyr and Vcr are not toxic to DRGs and intervertebral disc cells, and there is potential for repurposing these compounds for axonal dieback compounds to cause local denervation and alleviate pain.

Identification of compounds that cause axonal dieback without cytotoxicity in dorsal root ganglia explants and intervertebral disc cells with potential to treat pain via denervation.

Rebecca A. Wachs

Department of Biological Systems Engineering

University of Nebraska-Lincoln

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features