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1.  Positively Charged Oligo[Poly(Ethylene Glycol) Fumarate] Scaffold Implantation Results in a Permissive Lesion Environment after Spinal Cord Injury in Rat 
Tissue Engineering. Part A  2015;21(13-14):2099-2114.
Positively charged oligo[poly(ethylene glycol) fumarate] (OPF+) scaffolds loaded with Schwann cells bridge spinal cord injury (SCI) lesions and support axonal regeneration in rat. The regeneration achieved is not sufficient for inducing functional recovery. Attempts to increase regeneration would benefit from understanding the effects of the scaffold and transplanted cells on lesion environment. We conducted morphometric and stereological analysis of lesions in rats implanted with OPF+ scaffolds with or without loaded Schwann cells 1, 2, 3, 4, and 8 weeks after thoracic spinal cord transection. No differences were found in collagen scarring, cyst formation, astrocyte reactivity, myelin debris, or chondroitin sulfate proteoglycan (CSPG) accumulation. However, when scaffold-implanted animals were compared with animals with transection injuries only, these barriers to regeneration were significantly reduced, accompanied by increased activated macrophages/microglia. This distinctive and regeneration permissive tissue reaction to scaffold implantation was independent of Schwann cell transplantation. Although the tissue reaction was beneficial in the short term, we observed a chronic fibrotic host response, resulting in scaffolds surrounded by collagen at 8 weeks. This study demonstrates that an appropriate biomaterial scaffold improves the environment for regeneration. Future targeting of the host fibrotic response may allow increased axonal regeneration and functional recovery.
PMCID: PMC4507127  PMID: 25891264
2.  A Safety Study on Intrathecal Delivery of Autologous Mesenchymal Stromal Cells in Rabbits Directly Supporting Phase I Human Trials 
Transfusion  2014;55(5):1013-1020.
There are no effective treatments that slow the progression of neurodegenerative diseases. A major challenge of treatment in neurodegenerative diseases is appropriate delivery of pharmaceuticals into the cerebrospinal fluid (CSF) of affected individuals. Mesenchymal stromal cells (MSCs – either naïve or modified) are a promising therapy in neurodegenerative diseases and may be delivered directly into the CSF where they can reside for months. In this preclinical study, we evaluated the safety of intrathecal autologous MSCs in a rabbit model.
Autologous adipose-derived MSCs (or a-CSF) were delivered intrathecally, either with single or repeated injections into the foramen magnum of healthy rabbits, and monitored for 4 and 12 weeks, respectively.
Rabbits tolerated injections well and no definitive MSC-related side effects were observed apart from three rabbits that had delayed death secondary to traumatic foramen magnum puncture. Functional assessments and body weights were equivalent between groups. Gross pathology and histology did not reveal any abnormalities or tumor growth. Complete blood count (CBC) data were normal and there were no differences in CSF IL-6 levels in all groups tested.
Our data suggest that intrathecal delivery of autologous MSCs is safe in a rabbit model. Data from this study has supported two successful Investigational New Drug (IND) applications to the FDA, resulting in the initiation of two clinical trials using autologous MSCs in amyotrophic lateral sclerosis and multiple system atrophy.
PMCID: PMC4428970  PMID: 25413276
Amyotrophic lateral sclerosis; cell therapy; intrathecal injection; multiple system atrophy; mesenchymal stromal cell; MSC; rabbit; safety study
3.  Comparison of Cellular Architecture, Axonal Growth, and Blood Vessel Formation Through Cell-Loaded Polymer Scaffolds in the Transected Rat Spinal Cord 
Tissue Engineering. Part A  2014;20(21-22):2985-2997.
The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF+) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF+ scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF+ scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.
PMCID: PMC4229864  PMID: 24854680
4.  Candidate pathway-based genetic association study of Platinum and Platinum-Taxane Related Toxicity in a Cohort of Primary Lung Cancer Patients 
Chemotherapy-induced peripheral neuropathy (CIPN) is a common toxicity secondary to chemotherapy. Genetic factors may be important in predisposing patients to this adverse effect.
Patients and Methods
We studied 950 primary lung cancer patients, who received platinum or platinum-combination drug chemotherapy and who had DNA available for study. We analyzed epidemiological risk factors in 279 CIPN patients and 456 non-CIPN patients and genetic risk factors in 141 CIPN patients and 259 non-CIPN patients. The risk factors studied included demographic, diagnostic, and treatment data, as well as 174 tag SNPs (single nucleotide polymorphisms) across 43 candidate genes in the glutathione, cell cycle, DNA repair, cell signaling, and apoptosis pathways.
Patients who had diabetes mellitus were more likely to have CIPN (p=0.0002). Other epidemiologic risk factors associated with CIPN included number of cycles (p=0.0004) and type of concurrent chemotherapy (p<0.001) . SNPs most associated with CIPN were in glutathione peroxidase 7 (GPX7) gene (p values 0.0015 and 0.0028, unadjusted and adjusted) and in ATP-binding cassette sub-family C member 4 (ABCC4) gene (p values 0.037 and 0.006, unadjusted and adjusted). We also found other suggestive associations in methyl-o-guanine-methyl-transferase (MGMT) and glutathione-S-transferase (GST) isoforms.
Epidemiological and genetic risk factors associated with CIPN in this cohort, included the type of chemotherapy drug, intensity of chemotherapy treatment, and genes known to be associated with chemotherapy resistance. These findings suggest that differentiating between cytotoxic and neurotoxic mechanisms of chemotherapy drugs is challenging but represents an important step toward individualized therapy and improving quality of life for patients.
PMCID: PMC4334320  PMID: 25586538
Chemotherapy induced peripheral neuropathy; genetic; single nucleotide polymorphism; platinum drugs; paclitaxel; diabetes
5.  An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster 
Fly  2013;7(3):187-192.
We have developed a novel model system in Drosophila melanogaster to study chemotherapy-induced neurotoxicity in adult flies. Neurological deficits were measured using a manual geotactic climbing assay. The manual assay is commonly used; however, it is laborious, time-consuming, subject to human error and limited to observing one sample at a time. We have designed and built a new automated fly-counting apparatus that uses a “video capture-particle counting technology” to automatically measure 10 samples at a time, with 20 flies per sample. Climbing behavior was assessed manually, as in our previous studies, and with the automated apparatus within the same experiment yielding statistically similar results. Both climbing endpoints as well as the climbing rate can be measured in the apparatus, giving the assay more versatility than the manual assay. Automation of our climbing assay reduces variability, increases productivity and enables high throughput drug screens for neurotoxicity.
PMCID: PMC4049852  PMID: 23695893
automated; Drosophila; geotactic; climbing; neurotoxicity; apparatus; chemotherapy; cisplatin
6.  Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy 
Annals of neurology  2014;76(5):727-737.
Mutations in Charcot-Marie-Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy—considered “acquired” in medical parlance—is unknown. Chemotherapy induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as clinical model to investigate the association of non-CMT polyneuropathy with CMT genes.
269 neurologically asymptomatic cancer patients were enrolled in the clinical trial Alliance N08C1 to receive the neurotoxic drug paclitaxel, while undergoing prospective assessments for polyneuropathy. 49 CMT genes were analyzed by targeted massively parallel sequencing of genomic DNA from patient blood.
119 (of 269) patients were identified from the two ends of the polyneuropathy phenotype distribution: patients that were most- and least susceptible to paclitaxel polyneuropathy. The CMT gene PRX was found to be deleteriously mutated in patients who were susceptible to CIPN but not in controls (p=8×10−3). Genetic variation in another CMT gene, ARHGEF10, was highly significantly associated with CIPN (p=5×10−4). Three non-synonymous recurrent single nucleotide variants contributed to the ARHGEF10 signal: rs9657362, rs2294039, and rs17683288. Of these, rs9657362 had the strongest effect (odds ratio of 4.8, p=4×10−4).
The results reveal an association of CMT gene allelic variability with susceptibility to CIPN. The findings raise the possibility that other acquired polyneuropathies may also be co-determined by genetic etiological factors, of which some may be related to genes already known to cause the phenotypically related Mendelian disorders of CMT.
PMCID: PMC4388308  PMID: 25164601
7.  Peripheral Neuropathy Due to Vitamin Deficiency, Toxins, and Medications 
Continuum : Lifelong Learning in Neurology  2014;20(5 Peripheral Nervous System Disorders):1293-1306.
Purpose of Review:
Peripheral neuropathies secondary to vitamin deficiencies, medications, or toxins are frequently considered but can be difficult to definitively diagnose. Accurate diagnosis is important since these conditions are often treatable and preventable. This article reviews the key features of different types of neuropathies caused by these etiologies and provides a comprehensive list of specific agents that must be kept in mind.
Recent Findings:
While most agents that cause peripheral neuropathy have been known for years, newly developed medications that cause peripheral neuropathy are discussed.
Peripheral nerves are susceptible to damage by a wide array of toxins, medications, and vitamin deficiencies. It is important to consider these etiologies when approaching patients with a variety of neuropathic presentations; additionally, etiologic clues may be provided by other systemic symptoms. While length-dependent sensorimotor axonal peripheral neuropathy is the most common presentation, several examples present in a subacute severe fashion, mimicking Guillain-Barré syndrome.
PMCID: PMC4208100  PMID: 25299283
8.  Hemisection spinal cord injury in rat: The value of intraoperative somatosensory evoked potential monitoring 
Journal of neuroscience methods  2012;211(2):179-184.
Techniques used to produce partial spinal cord injuries in animal models have the potential for creating variability in lesions. The amount of tissue affected may influence the functional outcomes assessed in the animals. The recording of somatosensory evoked potentials (SSEPs) may be a valuable tool for assessing the extent of lesion applied in animal models of traumatic spinal cord injury (SCI). Intraoperative tibial SSEP recordings were assessed during surgically induced lateral thoracic hemisection SCI in Sprague-Dawley rats. The transmission of SSEPs, or lack thereof, was determined and compared against the integrity of the dosal funiculi on each side of the spinal cord upon histological sectioning. An association was found between the presence of an SSEP signal and presence of intact dorsal funiculus tissue. The relative risk is 4.50 (95% confidence interval: 1.83 to 11.08) for having an intact dorsal funiculus when the ipsilateral SSEP was present compared to when it was absent. Additionally, the amount of spared spinal cord tissue correlates with final functional assessments at nine weeks post injury: BBB (linear regression, R2 = 0.618, p <0.001) and treadmill test (linear regression, R2 = 0.369, p = 0.016). Therefore, we propose intraoperative SSEP monitoring as a valuable tool to assess extent of lesion and reduce variability between animals in experimental studies of SCI.
PMCID: PMC3491113  PMID: 22960163
electrophysiology; pathologic correlation; incomplete spinal cord injury; exercise; functional outcome
9.  Translating stem cell research to the clinic: a primer on translational considerations for your first stem cell protocol 
Over the last two decades, a new therapeutic paradigm has emerged which has changed the way debilitating diseases may be treated in the future. Instead of using small-molecule drugs and devices to ameliorate the symptoms of disease, clinicians may harness the therapeutic power of cells to regenerate and cure diseases which currently represent a major unmet medical need. Advancements in the scientific knowledge of stem cell biology, along with highly encouraging preclinical proof-of-concept studies, in the last several years have served as a launch pad for testing such therapeutics in humans with life-threatening diseases. However, translating basic research findings into human therapy has not been straightforward and has presented many scientific, clinical, and regulatory challenges for scientists and clinicians. In this article, we provide a guidance framework for investigators for the design of early-phase clinical studies using stem cell-based therapeutics. Furthermore, important trial parameters and design features which must be considered before regulatory submission of such studies are highlighted.
PMCID: PMC4546289  PMID: 26296990
10.  A systematic review of animal models used to study nerve regeneration in tissue-engineered scaffolds 
Biomaterials  2012;33(32):8034-8039.
Research on biomaterial nerve scaffolds has been carried out for 50 years. Only three materials (collagen, polycaprolactone and polyglycollic acid) have progressed to clinical use. Pre-clinical animal models are critical for testing nerve scaffolds prior to implementation in clinical practice. We have conducted a systematic review of 416 reports in which animal models were used for evaluation of nerve regeneration into synthetic conduits. A valid animal model of nerve regeneration requires it to reproduce the specific processes that take place in regeneration after human peripheral nerve injury. No distinct animal species meets all the requirements for an ideal animal model. Certain models are well suited for understanding regenerative neurobiology while others are better for pre-clinical evaluation of efficacy. The review identified that more than 70 synthetic materials were tested in eight species using 17 different nerves. Nerve gaps ranged from 1 to 90 mm. More than 20 types of assessment methodology were used with no standardization of methods between any of the publications. The review emphasizes the urgent need for standardization or rationalization of animal models and evaluation methods for studying nerve repair.
PMCID: PMC3472515  PMID: 22889485
Peripheral nerve injury; peripheral nerve repair; nerve tube; nerve scaffold; biodegradable
11.  Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair 
Biomaterials  2011;32(32):8077-8086.
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
PMCID: PMC3163757  PMID: 21803415
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
12.  Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes 
Neurosurgery  2008;63(1):144-155.
Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.
Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.
In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.
Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.
PMCID: PMC3463233  PMID: 18728579
misdirection; axon targeting; double labeling; peripheral nerve regeneration; rat sciatic nerve model; retrograde tracing
13.  Sustained Delivery of Dibutyryl Cyclic Adenosine Monophosphate to the Transected Spinal Cord Via Oligo [(Polyethylene Glycol) Fumarate] Hydrogels 
Tissue Engineering. Part A  2011;17(9-10):1287-1302.
This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.
PMCID: PMC3079174  PMID: 21198413
14.  Cisplatin induced Mitochondrial DNA Damage In Dorsal Root Ganglion Neurons 
Neurobiology of disease  2010;41(3):661-668.
Cisplatin is a platinum-based chemotherapeutic agent that induces peripheral neuropathy in 30% of patients. Peripheral neuropathy is the dose limiting side effect, which has no preventative therapy. We have previously shown that cisplatin induces apoptosis in dorsal root ganglion (DRG) sensory neurons by covalently binding to nuclear DNA (nDNA), resulting in DNA damage, subsequent p53 activation and Bax-mediated apoptosis via the mitochondria. We now demonstrate that cisplatin also directly binds to mitochondrial DNA (mtDNA) with the same binding affinity as nDNA. Cisplatin binds 1 platinum molecule per 2166 mtDNA base pairs and 1 platinum molecule per 3800 nDNA base pairs. Furthermore, cisplatin treatment inhibits mtDNA replication as detected by 5-bromo-2'-deoxy-uridine (BrdU) incorporation and inhibits transcription of mitochondrial genes. The relative reduction in mtDNA transcription is directly related to the distance the gene is located from the transcription initiation point, which implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradation in vitro and in vivo. Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity separate from the established nDNA damage pathway.
PMCID: PMC3031677  PMID: 21145397
Chemotherapy: Neuropathy; DRG Neurons; Cisplatin; Mitochondria: DNA and DNA damage
The Salk inactivated poliovirus vaccine is one of the most rapid examples of bench-to-bedside translation in medicine. In the span of 6 years, the key basic lab discoveries facilitating the development of the vaccine were made, optimization and safety testing was completed in both animals and human volunteers, the largest clinical trial in history of 1.8 million children was conducted, and the results were released to an eagerly awaiting public. Such examples of rapid translation can not only offer clues to what factors can successfully drive and accelerate the translational process but also what mistakes can occur (and thus should be avoided) during such a swift process. In this commentary, we explore the translational path of the Salk polio vaccine from the key basic science discoveries to the 1954 Field Trials and delve into the scientific and sociopolitical factors that aided in its rapid development. Moreover, we look at the Cutter and Wyeth incidents after the vaccine’s approval and the errors that led to them.
PMCID: PMC2928990  PMID: 20718820
16.  Rituximab for Non-Hodgkin’s Lymphoma: A Story of Rapid Success in Translation 
Translational stories range from straightforward to complex. In this commentary, the story of the rapid and successful translation of rituximab therapy for the treatment of non-Hodgkin’s lymphoma (NHL) is examined. Development of this monoclonal antibody therapy began in the late 1980s. In 1994, rituximab received its first approval for the treatment of NHL by the United States Food and Drug Administration (FDA). Rituximab has since been approved for additional indications and has transformed medical practice. However, the social and political implications of these rapid successes are only beginning to become clear. In this commentary, key events in the rapid translation of rituximab from the bench to bedside are highlighted and placed into this historical framework. To accomplish this, the story of rituximab is divided into the following six topics, which we believe to be widely applicable to case studies of translation: (1) underlying disease, (2) key basic science, (3) key clinical studies in translation, (4) FDA approval process, (5) changes to medical practice, and (6) the social and political influences on translation.
PMCID: PMC4264563  PMID: 24528902
oncology; hematology; hematopathology; lymphoma; leukemia; immunotherapy
17.  Bortezomib alters microtubule polymerization and axonal transport in rat dorsal root ganglion neurons 
Neurotoxicology  2013;0:10.1016/j.neuro.2013.09.001.
Bortezomib is part of a newer class of chemotherapeutic agents whose mechanism of action is inhibition of the proteasome-ubiquitination system. Primarily used in multiple myeloma, bortezomib causes a sensory-predominant axonal peripheral neuropathy in approximately 30% of patients. There are no established useful preventative agents for bortezomib-induced peripheral neuropathy (BIPN), and the molecular mechanisms of BIPN are unknown. We have developed an in vitro model of BIPN using rat dorsal root ganglia neuronal cultures. At clinically–relevant dosages, bortezomib produces a sensory axonopathy as evidenced by whole explant outgrowth and cell survival assays. This sensory axonopathy is associated with alterations in tubulin and results in accumulation of somatic tubulin without changes in microtubule ultrastructure. Furthermore, we observed an increased proportion of polymerized tubulin, but not total or acetylated tubulin, in bortezomib-treated DRG neurons. Similar findings are observed with lactacystin, an unrelated proteasome-inhibitor, which argues for a class effect of proteasome inhibition on dorsal root ganglion neurons. Finally, there is a change in axonal transport of mitochondria induced by bortezomib in a time-dependent fashion. In summary, we have developed an in vitro model of BIPN that recapitulates the clinical sensory axonopathy; this model demonstrates that bortezomib induces an alteration in microtubules and axonal transport. This robust model will be used in future mechanistic studies of BIPN and its prevention.
PMCID: PMC3844018  PMID: 24035926
bortezomib; chemotherapy-induced peripheral neuropathy; peripheral neuropathy; neurotoxicity; dorsal root ganglia; rat
18.  Importance of the vasculature in cyst formation after spinal cord injury 
Journal of neurosurgery. Spine  2009;11(4):432-437.
Glial scar and cystic formation greatly contribute to the inhibition of axonal regeneration after spinal cord injury (SCI). Attempts to promote axonal regeneration are extremely challenging in this type of hostile environment. The objective of this study was to examine the surgical methods that may be used to assess the factors that influence the level of scar and cystic formation in SCI.
In the first part of this study, a complete transection was performed at vertebral level T9–10 in adult female Sprague-Dawley rats. The dura mater was either left open (control group) or was closed using sutures or hyaluronic acid. In the second part of the study, complete or subpial transection was performed, with the same dural closure technique applied to both groups. Histological analysis of longitudinal sections of the spinal cord was performed, and the percentage of scar and cyst formation was determined.
Dural closure using sutures resulted in significantly less glial scar formation (p = 0.0248), while incorporation of the subpial transection surgical technique was then shown to significantly decrease cyst formation (p < 0.0001).
In this study, the authors demonstrated the importance of the vasculature in cyst formation after spinal cord trauma and confirmed the importance of dural closure in reducing glial scar formation.
PMCID: PMC2981802  PMID: 19929340
traumatic spinal cord injury; vascular injury; glial cell response to injury
19.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of Neurotrauma  2009;26(10):1759-1771.
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
20.  Neural Stem Cell– and Schwann Cell–Loaded Biodegradable Polymer Scaffolds Support Axonal Regeneration in the Transected Spinal Cord 
Tissue Engineering. Part A  2009;15(7):1797-1805.
Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), βIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n = 17), SCs (n = 17), and no cells (control) (n = 8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and βIII tubulin–positive cells. GFAP-positive cells were only seen at the spinal cord–scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.
PMCID: PMC2792101  PMID: 19191513
21.  Key changes in denervated muscles and their impact on regeneration and reinnervation 
Neural Regeneration Research  2014;9(20):1796-1809.
The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.
PMCID: PMC4239769  PMID: 25422641
nerve regeneration; denervation; reinnervation; fibrillation potential; muscle fiber conduction velocity; muscle fiber diameter; maximal isometric force; neuromuscular junction; gene expression; neural regeneration
22.  Rigid Fixation of the Spinal Column Improves Scaffold Alignment and Prevents Scoliosis in the Transected Rat Spinal Cord 
Spine  2008;33(24):E914-E919.
Study Design
A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging.
To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation.
Summary of Background Data
Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration.
Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery.
Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 ± 2.03° was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 ± 0.75° (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 ± 1.68°) compared with the unfixed group (10.13 ± 1.46°). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 ± 1.23°) compared with the unfixed group (11 ± 2.82°) (P = 0.0143).
Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.
PMCID: PMC2773001  PMID: 19011531
spine fixation; transection spinal cord injury; scaffold; scoliosis
23.  Axon Regeneration through Scaffold into Distal Spinal Cord after Transection 
Journal of neurotrauma  2009;26(10):1759-1771.
We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.
PMCID: PMC2763055  PMID: 19413501
axonal tracing; biodegradable polymers; Fast Blue; Schwann cells; spinal cord injury
24.  The Regulation of Apoptosis by the Downstream Regulatory Element Antagonistic Modulator/Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Through Interactions with Hexokinase I 
The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.
PMCID: PMC3640266  PMID: 23524266
Apoptosis; DREAM; calsenilin; KChIP3; hexokinase I; neuronal calcium sensor proteins; EF-hand proteins; calcium
25.  Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: In vitro characterization and application in polycaprolactone fumarate nerve conduits 
Acta biomaterialia  2011;8(2):511-518.
Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of VEGF encapsulating poly-lactic-co-glycolic acid (PLGA) microspheres and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of the microspheres with a diameter < 53 µm. VEGF content measured by ELISA indicated 93.79 ±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24 hours, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation confirming that VEGF retained its bioactivity through the 4-week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold, robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system.
PMCID: PMC3972821  PMID: 22019759
microsphere; poly-lactic co-glycolic acid; vascular endothelial growth factor; bioactivity; biodegradation; nerve guide

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