Stephen M. Selkirk

Apoptosis of oligodendrocytes in the central nervous system results in rapid focal demyelination

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that presents with variable pathologies that may reflect different disease‐causing mechanisms. Existing animal models of MS induce pathology using either local injection of gliotoxins or stimulation of the immune system with myelin‐related peptides. In none of these models is the primary cellular target well characterized, and although demyelination is a hallmark pathological feature in MS, it is unclear to what extent this reflects local oligodendrocyte loss. To unambiguously identify the effects of oligodendrocyte death in the absence of inflammatory stimulation, we developed a method for experimentally inducing programmed cell death selectively in mature oligodendrocytes and assessed the effects on demyelination, immunological stimulation, and gliosis. The resulting pathology is discussed relative to observed MS pathologies.

Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30–50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

The roles of blood-derived macrophages and resident microglia in the neuroinflammatory response to implanted intracortical microelectrodes.

Resident microglia and blood-borne macrophages have both been implicated to play a dominant role in mediating the neuroinflammatory response affecting implanted intracortical microelectrodes. However, the distinction between each cell type has not been demonstrated due to a lack of discriminating cellular markers. Understanding the subtle differences of each cell population in mediating neuroinflammation can aid in determining the appropriate therapeutic approaches to improve microelectrode performance. Therefore, the goal of this study is to characterize the role of infiltrating blood-derived cells, specifically macrophages, in mediating neuroinflammation following intracortical microelectrode implantation. Interestingly, we found no correlation between microglia and neuron populations at the microelectrode-tissue interface. On the other hand, blood-borne macrophages consistently dominated the infiltrating cell population following microelectrode implantation. Most importantly, we found a correlation between increased populations of blood-derived cells (including the total macrophage population) and neuron loss at the microelectrode-tissue interface. Specifically, the total macrophage population was greatest at two and sixteen weeks post implantation, at the same time points when we observed the lowest densities of neuronal survival in closest proximity to the implant. Together, our results suggest a dominant role of infiltrating macrophages, and not resident microglia, in mediating neurodegeneration following microelectrode implantation.

An organotypic spinal cord slice culture model to quantify neurodegeneration

Activated microglia cells have been implicated in the neurodegenerative process of Alzheimers disease, Parkinsons disease, Huntingtons disease, amyotrophic lateral sclerosis, and multiple sclerosis; however, the precise roles of microglia in disease progression are unclear. Despite these diseases having been described for more than a century, current FDA approved therapeutics are symptomatic in nature with little evidence to supporting a neuroprotective effect. Furthermore, identifying novel therapeutics remains challenging due to undetermined etiology, a variable disease course, and the paucity of validated targets. Here, we describe the use of a novel ex vivo spinal cord culture system that offers the ability to screen potential neuroprotective agents, while maintaining the complexity of the in vivo environment. To this end, we treated spinal cord slice cultures with lipopolysaccharide and quantified neuron viability in culture using measurements of axon length and FluoroJadeC intensity. To simulate a microglia-mediated response to cellular debris, antigens, or implanted materials/devices, we supplemented the culture media with increasing densities of microspheres, facilitating microglia-mediated phagocytosis of the particles, which demonstrated a direct correlation between the phagocytic activities of microglia and neuronal health. To validate our models capacity to accurately depict neuroprotection, cultures were treated with resveratrol, which demonstrated enhanced neuronal health. Our results successfully demonstrate the use of this model to reproducibly quantify the extent of neurodegeneration through the measurement of axon length and FluoroJadeC intensity, and we suggest this model will allow for accurate, high-throughput screening, which could result in expedited success in translational efficacy of therapeutic agents to clinical trials.

Foamy virus as a gene transfer vector to the central nervous system

Engineered foamy virus (FV) vectors have been lauded for their superior safety profiles and stable integration patterns compared to their gammaretroviral counterparts. The drawback has been the belief that FV incorporation is cell cycle-dependent, thereby limiting its utility in post-mitotic tissues such as the central nervous system. In this brief communication, we challenged this theory by examining FV in vivo. We injected equal titers of FV and lentivirus (LV) into the adult rat brain and found that at 1 week, FV transduced a significantly greater volume of bromodeoxyuridine (BrdU)-negative brain parenchyma than did LV. By 8 weeks, however, the volume of transduced tissue was greatly reduced—comparable to LV—and restricted to BrdU+. Taken together, these data implicate a role for FV in short-term gene delivery strategies to the CNS.

Early and innovative symptomatic care to improve quality of life of ALS patients at Cleveland VA ALS Center.

INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology. However, there is good evidence to validate the theory that military service predisposes the individual to the future development of the disease [1]. As a direct result of these data, in 2008 ALS became a presumptively compensable illness for all veterans with 90 days or more of continuously active service in the military. This decision led to an influx of ALS patients into the Department of Veterans Affairs (VA) healthcare system and the development of a nationwide plan of care [2]. The implementation of this plan at the Cleveland VA Spinal Cord Injury (SCI) Division has been challenging. However, it has offered the opportunity to review current practice parameters that dictate the care of ALS patients in a new context. Using these parameters and considering the strengths and weakness of the VA healthcare system, we have made significant improvements to current practice and applied novel technology to care for ALS patients. Herein we describe these innovations and the foundation we have established to provide high quality ALS care. BACKGROUND ALS is a terminal neurodegenerative disorder. The management of patients with ALS centers almost entirely around symptomatic care. Although many studies have examined the role of particular interventions in prolonging survival after diagnosis or decreasing the rate of decline in physical ability, perhaps the most important studies guiding intervention have investigated quality of life (QoL) over the course of the disease. In 1999, the American Academy of Neurology (AAN) published the first set of formal practice parameters based on a review of the literature [3]. These evidence- based practice parameters were revised in 2009 [4] and included important updates on how care is administered, as well as recommendations regarding the use of the first disease-modifying agent available. The European Federation of Neurological Societies (EFNS) published parallel recommendations in 2005 [5], with revised and updated practice guidelines published in 2012 [6]. A third publication in 2007 also summarized current evidence-based recommendations for care of ALS patients [7]. These three sets of recommendations are nearly identical and in many instances lack precise details in terms of when and how to implement care. This reveals a need for further studies to answer basic questions regarding interventions, such as when a percutaneous endoscopic gastrostomy (PEG) tube should be placed in a patient or how early respiratory support should be provided. This information would help standardize practice parameters and allow for a higher level of symptomatic care in patients with ALS. INNOVATIVE CARE IN VA SYSTEM Early Noninvasive Positive Pressure Ventilation for ALS Patients The majority of patients with ALS will die from respiratory failure [8]. Furthermore, during the course of the disease, respiratory function correlates with QoL and patient survival [9-11]. The survival benefits from noninvasive positive pressure ventilation (NIPPV) supersede those obtained from riluzole or PEG tube insertion [12]. It follows, logically, that aggressive management of respiratory compromise should be part of a comprehensive plan not simply to prolong life (because some patients prefer not to have their life prolonged) but also as a mechanism to improve QoL. The latter goal, which is more consistent with our mission as an ALS center, is supported by several well-designed studies [12-13]. These studies suggest that NIPPV improves QoL by improving a patients energy, concentration, and sleep quality, decreasing daytime somnolence and physical fatigue. NIPPV also improves lung compliance [14] and decreases the rate of decline in vital capacity [15-16]. Despite this evidence, the ALS Clinical Assessment Research and Education database has documented underutilization of NIPPV by patients with ALS [17]. …

Guest Editorial: Developing a system of care for ALS patients at the Louis Stokes Cleveland VA Medical Center, Spinal Cord Injury Division

INTRODUCTION Amyotrophic lateral sclerosis (ALS), also referred to as Lou Gehrigs disease, is a fatal motor neuron disease characterized by rapidly progressive weakness, dysarthria, dysphagia, and respiratory failure. Fifty percent of patients will die within 3 years of diagnosis and 95 percent within 5 years-- mainly from respiratory failure [1]. The only available treatment, riluzole, has limited efficacy, prolonging survival by 3 months [2-3]. Although familial cases have led to the identification of associated genetic loci, the etiology of sporadic cases is unknown. There is, however, an association between military service and the development of ALS [4]. Hence, in 2008, ALS became a presumptively compensable illness for all Veterans with 90 days or more of continuously active service in the military. This provides the Veteran full access to medical care, medication management, durable medical equipment (DME), and monthly financial compensation. Since this decision in 2008, there has been a growing appreciation for the level of Department of Veterans Affairs (VA) benefits associated with the diagnosis of ALS, and subsequently more Veterans have entered the VA medical system with a diagnosis of ALS, both at the Cleveland VA Medical Center and in Veterans Integrated Service Network (VISN) 10 in Ohio. Between 2005 and 2010, the spinal cord injury (SCI) outpatient clinic treated 19 patients with ALS, compared with 60 patients in years 2011 to 2012 alone. This increased awareness of VA benefits at the outset resulted in Veterans presenting at their first evaluation with advanced stages of ALS, characterized by speech, swallowing, and breathing problems. We found many of the Veterans and their family members psychologically unprepared to cope with the disease progression, often times expressing anger or fear during the initial evaluation. Most patients lacked a clearly defined plan for their future care. The rapid progression of this disease resulted in both the Veteran and the staff being unprepared to cope with the need to make imminent medical decisions regarding percutaneous nutrition, noninvasive versus invasive ventilation, and other complex end-of-life decisions. The processes that were in place to provide care for patients with SCI, although interdisciplinary in nature, lacked the speed and complexity required to care for patients with a rapidly progressive neurodegenerative disorder. The realization that our current paradigm of care needed to be modified to provide excellent medical services to patients with ALS led to the development of a joint workgroup between the SCI and the Neurology Services. The overarching goal of this workgroup was to address care needs and improve processes for Veterans with ALS. This team recognized the complexity of ALS care and looked for solutions to address the unique needs of Veterans with ALS, which included rapid medical management of respiratory issues, nutritional needs, psychological support, education, and providing end-of-life care while expanding the interdisciplinary approach already in place on the SCI service. BACKGROUND ALS and Veterans ALS occurs in all major countries worldwide, with higher rates in the United States and England [5]. Approximately 5,600 new cases are diagnosed in the United States each year. The estimated prevalence in the United States is 5 per 100,000 people [6]. About 5 percent of cases are attributed to the inheritance of genetic defects, many of which have been identified; most of these follow an autosomal dominant inheritance pattern. The remaining 95 percent of cases are considered sporadic (nonfamilial) and of unknown etiology. Interestingly, cohorts have been identified suggesting possible causative relationships between the disease and various factors, including pesticide exposure [7], head trauma [8], strenuous physical activity [9], and heavy metal exposure [10]. …

Targeting CD14 on blood derived cells improves intracortical microelectrode performance

Intracortical microelectrodes afford researchers an effective tool to precisely monitor neural spiking activity. Additionally, intracortical microelectrodes have the ability to return function to individuals with paralysis as part of a brain computer interface. Unfortunately, the neural signals recorded by these electrodes degrade over time. Many strategies which target the biological and/or materials mediating failure modes of this decline of function are currently under investigation. The goal of this study is to identify a precise cellular target for future intervention to sustain chronic intracortical microelectrode performance. Previous work from our lab has indicated that the Cluster of Differentiation 14/Toll-like receptor pathway (CD14/TLR) is a viable target to improve chronic laminar, silicon intracortical microelectrode recordings. Here, we use a mouse bone marrow chimera model to selectively knockout CD14, an innate immune receptor, from either brain resident microglia or blood-derived macrophages, in order to understand the most effective targets for future therapeutic options. Using single-unit recordings we demonstrate that inhibiting CD14 from the blood-derived macrophages improves recording quality over the 16 week long study. We conclude that targeting CD14 in blood-derived cells should be part of the strategy to improve the performance of intracortical microelectrodes, and that the daunting task of delivering therapeutics across the blood-brain barrier may not be needed to increase intracortical microelectrode performance.

Inhibition of the cluster of differentiation 14 innate immunity pathway with IAXO-101 improves chronic microelectrode performance

OBJECTIVE Neuroinflammatory mechanisms are hypothesized to contribute to intracortical microelectrode failures. The cluster of differentiation 14 (CD14) molecule is an innate immunity receptor involved in the recognition of pathogens and tissue damage to promote inflammation. The goal of the study was to investigate the effect of CD14 inhibition on intracortical microelectrode recording performance and tissue integration. APPROACH Mice implanted with intracortical microelectrodes in the motor cortex underwent electrophysiological characterization for 16 weeks, followed by endpoint histology. Three conditions were examined: (1) wildtype control mice, (2) knockout mice lacking CD14, and (3) wildtype control mice administered a small molecule inhibitor to CD14 called IAXO-101. MAIN RESULTS The CD14 knockout mice exhibited acute but not chronic improvements in intracortical microelectrode performance without significant differences in endpoint histology. Mice receiving IAXO-101 exhibited significant improvements in recording performance over the entire 16 week duration without significant differences in endpoint histology. SIGNIFICANCE Full removal of CD14 is beneficial at acute time ranges, but limited CD14 signaling is beneficial at chronic time ranges. Innate immunity receptor inhibition strategies have the potential to improve long-term intracortical microelectrode performance.

Delivering tertiary centre specialty care to ALS patients via telemedicine: a retrospective cohort analysis

Abstract Objective: This study was undertaken to determine if ALS patients evaluated via telemedicine received the same quality of care as patients evaluated by traditional face-to-face encounters. Methods: A retrospective cohort study design was used. Participants were patients diagnosed with ALS that received multidisciplinary care at the tertiary Cleveland VA ALS Centre between 1 March 2008- and 31 anuary 2015. Participants were not randomised, but chose telemedicine based on preference, disability level or distance from the clinic. Telemedicine in this study consisted of a video conferencing platform enabling remote rather than face-to-face encounters with participants. Results: There was no significant association between receiving quality ALS care and the mode of care. There was a trend for telemedicine patients to utilise home health care less often than those that received clinic care (AOR 0.50; 95% CI 0.16–1.59). There was no significant difference in survival time between the two groups (log-rank test χ2 = 3.62, df = 1, p = 0.05). Patients receiving telemedicine had a higher probability of remaining stable or having <30% decrease in ALSFRS-R over time (log-rank test χ2 = 4.46, df = 1, p = 0.03). There was a significantly lower risk of disease progression for patients receiving telemedicine (HR = 0.39, 95% CI = 0.16–0.93). Conclusions: Patients managed by telemedicine received the same quality of care and had similar outcomes to those patients seen via traditional face-to-face encounters. Telemedicine is an effective platform for delivering high quality tertiary ALS care.