Large-gap peripheral nerve repair using xenogeneic transplants in rhesus macaques.

Surgical intervention is required to successfully treat severe, large-gap (≥4 cm) peripheral nerve injuries. However, all existing treatments have shortcomings and an alternative to the use of autologous nerves is needed. Human and porcine nerves are physiologically similar, with comparable dimensions and architecture, presence and distribution of Schwann cells, and conserved features of the extracellular matrix (ECM). We report the repair of fully transected radial nerves in 10 Rhesus Macaques using viable, whole sciatic nerve from genetically engineered (GalT-KO), designated pathogen free (DPF) porcine donors. This resulted in the regeneration of the transected nerve, and importantly, recovery of wrist extension function, distal muscle reinnervation, and recovery of nerve conduction velocities and compound muscle action potentials similar to autologous controls. We also demonstrate the absence of immune rejection, systemic porcine cell migration, and detectable residual porcine material. Our preliminary findings support the safety and efficacy of viable porcine nerve transplants, suggest the interchangeable therapeutic use of cross-species cells, and highlight the broader clinical potential of xenotransplantation.

Preclinical nerve conduction: Nerve battery options for primate studies.

Electrophysiological neurodiagnostic tests of nerve conduction (NC) are key assays included in preclinical safety and toxicology programs to assess the peripheral neuropathy (PN) liability of a new drug. Despite their increased use, standardization of nerve conduction studies (NCS) is lacking in the preclinical space, with limited regulatory guidelines stipulating type and number of nerves or minimum combinations appropriate for each stage of drug development or indication. Detection of subtle peripheral toxicities depends on choosing appropriate nerve targets for testing, especially when functional changes remain above the lower limit of normal values. To support robust preclinical toxicology study designs, the current short communication provides options and recommendations for selecting peripheral nerves for clinically translatable nerve conduction batteries applicable to toxicology and gene therapy, with a focus on primate models. A comprehensive compilation of accessible nerve locations is offered including lower and upper extremity motor nerves, and sensory nerves with origin at multiple DRG levels. Rankings of technique difficulty and repeatability across serial collections are presented for each assay informed by serial nerve conduction from 500 adult primates. The goal of this communication is to support the standardization and preclinical implementation of this important assay.

Characterization of AAV-mediated dorsal root ganglionopathy.

Recent studies in non-human primates administered recombinant adeno-associated viruses (rAAVs) have shown lesions in the dorsal root ganglia (DRG) of unknown pathogenesis. In this study, rAAV9s manufactured using different purification methods alongside a non-expressing Null AAV9 vector was administered to groups of cynomolgus monkeys followed by neuropathological evaluation after 4 weeks. Lesions, including neuronal degeneration, increased cellularity, and nerve fiber degeneration, were observed in the DRG, regardless of purification methods. Animals did not develop any neurological signs throughout the study, and there was no loss of function observed in neuro-electrophysiological endpoints or clear effects on intraepidermal nerve fiber density. However, magnetic resonance imaging (MRI) of animals with axonopathy showed an increase in short tau inversion recovery (STIR) intensity and decrease in fractional anisotropy. In animals administered the Null AAV9 vector, DRG lesions were not observed despite vector DNA being detected in the DRG at levels equivalent to or greater than rAAV9-treated animals. This study further supports that DRG toxicity is associated with transgene overexpression in DRGs, with particular sensitivity at the lumbar and lumbosacral level. The data from this study also showed that the nerve fiber degeneration did not correlate with any functional effect on nerve conduction but was detectable by MRI.

In Vitro Screening for Seizure Liability Using Microelectrode Array Technology.

Drug-induced seizure liabilities produce significant compound attrition during drug discovery. Currently available in vitro cytotoxicity assays cannot predict all toxicity mechanisms due to the failure of these assays to predict sublethal target-specific electrophysiological liabilities. Identification of seizurogenic and other electrophysiological effects at early stages of the drug development process is important to ensure that safe candidate compounds can be developed while chemical design is taking place, long before these liabilities are discovered in costly preclinical in vivo studies. The development of a high throughput and reliable in vitro assay to screen compounds for seizure liabilities would de-risk compounds significantly earlier in the drug discovery process and with greater dependability. Here we describe a method for screening compounds that utilizes rat cortical neurons plated onto multiwell microelectrode array plates to identify compounds that cause neurophysiological disruptions. Changes in 12 electrophysiological parameters (spike train descriptors) were measured after application of known seizurogenic compounds and the response pattern was mapped relative to negative controls, vehicle control and neurotoxic controls. Twenty chemicals with a variety of therapeutic indications and targets, including GABAA antagonists, glycine receptor antagonists, ion channel blockers, muscarinic agonist, δ-opioid receptor agonist, dopaminergic D2/adrenergic receptor blocker and nonsteroidal anti-inflammatory drugs, were tested to assess this system. Sixteen of the seventeen seizurogenic/neurotoxic compounds tested positive for seizure liability or neurotoxicity, moreover, different endpoint response patterns for firing rate, burst characteristics and synchrony that distinguished the chemicals into groups relating to target and seizurogenic response emerged from the data. The negative and vehicle control compounds had no effect on neural activity. In conclusion, the multiwell microelectrode array platform using cryopreserved rat cortical neurons is a highly effective high throughput method for reliably screening seizure liabilities within an early de-risking drug development paradigm.

Evaluation of respiratory function in freely moving Beagle dogs using implanted impedance technology.

INTRODUCTION: The Safety Pharmacology ICH S7A guidelines mandate the preclinical evaluation of drug effects on respiratory function. Chronic measurements of potential drug effects are commonly performed in rodents due to lack of a viable alternative in large animals. Presently, although the value and validity of these standard methods cannot be refuted, each method presents inherent limitations; such as the introduction of restraint stress (e.g. head-out rodent plethysmography and the pneumotachograph-equipped dog face mask), or sensitivity issues (e.g. whole body plethysmography). Since these approaches may limit the number of time points tested or affect respiratory parameters, new and accurate methods are needed for assessing respiratory function in conscious, freely moving animals. METHODS: We evaluated a new surgically implanted telemetry device, which adds an impedance sensor for the chronic measurement of respiratory parameters to the standard device used for safety pharmacology cardiovascular studies. The feasibility of the implantable device was assessed based on concordance of respiratory data with pneumotachograph-recorded parameters in conscious Beagle dogs following intravenous administration of a positive control (4 mg/kg doxapram). RESULTS: Linear regression analysis of data collected under restrained conditions showed a high correlation (R(2) 0.95) between impedance-derived respiratory parameters (tidal volume and respiratory frequency) and direct measurements of respiration via pneumotachograph. The correlation was reproduced when animals were challenged under the same dosing regimen. Volume changes similar to those obtained during the restrained collection were observed during the ambulatory collection following doxapram administration. Calibration of impedance-based values was adequate using both individual and population-based baseline conversion factors, both approximating actual mean respiratory variables collected with the pneumotachograph. DISCUSSION: The benefit of this model is the accurate, continuous measurement of respiratory endpoints in restrained, as well as ambulatory settings. Assessment of multiple physiological parameters collected concurrently and the use of population-based calibrations may enable the maximization of resources and shortened timelines in drug development.

Signalling within the neurovascular unit in the mammalian retina.

Neuronal activity in the central nervous system evokes localized changes in blood flow, a response termed neurovascular coupling or functional hyperaemia. Modern functional imaging methods, such as functional magnetic resonance imaging (fMRI), measure signals related to functional hyperaemia in order to determine localization of brain function and to diagnose disease. The cellular mechanisms that underlie functional hyperaemia, however, are not well understood. Glial cells have been hypothesized to be intermediaries between neurons and blood vessels in the control of neurovascular coupling, owing to their ability to release vasoactive factors in response to neuronal activity. Using an in vitro preparation of the isolated, intact rodent retina, we have investigated two likely mechanisms of glial control of the vasculature: glial K(+) siphoning and glial induction of vasoactive arachidonic acid metabolites. Potassium siphoning is a process by which a K(+) current flowing through glial cells transfers K(+) released from active neurons to blood vessels. Since slight increases in extracellular K(+) can cause vasodilatation, this mechanism was hypothesized to contribute to neurovascular coupling. Our data, however, suggest that glial K(+) siphoning does not contribute significantly to neurovascular coupling in the retina. Instead, we suggest that glial cells mediate neurovascular coupling by inducing the production of two types of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which dilate and constrict vessels, respectively. We show that both light flashes and direct glial stimulation produce vasodilatation or vasoconstriction mediated by EETs and 20-HETE, respectively. Further, we show that the type of vasomotor response observed (dilatation or constriction) depends on retinal levels of nitric oxide. Our data also demonstrate that glial cells are necessary intermediaries for signalling from neurons to blood vessels, since functional hyperaemia does not occur when neuron-to-glia communication is interrupted. These results indicate that glial cells play an important role in mediating functional hyperaemia and suggest that the regulation of blood flow may involve both vasodilating and vasoconstricting components.

Neurovascular coupling is not mediated by potassium siphoning from glial cells.

Neuronal activity evokes localized changes in blood flow, a response termed neurovascular coupling. One widely recognized hypothesis of neurovascular coupling holds that glial cell depolarization evoked by neuronal activity leads to the release of K+ onto blood vessels (K+ siphoning) and to vessel relaxation. We now present two direct tests of this glial cell-K+ siphoning hypothesis of neurovascular coupling. Potassium efflux was evoked from glial cells in the rat retina by applying depolarizing current pulses to individual cells. Glial depolarizations as large as 100 mV produced no change in the diameter of adjacent arterioles. We also monitored light-evoked vascular responses in Kir4.1 knock-out mice, where functional Kir K+ channels are absent from retinal glial cells. The magnitude of light-evoked vasodilations was identical in Kir4.1 knock-out and wild-type animals. Contrary to the hypothesis, the results demonstrate that glial K+ siphoning in the retina does not contribute significantly to neurovascular coupling.

Calcium signaling in specialized glial cells.

This article reviews calcium signaling in three specialized types of glial cells: Müller cells of the retina, Bergmann glial cells of the cerebellum, and radial glial cells of the developing cortex. Müller cells generate spontaneous and neuronal activity-evoked increases in Ca(2+). Neuron to Müller cell signaling is mediated by neuronal release of ATP and activation of glial P2Y receptors. Müller cells, in turn, modulate neuronal excitability and mediate vasomotor responses. Bergmann glial cells also generate spontaneous and activity-evoked Ca(2+) increases. Neuron to Bergmann glia signaling is mediated by neuronal release of nitric oxide, noradrenaline, and glutamate. In Bergmann glia, Ca(2+) increases control the structural and functional interactions between these cells and Purkinje cell synapses. In the ventricular zone of the developing cortex, radial glial cells generate spontaneous Ca(2+) increases that propagate as Ca(2+) waves through clusters of neighboring glial cells. These Ca(2+) increases control cell proliferation and neurogenesis.

Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling.

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.