Microdosing of a kappa opioid receptor agonist within proximal nucleus accumbens shell microstructures revealing opposing behavioral outcomes.

Targeted intracranial delivery of molecularly-specific therapies within intricate brain structures poses a formidable challenge due to the heterogeneity of neuronal phenotypes and functions. Here we report the use of an implantable, miniaturized neural drug delivery system permitting dynamic adjustment of pharmacotherapies. Specifically, we exploit the spatial accuracy afforded by this method for targeting modulation of neuronal microstructures. Kappa opioid receptors (KOR) within the dorsal medial nucleus accumbens shell (NASh) are selectively activated through micro infusions of the KOR agonist, U-50488. Remarkably, we demonstrate that micro infusions of U-50488 into the dorsal NASh induces reward-like conditioned place preferences, whereas a mere 1 mm shift ventrally results in conditioned place aversions. The striking precision afforded by this method may prove useful in other neurotherapeutic interventions.

Single-sided magnetic resonance-based sensor for point-of-care evaluation of muscle.

Magnetic resonance imaging is a widespread clinical tool for the detection of soft tissue morphology and pathology. However, the clinical deployment of magnetic resonance imaging scanners is ultimately limited by size, cost, and space constraints. Here, we discuss the design and performance of a low-field single-sided magnetic resonance sensor intended for point-of-care evaluation of skeletal muscle in vivo. The 11 kg sensor has a penetration depth of >8 mm, which allows for an accurate analysis of muscle tissue and can avoid signal from more proximal layers, including subcutaneous adipose tissue. Low operational power and shielding requirements are achieved through the design of a permanent magnet array and surface transceiver coil. The sensor can acquire high signal-to-noise measurements in minutes, making it practical as a point-of-care tool for many quantitative diagnostic measurements, including T2 relaxometry. In this work, we present the in vitro and human in vivo performance of the device for muscle tissue evaluation.

Single-sided magnetic resonance-based sensor for point-of-care evaluation of muscle.

Magnetic resonance (MR) imaging is a powerful clinical tool for the detection of soft tissue morphology and pathology, which often provides actionable diagnostic information to clinicians. Its clinical use is largely limited due to size, cost, time, and space constraints. Here, we discuss the design and performance of a low-field single-sided MR sensor intended for point-of-care (POC) evaluation of skeletal muscle in vivo. The 11kg sensor has a penetration depth of > 8 mm, which allows for an accurate analysis of muscle tissue and can avoid signal from more proximal layers, including subcutaneous adipose tissue. Low operational power and minimal shielding requirements are achieved through the design of a permanent magnet array and surface transceiver coil. We present the in vitro and human in vivo performance of the device for muscle tissue evaluation. The sensor can acquire high signal-to-noise (SNR > 150) measurements in minutes, making it practical as a POC tool for many quantitative diagnostic measurements, including T2 relaxometry.

Diffusion Tensor Imaging Detects Acute and Subacute Changes in Corpus Callosum in Blast-Induced Traumatic Brain Injury.

There is a critical need for understanding the progression of neuropathology in blast-induced traumatic brain injury using valid animal models to develop diagnostic approaches. In the present study, we used diffusion imaging and magnetic resonance (MR) morphometry to characterize axonal injury in white matter structures of the rat brain following a blast applied via blast tube to one side of the brain. Diffusion tensor imaging was performed on acute and subacute phases of pathology from which fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity were calculated for corpus callosum (CC), cingulum bundle, and fimbria. Ventricular volume and CC thickness were measured. Blast-injured rats showed temporally varying bilateral changes in diffusion metrics indicating persistent axonal pathology. Diffusion changes in the CC suggested vasogenic edema secondary to axonal injury in the acute phase. Axonal pathology persisted in the subacute phase marked by cytotoxic edema and demyelination which was confirmed by ultrastructural analysis. The evolution of pathology followed a different pattern in the cingulum bundle: axonal injury and demyelination in the acute phase followed by cytotoxic edema in the subacute phase. Spatially, structures close to midline were most affected. Changes in the genu were greater than in the body and splenium; the caudal cingulum bundle was more affected than the rostral cingulum. Thinning of CC and ventriculomegaly were greater only in the acute phase. Our results reveal the persistent nature of blast-induced axonal pathology and suggest that diffusion imaging may have potential for detecting the temporal evolution of blast injury.

Mechanical considerations for design and implementation of peripheral intraneural devices.

OBJECTIVE: Neural interfaces designed to stimulate or record electrical activity from peripheral nerves have applications ranging from the electrical modulation of nerve activity as a therapeutic option (e.g. epilepsy and depression) to the design of prosthetics. Currently, most peripheral nerve interfaces are either cuff-style devices that wrap around the target nerve or intraneural devices that are implanted within the nerve. While the latter option offers higher specificity and signal-to-noise ratio, penetrating devices can cause significant damage to the nerve due to the high degree of mechanical mismatch. Because of this, there is interest in developing penetrating devices fabricated from soft or softening materials (materials having a low elastic modulus). However, there is currently a lack of understanding regarding implantation forces required for successful insertion, which is a constraint for soft device design. Softer devices require robust designs to achieve a critical buckling force that is larger than forces experienced during device insertion. APPROACH: This study comprehensively assesses insertion force under different implantation conditions, with three variations for implantation speed, angle, and device tip angle, during insertion of silicon shanks in rat sciatic nerve. Additionally, we report compression moduli for rat sciatic nerve at different compression rates to inform computational modeling. MAIN RESULTS: We found that insertion speed and angle had significant effects on peak insertion force. We observed lower insertion forces (10-60 mN) when the device was implanted at higher angles relative to perpendicular insertion (80-125 mN). We also demonstrate the use of a nerve-stabilizing device to keep the nerve immobile during implantation. Additionally, we found that compression moduli were significantly different in small and large strain regions of the stress-strain curve with values between 1500-4500 Pa depending on compression rate. SIGNIFICANCE: This study provides information imperative to the design and successful implementation of soft penetrating peripheral nerve interfaces.

Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy.

BACKGROUND: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. NEW METHOD: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. RESULTS: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. COMPARISON WITH EXISTING METHODS: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. CONCLUSIONS: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Chronic softening spinal cord stimulation arrays.

OBJECTIVE: We sought to develop a cervical spinal cord stimulator for the rat that is durable, stable, and does not perturb the underlying spinal cord. APPROACH: We created a softening spinal cord stimulation (SCS) array made from shape memory polymer (SMP)-based flexible electronics. We developed a new photolithographic process to pattern high surface area titanium nitride (TiN) electrodes onto gold (Au) interconnects. The thiol-ene acrylate polymers are stiff at room temperature and soften following implantation into the body. Durability was measured by the duration the devices produced effective stimulation and by accelerated aging in vitro. Stability was measured by the threshold to provoke an electromyogram (EMG) muscle response and by measuring impedance using electrochemical impedance spectroscopy (EIS). In addition, spinal cord modulation of motor cortex potentials was measured. The spinal column and implanted arrays were imaged with MRI ex vivo, and histology for astrogliosis and immune reaction was performed. MAIN RESULTS: For durability, the design of the arrays was modified over three generations to create an array that demonstrated activity up to 29 weeks. SCS arrays showed no significant degradation over a simulated 29 week period of accelerated aging. For stability, the threshold for provoking an EMG rose in the first few weeks and then remained stable out to 16 weeks; the impedance showed a similar rise early with stability thereafter. Spinal cord stimulation strongly enhanced motor cortex potentials throughout. Upon explantation, device performance returned to pre-implant levels, indicating that biotic rather than abiotic processes were the cause of changing metrics. MRI and histology showed that softening SCS produced less tissue deformation than Parylene-C arrays. There was no significant astrogliosis or immune reaction to either type of array. SIGNIFICANCE: Softening SCS arrays meet the needs for research-grade devices in rats and could be developed into human devices in the future.

Reduction in Temporary and Permanent Audiological Injury Through Internal Jugular Vein Compression in a Rodent Blast Injury Model.

HYPOTHESIS: Internal jugular vein (IJV) compression influences not only intracranial but also intracochlear physiology and has demonstrated preclinical effectiveness in reducing acute audiological injury in a rodent blast model. However, the long-term effects in this model are unknown. BACKGROUND: Blast wave-induced audiological injury from an improvised explosive device is a leading cause of morbidity among service members in theater but there are limitations to the current protective measures. METHODS: For this study, we exposed 20 Sprague Dawley rats to a 16.8 ±â€Š0.3 PSI (195.3 dB SPL) right-sided shock wave in which 10 had application of a custom IJV compression collar in place at the time of injury. RESULTS: IJV compression at the time of injury was shown acutely to significantly reduce the incidence of tympanic membrane rupture and the initial temporary threshold shift on otoacoustic emissions in both the right and left ears of animals who had collar application immediately after and 7 days post injury. At 28 days from injury, collared animals demonstrated a return to baseline of otoacoustic emission values while the noncollared animals had persistent threshold shifts, signifying the presence of a permanent threshold shift only in those animals without collar application. IJV compression was also found to significantly reduce hair cell loss at the base of the cochlea secondary to mechanical trauma from the blast wind. CONCLUSION: Previously observed acute protective effects of IJV compression are sustained at chronic time points. IJV compression can potentially be used to reduce long-term permanent morbidity from blast-induced audiological trauma.

Internal Jugular Vein Compression: A Novel Approach to Mitigate Blast Induced Hearing Injury.

HYPOTHESIS: Internal jugular vein (IJV) compression before blast injury will lead to reduced risk of traumatic hearing injury following exposure to a blast injury. BACKGROUND: IJV compression and its effects on not only intracranial, but also intracochlear pressure may potentiate blast induced hearing injury, therefore, precluding its use as a prophylactic therapy for blast induced traumatic brain injury. METHODS: Twenty Sprague Dawley rats were exposed to a 17.9 ±â€Š0.4 PSI (195.8 dB SPL) right sided shock wave in which 10 had application of a custom IJV compression collar before injury. All rodents received baseline and post blast injury otoacoustic emission (OAE) and auditory brainstem response (ABR) testing followed by cochlear histology. RESULTS: IJV compression was shown to significantly reduce ABR and OAE threshold shifts in comparison to the non-intervention group by: 14.9 ±â€Š4.8 dB (right ear ABR 0.5 kHz Day 1 post blast, p = 0.01), 13.1 ±â€Š4.9 dB (right ear ABR 4 kHz Day 1 post blast, p = 0.04), 16.5 ±â€Š4.5 dB (right ear ABR click Day 1 post blast, p = 0.003), 12.1 ±â€Š4.6 dB (right ear ABR click Day 6 post blast, p = 0.04), and 14.0 ±â€Š3.2 dB (both ears OAE 3.2-10 kHz, p < 0.0001). Also, those animals with collar application had a greater number of total hair cells per mm from 70 to 100% distance from the cochlear apex following blast injury in comparison to those without intervention (blast: 211.8 ±â€Š27.5 versus blast+collar: 355.5 ±â€Š39.5 [p = 0.0002]). CONCLUSION: This study supports the use of IJV compression in a pre-clinical model as a new prophylactic mechanism to combat blast induced hearing injury.

Effect of Internal Jugular Vein Compression on Intracranial Hemorrhage in a Porcine Controlled Cortical Impact Model.

Internal jugular vein (IJV) compression has been shown to reduce axonal injury in pre-clinical traumatic brain injury (TBI) models and clinical concussion studies. However, this novel approach to prophylactically mitigating TBI through venous congestion raises concerns of increasing the propensity for hemorrhage and hemorrhagic propagation. This study aims to test the safety of IJV compression in a large animal controlled cortical impact (CCI) injury model and the resultant effects on hemorrhage. Twelve swine were randomized to placement of a bilateral IJV compression collar (CCI+collar) or control/no collar (CCI) prior to CCI injury. A histological grading of the extent of hemorrhage, both subarachnoid (SAH) and intraparenchymal (IPH), was conducted in a blinded manner by two neuropathologists. Other various measures of TBI histology were also analyzed including: ß-amyloid precursor protein (ß-APP) expression, presence of degenerating neurons, extent of cerebral edema, and inflammatory infiltrates. Euthanized 5 h after injury, the CCI+collar animals exhibited a significant reduction in total SAH (p = 0.024-0.026) and IPH scores (p = 0.03-0.05) compared with the CCI animals. There was no statistically significant difference in scoring for the other markers of TBI (ß-APP, neuronal degeneration, cerebral edema, or inflammatory infiltration). In conclusion, IJV compression was shown to reduce hemorrhage (SAH and IPH) in the porcine CCI model when applied prior to injury. These results suggest the role of IJV compression for mitigation of not only axonal, but also hemorrhagic injury following TBI.

Dual behavior of N-acetylcysteine during ethanol-induced oxidative stress in embryonic chick brains.

OBJECTIVES: Ethanol (EtOH) causes oxidative stress in embryos. Because N-acetylcysteine (NAC) failures and successes in ameliorating EtOH-induced oxidative stress have been reported, the objective was to determine if exogenous NAC ameliorated EtOH-induced oxidative stress within embryonic chick brains. METHODS: Control eggs were injected with approximately 25 µl of water on day 0, 1, and 2 of development (E0-2). Experimental eggs were injected with dosages of either 3.0 mmol EtOH/kg egg; 747 µmol NAC/kg egg; 3.0 mmol EtOH and 747 µmol NAC/kg egg; 1000 µmol NAC/kg egg; or 3.0 mmol EtOH and 1000 µmol NAC/kg during the first 3 days of development (E0-2). At 11 days of development (E11; late embryogenesis), brains were harvested and subsequently assayed for oxidative stress markers including the loss of long-chain membrane polyunsaturated fatty acids (PUFAs); the accumulation of lipid hydroperoxides (LPO); decreased glutathione (GSH) and glutathione/glutathione disulfide (GSSG) levels; and decreased glutathione peroxidase (GPx) activities. RESULTS: EtOH (3 mmol/kg egg), medium NAC (747 µmol/kg egg), and EtOH and medium NAC promoted oxidative stress. These treatments caused decreased brain membrane long-chain PUFAs; increased LPO levels; decreased GSH levels and GSH/GSSG levels; and decreased Se-dependent GPx activities. High NAC dosages (1000 µmol/kg egg) attenuated EtOH-induced oxidative stress within EtOH and high NAC-treated chick brains. DISCUSSION: Exogenous EtOH and/or medium NAC propagated oxidative stress. Meanwhile, high NAC ameliorated EtOH-induced oxidative stress.

Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format.

Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.