Chronic sensory-motor activity in behaving animals using regenerative multi-electrode interfaces.

Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control and feel of robotic prosthetic limbs. We have developed a Regenerative Multi-electrode Interface (REMI) that guides re-growing axons through an electrode array deployed in the lumen of a nerve guide. While acute studies have shown the use of the REMI in the rat sciatic nerve, the quality of chronic signal recording has not been reported. Here we show that implantation of this interface in the sciatic nerve is stable with high quality recordings up to 120 days and failures mainly attributable to abiotic factors related to pedestal detachment and wire breakage. We further tested the interfacing of REMI with fascicles of the sciatic nerve that primarily innervate muscles (tibial) and skin (sural). When implanted into the tibial nerve, bursting activity was observed synchronous to stepping. However, implantation of REMI into the sural nerve failed due to its small size. While fascicles smaller than 300 µm are a challenge for regenerative interfacing, we show that a modified REMI can be used in an insertion mode to record sensory signals from skin. In summary, the REMI represents an effective tool for recording firing patterns of specific axon types during voluntary movement, which may be used to improve the motor control and sensory feedback in closed loop control systems for robotic prosthesis.

Peripheral nerve repair through multi-luminal biosynthetic implants.

Peripheral nerve damage is routinely repaired by autogenic nerve grafting, often leading to less than optimal functional recovery at the expense of healthy donor nerves. Alternative repair strategies use tubular scaffolds to guide the regeneration of damaged nerves, but despite the progress made on improved structural materials for the nerve tubes, functional recovery remains incomplete. We developed a biosynthetic nerve implant (BNI) consisting of a hydrogel-based transparent multichannel scaffold with luminar collagen matrix as a 3-D substrate for nerve repair. Using a rat sciatic nerve injury model we showed axonal regeneration through the BNI to be histologically comparable to the autologous nerve repair. At 10 weeks post-injury, nerve defects repaired with collagen-filled, single lumen tubes formed single nerve cables, while animals that received the multi-luminal BNIs showed multiple nerve cables and the formation of a perineurial-like layer within the available microchannels. Total numbers of myelinated and unmyelinated axons in the BNI were increased 3-fold and 30%, respectively, compared to collagen tubes. The recovery of reflexive movement confirmed the functional regeneration of both motor and sensory neurons. This study supports the use of multi-luminal BNIs as a viable alternative to autografts in the repair of nerve gap injuries.

Characterization of a novel bidirectional distraction spinal cord injury animal model.

Scoliosis corrective surgery requires the application of significant multidirectional stress forces, including distraction, for correction of the curved spine deformity and the application of fixation rods. If excessive, spine distraction may result in the development of new neurological deficits, some as severe as permanent paralysis. Current animal models of spinal cord injury, however, are limited to contusion, transection, or unidirectional distraction injuries, which fail to replicate the multidirectional forces that occur during spine corrective surgery. To address such limitation, we designed a novel device that relies on intervertebral grip fixation and linear actuators to induce controllable bidirectional distraction injuries to the spine. The device was tested in three (i.e., 3, 5, and 7 mm) distention paradigms of the rat T9-T11 vertebra, and the resulting injuries were evaluated through electrophysiological, behavioral, and histological analysis. As expected, 3mm bilateral spine distractions showed no neurological deficit. In contrast, those with 5 and 7 mm showed partial and complete paralysis, respectively. The relationship between the severity of the spine distraction and injury to the spinal cord tissue was determined using glial fibrillary acidic protein immunocytochemistry for visualization of reactive astrocytes and labeling of ED1-positive activated macrophages/microglia. Our results demonstrate that this device can produce bidirectional spine distraction injuries with high precision and control and, thus, may be valuable in contributing to the testing of neuroprotective strategies aimed at preventing unintended new neurological damage during corrective spine surgery.

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.

Diabetes related cardiomyopathy time dependent echocardiographic evaluation in an experimental rat model.

Type I diabetes is associated with a unique form of cardiomyopathy in the absence of atherosclerosis. The mechanisms involved in this phenomenon are not defined, but in humans this is associated with initial diastolic dysfunction followed by altered contractile performance. A relevant animal model would provide opportunities for mechanistic studies and experimental therapeutics, but none have been previously established for this unique form of cardiac pathophysiology, particularly with respect to clinically relevant and time-dependent diastolic and systolic assessments. Here we tested the hypothesis that the streptozotocin rat model mimics human phenomena with respect to time-dependent diastolic and systolic performance deficits, and investigated a role for cardiac hypertrophy and/or fibrosis. Streptozotocin was dosed 65 mg/kg i.p. and cardiac performance was assessed longitudinally for 56 days using noninvasive echocardiographic techniques. Significant hyperglycemia was detected within 3 days and remained elevated throughout the study (p<0.05). Significant reductions in HR and diastolic performance (transmitral flow velocities and slopes) were observed within 3 days relative to age matched controls, and these reductions progressed throughout the 56 day study. In contrast, statistically significant systolic dysfunction (LV fractional shortening, cardiac output) and LV dilation were detected only after 35 days. Increases in LV size and/or extent of fibrosis were not observed at any time. These results demonstrate the value of echocardiographic methods for time-dependent diastolic and systolic assessments in rodent models. Furthermore, diastolic dysfunction precedes contractile abnormalities in the streptozotocin model, similar to events that occur in humans.

Perineurium inflammation and altered connexin isoform expression in a rat model of diabetes related peripheral neuropathy.

Diabetes related peripheral neuropathy involves both somatic and autonomic nerves and leads to an array of debilitating abnormalities. Mechanisms may include decreased neuronal conductance, reactive oxygen species, and decreased performance of the perineurium blood-nerve barrier. Here we studied the perineurium characteristics of the dorsal penile nerve in a rat model of diabetes related peripheral neuropathy. Immunohistochemistry showed extensive and perineurial cell-specific nitric oxide synthase2 staining in diabetic animals as compared to age matched controls (P<0.05); however no apparent difference in immunostaining pattern was observed for 3-nitrotyrosine (a stable biomarker of peroxynitrite formation). Significant reductions in connexins 32 and 26 were seen in the diabetic perineurium with no detectable levels of connexin 43 in either control or diabetic dorsal nerve. These data provide new evidence of perineurial cell inflammatory responses and altered gap junction protein expression during diabetes related neuropathies and suggests that strategies to protect this cell type may have therapeutic value.