Development and characterization of in vivo flexible electrodes compatible with large tissue displacements.

Electrical activity is the ultimate functional measure of neuronal tissue and recording that activity remains a key technical challenge in neuroscience. The mechanical mismatch between rigid electrodes and compliant brain tissue is a critical limitation in applications where movement is an inherent component. An electrode that permits recording of neural activity, while minimizing tissue disruption, is beneficial for applications that encompass both normal physiological movements and those which require consistent recording during large tissue displacements. In order to test the extreme of this range of movement, flexible electrodes were developed to record activity during and immediately following cortical impact in the rat. Photolithography techniques were used to fabricate flexible electrodes that were readily insertable into the brain using a parylene C base and gold conduction lines and contact pads, permitting custom geometry. We found that this electrode configuration retained mechanical and electrical integrity following both durability studies and large movements within the cortex. This novel flexible electrode configuration provides a novel platform for experimentally examining neuronal activity during a range of brain movements.

CNS injury biomechanics and experimental models.

Traumatic brain injury (TBI) and traumatic spinal cord injury (SCI) are acquired when an external physical insult causes damage to the central nervous system (CNS). Functional disabilities resulting from CNS trauma are dependent upon the mode, severity, and anatomical location of the mechanical impact as well as the mechanical properties of the tissue. Although the biomechanical insult is the initiating factor in the pathophysiology of CNS trauma, the anatomical loading distribution and the resulting cellular responses are currently not well understood. For example, the primary response phase includes events such as increased membrane permeability to ions and other molecules, which may initiate complex signaling cascades that account for the prolonged damage and dysfunction. Correlation of insult parameters with cellular changes and subsequent deficits may lead to refined tolerance criteria and facilitate the development of improved protective gear. In addition, advancements in the understanding of injury biomechanics are essential for the development and interpretation of experimental studies at both the in vitro and in vivo levels and may lead to the development of new treatment approaches by determining injury mechanisms across the temporal spectrum of the injury response. Here we discuss basic concepts relevant to the biomechanics of CNS trauma, injury models used to experimentally simulate TBI and SCI, and novel multilevel approaches for improving the current understanding of primary damage mechanisms.

The DETECT system: portable, reduced-length neuropsychological testing for mild traumatic brain injury via a novel immersive environment.

Undiagnosed mild traumatic brain injury (mTBI) often leads to poor patient management and significant morbidity. The lack of an efficient screening tool is especially apparent in the athletic setting, where repetitive injuries can lead to prolonged disability. We have developed the Display Enhanced Testing for Concussions and mTBI system (DETECT), in order to create a portable immersive environment that could eliminate visual and audio distractions. Neuropsychological tests sensitive to mTBI were modified for use with the system and allow rapid neurological assessment independent of the environment or trained personnel. We evaluated the immersive qualities of the DETECT system in 42 uninjured controls. The system was successful in blocking out external audiovisual stimuli. The neuropsychological test results obtained in a stimulus rich environment were equivalent to those obtained in a controlled quiet environment. The immersive environment, portability, and brevity of the DETECT system allow for real-time cognitive testing in situations previously deemed impractical or unavailable for mTBI patients.

Pharmacologic inhibition of poly(ADP-ribose) polymerase is neuroprotective following traumatic brain injury in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which has been shown to be activated following experimental traumatic brain injury (TBI), binds to DNA strand breaks and utilizes nicotinamide adenine dinucleotide (NAD) as a substrate. Since consumption of NAD may be deleterious to recovery in the setting of CNS injury, we examined the effect of a potent PARP inhibitor, GPI 6150, on histological outcome following TBI in the rat. Rats (n = 16) were anesthetized, received a preinjury dose of GPI 6150 (30 min; 15 mg/kg, i.p.), subjected to lateral fluid percussion (FP) brain injury of moderate severity (2.5-2.8 atm), and then received a second dose 3 h postinjury (15 mg/kg, i.p.). Lesion area was examined using Nissl staining, while DNA fragmentation and apoptosis-associated cell death was assessed with terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labeling (TUNEL) with stringent morphological evaluation. Twenty-four hours after brain injury, a significant cortical lesion and number of TUNEL-positive/nonapoptotic cells and TUNEL-positive/apoptotic cells in the injured cortex of vehicle-treated animals were observed as compared to uninjured rats. The size of the trauma-induced lesion area was significantly attenuated in the GPI 6150-treated animals versus vehicle-treated animals (p < 0.05). Treatment of GPI 6150 did not significantly affect the number of TUNEL-positive apoptotic cells in the injured cortex. The observed neuroprotective effects on lesion size, however, offer a promising option for further evaluation of PARP inhibition as a means to reduce cellular damage associated with TBI.

Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury.

Tissue engineering in the post-injury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. We have characterized the use of methylcellulose (MC) as a scaffolding material, whose concentration and solvent were varied to manipulate its physical properties. MC solutions were produced to exhibit low viscosity at 23 degrees C and form a soft gel at 37 degrees C, thereby making MC attractive for minimally invasive procedures in vivo. Degradation and swelling studies in vitro demonstrated a small amount of initial polymer erosion followed by relative polymer stability over the 2-week period tested as well as increased hydrogel mass due to solvent uptake. Concentrations up to 8% did not elicit cell death in primary rat astrocytes or neurons at 1 or 7 days. Acellular 2% MC (30 microl) was microinjected into the brains of rats 1 week after cortical impact injury (velocity = 3 m/s, depth = 2 mm) and examined at 2 days (n = 8; n = 3, vehicle injected) and 2 weeks (n = 5; n = 3, vehicle injected). The presence of MC did not alter the size of the injury cavity or change the patterns of gliosis as compared to injured, vehicle-injected rats (detected using antibodies against GFAP and ED1). Collectively, these data indicate that MC is well suited as a biocompatible injectable scaffold for the repair of defects in the brain.

Regional and temporal alterations in DNA fragmentation factor (DFF)-like proteins following experimental brain trauma in the rat.

DNA fragmentation, an early event in neuronal death following traumatic brain injury, may be triggered by the 40-kDa subunit of DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45-kDa subunit of DFF (DFF45), and activation of DFF40 may occur as a result of caspase-3-mediated cleavage of DFF45 into 30- and 11-kDa fragments. In this study, the intracellular distribution of DFF45 and DFF40 was examined following lateral fluid percussion brain injury of moderate severity (2.4-2.7 atm) in male Sprague-Dawley rats. In the cytosolic fraction (S1) of the injured cortex at 2 and 24 h postinjury, significant decreases in the intensities of DFF45-like proteins at 45- and 32-kDa bands and a concomitant increase in the 11-kDa bands were observed (p < 0.05 vs. uninjured controls). A significant decrease in the intensities of the 32-kDa band in the nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction (p < 0.05) and recovered by 24 h postinjury, whereas DFF40 was significantly decreased in the S1 and increased in the P1 fraction at both 2 and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase-3-DFF45/DFF40-DNA cleavage observed following traumatic brain injury.

Temporal patterns of poly(ADP-ribose) polymerase activation in the cortex following experimental brain injury in the rat.

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.

Dynamic mechanical deformation of neurons triggers an acute calcium response and cell injury involving the N-methyl-D-aspartate glutamate receptor.

A biomechanical in vitro model of traumatic brain injury was used to examine cellular response to physical insults and the underlying mechanisms that lead to cell dysfunction. A cell shearing injury device was used to deform human NTera-2 neurons at high loading rates during the investigation of mechanisms of cytosolic free calcium increases, which may be detrimental to a cell. Cytosolic free calcium rose immediately to almost three times baseline and was associated with lactate dehydrogenase release at 24 hr, indicating significant cell injury. Low loading rates did not elicit these responses. A major portion of the calcium increase and subsequent cell injury was dependent on the presence of extracellular free calcium. Blocking the N-methyl-D-aspartate glutamate receptor complex with dizocilipine maleate attenuated calcium increases by 45% in injured neurons and blocked a significant part (50%) of the lactate dehydrogenase release. In addition, pretreatment with nifedipine or riluzole also significantly reduced cytosolic free calcium but did not affect cell injury, whereas tetrodotoxin had no affect on either outcome parameter. These results suggest that the increased membrane permeability and immediate calcium influx associated with this model of mechanical injury trigger several cellular pathways, including N-methyl-D-aspartate receptor-mediated cell damage.

An in vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation.

A novel in vitro system was developed to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device (CSID) is a parallel disk viscometer that applies fluid shear stress with variable onset rate. The CSID was used in conjunction with microscopy and biochemical techniques to obtain a quantitative expression of the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows. A significant portion of the shear stress was directed in the r-direction during start-up, and therefore the full Navier-Stokes equation was necessary to accurately describe the transient shear stress. When shear stress was applied at a high rate (800 dyne cm-2 sec-1) to cultured neurons, a range of cell membrane strains (0.01 to 0.53) was obtained, suggesting inhomogeneity in cellular response. Functionally, cytosolic calcium and extracellular lactate dehydrogenase levels increased in response to high strain rate (> 1 sec-1) loading, compared with quasistatic (< 1 sec-1) loading. In addition, a subpopulation of the culture subjected to rapid deformation subsequently died. These strain rates are relevant to those shown to occur in traumatic injury, and, as such, the CSID is an appropriate model for studying the biomechanics and pathophysiology of neuronal injury.

An in vitro model of traumatic neuronal injury: loading rate-dependent changes in acute cytosolic calcium and lactate dehydrogenase release.

We developed a new in vitro model of neuronal injury using NT2-N cells to examine the effects of hydrodynamic loading rate on intraneuronal calcium dynamics and lactate dehydrogenase (LDH) release. Our apparatus consisted of a parallel disk viscometer which induced fluid shear stress with well-defined magnitudes and loading rates to cultured cells. We found that the deformation response of the cells was dependent on the severity of the insult, with increased cellular strains generated for higher shear stresses at a constant loading rate. Peak intracellular free calcium concentration correlated with strain, suggesting that mechanical deformation may regulate calcium response. Slowly applied fluid shear stress elicited no response, whereas high loading rates resulted in peak calcium increases 2.9 to 3.6 times baseline values as injury severity was increased. LDH release measured within 5 min after the insult correlated with loading rate. In addition, LDH release continued to increase out to 24 h following high loading rate conditions, demonstrating that the application of fluid shear stress led to prolonged cell damage. The acute response in NT2-N cells subjected to an insult with the CSID is dependent on the loading rate, and these results suggest that initial membrane deformation may trigger subsequent events.

The effect of N-acetylaspartate on the intracellular free calcium concentration in NTera2-neurons.

We have studied the effect of relatively high concentrations of extracellular N-acetylaspartate (NAA) on the intracellular free calcium concentration [Ca2+]i in NTera2-neurons. While low concentrations of extracellular NAA (0.1, 1 mM) had no effect on the [Ca2+]i, high concentrations of extracellular NAA (3, 10 mM) elicited sharp and statistically significant elevations of [Ca2+]i. Different classes of antagonists of the N-methyl-D-aspartate (NMDA) receptor abolished the NAA induced elevations of the [Ca2+]i, indicating the involvement of the NMDA receptor in NAA-induced elevations of [Ca2+]i.

Norepinephrine-stimulated phosphatidylinositol metabolism in genetically epilepsy-prone and kindled rats.

Genetically epilepsy-prone rats (GEPR-9) and kindled rats have reduced noradrenergic function. In the present study, norepinephrine-stimulated accumulation of inositol phosphates was reduced in cerebral cortex of GEPR-9 and kindled rats when compared to control and non-kindled rats, respectively. No such reduction was found in amygdala/pyriform cortex and hippocampus. These results support the hypothesis that cortical noradrenergic and associated second messenger systems are impaired in epilepsy.