1.Traumatic Brain Injury (TBI) is a major public health problem. Since 2001, over 150,000 US military personnel have been diagnosed with a mild form of TBI, often after exposure to an explosive blast (bTBI), with a spectrum of neurological and psychological deficits. The mechanisms of the primary injury phase, a direct result of the shockwave generated by an explosion, are the least understood. The blast shock wave (BSW) of primary bTBI is a transient, solitary supersonic pressure wave with a rapid (sub-msec) increase in pressure (i.e. compression) followed by a more slowly developing (msec) rarefraction phase of low pressure (i.e. tension). In the majority of bTBI, the peak pressure is low;exposure to blasts estimated to create 10 atm peak pressure in the skull for a few milliseconds can result in death for unprotected persons. We asked the central question: does the BSW itself act directly on human brain cells, or does the BSW act indirectly through secondary shear stresses? We attached a pneumatic device to one of 96 wells positioned on a microscope and vary the amplitude of the pressure transient with an adjustable quick release plug. The pressure waveform characteristics are comparable to those recorded in open field blasts;the pressure waveform profile closely resembles a classic Friedlander curve. The simulated blasts were generated with rise times in the 0.1 msec range and a two component falling phase: a fast component dropping below ambient pressure within 0.5 msec, and a slower component returning to ambient pressure within 2 msecs;pressures from 514 atm above ambient pressure were examined. Our findings show that human brain cells in culture are indifferent to blast induced fast transient pressure waves (BSW) consisting of sub-msec rise time, positive peaks of up to 15 atm, followed by tensions of 0.2 atm, of msec total duration. Furthermore, we have shown that the cells only respond with global elevations in intracellular free Ca2+ when sufficient shear forces are simultaneously induced with the pressure profiles. These results makes it unlikely that the primary effect of a BSW on brain cells in vivo is a direct effect of the compression and tension forces created by the pressure transient per se. While the pressure transient that is created in our system is very similar to the classic Friedlander curve, it is possible that significant differences exist between the nature of the shear forces created by our system and those induced during an actual blast in vivo. In addition, we do not know the magnitudes of the low-pressure components that develop at brain cells during an actual blast. However, the observed correlations between cellular response and shear forces, and the lack of correlation to pressure, per se, suggest that shear forces are likely involved in the primary injury phase of bTBI. The influence of a controlled shear stress on cells in general and neurons in particular has been investigated in a variety of model systems including a rotating cone, linear actuator, and micro-fluidic-vacuum transfection. Calcium has been implicated in the induction of neuronal death during TBI and stroke;calcium is elevated for long periods of time (days, in cells surviving TBI and stroke). In our experiments, calcium is elevated transiently for short periods of time (seconds to a few minutes), and cell death does not occur even after 20 hours following this excitation. Thus the mechanism of mild bTBI may differ from that in TBI and stroke injuries, which do lead to cell death. Brain cells exposed to blast wave profiles lacking shear forces had no calcium response, even at peak pressures up to 15 atm and trough pressures of 0.2 atm, suggesting that a shear dependent mechanism of primary bTBI may involve mechano-sensitive channels, lipidic pores, or uniquely vulnerable regions of the neuronal plasma membrane, leading to activation of a small population of cells and subsequent amplification through cell-cell signaling. The high curvature stress at the necks of pre-synaptic and post-synaptic boutons or fine processes of astrocytes may be an example of vulnerable regions since the curvature stress would add to the shear stress at those points, known to disassemble during homogenization. Using primary human brain cell cultures at the level of single and small networks of cells, we found that shear forces acting at cellular length scales, rather than changes in pressure per se control the major activation parameter of CNS derived cell culture, intracellular calcium. Rapid compression and positive tension alone have been ruled out as the origin of calcium dependent cell-cell signaling following a BSW. It is now possible to evaluate both the pharmacology of the propagated calcium response associated with a blast in the presence of shear forces and the behavior of other cellular markers during varied blast conditions. 2. Limb girdle muscular dystrophy 2b (LGMD2b) and Myoshi Myopathy (MM) are late-onset muscular dystrophies caused by point mutations and deletions resulting in reduced levels, or absence, of the protein dysferlin. In dysferlinopathy patients, the sarcolemma displays characteristic abnormalities, including 0.12.0 μm discontinuities, thickened basal lamina, and accumulations of small vesicles at the sarcolemma;these features suggest that dysferlin is required for maintenance of sarcolemmal integrity. Dysferlin is a large ( 200 kDa) membrane-anchored protein with six C2 domains, having sequence similarity to synaptotagmins. By analogy to synaptotagmin's function as a possible calcium sensor in exocytosis, it has been proposed that dysferlin may serve as a calcium sensor for membrane repair. Isolated wild-type mouse muscle fibers can reseal sarcolemmal wounds in the presence of 1 mM Ca2 +free, but fibers from a dysferlin knock-out mouse are defective in resealing, based on the unimpeded uptake of FM143 fluorescent dye following laser wounding. Studies of muscle damage and repair in vivo suggest that dysferlin may have other functions in addition to membrane resealing. The A/J mouse strain has a spontaneous dysferlin mutation, due to a retrotransposon insertion in intron 4 of the dysf gene, and no detectable dysferlin protein expression. A/J mice exhibit a progressive muscular dystrophy, appearing 2 months in the proximal muscles and spreading to the distal muscles by 5 months. A/J mice exhibit a defect in recovery from muscle injury caused by a single large strain lengthening contraction. Large strain lengthening contractions produce microtears in muscle fibers which spontaneously reseal, based on retention of fluorescent dextran. A/J muscle fibers appear to reseal normally following injury, but become necrotic a few days later, and must be replaced by new myogenesis. In order to study membrane resealing using a model cell system, we have developed a dysferlin-deficient cell line from the A/J mouse strain. These cells differentiate into morphologically typical myotubes which do not express detectable levels of dysferlin. GREG myotubes exhibit a heterogeneous membrane resealing deficiency which varies in severity between individual myotubes, suggesting they possess both dysferlin-dependent and independent modes of membrane repair. Dysferlin-independent membrane repair could represent a compensatory process operant in the presence of dysferlin deficiency. Dysferlin is not required for either myoblast proliferation or fusion into myotubes in this cell line. GREG myotubes are defective in plasma membrane repair, as previously observed in dysferlin-deficient muscle fibers. Under the wounding conditions used, the extent of the membrane repair defect in GREG myotubes is variable;approximately 34% of the GREG myotubes exhibit membrane repair, compared with 100% of dysferlin-positive C2C12 myotubes.
Showing the most recent 10 out of 52 publications
