Betty F. Sisken

Gait-stance duration as a measure of injury and recovery in the rat sciatic nerve model

The rat sciatic nerve is a well-established animal model for the study of peripheral nerve crush injury. Footprint analysis is the most widely used non-invasive method of measuring functional recovery after injury in this model. However, this method has significant limitations due to inability to obtain clear reproducible prints, especially when the injury is severe, and variation of these prints with gait velocity. In the case of contracture or toe loss, footprint analysis is unreliable. We describe a new technique, gait-stance duration, which is capable of non-invasively quantitating functional recovery in the rat model. This method is not dependent on accurate foot positioning during gait. It utilizes video recording of the animal walking and measures the time each hind foot is in contact with the floor by counting the number of frames that pass. By pairing consecutive steps, it minimizes variation due to changes in velocity and, by calculating a ratio of injured/uninjured hind feet, comparisons to normal gait can be made. This method shows recovery patterns similar to footprint analysis with small inter-animal variability. We believe it has significant advantages over footprint analysis for the measurement of functional recovery in the crushed sciatic nerve rat model.

EMF Signals and Ion/Ligand Binding Kinetics: Prediction of Bioeffective Waveform Parameters

The kinetics of an electromagnetic field (EMF) target pathway are used to estimate frequency windows for EMF bioeffects. Ion/ligand binding is characterized via first order kinetics from which a specific electrical impedance can be derived. The resistance/capacitance properties of the binding pathway impedance, determined by the kinetics of the rate-determining step, define the frequency range over which the target pathway is most sensitive to external EMF. Applied signals may thus be configured such that their spectral content closely matches that of the target, using evaluation of the signal to thermal noise ratio to optimize waveform parameters. Using the approach proposed in this study, a pulsed radio frequency (PRF) waveform, currently employed clinically for soft tissue repair, was returned by modulation of burst duration, producing significant bioeffects at substantially reduced signal amplitude. Application is made to Ca2+/Calmodulin-dependent myosin phosphorylation, for which the binding time constants may be estimated from reported kinetics, neurite outgrowth from embryonic chick dorsal root explants and bone repair in a fracture model. The results showed that the retuned signal produced increased phosphorylation rates, neurite outgrowth and biomechanical strength that were indistinguishable from those produced by the clinical signal, but with a tenfold reduction in peak signal amplitude, approximately 800-fold reduction in average amplitude and approximately 10(6)-fold reduction in average power.

Pulsed Electromagnetic Fields and Normal Chick Development

The effects of two clinical-type signals of pulsed electromagnetic fields (PEW) were tested on a large sample of developing chick embryos by two independent laboratories. The embryos were examined after seven days of incubation to avoid the early stages when most spontaneous abnormalities occur. The data were pooled and analyzed statistically. Neither signal, when administered continuously for the first seven days of development, or for the first 24 hours, produced a significant increase in the incidence of malformations. These results are discussed in relation to those of other investigators claiming PEW-related effects on normal development.

Effects of pulsed magnetic fields on neurite outgrowth from chick embryo dorsal root ganglia

We have previously shown that neurite outgrowth from 6-day chick embryo dorsal root ganglia (DRG) in vitro was stimulated when nerve growth factor (NGF) and pulsed magnetic fields (PMF) are used in combination. 392 DRGs were studied in a field excited by a commercial PMF generator. We have now analyzed an additional 416 DRGs exposed to very similar PMFs produced by an arbitrary wavefrom generator and power amplifier. We reproduced our previous findings that combination of NGF and bursts of asymmetric, 220 microsecond-wide, 4.0 mT-peak pulses induced significantly (p < 0.05) greater outgrowth than NGF alone, that fields without NGF do not significantly alter outgrowth, and that, unlike NGF alone, 4.0 mT fields and NGF can induce asymmetric outgrowth. The asymmetry does not seem to have a preferred orientation with respect to the induced electric field. Analysis of the data for the entire 808 DRGs confirms these findings. Importantly, we find similar results for pulse bursts repeated at 15 or 25 Hz.

Improved footprint analysis using video recording to assess functional recovery following injury to the rat sciatic nerve

Footprint analysis is a non-invasive method to quantitate functional recovery after crush injury in the rat sciatic nerve model. Traditional methods of producing the footprints for measurement are limited by inability to reliably produce clear prints when the injury is severe. We describe the use of video technique with image analysis to record and measure these prints. Video had fewer unmeasurable prints than ink. For the 1-5 and 2-4 toe spreads, there was good correlation of video measurements with ink method and better repeatability using video as compared with ink. However, the print length parameter determined by video had poorer repeatability and poorly correlated with that measured by ink. Therefore, calculation of a Sciatic Function Index by video is not appropriate. Since the print length also varies with gait velocity, we believe that a ratio of injured:uninjured hindfoot 1-5 toe spreads as measured by video is a more reliable and repeatable measure of functional recovery in this model.

PEMF, Direct Current and Nedronal Regeneration: Effect of Field Geometry and Current Density

Sensory ganglia from 7-8 day chick embryos were exposed to pulsed electromagnetic fields (PEMF) or direct current(DC) in order to correlate stimulation of neurite outgrowth with current density as a function of field geometry. Growth scores were obtained on ganglia growing in the inner and outer rings of 50 mm culture dishes. Control cultures and cultures treated with nerve growth factor served as standards. In PEMF experiments with the coil pair oriented horizontally, no correlation was observed between ganglia growth and current density in contrast to our previous findings with the coils oriented vertically. Comparison of current density for vertical and horizontal coils driven identically suggests a dose-saturation effect for the induced current with a threshold at approximately 0.4 uA/cm2. Application of DC elicited significantly greater growth as a function of location with current density levels above 9 nA/cm2. Interestingly, the total charge input for PEMF and DC stimulation was nearly identical, 1...

Quantitation of neurite growth parameters in explant cultures using a new image processing program.

An interactive image processing program was developed to quantify the effects of various biochemical and physical factors on cultured explants of nerve tissue. We used this method to obtain a growth curve of chick embryo dorsal root ganglia (DRG) in media containing various concentrations of nerve growth factor (NGF). In the past, neurite lengths and numbers were measured manually using collages of 35 mm color photographs or made directly under the microscope. Our new program makes it possible to quantify the growth of whole live, unstained DRGs on photograph collages or digital images with respect to center area, neurite area, total explant area, and the number and length of neurites almost exclusive of background artifacts. After comparing the old and new methods, we conclude that our analysis algorithm correlates well with previously accepted protocols for assessing stimulation and inhibition of growth. It rapidly measures several biologically-relevant properties and provides a means to obtain information on six parameters (neurite area, neurite length, neurite number, center area, total area, neurite density) using a single quantitative method. Neurite area in the presence of 10 ng/ml or 20 ng/ml NGF was the most significantly increased parameter as was expected from previous studies since it includes both neurite length and number as well as any crossing fibers.

Triethanolamine, tris, hepes, and cytosine arabinoside show neuritogenic activity in cultured chick embryo ganglia

Neuritogenesis, which occurs to a slight extent in chick embryo ganglia maintained under standard conditions and which is maximally stimulated by nerve growth factor, also was enhanced by presence in the medium of buffers (triethanolamine, Tris, and Hepes) and cytosine arabinoside and by the passage of direct electric current. The major effect of the buffers probably was to remove protons from cell membranes, that of the current to produce accelerated movement of ions through membranes of the ganglionic cells, and that of cytosine arabinoside to decrease the numbers of nonneural cells by inhibiting DNA synthesis. The buffers were neuritogenically ineffective on nerve growth factor-sensitive PC12 pheochromocytoma cells in culture. Media from ganglia in which triethanolamine or passage of electric current had elicited outgrowth of neurites produced no observable effect on PC12 cells under our experimental conditions. Current data fit the hypothesis that, whereas nerve growth factor exerts direct neuritogenic effects on neurons, the other treatments affect neural-nonneural interactions, possibly by way of gap junctions or changes in direct physical contact, so as to disinhibit inherent neural neuritogenic potential and/or to stimulate it.

233 - Comparison of the effects of minute levels of direct current and nerve growth factor on embryonic sensory ganglia in vitro

Abstract In this report, direct current and nerve growth factor (NGF) were used to stimulate neuritic outgrowth from eight day chick embryo sensory ganglia. The effects of both agents have been analyzed and compared to control cultures as well as to each other on a morphological and biochemical basis. The morphological study was done on the scanning electron microscope which affords a three-dimensional view of the complex arrangement of nerve fibers and glial cells as they relate to each other and to the underlying fibroblast mat upon which they grow. In order of complexity, control cultures show minimal outgrowth, cultures treated with direct current exhibit stimulated growth and NGF produces the maximal growth response in terms of length, density and branching of the neurites. NGF also produces a maximal effect on the glial cell component and on the expansion of growth cones found at the end of the elongating neurites. Direct current and NGF treatment cause a two fold increase over controls in uptake and incorporation of leucine-3H into trigeminal ganglia after 20 hours of incubation. Thus, the effects of direct current and NGF though similar are not equal. The mechanisms by which either exert their influence are proposed.

Responses of host motor and sensory neurons to a neural tube implant in amputated chick limbs.

The right wingbuds of stage 23-25 chick embryos were amputated at the future elbow region and a segment of 2-day neural tube was implanted longitudinally into the limb stump of experimental embryos to induce limb regeneration. Control embryos had no implant in the amputated limb stump. To analyze effects of the neural tube implant (NTI) upon the host nervous system, quantitative determinations were made of the peripheral limb field (PLF), dorsal root ganglia (DRG) and spinal cord lateral motor column (LMC) of the amputated side for comparison with similar determinations of the unamputated side in all embryos. The PLF was estimated by determining the area of the skeletal elements of the amputated and unamputated limb of each embryo. The size of the DRG was estimated by determining the sectional-profile area of a pair of ganglia; the LMC was determined by counting the neurons on both sides of a single spinal cord segment. The PLF was less on the amputated than on the unamputated side but was significantly greater in amputated limbs that received a NTI. The size of the DRG was positively correlated with the size of the PLF in all groups, indicating the DRG was not directly affected by the NTI but did respond to target structures. The number of neurons in the LMC was not positively correlated with the PLF and was not reduced by limb amputation in embryos with a NTI. The data suggest that the NTI may have protected host spinal cord cells from the induced-cell death expected to follow limb amputation; perhaps the implant produces a neuronal survival factor.