Tissue plasminogen activator (rt-PA) in acute ischemic stroke: Outcomes associated with ambulation.

PURPOSE: Several clinical trials have highlighted general favorable outcomes of intravenous tissue type plasminogen activator (rt-PA) in acute ischemic stroke using different measures including, National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). Findings from most of these measures indicate that the benefits of rt-PA are time dependent, thus, supporting intensive efforts to fast-track hospital thrombolytic treatment in patients with stroke. Despite the widespread benefits of rt-PA, the effectiveness of this therapy on specific functions such as ambulatory performance of the recovering stroke patient is not fully investigated. We aim to investigate this issue in the current study. METHODS: We analyzed data from a retrospective cohort of acute ischemic stroke patients admitted to Greenville Health System (GHS) between 2010-2013. We identified patients who received rt-PA within a 4.5 hour time frame following the onset of acute ischemic stroke symptoms. Our analysis compared ambulatory variables and hospital-level characteristics in proportions of patients receiving rt-PA with those not receiving rt-PA. This analysis determined whether early treatment with rt-PA is associated with favorable changes in ambulatory status from admission to discharge following acute ischemic stroke. RESULTS: Among 663 patients with ischemic stroke who were eligible to receive rt-PA, 241 patients received rt-PA and 422 patients did not due to several risk factors. We found a statistically significant difference (P < 0.001) for changes in ambulation status from hospital admission to discharge between patients receiving rt-PA and patients who did not receive rt-PA. Among patients who received rt-PA, 27.8% improved in their ambulation status, 41.9% saw no change in their ambulation status, 0.4% worsened in their ambulation status, and 29.9% were unable to be determined. Of the patients who did not receive rt-PA, 20.1% improved in their ambulation status, 61.8% saw no change in their ambulation status, 1.4% worsened in their ambulation status, and 16.6% were unable to be determined. CONCLUSION: Our current study indicates that early treatment with rt-PA may be associated with favorable changes in ambulatory status from admission to discharge following acute ischemic stroke.

Peripheral survival of naïve CD8+ T cells.

Maintenance of a sufficient population of naive CD8+ T cells in the peripheral lymphoid compartment is critical for immunocompetence. Peripheral T cell number is a function of T cell generation, survival, and death. Homeostasis, a critical balance between survival and death, must exist to prevent either lymphopenia or lymphocytosis. In the current review, we discuss known requirements for the survival of naive peripheral CD8+ T cells as well as mechanisms of death when survival signals are lost. We also discuss associations between survival and homeostasis-driven proliferation, and highlight the gaps in our knowledge of these critical processes.

Advanced modeling environment for developing and testing FES control systems.

Realistic models of neuromusculoskeletal systems can provide a safe and convenient environment for the design and evaluation of controllers for functional electrical stimulation (FES) prior to clinical trials. We have developed a set of integrated musculoskeletal modeling tools to facilitate the model building process. Simulink models of musculoskeletal systems are created using two software packages developed in our laboratory, Musculoskeletal Modeling in Simulink (MMS) and virtual muscle, in addition to one software package available commercially, SIMM (Musculographics Inc., USA). MMS converts anatomically accurate musculoskeletal models generated by SIMM into Simulink(R) blocks. It also removes run-time constraints on kinetic simulations in SIMM, and allows the development of complex musculoskeletal models without writing a line of code. Virtual muscle builds realistic Simulink models of muscles responding to either natural recruitment or FES. Models of sensorimotor control systems can be developed using various Matlab (Mathworks Inc., USA) toolboxes and integrated easily with these musculoskeletal blocks in the graphical environment of Simulink.

Characterization of a novel pectate lyase, Pel10A, from Pseudomonas cellulosa.

Biological recycling of plant material is essential for biosphere maintenance. This perpetual task involves a complex array of enzymes, including extracellular polysaccharide hydrolases and lyases. Whilst much is known about the structure and function of the hydrolases, relatively little is known about the structures and mechanisms of the corresponding lyases. To this end, crystals of the catalytic module of a novel family 10 pectate lyase, Pel10A from Pseudomonas cellulosa, were obtained using polyethylene glycol 2000 monomethylether as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 47.7, b = 106.1, c = 55.4 A, beta = 92.0 degrees, and have two molecules in the asymmetric unit. The crystals diffract beyond 1.5 A using synchrotron radiation.

Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module.

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2 kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2 kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8 kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH 10 and in the presence of 2 mM Ca2+. Highest enzyme activity was detected at 62 degrees C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167-177], are the first CBM-containing pectinases described to date.

Congruence of mossy fiber and climbing fiber tactile projections in the lateral hemispheres of the rat cerebellum.

We have examined the spatial relationship between the mossy fiber and climbing fiber projections to crus IIa in the lateral hemispheres of the rat cerebellum. Experiments were performed in ketamine/xylazine anesthetized rats using extracellular recordings and high-density micromapping techniques. Responses were elicited using small, tactile stimuli applied to the perioral and forelimb regions at a rate of 0.5 Hz. In our first series of experiments we demonstrate that the primary (i.e., strongest) receptive field for a single Purkinje cell's complex spike is similar to the primary receptive field of the granule cells immediately subjacent to that Purkinje cell. In our second series of experiments we demonstrate that the granule cell region most strongly activated by a particular peripheral stimulus is immediately subjacent to the Purkinje cells whose complex spikes are also activated most strongly by the same stimulus. The region of climbing fibers activated by a localized peripheral stimulus is "patchy"; it clearly does not conform to the notion of a continuous microzone. These results support original observations first reported in the 1960s using evoked potential recording techniques that the mossy fiber and climbing fiber pathways converge in cerebellar cortex. However, we extend this earlier work to show that the two pathways converge at the level of single Purkinje cells. Many cerebellar theories assume that mossy fiber and climbing fiber pathways carry information from different peripheral locations or different modalities to cerebellar Purkinje cells. Our results appear to contradict this basic assumption for at least the tactile regions of the lateral hemispheres.

Virtual muscle: a computational approach to understanding the effects of muscle properties on motor control.

This paper describes a computational approach to modeling the complex mechanical properties of muscles and tendons under physiological conditions of recruitment and kinematics. It is embodied as a software package for use with Matlab and Simulink that allows the creation of realistic musculotendon elements for use in motor control simulations. The software employs graphic user interfaces (GUI) and dynamic data exchange (DDE) to facilitate building custom muscle model blocks and linking them to kinetic analyses of complete musculoskeletal systems. It is scalable in complexity and accuracy. The model is based on recently published data on muscle and tendon properties measured in feline slow- and fast-twitch muscle, and incorporates a novel approach to simulating recruitment and frequency modulation of different fiber-types in mixed muscles. This software is distributed freely over the Internet at http://ami.usc.edu/mddf/virtualmuscle.

Measured and modeled properties of mammalian skeletal muscle: III. the effects of stimulus frequency on stretch-induced force enhancement and shortening-induced force depression.

Stretch-induced force enhancement and shortening-induced force depression were examined in fast-twitch feline caudofemoralis muscle at 37 degrees C. These phenomena were induced by applying ramp length changes during the first 100--200 ms of an otherwise isometric contraction. The effects of various stimulus frequencies ranging from 30 to 120 pps were investigated over lengths ranging from 0.85 to 1.15 L0. Distributed asynchronous stimulation of bundles of ventral roots was employed to produce smooth contractions at sub-tetanic stimulus frequencies in whole muscle. Of the two components of force enhancement identified by Noble (1992) we observed only the transient component that decays with time; we did not observe residual force enhancement. The force depression that we observed was symmetrical in almost all respects to the transient force enhancement, and was unlike the shortening-induced de-activation and residual force depression identified by Edman (Edman. 1975; Edman et al., 1993). Both transient force enhancement and depression were independent of work, load and activation. Reversals in the direction of ramp length changes following either an initial stretch or initial shortening were shown to cancel the effects of both transient force enhancement and transient force depression. The distances over which these cancellations could be achieved were different for the lengthening and shortening effects. This asymmetry can be reconciled with the predictions of Huxley's original cross-bridge mechanism by incorporating the recent suggestion that myosin heads can interact with multiple actin binding sites during a single 'working' stroke. We conclude that the types of force enhancement/ depression that are most likely to be encountered under physiological conditions are the transient effects observed here, but that even these will have relatively little effect on force production during most natural behaviors.

Measured and modeled properties of mammalian skeletal muscle: IV. dynamics of activation and deactivation.

The interactive effects of length and stimulus frequency on rise and fall times and on sag were investigated in fast-twitch feline caudofemoralis at normal body temperature. The length and stimulus frequency ranges studied were 0.8 1.2 L0 and 15 60 pps. Isometric rise times were shortest under two sets of conditions: short lengths + low stimulus frequencies and long lengths + high stimulus frequencies. In contrast the isometric fall time relationship showed a single minimum at short lengths + low stimulus frequencies. Velocity was shown to have an additional effect on fall time, but only at higher stimulus frequencies (40 60 pps): fall times were shorter during movement in either direction as compared to isometric. The effects of sag were greatest at shorter lengths and lower stimulus frequencies during isometric stimulus trains. Potential mechanisms underlying this last effect were investigated by comparing isometric twitches elicited prior to and immediately following a sag-inducing stimulus train. Post-sag twitches produced less force, reached peak force earlier and initially decayed more quickly compared to pre-sag twitches. However, the final rate of force decay and the initial rate of force rise (during the first 15 ms) were unaffected by sag. We construct a logical argument based on these findings to hypothesize that the predominant mechanism underlying sag is an increase in the rate of sarcoplasmic calcium ion removal. All of the above findings were used to construct a model of activation dynamics for fast-twitch muscle, which was then extrapolated to slow-twitch muscle. When coupled with a previous model of kinematic dynamics, the complete model produced accurate predictions of the forces actually recorded during experiments in which we applied concurrent dynamic changes in length. velocity and stimulus frequency.

Measured and modeled properties of mammalian skeletal muscle. I. The effects of post-activation potentiation on the time course and velocity dependencies of force production.

Activation of mammalian fast-twitch skeletal muscle induces a persistent effect known as post-activation potentiation (PAP), classically defined as an increase in force production at sub-maximal levels of activation. The underlying mechanism is thought to be phosphorylation of the myosin regulatory light chain (MRLC), which leads to an increase in the rate constant for cross-bridge attachment (Sweeney et al., 1993). If true, this suggests the hypothesis that other contractile properties should be affected during PAP. Using a feline fast-twitch whole-muscle preparation (caudofemoralis) at 37 degrees C, we observed that PAP greatly increased tetanic forces during active lengthening decreased isometric tetanic rise times and delayed isometric tetanic force relaxation. The first two of these effects were length dependent with a greater effect occurring at shorter lengths. These findings confirmed that PAP has other functionally important effects beyond a simple increase in sub-maximal isometric forces. Furthermore, length was found to have an effect independent of PAP on the shortening half of the FV relationship (less force was produced at longer lengths) and on the rate of force relaxation during the later stages of isometric tetanic force decay (slower relaxation at longer lengths). All of these findings can be explained with a simplified, two-state model of cross-bridge dynamics that accounts for the interaction of both interfilament spacing and MRLC phosphorylation on the apparent rate constants for cross-bridge attachment and detachment. These findings are largely consistent with data collected previously from reduced preparations such as skinned fibers at cold, unphysiological temperatures (e.g. 5 degrees C). One finding that could not be explained by our model was that twitch fall times in the dispotentiated state were parabolically correlated with length, whereas in the potentiated state the relationship was linear. The time course of decay of this effect did not follow the time course of force dispotentiation, suggesting that there are other activation-dependent processes occurring in parallel with MRLC phosphorylation.

A hierarchical foundation for models of sensorimotor control.

Successful performance of a sensorimotor task arises from the interaction of descending commands from the brain with the intrinsic properties of the lower levels of the sensorimotor system, including the dynamic mechanical properties of muscle, the natural coordinates of somatosensory receptors, the interneuronal circuitry of the spinal cord, and computational noise in these elements. Engineering models of biological motor control often oversimplify or even ignore these lower levels because they appear to complicate an already difficult problem. We modeled three highly simplified control systems that reflect the essential attributes of the lower levels in three tasks: acquiring a target in the face of random torque-pulse perturbations, optimizing fusimotor gain for the same perturbations, and minimizing postural error versus energy consumption during low- versus high-frequency perturbations. The emergent properties of the lower levels maintained stability in the face of feedback delays, resolved redundancy in over-complete systems, and helped to estimate loads and respond to perturbations. We suggest a general hierarchical approach to modeling sensorimotor systems, which better reflects the real control problem faced by the brain, as a first step toward identifying the actual neurocomputational steps and their anatomical partitioning in the brain.

Measured and modeled properties of mammalian skeletal muscle. II. The effects of stimulus frequency on force-length and force-velocity relationships.

Interactions between physiological stimulus frequencies, fascicle lengths and velocities were analyzed in feline caudofemoralis (CF), a hindlimb skeletal muscle composed exclusively of fast-twitch fibers. Split ventral roots were stimulated asynchronously to produce smooth contractions at sub-tetanic stimulus frequencies. As described previously, the peak of the sub-tetanic force-length relationship was found to shift to longer lengths with decreases in stimulus frequency, indicating a length dependence for activation that is independent of filament overlap. The sub-tetanic force-velocity (FV) relationship was affected strongly both by stimulus frequency and by length; decreases in either decreased the slope of the FV relationship around isometric. The shapes of the force transients following stretch or shortening revealed that these effects were not due to a change in the instantaneous FV relationship; the relative shape of the force transients following stretch or shortening was independent of stimulus frequency and hardly affected by length. The effects of stimulus frequency and length on the sub-tetanic FV relationship instead appear to be caused by a time delay in the length-dependent changes of activation. In contrast to feline soleus muscle, which is composed exclusively of slow-twitch fibers, CF did not yield at sub-tetanic stimulus frequencies for the range of stretch velocities tested (up to 2 L0/s). The data presented here were used to build a model of muscle that accounted well for all of the effects described. We extended our model to account for slow twitch muscle by comparing our fast-twitch model with previously published data and then changing the necessary parameters to fit the data. Our slow-twitch model accounts well for all previous findings including that of yielding.

Feline caudofemoralis muscle. Muscle fibre properties, architecture, and motor innervation.

Feline caudofemoralis (CF) is a promising preparation in which to study the properties of mammalian fast-twitch skeletal muscle, but little is known about its muscle fiber properties, architecture, and motor innervation. We used histochemical techniques to confirm that it contained predominantly type IIB fibers (95+/-2%, n=8, with six of eight muscles composed exclusively of type IIA and IIB fibers), but physiological experiments showed less fatiguability than for the type IIB component of medial gastrocnemius. This may be related to the surprisingly strong and regular recruitment of CF during repetitive tasks such as walking and trotting, which we demonstrated electromyographically. We measured muscle length over the anatomical range of motion for CF (approximately 0.6-1.2 L0) and estimated working length during walking and trotting (approximately 0.95-1.15 L0). The specific tension was similar to that of the exclusively slow-twitch soleus muscle (31.2+/-4.7 N/cm2 compared with 31.8+/-4.1 N/cm2; P>0.8). Single fiber dissections of CF revealed a series-fibered architecture with a mean of 2.3 fibers, each 2.5 cm long, required to span the fascicle length. We identified two neuromuscular compartments in CF by cutting one of the two nerve branches innervating CF and depleting the glycogen stores in the intact motor units. These compartments were in parallel and extended the length of the muscle; their electromyographic activity was similar during various natural behaviors. CF and gluteus maximus motoneurons were labeled concurrently with a combination of fluorescent, retrograde tracers including Fluororuby, Fluorogold and Fast Blue. The CF motor nucleus was located in L7-S1, overlapping and intermingling extensively with the nucleus of the adjacent gluteus maximus muscle. Distributions of CF motoneuron diameter revealed one large peak around 50-55 microm, with relatively few small-diameter (less than 35 microm) cells. Using estimates of the total number of fibers in three muscles and the estimated number of alpha-motoneurons for those same muscles, we calculated a mean innervation ratio of approximately 270, which is at the low end of the innervation ratios for type IIB motor units from other feline muscles and more similar to type IIA motor units. In general, CF appears to be a useful preparation in which to study the properties of fast-twitch muscle, but these properties may vary somewhat from type IIB fibers from different muscles.

The effect of sarcomere length on triad location in intact feline caudofeomoralis muscle fibres.

The location of triads within a mammalian skeletal muscle sarcomere has traditionally been defined as 'at the A-I junction'. We attempted to verify this statement by examining systematically the location of triads within the sarcomere over the physiological range of sarcomere lengths. This study was conducted using intact feline muscle fibres from caudofemoralis and exclusively fast-twitch muscle from the hindlimb. Our results intact fibres indicate that the distance between the Z-band and triad (ZT) is relatively constant over the range of sarcomere lengths (SLs)examined in this study (1.8-3.4 micron). The slope between ZT and SL was measured to be 0.06 +/- 0.01 (r= 0.36, p < 0.001) while the slope between the M-line to triad distance (MT) and SL was measured to be 0.44 +/- 0.01 (r > 0.9, p < 0.001). The mean ZT was 0.52 +/- 0.07 micron, which corresponds to a triad location approximately halfway along the thin filaments. These results do not support the traditional statement regarding triad location. Nor do these results support a similar recent study conducted using chemically skinned muscle fibres from rat extensor digitorum longus (also a homogeneously fast-twitch muscle of the hindlimb), in which a slope of 0.25 was observed between ZT and SL (r > 0.9, p < 0.01). These results are, however, in qualitative agreement with results using intact fibres from fast-twitch rat semitendinosus. Based upon known morphology, we suggest that the only structure supporting triad position is the SR itself, and that a non-homogeneous distribution of the SR within the sarcomere might be responsible for maintaining triad location near the mid-region of the thin filaments. We also suggest that there might be optimal design reasons for locating the triads at the mid-region of the thin filaments.

Relationships between range of motion, lo, and passive force in five strap-like muscles of the feline hind limb.

The relationships between range of motion, optimal length for force production (lo), and passive force provide useful insights into the structure and function of muscles but are unknown for most individual muscles. We measured these values and examined their relationships in five strap-like muscles of the cat hind limb: caudofemoralis, semitendinosus, sartorius anterior, tenuissimus, and biceps femoris anterior. The range of motion relative to lo was found to vary significantly between different muscles and even between different specimens of the same muscle. The passive force-length (FL) curve was found to be correlated with both lo and lmax (maximal in situ muscle length) but was correlated more strongly with lmax. The mean passive force produced by these muscles at lmax was less than 7% of estimated maximal isometric force, suggesting that passive force may not be important in these muscles during normal activation patterns. The variance in passive FL curves between specimens of the same muscle was found to be significantly lower when length was scaled by lmax as opposed to lo. These results suggest that lmax may provide a more useful scaling factor for generic models of muscle. However, the passive length-tension properties of mammalian muscle appear to reflect a complex mix of structures at both the myofilament and connective tissue levels that may differ depending on muscle-fiber architecture and perhaps on the history of trophic influences on a particular specimen.

Mechanics of feline soleus: I. Effect of fascicle length and velocity on force output.

The aim of the present study was to quantify how fascicle length and velocity modify force production in cat soleus. A computerized muscle puller controlled the length and velocity of the whole-muscle. We recorded the force output at the tendon and the length of muscle fascicles using sonomicrometry during whole-muscle isometric and isokinetic contractions. Peak muscle stress was estimated as 31.8 +/- 4.1 N cm-2 (mean and SD) and optimal fascicle length, Lo, was estimated as 3.8 +/- 0.6 cm which corresponds to an optimal sarcomere length of 2.49 +/- 0.08 microns. The isometric force-length data followed closely the expected force-length relationship for cat sarcomeres. The force-velocity relationship was found to be similar in shape between cats, but the per cent increment of force over isometric levels for lengthening contractions was highly variable. Estimates of the kinematics of the fascicles based on whole-muscle length were systematically incorrect; whole-muscle velocity was 21% greater than fascicle velocity. The force-velocity data demonstrated consistent dependencies on fascicle length. At lengths below 0.7 Lo (1.74 microns), the shape of the force-velocity relationship was altered by the inclusion of a passive, repulsive force in the estimate of active isometric force. The shape of the force-velocity relationship changed at lengths greater than 0.7 Lo, but was restricted to lengthening velocities where the increment of force with respect to isometric levels was found to increase with fascicle length. This change in shape in the force-velocity relationship for lengthening contractions reveals a systematic, but previously unknown interdependence between fascicle length and velocity on muscle force production.

Mechanics of feline soleus: II. Design and validation of a mathematical model.

We have developed a mathematical model to describe force production in cat soleus during steady-state activation over a range of fascicle lengths and velocities. The model was based primarily upon a three element design by Zajac but also considered the many different features present in other previously described models. We compared quantitatively the usefulness of these features and putative relationships to account for a set of force and length data from cat soleus wholemuscle described in a companion paper. Among the novel features that proved useful were the inclusion of a short-length passive force resisting compression, a new normalisation constant for connective-tissue lengths to replace the potentially troublesome slack length, and a new length dependent term for lengthening velocities in the force-velocity relationship. Each feature of this model was chosen to provide the most accurate description of the data possible without adding unneeded complexity. Previously described functions were compared with novel functions to determine the best description of the experimental data for each of the elements in the model.

A novel method to identify migration of small implantable devices.

A histologic method has been developed to assess the migration of chronically-implanted intramuscular devices. Bullet-shaped glass devices with varying tip configurations and glass-encapsulated microstimulators of a similar size were loaded with the fluorescent tracer Procion Yellow dissolved in molten glucose. Dissolution of the hardened glucose soon after contact with body fluids released the Procion Yellow, which binds irreversibly to local tissues, thereby marking the initial site of implantation with a localized fluorescent spot. After survival times of 2-7 weeks, histologic analysis usually showed a close physical relationship between the fluorescent spot and the connective-tissue capsule from which the device was extracted. In one case, migration of a sharply pointed device from the deep surface of an implanted muscle was recognized by differences in the location of the dye spot and the site of explantation from nearby fascia. Results suggested that this method could measure migratory distances as small as 5-10 mm.

Pyramidal neurones in human precentral gyrus contain nitric oxide synthase.

Pyramidal cells in the mammalian neocortex do not normally contain detectable levels of the enzyme nitric oxide synthase. However one region of the human neocortex contains pyramidal neurones that express neuronal nitric oxide synthase activity. These neurons are mainly located in layer V of the precentral gyrus and frontal cortex and are predominantly Betz cells. The proportion of Betz cells stained in the eight brains examined varied from 5 to 80%. The brains of eight rats that had received a stab wound to the parietal cortex were also examined. Following a survival period of 7 or 14 days, small groups of pyramidal neurones surrounding the lesion contained moderate levels of neuronal nitric oxide synthase. We suggest that human pyramidal neurones may start expressing nitric oxide synthase as a response to damage or age-related stress and that the nitric oxide released may have a neuroprotective role.