Effect of Acute Administration of Angiopoietin-1 in Experimental Traumatic Spinal Cord Injury: Magnetic Resonance Imaging and Neurobehavioral Studies

Spinal cord injury (SCI) is a devastating condition that affects people in the prime of their lives. A myriad of vascular events occur after SCI, each of which contributes to the evolving pathology. The primary trauma causes mechanical damage to blood vessels, resulting in hemorrhage. The blood-spinal cord barrier (BSCB), a neurovascular unit that limits passage of most agents from systemic circulation to the central nervous system, breaks down, resulting in inflammation, scar formation, and other sequelae. Protracted BSCB disruption may exacerbate cellular injury and hinder neurobehavioral recovery in SCI. In these studies, angiopoietin-1 (Ang1), an agent known to reduce vascular permeability, was hypothesized to attenuate the severity of secondary injuries of SCI. Using longitudinal magnetic resonance imaging (MRI) studies (dynamic contrast-enhanced [DCE]-MRI for quantification of BSCB permeability, highresolution anatomical MRI for calculation of lesion size, and diffusion tensor imaging for assessment of axonal integrity), the acute, subacute, and chronic effects of Ang1 administration after SCI were evaluated. Neurobehavioral assessments were also performed. These non-invasive techniques have applicability to the monitoring of therapies in patients with SCI. In the acute phase of injury, Ang1 was found to reduce BSCB permeability and improve neuromotor recovery. Dynamic contrast-enhanced MRI revealed a persistent compromise of the BSCB up to two months post-injury. In the subacute phase of injury, Ang1’s effect on reducing BSCB permeability was maintained and it was found to transiently reduce axonal integrity. The SCI lesion burden was assessed with an objective method that compared favorably with segmentations from human raters. In the chronic phase of injury, Ang1 resulted in maintained reduction in BSCB permeability, a decrease in lesion size, and improved axonal integrity. Finally, longitudinal correlations among data from the MRI modalities and neurobehavioral assays were evaluated. Locomotor recovery was negatively correlated with lesion size in the Ang1 cohort and positively correlated with diffusion measures in the vehicle cohort. In summary, the results demonstrate a possible role for Ang1 in mitigating the secondary pathologies of SCI during the acute and chronic phases of injury.

Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates.

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of

Modulatory effects of bag cell peptides in {\it Aplysia}: Behavioral and electrophysiological studies

Reproductive hormones have effects on the nervous system not directly related to reproductive function. In the rat, for example, luteinizing hormone releasing hormone has dramatic effects on learning and memory. The present work attempts to examine the effects of reproductive hormones on non-reproductive behaviors and the neural loci and mechanisms underlying these effects in Aplysia, an animal whose behaviors, reproductive hormones and neural circuitry have been well characterized.^ In Aplysia, the neurosecretory bag cells release several peptides that are responsible for eliciting egg laying. The effects of these peptides on the defensive tail-siphon withdrawal reflex, as well as sensitization of this reflex, were examined. Sensitization, a simple form of nonassociative learning, refers to the behavioral enhancement of a response to a test stimulus after the presentation of a strong stimulus, that may last minutes (short-term) or days (long-term). An extract of the bag cells (BCE) inhibited the baseline siphon component of the tail-siphon withdrawal reflex and suppressed long-term, but not short-term, sensitization of the reflex. Preliminary experiments suggest that BCE also affects the tail component of the tail-siphon withdrawal reflex.^ To determine the neural mechanisms underlying the inhibition of the baseline reflex, electrophysiological studies were performed using an in vitro analogue of the tail-siphon withdrawal reflex to examine the ability of BCE, as well as the individual bag cell peptides (BCPs), to modulate the circuitry of the reflex. Bag cell extract attenuated the synaptic strength of the monosynaptic connections between tail sensory neurons and tail motor neurons. When individually applied only $\beta$-BCP produced a similar attenuation. This effect of $\beta$-BCP was not dependent on changes in duration of the presynaptic action potential.^ An in vitro analogue of long-term sensitization training was developed to examine the mechanisms by which the BCPs may affect long-term sensitization of the tail-siphon withdrawal reflex. This analogue exhibited both short- and long-term facilitation of the connections between the tail sensory and motor neurons.^ The results of these behavioral and electrophysiological experiments suggest that the BCPs inhibit the tail-siphon withdrawal reflex, at least in part, by modulating the synaptic strength of the connections between the sensory neurons and motor neurons underlying the reflex. One candidate for this effect is $\beta$-BCP. Thus, the peptides which elicit egg laying may also serve other functions such as the inhibition of defensive reflexes. In addition, these experiments raise the possibility that BCPs may exert a long lasting effect ($>$24 hr), suppressing long-term sensitization of the tail-siphon withdrawal reflex. ^

Convergent and divergent modulatory pathways in {\it Aplysia\/}: Cellular and network studies

Neuromodulation is essential to many functions of the nervous system. In the simple gastropod mollusk Aplysia californica, neuromodulation of the circuits for the defensive withdrawal reflexes has been associated with several forms of learning. In the present work, the neurotransmitters and neural circuitry which contribute to the modulation of the tail-siphon withdrawal reflex were examined.^ A recently-identified neuropeptide transmitter, buccalin A was found to modulate the biophysical properties of the sensory neurons that mediate the reflex. The actions of buccalin A on the sensory neurons were compared with those of the well-characterized modulatory transmitter serotonin, and convergence and divergence in the actions of these two transmitters were evaluated. Buccalin A dramatically increased the excitability of sensory neurons and occluded further enhancement of excitability by serotonin. Buccalin A produced no significant change in spike duration, and it did not block serotonin-induced spike broadening. Voltage-clamp analysis revealed the currents that may be involved in the effects on spike duration and excitability. Buccalin A decreased an outward current similar to the S-K$\sp+$ current (I$\sb{\rm K,S}$). Buccalin A appeared to occlude further modulation of I$\sb{\rm K,S}$ by serotonin, but did not block serotonin-induced modulation of the voltage-dependent delayed rectifier K$\sp+$ current (I$\sb{\rm K,V}$). These results suggest that buccalin A converges on some, but not all, of the same subcellular modulatory pathways as serotonin.^ In order to begin to understand neuromodulation in a more physiological context for the tail-siphon withdrawal reflex, the modulatory circuitry for the tail-withdrawal circuit was examined. Mechanoafferent neurons in the J cluster of the cerebral ganglion were identified as elements of a modulatory circuit for the reflex. Excitatory and inhibitory connections were observed between the J cells and the pleural sensory neurons, the tail motor neurons, and several classes of interneurons for the tail-siphon withdrawal circuit. The J cells produced both fast and slow PSPs in these neurons. Of particular interest was the ability of the J cells to produce slow EPSPs in the pleural sensory neurons. These slow EPSPs were associated with an increase in the excitability of the sensory neurons. The J cells appear to mediate both sensory and modulatory inputs to the circuit for the tail-siphon withdrawal reflex from the anterior part of the animal. ^

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Long-term injury induced plasticity in {\it Aplysia\/} neurons: Behavioral, electrophysiological, and morphological studies

Nerve injury is known to produce a variety of electrophysiological and morphological neuronal alterations (reviewed by Titmus and Faber, 1990; Bulloch and Ridgeway, 1989; Walters, 1994). Determining if these alterations are adaptive and how they are activated and maintained could provide important insight into basic cellular mechanisms of injury-induced plasticity. Furthermore, characterization of injury-induced plasticity provides a useful assay system for the identification of possible induction signals underlying these neuronal changes. Understanding fundamental mechanisms and underlying induction signals of injury-induced neuronal plasticity could facilitate development of treatment strategies for neural injury and neuropathic pain in humans.^ This dissertation characterizes long-lasting, injury-induced neuronal alterations using the nervous system of Aplysia californica as a model. These changes are examined at the behavioral, electrophysiological, and morphological levels. Injury-induced changes in the electrophysiological properties of neurons were found that increased the signaling effectiveness of the injured neurons. This increase in signalling effectiveness could act to compensate for partial destruction of the injured neuron's peripheral processes. Recovery of a defensive behavioral response which serves to protect the animal from further injury was found within 2 weeks of injury. For the behavioral recovery to occur, new neural pathways must have been formed between the denervated area and the CNS. This was found to be mediated at least in part by new axonal growth which extended from the injured cell back along the original pathway (i.e. into the injured nerve). In addition, injury produced central axonal sprouting into different nerves that do not usually contain the injured neuron's axons. This could be important for (i) finding alternative pathways to the periphery when the original pathways are impassable and (ii) the formation of additional synaptic connections with post-synaptic targets which would further enhance the signalling effectiveness of the injured cell. ^