Excitability of Aβ sensory neurons is altered in an animal model of peripheral neuropathy

<p>Abstract</p> <p>Background</p> <p>Causes of neuropathic pain following nerve injury remain unclear, limiting the development of mechanism-based therapeutic approaches. Animal models have provided some directions, but little is known about the specific sensory neurons that undergo changes in such a way as to induce and maintain activation of sensory pain pathways. Our previous studies implicated changes in the Aβ, normally non-nociceptive neurons in activating spinal nociceptive neurons in a cuff-induced animal model of neuropathic pain and the present study was directed specifically at determining any change in excitability of these neurons. Thus, the present study aimed at recording intracellularly from Aβ-fiber dorsal root ganglion (DRG) neurons and determining excitability of the peripheral receptive field, of the cell body and of the dorsal roots.</p> <p>Methods</p> <p>A peripheral neuropathy was induced in Sprague Dawley rats by inserting two thin polyethylene cuffs around the right sciatic nerve. All animals were confirmed to exhibit tactile hypersensitivity to von Frey filaments three weeks later, before the acute electrophysiological experiments. Under stable intracellular recording conditions neurons were classified functionally on the basis of their response to natural activation of their peripheral receptive field. In addition, conduction velocity of the dorsal roots, configuration of the action potential and rate of adaptation to stimulation were also criteria for classification. Excitability was measured as the threshold to activation of the peripheral receptive field, the response to intracellular injection of depolarizing current into the soma and the response to electrical stimulation of the dorsal roots.</p> <p>Results</p> <p>In control animals mechanical thresholds of all neurons were within normal ranges. Aβ DRG neurons in neuropathic rats demonstrated a mean mechanical threshold to receptive field stimulation that were significantly lower than in control rats, a prolonged discharge following this stimulation, a decreased activation threshold and a greater response to depolarizing current injection into the soma, as well as a longer refractory interval and delayed response to paired pulse electrical stimulation of the dorsal roots.</p> <p>Conclusions</p> <p>The present study has demonstrated changes in functionally classified Aβ low threshold and high threshold DRG neurons in a nerve intact animal model of peripheral neuropathy that demonstrates nociceptive responses to normally innocuous cutaneous stimuli, much the same as is observed in humans with neuropathic pain. We demonstrate further that the peripheral receptive fields of these neurons are more excitable, as are the somata. However, the dorsal roots exhibit a decrease in excitability. Thus, if these neurons participate in neuropathic pain this differential change in excitability may have implications in the peripheral drive that induces central sensitization, at least in animal models of peripheral neuropathic pain, and Aβ sensory neurons may thus contribute to allodynia and spontaneous pain following peripheral nerve injury in humans.</p

Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4+ T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate

Low-Cost Strategy to Detect Faults Affecting Scrubbers in SRAM-Based FPGAs

SRAM-based Field Programmable Gate Arrays (FPGAs) are vulnerable to SEUs. For applications demanding high reliability this problem is often solved by integrating in the system a scrubber, a circuit that periodically scans the FPGA configuration memory and reconfigures it if an error is detected. Since the scrubber is usually implemented in the same FPGA device, it is also vulnerable to SEUs, thus the scrubber reliability is increased by adopting standard fault tolerance techniques. These solutions guarantee the scrubber reliability, but generally require a large area overhead. In this paper, we present a novel low-cost strategy capable to detect faults in the FPGA configuration memory implementing the scrubber. The proposed technique is based on time redundancy, forcing the scrubber output to produce an error indication for each word read from the FPGA memory, in order to detect the faults affecting the portion of FPGA memory implementing the scrubber. The implementation of our proposed strategy presents a negligible impact in terms of area overhead (4.17%) and a limited increase in power consumption (22.9%) over the original (unprotected) scrubber. As for the impact on system performance introduced by our strategy, it is of approximately the 38.2% over the unprotected scrubber, but it can be significantly lowered by reducing the frequency at which the scrubber is applied to test the FPGA

Towards a Non-Human Primate Model of Alpha-Synucleinopathy for Development of Therapeutics for Parkinson’s Disease: Optimization of AAV1/2 Delivery Parameters to Drive Sustained Expression of Alpha Synuclein and Dopaminergic Degeneration in Macaque

<div><p>Recent failures in clinical trials for disease modification in Parkinson’s disease have highlighted the need for a non-human primate model of the synucleinopathy underpinning dopaminergic neuron degeneration. The present study was defined to begin the development of such a model in cynomolgus macaque. We have validated surgical and vector parameters to define a means to provide a robust over-expression of alpha-synuclein which is associated with Lewy-like pathology and robust degeneration of the nigrostriatal pathway. Thus, an AAV1/2 vector incorporating strong transcription and transduction regulatory elements was used to deliver the gene for the human A53T mutation of alpha-synuclein. When injected into 4 sites within each substantia nigra (7 μl per site, 1.7 x 10¹² gp/ml), this vector provided expression lasting at least 4 months, and a 50% loss of nigral dopaminergic neurons and a 60% reduction in striatal dopamine. Further studies will be required to develop this methodology into a validated model of value as a drug development platform.</p></div

Summary of results from Experiment 2.

<p>Summary of results from Experiment 2.</p

No significant loss of substantia nigra dopamine neurons.

<p>Cell counting stereology was performed on tyrosine hydroxylase immunoreactive neurons within the borders of the substantia nigra from animals exposed to 17 weeks of either AAV1/2 driven overexpression of human A53T alpha-synuclein (A) or GFP (B) (both 20 μl of 1.7 x 10¹² gp/ml). No significant difference in total estimated numbers was observed, while a 15% reduction between the groups was calculated (P>0.05, NS) (C, mean ±SEM).</p

Transgene expression in the substantia nigra and putamen 17 weeks post surgical delivery of AAV1/2 hA53T alpha synuclein.

<p>Panel A shows wide spread overexpression of alpha synuclein (aSyn) in dopamine neurons within the macaque substantia nigra (SN). This was evident throughout the anatomical boundaries of the SN (Panels A-D). The inset of Panel A and Panel E shows high power magnification of the accumulation of aSyn in SN cell bodies. Transport and expression of aSyn was observed throughout the putamen in animals that received SN injections of AAV1/2 hA53T alpha synuclein (Panel G) compared to endogenous expression of aSyn in the SN (Panel F) and putamen (Panel H) of AAV1/2 GFP injected controls. Scale bars: in inset of panel A, 50 μm; in panel E, 25 μm and in panel F-H, 50 μm.</p