Evaluation of neurotoxicity and long-term function and behavior following intrathecal 1 % 2-chloroprocaine in juvenile rats

Spinally-administered local anesthetics provide effective perioperative anesthesia and/or analgesia for children of all ages. New preparations and drugs require preclinical safety testing in developmental models. We evaluated age-dependent efficacy and safety following 1 % preservative-free 2-chloroprocaine (2-CP) in juvenile Sprague-Dawley rats. Percutaneous lumbar intrathecal 2-CP was administered at postnatal day (P)7, 14 or 21. Mechanical withdrawal threshold pre- and post-injection evaluated the degree and duration of sensory block, compared to intrathecal saline and naive controls. Tissue analyses one- or seven-days following injection included histopathology of spinal cord, cauda equina and brain sections, and quantification of neuronal apoptosis and glial reactivity in lumbar spinal cord. Following intrathecal 2-CP or saline at P7, outcomes assessed between P30 and P72 included: spinal reflex sensitivity (hindlimb thermal latency, mechanical threshold); social approach (novel rat versus object); locomotor activity and anxiety (open field with brightly-lit center); exploratory behavior (rearings, holepoking); sensorimotor gating (acoustic startle, prepulse inhibition); and learning (Morris Water Maze). Maximum tolerated doses of intrathecal 2-CP varied with age (1.0 μL/g at P7, 0.75 μL/g at P14, 0.5 μL/g at P21) and produced motor and sensory block for 10−15 min. Tissue analyses found no significant differences across intrathecal 2-CP, saline or naïve groups. Adult behavioral measures showed expected sex-dependent differences, that did not differ between 2-CP and saline groups. Single maximum tolerated in vivo doses of intrathecal 2-CP produced reversible spinal anesthesia in juvenile rodents without detectable evidence of developmental neurotoxicity. Current results cannot be extrapolated to repeated dosing or prolonged infusion

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain

Measurement and Modeling of Particle Radiation in Coal Flames

This work aims at developing a methodology that can provide information of in-flame particle radiation in industrial-scale flames. The method is based on a combination of experimental and modeling work. The experiments have been performed in the high-temperature zone of a 77 kWth swirling lignite flame. Spectral radiation, total radiative intensity, gas temperature, and gas composition were measured, and the radiative intensity in the furnace was modeled with an axisymmetric cylindrical radiation model using Mie theory for the particle properties and a statistical narrow-band model for the gas properties. The in-flame particle radiation was measured with a Fourier transform infrared (FTIR) spectrometer connected to a water-cooled probe via fiber optics. In the cross-section of the flame investigated, the particles were found to be the dominating source of radiation. Apart from giving information about particle radiation and temperature, the methodology can also provide estimates of the amount of soot radiation and the maximum contribution from soot radiation compared to the total particle radiation. In the center position in the flame, the maximum contribution from soot radiation was estimated to be less than 40% of the particle radiation. As a validation of the methodology, the modeled total radiative intensity was compared to the total intensity measured with a narrow angle radiometer and the agreement in the results was good, supporting the validity of the used approach

From descriptive to predictive distribution models: a working example with Iberian amphibians and reptiles

BACKGROUND: Aim of the study was to identify the conditions under which spatial-environmental models can be used for the improved understanding of species distributions, under the explicit criterion of model predictive performance. I constructed distribution models for 17 amphibian and 21 reptile species in Portugal from atlas data and 13 selected ecological variables with stepwise logistic regression and a geographic information system. Models constructed for Portugal were extrapolated over Spain and tested against range maps and atlas data. RESULTS: Descriptive model precision ranged from 'fair' to 'very good' for 12 species showing a range border inside Portugal ('edge species', kappa (k) 0.35–0.89, average 0.57) and was at best 'moderate' for 26 species with a countrywide Portuguese distribution ('non-edge species', k = 0.03–0.54, average 0.29). The accuracy of the prediction for Spain was significantly related to the precision of the descriptive model for the group of edge species and not for the countrywide species. In the latter group data were consistently better captured with the single variable search-effort than by the panel of environmental data. CONCLUSION: Atlas data in presence-absence format are often inadequate to model the distribution of species if the considered area does not include part of the range border. Conversely, distribution models for edge-species, especially those displaying high precision, may help in the correct identification of parameters underlying the species range and assist with the informed choice of conservation measures

In Vivo Gene Knockdown in Rat Dorsal Root Ganglia Mediated by Self-Complementary Adeno-Associated Virus Serotype 5 Following Intrathecal Delivery

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4–L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons