Psychophysical studies of the itch sensation and itchy skin ("alloknesis") produced by intracutaneous injection of histamine.

Psychophysical measurements of itch and itchy skin ("alloknesis"--itch produced by innocuous mechanical stimulation) were obtained in human volunteers following intracutaneous or subcutaneous injections of histamine or papain into the volar forearm. Histamine and papain were given in doses of 0.1, 1, or 10 micrograms in 10 microliters of saline. The effects of the depth of injection and of skin temperature on the latency, magnitude, and duration of itch were examined. Also, dose-response functions were obtained for the area of alloknesis produced by intracutaneous injections of histamine. Finally, the neural mechanisms underlying the spread of alloknesis were investigated via local anesthesia of the skin. Intracutaneous and subcutaneous injections of histamine, but not papain, produced a sensation of itch without pain. The latency of itch was shorter after an intracutanous than after a subcutaneous injection of histamine. The mean latencies of itch produced by a 1-microgram dose were 9.5 and 23.0 sec for intracutaneous and subcutaneous injections, respectively. No differences were observed in the magnitude or duration of itch. Similarly, the latency of itch was increased when the skin temperature at injection site was lowered to 15 degrees C, whereas the magnitude and duration of itch were unaffected. Intracutaneous and subcutaneous injections of histamine produced similar areas of alloknesis. However, the magnitude and duration of alloknesis were dependent on dose. The mean maximum areas of alloknesis produced by intracutaneous injections of 0.1, 1, and 10 micrograms of histamine were 28.3, 47.2, and 43.8 cm2, respectively. Alloknesis was present at 2 min after injection, increased to a maximum area without 10 min, and then gradually decreased during the next 25-40 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of interferon responsive genes in rodent models of transmissible spongiform encephalopathy disease

Background

Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD.

Results

Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits.

Conclusion

The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Aβ42 alone is sufficient to initiate Aβ deposition, both Aβ40 and Aβ42 may contribute to cognitive deficits.     

Chronic Compression of the Dorsal Root Ganglion Enhances Mechanically Evoked Pain Behavior and the Activity of Cutaneous Nociceptors in Mice

Radicular pain in humans is usually caused by intraforaminal stenosis and other diseases affecting the spinal nerve, root, or dorsal root ganglion (DRG). Previous studies discovered that a chronic compression of the DRG (CCD) induced mechanical allodynia in rats and mice, with enhanced excitability of DRG neurons. We investigated whether CCD altered the pain-like behavior and also the responses of cutaneous nociceptors with unmyelinated axons (C-fibers) to a normally aversive punctate mechanical stimulus delivered to the hairy skin of the hind limb of the mouse. The incidence of a foot shaking evoked by indentation of the dorsum of foot with an aversive von Frey filament (tip diameter 200 μm, bending force 20 mN) was significantly higher in the foot ipsilateral to the CCD surgery as compared to the contralateral side on post-operative days 2 to 8. Mechanically-evoked action potentials were electrophysiologically recorded from the L3 DRG, in vivo, from cell bodies visually identified as expressing a transgenically labeled fluorescent marker (neurons expressing either the receptor MrgprA3 or MrgprD). After CCD, 26.7% of MrgprA3⁺ and 32.1% MrgprD⁺ neurons exhibited spontaneous activity (SA), while none of the unoperated control neurons had SA. MrgprA3⁺ and MrgprD⁺ neurons in the compressed DRG exhibited, in comparison with neurons from unoperated control mice, an increased response to the punctate mechanical stimuli for each force applied (6, 20, 40, and 80 mN). We conclude that CCD produced both a behavioral hyperalgesia and an enhanced response of cutaneous C-nociceptors to aversive punctate mechanical stimuli.     

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background  

The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).     

Cotton boll age influences feeding preference by brown stink bug (Heteroptera: Pentatomidae)

Cotton plants were infested with brown stink bug, Euschistus servus (Say), to define cotton boll age classes (based on heat unit accumulation beyond anthesis) that are most frequently injured during each of the initial 5 wk of flowering. Bolls from each week were grouped into discrete age classes and evaluated for the presence of stink bug injury. Brown stink bug injured significantly more bolls of age class B (approximately 165-336 heat units), age class C (approximately 330-504 heat units), and age class D (approximately 495-672 heat units) during the initial 3 wk in both years and in week 5 in 2002 compared with other boll ages. Generally, the frequency of injured bolls was lowest in age class A (< or = 168 heat units) during these periods. The preference by brown stink bug for boll age classes B, C, and D within a week was similar when ages were combined across all 5 wk. Based on these data, bolls that have accumulated 165.2 through 672 heat units beyond anthesis (approximately 7-27-d-old) are more frequently injured by brown stink bug when a range of boll ages are available. The boll ages in our studies corresponded to a boll diameter of 1.161-3.586 cm with a mid-range of 2.375 cm. A general protocol for initiating treatments against stink bugs is to sample bolls for evidence of injury as an indicator of presence of infestations in cotton. Sampling bolls within a defined range, which is most likely to be injured, should improve the precision of this method in detecting economic stink bug infestations in cotton.     

Quantitative relationship between indole-3-acetic acid and abscisic acid during leaf growth in Coleus blumei

Quantitative determinations by gas chromatography-mass spectrometry ofindole-3-acetic acid (IAA) and abscisic acid (ABA) in growing leaves ofColeusblumei plants show parallel declines in leaf concentrations of bothhormones,except in leaf number 3 (about three-fourths of full size) where IAA level wasthe lowest of those measured. Expansion of the most recently unfurled leaf tofull size serves, in effect, to dilute both IAA and ABA about 1.7 to 1.Althoughabsolute levels of leaf IAA varied as much as an order of magnitude from onebatch of plants to another, ABA levels were proportional to the IAA level withan overall correlation coefficient of 0.91. Evidence, both correlative andcausal, for the determination of ABA status by IAA—and of IAA status byABA—in leaves and other developing organs is summarized.     

Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.     

Routes to improving the reliability of low level DNA analysis using real-time PCR

Background  

Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.