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Summary Aim To investigate the effect of ablative fractional CO2 laser (AFCO2L) on burns scar appearance and dermal architecture at 6 weeks and up to 3-years post-treatment. Methods Twenty adult patients with a burn-related scar were recruited. Inclusion criteria were a minimum scar area of 10 × 10 cm and Vancouver scar scale (VSS) score of >5 and ≥6 months since the time of injury. The region of scar was randomised to treatment/control zones. Treatment zones received 3 standardised laser treatments at 4- to 6-week intervals. All areas of scar received standard scar care. Outcome measures were recorded at baseline, 6-weeks post final treatment and up to 3 years post-treatment. Measures included blinded assessor VSS, Patient Scar Assessment Scale and histological tissue analysis. Results Nineteen and nine patients completed the short- and long-term studies, respectively. Clinical results revealed improvement in all scar areas over time. There was a statistically significant improvement in pain and itch in the treatment zone compared to the control zone at 6 weeks. Histological data revealed a significant increase in medium-sized collagen fibres at 6 weeks relative to the control site. Sub-group analysis according to scar age revealed greater histological improvement following laser treatment in immature scars relative to more mature scar. Conclusions Results demonstrate that 3 treatments of AFCO2L significantly improve scar pain, itch and dermal architecture at 6 weeks post-treatment. Histological results suggest greater potential in treating immature scar. Further investigation into the timing of laser treatment could help assist treatment protocols.

Insulin detemir has a different profile of action on the central nervous system (CNS) than human insulin. It has been hypothesized that this is caused by an altered ability of insulin detemir to cross the blood-brain barrier (BBB). Here, we measured the permeability of the BBB to insulin detemir. We labeled insulin detemir with radioactive iodine (I-Det) and examined its ability to cross the BBB of the mouse. Permeation was assessed after intravenous injection and by brain perfusion in the presence or absence of excess insulin detemir. The ability of insulin detemir to inhibit human insulin transport across the BBB was also assessed. I-Det did not cross the BBB either after intravenous injection or when studied by brain perfusion, a method which removes or reduces the influence of circulating proteins. Unlabeled detemir was about 10 times less potent than human insulin at inhibiting the transport of radioactive human insulin across the BBB. The altered CNS profile of insulin detemir may be caused by its poor access to CNS receptors and by a block of human insulin from crossing the BBB.

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with 131I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine 131I-IL-1β and 131I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine 131I-IL-1β or 131I-IL-6, respectively, across the BBB. Transport of both ovine 131I-IL-1β and 131I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine 131I-IL-1β or 131I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimer's disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted.

Alzheimer's disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Although pain was previously not considered an important element of multiple sclerosis (MS), recent evidence indicates that over 50% of MS patients suffer from chronic pain. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine whether changes in nociception occur during disease progression and to investigate whether sex influences the development of nociception or disease-associated neurological symptoms. Using the rotarod assay, TMEV infected male mice displayed increased neurological deficits when compared to TMEV infected female mice, which mimics what is observed in human MS. While both male and female TMEV infected mice exhibited thermal hyperalgesia and mechanical allodynia, female mice developed mechanical allodynia at a faster rate and displayed significantly more mechanical allodynia than male mice. Since neuropathic symptoms have been described in MS patients, we quantified sensory nerve fibers in the epidermis of TMEV-infected and non-infected mice to determine if there were alterations in epidermal nerve density. There was a significantly higher density of PGP9.5 and CGRP-immunoreactive axons in the epidermis of TMEV-infected mice versus controls. Collectively these results indicate that the TMEV model is well suited to study the mechanisms of MS-induced nociception and suggest that alterations in peripheral nerve innervation may contribute to MS pain.

Multiple sclerosis patients typically experience increased pain that is relatively insensitive to opiate treatment. The mechanistic basis for this increased nociception is currently poorly understood. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine possible changes in spinal cord opioid receptor mRNA over the course of disease progression. TMEV infection led to significantly decreased mu, delta and kappa opioid receptor mRNA expression as analyzed by quantitative real-time PCR in both male and female mice at days 90, 150 and 180 post-infection (PI). Since opioid receptor mRNA expression decreased in TMEV mice, we examined whether opiate analgesia is also altered. TMEV infected female mice had significantly decreased opiate analgesia in thermal nociceptive tests beginning at day 90 PI, while TMEV-infected male mice did not display significantly decreased opiate analgesia until day 120 PI. The novel finding that opioid receptor expression is significantly decreased in the spinal cord of TMEV mice could explain the increased nociception and loss of opiate analgesia observed in both TMEV mice and multiple sclerosis patients.

Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy3 and Cy5, are not ideal due to the decreased stability and fluorescence intensity of the Cy5 dye relative to the Cy3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor 555 and Alexa Fluor 647 are increasingly promoted as replacements for CyDye in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye resulted in significantly higher signal intensities (P0.05) and signal minus background levels (P0.05) with no significant difference in background values (P0.05). This translated to greater levels of differential gene expression with CyDye than with the Alexa Fluor counterparts. However, CyDye fluorophores and in particular Cy5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.

Microarray is a widely used technique to study gene expression. The increasing interest in the technology has resulted in increased availability of commercial accessory reagents and instrumentation. In principle, commercial advances in reagents, kits and equipment should greatly improve assay performance, since they each bring a measure of quality assurance and uniformity to the data above that which may be obtained from the original manual hybridization processes. However, independent validation of this perceived benefit remains an essential part of the adoption process and, in microarrays, this has often been overlooked for want of immediate convenience. We describe here the comparative evaluation of two automated hybridization instruments, namely the MAUI(R) (microarray user interface) hybridization system and the GeneTAC HybStation, against the conventional manual coverslip hybridization methodology. Our results show that there is a significant advantage in using automated hybridization instrumentation over the diffusion-based coverslip methodology. We observed an enhancement of the mean signal, signal-to-noise ratio, and reproducibility between replicates when using both the MAUI(R) hybridization system and the GeneTAC HybStation. Automation further reduced labour time, offered simplicity and greater reproducibility and accuracy in the results. The present study has independently validated the benefits automation brings to the microarray hybridization process and highlights the differences between the instruments examined. We further comment on the higher quality of spot morphology when hybridization is performed at a lower temperature in combination with our buffer of choice.