Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre- and post-photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high-sensitivity LX-200 system using photobleached magnetic beads, human interleukin-8 is detected with a threefold improvement in detection limit and signal-to-noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence-based assays.

Histological quantitation of brain injury using whole slide imaging: a pilot validation study in mice.

Quantitative assessment of serial brain sections provides an objective measure of neurological events at cellular and molecular levels but is difficult to implement in experimental neuroscience laboratories because of variation from person-to-person and the time required for analysis. Whole slide imaging (WSI) technology, recently introduced for pathological diagnoses, offers an electronic environment and a variety of computational tools for performing high-throughput histological analysis and managing the associated information. In our study, we applied various algorithms to quantify histologic changes associated with brain injury and compared the results to manual assessment. WSI showed a high degree of concordance with manual quantitation by Pearson correlation and strong agreement using Bland-Altman plots in: (i) cortical necrosis in cresyl-violet-stained brain sections of mice after focal cerebral ischemia; (ii) intracerebral hemorrhage in ischemic mouse brains for automated annotation of the small regions, rather than whole hemisphere of the tissue sections; (iii) Iba1-immunoreactive cell density in the adjacent and remote brain regions of mice subject to controlled cortical impact (CCI); and (iv) neuronal degeneration by silver staining after CCI. These results show that WSI, when appropriately applied and carefully validated, is a highly efficient and unbiased tool to locate and identify neuropathological features, delineate affected regions and histologically quantify these events.

Selective inhibition of matrix metalloproteinase-9 attenuates secondary damage resulting from severe traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability. Following the initial insult, severe TBI progresses to a secondary injury phase associated with biochemical and cellular changes. The secondary injury is thought to be responsible for the development of many of the neurological deficits observed after TBI and also provides a window of opportunity for therapeutic intervention. Matrix metalloproteinase-9 (MMP-9 or gelatinase B) expression is elevated in neurological diseases and its activation is an important factor in detrimental outcomes including excitotoxicity, mitochondrial dysfunction and apoptosis, and increases in inflammatory responses and astrogliosis. In this study, we used an experimental mouse model of TBI to examine the role of MMP-9 and the therapeutic potential of SB-3CT, a mechanism-based gelatinase selective inhibitor, in ameliorating the secondary injury. We observed that activation of MMP-9 occurred within one day following TBI, and remained elevated for 7 days after the initial insult. SB-3CT effectively attenuated MMP-9 activity, reduced brain lesion volumes and prevented neuronal loss and dendritic degeneration. Pharmacokinetic studies revealed that SB-3CT and its active metabolite, p-OH SB-3CT, were rapidly absorbed and distributed to the brain. Moreover, SB-3CT treatment mitigated microglial activation and astrogliosis after TBI. Importantly, SB-3CT treatment improved long-term neurobehavioral outcomes, including sensorimotor function, and hippocampus-associated spatial learning and memory. These results demonstrate that MMP-9 is a key target for therapy to attenuate secondary injury cascades and that this class of mechanism-based gelatinase inhibitor-with such desirable pharmacokinetic properties-holds considerable promise as a potential pharmacological treatment of TBI.