Neurologic disorders such as Alzheimer's, Parkinson's disease, and Restless Legs Syndrome involve a loss of brain iron homeostasis. Moreover, iron deficiency is the most prevalent nutritional concern worldwide with many associated cognitive and neural ramifications. Therefore, understanding the mechanisms by which iron enters the brain and how those processes are regulated addresses significant global health issues. The existing paradigm assumes that the endothelial cells (ECs) forming the blood-brain barrier (BBB) serve as a simple conduit for transport of transferrin-bound iron. This concept is a significant oversimplification, at minimum failing to account for the iron needs of the ECs. Using an in vivo model of brain iron deficiency, the Belgrade rat, we show the distribution of transferrin receptors in brain microvasculature is altered in luminal, intracellular, and abluminal membranes dependent on brain iron status. We used a cell culture model of the BBB to show the presence of factors that influence iron release in non-human primate cerebrospinal fluid and conditioned media from astrocytes; specifically apo-transferrin and hepcidin were found to increase and decrease iron release, respectively. These data have been integrated into an interactive model where BBB ECs are central in the regulation of cerebral iron metabolism.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

We dissected and perfused outer medullary vasa recta (OMVR) from vascular bundles in the rat. Permeabilities of sodium (PNa) and urea (P.) were simultaneously determined from the lumen-to-bath efflux of 22Na and [ '4C Iurea. PNa and P.were also measured by in vivo microperfusion of descending (DVR) and ascending vasa recta (AVR) at the papillary tip of Munich-Wistar rats. In some OMVR PN. was indistinguishable from zero. The mean±SE of PNa(X10-, cm/s) in OMVR was 76±9. P. in OMVR was always very high (X10-5, cm/s), 360±14. There was no correlation between OMVR PN. and P,. Inner medullary AVR and DVR had PN. of 115±10 and 75±10, respectively, and P. of 121±10 and 76±11, respectively. PN. and P, in papillary vasa recta were always nearly identical and highly correlated. Transport of ["4C] urea in OMVR was reversibly inhibited by addition of unlabeled urea or phloretin to the bath and lumen, providing evidence for carrier-mediated transport. These data suggest that sodium and urea might traverse the wall of inner medullary vasa recta by a paracellular pathway while urea also crosses by a transcellular route in OMVR. Electron microscopic examination of seven in vitro perfused OMVR revealed no fenestrations and exposure of these vessels to 10 ,M calcium ionophore A23187 or 1 nM angiotensin II resulted in reversible contraction, suggesting that in vitro perfused OMVR are DVR only. (J. Clin. Invest. 1994. 93:212-222.)

Hypothesis: Fibroblasts, not myofibroblasts, are responsible for wound contraction. Only myofibroblasts express a smooth muscle actin for which vanadate blocks its expression. Wound contraction in vanadate-treated rats will proceed normally in the absence of myofibroblasts. Design: Laboratory study using rats. Methods: Wound healing in rats receiving vanadate parenterally, an inhibitor of tyrosine phosphate phosphatases, was investigated. For 21 days, treated rats received drinking water containing vanadate, 0.2 mg/mL, in isotonic sodium chloride solution, and the control rats received isotonic sodium chloride solution alone. On day 7, 4 square, full-excision wounds were made dorsally and measured, then 2 polyvinyl alcohol sponges were placed ventrally in subcutaneous pockets. Results: After 2 weeks, the wound area in the rats receiving vanadate measured 7.1 ± 1.8 U (mean ± SD), and the wound area in the control rats measured 7.2 ± 2.2 U. The control rats' granulation tissue (GT) had myofibroblasts, or ␣-smooth muscle (␣-SM) actin-positive fibroblasts, whereas the vanadate-treated group's fibroblasts were devoid of ␣-SM actin. By Western blot analysis, GT homogenates in the vanadate-treated group contained less ␣-SM actin. By electron microscopy, control rats' GT showed classic myofibroblast populations, and the collagen fiber bundles were randomly organized. In contrast, the wounds in the vanadate-treated group showed unencumbered fibroblast populations and neatly ordered, parallel collagen fiber bundles. By polarized light microscopy, the GT of the vanadate-treated group displayed orderly collagen fiber bundles. Conclusions: The differentiation of fibroblasts into myofibroblasts requires the dephosphorylation of selected tyrosine phosphate residues. In the absence of myofibroblasts, the rate of rat wound contraction is normal, and collagen fiber bundles have a more orderly arrangement. Myofibroblasts are not required for wound contraction.