Imaging of Estrogen Receptor-α in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -α and β),estrogen receptor-α has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-α is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-α using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 μm cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope

Reproductive senescence blunts response of estrogen receptor-α expression to estrogen treatment in rat post-ischemic cerebral microvessels.

Several studies demonstrate that estrogen treatment improves cerebral blood flow in ischemic brain regions of young ovariectomized (OVX) rats. Estrogen receptor-α (ER-α) may mediate estrogen's beneficial actions via its effects on the cerebral microvasculature. However, estrogen-derived benefit may be attenuated in aged, reproductively senescent (RS) rats. Our goal was to determine the effects of aging, estrogen deprivation and estrogen repletion with oral conjugated estrogens (CE) on postischemic cerebral microvascular protein expression of ER-α and ER-β.Fisher-344 (n = 37) female rats were randomly divided into the following groups: OVX, OVX CE-treated, RS untreated, and RS CE-treated. After 30 days pretreatment with CE (0.01 mg/kg) rats were subjected to 15 min. transient global cerebral ischemia. Non-ischemic naïve, OVX and RS rats were used as controls. Expression of ER-α and ER-β in isolated cortical cerebral microvessels (20 to 100 µm in diameter) was assessed using Western blot and immunohistochemistry techniques.Age and reproductive status blunted nonischemic ER-α expression in microvessels of OVX rats (0.31 ± 0.05) and RS rats (0.33 ± 0.06) compared to naïve rats (0.45 ± 0.02). Postischemic microvascular expression of ER-α in OVX rats (0.01 ± 0.0) was increased by CE treatment (0.04 ± 0.01). Expression of ER-α in microvessels of RS rats (0.03 ± 0.02) was unaffected by CE treatment (0.01 ± 0.02). Western blot data are presented as a ratio of ER-α or ER-β proteins to β-actin and. Oral CE treatment had no effect on ER-β expression in postischemic microvessels of OVX and RS rats. Statistical analysis was performed by One-Way ANOVA and a Newman-Keuls or Student's post-hoc test.Chronic treatment with CE increases ER-α but not ER-β expression in cerebral microvessels of OVX rats. Aging appears to reduce the normal ability of estrogen to increase ER-α expression in postischemic cerebral microvessels

Representative slides obtained from immunohistochemistry staining (A) and Western blotting (B) demonstrate changes in ER-α protein expression in cerebral microvessels isolated from rats at baseline and at 2 hours of reperfusion.

<p>Young ovariectomized (OVX) and old reproductively senescent (RS) rats had lower levels of ER-α protein expression in the cerebral microvessels than did young, naive rats. Chronic oral conjugated equine estrogen (CE) treatment resulted in increased expression levels of ER-α protein in OVX, but not in RS rats. Arrows indicate ER-α protein (A). Values of ER-α are expressed as a ratio to β-actin and presented as mean ± SD; *<i>p</i><0.05 vs. corresponding control. Data are from four independent gels, with n = 4 per group.</p

Filtration of cerebral microvessel samples improves purity.

<p>(A) Representative microscopy images (50×magnification) of suspended isolated cerebral microvessels stained with a 1% solution of Evans Blue in 0.9% NaCl. Arrows point to cerebral microvessels. Samples filtered three times through a 60-µm nylon mesh show substantially less cellular debris than those filtered one or two times. (B) Western blotting illustrates that the concentration of von Willebrand factor (vWF) protein increased significantly in samples filtered three times compared to samples filtered one time. Values of vWF expression are presented as a ratio to β-actin. All values are means ± SD; **<i>p</i><0.01 vs. homogenized brain or cerebral microvessel samples filtered one time. Western blot data are based on three independent gels, with n = 3 per group.</p

Estrogen treatment did not have any effect on ER-β expression in post-ischemic cerebral microvessels of young ovariectomized (OVX) or old reproductively senescent (RS) rats.

<p>Values of ER-β are expressed as a ratio to β-actin and presented as mean ± SD. Data are from three independent gels, with n = 3 per group.</p

Evidence that opioids may have toll-like receptor 4 and MD-2 effects

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.Mark R. Hutchinson, Yingning Zhang, Mitesh Shridhar, John H. Evans, Madison M. Buchanan, Tina X. Zhao, Peter F. Slivka, Benjamen D. Coats, Niloofar Rezvani, Julie Wieseler, Travis S. Hughes, Kyle E. Landgraf, Stefanie Chan, Stephanie Fong, Simon Phipps, Joseph J. Falke, Leslie A. Leinwand, Steven F. Maier, Hang Yin, Kenner C. Rice and Linda R. Watkinshttp://www.elsevier.com/wps/find/journaldescription.cws_home/622800/description#descriptio