Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

Collagen in the scarless fetal skin wound : detection with picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Differential expression of VEGF ligands and receptors in prostate cancer

BACKGROUND Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. METHODS The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. RESULTS Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. CONCLUSIONS These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

Putting vital stains in context

While vital staining remains a cornerstone in the diagnosis of ocular disease and contact lens complications, there are many misconceptions regarding the properties of commonly used dyes by eye-care practitioners and what is and what is not corneal staining after instillation of sodium fluorescein. Similarly, the proper use and diagnostic utility of rose Bengal and lissamine green B, the other two ophthalmic dyes commonly used for assessing ocular complications, have similarly remained unclear. Due to the limitations of vital stains for definitive diagnosis, concomitant signs and symptoms in addition to a complete patient history are required. Over the past decade, there have been many reports of a type of corneal staining—often referred to as solution-induced corneal staining (SICS)—that is observed with the use of multipurpose solutions in combination with soft lenses, more specifically silicone hydrogel lenses. Some authors believe that SICS is a sign of lens/solution incompatibility; however, new research shows that SICS may be neither a measure of lens/solution biocompatibility nor ‘true’ corneal staining, as that observed in pathological situations. A large component of SICS may be a benign phenomenon, known as preservative-associated transient hyperfluorescence (PATH). There is a lack of correlated signs and/or symptoms with SICS/PATH. Several properties of SICS/PATH, such as appearance and duration, differentiate it from pathological corneal staining. This paper reviews the properties of vital stains, their use and limitations in assessment of the ocular surface, the aetiology of corneal staining, characteristics of SICS/PATH that differentiate it from pathological corneal staining and what the SICS/PATH phenomenon means for contact lens-wearing patients.

Response to re : putting vital stains in context

A response to: "Re: Putting vital stains in context" by Eric Papas & Lyndon Jones, published in the same issue of this journal. "There has been considerable discussion in recent times about the origins of solution-induced corneal staining (SICS) and I welcome this opportunity to further clarify some points raised in my paper1 in relation to certain issues highlighted by Drs Papas and Jones.2 Part of the difficulty in understanding these phenomena relates to the imprecise terminology used. For example, Drs Papas and Jones state ‘. . . SICS..."

Response to re : putting vital stains in context

A response to "Re: Putting vital stains in context" by Charles W McMonnies, published in the same issue of this journal. "I thank Professor McMonnies for his thoughtful comments,1 which rightly forcemeto more directly address the clinical ramifications of solution-induced corneal staining (SICS). I concur with his observation that determining whether the staining can be attributed to preservative-associated transient hyperfluorescence (PATH) or true pathology can be difficult in a typical clinical situation, perhaps requiring two visits in a single day. There is no easy answer to this dilemma..."

Experimental verification of the modified spring-mass theory of fiber Bragg grating accelerometers using transverse forces

A fiber Bragg grating (FBG) accelerometer using transverse forces is more sensitive than one using axial forces with the same mass of the inertial object, because a barely stretched FBG fixed at its two ends is much more sensitive to transverse forces than axial ones. The spring-mass theory, with the assumption that the axial force changes little during the vibration, cannot accurately predict its sensitivity and resonant frequency in the gravitational direction because the assumption does not hold due to the fact that the FBG is barely prestretched. It was modified but still required experimental verification due to the limitations in the original experiments, such as the (1) friction between the inertial object and shell; (2) errors involved in estimating the time-domain records; (3) limited data; and (4) large interval ∼5 Hz between the tested frequencies in the frequency-response experiments. The experiments presented here have verified the modified theory by overcoming those limitations. On the frequency responses, it is observed that the optimal condition for simultaneously achieving high sensitivity and resonant frequency is at the infinitesimal prestretch. On the sensitivity at the same frequency, the experimental sensitivities of the FBG accelerometer with a 5.71 gram inertial object at 6 Hz (1.29, 1.19, 0.88, 0.64, and 0.31 nm/g at the 0.03, 0.69, 1.41, 1.93, and 3.16 nm prestretches, respectively) agree with the static sensitivities predicted (1.25, 1.14, 0.83, 0.61, and 0.29 nm/g, correspondingly). On the resonant frequency, (1) its assumption that the resonant frequencies in the forced and free vibrations are similar is experimentally verified; (2) its dependence on the distance between the FBG’s fixed ends is examined, showing it to be independent; (3) the predictions of the spring-mass theory and modified theory are compared with the experimental results, showing that the modified theory predicts more accurately. The modified theory can be used more confidently in guiding its design by predicting its static sensitivity and resonant frequency, and may have applications in other fields for the scenario where the spring-mass theory fails.

An evidence-based brochure to educate contact lens wearers about safe contact lens wear

Contact lenses are a successful and popular means to correct refractive error and are worn by just under 700,000 Australians1 and approximately 125 million people worldwide. The most serious complication of contact lens wear is microbial keratitis, a potentially sight-threatening corneal infection most often caused by bacteria. Gram-negative bacteria, in particular pseudomonas species, account for the majority of severe bacterial infections. Pathogens such as fungi or amoebae, which feature less often, are associated with significant morbidity. These unusual pathogens have come into the spotlight in recent times with an apparent association with specific lens cleaning solutions...

The science behind the “stain”

It has been almost fi ve years since I fi rst published the article entitled “Much Ado About Staining” in Review of Optometry, which explored what we really knew in 2006 about the relationship between “corneal staining” and contact lens multipurpose solutions (MPS). This was published just prior to the controversial “staining grid.” While the Grid showed MPS-associated hyperfl uorescence under the slitlamp at two hours, it did not explain the “what” or “why” behind it; even so, many proponents of the Grid continue to suggest that it shows us which solution/lens combinations are “biocompatible” and which are not. New evidence suggests that the preservative-associated transient hyperfl uorescence (or PATH) observed at two hours after lens insertion is a benign phenomenon due to an interaction between fl uorescein, MPS preservatives, and corneal cell membranes. The misinterpretation of PATH as “real” corneal staining, like that observed in pathological conditions, may be due in part to the fact that there is not a lot of teaching regarding the true properties of fl uorescein and what is actually occurring when we see either PATH or corneal staining. To discuss the science of fl uorescein, corneal staining, and PATH, I have asked some of the preeminent research experts in the study of fl uorescence spectroscopy and corneal staining from around the world to share their new research and personal opinions on these topics...

The role of RGPs in modern practice

Has the 1998 prediction of a well-known contact lens researcher – that rigid contact lenses will be obsolete by the year 2010 – come to fruition? This Eulogy to RGPs will demonstrate why it has. A recent survey of international contact lens prescribing trends shows that rigid lenses constituted less than 5% of all contact lenses prescribed in 16 out of 27 nations surveyed. This compares with rigid lenses representing 100% of all lenses prescribed 1965 and about 40% in 1990). With the wide range of sophisticated soft lens materials available today, including super-permeable silicone hydrogels, and designs capable of correcting astigmatism and presbyopia, there is now no need to fit cosmetic patients with rigid lenses, with the associated intractable problems of rigid lens-induced ptosis, 3 and 9 o’clock, staining, lens binding, corneal warpage and adaptation discomfort. Orthokeratology is largely a fringe application of marginal efficacy, and the notion that rigid lenses arrest myopia progression is flawed. That last bastion of rigid lens practice – fitting patients with severely distorted corneas as in keratoconus – is about to crumble in view of a number of demonstrations by independent research groups of the efficacy of custom-designed wavefront-corrected soft contact lenses for the correction of keratoconus. It is concluded that rigid contact lenses now have no place in modern contact lens practice.

Oxygen; my view

Oxygen has been the “holy grail” of contact lens wear for over 100 years, but it is just one piece of a complex jigsaw puzzle. Clearly, high oxygen transmissibility (Dk/t) silicone hydrogel lenses meet the oxygen needs of the cornea. The Dk/t of these lenses is over 75 Dk units, which is far above that of the “best” hydrogel lenses (30 Dk units). Clinical trials have failed to reveal any hypoxic problemswith silicone hydrogel lenses. Thus, conditions such as epithelial microcysts, limbal redness, hypoxic staining, stromal neovascularisation, oedema and endothelial polymegethism do not occur with these lenses. My view is that – looking at the “big picture” – we are far better off now that we have silicone hydrogel lenses.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Antibacterial anthranilic acid derivatives from Geijera parviflora

Five anthranilic acid derivatives, a mixture I of three new compounds 11′-hexadecenoylanthranilic acid (1), 9′-hexadecenoylanthranilic acid (2), and 7′-hexadecenoylanthranilic acid (3), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid (4) together with a new natural product, hexadecanoylanthranilic acid (5), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1-3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains. © 2013 Elsevier B.V.