Melanocytes are a major cellular component of the choroid which aids in the maintenance of choroidal integrity and vision. Unfortunately, our knowledge regarding the cell autonomous melanocyte function, in preserving choroidal health and the ocular pathologies associated with choroidal dysfunction, remain largely unknown. The ability to culture melanocytes has advanced our knowledge regarding the origin and function of these cells in choroidal homeostasis and vision. However, the culture of murine choroid melanocytes has not been previously reported. Here, we describe a method for the isolation of melanocytes from the mouse choroid, as well as the delineation of many of their cellular characteristics, including the expression of various cell-specific markers, cell adhesion molecules, melanogenic capacity, and inflammatory responses to various extracellular stressors. Unraveling the molecular mechanisms that regulate melanocyte functions will advance our understanding of their role in choroidal homeostasis and how alterations in these functions impact ocular diseases that compromise vision.

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.

Intramyocardial injection of adipose-derived stem cells (ASC) with other cell types in acute myocardial infarction (AMI) animal models has consistently shown promising clinical regenerative capacities. We investigated the effects of intramyocardial injections of mouse ASC (mASC) with mouse endothelial cells (mEC) on left ventricular function and generation of pericardial fat in AMI rats. AMI rat models were created by ligating left anterior descending coronary artery and were randomly assigned into four groups: control (n = 10), mASC (n = 10), mEC (n = 10) and mASC+mEC (n = 10) via direct intramyocardial injections, and each rat received 1x106 cells around three peri-infarct areas. Echocardiography and cardiac positron emission tomography (PET) were compared at baseline and on 28 days after AMI. Changes in left ventricular ejection fraction measured by PET, increased significantly in mASC and mASC+mEC groups compared to mEC and control groups. Furthermore, significant decreases in fibrosis were confirmed after sacrifice on 28 days in mASC and mASC+mEC groups. Successful cell engraftment was confirmed by positive Y-Chromosome staining in the transplantation region. Pericardial fat increased significantly in mASC and mASC+mEC groups compared to control group, and pericardial fat was shown to originate from the AMI rat. mASC group expressed higher adiponectin and lower leptin levels in plasma than control group. In addition, pericardial fat from AMI rats demonstrated increased phospho-AMPK levels and reduced phospho-ACC levels. Intramyocardial mASC transplantation after AMI in rats increased pericardial fat, which might play a protective role in the recovery of myocardial function after ischemic myocardial damage.

Vapocoolant sprays are commonly used to minimize pain following minor interventions such as venipuncture, shave biopsy or needle insertion. Although these sprays have been widely used in clinical practice, little is known about their effect on microcirculation or cutaneous blood flow.
To evaluate the real-time effect of a topical vapocoolant using a well-established, rat cremaster muscle microcirculatory model, allowing direct measurement of changes in vessel diameter, capillary density and leukocyte behaviour.
Fifty rats were divided into a control and four experimental groups: group 1: 4 s spray with vapocoolant at 18 cm distance; group 2: 10 s spray at 18 cm distance; group 3: 4 s spray at 8 cm distance; and group 4: 10 s spray at 8 cm distance. Vessel diameters, capillary density and leukocyte behaviour were monitored for 1 h thereafter. Muscle was harvested for immunohistochemistry analysis of proangiogenic markers (vascular endothelial growth factor and von Willebrand factor), leukocyte behaviour markers (E-selectin, vascular cell adhesion molecule, intercellular adhesion molecule), pimonidazole-hypoxia staining and ApopTag (Millipore, USA) staining for apoptosis. Gene expression for inflammatory markers (interleukin [IL]-1β, IL-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha and interferon-gamma) was evaluated using polymerase chain reaction and myeloperoxidase assay for inflammation was performed.
The use of refrigerant spray decreased vessel diameter and capillary density initially, although none of these decreases were statistically significant. Polymerase chain reaction showed no significant changes. The myeloperoxidase assay showed statistically significant increase in myeloperoxidase activity in groups 2, 3 and 4. Immunohistochemistry was negative for angiogenic and proinflammatory markers.
The lack of statistically significant changes in vessel diameter and inflammatory markers corroborated the safety on microcirculation.

Adult mesenchymal progenitor cells (MPCs) are adherent stromal cells of non-haematopoietic origin derived from bone marrow and other tissues. Upon limited in vitro expansion, they retain their self-renewal capacity as well as their potential to differentiate into tissues of mesenchymal lineage, such as bone, cartilage, muscle, tendon and connective tissues. Amongst these tissues, cartilage is the only one with insufficient self-renewal capacity, thus MPCs would qualify as an excellent tool for therapeutic regeneration of focal cartilage lesions. However, optimal in vitro manipulation of MPCs is a prerequisite; identification and a better understanding of the molecular mechanisms regulating their differentiation pathways are needed. Despite wide usage of rats as a mammalian experimental model for preclinical fracture healing and orthopaedic tissue regeneration studies, basal gene and protein expression profiles of the osteo-chondrogenic differentiation lineages of adult rat MPCs have rarely been investigated. Therefore, this study was carried out for a quantitative RT-PCR based time-course profiling of osteo- and chondrogenesis related gene expression in undifferentiated and differentiated rat adult MPCs. In addition, with an antibody array analysis TIMP-1, MCP-1 and VEGFalpha-164 were detected in the culture supernatant and CINC-2 and beta-NGF in the cell lysate of MPCs according to their differentiation commitment. Identification of differentially expressed genes and proteins along the osteo-chondrogenic lineage provides a foundation for a more reproducible and reliable quality and differentiation control of rat bone marrow-derived MPCs used for osteochondrogenic differentiation studies.