A novel femur window chamber for in vivo studies of bone microcirculation

Background: Due to technical difficulties of in vivo observation of blood flow and microvessels in bone, no study has been done concerning the role of blood flow in bone remodeling. Objective: To develop a new window chamber for microscopic observation of the microcirculation in living bone, and to examine the utility of the chamber using rat femur in health and diseases. Methods: A stainless chamber (19 mm in diameter and 5 mm in height) with a circular window (7.5 mm in diameter) for microscopic observation was developed. The chamber was put on rat femur which was exposed for direct observation of the microvasculature. Intravital observation was made of bone blood flow and microvessels, using fluorescence videomicroscopy and confocal laser microscopy. The utility of the chamber was examined based on images of microcirculation (normal and abnormal) in the femur bone. Results and conclusions: Images of rat femur microvasculature were enhanced in the quality by use of the femur window chamber. The new chamber provides a powerful tool for in vivo studies of the bone microcirculation in health and diseases.

Long-term oral vitamin C administration improves cerebral microvascular vasodilatory impairment in diabetes: in vivo evidence using diabetic rats

Background: Many clinical reports have indicated that ascorbic acid (vitamin C) improves vasodilatory impairments in patients with diabetes mellitus, but there is very little in vivo evidence to demonstrate its effectiveness on the brain. Objective: To investigate long-term effects of oral vitamin C administration on the cerebral microvascular vasodilation in diabetes, using streptozotocin (STZ)-induced diabetic rats. Materials and methods: Diabetes was induced in male Wistar Furth rats by a single intravenous injection of STZ (55 mg/kg b.w). Ascorbic acid (vitamin C) was administered in drinking water (1g/l). The rats were divided into control and diabetic groups with or without administration of vitamin C. The cerebral microcirculation was observed at different times (12, 24 and 36 weeks) after vitamin C supplementation, using fluorescence videomicroscopy. Responses of cerebral arterioles to acetylcholine (ACh), adenosine-5 diphosphate (ADP) and nitroglycerine (NTG) were studied by measuring diameters of cerebral arterioles before and after topical application on the cortical surface. Results: The vasodilatory responses of cerebral arterioles to ACh and ADP were significantly decreased in diabetic rats, compared with non-diabetic (control) rats. The response to NTG was not altered in diabetic rats, indicating that the vasodilatory impairment involves at the endothelium. The impaired endothelium-dependent vasodilation was prevented by long-term vitamin C administration. Conclusion: Long-term oral vitamin C administration might be of clinical relevance in improving cerebral microvascular vasodilatory impairment in diabetes.

Investigation of Chemotactic Activities in Differentiated HL-60 Cells by a Time-lapse Videomicroscopic Assay

BACKGROUND: Chemotaxis is one of the cardinal functions of leukocytes, which enables them to be recruited efficiently to the right place at the right time. Analyzing chemotactic activities is important not only for the study on leukocyte migration but also for many other applications including development of new drugs interfering with the chemotactic process. However, there are many technical limitations in the conventional in vitro chemotaxis assays. Here we applied a new optical assay to investigate chemotactic activities induced in differentiated HL-60 cells. METHODS: HL-60 cells were stimulated with 0.8% dimethylformamide (DMF) for 4 days. The cells were analyzed for morphology, flow cytometry as well as chemotactic activities by a time-lapse videomicroscopic assay using a chemotactic microchamber bearing a fibronectin-coated cover slip and an etched silicon chip. RESULTS: Videomicroscopic observation of the real cellular motions in a stable concentration gradient of chemokines demonstrated that HL-60 cells showed chemotaxis to inflammatory chemokines (CCL3, CCL5 and CXCL8) and also a homeostatic chemokine (CXCL12) after DFM-induced differentiation to granulocytic cells. The cells moved randomly at a speed of 6.99+/-1.24 micrometer/min (n=100) in the absence of chemokine. Chemokine stimulation induced directional migration of differentiated HL-60 cells, while they still wandered very much and significantly increased the moving speeds. CONCLUSION: The locomotive patterns of DMF-stimulated HL-60 cells can be analyzed in detail throughout the course of chemotaxis by the use of a time-lapse videomicroscopic assay. DMF-stimulated HL-60 cells may provide a convenient in vitro model for chemotactic studies of neutrophils.

Role of Nitric Oxide in Leukocyte-Endothelial Interaction in Cerebral Venules during Reperfusion after Global Ischemia

OBJECTIVE: Reactive oxygen metabolites and polymorphonuclear leukocytes have been implicated in the pathophysiology of reperfusion injury. The mechanisms involved in superoxide-mediated leukocyte adherence remain unclear, however, nitric oxide(NO) may contribute to this response. The present study is undertaken to elucidate mechamisms controlling NO based mechanisms that regulated leukocyte-endothelial interactions in the cerebral vasculature after global cerebral ischemia and reperfusion. METHODS: Pial venular leukocyte adherence of anesthetized newborn piglets was quantified by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during 2hours of reperfusion after global ischemia induced by 9minutes of asphyxia. Nitric oxide synthase(NOS) was inhibited by local window superfusion of L-nitroarginine(NA); superfusion of sodium nitroprusside(SNP) was used to donate NO. RESULTS: The mean number of adherent leukocytes to cerebral venules in the 9minutes asphyxia and 2hours reperfusion group were 161+/-19 compared with 13+/-4 in the nonasphyxial group. Superfusion of L-NA through the cranial window for 2hours resulted in leukocyte adherence similar to that observed during the initial 2hours of reperfusion after asphyxia. Leukocyte adherence was not additionally increased in asphyxic animal treated with L-NA. SNP inhibited asphyxia induced leukocyte adherence back to control levels. CONCLUSIONS: Nitric oxide inhibits leukocyte adherence to cerebral venules during the initial hours of reperfusion after asphyxia, and that NO supplementation inhibit asphyxia induced leukocyte adherence back to control levels. These results indicate that NO is an important factor in ischemia-reperfusion induced leukocyte adherence.

Comparative study on motility of the cultured fetal and neonatal dermal fibroblasts in extracellular matrix

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).

Clival Window Videomicroscopy of Basilar Artery Reactivity in Newborn Piglet

This report is the author's in vivo study of basilar artery reactivity to physiologic and pharmacologic stimuli in a newborn animal model. Isoflurane-anesthetized and ventilated piglets(less than 3 days of age) were placed supinely, the pharyngeal muscles retracted, and a 9 mm diameter hole was drilled in the clivus. The dura was incised in a circular fashion and coagulated, and a stainless steel and glass window with inflow and outflow ports was affixed to the clivus with dental cement. Using a color camera mounted on a triocular stereomicroscope, images of the basilar artery were recorded to Super-VHS tape, and stimulus-induced changes in vessel diameter were determined off-line from the videotape record using an image analysis system. Induction of moderate hypercapnia(PaCO2=69+/-3 mmHg) dilated the basilar artery 21+/-4%(p=0.002;n=5);hypocapnia(PaCO2=21+/-0.5 mgHg) constricted the artery 10+/-3%(p=0.05;n=5). A reduction in cerebral perfusion pressure induced by hemorrhagic hypotension to a mean arterial pressure(M ABP) of 34+/-0.5 mmHg was associated with a 9+/-1.6% reduction(p=0.0005;n=11) in basilar artery diameter. Superfusion of the clival window with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine(L-NMMA)(100 microM) decreased artery diameter 20+/-5.6%(p=0.01;n=6). Superfusion of 10 microM and 100 microM adenosine caused dose-dependent increases in artery diameter of 9.3+/-4%(p=0.02;n=4) and 13+/-5%(p=0.03;n=5), respectively. These results indicate that, while reactivity to CO2 is intact in the newborn basilar artery, autoregulatory adjustment in brainstem blood flow in newborns may be limited due to basilar artery constriction in response to systemic hypotension. Nitric oxide provides a tonic vasodilative influence on basilar artery tone in the neonate.