Regional variations in capillary hemodynamics in the cat retina. Invest Ophthalmol Vis Sci.

PURPOSE. The behavior of the retinal microcirculation and its role in the progression of ocular disease is of considerable interest, yet few details are known about the flow of blood through die capillary networks of the retina. Although retinal vessels may be viewed through the pupil using standard optics, the optical limitations of the cornea and lens prevent the resolution of retinal features smaller than approximately 10 /xm in size. Because red blood cells are smaller than this, fluorescent techniques such as angiography, specific cell labeling, and fluorescein-encapsulated liposomes have typically been used to observe the retinal microcirculation in vivo. Here the authors report a study of in vivo retinal capillary hemodynamics using white light GRadient INdex of refraction (GRIN) lens endoscopy. METHODS. GRIN lens endoscopy and robotic manipulation were used to directly observe and record the motion of erythrocytes within retinal capillary networks. Video images from the endoscope were analyzed to determine the regional variation of erythrocyte velocity and normalized optical density (an index of relative capillary hematocrit) in the superficial retinal capillaries of the cat. RESULTS. A significant decrease in mean retinal capillary velocity coupled with a corresponding increase in red blood cell density was observed in peripheral regions of the retina when compared with regions of the retina near the optic disc. Stasis or intermittent flow was not observed in the unstained retina, nor were capillaries noted that contained only plasma. CONCLUSIONS. Quantification of the bloodflow in retinal microcirculation was possible using GRIN lens endoscopy and showed significant regional heterogeneity in the cat retina. (Invest Ophthalmol VisSci. 1998;39:407-4l5) T he healthy retina regulates both flow 1 ' 2 and perfusion pressure 3 over a wide range of intraocular pressure, and the time course of this vascular response points to atitoregulation by local myogenic factors

Chapter 10: Some Cognitive Issues in Engineering Design

Design is an important outcome in the engineering curricula and profession. Design courses may first appear to students as freshman or late as seniors depending on universities. With the goals to solve the design process, enhance teamwork, and enhancing communication within and between the team and client, design is integrative, requiring students to engage in a more holistic kind of thinking and resourcefulness. Students are familiar with the domain and the logical progression in textbooks from concept to concept, but the design problem is often not well formulated requiring students to formulate both the problem and consideration before working toward a solution. Teaching design can also be difficult as it requires moving students out of their comfort zone into self-directed and independent world of design. Mathematical modeling is an important aspect of design, and scaffolding appears to be helpful in improving students\u27 abilities to generate and use mathematical models in biomedical engineering senior design. A study of student capabilities followed by a classroom intervention in biomedical engineering design is discussed.https://openscholarship.wustl.edu/circle_book/1001/thumbnail.jp

Schematic of the pressure chamber set-up.

<p>Chambers depicted with the lids off. Lids were tightly screwed into place and sealed with a silicone O-ring prior to pressurization. Tubing colored in blue represents the inlet gas streams while tubing colored in red represents the outlet gas streams. Dotted vs. solid colored lines are used to distinguish tubing attached to the left vs. right pressure chambers respectively. The two black dots near the top and bottom of each pressure chamber represent the positions of the outlet and inlet gas streams, respectively. The two large rectangles within each chamber represent the shelves used to hold cell culture plates, while the smaller rectangles at the bottoms of each chamber represent the water reservoirs. The dotted black line represents the 37°C incubator.</p

The effect of the rate of hydrostatic pressure depressurization on cells in culture

<div><p>Changes in hydrostatic pressure, at levels as low as 10 mm Hg, have been reported in some studies to alter cell function in vitro; however, other studies have found no detectable changes using similar methodologies. We here investigate the hypothesis that the rate of depressurization, rather than elevated hydrostatic pressure itself, may be responsible for these reported changes. Hydrostatic pressure (100 mm Hg above atmospheric pressure) was applied to bovine aortic endothelial cells (BAECs) and PC12 neuronal cells using pressurized gas for periods ranging from 3 hours to 9 days, and then the system was either slowly (~30 minutes) or rapidly (~5 seconds) depressurized. Cell viability, apoptosis, proliferation, and F-actin distribution were then assayed. Our results did not show significant differences between rapidly and slowly depressurized cells that would explain differences previously reported in the literature. Moreover, we found no detectable effect of elevated hydrostatic pressure (with slow depressurization) on any measured variables. Our results do not confirm the findings of other groups that modest increases in hydrostatic pressure affect cell function, but we are not able to explain their findings.</p></div

Level of BAEC death following pressurization as indicated by CellTox.

<p>Cell viability was monitored over the course of 24 hrs for an ambient atmospheric pressure negative control, a vehicle negative control (0.1% DMSO), two experimental groups that received a 100 mm Hg hydrostatic pressure treatment for 3 hrs and were then either slowly or rapidly depressurized, and finally a staurosporine treated (1μM for 24 hrs before measurements) positive control. Cell viability was monitored by measuring fluorescence on a plate reader (RFU = relative fluorescence units). Error bars represent SE. Data is the mean of three to five replicates per experimental group.</p

PC12 viability following exposure to elevated hydrostatic pressure for 3 hours, rapid or slow depressurization, and additional culture time from 0 to 24 hours.

<p>Comparisons with two negative controls (atmospheric pressure, and 0.1% DMSO vehicle controls) and positive control (1 μM staurosporine prior to the first timepoint) are shown. Cell death as measured by the CellTox assay is shown in relative fluorescence units (RFUs). Error bars represent standard error.</p

F-actin distribution in BAECs after 3 hrs of exposure to elevated hydrostatic pressure followed by rapid depressurization or slow depressurization as compared with a negative control (atmospheric pressure).

<p>F-actin is stained in red; nuclear DNA is stained in blue. Subconfluent regions of each sample are depicted in the top row while confluent regions of each sample are depicted in the bottom row. Scale bar = 40 μm.</p

Percentages of BAEC populations in each of the four Annexin V/DAPI quadrants indicated in the flow cytometry plots in Fig 6.

<p>Percentages of BAEC populations in each of the four Annexin V/DAPI quadrants indicated in the flow cytometry plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189890#pone.0189890.g006" target="_blank">Fig 6</a>.</p

Flow cytometry data for BAECs stained with Annexin V and counterstained with DAPI.

<p>Staining was done immediately after subjecting cells to rapid depressurization (A) or slow depressurization (B) following a 3 hr application of elevated hydrostatic pressure. Results for a negative control (C: atmospheric pressure) and a positive control (D: treatment with 1 μM staurosporine for 24 hrs) are also shown. Annexin V and DAPI fluorescence intensity, in arbitrary units (A.U.), are given on the abscissa and ordinate, respectively. Cytometry data is presented as four quadrants superimposed over the resulting DAPI vs. Annexin V plots to distinguish between necrotic cells (DAPI positive/Annexin negative), late stage apoptotic cells (DAPI positive/Annexin positive), healthy cells (DAPI negative/Annexin negative), and early stage apoptotic cells (DAPI negative, Annexin positive).</p

Confocal images of F-actin distribution in BAECs after exposure to elevated hydrostatic pressure for 24 hours followed by either rapid (A) or slow (B) depressurization as compared with a negative control (C: atmospheric pressure).

<p>F-actin is stained in red; nuclear DNA is stained in blue. The first two columns, which both contain images of confluent monolayers within each sample, were taken at z heights approximately 0.5–1 μm apart, moving from the surface (first column) down into the interior of the cell (second column). The third column contains images of cell multilayers which were found in all samples. Scale bar = 40 μm.</p