Biomechanical strain as a trigger for pore formation in Schlemm's canal endothelial cells.

The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (∼65,000 µm(2) per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm(2) at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular "I-pores" and paracellular "B-pores" both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 µm for I-pores (N = 79 pores) and 0.67 µm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

Simultaneous Measurement of Endothelial Cell Proliferation and Cell Cycle Stage Using Flow Cytometry.

Endothelial cell proliferation rate is an important indicator of vascular health. Being able to detect the rate of endothelial cell proliferation, or cell cycle disturbances after intervention is a valuable tool for analysing any beneficial or detrimental effects of treatments in vitro. Here, we describe a straightforward flow cytometric-based method of proliferation and cell cycle tracking that can be performed on human endothelial cells in culture over several days.

Use of a Thin Layer Assay for Assessing the Angiogenic Potential of Endothelial Cells In Vitro.

Angiogenesis is essential for wound healing and regeneration and plays a significant role in several pathologies including cancer and atherosclerosis. In vitro assays offer simple and powerful tools for investigating the regulation of the angiogenic functions of primary endothelial cells (ECs) before moving to in vivo studies. The classic in vitro two-dimensional angiogenesis assay utilizes Basement Membrane Extract (BME) to study the differentiation and sprouting of ECs over a 24-h period. The protocol described here details a thin layer BME adaptation of the angiogenesis assay requiring significantly less BME and carried out in 96-well plates, allowing for a larger data yield at a greatly reduced cost, while maintaining the robustness of an assay used extensively over the past three decades.

CD25+ natural regulatory T cells are critical in limiting innate and adaptive immunity and resolving disease following respiratory syncytial virus infection.

Regulatory CD4(+) T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4(+) Foxp3(+) CD25(+) natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25(+) natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-gamma). CD25(+) natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures.

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.

Communicating hydrocephalus. Cisternographic and neuropathologic studies.

Chronic communicating hydrocephalus was produced in adult dogs by injection of silastic into the subarachnoid space. Electron microscopy was used to verify the sequence of pathologic changes in the ventricular wall. The pathologic findings were correlated with cisternographic images and measurements of cerebrospinal fluid (CSF) pressure. Early in hydrocephalus, the CSF pressure was increased and cisternograms showed ventricular entry and clearing; the ependyma was stretched and fluid accumulated in subependymal regions. In animals with chronic hydrocephalus, the CSF pressure was normal and cisternograms disclosed radioactivity persisting in the ventricles. At this time the ependyma was severely damaged, the subependymal white matter showed enlargement of the extracellular space, and degenerative changes were present in axons and myelin sheaths.

Normal-pressure hydrocephalus: diagnosis and patient selection for shunt surgery.

Currently accepted modes of clinical and radiologic evaluation were analyzed retrospectively in 55 patients with "normal-pressure" hydrocephalus on whom a cerebrospinal fluid shunting procedure was done. When applied alone, each criterion neither reliably differentiated normal-pressure hydrocephalus from cortical atrophy nor indicated in a significant number of cases which patients would benefit from shunting. Therefore, future prospective evaluations should include clinical history, physical and neurologic examination, skull radiography, echoencephalography, psychometric testing, brain scanning, lumbar puncture with cerebrospinal fluid laboratory analysis, isotope cisternography, pneumoencephalography, and constant-infusion manometric testing. Cerebral angiography may add optional support to the diagnosis of cortical atrophy but always should be done before lumbar puncture if there is evidence of intracranial mass and/or increased pressure is revealed on neurologic examination, skull radiographs, echograms, or brain scans. Patients with seizures should undergo electroencephalography. Postoperative improvement should be evaluated using serial neurologic and psychometric examinations. Echoencephalography may confirm postshunt reductions in ventricular size.

The effect of cerebrospinal fluid pressure on the size of drainage pathways.

The cerebrospinal fluid drainage pathways were studied using labeled molecules of different sizes. Following determination of the limiting size, the cerebrospinal fluid pressure was raised above the normal range and changes in transfer were measured. These data show that molecules smaller than the limiting size will transfer at an increased rate in response to elevated pressures. Larger molecules did not demonstrate an increased transfer with raised cerebrospinal fluid pressures, suggesting that the pathways did not enlarge.

The effect of percutaneous oestradiol on atheroma formation in ovariectomized cholesterol-fed rabbits.

OBJECTIVE: The aim of this study was to examine the effect of percutaneous oestradiol on the lipid profile and on atheroma formation using an animal model. METHODS: The study was of 12 weeks duration. Fifty sexually mature female New Zealand White rabbits were divided into five groups of equal size. Two groups acted as controls and received normal rabbit chow. Rabbits in one of these groups were ovariectomized. The remaining three groups were ovariectomized but received 1% cholesterol enriched rabbit chow. One of these cholesterol-fed groups received 0.3 mg/kg percutaneous oestradiol daily whilst another received 0.1 mg/kg oral oestradiol daily. Measurements of concentrations of total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were made at the beginning and end of the study. Aortic atheroma formation was measured using computerized image analysis of uptake of Sudan III staining. RESULTS: After 12 weeks there were significant increases in the mean concentrations of TC in the three cholesterol-fed groups compared with controls (P < 0.001). Changes in HDL-C and TG concentrations were less consistent. The mean area of aortic atheroma formation was significantly less in both the percutaneous oestradiol group (4.9%) and the oral oestradiol group (8.6%) compared with the non-oestrogen-treated cholesterol-fed group (19.5%) (P < 0.001, < 0.01 respectively). CONCLUSION: These results suggest that percutaneous oestradiol has a direct protective effect on atheroma formation independent of serum concentrations of total cholesterol.

Clinical magnetic resonance imaging with nuclear medicine correlation.

The current role of magnetic resonance imaging (MRI) in different organ systems is discussed and compared to nuclear medicine and to other available clinical diagnostic modalities. The value of optimizing radiofrequency pulse sequence selection to provide additional tissue characterization is also described. The results of nuclear medicine and MRI studies in 56 patients are compared to evaluate the clinical diagnostic contribution of each imaging modality for various pathological processes. In addition, the state-of-the-art MRI systems and future development in MRI technology with its potential contribution is defined.

Rhinorrhea, ventricular radiopharmaceutical stasis and communicating hydrocephalus: evaluation by serial cisternography.

Spontaneous cerebrospinal fluid rhinorrhea has been known to occur in association with hydrocephalus. The specific pathophysiology which results in a potential communication between the cerebrospinal fluid space (subarachnoid space) and the nasopharynx is unknown. The relationship of CSF movement and rhinorrhea was evaluated in ten random source mongrel dogs. These data suggest that spontaneous CSF rhinorrhea may occur during the early developmental phase of communicating hydrocephalus in dogs. At this time radiopharmaceutical movement showed ventricular entry and clearing. When the lateral ventricles enlarged, ventricular radiopharmaceutical stasis was seen and the rhinorrhea disappeared. This suggests that CSF rkinorrhea may act as a compensatory mechanism which partially protects the CSF compartment to withstand the extra CSF during the early development of communicating hydrocephalus.

Physical characteristics of modern radiographic screen-film systems.

The most important characteristics that determine the performance of a screen-film system are system sensitivity (speed), slope or the average gradient of the characteristic curve, latitude of the film, system resolution (MTF) and system noise. In addition to these, one has to consider secondary characteristics such as the base plus fog level, the base tint, safelight sensitivity, reciprocity law failure and film granularity while evaluating performance. A comprehensive study of many of these characteristics has been made for a number of film-screen systems on the market. The data has been tabulated in the form of what are called "characteristic tables of radiographic film-screen systems." In these tables, characteristics such as average gradient, base plus fog level, etc. which depend on film alone, appear along the horizontal axis and MTF values which essentially depend on screens only appear along the vertical axis. The body of the table contains the absolute speeds in units of mR-1 at 80 KVP (heavy filtration) and a quantum mottle index of the various film-screen combinations tested, since these depend both on the film as well as on the screens.

In vitro measurement of respiration of choroid plexus cells in communicating hydrocephalus.

Communicating hydrocephalus occurs as a result of inappropriate CSF production in the circumstance of diminished absorption. Ventricular enlargement does not progress as rapidly as the measured normal production of CSF would suggest. Thus, compensatory mechanisms must exist to respond in some manner to the altered pathophysiology. In this experiment the metabolic activity (O2 consumption) of the choroid plexus cells in animals with communicating hydrocephalus was compared with that of normals. The modified Cartesian diver technique of Zeuthen13 was employed. These preliminary measurements show that the metabolic activity of the choroid plexus cells was the same in hydrocephalic animals and normals. Response to experimentally induced increased CSF pressure also showed no difference.

Magnetic resonance imaging of ischemic bowel in rabbit model.

Acute mesenteric ischemic bowel disease is a common yet complex disorder with high morbidity and mortality rates predominantly caused by delayed diagnosis. We examined the role of magnetic resonance imaging (MRI) in the early detection of small bowel ischemia using the rabbit model. Surgical ligation of the appropriate arterial vascular supply to the ileum of 10 rabbits produced the ischemic compromise. The animals were imaged at different time intervals after the arterial occlusion. Multislice, T1 and T2-weighted images were obtained in axial and coronal planes. Abnormal findings of the dearterialized segment of bowel were visualized as early as 45 minutes after vascular occlusion. The findings consisted of: (1) bowel wall thickening, (2) two- to three-fold increase in signal intensity from bowel on T2-weighted images, and (3) isointensity or slightly increased signal intensity within the bowel wall on T1-weighted images. MRI appears to be a sensitive, noninvasive technique for the early detection of bowel ischemia in the rabbit animal model.

AUR memorial award--1988. MRI enhancement of perfused tissues using chromium labeled red blood cells as an intravascular contrast agent.

It has been demonstrated that chromium (Cr) labeling significantly decreases the relaxation times of packed red blood cells (RBCs). In this study, the spin-lattice relaxation time (T1) of human red cells was shortened from 836 ms to 29 ms and the spin-spin relaxation time (T2) shortened from 134 ms to 18 ms, when the cells were labeled at a Cr incubation concentration of 50 mM. Labeling of canine cells at 50 mM resulted in a T1 of 36 ms and a T2 of 26 ms. A labeling concentration of 10 mM produced similar relaxation enhancement, with uptake of 47% of the available Cr, and was determined to be optimal. The enhancement of longitudinal and transverse relaxation rates (1/T1,-1/T2) per amount of hemoglobin-bound Cr are 6.9 s-1 mM-1 and 9.8 s-1 mM-1 respectively, different from those of a pure Cr+3 solution. Labeling cells at 10 mM decreased the survival half-time in vivo from 16.6 days to 4.7 days in dogs. No difference in red cell survival was found with the use of hetero-transfusion versus auto-transfusion of labeled RBCs. Significant shortening of the T1 (912 ms to 266 ms, P = .03) and T2 (90 ms to 70 ms, P = .006) of spleen and the T1 (764 ms to 282 ms, P = .005) and the T2 (128 ms to 86 ms, P = .005) of liver occurred when 10% of the RBC mass of dogs was exchanged with Cr labeled cells. Liver and spleen spin density changes (P greater than 0.23) and muscle spin density and relaxation changes (P greater than 0.4) were insignificant. The in vivo T1 of a canine spleen which had been infarcted did not change following transfusion with labeled cells, where the T1 of liver did shorten. We believe this preliminary study suggests that Cr labeled red cells may have the potential to become an intravascular magnetic resonance imaging contrast agent.

Paramagnetic NMR contrast agents. Development and evaluation.

Paramagnetic ions could be theoretically used as NMR contrast agents because of their effect upon T1. However, the toxicity of these ions prevents their application. By the formation of appropriate chemical complexes with these ions, the toxicity of these agents can be substantially reduced while maintaining the paramagnetic effect. Two potential NMR contrast agents, one for oral use and one for intravenous administration, were developed and evaluated both in vitro and in vivo. The effect upon T1 in vitro of these paramagnetic compounds was determined using a JEOL FX-90Q NMR spectrometer. These agents were evaluated in vivo in dogs with a Technicare 0.3 tesla superconducting magnet system and in rabbits with the Aberdeen 0.04 tesla resistive NMR imager. Using calculated T1 NMR images, a nontoxic dose of gadolinium oxalate provided visualization of the gastrointestinal tract. Intravenous administration of chromium EDTA provided enhancement of the kidneys, ureters, and bladder, thereby potentially allowing for the evaluation of renal function with magnetic resonance imaging. Stable paramagnetic complexes can serve as effective, nontoxic, oral and intravenous NMR contrast agents.

Enhancement of red blood cell proton relaxation with chromium labeling.

Nuclear medicine has utilized chromium (Cr) for decades to label red blood cells (RBCs). The purpose of this project was to determine whether sufficient paramagnetic Cr could be bound to red cells to influence proton relaxation significantly. We demonstrated that the T1 and T2 of RBCs can be substantially shortened by labeling them with paramagnetic Cr. Proton relaxation enhancement occurs when red cells are incubated with sodium chromate (VI) over a concentration range of 0.10 mM to 31.6 mM. Labeling with Cr at a concentration of 31.6 mM shortened the T1 of packed cells from 714 msec to 33 msec, and the T2 from 117 msec to 24 msec, as compared with nonlabeled red cells. In vitro hemolysis was significantly increased after labeling at 31.6 mM, but not at lower concentrations. Cr-induced proton relaxation enhancement varied with RBCs from different species, temperature, pH, and length of incubation. T1 values of kidneys containing labeled red cells (303 msec), or labeled cells diluted 10-fold with nonlabeled cells (479 msec), were decreased compared with kidneys containing only nonlabeled cells (600 msec). Finally, preliminary data indicate that the signal intensity of perfused renal tissue is significantly influenced in vivo by infusion of Cr-labeled RBCs. This study demonstrated that Cr labeling of RBCs sufficiently enhances red cell proton relaxation to provide excised organs containing red cells, of which 10% have been Cr-labeled, with shorter T1 and T2 values than organs containing nonlabeled cells. In addition, the ability of labeled cells to alter signal intensity in vivo suggests that Cr may have the potential to become an MRI contrast agent.

Early changes in experimental hydrocephalus.

In acute obstruction of the cerebral spinal fluid (CSF) absorption pathways, fluid is produced more rapidly than it is absorbed, and the ventricles enlarge proximal to the obstructions. Communicating hydrocephalus results from a difference between the rates of production and absorption of cerebrospinal fluid. In animals with chronic communicating hydrocephalus, the initial pathologic changes appear to involve the periventricular tissue near the angles of the lateral ventricles. The present investigation was designed to identify the various changes associated with the production of communicating hydrocephalus in acutely hydrocephalic preparations and to relate these findings to those found in experimental animals with chronic communicating hydrocephalus. The results of this study seem to confirm that the changes noted in the chronically hydrocephalic animals occur as early as 12 hours after the restriction of the normal flow of CSF.

Cobblestone area-forming cells, long-term culture-initiating cells and NOD/SCID repopulating cells in human neonatal blood: a comparison with umbilical cord blood.

Our prior study demonstrated that neonatal blood (NB) contained hematopoietic stem and progenitor cells that declined rapidly after birth. To validate that NB is a source of functional stem cells, we characterized this population in terms of cobblestone area-forming cells (CAFC), long-term culture-initiating cells (LTC-IC) and NOD/SCID mouse repopulating cells (SRC) in NB and umbilical cord blood (CB). Our data demonstrated that the frequencies of CAFC (30.2 vs 37.1, P = 0.14) and LTC-IC (28.6 vs 31.0, P = 0.49) in 1 x 10(5) mononuclear cells (MNC) of NB and CB were similar, suggesting that these cells were preserved in the circulation of the neonates shortly after birth. Sublethally irradiated NOD/SCID mice were transplanted with CD34(+) cells enriched from thawed NB and CB. At 6 weeks post transplant, human (hu)CD45(+) cells were detected in the bone marrow (BM), spleen and peripheral blood (PB) of the mice as demonstrated by flow cytometric and DNA analysis. Levels of huCD45(+)cells and colony forming units (CFU) appeared to be dependent on the infusion cell dose and were higher in animals receiving CB cells when compared with those of the NB group. The transplanted cells were capable of differentiation into multi-lineage progenitor cells (CD34(+) cells and differential CFU), as well as mature myeloid (CD14(+), CD33(+)), B lymphoid (CD19(+)) and megakaryocytic (CD61(+)) cells in the recipients. NB cells, subjected to ex vivo culture in an optimized preclinical condition, were significantly expanded to early and committed progenitor cells. Expanded NB contained SRC at a reduced quantity but with high proportions of CD14(+) cells and CD33(+) cells. Our study confirms that NB contains pluripotent hematopoietic stem and progenitor cells capable of homing and engrafting the NOD/SCID mice.