Modulation of Rat Pial Arteriolar Responses to Flow by Glucose

Background: Pial arteriolar responses to flow contribute to regulation of cerebral perfusion and vary according to the transmural pressure to which the vessel is exposed. This study determined the effect of increased glucose concentration on the flow responses of pial arterioles at low and high levels of transmural pressure.

Methods: Pial arterioles from Sprague-Dawley rats were mounted in a perfusion myograph. In some arterioles, the endothelium was removed by perfusion with air. Diameters were recorded at transmural pressures of 60 and 120 mmHg during superfusion with physiologic saline containing 5 mm D-glucose, 20 mm D-glucose, or 5 mm D-glucose and 15 mm L-glucose. Diameters during superfusion with saline containing 44 mm D-glucose were measured at an intraluminal pressure of 60 mmHg. Flow-diameter relationships (5-30 [mu]l/min) were recorded during perfusion with the same solutions.

Results: Increasing D-glucose concentration caused constriction (P < 0.05) in endothelium-denuded but not in endothelium-intact arterioles. Addition of L-glucose caused constriction in endothelium-intact and -denuded vessels (P < 0.05 for both). At a D-glucose concentration of 5 mm and at low intraluminal pressure, flow elicits endothelium-dependent dilation such that shear stress remains constant. At a D-glucose concentration of 20 or 44 mm, after addition of L-glucose (15 mm), and at high intraluminal pressures, flow elicits constriction and shear stress is unregulated.     

Comparison of Cytotoxic Effects of Bisphosphonates In Vitro and In Vivo

We compared the cytotoxic effects of alendronate (ALN) and incadronate (YM175) on isolated rabbit osteoclasts in vitro and on rats in vivo. In the in vitro experiment, each bisphosphonate was added to the culture of isolated osteoclasts at the final concentration of 3 × 10⁻⁵, 3 × 10⁻⁴, or 3 × 10⁻³ M, and the amount of creatine phosphokinase (CPK) released into the medium was taken as an index of cytotoxicity at 5, 10, and 24 hours after the treatment. Also viability of osteoclasts, measured in terms of trypan blue exclusion, was assessed at 24 hours after the treatment. In YM175-treated groups, CPK activity in the medium increased in a concentration-dependent manner with time, and phase-contrast microscopic observation revealed damaged cell membranes and nuclear deterioration in YM175-treated osteoclasts. As a result, the viability of the osteoclasts was decreased at the concentrations of 3 × 10⁻⁴ and 3 × 10⁻³ M. However, in the ALN-treated groups, neither CPK activity nor viability of isolated osteoclasts changed significantly compared with control levels even at 3 × 10⁻³ M for up to 24 hours. In the in vivo experiment, each bisphosphonate was administered separately to normal rats (7 weeks old, Sprague-Dawley) by intravenous injection at 1, 5, or 25 mmol/kg. Two days after the injection, the animals were euthanized, and the plasma Ca concentration and total CPK activity were measured. In YM175-injected rats, the CPK activity increased at 25 mmol/kg, and a slight decrease in the plasma Ca level was seen at this dose. In contrast, in ALN-injected rats, CPK activity did not increase even at 5 or 25 mmol/kg, and the plasma Ca level did decrease significantly compared with controls. Isozyme analysis revealed that, not only was CPK activity increased in the BB type in YM175-injected rats, it was also increased in the MB and MM types. In conclusion, alendronate, unlike YM175, does not have any cytotoxic effects on osteoclasts either in vitro or in vivo. Received: 14 May 1997 / Accepted: 27 January 1998     

The Effects of Losartan in Preserving the Structural Integrity of Decellularized Small Diameter Vascular Allograft Conduit Implants In Vivo

Decellularization is a proposed method of preparing nonautologous biological arterial vascular scaffolding; however, the fate of the supporting medial elastic fiber, which is important in preserving the vascular structural integrity, is uncertain. The influence of losartan on preserving the medial elastic fiber integrity in decellularized small diameter vascular conduits (SDVC) was investigated. Decellularized infrarenal abdominal aortic allografts were implanted in Sprague‐Dawley rats treated either with (study rats, n = 6) or without oral losartan (control rats, n = 6) and graded 8 weeks later according to a remodeling scoring system (1‐mild, 2‐moderate, 3‐severe) which we devised based on the intimal hyperplasia degree, morphologic changes, and elastic fiber fragmentation of the conduits. DAPI immunohistochemistry analysis was performed in 47 (25 decellularization only and 22 losartan treatment) cross‐sectional slide specimens. The losartan versus decellularization only SDVC showed a significantly lower medial elastic fragmentation score (1.32 vs. 2.24, P < 0.001), superior medial layer preservation, and relatively more normal appearing intimal cellular morphology. The results suggested rats receiving decellularized SDVCs treated with losartan may yield superior medial layer elastic fiber preservation.     

Development of an Axial Flow Ventricular Assist Device: In Vitro and In Vivo Evaluation

Abstract: A collaborative effort between Baylor College of Medicine and NASA/Johnson Space Center is underway to develop an axial flow ventricular assist device (VAD). We evaluated inducer/impeller component designs in a series of in vitro hemolysis tests. As a result of computational fluid dynamic analysis, a flow inducer was added to the front of the pump impeller. According to the surface pressure distribution, the flow inducer blades were connected to the impeller long blades. This modification eliminated high negative pressure areas at the leading edge of the impeller. Comparative studies were performed between inducer blade sections that flowed smoothly into the impeller blades (continuous blades) and those that formed discrete separate pumping sections (discontinuous blades). The inducer/impeller with continuous blades showed significantly (p < 0.003) lower hemolysis with a normalized index of hemolysis (NIH) of 0.018 ±< 0.007 g/100 L (n = 3), compared with the discontinuous model, which demonstrated an NIH of 0.050 ± 0.007 g/100 L (n = 3). The continuous blade model was evaluated in vivo for 2 days with no problems. One of the pumps evaluated ran for 5 days in vivo although thrombus formation was recognized on the flow straightener and the inducer/impeller. As a result of this study, the pump material was changed from polyether polyurethane to polycarbonate. The fabrication method was also changed to a computer numerically controlled (CNC) milling process with a final vapor polish. These changes resulted in an NIH of 0.0029 ± 0.0009 g/100 L (n = 4). which is a significant (p < .0001) value 6 times less than that of the previous model. This model was used for in vivo studies and achieved 9 days of operation with a sufficient flow between 3.6 and 4.7 L/min against 80 to 100 mm Hg mean arterial pressure. Plasma free hemoglobin levels remained at 2–3 mg/dl with a hematocrit of 20%.     

Immunosuppressive Effects of Emodin: An In Vivo and In Vitro Study

Objective

We sought to investigate the immunosuppressive effects of emodin and its potential in vivo and in vitro mechanisms.

Methods

In vitro immunosuppressive effects of emodin were analyzed by its ability to suppress the response of human peripheral blood mononuclear cells to phytohemagglutinin (PHA) and to mixed lymphocyte culture (MLC). We examined changes in interleukin (IL)-2 and IL-4 in within MLC supernates. The in vivo immunosuppressive effects of emodin were analyzed using a skin transplantation model in mice. We also investigated the mean survival time (MST) and plasma IL-2 levels.

Results

In vitro experiments: Responses of mononuclear cells to PHA and MLC were suppressed by emodin treatment. Decreased production of IL-2 along with promoted secretion of IL-4 was also observed by emodin treatment during MLC. In vivo experiments: The emodin-treated group showed prolonged MST of skin grafts and decreased serum IL-2 production.

Conclusions

Emodin showed immunosuppressive activities both in vivo and in vitro. The potential immunosuppressive mechanism of emodin's may be suppression of lymphocyte proliferation and influences on cytokines.     

Cutaneous Effects of Cryogen Spray Cooling on In Vivo Human Skin

BACKGROUND: Despite widespread clinical use of cryogen spray cooling (CSC) in conjunction with laser dermatologic surgery, in vivo cutaneous effects have not been systematically evaluated. OBJECTIVE: The authors characterize the in vivo cutaneous effects for Fitzpatrick skin types I through VI after CSC exposures of varying spurt durations and spurt delivery patterns (single vs. multiple spurts). MATERIALS AND METHODS: Twenty-seven normal human subjects were exposed to single cryogen spurts from 10 to 80 milliseconds, and multiple spurt patterns consisting of two 20-millisecond spurts, four 10-millisecond spurts, and eight 5-millisecond spurts. Subjects were evaluated by clinical observation and photography at 1 hour, 1 day, and 1, 4, 8, and 12 weeks after CSC exposure. RESULTS: Acute erythema and urticaria (1-24 hours) were noted in 14 of 27 and 3 of 27 subjects, respectively. Transient hyperpigmentation occurred in 4 of 27 subjects (skin types III-VI) but resolved spontaneously without medical intervention in all subjects by 8 weeks. No permanent skin changes were noted in any subjects. Skin reactions were more common with longer single-spurt durations (50 milliseconds or greater) and multiple spurt patterns. CONCLUSION: Acute erythema, urticaria, and, less commonly, transient hyperpigmentation were observed after CSC exposure. Permanent skin injury was not observed and is unlikely.     

Beneficial Effects of Desferrioxamine on Encapsulated Human Islets—In Vitro and In Vivo Study

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 μM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose‐induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF‐1α (Hypoxia inducible factor‐1 alpha), caspase‐3 and caspase‐8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase‐3 and caspase‐8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6‐ and 2.5‐fold at 24 and 72 h, respectively, with a concomitant increase in HIF‐1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF‐1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.     

Effects of Mycophenolic Acid on Human Fibroblast Proliferation, Migration and Adhesion In Vitro and In Vivo

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novo synthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.     

Flow Characteristics of the St. Jude Prosthetic Valve: An In Vitro and In Vivo Study

The St. Jude cardiac prosthetic aortic valve was evaluated in vitro and in vivo in an attempt to establish flow characteristics and to correlate them with clinical findings. In vitro, a fluid vehicle (6% Polyol V-10, 32°C) with viscosity similar to blood (0.035 dyne-sec/cm²) was used under conditions of steady flow through a flow chamber simulating the aortic root. Gradient, velocity, and shear stress were measured 5.79 mm, 26.79 mm, 44.79 mm, and 77.79 mm downstream from 25-mm and 27-mm valves using a laser-Doppler anemometer. At 417 ml/sec, the valve gradient was 6.2 mmHg with the 25-mm valve, and 5.2 mmHg with the 27-mm prosthesis. Velocity was maximum at the orifice center, and wall shear stress was low (maximum 600 dyne/cm²). In vivo, six patients with 25-mm St. Jude aortic valves were studied within 48 hours after surgery to determine cardiac output, valve flow, and gradient. The gradient was 3.3 ± 1.9 mmHg (M ± SD) at 249 ± 96 ml/sec and the effective valve area was as large as the geometric area (2.58 vs. 3.09 cm²). Thus, flow through the St. Jude valve is unobstructed and central, has low turbulence, and achieves optimal effective valve area for a given available orifice area.     

Effects of Testicular and Ovarian Inhibin-Like Activity, Using In Vitro and In Vivo Systems

Inhibin-like activities in charcoal-treated bovine follicular fluid (FF) and medium from cultured Sertoli cells (SCCM) were assayed in an in vitro bioassay system, using cultured pituitary cells. Addition of both fluids resulted in parallel dose-dependent decreases of the concentration of follicle-stimulating hormone (FSH) in the medium, both in the presence or absence of luteinizing hormone-releasing hormone (LH-RH).
A single injection of FF into immature and adult male and female rats resulted in decreased peripheral levels of FSH, but not of LH, after 4 or 8 h. This decrease was larger and occurred faster in adult female rats than in prepubertal female animals. Injection of FF into adult female rats, immediately after unilateral ovariectomy (ULO) prevented the specific increase of FSH levels, occurring in control animals. This suppression could not be obtained after treatment with steroids.
Daily treatment of adult female or immature male rats for periods longer than 5 days did not result in prolonged suppression of circulating FSH concentrations; LH levels were significantly increased. The female animals showed cyclic vaginal smear changes and normal ovulation; in the male rats testis weight and numbers of spermatogenic cells were reduced.
It is concluded that testicular and ovarian inhibin-like activities have similar properties. Injections of FF into male and female rats cause similar effects on FSH and LH. The effect of FF in ULO-animals suggests that inhibin could play a role in the short-term regulation of the number of developing follicles in the ovary. Injection of FF into male rats causes a probably transient impairment of spermatogenesis through an initial suppression of FSH.     

Impact of In Vivo Ranges of the Variances in the Flow Velocity Waveforms and Flow Split Ratio on the Hemodynamic Effects of the Anastomotic Cuff at Distal End-to-Side Anastomosis

Purpose The blood flow inside distal end-to-side anastomoses may be affected by inlet flow velocity profiles and outlet blood distribution ratios. This study investigated the in vivo range of these variables and their impact on the hemodynamic effects of an anastomotic cuff using a computational fluid dynamics approach. Materials and Methods Predesigned expanded polytetrafluoroethylene cuffed grafts were used in 22 femoropopliteal bypasses and straight grafts were used for 10 cases. The flow distribution was examined by angiographic techniques and the inlet flow velocity was determined by a spectral Doppler method. Results The caudal outflow distribution ratio was 95.6% ± 7.2% (75%–100%). The positive peak flow velocity was 1.66 ± 0.25 m/s. The ratio of the absolute value of the negative to positive peak velocity was 0.32 ± 0.12. The cuff increased wall shear stress along the artery floor regardless of the flow velocity profiles at 100% of the caudal distribution ratio but not at 75%. Conclusion The hemodynamics inside the anastomosis were thus found to be affected by these flow variables. The proper indications for the cuff should therefore not be decided based simply on the location of the anastomosis without taking the fluid dynamic factors in the vicinity of the anastomosis into full consideration.     

Effects of Human Tumor Cell Lines on Local New Bone Formation In Vivo

Although some tumors cause osteolytic lesions, there are some that stimulate new bone formation. This is an important phenomenon because the responsible mechanisms probably represent an aberration of normal physiological bone formation, and identifying the factors involved in the process may lead to new therapies for various bone diseases. To clarify our understanding of the potential mechanism responsible, we compared and quantitated the extent of new bone formation stimulated by human tumors (HeLa, Hep-2, AV-3, FL, WISH and KB), some of which have osteogenic activity in vivo [2]. Tumor cells were injected over the calvaria of nude mice to examine formation of new bone. The tumor cells produced three histologically distinct patterns of new bone growth: (1) WISH and KB stimulated appositional bone growth adjacent to periosteal bone surfaces; (2) HeLa and Hep2 induced new bone growth over calvarial surface even when distant from the tumor mass; (3) FL stimulated bone formation adjacent to periosteum as well as ectopic bone formation in sites distant from bone. All tumors except AV3 induced mean new bone thickness >100 μm, and Hep-2 cells produced bone 330 μm thick. PCR and Northern blot analysis of mRNA isolated from cultured tumor cells revealed that all cell lines expressed mRNA for TGFβ, (fibroblast growth factor) FGF-1, FGF-2, and IGF-I, and most cell lines produced mRNA for PDGF. Only FL expressed large amounts of mRNA for BMP2. In serum-free conditioned media from Hep2 and HeLa cells purified by heparin affinity chromatography, we have identified FGF-1, FGF-2, and PDGF by immunodetection with specific antibodies. Our results show that new bone growth caused by these tumors is likely due to the production of bone growth factors by the tumor cells, and that the overall effects on bone may be due to several factors working in concert. Received: 15 January 1996 / Accepted: 3 May 1996     

Genomic Analysis of Pterostilbene Predicts Its Antiproliferative Effects Against Pancreatic Cancer In Vitro and In Vivo

Background

To investigate the inhibitory role of pterostilbene in pancreatic cancer, we conducted a genomic analysis of pterostilbene-treated pancreatic cancer cells. We also investigated the effect of pterostilbene upon the carcinogenic markers, manganese superoxide dismutase, cytochrome C, Smac/DIABLO, and STAT3 phosphorylation in vitro. The antiproliferative effects of pterostilbene were further evaluated in an in vivo model.

Methods

Pancreatic cancer cells were treated with pterostilbene and evaluated with DNA microarray analysis. Pterostilbene-treated cells were analyzed for cytochrome C, Smac/DIABLO, manganese superoxide dismutase (MnSOD)/antioxidant activity, and STAT3 phosphorylation using ELISA. Data were statistically analyzed using ANOVA. Pterostilbene was then administered to nude mice for 8?weeks, and tumor growth rates were recorded and statistically analyzed.

Results

Microarray analysis of pterostilbene-treated cells revealed upregulation of pro-apoptosis genes. In vitro, pterostilbene treatment altered levels of phosphorylated STAT3, MnSOD/antioxidant activity, cytochrome C, and Smac/DIABLO. In nude mice, oral pterostilbene inhibited tumor growth rates.

Conclusion

Pterostilbene alters gene expression in pancreatic cancer and increases the antiproliferative markers cytochrome C, Smac/DIABLO, and MnSOD/antioxidant activity. It was also shown to inhibit phosphorylated STAT3, a marker of accelerated tumorigenesis, and decrease pancreatic tumor growth in vivo. Further studies are warranted to elucidate the effects of pterostilbene in humans.     

Laser-Doppler Examination Shows High Flow in Some Common Telangiectasias of the Lower Limb

BACKGROUND: The accepted pathophysiology of telangiectasias is reflux from superficial or deep veins. There are physical signs and scientific findings that do not fit this theory but support the possibility of arteriovenous (AV) shunt origin. OBJECTIVE: If there is a higher flow in spider veins than in the surrounding skin, it means that AV shunts participate in the circulation of the telangiectasia. On the other hand, slow flow indicates reflux as the etiologic factor. METHOD: Telangiectasias and the surrounding skin of 22 legs of 19 patients were examined with laser-Doppler equipment. RESULTS: The probe over the spider vein found a higher flow value (average 28.2 perfusion units [PU]) than in the surrounding skin (15.6 PU) in 13 limbs, but it was significantly higher only in 5 cases. In 9 limbs, the flow was slower. CONCLUSION: We interpret the higher flow values as a consequence of open AV shunts. This means that AV shunt pathophysiology was present in some of our cases.     

In Vivo and In Vitro Effects of a Pulsed Electromagnetic Field on Net Calcium Flux in Rat Calvarial Bone

Although PEMF's have been found to promote fracture healing and to modulate the activity of bone cells in vitro, effects on bone metabolism are largely unexplored. A bioassay using neonatal rat calvarial bone was used to determine the early effects of a pulsing electromagnetic field (PEMF) exposure in vivo and in vitro on bone metabolic calcium exchange. Bone discs taken from whole body exposed animals (0-4 hours) show a log exposure time-dependent average increase in net Ca uptake in the 0-50% range (r2 = 0.83). This increase could be detected immediately after exposure and also after 24 hours, but not 48 hours later. Animals given whole body PEMF exposure also showed a decrease in serum calcium and did not elevate serum Ca after administration of exogenous parathyroid hormone (PTH). Bone discs from untreated rats, exposed to PEMF for 15 minutes in vitro and then assayed, showed net Ca uptake increases of a similar magnitude and also were refractory to the Ca-releasing effect of PTH. Unexposed discs responded normally to PTH by decreasing net Ca uptake. Treatment of calvarial discs with calcitonin or acetazolamide, both of which inactivate osteoclasts, made the bone refractory to further increases in Ca uptake by PEMF. These results suggest that PEMF exposure produces PTH-refractory osteoclastics and has a relatively rapid effect on increasing net bone Ca uptake, putatively due to a decrease in PTH/paracrine-mediated bone resorption.