Blue Dye Staining Research Articles

Vascular endothelial growth factor induced functional and morphologic signs of endothelial dysfunction in isolated arteries from normal pregnant women

Objective: The purpose of this study was to determine the effects of vascular endothelial growth factor on basal tone, endothelium-dependent dilatation, permeability, and morphologic features of endothelium in isolated arteries from normal pregnant women. We hypothesized that vascular endothelial growth factor might induce signs of endothelial dysfunction. Study Design: Arteries (approximately 200 μm) were dissected from subcutaneous fat biopsy specimens that were obtained at cesarean delivery and mounted on a pressure arteriograph. Changes in basal tone, dilatation to bradykinin (1 nmol/L to 3 μmol/L) before, during, and after 3 hours of incubation with vascular endothelial growth factor (0.5 or 1 nmol/L), vascular endothelial growth factor (0.5 nmol/L) plus bosentan (a nonselective endothelin receptor A and B antagonist, 1 μmol/L), or vehicle were compared. Scanning electron microscopy was applied for endothelial morphologic features. Permeability to Evans blue dye was evaluated in arteries after incubation with vascular endothelial growth factor, vascular endothelial growth factor plus angiopoietin-1, or vehicle, and in arteries that were obtained from women with preeclampsia. Results: Basal tone was higher after 60 minutes of incubation with vascular endothelial growth factor (0.5 nmol/L) compared with vehicle (29% ± 5% [n = 10] vs 10% ± 4% [n = 7], P = .006). Combination of vascular endothelial growth factor with bosentan failed to increase the tone (n = 4). Bradykinin-mediated dilatation was impaired in arteries that were incubated with vascular endothelial growth factor 0.5 nmol/L (max dilatation: 287% ± 16% vs 160% ± 23% [n = 10], P = .0001) or vascular endothelial growth factor 1 nmol/L (max dilatation: 207% ± 21% vs 88% ± 4% [n = 3], P = .003). Bradykinin-mediated dilatation was similar after incubation with vehicle (n = 7) or the combination of vascular endothelial growth factor plus bosentan (n = 4). Evans blue dye staining was higher after incubation with vascular endothelial growth factor but was reversed by the addition of angiopoietin-1. Scanning electron microscopy demonstrated the development of intercellular gaps. Conclusion: Vascular endothelial growth factor impaired bradykinin-mediated dilatation and enhanced basal tone and permeability. This might indicate a potential role for vascular endothelial growth factor in the development of endothelial dysfunction in pregnancy. Angiopoietin-1 inhibited the vascular endothelial growth factor–induced vascular leakage, which may have therapeutic implications in preeclampsia. (Am J Obstet Gynecol 2003;188:168-76.)

Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists

In the mouse and several other species, including man, capacitated acrosome-intact spermatozoa interact with natural [soluble zona pellucida (ZP) and progesterone (P4)] and synthetic [neoglycoproteins (ngps) and calcium (Ca 2+) ionophore] agonists, prior to the initiation of a Ca 2+-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane overlying the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents [i.e., induction of the acrosome reaction (AR)]. This step is believed to be a prerequisite that enables the acrosome-reacted spermatozoon to penetrate the ZP and fertilize the egg. Although the rat is one of the most commonly used laboratory animals, very little is known about the chemical nature of agonists that induce the AR in this species. The lack of this information is primarily due to the fact that the rat sperm acrosome is a relatively thin structure. Thus, it is difficult to assess the status of the sperm acrosome in this species. In this report, we describe the use of a Coomassie brilliant blue dye staining procedure to assess the status of the rat sperm acrosome by light microscopy. The procedure is highly reproducible and has allowed us to determine the effects of carbohydrate (ngps and mouse ZP) and noncarbohydrate (P4 and Ca 2+ ionophore) agonists on capacitated spermatozoa. In addition, we have used a pharmacological approach to examine the functional significance of calmodulin (CaM), a Ca 2+-binding protein, in induction of the AR in spermatozoa. Data presented in this report demonstrate that several ngps, solubilized mZP, P4, and Ca 2+ ionophores induce the AR in rat spermatozoa. Furthermore, we demonstrate that, whereas CaM antagonists blocked P4-induced AR, most of the inhibitors used had no significant effect on the Ca 2+ ionophore-induced (nonphysiological) AR.

Cardiac myocyte-specific excision of the beta1 integrin gene results in myocardial fibrosis and cardiac failure.

Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of beta1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of beta1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the beta1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte beta1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of beta1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old beta1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt(max) and LV dP/dt(min)) as compared with control groups (n=8 to 10 of each, P<0.01). Hemodynamic loading imposed by 7 days of transverse aortic constriction showed that the beta1 integrin knockout mice were intolerant of this stress as they had 53% survival versus 88% in controls (n=15 each). By 6 months of age, mice with depressed ventricular expression of beta1 integrin developed a dilated cardiomyopathy that was not evident in any control animals and had patchy decrease in glucose metabolism as determined by positron emission tomography. Myocyte membrane integrity as determined via Evan's blue dye staining was disrupted in the beta1 integrin knockout mice. This model provides strong evidence for the importance of beta1 integrin in cardiac form and function and indicates that integrins can be linked to development of cardiomyopathies.

In vivo blockade of tumor necrosis factor-alpha accelerates functional endothelial recovery after balloon angioplasty.

Tumor necrosis factor-alpha (TNF) is expressed locally in arteries at sites of balloon injury. In vitro studies have shown that TNF inhibits cell cycle progression and induces apoptosis in endothelial cells. Accordingly, we performed a series of experiments to test the hypothesis that inhibiting TNF could accelerate endothelial recovery after angioplasty. TNF soluble receptor (TNFsr) has been shown to neutralize the actions of TNF in vitro and in vivo. Sprague-Dawley rats received TNFsr versus control IgG through an intraperitoneal injection. De-endothelializing balloon injury was then performed, and animals were killed after 1 week to evaluate re-endothelialization (Evans blue dye staining) and after 2 weeks to evaluate re-endothelialization and endothelial function. At both time points, blockade of TNF using TNFsr resulted in an increase in re-endothelialization, as measured as absolute area and percent area re-endothelialized. TNFsr also accelerated functional endothelial recovery, which manifest as an increase in nitric oxide production. Neointimal thickening was also shown inhibited. In vivo blockade of TNF accelerates functional endothelial recovery after barotraumatic de-endothelializing injury. These findings suggest that locally expressed TNF acts to inhibit functional endothelial recovery after angioplasty and that transient blockade of TNF may improve the long-term success of angioplasty.

Regional topical hypothermia of the beating heart: preservation of function and tissue

Background. Protection of the myocardium during beating heart operations is paramount. The goal of this study is to determine if regional topical hypothermia (RTH) preserves myocardial viability and function during periods of temporary coronary artery occlusion. Methods. Sixteen pigs were divided into two groups (RTH and control). Each group received 40 minutes of midleft anterior descending coronary occlusion followed by 3 hours of reperfusion. The RTH group (n = 10) received RTH and the control group (n = 6) received no cooling. Myocardial and core temperatures were measured with thermistors. Sonomicrometers and micromonameters were used to determine load independent indices of myocardial function. These indices were measured at base line, during coronary occlusion, and at 3 hours of reperfusion. The myocardium at risk and the infarct area were determined with monastral blue dye and triphenyl tetrazolium chloride staining. Results. The mean myocardial temperature in the risk zone during coronary occlusion was significantly less in the RTH group (29.4°C ± 5.6°C versus 35.7°C ± 1.1°C, p < 0.05). After 40 minutes of coronary occlusion, both the RTH group and control had a significant reduction in regional elastance (9.38 ± 3.54 and 11.05 ± 1.67 mm Hg/mm) compared with base line measurements (14.70 ± 2.42 and 16.80 ± 4.79 mm Hg/mm), p < 0.05. However, after 3 hours of reperfusion, the elastance returned to base line levels in the RTH group (15.83 ± 3.06 mm Hg/mm) but remained significantly depressed in the control group (9.97 ± 3.63 mm Hg/mm, p < 0.04). Myocardial necrosis as a percentage of the risk zone was significantly less in the hypothermia group (25% ± 2% versus 62% ± 5%, p < 0.001). Conclusions. Regional topical hypothermia during isolated temporary coronary occlusion provides regional myocardial protection expressed as a return of function and decreased necrosis. Regional topical hypothermia may be clinically applicable to myocardial preservation during beating heart operations.

Sentinel lymph node biopsy for melanoma: how many radioactive nodes should be removed?

Sentinel lymph node (SLN) biopsy has become a standard method of staging patients with cutaneous melanoma. Sentinel lymph node biopsy usually is performed by intradermal injection of a vital blue dye (isosulfan blue) plus radioactive colloid (technetium sulfur colloid) around the site of the tumor. Intraoperative gamma probe detection has been shown to improve the rate of SLN identification compared to the use of blue dye alone. However, multiple sentinel nodes often are detected using the gamma probe. It is not clear whether these additional lymph nodes represent true sentinel nodes, or second-echelon lymph nodes that have received radiocolloid particles that have passed through the true sentinel node. This analysis was performed to determine the frequency with which these less radioactive lymph nodes contain metastatic disease when the most radioactive, or "hottest," node does not. In the Sunbelt Melanoma Trial, 1184 patients with cutaneous melanoma of Breslow thickness 1.0 mm or more had sentinel lymph nodes identified. Sentinel lymph node biopsy was performed by injection of technetium sulfur colloid plus isosulfan blue dye in 99% of cases. Intraoperative determination of the degree of radioactivity of sentinel nodes (ex vivo) was measured, as well as the degree of blue dye staining. Sentinel nodes were identified in 1373 nodal basins in 1184 patients. A total of 288 of 1184 patients (24.3%) were found to have sentinel node metastases detected by histology or immunohistochemistry. Nodal metastases were detected in 306 nodal basins in these 288 patients. There were 175 nodal basins from 170 patients in which at least one positive sentinel node was found and more than one sentinel node was harvested. Blue dye staining was found in 86.3% of the histologically positive sentinel nodes and 66.4% of the negative sentinel nodes. In 40 of 306 positive nodal basins (13.1%), the most radioactive sentinel node was negative for tumor when another, less radioactive, sentinel node was positive for tumor. In 20 of 40 cases (50%), the less radioactive positive sentinel node contained 50% or less of the radioactive count of the hottest lymph node. The cervical lymph node basin was associated with an increased likelihood of finding a positive sentinel node other than the hottest node. If only the most radioactive sentinel node in each basin had been removed, 13.1% of the nodal basins with positive sentinel nodes would have been missed. It is recommended that all blue lymph nodes and all nodes that measure 10% or higher of the ex vivo radioactive count of the hottest sentinel node should be harvested for optimal detection of nodal metastases.

Methylene blue staining: Is it really useful in Barrett's esophagus?

Background: If areas of specialized intestinal metaplasia (SIM) or dysplasia in Barrett's esophagus can be identified at endoscopy, the number of biopsies could be reduced and the sensitivity of biopsy surveillance would increase. It has been suggested that methylene blue (MB) dye staining may be useful for this purpose. Methods: Nine patients were prospectively studied with Barrett's esophagus. Staining involved sequential spraying of 10% N-acetylcysteine, 0.5% MB and water. Quadrantic biopsies were obtained from Barrett's epithelium and collected in separate containers depending on whether they were taken from stained or unstained areas. Seven patients undergoing yearly surveillance were asked to compare the discomfort of this endoscopy with that of previous surveillance endoscopies. Biopsies were analyzed for the presence and the percentage of SIM and dysplasia by a nonblinded pathologist. Results: MB staining prolonged endoscopy by a mean of 8 minutes (47% increase in procedure time) and was associated with significant vomiting during the procedure in 2 patients. Staining was observed in all 9 patients. All 7 patients undergoing yearly endoscopic surveillance indicated more discomfort with endoscopy plus MB staining. Of 37 biopsies from stained mucosa, 20 contained SIM; of 23 from unstained mucosa, 15 contained SIM (57% sensitivity, 32% specificity for MB staining). Conclusions: In this small, nonblinded study MB staining was associated with prolongation of endoscopy, increased patient discomfort, and potentially serious complications and was neither very sensitive nor specific for SIM. It is our recommendation that this technique should not be routinely used in endoscopic surveillance of patients with Barrett's esophagus. Further studies of MB staining are needed. (Gastrointest Endosc 2001;53:333-5.)

Contact areas and pressure distribution on the proximal articular surface of the proximal phalanx under sagittal plane loading.

The objective of this study was to map topographically contact areas and pressure distributions on the proximal articular surface (PAS) of the proximal phalanx (PI) under various clinically relevant loading conditions. Left and right forelimbs of 13 mature horses were transected halfway down the radius and loaded in a position mimicking the weightbearing attitude close to the midstance phase. Five loads were used which corresponded with loads that can be expected in different gaits or during athletic performance (stance: 1800 N, walk: 3600 N, trot: 5400 N, gallop: 10,500 N and jumping: 12,000 N). Contact areas and pressure distributions at the PAS of PI were determined using a methylene blue dye staining technique and 2 pressure sensitive films (low pressure: range 2.5-10 MPa and medium pressure: range 10-50 MPa). The contact area of PI was positively correlated (r = 0.86; P<0.01) with the applied load. The contact area increased from 63% at 1800 N to 95% at 12,000 N and gradually shifted to include more of the edges of the articular surface, but especially the dorsal articular margin of PI. Pressure distribution patterns were similar under the different loading conditions. Pressure was less at the palmar margin and in the central depression and highest at the dorsal articular margin. With increasing load, the highest peak pressures were measured at sites of the dorsal articular margin that are not loaded in the standing or walking horse. The results of this study suggest that the frequent occurrence of osteochondral lesions at the dorsal articular margin of PI is caused by the combination of the intermittent character and the high absolute values of loads at this site as they occur during athletic performance.

Practical guidelines for optimal gamma probe detection of sentinel lymph nodes in breast cancer: Results of a multi-institutional study

Introduction: Multiple radioactive lymph nodes are often removed during the course of sentinel lymph node (SLN) biopsy for breast cancer when both blue dye and radioactive colloid injection are used. Some of the less radioactive lymph nodes are second echelon nodes, not true SLNs. The purpose of this analysis was to determine whether harvesting these less radioactive nodes, in addition to the “hottest” SLNs, reduces the false-negative rate. Methods: Patients were enrolled in this multicenter (121 surgeons) prospective, institutional review board–approved study after informed consent was obtained. Patients with clinical stage T1-2, N0, M0 invasive breast cancer were eligible. This analysis includes all patients who underwent axillary SLN biopsy with the use of an injection of both isosulfan blue dye and radioactive colloid. The protocol specified that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest node should be removed and designated SLNs. All patients underwent completion level I/II axillary dissection. Results: SLNs were identified in 672 of 758 patients (89%). Of the patients with SLNs identified, 403 patients (60%) had more than 1 SLN removed (mean, 1.96 SLN/patient) and 207 patients (31%) had nodal metastases. The use of filtered or unfiltered technetium sulfur colloid had no impact on the number of SLNs identified. Overall, 33% of histologically positive SLNs had no evidence of blue dye staining. Of those patients with multiple SLNs removed, histologically positive SLNs were found in 130 patients. In 15 of these 130 patients (11.5%), the hottest SLN was negative when a less radioactive node was positive for tumor. If only the hottest node had been removed, the false-negative rate would have been 13.0% versus 5.8% when all nodes with 10% or more of the ex vivo count of the hottest node were removed (P =.01). Conclusions: These data support the policy that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest SLN should be harvested for optimal nodal staging. (Surgery 2000;128:139-44.)

Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters.

A mutation in the delta-sarcoglycan (SG) gene with absence of delta-SG protein in the heart has been identified in the BIO14.6 cardiomyopathic (CM) hamster, but how the defective gene leads to myocardial degeneration and dysfunction is unknown. We correlated left ventricular (LV) function with increased sarcolemmal membrane permeability and investigated the LV distribution of the dystrophin-dystroglycan complex in BIO14.6 CM hamsters. On echocardiography at 5 wk of age, the CM hamsters showed a mildly enlarged diastolic dimension (LVDD) with decreased LV percent fractional shortening (%FS), and at 9 wk further enlargement of LVDD with reduction of %FS was observed. The percent area of myocardium exhibiting increased membrane permeability or membrane rupture, assessed by Evans blue dye (EBD) staining and wheat germ agglutinin, was greater at 9 than at 5 wk. In areas not stained by EBD, immunostaining of dystrophin was detected in CM hamsters at sarcolemma and T tubules, as expected, but it was also abnormally expressed at the intercalated discs; in addition, the expression of beta-dystroglycan was significantly reduced compared with control hearts. As previously described, alpha-SG was completely deficient in CM hearts compared with control hearts. In myocardial areas showing increased sarcolemmal permeability, neither dystrophin nor beta-dystroglycan could be identified by immunolabeling. Thus, together with the known loss of delta-SG and other SGs, abnormal distribution of dystrophin and reduction of beta-dystroglycan are associated with increased sarcolemmal permeability followed by cell rupture, which correlates with early progressive cardiac dysfunction in the BIO14.6 CM hamster.

6953 The utility of methylene blue dye staining in detecting adenocarcinoma in patients with barrett's esophagus.

Chromoendoscopy of the gastrointestinal tract has been used recently in the United States to better delineate pathology. Methylene blue (MB) stains intestinal metaplasia blue, and has recently been evaluated in screening protocols in patients with Barrett's esophagus. The clinical utility of MB, however, has not fully been assessed. Aim: The aim of our study was to determine if methylene blue can enhance endoscopic detection of adenocarcinoma in patients with Barrett's esophagus. Methods: We retrospectively reviewed all patients referred to Georgetown University Hospital for endoscopic ultrasound over the last three years with Barrett's and adenocarcinoma of the esophagus. MB was administered after acetic acid in patients who received staining.A gastroenterologist that was blinded to prior staging reviewed videotapes of all procedures. Cases were classified as to whether they were endoscopically obvious (O) or not obvious (NO) prior to MB staining. After staining with MB was performed, cases were classified as to whether there was complete clearance (CC) of the tumor, partial clearance (PC), or minimal clearance (MC). Results: Overall, 36 patients were referred for tumor staging of their adenocarcinoma. All patients underwent endoscopic ultrasound. MB staining was performed in 11 patients. Of the 36 patients, 34 patients were determined to have endoscopically obvious lesions. All lesions that were not endoscopically obvious had been classified as T1 lesions on EUS. This included 20% (2/10) of the T1 lesions. After MB staining, no patient had CC of their tumor, 8 patients had PC, and 3 patients had MC. Both patients whose cancers were not obvious on visualization had minimal clearance with MB. Conclusions: 1. Methylene blue does not enhance endoscopic visualization of adenocarcinoma of the esophagus in patients with Barrett s esophagus and known adenocarcinoma. 2. Ninety four percent of patients referred with adenocarcinoma had tumors that were easily visualized endoscopically. 3. Patients whose tumors are difficult to visualize endoscopically are likely to be T1 lesions. 4. Endoscopic visualization may be difficult in up to 20% of patients with T1 lesions.

Preferential Flow and Pedotransfer Functions for Transport Properties in Sandy Kandiudults

Soils with differences in argillic and kandic horizon clay content (12–28% clay), thickness of overlying sandy eluvial horizons (ranging from &lt;0.50 to &gt;1.0 m), and degree of structural development occur in upland Kandiudult soils in the Upper Coastal Plain of Georgia. Interest in agricultural site‐specific management necessitates more adequate characterization of solute transport properties between and within these soils. Undisturbed columns (15‐cm diam., ) were collected by horizon for three pedons typifying the extremes of the clay content in the argillic horizon. Breakthrough curves (BTCs) were conducted using a Br− tracer and were evaluated by fitting the single‐ and two‐region adaption of the convection–dispersion equation (CDE) to outflow measurements. Saturated hydraulic conductivity (Ks) measurements and methylene blue dye staining of conducting voids were also performed on the cores. Dye staining indicated differences in preferential flow occurred between surface (Ap), eluvial (E), and argillic and kandic (Bt) horizons. Retardation factors (R) were positively correlated with clay quantities, and horizons possessing relatively less clay (A and E horizons) possessed the highest α values (quickest solute exchange rates between mobile and immobile regions). Horizons with relatively higher clay quantities (Bts) had the lowest α and β (ratio of mobile water to the volumetric water content) values, but the highest effective dispersivity (λeff) values. Dye‐stained areas were correlated with β, which suggested β may be an approximation of the degree of preferential flow for these soils. For the three pedons studied, significant differences in α existed between the extremes, and thus they are interpreted to behave differently from a solute transport standpoint. Pedotransfer functions (PTFs) were developed for grossly estimating hydraulic and transport parameters.

Antibody blockade of thrombospondin accelerates reendothelialization and reduces neointima formation in balloon-injured rat carotid artery.

Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery. Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen-positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen-positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points. Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation.

Pathological analysis of transmyocardial laser revascularization for acute myocardial ischemia.

Transmyocardial laser revascularization (TMLR) is an alternative surgical procedure for the patients with intractable coronary artery disease. Efficacy of the treatment has been established, however, the mechanism of TMLR is still controversial. In this study, we investigated the effect of TMLR on acute myocardial ischemia with pathological analysis. Under general anesthesia, the hearts of mongrel dogs were exposed. Then, the anterior descending branch of the left coronary artery was ligated to make the ischemic area on the left ventricle. Laser punctures were made 30 minutes after coronary ligation in the TMLR group (n = 5), and no further procedure was performed after coronary ligation in the AMI group (n = 5). One month after these operations, the hearts were extirpated for pathological studies. The avascular area and the viable area in the infarcted area were macroscopically separated by Evans blue dye and triphenyltetrazolium chloride (TTC) staining. Thickness of the left ventricular wall in the infarcted area was also measured and compared. Furthermore, all of the infarcted area and the lased area were microscopically examined with Masson's trichrome stain. The size of the infarcted area in the TMLR group was smaller than that in the AMI group. It was significantly different (p < 0.05) in the basal and apical regions. As a result, the ratio of the viable area by the avascular area was larger in the TMLR group than in the AMI group. It was significantly different (p < 0.05) in the apical region. In the basal region, the thickness of the left ventricle in the AMI group was thinner than that of untreated dogs (normal group: n = 5), and there was no difference between the normal group and the TMLR group. Whereas in the apical region, significant difference of the thickness was found among AMI, TMLR, and normal groups (p < 0.05). In conclusion, our study supported; 1) TMLR reduced overall infarcted size, and increased the viable area in the infarcted area, 2) TMLR prevented the thinning of the left ventricular wall.

Ultra-early clot aspiration after lysis with tissue plasminogen activator in a porcine model of intracerebral hemorrhage: edema reduction and blood-brain barrier protection.

Ultra-early hematoma evacuation (< 4 hours) after intracerebral hemorrhage (ICH) may reduce mass effect and edema development and improve outcome. To test this hypothesis, the authors induced lobar hematomas in pigs. The authors infused 2.5 ml of blood into the frontal cerebral white matter in pigs weighing 8 to 10 kg. In the treatment group, clots were lysed with tissue plasminogen activator ([tPA], 0.3 mg) and aspirated at 3.5 hours after hematoma induction. Brains were frozen in situ at 24 hours post-ICH and hematomal and perihematomal edema volumes were determined on coronal sections by using computer-assisted morphometry. Hematoma evacuation rapidly reduced elevated cerebral tissue pressure from 12.2+/-1.3 to 2.8+/-0.8 mm Hg. At 24 hours, prior clot removal markedly reduced hematoma volumes (0.40+/-0.10 compared with 1.26+/-0.13 cm3, p < 0.005) and perihematomal edema volumes (0.28+/-0.05 compared with 1.46+/-0.24 cm3, p < 0.005), compared with unevacuated control lesions. Furthermore, no Evans blue dye staining of perihematomal edematous white matter was present in brains in which the hematomas had been evacuated, compared with untreated controls. Hematomas were quickly and easily aspirated after treatment with tPA, resulting in significant reductions in mass effect. Hematoma aspiration after fibrinolysis with tPA enabled removal of the bulk of the hematoma (> 70%), markedly reduced perihematomal edema, and prevented the development of vasogenic edema. These findings in a large-animal model of ICH provide support for clinical trials that include the use of fibrinolytic agents and ultra-early stereotactically guided clot aspiration for treating ICH.

Technique of, and complications attributable to, repeated hyperosmotic blood-brain barrier disruption in dogs

Abstract Objective To design a repeatable technique for reversible, hyperosmotic blood-brain barrier disruption (BBBD) in dogs and evaluate clinical effects of multiple BBBD. Animals 10 healthy adult dogs. Procedure Using fluoroscopic guidance, an arterial catheter was directed into the internal carotid artery via the femoral artery of 10 dogs. Blood-brain barrier disruption was achieved in 5 dogs, using intracarotid infusion of mannitol. Five control dogs received only saline solution. After recovery, dogs were monitored for clinical signs of disease before a second, nonsurvival procedure was performed 2 to 3 weeks later. BBBD was estimated, using computed tomographic (CT) densitometry values, as well as Evan’s blue dye staining on necropsy specimens. Results Seven dogs completed the entire study. Two treatment dogs were lost after the first infusion because of deteriorating neurologic function attributed to CNS edema and increased intracranial pressure. One control dog was lost because of vessel wall damage during catheterization. The remaining dogs had only transient neurologic, ocular, and vasculature injuries. Successful BBBD was documented in all treated dogs by use of CT and Evan’s blue dye evaluation. Conclusion Repeated catheterization of the internal carotid artery and disruption of the blood-brain barrier is possible in dogs. Clinical Relevance Refinement of this technique would be useful not only for improved delivery of chemotherapeutic agents in patients with brain tumors, but also would allow further investigation of new treatments involving genetically engineered retroviruses and monoclonal antibodies. (Am J Vet Res 1998;59:1503–1510)

MORPHOLOGIC AND HYDRAULIC PROPERTIES OF SOILS WITH WATER RESTRICTIVE HORIZONS IN THE GEORGIA COASTAL PLAIN

Hydraulic and morphological properties of soils from a 0.36-ha site in the Georgia Coastal Plain were evaluated. Objectives included characterizing the morphological and hydraulic properties ofmajor horizons in soils with plinthite, determining the extent of preferential flow, and relating flow/transport parameters derived from breakthrough curve analyses to morphological properties. These soils have developed from Miocene aged sediments and are classified in fine-loamy, siliceous, thermic families of Plinthaquic, Aquic, Arenic Plinthic, Plinthic, and Typic Kandiudults. Morphological evidence indicates that BC horizons are restrictive to vertical percolation of drainage water. Methylene blue dye staining, K sat and breakthrough curves (analyzed by two region/MIM using CXTFIT) were measured on 15-cm-diameter undisturbed cores to determine the effects ofargillic horizons, argillic horizons with plinthite, and subjacent BC horizons on hydraulic properties of the soils. K sat for seven sampled pedons averaged 1.6 X 10 -2 , 1.1. X 10 -2 , and 3.8 X 10 -3 mm s -l for Bt, Btv, and BC horizons, respectively. Dye staining and model output (MIM) indicate a greater degree of preferential flow with depth and subsequent less mobile area contributing to water flow. Analyses of the flowpaths indicate water is translocated in regions with relatively higher porosity that also contain a higher proportion of coarser pores. Micrographs indicate that flowpaths are associated with bio-pores and areas ofbetter aggregation in the Bt horizons and structural voids in the BC horizons. BC horizons in these soils are less permeable because ofincreased clay content, differences in pore characteristics, and less cross-sectional area contributing to flow.

Regulation of osteoclast function.

Regulation of osteoclast function.

The lazaroid U74389G protects normal brain from stereotactic radiosurgery-induced radiation injury

Purpose : To test an established model of stereotactic radiosurgery-induced radiation injury with pretreatments of either methylprednisolone or the lazaroid U74389G. Methods and Materials : Nine cats received stereotactic radiosurgery with a linear accelerator using an animal radiosurgery device. Each received a dose of 125.0 Gy prescribed to the 84% isodose shell to the anterior limb of the right internal capsule. One animal received no pretreatment, two received citrate vehicle, three received 30 mg/kg of methylprednisolone, and three received 5 mg/kg of U74389G. After irradiation, the animals had frequent neurologic examinations, and neurologic deficits developed in all of them. Six months after the radiation treatment, the animals were anesthetized, and had gadolinium-enhanced magnetic resonance (MR) scans, followed by Evans blue dye perfusion, euthanasia, and brain fixation. Results : Magnetic resonance scans revealed a decrease in the size of the lesions from a mean volume of 0.45 ± 0.06 cm 3 in the control, vehicle-treated, and methylprednisolone-treated animals to 0.22 ± 0.14 cm 3 in the U74389G-treated group. The scans also suggested the absence of necrosis and ventricular dilatation in the lazaroid-treated group. Gross pathology revealed that lesions produced in the untreated, vehicle-treated, and methylprednisolone-treated cats were similar and were characterized by a peripheral zone of Evans blue dye staining with a central zone of a mature coagulative necrosis and focal hemorrhage. However, in the U74389G-treated animals, the lesions were found to have an area of Evans blue dye staining, but lacked discrete areas of necrosis and hemorrhage. Conclusion : These results suggest that the lazaroid U74389G protects the normal brain from radiation injury produced by stereotactic radiosurgery.

Intraoperative Radiolymphoscintigraphy Improves Sentinel Lymph Node Identification for Patients with Melanoma

The sentinel lymph node (SLN), the first node draining the primary tumor site, has been shown to reflect the histologic features of the remainder of the lymphatic basin in patients with melanoma. Intraoperative localization of the SLN, first proposed by Morton and colleagues, has been accomplished with the use of a vital blue dye mapping technique. Technical difficulties resulting in unsuccessful explorations have occurred in up to 20% of the dissections. The authors aimed to define the SLN using gamma detection probe mapping and to determine whether intraoperative radiolymphoscintigraphy using technetium sulfur colloid and a hand-held gamma-detecting probe could be used to improve detection of all SLNs for patients with melanoma. To ensure that all initial nodes draining the primary site were removed at the time of selective lymphadenectomy, the authors used intraoperative radiolymphoscintigraphy to confirm the location of the SLN, which was determined initially with the preoperative lymphoscintigram and the intraoperative vital blue dye injection. The patient population consisted of 106 consecutive patients who presented with cutaneous melanomas larger than 0.75 mm in all primary site locations. The preoperative lymphoscintigram revealed that 22 patients had more than one lymphatic basin sampled. Two hundred SLNs and 142 neighboring non-SLNs were harvested from 129 basins in 106 patients. After the skin incision was made, the mean ratio of hot spot to background activity was 8.5:1. The mean ratio of ex vivo SLN-to-non-SLN activity for 72 patients who had SLNs harvested was 135.6:1. When correlated with the vital blue dye mapping, 139 of 200 (69.5%) SLNs demonstrated blue dye staining, whereas 167 of 200 (83.5%) SLNs were hot according to radioisotope localization. With the use of both intraoperative mapping techniques, identification of the SLN was possible for 124 of the 129 (96%) basins sampled. Micrometastases were identified in SLNs of 16 of the 106 (15%) patients by routine histologic analysis. The use of intraoperative radiolymphoscintigraphy can improve the identification of all SLNs during selective lymphadenectomy.