Characteristics of the New Mast Cell-Rich Nodal Structure in the Rat Skin Surface.

Background: : Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats. Objectives: : To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints. Methods: : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats. Results: : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues. Conclusion: : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

Combined 68Ga-NOTA-Evans Blue Lymphoscintigraphy and 68Ga-NOTA-RM26 PET/CT Evaluation of Sentinel Lymph Node Metastasis in Breast Cancer Patients.

In this study, we applied a new strategy to identify sentinel lymph node (SLN) metastasis by combining 68Ga-NOTA-Evans Blue (68Ga-NEB) for SLN mapping and 68Ga-NOTA-RM26 for LN metastasis detection in breast cancer patients. A total of 24 female patients with breast cancer diagnosed by core biopsy or suspected by mammography or ultrasonography were recruited and provided informed consent. All patients underwent 68Ga-NEB and 68Ga-NOTA-RM26 PET/CT imaging. Visual analysis of 68Ga-NEB PET/CT images was used to determine SLNs, and then compared with the 68Ga-NOTA-RM26 results and histopathological findings. SLNs were visualized in 24 of 24 patients (100.0%) within 4.0-10.0 (5.6 ± 1.4) min. All patients were pathologically diagnosed with breast cancer, and 12 patients had ipsilateral lymph node metastasis. By combining 68Ga-NEB and 68Ga-NOTA-RM26 images, 7/12 (58.3%) patients showed mild to intense uptake of 68Ga-NOTA-RM26 in SLNs, 1/12 patient (8.3%) had moderate uptake of 68Ga-NOTA-RM26 in the non-SLNs rather than SLN, indicating possible bypass lymphatic drainage, partially accounting for the false negatives in SLN biopsy during surgery. No false positives were found. The SUVmax of 68Ga-NOTA-RM26 activity in metastatic SLNs was significantly higher than that in non-metastatic SLNs (2.2 ± 2.3 vs 0.7 ± 0.1, P = 0.047). This study manifests the value of combination of 68Ga-NEB and 68Ga-NOTA-RM26 dual tracer PET/CT in preoperative evaluation of SLN metastasis in breast cancer patients, especially in those patients with lymphatic obstruction and bypass drainage. In general, positive 68Ga-NOTA-RM26 uptake in either SLN or other lymph nodes can apply lymph node dissection rather than intraoperative SLN biopsy.

Impaired Peripheral Lymphatic Function and Cerebrospinal Fluid Outflow in a Mouse Model of Alzheimer's Disease.

Cerebrospinal fluid (CSF) outflow from the brain occurs through absorption into the arachnoid villi and, more predominantly, through meningeal and olfactory lymphatics that ultimately drain into the peripheral lymphatics. Impaired CSF outflow has been postulated as a contributing mechanism in Alzheimer's disease (AD). Herein we conducted near-infrared fluorescence imaging of CSF outflow into the peripheral lymph nodes (LNs) and of peripheral lymphatic function in a transgenic mouse model of AD (5XFAD) and wild-type (WT) littermates. CSF outflow was assessed from change in fluorescence intensity in the submandibular LNs as a function of time following bolus, an intrathecal injection of indocyanine green (ICG). Peripheral lymphatic function was measured by assessing lymphangion contractile function in lymphatics draining into the popliteal LN following intradermal ICG injection in the dorsal aspect of the hind paw. The results show 1) significantly impaired CSF outflow into the submandibular LNs of 5XFAD mice and 2) reduced contractile frequency in the peripheral lymphatics as compared to WT mice. Impaired CSF clearance was also evidenced by reduction of fluorescence on ventral surfaces of extracted brains of 5XFAD mice at euthanasia. These results support the hypothesis that lymphatic congestion caused by reduced peripheral lymphatic function could limit CSF outflow and may contribute to the cause and/or progression of AD.

Relationship between Plasma Albumin Concentration and Plasma Volume in 5 Inbred Rat Strains.

Using the Evans Blue procedure, we previously found strain-related differences in plasma volumes in 5 inbred rat strains. Because albumin binds strongly with Evans blue, this protein is important in the Evans blue method of plasma volume determination. Therefore, we speculated that interstrain differences in plasma albumin concentration (PAC) could distort calculated plasma volumes. To address this concern, we used ELISA techniques to measure PAC in these inbred rat strains. In study A, the blood volume was measured by using Evans blue dye, and albumin was measured at the start of hemorrhage. In study B, blood volume was not measured, and albumin was measured twice, near the start and end of hemorrhage (approximately 14 min apart). Neither study revealed any interstrain differences in PAC, which decreased after hemorrhage in all 5 strains. No correlation was found between PAC and plasma volume, survival time, blood lactate, or blood base excess. Percentage changes in PAC during hemorrhage were greater in salt-sensitive compared with Lewis rats. Moreover, these percentage changes were associated with survival time in Fawn hooded hypertensive rats. Our data show that the plasma volumes we measured previously were not misrepresented due to variations in PAC.

Permeability Parameters Measured with Dynamic Contrast-Enhanced MRI: Correlation with the Extravasation of Evans Blue in a Rat Model of Transient Cerebral Ischemia.

OBJECTIVE: The purpose of this study was to correlate permeability parameters measured with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a clinical 3-tesla scanner with extravasation of Evans blue in a rat model with transient cerebral ischemia. MATERIALS AND METHODS: Sprague-Dawley rats (n = 13) with transient middle cerebral artery occlusion were imaged using a 3-tesla MRI with an 8-channel wrist coil. DCE-MRI was performed 12 hours, 18 hours, and 36 hours after reperfusion. Permeability parameters (K(trans), ve, and vp) from DCE-MRI were calculated. Evans blue was injected after DCE-MRI and extravasation of Evans blue was correlated as a reference with the integrity of the blood-brain barrier. Correlation analysis was performed between permeability parameters and the extravasation of Evans blue. RESULTS: All permeability parameters (K(trans), ve, and vp) showed a linear correlation with extravasation of Evans blue. Among them, K(trans) showed highest values of both the correlation coefficient and the coefficient of determination (0.687 and 0.473 respectively, p < 0.001). CONCLUSION: Permeability parameters obtained by DCE-MRI at 3-T are well-correlated with Evans blue extravasation, and K(trans) shows the strongest correlation among the tested parameters.

Permeability Parameters Measured with Dynamic Contrast-Enhanced MRI: Correlation with the Extravasation of Evans Blue in a Rat Model of Transient Cerebral Ischemia

OBJECTIVE: The purpose of this study was to correlate permeability parameters measured with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a clinical 3-tesla scanner with extravasation of Evans blue in a rat model with transient cerebral ischemia. MATERIALS AND METHODS: Sprague-Dawley rats (n = 13) with transient middle cerebral artery occlusion were imaged using a 3-tesla MRI with an 8-channel wrist coil. DCE-MRI was performed 12 hours, 18 hours, and 36 hours after reperfusion. Permeability parameters (K(trans), v(e), and v(p)) from DCE-MRI were calculated. Evans blue was injected after DCE-MRI and extravasation of Evans blue was correlated as a reference with the integrity of the blood-brain barrier. Correlation analysis was performed between permeability parameters and the extravasation of Evans blue. RESULTS: All permeability parameters (K(trans), v(e), and v(p)) showed a linear correlation with extravasation of Evans blue. Among them, K(trans) showed highest values of both the correlation coefficient and the coefficient of determination (0.687 and 0.473 respectively, p < 0.001). CONCLUSION: Permeability parameters obtained by DCE-MRI at 3-T are well-correlated with Evans blue extravasation, and K(trans) shows the strongest correlation among the tested parameters.

An in vivo assay to test blood vessel permeability.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

A new method of quantitatively assessing the opening of the blood-brain barrier in murine animal models.

The blood-brain barrier (BBB) restricts the delivery of drugs into the brain. Different strategies have been developed to circumvent this obstacle. One such approach, the osmotic BBB disruption (BBBD), has been under pre-clinical study since the 70's. Typically, qualitative ex vivo assessment of the extent of BBBD has been performed using Evan's blue staining technique. In this study, we describe a simple quantitative technique based on albumin indirect immunohistochemistry to measure the extent of BBB breach. Thirty Fischer rats were assigned to one of 6 groups: a control group, and BBBD groups with escalation in IA mannitol infusion rate: 0.06, 0.08, 0.10, 0.12 and 0.15 cc/s. Fifteen minutes after the BBBD procedure, the animals were sacrificed, brain harvested and sections stained for albumin. Using an image analysis software, isolated albumin staining pixels were expressed as a fraction of the treated hemisphere. This ratio was used as a percentage value in the intensity of the BBB permeabilization. All sections studied harbored staining, averaging 0.37% for the controls (group 1), 5.69% for group 2 (0.06 cc/s), 10.44% for group 3 (0.08 cc/s), 6.99% for group 4 (0.1 cc/s), 18.50% for group 5 (0.12 cc/s) and reaching 61.70% for group 6 (0.15 cc/s). Important variations were observed between animals. A threshold effect was observed, and animals in group 6 presented a significant increase in BBB permeabilization compared to the other groups. We hereby detail a simple technique that can be applied to quantitatively measure the extent of the BBB breach notwithstanding the pathological process.

Permeability of endothelial barrier: cell culture and in vivo models.

The methods for assessment of endothelial barrier permeability are vital tools of experimental biology. They allow us to measure permeability of endothelial monolayer in cell culture and in lung vessels or to determine formation of tissue edema resulting from increased permeability of vasculature. This chapter provides an overview of the most common protocols.

Assessment of permeability in barrier type of endothelium in brain using tracers: Evans blue, sodium fluorescein, and horseradish peroxidase.

Blood-brain barrier (BBB) constituted primarily by the capillary endothelial cells functions to maintain a constant environment for the brain, by preventing or slowing down the passage of a variety of blood-borne substances, such as serum proteins, chemical compounds, ions, and hormones from the circulation into the brain parenchyma. Various diseases such as brain tumors, epilepsy, and sepsis disturb the BBB integrity leading to enhanced permeability of brain microvessels. In animal models, a variety of experimental insults targeted to the BBB integrity have been shown to increase BBB permeability causing enhanced passage of molecules into the brain paranchyma by transcellular and/or paracellular pathways. This alteration can be demonstrated by intravascular infusion of exogenous tracers and subsequent detection of extravasated molecules in the brain tissue. A number of exogenous BBB tracers are available, and they can be used for functional and structural analysis of BBB permeability. In this chapter, we aimed to highlight the basic knowledge on the use of three most commonly performed tracers, namely Evans blue dye, sodium fluorescein, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, and electron microscopy are also discussed. While tracing studies at the morphological level are mainly aimed at the identification and characterization of the tracers both in the barrier related cells and brain parenchyma, spectrophotometric and spectrophotofluorometric assays enable quantification of BBB permeability. The results of our studies that we performed using the mentioned tracers indicate that barrier type of endothelial cells in brain play an important role in paracellular and/or transcytoplasmic trafficking of macromolecules across BBB under various experimental settings, which may provide new insights in both designing approaches for the management of diseases with BBB breakdown and developing novel trans-BBB drug delivery strategies.

[Mast cell and substance P are involved in the process of acupoint sensitization induced by acute gastric mucosal injury].

OBJECTIVE: To observe changes of mast cells (MCs) number and morphology, and substance P (SP) expression in Evans blue (EB) extravasated region around acupoint "Pishu" (BL 20) and "Weishu" (BL 21) after acute gastric mucosal injury (AGMI) so as to investigate the mechanism underlying visceral problems-induced acupoint activation. METHODS: Thirty adult Wistar rats were randomly divided into normal control (n = 15) and AGMI groups (n = 15). AGMI model was duplicated by perfusing the rats with 0.5 mol/L HCl (1 mL/100 g) after fasting for 20 h. Five hours after AGMI, the rats were treated by tail-intravenous injection of EB dye (5 mg/100 g, 50 mg/mL in normal saline) for inducing dye-plasma extravasation in the skin around BL 20, BL 21 regions, etc. at the back. The rats of the normal control group were treated with tail-intravenous injection of 0.9% NaCl. The skin and subcutaneous tissues (2 mmx 2 mm) of extravasated EB dye points (BL 20 or BL 21 region) and those 2 mm lateral to the extravasated EB dye points in the model group and the corresponding points in the normal control group were sampled (followed by fixing them in 4% paraformaldehyde), sectioned and stained by toluidine blue (for labeling MCs). The expression of SP in the extravasated EB dye skin and subcutaneous tissues was detected by immunohistochemistry (n = 5) and western blot (n = 5) respectively. The number of MCs in these samples was counted and the degranulation rate of MCs calculated. RESULTS: The total number of MCs and the number of degranulated MCs were significantly more in the EB extravasation points (corresponding to BL 20/BL 21 area) of AGMI group than those in the control spots of AGMI group and than those in the normal control group (P < 0.05, P < 0.001). The degranulation rate of MCs was significantly higher in the EB extravasation points of AGMI group than those in the control spots of AGMI group and in the normal control group (P < 0.01). In comparison with normal control group, the SP expression level was increased consideraly in the control spots of AGMI group and AGMI group (P < 0.01). CONCLUSION: After AGMI, the numbers of MCs and the degranulated MCs, and the SP expression level in BL 20/BL 21 area were increased significantly, suggesting an involvement of MCs and SP in the process of AGMI-induced activation of acupoints.

Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD(+) muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra- and interlimb amounts of EBD in 2-7-month-old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse.

Effects of diagnostic contrast-enhanced ultrasound on permeability of placental barrier: a primary study.

OBJECTIVE: To evaluate the effects of contrast-enhanced ultrasound (CEU) on the permeability of placental barrier primarily. METHODS: A total of 60 pregnant Sprague Dawley (SD) rats were divided into 10 groups, including six groups of microbubbles-enhanced ultrasound (varied mechanical index (MI) of 0.13, 1.0 and 1.4 with continuous and intermittent insonation respectively) (US+MB), two groups of ultrasound insonation only (continuous and intermittent insonation respectively) (US), the group of microbubbles only (MB) and the control group. Evans blue (EB), as the tracer, was intravenously injected before treatment. The EB in placenta and fetus was observed under fluorescence microscope and analyzed quantitatively. The EB amount was compared between groups and between placenta and fetus. Lanthanum nitrate-tracing transmission electron microscope examination was performed to observe the distribution of lanthanum in the placenta and fetus. RESULTS: Observed by naked eye, the plancenta was dyed into deep blue while there was no sign of dyeing to the fetus in all groups. Under fluorescence microscope, the red fluorescence radiated by EB was observed in placenta but not in fetus. The EB amount in placenta, insonated by microbubbles-enhanced ultrasound of varied MI, was higher significantly than that in MB, US and control group (all P<0.01) while there was no difference between the latter three groups. And in each group, EB amount was much higher in placenta than that in fetus (P<0.01). The lanthanum particles deposited in the intercellular space in the syntrophoblast while there was no lanthanum presented in the cytotrophoblast in all groups. CONCLUSION: Our findings suggest that diagnostic CEU with SonoVue will not increase the permeability of placenta to the macromolecules larger than albumin, although it may affect placenta.

Re-evaluation of Evans Blue dye as a marker of albumin clearance in murine models of acute lung injury.

Quantifying the amount of albumin conjugated to Evans Blue dye (EBA) fluxing across organ-specific vascular barriers is a popular technique to measure endothelial monolayer integrity in rodent and murine models of human diseases. We have re-evaluated this technique with a specific focus of assessing the commonly used turbidity correction factors. These factors, originally developed and required in a spectrophotometric assay to quantify Evans Blue (EB) in human infant or dog serum, produced negative numbers when applied to murine models of acute lung injury. We next sought to determine tissue-specific correction factors for murine tissues and experimentally derived such factors, which allow estimation of the amount of EB in formamide extracts of murine tissues as positive numbers. Utilization of a best fit correction factor in a lipopolysaccharide (LPS)-induced murine model of acute lung injury resulted in significantly increased sensitivity and repeatability of the EB dye tissue extravasation assay. This factor may be of significant utility in animal models of inflammatory injury.

Propofol-induced vascular permeability change is related to the nitric oxide signaling pathway and occludin phosphorylation.

The present study was undertaken to elucidate the mechanism of intra-arterial propofol-induced vascular permeability change resulting in tissue edema. The mechanism of propofol-induced hyperpermeability was examined in a rat femoral artery injection model. Vascular permeability was determined by measuring the Evans blue content of the dorsal skin of the infused limb at 15, 30, 45 and 60 min after propofol injection. The total content of the tight junction proteins occludin, ZO-1 and claudin-5 under experimental conditions was also determined by western blotting. Intra-arterial injection with propofol resulted in a marked dose-dependent increase in vascular permeability of the rat hindpaw. Pretreatment with 10 mg/kg of N-nitro-L: -arginine methyl ester (L: -NAME) but not aminoguanidine significantly inhibited the change in vascular permeability after challenge with propofol. Pretreatment with L: -arginine and nitroprusside increased the propofol-induced permeability change. Intra-arterial injection of propofol significantly increased occludin phosphorylation after 15 min, which was consistent with the time profile of the vascular permeability change. L: -NAME partially reversed the change in occludin phosphorylation, whereas aminoguanidine had no effect compared with that in the controls. Our observations indicate that nitric oxide (NO) is an important mediator in the induction of vascular permeability induced by propofol. Occludin phosphorylation is a determining factor in the vascular permeability change induced by propofol. NO synthase (NOS) inhibitors might be useful in the treatment of accidental intra-arterial injection of propofol, in the reduction of any adverse effects.

Rats exhibit aldosterone-dependent sodium appetite during 24 h hindlimb unloading.

Hindlimb unloading (HU) is an animal model of microgravity and bed rest. In these studies, we examined the role of ingestive behaviours in regulating body fluid balance during 24 h HU. In the first experiment, all rats were given distilled water to drink while two groups were also given access to a sodium chloride solution (0.9% or 1.8%). Water and saline intakes were measured before, during and after 24 h of HU. Rats reduced water intake during 24 h HU in all conditions. During HU, rats increased their intakes of both saline solutions (0.9% NaCl (n= 11): control 7.8 +/- 3 ml; HU 18.2 +/- 4 ml; recovery 8.9 +/- 2.5 ml; 1.8% NaCl (n= 7): control 1.0 +/- 0.4 ml; HU 3.8 +/- 0.3 ml; recovery 1.2 +/- 0.5 ml). Although water intake decreased there was no reduction in total fluid intake when saline was available. Plasma volumes were reduced during HU compared to rats in a normal posture when only water was available to drink (control (n= 11) versus HU (n= 11): 4.0 +/- 0.2 versus 3.4 +/- 0.2 ml (100 g body weight)(-1)). When 0.9% saline was available in addition to water, plasma volumes after 24 h HU were not different from rats in a normal posture (control (n= 11) versus HU (n= 12): 4.3 +/- 0.4 versus 4.3 +/- 0.1 ml (100 g body weight)(-1)). Plasma aldosterone but not plasma renin activity was significantly elevated after 24 h HU. Central infusions of spironolactone blocked the increased intake of 1.8% saline that was associated with 24 h HU. Thus, HU results in an aldosterone-dependent sodium appetite and the ingestion of sodium may help maintain plasma volume.

Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.

Evans Blue Dye (EBD) is widely used to study cellular membrane permeability and has recently been utilised in mdx mice to identify permeable skeletal myofibres that have become damaged as a result of muscular dystrophy. EBD has the potential to be a useful vital stain of myofibre permeability in other models of skeletal muscle injury and membrane-associated fragility. The parameters for its use for such purposes were optimised in the present study, of particular interest is the use of EBD to identify the onset of muscle damage. This study compared intravenous vs. intraperitoneal injection; tissue fixation; volume of EBD; time of availability in tissue; and persistence after injection in mdx mice (with endogenous muscle damage) and control mice. Satisfactory labelling of permeable myofibres was seen in frozen sections viewed with fluorescence microscopy when intraperitoneal injection of a 1% EBD solution injected at 1% volume relative to body mass was administered between 16 and 24 h prior to tissue sampling. EBD labelling was then assessed in three mouse models of experimental injury and repair-cut injury, whole muscle grafts, and exercise-induced muscle damage. These experiments demonstrated that (i) following a cut injury across myofibres, EBD penetrated up to 150 microm from the injury site over a 20-h period; (ii) EBD was present throughout myofibres of avascular whole muscle graft by one day after transplantation; and (iii) damaged myofibres were detected within 20 min after controlled lengthening-contraction exercise. This simple and inexpensive technique has sensitivity for the detection of increased myofibre permeability and/or sublethal damage that has advantages over other traditional histological techniques at the light microscopy level.

Sentinel node biopsy and lymphoscintigraphy with a technetium 99m labeled blue dye in a rabbit model.

BACKGROUND: Lymphatic mapping for sentinel node biopsy in breast cancer and melanoma usually involves initial peritumoral injection of a radioisotope, gamma camera detection of the sentinel lymph node several hours prior to the operation, and separate perioperative injection of a blue dye. We have developed a combined approach using technetium 99m labeled blue dye (Evans Blue) for use in lymphoscintigraphy that may be injected as a single dose just prior to the operation. METHODS: In an anesthetized rabbit model we dissected a hind limb to display the popliteal node and afferent lymphatic. Technetium 99m Evans Blue ((99m)Tc-EB) (22 MBq; 0.5 mL) was injected subdermally in the dorsum of the paw. Simultaneous digital and gamma camera images were obtained at 14 time intervals to 30 minutes post injection. For each of these time intervals the percentage of radioactivity and percentage blueness of the popliteal node were determined. Urine and afferent lymphatic fluid were analyzed by chromatography. The popliteal node was excised post mortem, placed into solvent solutions and analyzed for blueness and radioactivity. RESULTS: Time-activity curves for radioactivity and time-blueness curves for Evans Blue uptake showed strong correlation (r = 0.958). Lymph analysis suggested (99m)Tc-EB is mainly bound to endogenous proteins. Urine was radioactive but not colored, (99m)Tc-EB being metabolized and excreted in the urine as 1,7-diamino-8-naphthol-2,4-disulfonic acid. Prolonged exposure of node to solvents did not dissociate any blue coloration or radioactivity. CONCLUSIONS: (99m)Tc-EB and Evans Blue are simultaneously retained and concentrated in the sentinel lymph node. This process is rapid and reproducible. (99m)Tc-EB migrates at the same rate as Evans Blue in lymph, where it is transported as bound to endogenous proteins. These dye molecules are metabolized by reductive cleavage in the liver and then excreted renally as colorless, radioactive metabolites. This novel agent has the potential to facilitate lymphatic mapping and subsequent sentinel node biopsy for a range of solid malignancies including breast cancer and melanoma.

Determination of total blood volume by indicator dilution: a comparison of mean transit time and mass conservation principle.

OBJECTIVE: Using indocyanine green (ICG), blood volume can be determined within minutes according to the mass conservation principle by back-extrapolation of the concentration/time curve to the time of injection (BVTinj) or by the transit time approach (BVMTT) as the product of cardiac output and mean transit time (MTT) of ICG through the circulation. To see which factor accounts for the difference between the two methods we measured cardiac output and MTT independently and compared the volumes with those obtained by dilution of Evans blue (BVEB). DESIGN: Prospective animal study. SETTINGS: University department of experimental anaesthesiology. ANIMALS: Six anaesthetised, spontaneously breathing dogs with chronically implanted ultrasound flow probes around the pulmonary artery. MEASUREMENTS AND RESULTS: BVMTT and BVTinj agreed closely (48 +/- 2 ml.kg-1 and 49 +/- 2 ml.kg-1), but underestimated blood volume by about 40% compared with BVEB (75 +/- 1 ml.kg-1). Transit times measured were 33 +/- 1 s and should be about 50 s as calculated from the quotient of BVEB and cardiac output. CONCLUSIONS: Both methods underestimate blood volume by about the same extent compared with BVEB, probably because slowly perfused compartments are not detected during the short measurement period of 4 min. In the case of the transit time approach, rather short transit times result and in the case of the mass conservation principle, back-extra-polation yields rather high plasma concentrations of ICG at the time of injection. Accordingly, the two methods seem to be equivalent for measuring blood volume rapidly, although the absolute volume is underestimated by about 40%.

Determination of intracranial tumor volumes in a rodent brain using magnetic resonance imaging, Evans blue, and histology: a comparative study.

The measurement of tumor volumes is a practical and objective method of assessing the efficacy of a therapeutic agent. However, the relative accuracy of different methods of assessing tumor volume has been unclear. Using T1-weighted, gadolinium-enhanced magnetic resonance Imaging (T1-MRI), Evans Blue infusion and histology we measured intracranial tumor volumes in a rodent brain tumor model (RT2) at days 10, 16 and 18 after implantation of cells in the caudate putamen. There is a good correlation between tumor volumes comparing T1-MRI and Evans Blue (r2 = 0.99), T1-MRI and Histology (r2 = 0.98) and histology and Evans Blue (r2 = 0.93). Each of these methods is reliable in estimating tumor volumes in laboratory animals. There was significant uptake of gadolinium and Evans Blue in the tumor suggesting a wide disruption of the blood-brain barrier.