Lothar Schilling

Application of micro-CT in small animal imaging.

Over the past decade, the number of publications using micro-computed tomography (muCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the key technical advancements that have allowed researchers to capture increasingly detailed anatomical images of small animals and to monitor the progression of disease in small animal models. The purpose of this review is to present the technical aspects of muCT, as well as current research applications. Our objectives are threefold: to familiarize the reader with the basics of muCT techniques; to present the type of experimental designs currently used; and to highlight limitations, future directions, in muCT-scanner research applications, and experimental methods. As a first step we present different muCT setups and components, as well as image contrast generation principles. We then present experimental approaches in order of the evaluated organ system. Finally, we provide a short summary of some of the technical limitations of muCT imaging and discuss potential future developments in muCT-scanner techniques and experimental setups.

HYPERTONIC SALINE SOLUTION FOR CONTROL OF ELEVATED INTRACRANIAL PRESSURE IN PATIENTS WITH EXHAUSTED RESPONSE TO MANNITOL AND BARBITURATES

Critically elevated intracranial pressure (ICP) represents the most important cause of morbidity and mortality in patients suffering from severe traumatic brain injury (TBI) and is a serious complication after subarachnoid hemorrhage (SAH). Thus new strategies for the control of ICP are required. Based on the evidence available hypertonic saline solution (HSS) may be a promising approach. It was therefore the aim of the present study to evaluate in a prospective manner the effects of HSS on ICP and cerebral perfusion pressure (CPP) in patients with therapy-resistant elevation of ICP. A total of 48 bolus infusions of HSS (7.5%, 2 ml kg-1 b.w.; infusion rate 20 ml min-1) were given intravenously (range 1-15 per patient) to 10 patients (age 41 +/- 6 years) with TBI and SAH. Only patients with ICP > 25 mmHg not responding to standard ICP-management protocol and plasma sodium (Na+) concentration < 150 mmol l-1 were included in the study. Within the first hour after HSS application, ICP decreased from 33 +/- 9 mmHg to 19 +/- 6 mmHg (p < 0.05) and further to 18 +/- 5 mmHg at the time of maximum effect (98 +/- 11 min post bolus). Decrease of ICP was accompanied by a rise of CPP from 68 +/- 11 mmHg to 79 +/- 11 mmHg (p < 0.05) after 1 h and further to 81 +/- 11 mmHg at the time of maximum effect. Plasma Na+ concentration was 141 +/- 6 mmol l-1 before and 143 +/- 5 mmol l-1 1 h after HSS bolus. Corresponding values for plasma osmolality were 302 +/- 11 and 308 +/- 12 mOsm l-1. When the ICP lowering effect was transient, subsequent HSS bolus was necessary 163 +/- 54 min after previous dosing. The present results indicate that repeated bolus application of HSS (7.5% NaCl, 2 ml kg-1 b.w.) is an effective measure to decrease ICP which is otherwise refractory to standard therapeutic approaches. Whether or not the therapy scheme is also suited as primary measure for the control of ICP remains to be established.

Characterization of Angiogenesis and Microcirculation of High–Grade Glioma: An Intravital Multifluorescence Microscopic Approach in the Athymic Nude Mouse:

The current study follows angiogenesis and microcirculatory changes associated with malignant glioma growth by means of an intravital fluorescence microscopic approach, which allows for the direct and continuous visualization of the glioma microvasculature and its quantitative analysis. Fluorescently labeled C6 rat glioma cells (5 × 105) were implanted into dorsal skinfold chamber preparations of athymic nude mice. Glioma growth, vascularization, microhemodynamics, vascular permeability, and leukocyte–endothelial cell interactions were simultaneously followed over a 22-day observation period using intravital epiillumination microscopy and a multifluorescent labeling technique. Analysis of the process of glioma vascularization revealed three stages with distinct microvascular characteristics: avascular stage (days 0 to 6), lag of glioma growth but initial glioma-induced angiogenesis within the host tissue in peritumoral areas; early vascular stage (days 6 to 14), glioma cell proliferation associated with a spatially homogeneous development of a glioma microvasculature; and late vascular stage (days 14 to 22), exponential tumor growth and expansion (> 400 mm3) with high vascular densities in the peritumoral region and reduced vascularization (microvascular perfusion) in the glioma center. Within the center, the functional vessel length per area correlated inversely with glioma size (P<0.01). In the peritumoral region, functional vessel length per area was independent of glioma size, indicating persistent, high angiogenic activity throughout the observation period. Thus, the microvasculature of mature gliomas revealed a microvascular zonal division with a progressive reduction of the functional vessel length per area within the tumor center. The perfusion failure of individual microvessels within the glioma center was partly compensated by an increase of diameters (P<0.05), and thus by an increase of blood flow in these functional microvessels (P<0.05) over time. Histologic analysis demonstrated both expanding and infiltrating growth patterns, as well as focal necroses on day 22. These are the first data from repeated in vivo analysis of glioma growth, vascularization, and microcirculation.

Expression of Growth Differentiation Factor-15/ Macrophage Inhibitory Cytokine-1 (GDF-15/MIC-1) in the Perinatal, Adult, and Injured Rat Brain

We and others have recently cloned a new member of the transforming growth factor‐β superfamily, growth differentiation factor‐15/ macrophage inhibitory cytokine‐1 (GDF‐15/MIC‐1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF‐15/MIC‐1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF‐15/MIC‐1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF‐15/MIC‐1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF‐15/MIC‐1‐producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Flourescent microscopy revealed both intra‐ and extracellular GDF‐15/MIC‐1 ir. Up‐regulation of GDF‐15/MIC‐1 in activated macrophages (Mϕ) is also supported by RT‐PCR, ICC, and Western blot experiments showing pronounced induction of GDF‐15/MIC‐1 expression (mRNA and protein) in retinoic acid/phorbol ester‐stimulated human Mϕ. Our data suggest that 1) GDF‐15/MIC‐1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme–epithelial interactions. Finally, GDF‐15/MIC‐1 may also act within the antiinflammatory cytokine network activated in CNS lesions. J. Comp. Neurol. 439:32–45, 2001.

Endothelin-Induced Contraction and Relaxation of Rat Isolated Basilar Artery: Effect of BQ-123:

In ring segments from rat basilar artery (BA) the endothelin (ET) peptides ET-1, ET-2, and ET-3 induced concentration-related contractions. The order of potency was ET-1 = ET-2 > ET-3, while no differences occurred in the maximum contraction. The selective ETA receptor antagonist, BQ-123 (10−10-10−4 M) alone elicited a small contraction only at 10−4 M. In the presence of BQ-123 (10−7-10−5 M), the concentration-response curve for ET-1 was shifted to the right without any decrease in maximum contraction, indicating competitive inhibition of ET-1 binding to the ETA receptor by BQ-123. The pA2 value calculated for BQ-123 was 6.935; the slope of the regression curve was 0.734. In contrast to ET-1, the contractile action of ET-3 was abolished by 10−5 M BQ-123. In segments precontracted with 10−6 M serotonin, ET-3, but not ET-1, induced relaxation at low concentrations (10−11-10−8 M), with maximum relaxation amounting to 17.8 ± 14.7% of precontraction (mean ± SD; n = 16). The relaxant action of ET-3 was abolished in vessels incubated with NG-nitro-l-arginine (10−5 M), an inhibitor of nitric oxide synthase. These results indicate that the ET-induced contraction of the isolated rat BA involves activation of the ETA receptor. The ET-3-induced relaxation of precontracted rat BA is apparently mediated by release of nitric oxide from the endothelium.

Angiotensin converting enzyme inhibition for arterial hypertension reduces the risk of recurrence in patients with chronic subdural hematoma possibly by an antiangiogenic mechanism.

OBJECTIVEChronic subdural hematoma (CSH) is characterized by pathological vascularization of the parietal membrane. Plasma leakage from immature vessels may be involved in hematoma enlargement and recurrence. We tested the hypothesis that the antiangiogenic side-effect of angiotensin converting enzyme (ACE)-inhibitor treatment for the control of arterial hypertension reduces the risk of recurrence in CSH. METHODSWe analyzed the data of 438 patients with CSH treated by a standard surgical procedure for hematoma evacuation in our department between 1995 and 2003. Patients with coagulopathies, malignancies, and independent neurological disorders were excluded from this study. Patient records were screened for age, sex, pre- and postoperative Markwalder score, arterial hypertension, medication with ACE-inhibitors, and recurrence of CSH. The rate of ACE-inhibitor treatment in our CSH patients was compared with an age-matched control group treated for herniated lumbar disc at the same time. The concentration of vascular endothelial growth factor was analyzed in hematoma samples and corresponding venous blood in 40 consecutive patients. RESULTSA total of 310 patients were included in this study. The demographic data of Group A (with ACE-inhibition) and Group B (without ACE-inhibition) were comparable. In Group A, 5% (four out of 81) of the patients experienced recurrence as opposed to 18% (42 out of 229) in Group B (P = 0.00345). A negative correlation was found between the yearly rates of medication with ACE-inhibitors and recurrence (r = −0.8488; P = 0.0044). The rate of ACE-inhibitor treatment was lower in the CSH patients (25%) than in the control group (40%). The VEGF content was significantly lower in the hematoma in patients with ACE-inhibition (mean, 8891 pg/ml; range, 4300–18,300 pg/ml) than in patients without (mean, 22,565 pg/ml; range, 4200–89,650 pg/ml; P = 0.0116). CONCLUSIONOur data suggest that ACE-inhibitor treatment for the control of arterial hypertension lowers the risk of recurrence in patients undergoing operation for CSH and possibly even the development of CSH. This effect might be the result of an antiangiogenic mechanism of ACE-inhibitors.

Targeting angiogenesis inhibits tumor infiltration and expression of the pro‐invasive protein SPARC

The solid growth of high‐grade glioma appears to be cri‐tically dependent on tumor angiogenesis. It remains unknown, however, whether the diffuse infiltration of glioma cells into healthy adjacent tissue is also dependent on the formation of new tumor vessels. Here, we analyze the relationship between tumor angiogenesis and tumor cell infiltration in an experimental glioma model. C6 cells were implanted into the dorsal skinfold chamber of nude mice, and tumor angiogenesis was monitored by intravital fluorescence videomicroscopy. Glioma infiltration was assessed by the extent of tumor cell invasion into the adjacent chamber tissue and by expression of SPARC, a cellular marker of glioma invasiveness. To test the hypothesis that glioma angiogenesis and glioma infiltration are codependent, we assessed tumor infiltration in both the presence and the absence of the angiogenesis inhibitor SU5416. SU5416 is a selective inhibitor of the VEGF/Flk‐1 signal‐transduction pathway, a critical pathway implicated in angiogenesis. Control tumors demonstrated both high angiogenic activity and tumor cell invasion accompanied by strong expression of SPARC in invading tumor cells at the tumor–host tissue border. SU5416‐treated tumors demonstrated reduced vascular density and vascular surface in the tumor periphery accompanied by marked inhibition of glioma invasion and decreased SPARC expression. A direct effect of SU5416 on glioma cell motility and invasiveness was excluded by in vitro migration and invasion assays. These results suggest a crucial role for glioma‐induced angiogenesis as a prerequisite for diffuse tumor invasion and a possible therapeutic role for anti‐angiogenic compounds as inhibitors of both solid and diffuse infiltrative tumor growth. Int. J. Cancer 87:261–268, 2000.

Specific pattern of growth factor distribution in chronic subdural hematoma (CSH): evidence for an angiogenic disease.

Summary.Summary.Background: The aim of this prospective study was to evaluate the significance of growth factors as determinants of the pathological degree of neovascularisation found in the parietal neomembrane of chronic subdural hematoma (CSH). Thus far the pathogenesis of the vascularisation has not been elucidated.Method: The concentrations of growth factors, i.e. vascular endothelial derived growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) were determined using ELISA technique in hematoma fluid and serum of 20 patients with uni- or bilateral CSH. For comparison, growth factor concentrations were determined in cerebrospinal fluid (CSF) of patients undergoing diagnostic myelography.Findings: Concentrations of VEGF and bFGF were significantly (p<0.001) increased in the hematoma fluid as compared with serum (VEGFh=8,142 pg/ml, bFGFh=8.7 pg/ml versus VEGFs=368 pg/ml, bFGFs=1.8 pg/ml). In contrast, PDGF concentration was significantly (p<0.001) lower in the hematoma (PDGFh=3,456 pg/ml versus PDGFs=31,937 pg/ml). The serum levels for VEGF, bFGF and PDGF in CSH patients lay within the range of normal volunteers. No growth factors were found in normal CSF.Interpretation: These results reveal a specific distribution pattern of growth factors in CSH patients. This pattern suggests that CSH may be considered a member of the angiogenic disease family.

Endothelin-3-Induced Relaxation of Isolated Rat Basilar Artery is Mediated by an Endothelial ETB-Type Endothelin Receptor:

The endothelin (ET) receptor mediating relaxation of cerebral arteries was characterized using ring segments obtained from the rat basilar artery. Under resting tension, ET-3 (>10−8 M) but not the specific ETB receptor agonist IRL 1620 induced contraction. In ring segments precontracted with 3 × 10−6 M prostaglandin (PG) F2α, ET-3 (10−12–10−8 M) and IRL 1620 (10−14–10−6 M) induced concentration-related relaxation. IRL 1620 was more potent than ET-3, the pD2 (-log10EC50) values being 10.002 ± 0.751 (mean ± SD) for IRL 1620 and 8.836 ± 0.415 for ET-3. Relaxation was abolished after preincubation with the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine (10−5 M) as well as in segments devoid of a functionally intact endothelium. At a concentration above 10−8 M, ET-3 resulted in a further increase of PGF2α-induced contraction that was not observed with IRL 1620. The presumably specific ETB receptor antagonist IRL 1038 (10−7–3 × 10−6 M) diminished or even abolished (3 × 10−6 M) the relaxation induced by ET-3 or IRL 1620. IRL 1038 did not exert any vasomotor effect by itself, and it did not significantly affect ET-3-induced contraction. These results indicate that in the rat isolated basilar artery, the ET-3-induced relaxation is probably due to activation of an ETB-type receptor located on the endothelial cells and mediated by release of nitric oxide.

Vascular imaging in small rodents using micro-CT.

In vivo animal models of neoplasm, stroke, subarachnoid hemorrhage, and other diseases involving alterations in vessel anatomy and diameter, require a fast and easy-to-use imaging tool that captures anatomical structure and biologic function data. Micro-computed tomography angiography (muCTA) offers high spatial and temporal resolution and is suitable to perform this task. However, conducting muCTA in small rodents, especially in mice, requires a high degree of accuracy and precision. This article describes a setup for in vivo muCTA in mice using both a bolus technique with a conventional contrast agent, as well as, angiography with a blood-pool contrast agent. Our setup in mice is at isotropic resolutions up to 16 microm with scanning times less than 1 min. The described protocol also addresses some of the technical challenges associated with the imaging of vascular structures in mice models.