Metastasis into the skeleton is a serious complication of certain neoplastic diseases such as breast, prostate and lung cancer, but the reasons for this osteotropism are poorly understood. Our aim was to establish a physiologically relevant animal model that is characterized by osteolytic lesions confined to the hind leg of nude rats. For this purpose, we injected 1310 5 MDA-MB-231 human breast cancer cells transfected with GFP into the superficial epigastric artery, which is an anastomosing vessel between the femoral and iliac arteries. As assessed with the aid of X-rays, computed tomography and immunohistochemisty, osteolytic lesions occurred exclusively in the femur, tibia and fibula of the animals. The tumor take rate was 93% in a series of 96 rats and the increase in lesion size was observed up to 110 days after tumor cell inoculation. When applying this animal model to the effects of an antibody against bone sialoprotein (BSP), a significantly reduced osteolytic lesion size was observed after preincubation of cells (2 hr, 600 mg/ml anti-BSP) prior to intra-arterial tumor cell injection resulting in 19 T/C% at day 60 after tumor implantation (p < 0.05). In addition, the osteolytic lesion size was also significantly reduced after s.c. treatment of the animals with the antibody (20 mg/kg anti-BSPx3 within 5 days after tumor implantation), resulting in 30 T/C% at day 60 after tumor cell implantation (p < 0.05). In conclusion, the novel rat model for site-specific osteolytic lesions provides in vivo evidence that preincubation of MDA-MB-231 GFP cells and treatment of rats after tumor implantation with an antibody against BSP significantly reduces the size of lytic lesions in bone. ' 2005 Wiley-Liss, Inc.Key words: nude rat model; MDA-MB-231 human breast cancer cell line; osteolysis; bone metastasis; bone sialoprotein (BSP); anti-BSP antibody Metastasis is a common phenomenon in the course of malignant tumor disease and the major reason for cancer-associated morbidity and mortality. In osteotropic malignancies such as breast, prostate, lung, kidney and thyroid cancer, the skeleton is frequently affected as metastatic site. From these cancers, the prevalence of skeletal disease is greatest in patients with breast or prostate carcinoma who in the advanced stage develop skeletal metastases in 70%. 1 Breast carcinoma patients experience a median survival time of 20 month after the first appearance of a bone metastasis. 2 Such a lesion results in a number of skeletal complications including pathological fractures, bone pain, hypocalcaemia and spinal cord compression. The reasons for the skeleton being the preferential localization site of metastasis in osteotropic cancers are poorly understood.In patients with primary breast cancer, elevated serum bone sialoprotein (BSP) was recognized as prognostic marker of subsequent bone metastasis 3 and was associated with poor survival. 4 BSP is a noncollagenous protein of the extracellular bone matrix and a member of the SIBLING (Small Integrin-Binding Ligand, N-linked Glycop...

The effects of vascular factors on the nervous system are still poorly investigated. Angiopoietin-1 (Ang-1), an endothelial cell growth factor with influences on blood vessel stabilization, has been recently reported to prevent apoptosis in a neuroblastoma cell line via a pathway dependent on Tie-2 receptor. The present study focuses on the effect of Ang-1 on cultured dorsal root ganglion (DRG) cells isolated from 1-day-old rats. Three-day-old DRG cultures were exposed to Ang-1 treatment under serum-free condition for another 5 days and stained with antibodies against neurofilament (NF) 200 protein. Neurite length and density increased compared with those of controls. Double-immunofluorescence staining demonstrated the co-localization of the Tie-2 receptor in some NF-200-positive perikarya. The reverse transcription/polymerase chain reaction technique identified Tie-2 receptor mRNA in intact DRG and in Ang-1-stimulated DRG cell cultures, but not in a Schwann cell line or in primary astrocyte cultures. Western blotting confirmed that the expression of NF 68 protein in cultures treated with Ang-1 or nerve growth factor was higher than that in cultures treated with medium alone. When the Tie-2 receptor was blocked with anti-Tie-2 receptor antibody, neurite outgrowth was severely impeded. Induction of trkA-receptor protein expression was observed to be dependent on the presence of Tie-2 receptors. We conclude that Ang-1 promotes neurite outgrowth from DRG cells positive for Tie-2 receptor. The signalling pathway appears to involve transactivation of the trkA receptor.

This study provides a morphologic characterization of the human superior olivary complex as revealed by immunohistochemistry by using antibodies against the calcium binding proteins parvalbumin, calbindin, calretinin, and the nonphosphorylated neurofilament H SMI-32. By combining these markers, it was possible to establish the neuronal architecture and details of the morphologic organization (including axonal terminals) of the different nuclei. The medial superior olivary nucleus is formed by a sheet of parallel-oriented cells. A clear segregation of axon terminals was noticed on the medially and laterally oriented dendrites of the mostly bipolar neurons. The lateral superior olivary nucleus lacked a distinct nuclear shape but was formed by several patches of rather irregularly arranged neurons. Calretinin or parvalbumin immunoreactive afferent terminals were observed which contacted somata or dendrites of these neurons. The immunolabeling also revealed the boundaries of the dorsal periolivary nucleus and morphologic detail of its neurons. A coherent nuclear structure that could be addressed as the medial nucleus of the trapezoid body was not identified by any single one or by combinations of the markers used. The data were also used to establish a three-dimensional-reconstruction of the three major subnuclei of the superior olivary complex. The results are discussed with respect to the possible role of the superior olivary complex in the processing of spatial acoustic information in the azimuthal plane.

Experimental human fetal neural progenitor cell (hfNPC) transplantation has proven to be a promising therapeutic approach after traumatic brain injury (TBI). However, the long-term efficacy and safety, which are both highly important for clinical translation of this approach, have thus far not been investigated. This study investigated the effect of local (L, 1 × 10(5) cells) and systemic (S, 5 × 10(5) cells) administration of PKH-26-labeled pre-differentiated hfNPCs over a period of 12 weeks, beginning 24 h after severe controlled cortical impact TBI in Sprague-Dawley rats. Accelerating rotarod testing revealed a trend toward functional improvement beginning 1 week after transplantation, and persisting until the end of the experiment. The traumatic lesion volume as quantified by magnetic resonance imaging was smaller in both treatment groups compared to control (C) animals (C = 54.50 mm(3), L = 32 mm(3), S = 37.50 mm(3)). Correspondingly, neuronal (NeuN) staining showed increased neuronal survival at the border of the lesion in both transplanted groups (S = 92.4%; L = 87.2%; 72.5%). Histological analysis of the brain compartments revealed transiently increased angiogenesis and reduced astroglial reaction during the first 4 weeks post-transplantation. PKH-26-positive cells were detected exclusively after local transplantation without any evidence of tumor formation. However, graft differentiation was seen only in very rare cases. In conclusion, transplantation of hfNPCs improved the long-term functional outcome after TBI, diminished trauma lesion size, and increased neuronal survival in the border zone of the lesion. This therapeutic effect was not likely due to cell replacement, but was associated with transiently increased angiogenesis and reduced astrogliosis.

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The distribution of perineuronal nets and the potassium channel subunit Kv3.1b was studied in the subdivisions of the cochlear nucleus, the medial nucleus of the trapezoid body, the medial and lateral superior olivary nuclei, the lateral lemniscal nucleus and the inferior colliculus of the rhesus monkey. Additional sections were used for receptor autoradiography to visualize the patterns of GABA A and GABA B receptor distribution. The Kv3.1b protein and perineuronal nets [visualized as Wisteria floribunda agglutinin (WFA) binding] were revealed, showing corresponding region-specific patterns of distribution. There was a gradient of labelled perineuronal nets which corresponded to that seen for the intensity of Kv3.1b expression. In the cochlear nucleus intensely and faintly stained perineuronal nets were intermingled, whereas in the medial nucleus of the trapezoid body the pattern changed to intensely stained perineuronal nets in the medial part and weakly labelled nets in its lateral part. In the inferior colliculus, intensely labelled perineuronal nets were arranged in clusters and faintly labelled nets were arranged in sheets. Using receptor autoradiography, GABA B receptor expression in the anterior ventral cochlear nucleus was revealed. The medial part of the medial nucleus of the trapezoid body showed a high number of GABA A binding sites whereas the lateral part exhibited more binding sites for GABA B . In the inferior colliculus, we found moderate GABA B receptor expression. In conclusion, intensely WFA-labelled structures are those known to be functionally involved in high-frequency processing.

The aim of this study was to evaluate the effect of an antiangiogenic treatment with the vascular endothelial growth factor antibody bevacizumab in an experimental model of breast cancer bone metastasis and to monitor osteolysis, soft tissue tumor, and angiogenesis in bone metastasis noninvasively by volumetric computed tomography (VCT) and magnetic resonance imaging (MRI). After inoculation of MDA-MB-231 human breast cancer cells into nude rats, bone metastasis was monitored with contrast-enhanced VCT and MRI from day 30 to day 70 after tumor cell inoculation, respectively. Thereby, animals of the treatment group (10 mg/kg bevacizumab IV weekly, n = 15) were compared with sham-treated animals (n = 17). Treatment with bevacizumab resulted in a significant difference versus control in osteolytic as well as soft tissue lesion sizes (days 50 to 70 and 40 to 70 after tumor cell inoculation, respectively; P < .05). This observation was paralleled with significantly reduced vascularization in the treatment group as shown by reduced increase in relative signal intensity in dynamic contrast-enhanced MRI from days 40 to 70 (P < .05). Contrast-enhanced VCT and histology confirmed decreased angiogenesis as well as new bone formation after application of bevacizumab. In conclusion, bevacizumab significantly inhibited osteolysis, surrounding soft tissue tumor growth, and angiogenesis in an experimental model of breast cancer bone metastasis as visualized by VCT and MRI.