Going against the tide--how encephalitogenic T cells breach the blood-brain barrier

During multiple sclerosis or its animal model, experimental autoimmune encephalomyelitis, circulating immune cells enter the central nervous system (CNS) causing neuroinflammation. Extravasation from the blood circulation across the vessel wall occurs through a multistep process regulated by adhesion and signal transducing molecules on the immune cells and on the endothelium. Since the CNS is shielded by the highly specialized blood-brain barrier (BBB), immune cell extravasation into the CNS requires breaching this particularly tight endothelial border. Consequently, travelling into the CNS demands unique adaptations which account for the extreme tightness of the BBB. Modern imaging tools have shown that after arresting on BBB endothelium, in vivo or in vitro encephalitogenic effector/memory T cells crawl for long distances, possibly exceeding 150 µm along the surface of the BBB endothelium before rapidly crossing the BBB. Interestingly, in addition to the distance of crawling, the preferred direction of crawling against the flow is unique for T cell crawling on the luminal surface of CNS microvessels. In this review, we will summarize the cellular and molecular mechanisms involved in the unique T cell behavior that is obviously required for finding a site permissive for diapedesis across the unique vascular bed of the BBB.

Immunologic privilege in the central nervous system and the blood-brain barrier

The brain is in many ways an immunologically and pharmacologically privileged site. The blood-brain barrier (BBB) of the cerebrovascular endothelium and its participation in the complex structure of the neurovascular unit (NVU) restrict access of immune cells and immune mediators to the central nervous system (CNS). In pathologic conditions, very well-organized immunologic responses can develop within the CNS, raising important questions about the real nature and the intrinsic and extrinsic regulation of this immune privilege. We assess the interactions of immune cells and immune mediators with the BBB and NVU in neurologic disease, cerebrovascular disease, and intracerebral tumors. The goals of this review are to outline key scientific advances and the status of the science central to both the neuroinflammation and CNS barriers fields, and highlight the opportunities and priorities in advancing brain barriers research in the context of the larger immunology and neuroscience disciplines. This review article was developed from reports presented at the 2011 Annual Blood-Brain Barrier Consortium Meeting.

Lowered microvascular vessel wall oxygen consumption augments tissue pO2 during PgE1-induced vasodilation

Continuous infusion of intravenous prostaglandin E1 (PgE1, 2.5 mug/kg/min) was used to determine how vasodilation affects oxygen consumption of the microvascular wall and tissue pO(2) in the hamster window chamber model. While systemic measurements (mean arterial pressure and heart rate) and central blood gas measurements were not affected, PgE1 treatment caused arteriolar (64.6 +/- 25.1 microm) and venular diameter (71.9 +/- 29.5 microm) to rise to 1.15 +/- 0.21 and 1.06 +/- 0.19, respectively, relative to baseline. Arteriolar (3.2 x 10(-2) +/- 4.3 x 10(-2) nl/s) and venular flow (7.8 x 10(-3) +/- 1.1 x 10(-2)/s) increased to 1.65 +/- 0.93 and 1.32 +/- 0.72 relative to baseline. Interstitial tissue pO(2) was increased significantly from baseline (21 +/- 8 to 28 +/- 7 mmHg; P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the microvascular wall decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the vascular wall, decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). This reduction reflects a 20% decrease in oxygen consumption by the vessel wall and up to 50% when cylindrical geometry is considered. The venular vessel wall gradient decreased from 12 +/- 4 to 9 +/- 4 mmHg (P < 0.001). Thus PgE1-mediated vasodilation has a positive microvascular effect: enhancement of tissue perfusion by increasing flow and then augmentation of tissue oxygenation by reducing oxygen consumption by the microvascular wall.

A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

High expression of neuropeptide Y1 receptors in ewing sarcoma tumors

PURPOSE: Peptide receptors are frequently overexpressed in human tumors, allowing receptor-targeted scintigraphic imaging and therapy with radiolabeled peptide analogues. Neuropeptide Y (NPY) receptors are new candidates for these applications, based on their high expression in specific cancers. Because NPY receptors are expressed in selected sarcoma cell lines and because novel treatment options are needed for sarcomas, this study assessed the NPY receptor in primary human sarcomas. EXPERIMENTAL DESIGN: Tumor tissues of 88 cases, including Ewing sarcoma family of tumors (ESFT), synovial sarcomas, osteosarcomas, chondrosarcomas, liposarcomas, angiosarcomas, rhabdomyosarcomas, leiomyosarcomas, and desmoid tumors, were investigated for NPY receptor protein with in vitro receptor autoradiography using (125)I-labeled NPY receptor ligands and for NPY receptor mRNA expression with in situ hybridization. RESULTS: ESFT expressed the NPY receptor subtype Y1 on tumor cells in remarkably high incidence (84%) and density (mean, 5,314 dpm/mg tissue). Likewise, synovial sarcomas expressed Y1 on tumor cells in high density (mean, 7,497 dpm/mg; incidence, 40%). The remaining tumors expressed NPY receptor subtypes Y1 or Y2 at lower levels. Moreover, many of the sarcomas showed Y1 expression on intratumoral blood vessels. In situ hybridization for Y1 mRNA confirmed the autoradiography results. CONCLUSIONS: NPY receptors are novel molecular markers for human sarcomas. Y1 may inhibit growth of specific sarcomas, as previously shown in an in vivo mouse model of human ESFT. The high Y1 expression on tumor cells of ESFT and synovial sarcomas and on blood vessels in many other sarcomas represents an attractive basis for an in vivo tumor targeting.

Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node

Lymph nodes are strategically localized at the interfaces between the blood and lymphatic vascular system, delivering immune cells and antigens to the lymph node. As cellular junctions of endothelial cells actively regulate vascular permeability and cell traffic, we have investigated their molecular composition by performing an extensive immunofluorescence study for adherens and tight junction molecules, including vascular endothelium (VE)-cadherin, the vascular claudins 1, 3, 5 and 12, occludin, members of the junctional adhesion molecule family plus endothelial cell-selective adhesion molecule (ESAM)-1, platelet endothelial cell adhesion molecule-1, ZO-1 and ZO-2. We found that junctions of high endothelial venules (HEV), which serve as entry site for naive lymphocytes, are unique due to their lack of the endothelial cell-specific claudin-5. LYVE-1(+) sinus-lining endothelial cells form a diffusion barrier for soluble molecules that arrive at the afferent lymph and use claudin-5 and ESAM-1 to establish characteristic tight junctions. Analysis of the spatial relationship between the different vascular compartments revealed that HEV extend beyond the paracortex into the medullary sinuses, where they are protected from direct contact with the lymph by sinus-lining endothelial cells. The specific molecular architecture of cellular junctions present in blood and lymphatic vessel endothelium in peripheral lymph nodes establishes distinct barriers controlling the distribution of antigens and immune cells within this tissue.

Role of the extraosseus blood supply in osteoarthritic femoral heads

Blood perfusion to the femoral head might be endangered during the surgical approach or the preparation of the femoral head or both in hip resurfacing arthroplasty. The contribution of the intramedullary blood supply to the femoral head in osteoarthritis is questionable. Therefore, the contribution of the extraosseous blood supply to osteoarthritic femoral heads was measured intraoperatively to question if there is measurable blood flow between the epiphysis and metaphysis in osteoarthritic hips in case of extraosseus vessel damage. At defined points during surgery we acquired the epiphyseal and metaphyseal femoral head perfusion by high-energy laser Doppler flowmetry. Complete femoral neck osteotomy sparing the retinacular vessels to simulate intraosseous blood disruption showed unchanged epiphyseal blood flow compared to initial measurement after capsulotomy. The pulsatile signal disappeared after transection of the retinacular vessels. Based on these acute measurements, we conclude intramedullary blood vessels to the femoral head do not provide measurable blood supply to the epiphysis once the medial femoral circumflex artery or the retinacular vessels have been damaged. We recommend the use of a safe surgical approach for hip resurfacing and careful implantation of the femoral component to respect blood supply to the femoral head and neck region in hip resurfacing arthroplasty.

Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain

PURPOSE Synchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. METHODS Tissue responses to MRT (two orthogonal arrays (2 × 400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. RESULTS In tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. CONCLUSION We provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.

Targeting the Blood-brain Barrier with a Non-canonical Iron-mimicry Mechanism

Treatment of central nervous system (CNS) diseases is limited by the blood-brain barrier (BBB), a selective vascular interface restricting passage of most molecules from blood into brain. Specific transport systems have evolved allowing circulating polar molecules to cross the BBB and gain access to the brain parenchyma. However, to date, few ligands exploiting such systems have proven clinically viable in the setting of CNS diseases. We reasoned that combinatorial phage-display screenings in vivo would yield peptides capable of crossing the BBB and allow for the development of ligand-directed targeting strategies of the brain. Here we show the identification of a peptide mediating systemic targeting to the normal brain and to an orthotopic human glioma model. We demonstrate that this peptide functionally mimics iron through an allosteric mechanism and that a non-canonical association of (i) transferrin, (ii) the iron-mimic ligand motif, and (iii) transferrin receptor mediates binding and transport of particles across the BBB. We also show that in orthotopic human glioma xenografts, a combination of transferrin receptor over-expression plus extended vascular permeability and ligand retention result in remarkable brain tumor targeting. Moreover, such tumor targeting attributes enables Herpes simplex virus thymidine kinase-mediated gene therapy of intracranial tumors for molecular genetic imaging and suicide gene delivery with ganciclovir. Finally, we expand our data by analyzing a large panel of primary CNS tumors through comprehensive tissue microarrays. Together, our approach and results provide a translational avenue for the detection and treatment of brain tumors.

Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

Vessel orientation-dependent sensitivity of optoacoustic imaging using a linear array transducer

For clinical optoacoustic imaging, linear probes are preferably used because they allow versatile imaging of the human body with real-time display and free-hand probe guidance. The two-dimensional (2-D) optoacoustic image obtained with this type of probe is generally interpreted as a 2-D cross-section of the tissue just as is common in echo ultrasound. We demonstrate in three-dimensional simulations, phantom experiments, and in vivo mouse experiments that for vascular imaging this interpretation is often inaccurate. The cylindrical blood vessels emit anisotropic acoustic transients, which can be sensitively detected only if the direction of acoustic radiation coincides with the probe aperture. Our results reveal for this reason that the signal amplitude of different blood vessels may differ even if the vessels have the same diameter and initial pressure distribution but different orientation relative to the imaging plane. This has important implications for the image interpretation, for the probe guidance technique, and especially in cases when a quantitative reconstruction of the optical tissue properties is required.

Oxidative Stress in Hypobaric Hypoxia and Influence on Vessel-Tone Modifying Mediators

Increased pulmonary artery pressure is a well-known phenomenon of hypoxia and is seen in patients with chronic pulmonary diseases, and also in mountaineers on high altitude expedition. Different mediators are known to regulate pulmonary artery vessel tone. However, exact mechanisms are not fully understood and a multimodal process consisting of a whole panel of mediators is supposed to cause pulmonary artery vasoconstriction. We hypothesized that increased hypoxemia is associated with an increase in vasoconstrictive mediators and decrease of vasodilatators leading to a vasoconstrictive net effect. Furthermore, we suggested oxidative stress being partly involved in changement of these parameters. Oxygen saturation (Sao2) and clinical parameters were assessed in 34 volunteers before and during a Swiss research expedition to Mount Muztagh Ata (7549 m) in Western China. Blood samples were taken at four different sites up to an altitude of 6865 m. A mass spectrometry-based targeted metabolomic platform was used to detect multiple parameters, and revealed functional impairment of enzymes that require oxidation-sensitive cofactors. Specifically, the tetrahydrobiopterin (BH4)-dependent enzyme nitric oxide synthase (NOS) showed significantly lower activities (citrulline-to-arginine ratio decreased from baseline median 0.21 to 0.14 at 6265 m), indicating lower NO availability resulting in less vasodilatative activity. Correspondingly, an increase in systemic oxidative stress was found with a significant increase of the percentage of methionine sulfoxide from a median 6% under normoxic condition to a median level of 30% (p<0.001) in camp 1 at 5533 m. Furthermore, significant increase in vasoconstrictive mediators (e.g., tryptophan, serotonin, and peroxidation-sensitive lipids) were found. During ascent up to 6865 m, significant altitude-dependent changes in multiple vessel-tone modifying mediators with excess in vasoconstrictive metabolites could be demonstrated. These changes, as well as highly significant increase in systemic oxidative stress, may be predictive for increase in acute mountain sickness score and changes in Sao2.

Novel insights into the development and maintenance of the blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β-catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.

Cognitive dysfunction in children with brain tumors at diagnosis

Background: Survivors of brain tumors have a high risk for a wide range of cognitive problems. These dysfunctions are caused by the lesion itself and its surgical removal, as well as subsequent treatments (chemo- and/or radiation therapy). Multiple recent studies have indicated that children with brain tumors (BT) might already exhibit cognitive problems at diagnosis, i.e., before the start of any medical treatment. The aim of the present study was to investigate the baseline neuropsychological profile in children with BT compared to children with an oncological diagnosis not involving the central nervous system (CNS). Methods: Twenty children with BT and 27 children with an oncological disease without involvement of the CNS (age range: 6.1 to 16.9 years) were evaluated with an extensive battery of neuropsychological tests tailored to the patient’s age. Furthermore, the child and his/her parent(s) completed self-report questionnaires about emotional functioning and quality of life. In both groups, tests were administered before any therapeutic intervention such as surgery, chemotherapy or irradiation. Groups were comparable with regard to age, gender and socioeconomic status. Results: Compared to the control group, patients with BTs performed significantly worse in tests of working memory, verbal memory and attention (effect sizes between 0.28 and 0.47). In contrast, the areas of perceptual reasoning, processing speed and verbal comprehension were preserved at the time of measurement. Conclusion: Our results highlight the need for cognitive interventions early in the treatment process in order to minimize or prevent academic difficulties as patients return to school.

Real-Time In Vivo Characterization of Primary Liver Tumors With Diffuse Optical Spectroscopy During Percutaneous Needle Interventions: Feasibility Study in Woodchucks

OBJECTIVE This study presents the first in vivo real-time optical tissue characterization during image-guided percutaneous intervention using near-infrared diffuse optical spectroscopy sensing at the tip of a needle. The goal of this study was to indicate transition boundaries from healthy tissue to tumors, namely, hepatic carcinoma, based on the real-time feedback derived from the optical measurements. MATERIALS AND METHODS Five woodchucks with hepatic carcinoma were used for this study. The woodchucks were imaged with contrast-enhanced cone beam computed tomography with a flat panel detector C-arm system to visualize the carcinoma in the liver. In each animal, 3 insertions were performed, starting from the skin surface toward the hepatic carcinoma under image guidance. In 2 woodchucks, each end point of the insertion was confirmed with pathologic examination of a biopsy sample. While advancing the needle in the animals under image guidance such as fluoroscopy overlaid with cone beam computed tomography slice and ultrasound, optical spectra were acquired at the distal end of the needles. Optical tissue characterization was determined by translating the acquired optical spectra into clinical parameters such as blood, water, lipid, and bile fractions; tissue oxygenation levels; and scattering amplitude related to tissue density. The Kruskal-Wallis test was used to study the difference in the derived clinical parameters from the measurements performed within the healthy tissue and the hepatic carcinoma. Kurtoses were calculated to assess the dispersion of these parameters within the healthy and carcinoma tissues. RESULTS Blood and lipid volume fractions as well as tissue oxygenation and reduced scattering amplitude showed to be significantly different between the healthy part of the liver and the hepatic carcinoma (P < 0.05) being higher in normal liver tissue. A decrease in blood and lipid volume fractions and tissue oxygenation as well as an increase in scattering amplitude were observed when the tip of the needle crossed the margin from the healthy liver tissue to the carcinoma. The kurtosis for each derived clinical parameter was high in the hepatic tumor as compared with that in the healthy liver indicating intracarcinoma variability. CONCLUSIONS Tissue blood content, oxygenation level, lipid content, and tissue density all showed significant differences when the needle tip was guided from the healthy tissue to the carcinoma and can therefore be used to identify tissue boundaries during percutaneous image-guided interventions.