Christian Schaefer

Neural driven angiogenesis by overexpression of nerve growth factor.

Mechanisms regulating angiogenesis are crucial in adjusting tissue perfusion on metabolic demands. We demonstrate that overexpression of nerve growth factor (NGF) in brown adipose tissue (BAT) of NGF-transgenic mice elevates both mRNA and protein levels of vascular endothelial growth factor (VEGF) and VEGF-receptors. Increased vascular permeability, leukocyte–endothelial interactions (LEI), and tissue perfusion were measured using intravital microscopy. NGF-stimulation of adipocytes and endothelial cells elevates mRNA expression of VEGF and its receptors, an effect blocked by NGF neutralizing antibodies. These data suggest an activation of angiogenesis as a result of both: stimulation of adipozytes and direct mitogenic effects on endothelial cells. The increased nerve density associated with vessels strengthened our hypothesis that tissue perfusion is regulated by neural control of vessels and that the interaction between the NGF and VEGF systems is the critical driver for the activated angiogenic process. The interaction of VEGF- and NGF-systems gives new insights into neural control of organ vascularization and perfusion.

Sequential changes in vessel formation and micro-vascular function during bone repair

Backgroundu2003Angiogenesis, the process of new vessel formation from a pre-existing vascular network, is essential for bone development and repair. New vessel formation and microvascular functions are crucial during bone repair, not only for sufficient nutrient supply, transport of macromolecules and invading cells, but also because they govern the metabolic microenvironment. Despite its central role, very little is known about the initial processes of vessel formation and microvascular function during bone repair. Methodsu2003To visualize and quantify the process of vessel formation and microvascular function during bone repair, we transplanted neonatal femora with a substantial defect into dorsal skin-fold chambers in severe combined immunodeficient (SCID) mice for continuous noninvasive in-vivo evaluation. We employed intravital microscopic techniques to monitor effective microvascular permeability, functional vascular density, blood flow rate and leukocyte flux repeatedly over 16 days. Oxytetracyclin and v. Kossa/v. Giesson staining was performed to quantify the calcification process in vivo and in vitro. Resultsu2003Development of a hematoma surrounding the defect area was the initial event, which was accompanied by a significant increase in microvascular permeability and blood flow rate. With absorption of the hematoma and vessel maturation, permeability decreased continuously, while vascular density and tissue perfusion increased. Histological evaluation revealed that the remodeling of the substantial defect prolonged the in-vivo monitored calcification process. Interpretationu2003The size of the initial substantial defect correlated positively with increased permeability, suggesting improved release of permeability-inducing cytokines. The unchanged permeability in the control group with boiled bones and a substantial defect corroborated these findings. The adaptation to increasing metabolic demands was initially mediated by increased blood flow rate, later with increasing vascular density through increased tissue perfusion rate. These insights into the sequence of microvascular alterations may assist in the development of targeted drug delivery therapies and caution against the use of permeability-altering drugs during bone healing.

Early microvascular complications of prediabetes in mice with impaired glucose tolerance and dyslipidemia

Microvascular complications are an important cause of morbidity in diabetic patients and can be detected in a significant number of patients at the time of diabetes diagnosis. However, little is known about the alterations in the microvasculature previous to the clinical manifestation of diabetes mellitus type 2. To obtain more insights into the early microvascular deterioration resulting from prediabetes, morphological and functional microvascular parameters were monitored using intravital fluorescence microscopy through a dorsal skin-fold chamber preparation in the uncoupling promotor-driven diphtheria toxin A chain (UCP1/DTA) mice. At the age of 12xa0weeks, the UCP1/DTA-mice were characterized by impaired glucose tolerance with concurrent unchanged fasting glucose, as well as dyslipidemia, hyperinsulinemia, hypertension and obesity. Prediabetic mice displayed combined hypertriglyceridemia and hypercholesterinemia. Associated with these prediabetic metabolic alterations, we demonstrate that microvascular density showed a dramatic decrease due to a reduction in perfused small vessels. A reduction in vascular density combined with unaltered blood flow in single vessels resulted in impaired tissue perfusion. Endothelial dysfunction with subsequently increased microvascular permeability and leukocyte–endothelium interactions were found. Our results of profound microvascular alterations at stages of normal fasting glucose underline the importance of early screening for prediabetes and associated microvascular complications.

Bilaterally increased VEGF-levels in muscles during experimental unilateral callus distraction

Angiogenesis is essential for wound healing and proliferative processes such as bone formation and repair. Since increased expression of the vascular endothelial growth factor (VEGF) stimulates bone formation, it can be hypothesized that surgical procedures leading to a systemic increase of VEGF for instance during wound healing, influence enchondral ossification processes and might be responsible for observed growth phenomena during callus distraction. To study the mechanisms of angiogenesis in soft tissue during unilateral callus distraction, lengthening of the right tibia was performed in 12 beagles. After osteotomy, application of a ring fixator and after five latency days, distraction was started for 25 days. A control group of four additional beagles underwent no surgical procedure. Subsequent to the distraction period (Group A), muscle samples from six beagles were taken from the distracted side (ds) and the contralateral non‐distracted side (n‐ds), six beagles underwent an additional consolidation period of 25 days (Group B). Samples were analyzed for VEGF, VEGFR‐1 and VEGFR‐2 mRNA expression using real‐time PCR and protein expression using Western Blot analysis. Muscles from both extremities showed significantly increased expression of VEGF and its cognate receptors VEGFR‐½. Expression decreased significantly after the consolidation period, whereby the level at the non‐distracted side decreased more than the level at the distracted side. Interestingly VEGF and VEGFR‐1 levels at the non‐distracted side were significantly higher than at the distracted side. In contrast VEGFR‐2, the receptor that mediates endothelial cell proliferation, showed higher levels at the distracted than at the non‐distracted side. These findings indicate that callus distraction results not only in locally increased expression of VEGF and its receptors, but leads also to increased VEGF and VEGFR‐½ levels at distant sides and might therefore be responsible for the observed growth phenomena during callus distraction.

Chemokine profile of disc degeneration with acute or chronic pain

OBJECTnDisc-related disorders such as herniation and chronic degenerative disc disease (DDD) are often accompanied by acute or chronic pain. Different mediators have been identified in the development of radicular pain and DDD. Previous studies have not analyzed individual cytokine profiles discriminating between acute sciatic and chronic painful conditions, nor have they distinguished between different anatomical locations within the disc. The aim of this study was to elucidate the protein biochemical mechanisms in DDD.nnnMETHODSnThe authors determined expression levels of matrix metalloproteinase-3, transforming growth factor-β (TGF-β), tumor necrosis factor-α, interleukin-1α, and pro-substance P using enzyme-linked immunosorbent assay and Western blot analyses in patients suffering from DDD (n = 7), acute back pain due to herniated discs with radiculopathy (n = 7), and a control group (n = 7). Disc tissue samples from the anulus fibrosus (AF) and nucleus pulposus (NP) were analyzed. Statistical analysis was performed using nonparametric tests.nnnRESULTSnA distinct distribution of cytokines was found in different anatomical regions of intervertebral discs in patients with DDD and herniated NP. Increased TGF-β levels were predominantly found in DDD. Matrix metalloproteinase-3 was increased in acute herniated disc material. Increased levels of substance P were found in patients suffering from DDD but not in patients with disc herniation. The data showed significantly higher levels of proinflammatory cytokines in the AF and NP of patients with DDD, and the expression levels in the AF were even higher than in the NP, suggesting that the inflammatory response initiates from the AF.nnnCONCLUSIONSnThese results highlight the complex mechanisms involved during disc degeneration and the need to distinguish between acute and chronic processes as well as different anatomical regions, namely the AF and NP. They also highlight potential problems in disc nucleus replacement therapies because the results suggest a biochemical link between AF and NP cytokine expression.

Accuracy and safety of fluoroscopic guided percutaneous pedicle screws in thoracic and lumbosacral spine: a review of 2000 screws.

Study Design. Retrospective study. Objective. To investigate the accuracy and safety of percutaneous pedicle screws placed using fluoroscopic guidance in the thoracic and lumbosacral spine. Summary of Background Data. Several studies had examined the accuracy and safety of percutaneous pedicle screws but provided large variations in their results with small number of patients or few number of pedicle screws evaluated. Methods. Computerized tomography of patients who had surgery with fluoroscopic guided percutaneous pedicle screws were chosen from 2 centers: (1) European patients from University Medical Center Hamburg-Eppendorf, Germany and (2) Asian patients from University Malaya Medical Centre, Malaysia. Screw perforations were classified into Grade 0, Grade 1 (<2 mm), Grade 2 (2–4 mm), and Grade 3 (>4 mm). Results. In total, 2000 percutaneous pedicle screws from 273 patients were analyzed: 1290 screws from 183 European patients and 710 screws from 90 Asian patients. The mean age was 59.1 ± 15.6. There were 140 male patients and 133 female patients. The total perforation rate was 9.4% with 151 (7.5%) Grade 1, 31 (1.6%) Grade 2, and 5 (0.3%) Grade 3 perforations. The total perforation rates among Europeans were 9.4% and among Asians were 9.3%. There was no difference between the 2 groups (P > 0.05). There were 3 distinct peaks in perforation rates (trimodal distribution) at T1, midthoracic region (T4–T7), and lumbosacral junction (L5 and S1). The highest perforation rates were at T1 (33.3%), S1 (19.4%), and T4 (18.6%). Conclusion. Implantation of percutaneous pedicle screws insertion using fluoroscopic guidance is safe and has the accuracy comparable to open techniques of pedicle screws insertion. Level of Evidence: 4

Percutaneous instrumentation of the cervical and cervico-thoracic spine using pedicle screws: preliminary clinical results and analysis of accuracy

The pedicle screw instrumentation represents the most rigid construct of the cervical and cervicothoracic spine and in spite of the risks to neurovascular structures clinical relevant complications do not occur frequently. The steep angles of the cervical pedicles result in a wide surgical exposure with extensive muscular trauma. The objective of this study was the evaluation of the accuracy of cervical pedicle screw insertion through a minimally invasive technique to reduce access-related muscular trauma. Therefore, percutaneous transpedicular instrumentation of the cervical and cervicothoracic spine was performed in 15 patients using fluoroscopy. All instrumentations from C2 to Th4 were inserted bilaterally through 2 to 3-cm skin and fascia incisions even in multilevel procedures and the rods were placed by blunt insertion through the incision. Thin-cut CT scan was used postoperatively to analyze pedicle violations. 76.4% of 72 screws were placed accurately. Most pedicle perforations were seen laterally towards the vertebral artery. Critical breaches >2xa0mm or narrowing of the transversal foramen occurred in 12.5% of screws; however, no revision surgery for screw displacement was needed in the absence of clinical symptoms. No conversion from percutaneous to open surgery was necessary. It was concluded that percutaneous transpedicular instrumentation of the cervical spine is a surgically demanding technique and should be reserved for experienced spine surgeons. The indications are limited to instrumentation-only procedures or in combination with anterior treatment, but with the potential to minimize access-related morbidity.

Dynamics of Microvascular Remodelling during Tumor Growth in Bone

Microcirculatory properties of tumors have been shown to play a pivotal role in tumor progression and inefficacy of therapies. Although the influence of the microenvironment on angiogenesis has been intensively investigated in soft tissue tumors, little is known about the microvascular properties in bone metastasis. To determine the impact of the bone microenvironment on tumor growth and microcirculation we performed intravital microscopy using the “femur window” after implantation of red‐fluorescent‐protein‐transduced breast cancer cells into the femura of severe‐combined‐immunodeficient mice. Tumor size, functional vascular density, vessel diameter, and vessel distribution were quantified over 14 days. Tumor growth and microcirculation could be quantified at a high spatial resolution. Tumor progression was associated with a rapid remodeling process of the microcirculation within the tumor and the surrounding tissue. Although the total functional vascular density remained unaltered, we found a significant loss in small vessels and a concomitant increase in vascular diameter. The presented study demonstrates for the first time dynamics of morphological microcirculatory alterations of tumor growth in bone. The observed changes in tumor vascularization exhibit strong similarities to soft tissue tumors; however, the dynamics of vascular alterations are more rapid in the bone microenvironment.

Microcirculation of secondary bone tumors in vivo: the impact of minor surgery at a distal site.

Microcirculatory properties of tumors play a pivotal role in tumor progression and inefficacy of therapies. It has been hypothesized that surgical interventions result in an accelerated tumor growth by increasing the level of pro‐angiogenic cytokines with subsequent amplification of tumor angiogenesis. To characterize the microvascular properties of secondary bone tumors in vivo and determine the impact of minor surgery on the microcirculation, we performed intravital microscopy over 25 days using a xenograft model of breast cancer tumor growth (MCF‐7) in bone. After engraftment of tumors the mice were treated with a mastectomy versus no surgery. Tumor growth was accompanied by angiogenic sprouting and decreased vascular diameters while blood flow rate and tumor perfusion remained constant. Mastectomy initially led to a significant reduction of functional vascular density, increased vascular diameter, and decreased blood flow velocities. However, neither tumor growth nor tissue perfusion was different between the groups. The presented study corroborates the assumption that tumor microcirculation in bone exhibits similar time‐dependent alterations compared to soft tissue tumors. A minor surgical intervention did not change tumor growth kinetics however microcirculatory properties were altered. Whether major surgery has an impact on tumor growth in bone should be clarified in further studies.

Comparison Between Minimally Invasive Surgery and Conventional Open Surgery for Patients With Spinal Metastasis: A Prospective Propensity Score-matched Study.

Study Design. Prospective propensity score-matched study. Objective. To compare the outcomes of minimal invasive surgery (MIS) and conventional open surgery for spinal metastasis patients. Summary of Background Data. There is lack of knowledge on whether MIS is comparable to conventional open surgery in treating spinal metastasis. Methods. Patients with spinal metastasis requiring surgery from January 2008 to December 2010 in two spine centers were recruited. The demographic, preoperative, operative, perioperative and postoperative data were collected and analyzed. Thirty MIS patients were matched with 30 open surgery patients using propensity score matching technique with a match tolerance of 0.02 based on the covariate age, tumor type, Tokuhashi score, and Tomita score. Results. Both groups had significant improvements in Eastern Cooperative Oncology Group (ECOG), Karnofsky scores, visual analogue scale (VAS) for pain and neurological status postoperatively. However, the difference comparing the MIS and open surgery group was not statistically significant. MIS group had significantly longer instrumented segments (5.5u200a±u200a3.1) compared with open group (3.8u200a±u200a1.7). Open group had significantly longer decompressed segment (1.8u200a±u200a0.8) than MIS group (1.0u200a±u200a1.0). Open group had significantly more blood loss (2062.1u200a±u200a1148.0u200amL) compared with MIS group (1156.0u200a±u200a572.3u200amL). More patients in the open group (76.7%) needed blood transfusions (with higher average units of blood transfused) compared with MIS group (40.0%). Fluoroscopy time was significantly longer in MIS group (116.1u200a±u200a63.3u200as) compared with open group (69.9u200a±u200a42.6u200as). Open group required longer hospitalization (21.1u200a±u200a10.8 days) compared with MIS group (11.0u200a±u200a5.0 days). Conclusion. This study demonstrated that MIS resulted in comparable outcome to open surgery for patients with spinal metastasis but has the advantage of less blood loss, blood transfusions, and shorter hospital stay. Level of Evidence: 3