Force-dependent development of neuropathic central pain and time-related CCL2/CCR2 expression after graded spinal cord contusion injuries of the rat.

Spinal cord injury (SCI) often results in intractable chronic central pain syndromes. Recently chemokines such as CCL2 were identified as possible key integrators of neuropathic pain and inflammation after peripheral nerve lesion. The focus of the current study was the investigation of time-dependent CCL2 and CCR2 expression in relation to central neuropathic pain development after spinal cord impact lesions of 100, 150, or 200 kdyn force on spinal cord level T9 in adult rats. Below-level pain was monitored with weekly sensory testing for 42 days after SCI. In parallel expression of CCL2/CCR2 on cervical, thoracic, and lumbar levels was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry early (7 days [7d]), intermediate (15d), and late (42d) after lesion. Cellular source and anatomical pain related expression was determined by double-immunohistochemistry. Force-defined SCI led to acute mechanical hypersensitivity in all lesion groups, and to persistent below-level pain in severely injured animals. While in the early post-operative time course, CCL2 and CCR2 were expressed in astroglia and granulocytes only on level T9; there was additional astroglial CCL2 expression in dorsal columns and dorsal horns above and below T9 of severely injured animals 42d after lesion. In dorsal horns (level L3-L5) of animals exhibiting chronic below-level pain CCL2 was co-expressed with transmitters and receptors that are involved in nociceptive processing like calcitonin gene-related peptide (CGRP), Substance-P, vanilloid-receptor-1, and its activated phosphorylated form. These data demonstrate lesion grade dependence of below-level pain development and suggest chemokines as potential candidates for integrating inflammation and central neuropathic pain after SCI.

No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes.

BACKGROUND: Recent studies have suggested the differentiation of human endothelial progenitor cells (huEPCs) isolated from peripheral blood into cardiomyocytes. This study investigates whether, when cocultured, neonatal rat cardiomyocytes (NRCMs) can induce transdifferentiation of huEPCs into cardiomyocytes. METHODS AND RESULTS: Coculture experiments with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled huEPCs and NRCMs have been performed. Cocultures have been analyzed by means of flow cytometry, 3D confocal laser microscopy, species-specific reverse transcriptase-polymerase chain reaction for the expression of human cardiac marker genes, and electron microscopy. Although fluorescence-activated cell sorting (FACS) analysis and conventional wide-field fluorescence microscopy suggested the existence of DiIpos cardiomyocytes in cocultures, no convincing evidence of cardiac differentiation of huEPCs has been obtained. Apparently, DiIpos cardiomyocytes were identified as necrotic NRCMs or NRCM-derived vesicles with high levels of autofluorescence or, alternatively, as NRCMs lying on top of or below labeled huEPCs or huEPC fragments. Accordingly, no expression of human Nkx2.5, GATA-4, or cardiac troponin I was detected. CONCLUSIONS: No convincing evidence of transdifferentiation of huEPCs into cardiomyocytes was obtained. Although we cannot exclude that recent contrary data may be due to slightly different culture protocols, our study has revealed that recently applied standard analysis tools including FACS and wide-field fluorescence microscopy are not sufficient to demonstrate transdifferentiation in coculture settings and can lead to misinterpretation of the data obtained solely with these methods.

Glioblastoma multiforme-report of 267 cases treated at a single institution.

OBJECTIVE: Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor in adults. We present 267 cases treated at a single institution and discuss clinical characteristics and prognostic factors with regard to the neurosurgical literature. METHODS: Included in this study were 267 patients who underwent craniotomy for newly diagnosed GBM between 1990 and 2001 at our department. Clinical charts and radiographic images were reviewed. Association to patient survival was estimated using log-rank test. RESULTS: Median patient age was 61 years (mean, 59.5; range, 22-86 years), the male-female ratio was 1.2:1. In 35 cases (13.1%) the tumor was multicentric. Most of the tumors were classified as primary GBM (87.6%). During follow-up,72 patients (26.4%) underwent recraniotomy for GBM recurrence and 3 patients (1.1%) developed spinal drop metastases. Overall median survival was 47 weeks (range, 5-305 weeks). The following parameters were significantly associated with prolonged survival: (1) age 61 years or younger, P < .001; (2) Karnofsky performance scale score of 70 or more, P < .001; (3) radiotherapy with a total dose of at least 54 Gy, P < .001; (4) chemotherapy, P < .001; (5) total tumor resection, P = .014; (6) recraniotomy for GBM recurrence, P = .012. CONCLUSIONS: Glioblastoma multiforme remains an important cause of morbidity and mortality from intracranial tumors. The overall prognosis is dismal, although interdisciplinary therapy can significantly prolong survival and allows a small subgroup of patients to survive 3 years or more.

Brachial plexus neurotization with donor phrenic nerves and its effect on pulmonary function.

OBJECT: To examine possible side effects of neurotizations in which the phrenic nerve was used, pulmonary function was analyzed pre- and postoperatively in patients with brachial plexus injury and root avulsions. METHODS: Twenty-three patients with complete brachial plexus palsy underwent neurotization of the musculocutaneous nerve, with the phrenic nerve as donor material. Patients who suffered lung contusions as part of the primary injury were excluded from this study. In 12 patients (five left-sided and seven right-sided neurotizations) pre- and postoperative functional parameters were compared and additional body plethysmography was performed more than 12 months postsurgery. Of the 23, no patient experienced pulmonary problems postoperatively. Nonetheless, pulmonary functional parameters showed a vital capacity in percent of the predicted value of 9.8 +/- 6.3% (mean +/- standard deviation [SD]) in all patients examined, which was a significant reduction (p = 0.0002). In right-sided phrenic nerve transfers this reduction was significant, at 14.3 +/- 3.3% (mean +/- SD), whereas left-sided transfers showed a nonsignificant reduction of 3.6 +/- 3.5% (mean +/- SD). The observed decrease in vital capacity (VC) correlates with the maximal inspiratory pressure (Pi(max)) as an indication of clinical significance. CONCLUSIONS: When the right phrenic nerve is used as a donor in neurotization of the musculocutaneous nerve, the patient incurs a higher risk of reduced pulmonary VC. If possible, the left phrenic nerve should be preferred. The Pi(max) has to be determined preoperatively to avoid any further decrease in the already reduced pulmonary function due to the initial injury.

Barotrauma as a possible cause of aneurysmal subarachnoid hemorrhage. Case report.

The authors report the case of a 47-year-old man who suffered a diving accident. After regaining consciousness he experienced severe headache. He was initially treated for barotrauma, but the persistent headache led to diagnostic imaging that revealed an aneurysmal subarachnoid hemorrhage. To the authors' knowledge, this is the first report of a ruptured brain aneurysm associated with barotrauma.

Invasive drug delivery.

The central nervous system is a very attractive target for new therapeutic strategies since many genes involved in neurological diseases are known and often only local low level gene expression is required. However, as the blood brain barrier on one hand prevents some therapeutic agents given systematically from exerting their activity in the CNS, it also provides an immune privileged environment. Neurosurgical technology meanwhile allows the access of nearly every single centre of the CNS and provides the surgical tool for direct gene delivery via minimal invasive surgical approaches to the brain. Successful therapy of the central nervous system requires new tools for delivery of therapeutics in vitro and in vivo (Fig. 1). The application of therapeutic proteins via pumps into the CSF was shown to be only of limited value since the protein mostly is not sufficiently transported within the tissue and the half life of proteins limits the therapeutic success. Direct gene delivery into the host cell has been a main strategy for years, and in the beginning the direct DNA delivery or encapsulation in liposomes or other artificial encapsulation have been applied with different success. For several years the most promising tools have been vectors based on viruses. Viruses are able to use the host cell machinery for protein synthesis, and some of them are able to stably insert into the host cell genome and provide long term transgene expression as long as the cell is alive. The increasing knowledge of viruses and their live cycle promoted the development of viral vectors that function like a shuttle to the cell, with a single round of infection either integrating or transiently expressing the transgene. Viral vectors have proven to be one of the most efficient and stable transgene shuttle into the cell and have gained increasing importance. The limitations of some viral vectors like the adenoviral vector and adeno-associated viral vector have been improved by new constructs like HIV-1 based lentiviral vectors. The immune response caused by expression of viral proteins, or the inability of some viral vectors like the retroviral vector to infect only dividing cells have been overcome by these new constructs. Lentiviral vectors allow an efficient and stable transgene expression over years in vivo without effecting transgene expression or immune response. In this Chapter we will describe synthetic vectors, give an overview of the most common viral vectors and focus our attention on lentiviral vectors, since we consider them to be the most efficient tool for gene delivery in the CNS.

Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors.

BACKGROUND: Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell-type-specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression. METHODS: Addressing this problem, we have integrated the human cytomegalovirus (CMV) immediate-early enhancer into an 'advanced' generation lentiviral vector. Expression cassettes with the reporter gene green fluorescent protein (GFP), combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under control of a ubiquitous phosphoglycerate kinase (mouse PGK), cardiomyocyte- (human atrial natriuretic factor (ANF), human ventricular myosin light chain (MLC2v)), or type II alveolar epithelial cell (AT-2)-specific human surfactant protein C (SP-C) promoter, were introduced. As insertion of an enhancing element can interfere with the promoter's specificity, expression levels conferred by our enhancer/promoter constructs were evaluated in target and non-target cells. RESULTS: Transduction of target cells with human CMV enhancer containing lentiviral vectors resulted in a multiple-log increase in GFP expression compared to corresponding vectors lacking the human CMV enhancer. In the case of the ANF, the MLC2v, and the SP-C promoters, tissue-specific reporter gene expression in cardiomyocytes and in lung AT-2 cells was maintained, as expression in non-target cells increased only up to 7-fold. CONCLUSIONS: The results of this study indicate that lentiviral vectors with the human CMV enhancer conferring efficient cell-type-specific gene expression may be useful tools for gene therapy purposes or cell tracing, e.g. to analyze stem cell differentiation in transplantation and co-culture settings.

Ex vivo and in vivo gene delivery to the brain.

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.

Matrix-degrading proteases ADAMTS4 and ADAMTS5 (disintegrins and metalloproteinases with thrombospondin motifs 4 and 5) are expressed in human glioblastomas.

Brain tumors, in particular glioblastomas, have a high morbidity and mortality, mainly due to their invasive nature. A prerequisite for this invasiveness is cell migration based on increased expression of proteases digesting the extracellular matrix. Brevican, an important extracellular proteoglycan that is upregulated in glioblastomas, can be degraded by certain proteases. We demonstrate that in human glioblastomas secretory proteases like ADAMTS4 and ADAMTS5 (aggrecanases 1 and 2; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs) are expressed on the mRNA and protein levels in considerable amounts. Real-time RT-PCR shows a higher levels of ADAMTS4 and 5 expressions in glioblastomas in situ, compared to cultured human glioblastoma cells. The upregulation of these proteases in vivo by cytokines may explain this difference. In vitro, transforming growth factor-beta induces ADAMTS4, but less ADAMTS5, and interleukin-1beta ADAMTS5, but not ADAMTS4. As demonstrated by immunohistochemistry and confocal microscopy in situ, ADAMTS5 expression is confined to proliferating glioblastoma cells of surgical tumor sections and with lower intensity to astroglial cells in normal brain sections, as opposed to brevican. In vitro, glioblastoma-derived ADAMTS5 degrades recombinant human brevican to several smaller fragments. Our results show that ADAMTS4 and 5 are upregulated on proliferating glioblastoma cells and these proteases may contribute to their invasive potential.

Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.

This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.