Potential stem cell therapy and application in neurotrauma.

Traumatic brain injury results from a sudden and external physical insult to the head, which is often accompanied by motor and cognitive impairment. Neurotrauma is characterized not only by focal abnormalities, but rather by multifocal, or even global structural and functional disturbances of the brain network. The impact initially causes necrotic cell death in the underlying tissue, followed by apoptotic cell death in the surrounding tissue due to multiple subsequent events, such as ischemia, excitotoxicity and altered gene expression. These pathological conditions are associated with high morbidity and mortality. Despite the high medical and economical relevance of neurotrauma there are currently no sufficient treatments. Supplementary therapeutic strategies have to be established. Many types of stem cells have the ability to engraft diffusely and become integral members of structures throughout the host CNS. Intrinsic factors appear to derive spontaneously from stem cells and seem to be capable of neuroprotective and/or neuroregenerative functions. Furthermore stem cells can be readily engineered to express specific genes. Such observations suggest that stem cells might participate in reconstructing the molecular and cellular milieu of traumatized brains. In this paper, the state of stem cell research is reviewed and its possible application in neurotrauma will be discussed.

Intrauterine growth restriction reduces nephron number and renal excretory function in newborn piglets.

To examine the effects of intrauterine growth restriction on nephron number, renal circulation, and renal excretory functions in newborns, studies were conducted on 1-day-old anaesthetized piglets, divided into normal weight (n = 6) and intrauterine growth restricted (n = 6) piglets. Renal blood flow was measured by coloured microspheres, glomerular filtration rate was measured by inulin clearance, and osmotic clearance and fractional sodium excretion were calculated. In addition, an estimation of the nephron number was performed by counting representative glomerular numbers in microscopic sections. Newborn intrauterine growth restricted piglets exhibited a reduced glomerular filtration rate and osmotic clearance (P < 0.05), whereas renal blood flow and the filtration fraction as well as fractional sodium excretion were similar in normal weight and intrauterine growth restricted piglets. The nephron number was markedly reduced in intrauterine growth restricted piglets even if the nephron number was related to body weight (P < 0.01). There was a positive correlation between nephron number and glomerular filtration rate (r = 0.69, P < 0.05). Reduced glomerular filtration rate of newborn intrauterine growth restricted piglets is associated with a reduced nephron number. Thus, at birth, compensatory response of renal function due to nephron deficit does not exist in intrauterine growth restricted piglets.

Underediting of glutamate receptor GluR-B mRNA in malignant gliomas.

In mammals, RNA editing by site-selective adenosine deamination regulates key functional properties of neurotransmitter receptors in the central nervous system. Glutamate receptor subunit B is nearly 100% edited at one position (the Q/R-site), which is essential for normal receptor function. Its significance is apparent from mouse models in which a slightly reduced rate of Q/R-site editing is associated with early onset epilepsy and premature death. Here we report that in tissues from malignant human brain tumors, this editing position of glutamate receptor subunit B is substantially underedited compared with control tissues. We also observe alterations in editing and alternative splicing of serotonin receptor 5-HT(2C) transcripts. These changes correlate with a decrease in enzymatic activity of the editing enzyme adenosine deaminase acting on RNA (ADAR) 2, as deduced from analysis of ADAR2 self-editing. Our results suggest a role for RNA editing in tumor progression and may provide a molecular model explaining the occurrence of epileptic seizures in association with malignant gliomas.

Short-term neuropathological aspects of in vivo suicide gene transfer to the F98 rat glioblastoma using liposomal and viral vectors.

To date, only few preclinical protocols on liposomal suicide gene transfer in tumors have been published, none of which directly compared viral to liposomal vectors in terms of immunoreactivity and efficacy. We thus studied the neuropathological alterations in 80 rats being treated for glioblastoma using liposomal and, for comparison, adenoviral and retroviral suicide gene transfer approaches to identify vector-associated efficacy and toxicity for further clinical studies. 62 rats served as controls. F98 tumors were established in Fisher rats and transfected in vivo with the thymidine kinase gene of herpes simplex virus (HSVtk) by a single intratumoral application and an implanted intratumoral continuous delivery system. Three days later ganciclovir was given intraperitoneally for 14 days. The animals were sacrificed 17 days post completed gene transfer. Brains were examined histologically and immunohistochemically using markers for immunocompetent cells. Ten animals showed complete tumor regression; they all belonged to the liposomal and adenoviral groups. In 6 of 10 experimental groups considerable numbers of lymphocytes along the margins of the regression cavities could be observed. Control animals of the liposomal and adenoviral groups showed only little lymphocytic infiltration, underlining the minimal immunogenicity of these carriers. In contrast, the retroviral control group featured a high lymphocyte infiltration. In summary, this study indicates that, in terms of both efficacy and immunoreaction, liposomes are as appropriate as adenoviruses in the treatment of rat glial tumors using suicide gene transfer strategies.

Immunomorphological sequelae of severe brain injury induced by fluid-percussion in juvenile pigs--effects of mild hypothermia.

Severe traumatic brain injury (TBI) often leads to a bad outcome with considerable neurological deficits. Secondary brain injuries due to a rise of intracranial pressure (ICP) and global hypoxia-ischemia are critical and may be reduced in extent by mild hypothermia. A porcine animal model was used to study the effect of severe TBI, induced by fluid percussion (FP; 3.5+/-0.3 atm) in combination with a secondary insult, i.e., temporary blood loss with hypovolemic hypotension. Six-week-old juvenile pigs were subjected to this kind of severe TBI; one group was then submitted to moderate hypothermia at 32 degrees C for 6 h, starting 1 h after brain injury. Animals were killed after 24 h. TBI and hypothermia-associated alterations in the brains were investigated by immunohistochemistry with antibodies against microtubule-associated protein 2 (MAP-2) and beta-amyloid precursor protein (betaAPP). In addition, DNA fragmentation was investigated by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) method. Seven of the 13 normothermic TBI animals developed a secondary increase in ICP (TBI-NT-ICP) after an interval of several hours. None of the animals in the hypothermic trauma (TBI-HT) group exhibited a secondary ICP increase, indicating a protective effect of the treatment. TBI-HT animals showed significantly higher levels of MAP-2 immunoreactivity, lower levels of betaAPP immunoreactivity and less DNA fragmentation than the TBI-NT-ICP animals. Differences between the TBI-HT group and normothermic animals without an ICP increase (TBI-NT) were less marked. A considerable decrease in MAP-2 outside the site of TBI-FP administration was seen only in the TBI-NT-ICP animals. MAP-2 immunohistochemistry was thus a reliable marker of diffuse brain damage. Axonal injury was present in all TBI groups, indicating its special significance in neurotrauma. Thus, severe TBI caused by FP, combined with temporary blood loss, consistently produced traumatic axonal injury and focal brain damage. Mild hypothermia was able to prevent a secondary increase in ICP and its sequelae of diffuse hypoxic-ischemic brain injury. However, hypothermia did not afford protection from traumatic axonal injury.

Matrix gene expression analysis and cellular phenotyping in chordoma reveals focal differentiation pattern of neoplastic cells mimicking nucleus pulposus development.

Chordoma is the fourth most common malignant primary neoplasm of the skeleton and almost the only one showing a real epithelial phenotype. Besides classic chordoma, so-called chondroid chordoma was described as a specific entity showing cartilage-like tissue within chordomatoid structures. However, since its first description, strongly conflicting results have been reported about the existence of chondroid chordoma and several studies suggested chondroid chordomas being in fact low-grade conventional chondrosarcomas. In the present study, we used cytoprotein expression profiling and molecular in situ localization techniques of marker gene products indicative of developmental phenotypes of chondrocytes to elucidate origin and biology of chondroid chordoma. We were able to demonstrate the chondrogenic potential of chordomas irrespectively of the appearance of overt cartilage formation by identifying the multifocal expression of type II collagen, the main marker of chondrocytic differentiation. Additionally, the cartilage-typical large aggregating proteoglycan aggrecan was present throughout all chordomas and, thus, a very characteristic gene product and marker of these neoplasms. Biochemical matrix composition and cell differentiation pattern analysis showed a high resemblance of classic chordomas and in chordoid areas of chondroid chordomas to the fetal chorda dorsalis, whereas chondroid areas of chondroid chordomas showed features similar to adult nucleus pulposus. This demonstrates on the cell function level the chondrocytic differentiation potential of neoplastic chordoid cells as a characteristic facet of chordomas, mimicking fetal vertebral development, ie, the transition of the chorda dorsalis to the nucleus pulposus. Our study firmly establishes a focal real chondrocytic phenotype of neoplastic cells in chordomas. Chondroid chordoma is neither a low-grade chondrosarcoma nor a misnomer as discussed previously.

Serum deprivation and NGF induce and modulate voltage-gated Na(+) currents in human astrocytoma cell lines.

Glial tumor cells derived from primary tissue express large voltage-gated Na(+) currents, whereas glioma cell lines usually lack this feature. We studied the effect of serum deprivation on the expression of Na(+) currents in two astrocytoma cell lines (1321N1 and A172). Serum deprivation for more than 2 days sufficed to induce large Na(+) currents in both cell lines; 300 nM of the specific blocker of voltage-gated Na(+) channels, tetrodotoxin, blocked these currents by about 85%. During serum deprivation, the cells also underwent morphological changes that were characterized by cell rounding and outgrowth of processes. Treatment with 100 ng/ml nerve growth factor (NGF) promoted these morphological changes and also accelerated the development of Na(+) currents. In 1321N1 cells, NGF increased the Na(+) current density after short serum deprivation (3--6 d) and changed several gating properties after longer serum deprivation (9--13 d). In comparison with cells from the early culture stage (3--6 d), the steady-state inactivation of the Na(+) current was shifted by -24 mV in NGF-treated cells from the late (9--13 d) culture stage. In untreated cells, this shift was only -13 mV. NGF accelerated the kinetics of inactivation and shifted the current-voltage relationship in cells from the late culture stage by -14 mV. In A172 cells, most of these effects were present already after short serum deprivation either in presence or absence of NGF. It is concluded that in astrocytoma cells, Na(+) currents are induced by serum deprivation and are modulated by NGF. This result supports the idea that NFG controls Na(+) currents in these cells by autocrine stimulation.

[The role of tumor infiltrating lymphocytes in the immunopathology of gliomas]. / Znaczenie nacieków limfocytarnych w immunopatologii glejaków mózgu.

Immune response was studied to human glioblastoma with an accumulation of lymphocytes at the tumour site. The anti-tumour activity of the tumour infiltrating lymphocytes was confirmed by results from numerous investigations. The role of lymphocytes in gliomas is still widely discussed. Recent studies suggest a potential role of infiltrating lymphocytes as cellular effectors of angiogenesis. In this paper the authors discuss the immune response abnormalities especially with regard to the role of lymphocytes in angiogenesis.

Source localization and possible causes of interictal epileptic activity in tumor-associated epilepsy.

Electrophysiological studies in gliomas have demonstrated action potentials in neoplastic cells. These "spiking tumor cells" are, however, an enigma. In attempt to find evidences for spikes within tumoral borders, 21 patients with different intracerebral tumors were preoperatively screened for the occurrence of epileptogenic discharges using multichannel MEG and EEG. A correlation between histopathology and the distance between dipole and tumor border could be found. Glioma patients showed epileptic activities closer to the border than those with mixed glioneuronal neoplasms and metastases. Four glioma patients demonstrated epileptic activity within the tumor boundary, however, not in the deep center of the tumor. Patch-clamping of cells from acute glioma slices did not yield a correlation between the presence of voltage-gated sodium channels in tumor cells and the MEG/EEG data. Our results demonstrate that the zone with the highest epileptogenic potential is different in gliomas and other brain tumors. However, our data do not strongly suggest that glioma cells are directly involved in the generation of tumor-associated epilepsy in vivo via their capability to generate action potentials.

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Large conductance Ca(2+)-activated K(+) channels in human meningioma cells.

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K(+) outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC(50) = 5.3 mm). Raising the internal Ca(2+) from 10 nm to 2 mm shifted the voltage of half-maximum activation (V(1/2)) of the K(+) current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca(2+)-activated (BK(Ca)) K(+) channel with a conductance of 296 pS (130 mm K(+) at both sides of the patch). V(1/2) of single-channel currents was +6, -12, -46, and -68 mV in the presence of 1, 10, 100, and 1000 microm Ca(2+), respectively, at the internal face of the patch. In cell-attached patches the open probability (P(o)) of BK(Ca) channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K(+) currents with 10 nm Ca(2+) in the pipette. Application of 20 microm cytochalasin D increased P(o) of BK(Ca) channels in cell-attached patches within minutes. These data suggest that the activation of BK(Ca) channels in meningioma cells does not only depend on voltage and internal Ca(2+) but is also controlled by the cytoskeleton.

The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.

The effect of region of interest variations on morphologic operations data and gray-level values extracted from digitized dental radiographs.

OBJECTIVE: To determine the correlations among morphologic operations (MO) values and the correlations among gray-level values for regions of interest (ROIs) placed at various locations on digital images of alveolar bone for 45 patients. STUDY DESIGN: As part of a larger study, up to 7 vertical bite-wing radiographs were taken and digitized for each of 45 patients. Sets of 2 rectangular ROIs were placed on the digitized images of interdental alveolar bone at 4 locations for each patient. The ROIs (1 crestal and 1 apical) were placed between second premolars and first molars in all 4 dental quadrants. Gray-level values were measured, and MO analysis was performed on each ROI. Descriptive statistics were calculated and correlations determined. RESULTS: Paired correlations (such as apical vs crestal, left vs right, maxillary apical vs mandibular apical) of MO values were weak (r = 0.01-0.21), but corresponding correlations for gray-level values were relatively strong (r = 0. 60-0.92). CONCLUSION: MO values varied with ROI location considerably more than did gray-level values. Additionally, ROI size and shape apparently affected MO data. Accurate placement and documentation of ROIs appear to be critical considerations in analyses that use MOs.

May 1999--16 year old male with an unexpected MRI finding.

Four years after resection of a supratentorial pilocytic astrocytoma this 16-year-old boy displayed widespread leptomeningeal seeding. Although the primary tumor lacked contrast enhancement, the multiple metastatic nodules were markedly contrast enhancing. Both the initial and disseminated tumor were consistent with a pilocytic astrocytoma. He was treated with vincristin and carboplatinum and the tumor was stable up to Dec. 1998. Dissemination of low-grade intracranial astrocytoma in children occurs in only 4%. It is not a sign of malignancy. The present case is similar to previously published cases. The prognosis of these patients might be quite favorable when treated with radiotherapy and/or chemotherapy.

PAR 1-type thrombin receptors are involved in thrombin-induced calcium signaling in human meningioma cells.

Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2-terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.

Presence of the proteinase-activated receptor-2 (PAR-2) in human brain tumor cells--trypsin- and SLIGRL-induced calcium response in primary cultured meningiomas.

This study focused on proteinase-activated receptor-2 (PAR-2) in primary cultured human meningioma cells. Stimulation of these cells with the serine proteinase trypsin resulted in a dose-dependent transient calcium response. Since the specific PAR-2 agonist peptide SLIGRL also induced [Ca2+]i mobilization in human meningioma cells and successive application of SLIGRL and trypsin elicited no new calcium signal we conclude that trypsin-induced calcium signaling is mediated by PAR-2 in human meningioma cells. To our knowledge, this is the first report describing functional PAR-2-type receptors in human brain tumor cells.

Neuropathological sequelae of traumatic injury in the brain. An overview.

The identification and interpretation of brain damage resulting from head injury is often not easy. The most obvious structural damage, which is identified post-mortem by neuropathologists, may not be the most reliable alteration with regard to clinico-pathological correlations. For example patients with a fracture of the skull, a severe cerebral contusion or a large intracerebral hematoma that is successfully treated can lead to a complete recovery if no other types of brain damage are present. Thus more subtle forms of pathology, which are often present and some of which can only be identified microscopically, may be more important. It is therefore necessary to get deeper insights into the consequences of brain injury. Though of course not exclusively, this aim can be reached by autopsy. Primary traumatic brain lesions result immediately from mechanical injury. Secondary alterations injuries develop through intracranial and extracranial trauma sequelae, which determine the course and outcome of brain damage. Traumatic brain damage can be classified as focal or diffuse. It may sometimes be difficult to distinguish traumatic from ischemic brain injury. One difference, however, is that the initial events of trauma involve mechanical distortion of the brain. Mechanoporation as traumatic defect in the cell membrane has recently been found to be one of the first steps which leads via ionic influxes to the activation of immediate early genes. Oxygen radicals and cell membrane lipid peroxidation occur also very early. Increased intracellular calcium, activation of phospholipases and calpains furthermore damage the membrane and cytoskeleton and block the axoplasmatic transport, by which delayed cell death can appear. For the description of the extent of traumatically induced brain damage and the possible clinico-pathological correlations it is necessary to take these alterations into consideration as specifically as possible. Neuropathology can contribute to this aim.

Functional GABA(A) receptors on human glioma cells.

Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABA(A) receptors. Currents were measured in whole-cell voltage clamp in response to gamma-aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl- concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABA(A) receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABA(A) receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca2+]i in the majority of the tumour cells due to the activation of Ca2+ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABA(A) receptors parallels unlimited growth typical for malignant tumours and immortal cell lines.

Glutamate receptor activation can trigger electrical activity in human glioma cells.

Cells from major types of gliomas, i.e. oligodendrogliomas and glioblastomas, are able to generate action potentials upon a current injection similar to neurons (Patt et al. (1996) Neuroscience, 71, 601-611; Labrakakis et al. (1997b) J. Neuropath. Exp. Neurol., 56, 243-254. Here, we report that activation of ionotropic glutamate receptors by the selective agonist, kainate, or by glutamate itself, depolarized the tumour cells in culture and living slices from tumour tissue, and can elicit volleys of action potentials, as recorded with the patch-clamp technique. Sixty-six percent of the glioblastoma cells, 44% of the astocytoma and 86% of the oligodendroglioma cells responded to glutamate and the specific agonist of AMPA/kainate receptors, kainate. The involvement of non-NMDA (N-methyl-D-aspartate) receptors is further supported by the observation that both kainate and glutamate currents were blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). The receptor activation was accompanied by an increase in cytosolic Ca2+, as recorded with a fura-2 microfluorometric system. The Ca2+ elevation was mediated by the activation of Ca2+ channels due to membrane depolarization. The presence of voltage-gated Ca2+ channels was confirmed by patch-clamp experiments. Taken together, these findings imply that the electrophysiological properties of glioma cells are more reminiscent of those of neurons than of glial cells.

Upregulation of vascular endothelial growth factor in severe chronic brain hypoxia of the rat.

The vascular endothelial growth factor (VEGF) has been shown to be upregulated in acute hypoxia. Although an increase in blood vessel number has been described in severe chronic brain hypoxia, it is unclear whether VEGF is upregulated in this condition. We therefore investigated male inbred Wistar rats, which were exposed for 9 to 13 weeks to decreasing amounts of oxygen, down to 7% O2 (15%: 15 days; 12%, 10%, respectively; 8%: 1 day, 3 weeks, respectively; 7%: 4 weeks). The expression of VEGF was studied by Northern analysis and in situ hybridization in frozen sections of cerebral cortex, hippocampus and cerebellum in six chronic hypoxic and two control rats. We found a marked upregulation of VEGF mRNA in all brain regions investigated, being strongest in cerebral cortex and cerebellum. Our results suggest a potential role of VEGF for vascular growth and vascular permeability observed in chronic cerebral hypoxia.