Western Agarose Native GeELution (WANGEL) with beta-amyloid and tau: Novel method to elute proteins or peptides using native agarose gels followed by Lumipulse assay.

Alzheimer´s disease is characterized by hyperphosphorylated tau neurofibrillary tangles and beta-amyloid plaques. Both molecules can be easily measured in human fluids or tissue extracts by immunoassays. However, the different molecular weight species can only be differentiated on Western Blot gels. Analysis of native proteins from polyacrylamide gels is also not well characterized. Hence, we developed a modified method to elute proteins or peptides from native agarose gels. Initially, full-length tau (60 kDa) and beta-amyloid(42) (4 kDa) were separated on a Western Blot gel and eluted from native agarose gels (WANGEL) using an elution system inside a polypropylene tube. The eluates were analyzed with the Lumipulse immunoassay. Both molecules were successfully eluted into 1% agarose gels to the cathode and were detected in the eluate. Additionally, tau was eluted from mouse cortical extracts, but was below the detection limit when eluted from human cerebrospinal fluid. Beta-amyloid(40) was eluted from CSF extracts and detected by Lumipulse. In cortical extracts taken from transgenic mice (APP_SweDI) beta-amyloid(42) was detectable as a native peptide and small oligomeric aggregates. Taken together, our novel WANGEL method enables fast, easy and cheap elution of protein/peptides from polyacrylamide/agarose gels with a subsequent analysis by Lumipulse immunoassay. Three bullet points:•Beta-amyloid and tau are major hallmarks in Alzheimer´s disease and are established cerebrospinal fluid biomarkers.•Lumipulse is a method to measure beta-amyloid and tau in cerebrospinal fluid in the pg/mL range.•Western Blot and our novel combined native agarose method (WANGEL) allows an easy and fast determination of the molecular size in combination with Lumipulse.

Collagen hydrogels loaded with fibroblast growth factor-2 as a bridge to repair brain vessels in organotypic brain slices.

Vessel damage is a general pathological process in many neurodegenerative disorders, as well as spinal cord injury, stroke, or trauma. Biomaterials can present novel tools to repair and regenerate damaged vessels. The aim of the present study is to test collagen hydrogels loaded with different angiogenic factors to study vessel repair in organotypic brain slice cultures. In the experimental set up I, we made a cut on the organotypic brain slice and tested re-growth of laminin + vessels. In the experimental set up II, we cultured two half brain slices with a gap with a collagen hydrogel placed in between to study endothelial cell migration. In the experimental set up I, we showed that the number of vessels crossing the cut was tendencially increased with the addition of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor, or platelet-derived growth factor-BB compared to the control group. In the experimental set up II, we demonstrated that a collagen hydrogel loaded with FGF-2 resulted in a significantly increased number of migrated laminin + cells in the gap between the slices compared to the control hydrogel. Co-administration of several growth factors did not further potentiate the effects. Taken together, we show that organotypic brain slices are good models to study brain vessels and FGF-2 is a potent angiogenic factor for endothelial cell proliferation and migration. Our results provide evidence that the collagen hydrogels can be used as an extracellular matrix for the vascular endothelial cells.

Brain Extracellular Interleukin-6 Levels Decrease Following Antipyretic Therapy with Diclofenac in Patients with Spontaneous Subarachnoid Hemorrhage.

In patients with aneurysmal subarachnoid hemorrhage (aSAH), increased brain extracellular interleukin (IL)-6 levels measured by cerebral microdialysis (CMD) were associated with disease severity, early brain injury, delayed cerebral infarction, and axonal injury. In this study, we analyzed brain extracellular IL-6 levels of aSAH patients following parenteral diclofenac. Twenty-four mechanically ventilated poor-grade aSAH patients were included. Changes in cerebral metabolism, brain/body temperature, and CMD-IL-6 levels following intravenous diclofenac infusion (DCF; 75 mg diluted in 100 cc normal saline) were retrospectively analyzed from prospectively collected bedside data (at 1 hour before DCF = baseline; and at 2, 4, and 8 hours after DCF). Statistical analysis was performed using generalized estimating equations. Seventy-two events in 24 aSAH patients were analyzed. Median age was 60 years (interquartile range [IQR]: 52-67), admission Hunt & Hess grade was 4 (IQR: 3-5), and modified Fisher grade (mFisher) was 4 (IQR: 3-4). Higher CMD-IL-6 levels at baseline were linked to fever, higher mFisher, delayed cerebral infarction, and metabolic distress (p < 0.05). CMD-IL-6 levels at baseline were 281.4 pg/mL (IQR: 47-1866) and significantly (p < 0.001; Wald-X2 = 106) decreased at 2 hours to 86.3 pg/mL (IQR: 7-1946), at 4 hours to 40.9 pg/mL (IQR: 4-1237), and at 8 hours to 53.5 pg/mL (IQR: 5-1085), independent of probe location or day after bleeding. Parenteral diclofenac may attenuate brain extracellular proinflammatory response in poor-grade aSAH patients.

Neuroinflammation is Associated with Brain Extracellular TAU-Protein Release After Spontaneous Subarachnoid Hemorrhage.

INTRODUCTION: Animal data suggest an association between neuroinflammation and secondary brain injury including axonal injury after aneurysmal subarachnoid hemorrhage (aSAH). We sought to study the association between brain extracellular interleukin (IL)-6 and TAU-protein levels as a surrogate marker for neuroinflammation and axonal injury in patients with poor grade aSAH. METHODS: Prospectively collected data from 26 consecutive poor-grade aSAH patients with multimodal neuromonitoring including cerebral microdialysis (CMD) were retrospectively analyzed. IL-6 and TAU-protein levels were analyzed using ELISA from a single CMD-sample every 24 hours and correlated with brain metabolic and hemodynamic parameters. Patients were dichotomized to highgrade (N=10) or low-grade (N=16) neuroinflammation according to their median CMD-IL-6 levels. Data were analyzed using generalized estimating equations to account for multiple within-subject measurements. RESULTS: Perilesional probe location (P=0.02) and aSAH related intracerebral hemorrhage (aICH) volume (P=0.003) at admission were associated with high-grade neuroinflammation. Brain extracellular TAU-protein levels (P=0.001), metabolic distress and delayed cerebral infarction (DCI; P=0.001) were linked to high-grade neuroinflammation. Relative or absolute phosphor-TAU levels were not correlated with CMD-IL-6 levels. High-grade neuroinflammation was a predictor for worse outcome three months after ictus, independently from probe location, initial Hunt&Hess grade and age (P=0.01). CONCLUSION: Neuroinflammation after aSAH is associated with intraparenchymal bleeding, deranged cerebral metabolism and TAU-protein release. The impact of potential anti-inflammatory treatment strategies on secondary brain injury after aSAH has to be investigated in future studies.

Early brain injury after aneurysmal subarachnoid hemorrhage: a multimodal neuromonitoring study.

INTRODUCTION: There is a substantial amount of evidence from animal models that early brain injury (EBI) may play an important role for secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). Cerebral microdialysis (CMD) allows online measurement of brain metabolites, including the pro-inflammatory cytokine interleukin-6 (IL-6) and matrix metalloproteinase-9 (MMP-9), which is indicative for disruption of the blood-brain barrier. METHODS: Twenty-six consecutive poor-grade aSAH patients with multimodal neuromonitoring were analyzed for brain hemodynamic and metabolic changes, including CMD-IL-6 and CMD-MMP-9 levels. Statistical analysis was performed by using a generalized estimating equation with an autoregressive function. RESULTS: The baseline cerebral metabolic profile revealed brain metabolic distress and an excitatory response which improved over the following 5 days (P <0.001). Brain tissue hypoxia (brain tissue oxygen tension of less than 20 mm Hg) was common (more than 60% of patients) in the first 24 hours of neuromonitoring and improved thereafter (P <0.05). Baseline CMD-IL-6 and CMD-MMP-9 levels were elevated in all patients (median = 4,059 pg/mL, interquartile range (IQR) = 1,316 to 12,456 pg/mL and median = 851 pg/mL, IQR = 98 to 25,860 pg/mL) and significantly decreased over days (P <0.05). A higher pro-inflammatory response was associated with the development of delayed cerebral ischemia (P = 0.04), whereas admission disease severity and early brain tissue hypoxia were associated with higher CMD-MMP-9 levels (P <0.03). Brain metabolic distress and increased IL-6 levels were associated with poor functional outcome (modified Rankin Scale of more than 3, P ≤0.01). All models were adjusted for probe location, aneurysm securing procedure, and disease severity as appropriate. CONCLUSIONS: Multimodal neuromonitoring techniques allow insight into pathophysiologic changes in the early phase after aSAH. The results may be used as endpoints for future interventions targeting EBI in poor-grade aSAH patients.

Bioprofiling of platelets in medicated patients with depression.

BACKGROUND: Changes in platelet bioactivity and aggregation are of interest when studying patients with depression as this could help to explain the statistically observed association of depression and chronic somatic, especially cardiovascular disease. This link could potentially be mediated through serotonergic signaling or immunological changes. METHODS: 38 medicated patients with major depressive disorder (MDD) and 30 mentally healthy controls, both without a diagnosis of cardiovascular disease, were included in this naturalistic study. Demographic and psychometric data were obtained. Platelet aggregability was measured by PFA-100 and bioactive compounds and serotonin levels were quantified in platelet sonicate. RESULTS: The comparison of patients with controls revealed no changes in platelet aggregability, but significant differences in platelet content of several bioactive compounds. In a second analysis, patients were grouped according to the receptors and transporters influenced by their medication and again compared to controls. A significant effect of MDD was found for platelet content of serotonin, CD40L, interleukin-1ß, and platelet factor-4, independent of medication. These markers can thus be classified as sensitive to MDD. The effect of medication on platelet parameters was also evaluated. Platelet content of matrix metalloproteinase-2 and ß-thromboglobulin was normalized in MDD patients by medication acting on the serotonin transporter. LIMITATIONS: Owing to the naturalistic study design, patients were on a variety of different medications and combination therapies. This was accounted for by a novel analysis method. CONCLUSION: Platelet serotonin levels and content of immunomodulatory compounds are significantly altered in patients with MDD, even if treatment effects are taken into account.

Nifedipine and nimodipine protect dopaminergic substantia nigra neurons against axotomy-induced cell death in rat vibrosections via modulating inflammatory responses.

Neurodegeneration of cholinergic and dopaminergic neurons is a major hallmark in Alzheimer's or Parkinson's disease, respectively. A dysregulation in calcium homeostasis may be part of this process and counteracting calcium influx may have neuroprotective properties in both diseases. Therefore, we investigated the putative neuroprotective or neurotoxic activity of L-type calcium channel (LTCC) inhibitors on cholinergic and dopaminergic neurons in a rat organotypic vibrosection model. Sagittal or coronal vibrosections (200 µm thick) of postnatal day 10 rats were cultured on 0.4 µm semipermeable membranes for 2 weeks with 10 ng/ml nerve growth factor (NGF) and/or glial-cell line derived neurotrophic factor (GDNF) to maintain survival of cholinergic or dopaminergic neurons, respectively. Thereafter, sections were incubated with 0.1, 1 or 10 µM isradipine, nicardipine or verapamil for 2 weeks to explore cytotoxicity. Alternatively, in order to explore neuroprotective activity, vibrosections were incubated without growth factors but with isradipine or verapamil or with nicardipine, nimodipine or nifedipine from the beginning for 4 weeks. Our data show that all LTCC inhibitors exhibited no neurotoxic effect on cholinergic and dopaminergic neurons. Further, LTCC inhibitors did not have any neuroprotective activity on cholinergic neurons. However, nimodipine and nifedipine significantly enhanced the survival of dopaminergic substantia nigra (SN) but not ventral tegmental area (VTA) neurons, while nicardipine, isradipine and verapamil had no effect. Nifedipine (and more potently GDNF) reduced inflammatory cytokines (macrophage inflammatory protein-2, tumor necrosis factor-α), but did not influence oxidative stress or caspase-3 activity and did not interfere with iron-mediated overload. Our data show that nifedipine and nimodipine are very potent to enhance the survival of axotomized SN neurons, possibly influencing inflammatory processes.

Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective.

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly "neuroactive" compounds, which we termed "enhancement of neuronal differentiation factors" (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.

Ethanol transiently suppresses choline-acetyltransferase in basal nucleus of Meynert slices.

The cholinergic system plays a major role in learning and cognition and cholinergic neurons appear to be particularly vulnerable to ethanol (EtOH) exposure. There are conflicting results if EtOH directly damages cholinergic neurons. Thus, the aims of the present study were (1) to investigate the effect of different EtOH concentrations on cholinergic neurons in organotypic brain slices of the nucleus basalis of Meynert (nbM) and (2) to study if the most potent cholinotrophic substance nerve growth factor (NGF) or inhibitors of mitogen activated kinase (MAPK) p38- and nitric-oxide synthase (NOS)-pathways may counteract any EtOH effect. Two-week old organotypic rat brain slices of the nbM were exposed to 1-100 mM EtOH for 7 days with or without drugs and the number of choline-acetyltransferase (ChAT)-positive neurons was counted. Our data show that EtOH significantly reduced the number of ChAT-positive neurons with the most potent effect at a concentration of 50 mM EtOH (54±5 neurons per slice, p<0.001), compared to control slices (120±13 neurons per slice). Inhibition of MAPK p38 (SB 203580, 10 µM) and NOS (L-thiocitrulline, 10 µM) counteracted the EtOH-induced decline of cholinergic neurons and NGF protected cholinergic neurons against the EtOH-induced effect. Withdrawal of EtOH resulted in a reversal of cholinergic neurons to nearly controls. In conclusion, EtOH caused a transient decline of cholinergic neurons, possibly involving MAPK p38- and NOS-pathways suggesting that EtOH does not induce direct cell death, but causes a transient downregulation of the cholinergic key enzyme, possibly reflecting a form of EtOH-associated plasticity.

Systemic proteasome inhibition triggers neurodegeneration in a transgenic mouse model expressing human α-synuclein under oligodendrocyte promoter: implications for multiple system atrophy.

Multiple system atrophy (MSA) is a progressive late onset neurodegenerative α-synucleinopathy with unclear pathogenesis. Recent genetic and pathological studies support a central role of α-synuclein (αSYN) in MSA pathogenesis. Oligodendroglial cytoplasmic inclusions of fibrillar αSYN and dysfunction of the ubiquitin-proteasome system are suggestive of proteolytic stress in this disorder. To address the possible pathogenic role of oligodendroglial αSYN accumulation and proteolytic failure in MSA we applied systemic proteasome inhibition (PSI) in transgenic mice with oligodendroglial human αSYN expression and determined the presence of MSA-like neurodegeneration in this model as compared to wild-type mice. PSI induced open field motor disability in transgenic αSYN mice but not in wild-type mice. The motor phenotype corresponded to progressive and selective neuronal loss in the striatonigral and olivopontocerebellar systems of PSI-treated transgenic αSYN mice. In contrast no neurodegeneration was detected in PSI-treated wild-type controls. PSI treatment of transgenic αSYN mice was associated with significant ultrastructural alterations including accumulation of fibrillar human αSYN in the cytoplasm of oligodendroglia, which resulted in myelin disruption and demyelination characterized by increased g-ratio. The oligodendroglial and myelin pathology was accompanied by axonal degeneration evidenced by signs of mitochondrial stress and dysfunctional axonal transport in the affected neurites. In summary, we provide new evidence supporting a primary role of proteolytic failure and suggesting a neurodegenerative pathomechanism related to disturbed oligodendroglial/myelin trophic support in the pathogenesis of MSA.

VIP, PACAP-38, BDNF and ADNP in NMDA-induced excitotoxicity in the rat retina.

PURPOSE: To evaluate the effect of intravitreal injection of N-methyl-D-aspartate (NMDA) on brain-derived neurotrophic factor (BDNF), pituitary adenylate cyclase-activating peptide-38 (PACAP-38), vasoactive intestinal peptide (VIP) and the VIP-associated glial protein activity-dependent neuroprotective protein (ADNP) in the rat retina. These elements have well-documented neuroprotective properties and may thus be integrated in endogenous neuroprotective mechanisms in the retina which break down in NMDA excitotoxicity. METHODS: A volume of 2 µl of 100 nmol NMDA was intravitreally injected into one eye of rats, the untreated eye served as a control. Time-dependent effects of NMDA on VIP, PACAP-38 and BDNF were detected by radioimmunoassay and ELISA, and the effect on the expression of VIP, PACAP-38 and ADNP was evaluated by quantitative RT-PCR 20 days after NMDA injection. Topical flunarizine served to find out whether the effect of NMDA is counteracted. RESULTS: Compared to PACAP-38, VIP levels significantly decreased on days 1, 7, 14, 28 and 56 after NMDA injection indicating that VIPergic cells are more vulnerable than PACAP-38-expressing cells. The expression of VIP and ADNP but not of PACAP-38 was found to be reduced, and application of topical flunarizine counteracted the decrease of VIP. BDNF levels significantly increased after days 1 and 3. CONCLUSION: The early upregulation of BDNF seems to act neuroprotectively and leads to a delay of ganglion cell loss. Although there is no direct evidence, the decrease of VIP and ADNP - the consequence of the presence of NMDA receptors on these peptide-expressing cells - might contribute to the breakdown of endogenous neuroprotective mechanisms given that the decrease of the VIP-related ADNP runs in parallel with the decrease of VIP. Activating and maintaining these mechanisms must be the primary aim in the therapy of diseases with retinal neuronal degeneration.

Two Blood Monocytic Biomarkers (CCL15 and p21) Combined with the Mini-Mental State Examination Discriminate Alzheimer's Disease Patients from Healthy Subjects.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder. In AD, monocytes migrate across the blood-brain barrier and differentiate into microglia, are linked to inflammatory responses and display age-dependent decreases in telomere lengths. METHODS: Six monocyte-specific chemokines and the (telomere-associated) tumor suppressor proteins p53 and p21 were determined by multiplex immunoassay in plasma and monocyte extracts of patients with AD or mild cognitive impairment, and levels were compared between patients and controls (without cognitive impairment). RESULTS: CCL15 (macrophage inflammatory protein-1δ), CXCL9 (monokine-induced by interferon-γ) and p21 levels were decreased in monocytes of AD patients compared with controls. CONCLUSION: The combination of monocytic CCL15 and p21 together with the Mini-Mental State Examination enables to differentiate AD patients from controls with high specificity and sensitivity.

Dkk-3 is elevated in CSF and plasma of Alzheimer's disease patients.

Biomarkers in CSF can offer improved diagnostic accuracy for Alzheimer's disease (AD). The present study investigated whether the glycoprotein and putative tumor suppressor Dickkopf homolog 3 (Dkk-3) is secreted into CSF and evaluated its applicability as a diagnostic marker for AD. Using our highly specific immunoenzymometric assay, Dkk-3 levels were measured in plasma and/or CSF of patients suffering from depression, mild cognitive impairment (MCI), or AD and compared with healthy subjects. Dkk-3 identity was verified by western blot and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS. High concentrations of Dkk-3 were detected in CSF compared with plasma (28.2 +/- 1.3 vs. 1.22 +/- 0.04 nmol/L, respectively). Consistently Dkk-3 expression was demonstrated in neurons of the cortex and epithelial cells of the choroid plexus, the major source of CSF. Significantly increased Dkk-3 levels in plasma and CSF were observed for AD patients compared with healthy subjects but not patients suffering from MCI or depression. In summary, our data indicate that elevated Dkk-3 levels are specifically associated with AD and might serve as a potential non-invasive AD biomarker in plasma.

Primary rat monocytes migrate through a BCEC-monolayer and express microglia-markers at the basolateral side.

Monocytes are pluripotent cells of the immune system, circulate in the blood and cross the blood-brain barrier continuously through life. The aim of this study was to explore if primary rat monocytes can adhere and transmigrate at a monolayer of brain capillary endothelial cells (BCEC) and if the monocytes undergo differentiation toward a microglial phenotype at the basolateral side. Monocytes and as a control primary microglia were immunohistochemically stained with markers for CD68 (clone ED-1), CD11b (clone OX-42) or CD11c (clone 8A2). The primary rat monocytes (100,000 cells added) adhered at the BCEC-monolayer (approx. 1200 cells/well) within 30 min and migrated to the basolateral side within 18 h (approx. 40,000 cells/well). The transmigrated monocytes partly differentiated and expressed microglia-markers at the basolateral side. Tumor necrosis factor-alpha as well as conditioned medium derived from BCEC stimulated the differentiation of monocytes in culture. In conclusion, monocytes adhere and migrate through a BCEC-monolayer and express microglia-markers at the basolateral side.

Vascular endothelial growth factor counteracts NMDA-induced cell death of adult cholinergic neurons in rat basal nucleus of Meynert.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in the brain and has recently been shown to possess neuroprotective activity. The aim of the present study was to observe if VEGF can counteract the excitotoxic N-methyl-D-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert in vivo in adult rats. Immunohistochemistry was used to detect vascular structures (RECA-1 or laminin staining) and cholinergic neurons (choline-acetyltransferase). To compare the in vivo VEGF effects also in vitro, VEGF was applied to axotomized cholinergic neurons in organotypic brain slices with or without NMDA. Our data provide evidence that VEGF counteracted cell death in vivo in adult rats but did not protect cholinergic neurons in developing brain slices. Nerve growth factor protected cholinergic neurons in vivo as well as in vitro. In conclusion, VEGF exhibits neuroprotective activity on adult cholinergic neurons of the basal nucleus of Meynert.

Protein delivery of caspase-3 induces cell death in malignant C6 glioma, primary astrocytes and immortalized and primary brain capillary endothelial cells.

Most brain tumors consist of transformed glia cells and are highly vascularized by capillary endothelial cells. The aim of the present study therefore was to deliver pro-apoptotic caspase-3 into malignant C6 glioma and immortalized rBCEC4 brain endothelial cells to induce cell death. Both cell lines were transfected with a reporter protein (beta-galactosidase) using lipid-mediated gene transfer (FuGENE6) or using the novel protein delivery reagent BioPORTER. beta-Galactosidase protein was successfully delivered into both cells, the protein expression peaked around day 2 and was transient. Delivery of caspase-3 induced TUNEL-positive cell death of both cell types. As a control, caspase-3 was also delivered to non-neoplastic primary astrocytes and endothelial cells and induced cell death. In conclusion BioPORTER-protein delivery of pro-apoptotic molecules may provide a potent tool to cause death of the cells in brain tumors, however, this method is limited due to its toxicity to non-malignant cells.

Brain capillary endothelial cells proliferate in response to NGF, express NGF receptors and secrete NGF after inflammation.

Nerve growth factor (NGF) is an important factor regulating survival in development and during regenerative or neuroinflammatory processes. The aim of this study was to investigate whether brain capillary endothelial cells (BCEC) respond to NGF and whether pro-inflammatory substances induce the secretion of NGF in these cells. Cells were incubated with the growth factors NGF or vascular endothelial growth factor or endothelial cell growth factor, and proliferation was observed by incorporation of 5-bromo-2'-deoxy-uridine. NGF-secretion was measured by ELISA and expression of the NGF-receptors trkA and p75(NTR) by Western blot. Proliferation of BCEC was enhanced by exogenous NGF (1-100 ng/ml.). BCEC expressed NGF receptors in vivo (P3, P10, P20, adult) and displayed secretion of endogenous NGF ( approximately 20 pg/ml) into the medium. Treatment of BCEC with the proinflammatory cytokine interleukin-1beta+lipopolysaccharide enhanced expression of p75(NTR) and the secretion of NGF ( approximately 35 pg/ml). The effects of NGF were blocked by anti-NGF antibodies (5 microg/ml). In summary, NGF shows proliferative activity in BCEC, and NGF is secreted after inflammation. Therefore, the NGF pathway can modulate BCEC and may influence blood-brain barrier functions.

Brain capillaries and cholinergic neurons persist in organotypic brain slices in the absence of blood flow.

Angiogenesis plays an important role during development of the brain and under pathological conditions. The aim of the present study was to observe interaction of brain capillaries and cholinergic neurons in organotypic brain slices. Immunohistochemistry was used to visualize brain capillary-like structures (RECA-1 antigen) and cholinergic neurons (choline acetyltransferase). Under normal culture conditions, a very low number of brain capillaries was found in 2- and 4-week-old cortex brain slices. Treatment of slices with acidic medium (pH 6) or hyperthermia (42 degrees C) markedly enhanced the number of brain capillaries. Incubation with 10 ng/mL vascular endothelial growth factor only enhanced angiogenesis in more developed slices. Cholinergic neurons survived in slices of the basal nucleus of Meynert; however, hyperthermia but not acidosis markedly decreased their number. In coslices of the basal nucleus of Meynert and cortex (pretreated with acidic medium), a high number of RECA-1-positive capillaries and cholinergic neurons persisted and displayed strong nerve fibre growth of cholinergic fibres into the cortex. In conclusion, we have demonstrated that RECA-1-positive capillaries and cholinergic neurons can be studied in slice cultures in the absence of blood perfusion, and that this model could provide a system to study mechanisms involved in vascular dementia.

Tumor necrosis factor-alpha triggers cell death of sensitized potassium chloride-stimulated cholinergic neurons.

Cell death of cholinergic neurons of the basal forebrain plays an important role in neurodegenerative disorders, such as Alzheimer's disease. Inflammatory cytokines, such as, for example, tumor necrosis factor-alpha (TNF-alpha), may be involved in these neurodegenerative processes. The aim of this project was to study the role of TNF-alpha in the survival and nerve fiber growth of cholinergic neurons of the basal nucleus of Meynert in organotypic brain slices and in adult rats. Cholinergic neurons were visualized by immunohistochemistry for the enzyme choline acetyltransferase and nerve fibers by histochemistry for the enzyme acetylcholinesterase. When co-slices of basal nucleus of Meynert and neocortex were sensitized for 15 min with 30 mM potassium chloride and subsequently incubated for 1 week with 20 ng/ml TNF-alpha, cholinergic neurons and nerve fibers markedly degenerated. Incubation with different growth factors rescued the loss of cholinergic cell bodies and cholinergic nerve fibers. Injection of 30 mM potassium chloride and 50 ng TNF-alpha into four defined cortical regions of anesthetized adult rats resulted in predominant cell death of cholinergic neurons on the ipsilateral side. In conclusion, our data show that TNF-alpha potentiated cell death of cholinergic neurons possibly via retrograde axonal damage in vitro and in vivo. Cortical overactivation combined with an increased expression of pro-inflammatory cytokines may contribute to the cell death observed in Alzheimer's disease and ageing.

Galanin and galanin receptors in human gliomas.

Galanin-like immunoreactivity (GAL-LI) and specific GAL binding sites have been shown to be widely distributed in the central nervous system (CNS) and in CNS tumors. GAL and its receptors have also been shown to be present in glial cells, but to date it is still unknown whether human gliomas produce GAL and express GAL receptors. In this study 20 brain tumors consisting of 15 glioblastomas, 4 meningiomas and 1 gliosarcoma were investigated for the presence of GAL-LI and GAL receptors. Immunofluorescence analysis revealed a dense network of GAL-LI positive cellular processes and cell bodies in 18 of the 20 tumors. In contrast, in vitro (125)I-labeled GAL receptor autoradiography showed substantial GAL binding in only 6 glioblastoma tissues. Reverse transcription-PCR analysis detected mRNA of all three known galanin receptors in the tumor tissues, with most tumors expressing multiple receptor subtypes. Pharmacological analysis of tumor membrane homogenates with GAL and the specific GAL receptor GalR2 agonist, AR-M1896, revealed that the GAL receptor GalR1 is most likely the receptor responsible for the observed GAL binding in the glioblastomas. No correlation could be found between GAL-LI, the level of GAL binding and proliferative activity as determined by immunostaining with the cell proliferation marker Ki-67.