Levels of matrix metalloproteinase-9 within cerebrospinal fluid in a rabbit model of coccidioidal meningitis and vasculitis.

Matrix metalloproteinase (MMP)-9 is produced by the central nervous system and inflammatory cells in a variety of inflammatory conditions in both animals and humans. MMP-9 promotes inflammation, breakdown of the blood-brain barrier, and vasculitis. Because vasculitis is seen frequently in patients with coccidioidal meningitis (CM), this study evaluated the presence of MMP-9 within the cerebrospinal fluid (CSF) of rabbits infected intracisternally with Coccidioides immitis arthroconidia. Infected rabbits demonstrated systemic and neurological sequelae to infection, including CSF pleocytosis. Levels of MMP-9 within CSF were assayed by use of zymography and compared with MMP-2 levels, which served as an internal control. Elevated levels of MMP-9 were detectable by day 3, continued to increase through day 10, and declined by day 15 after infection. MMP-9 may contribute to inflammation and vasculitis in this animal model. Future work can focus on evaluation of MMP inhibitors, to gain a better perspective of the role of this MMP in CM.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis

Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. In the context of neuro-inflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system. This review focuses on the role of MMPs in multiple sclerosis (MS) and bacterial meningitis (BM), two neuro-inflammatory diseases where current therapeutic approaches are insufficient to prevent severe disability in the majority of patients. Inhibition of enzymatic activity may prevent MMP-mediated neuronal damage due to an overactive or deviated immune response in both diseases. Downregulation of MMP release may be the molecular basis for the beneficial effect of IFN-beta and steroids in MS. Instead, synthetic MMP inhibitors offer the possibility to shut off enzymatic activity of already activated MMPs. In animal models of MS and BM, they efficiently attenuated clinical disease symptoms and prevented brain damage due to excessive metalloproteinase activity. However, the required target profile for the therapeutic use of this novel group of compounds in human disease is not yet sufficiently defined and may be different depending on the type and stage of disease. Currently available MMP inhibitors show little target-specificity within the MMP family and may lead to side-effects due to interference with physiological functions of MMPs. Results from human MS and BM indicate that only a restricted number of MMPs specific for each disease is up-regulated. MMP inhibitors with selective target profiles offer the possibility of a more efficient therapy of MS and BM and may enter clinical trials in the near future.

Pathogenesis of bacterial meningitis.

Bacterial meningitis is fatal in 5% to 40% of patients and causes neurologic sequelae in up to 30% of survivors. Much has been learned recently about the mechanisms that lead to brain injury during meningitis. Once bacteria have gained access to the central nervous system, their multiplication triggers a complex host response consisting of humoral and cellular immune mediators, reactive oxygen intermediates, matrix-metalloproteinases, and other host-derived factors. Alterations of the cerebral vasculature, with disruption of the blood brain barrier and global and focal ischemia, ultimately lead to functional and structural brain damage. This article reviews current concepts of the pathophysiology of bacterial meningitis and emphasizes possible therapeutic strategies to prevent its harmful consequences.

Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis.

BackgroundApproximately 7% of survivors from meningococcal meningitis (MM) suffer from neurological sequelae due to brain damage in the course of meningitis. The present study focuses on the role of matrix metalloproteinases (MMPs) in a novel mouse model of MM-induced brain damage.MethodsThe model is based on intracisternal infection of BALB/c mice with a serogroup C Neisseria meningitidis strain. Mice were infected with meningococci and randomised for treatment with the MMP inhibitor batimastat (BB-94) or vehicle. Animal survival, brain injury and host-response biomarkers were assessed 48 h after meningococcal challenge.ResultsMice that received BB-94 presented significantly diminished MMP-9 levels (p¿<¿0.01), intracerebral bleeding (p¿<¿0.01), and blood-brain barrier (BBB) breakdown (p¿<¿0.05) in comparison with untreated animals. In mice suffering from MM, the amount of MMP-9 measured by zymography significantly correlated with both intracerebral haemorrhage (p¿<¿0.01) and BBB disruption (p¿<¿0.05).ConclusionsMMPs significantly contribute to brain damage associated with experimental MM. Inhibition of MMPs reduces intracranial complications in mice suffering from MM, representing a potential adjuvant strategy in MM post-infection sequelae.

Can in-vitro chemoresponse assays help find new treatment regimens for malignant gliomas?

Various in-vitro chemosensitivity and resistance assays (CSRAs) have been demonstrated to be helpful decision aids for non-neurological tumors. Here, we evaluated the performance characteristics of two CSRAs for glioblastoma (GB) cells. The chemoresponse of fresh GB cells from 30 patients was studied in vitro using the ATP tumor chemoresponse assay and the chemotherapy resistance assay (CTR-Test). Both assay platforms provided comparable results. Of seven different chemotherapeutic drugs and drug combinations tested in vitro, treosulfan plus cytarabine (TARA) was the most effective, followed by nimustine (ACNU) plus teniposide (VM26) and temozolomide (TMZ). Whereas ACNU/VM26 and TMZ have proven their clinical value for malignant gliomas in large randomized studies, TARA has not been successful in newly diagnosed gliomas. This seeming discrepancy between in vitro and clinical result might be explained by the pharmacological behavior of treosulfan. Our results show reasonable agreement between two cell-based CSRAs. They appear to confirm the clinical effectiveness of drugs used in GB treatment as long as pharmacological preconditions such as overcoming the blood-brain barrier are properly considered.

Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders1, 2. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin3, 4 and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure5 and neurocognitive impairment6. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

Investigation of leptomeningeal enhancement in MS: A postcontrast FLAIR MRI study

OBJECTIVE To investigate possible leptomeningeal contrast enhancement using postcontrast fluid-attenuated inversion recovery (FLAIR) MRI as an additional marker of inflammation in patients with multiple sclerosis (MS). METHODS A cohort of 112 patients (73 women) with clinically definitive MS or a clinically isolated syndrome suggestive of CNS demyelination were included. A pathologic control group of 5 stroke patients was also examined. MRI was performed on a 3T system including FLAIR, T2-weighted, T1-weighted-contrast injection, followed by T1-weighted and FLAIR. RESULTS Of the 112 patients, 39 had an acute relapse at the time of MRI. In total, 96 contrast-enhancing lesions were identified on postcontrast T1-weighted images. The pathologic control group demonstrated the sensitivity of postcontrast FLAIR images demonstrating leptomeningeal enhancement in all cases. In contrast, only 1 out of 112 examined patients with MS showed a single area of abnormal leptomeningeal contrast enhancement. CONCLUSIONS In contrast to intraparenchymal blood-brain barrier (BBB) dysfunction that is frequently seen in patients with MS, BBB dysfunction of leptomeningeal vessels is usually not detectable in patients with early MS.

PSGL-1 and E/P-selectins are essential for T-cell rolling in inflamed CNS microvessels but dispensable for initiation of EAE

T-cell migration across the blood-brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P-selectin glycoprotein ligand-1 (PSGL-1) and its endothelial ligands E- and P-selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL-1 or E/P-selectins does not result in ameliorated EAE, we speculated that T-cell entry into the spinal cord is independent of PSGL-1 and E/P-selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL-1(-/-) proteolipid protein-specific T cells in inflamed spinal cord microvessels of WT or E/P-selectin(-/-) SJL/J mice during EAE. T-cell rolling but not T-cell capture was completely abrogated in the absence of either PSGL-1 or E- and P-selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T-cell invasion into the CNS parenchyma. Thus, PSGL-1 interaction with E/P-selectin is essential for T-cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T-cell rolling is not required for successful T-cell entry into the CNS and initiation of EAE.

Mutations in SLC1A4, encoding the brain serine transporter, are associated with developmental delay, microcephaly and hypomyelination

BACKGROUND L-serine plays an essential role in neuronal development and function. Although a non-essential amino acid, L-serine must be synthesised within the brain because of its poor permeability by the blood-brain barrier. Within the brain, its synthesis is confined to astrocytes, and its shuttle to neuronal cells is performed by a dedicated neutral amino acid transporter, ASCT1. METHODS AND RESULTS Using exome analysis we identified the recessive mutations, p.E256K, p.L315fs, and p.R457W, in SLC1A4, the gene encoding ASCT1, in patients with developmental delay, microcephaly and hypomyelination; seizure disorder was variably present. When expressed in a heterologous system, the mutations did not affect the protein level at the plasma membrane but abolished or markedly reduced L-serine transport for p.R457W and p.E256K mutations, respectively. Interestingly, p.E256K mutation displayed a lower L-serine and alanine affinity but the same substrate selectivity as wild-type ASCT1. CONCLUSIONS The clinical phenotype of ASCT1 deficiency is reminiscent of defects in L-serine biosynthesis. The data underscore that ASCT1 is essential in brain serine transport. The SLC1A4 p.E256K mutation has a carrier frequency of 0.7% in the Ashkenazi-Jewish population and should be added to the carrier screening panel in this community.

Morphological Brain Changes after Climbing to Extreme Altitudes-A Prospective Cohort Study.

BACKGROUND Findings of cerebral cortical atrophy, white matter lesions and microhemorrhages have been reported in high-altitude climbers. The aim of this study was to evaluate structural cerebral changes in a large cohort of climbers after an ascent to extreme altitudes and to correlate these findings with the severity of hypoxia and neurological signs during the climb. METHODS Magnetic resonance imaging (MRI) studies were performed in 38 mountaineers before and after participating in a high altitude (7126m) climbing expedition. The imaging studies were assessed for occurrence of new WM hyperintensities and microhemorrhages. Changes of partial volume estimates of cerebrospinal fluid, grey matter, and white matter were evaluated by voxel-based morphometry. Arterial oxygen saturation and acute mountain sickness scores were recorded daily during the climb. RESULTS On post-expedition imaging no new white matter hyperintensities were observed. Compared to baseline testing, we observed a significant cerebrospinal fluid fraction increase (0.34% [95% CI 0.10-0.58], p = 0.006) and a white matter fraction reduction (-0.18% [95% CI -0.32--0.04], p = 0.012), whereas the grey matter fraction remained stable (0.16% [95% CI -0.46-0.13], p = 0.278). Post-expedition imaging revealed new microhemorrhages in 3 of 15 climbers reaching an altitude of over 7000m. Affected climbers had significantly lower oxygen saturation values but not higher acute mountain sickness scores than climbers without microhemorrhages. CONCLUSIONS A single sojourn to extreme altitudes is not associated with development of focal white matter hyperintensities and grey matter atrophy but leads to a decrease in brain white matter fraction. Microhemorrhages indicative of substantial blood-brain barrier disruption occur in a significant number of climbers attaining extreme altitudes.

Successful transnasal delivery of stem cells in a rat model of perinatal hypoxic-ischemic brain injury

OBJECTIVE: New routes for cell transplantation into the brain need to be explored as intracerebral or intrathecal applications have a high risk to cause damage to the central nervous system. It has been hypothesized that transnasally administrated cells bypass the blood-brain barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and neural progenitor cells (NPC) to perinatal rats in a model of hypoxic-ischemic brain injury. STUDY DESIGN: Four-day-old Wistar rat pups, previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, either received WJ-MSC or green fluorescent protein-expressing NPC: The heads of the rat pups were immobilized and 3 ml drops containing the cells (50’000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600’000 cells. Animals were sacrificed 24h, 48h or 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin and sectioned. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSION: Our data show that transnasal delivery of WJ-MSC and NPC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials.

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger.

Brain barriers: Crosstalk between complex tight junctions and adherens junctions

Unique intercellular junctional complexes between the central nervous system (CNS) microvascular endothelial cells and the choroid plexus epithelial cells form the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB), respectively. These barriers inhibit paracellular diffusion, thereby protecting the CNS from fluctuations in the blood. Studies of brain barrier integrity during development, normal physiology, and disease have focused on BBB and BCSFB tight junctions but not the corresponding endothelial and epithelial adherens junctions. The crosstalk between adherens junctions and tight junctions in maintaining barrier integrity is an understudied area that may represent a promising target for influencing brain barrier function.