Inflammation by activated macrophage-like THP-1 cells increases human dura mater cell adhesion with alteration of integrin α2 ß1 and matrix metalloproteinase.

This study was designed to investigate (i) extracellular matrix to specify adhesive substrates to human dura mater cell (hDMC); (ii) the alteration on adhesion-related molecules in hDMC; and (iii) secreted matrix metalloproteinases (MMPs) linked with extracellular matrix remodeling after exposure to inflammation. The hDMC was cultured from human dura mater tissue, and the studies were performed with hDMC after co-culturing with macrophage like THP-1 cells (Mϕ). The adhesion of co-cultured hDMC through collagen I increased 6.4-fold and through collagen IV increased 5.0-fold compared with the adhesion of naïve cells (p < 0.001). Integrin subtype α2 ß1 expression was increased 6.3-fold (p < 0.001) and α1 expression was decreased 2.0-fold (p < 0.001) in the co-cultured cells compared with the naïve cells. Co-culturing induced significant increases in MMP-1 (13.9-fold, p < 0.01), MMP-3 (7.6-fold, p < 0.01), and VEGF (VEGF: 3.8-fold, p < 0.05) expression and decreases in MMP-9 (0.1-fold, p < 0.01) compared with the sum of naïve hDMC and Mϕ values. Increased hDMC adhesion under inflammatory conditions is caused by an increased cellular affinity for collagen I and IV mediated by increased hDMC levels of integrin subtype α2 ß1 and environmental MMP-1, -3 and decreased MMP-9. Selective integrin subtype α2 ß1 inhibition assay showed 37.8% and 35.7% reduction in adhesion of co-cultured hDMC to collagen I (p < 0.001) and IV (p = 0.057), respectively. The present study provides insight into the pathological conditions related to dura mater adhesion in inflammation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-11, 2019.

Valproate Plays a Protective Role against Migraine by Inhibiting Protein Kinase C Signalling in Nitroglycerin-treated Mice.

Migraine is a common disease with a high morbidity. Valproate (VP) is used as an anti-epilepsy drug in clinic. This study aimed to investigate the role of VP in nitroglycerin (NTG)-induced migraine using a mouse model. NTG was employed by intraperitoneal injection to induce a migraine model in mice. The NTG administration caused mouse head discomforts, decreased tolerance to cold or hot stimulation and increased content of nitric oxide, calcitonin gene-related peptide and neuropeptide Y in serum, which were ameliorated by intraperitoneal injection of VP. The levels of two inflammatory factors, interleukin (IL)-1ß and inducible nitric oxide synthase, in dura mater were increased by NTG treatment, while the increase was attenuated by application of VP. In addition, the phosphorylation levels of protein kinase C (PKC) α, γ, δ and ε were increased by NTG and decreased by VP. However, their total expression at the transcriptional and translational levels did not change significantly. Two substrates of PKC, cAMP-response element binding protein 1 and signal transducer and activator of transcription 1 were also phosphorylated by NTG application, and the phosphorylation level was attenuated by VP, consistent with the change of PKC informs. Together, we demonstrated that VP prevented damage due to migraine by inhibiting PKC signalling in NTG-injected mice, which may provide a basis for investigating the clinical treatment of migraine.

Activation of extracellular signal-regulated kinase in the trigeminal ganglion following both treatment of the dura mater with capsaicin and cortical spreading depression.

Extracellular signal-regulated kinase (ERK) is known to be phosphorylated after exposure to noxious stimuli. In this study, we investigated the response in the dura mater to nociceptive stimulation, which is thought to be responsible for the pathogenesis of headaches, including migraines. We also examined the level of ERK phosphorylation in the trigeminal ganglion following cortical spreading depression (CSD), which is thought to play an important role in migraine pathophysiology. Western blot and immunohistochemical analyses showed a significant increase in the ERK phosphorylation levels 3 min following an application of 10mM capsaicin to the dura mater. This increase was inhibited after an application of the TRPV1 antagonist capsazepine or a MEK inhibitor. An immunohistochemical analysis revealed that most of the small-sized trigeminal ganglion neurons with TRPV1-immunoreactivity that innervate the dura mater exhibited pERK-immunoreactivity, suggesting that these neurons had responded to nociceptive stimulation. CSD increased the level of ERK phosphorylation 30 min after its elicitation, and this response was inhibited by a prior intraventricular administration of TRPV1 antagonist. These results indicate that CSD can activate dural TRPV1 to send nociceptive signals to the trigeminal system, and they provide important clues regarding the relationship between CSD and the trigeminovascular system.

Possible role of cyclooxygenase-2 in developing chronic subdural hematoma.

Inflammatory cytokines are reportedly involved in the pathogenesis of chronic subdural hematomas (CSH), and the angiogenesis of hematomas has particularly been in focus. Cyclooxygenase-2 (COX-2) is an essential enzyme for the synthesis of prostaglandin E2 (PGE2). The COX-2-PGE2 pathway has been shown to influence angiogenic factors such as vascular endothelial growth factor (VEGF). We investigated the association of COX-2 expression in the dura mater and outer membrane with the pathogenesis of CSH, and suggested a treatment strategy on the basis of this association. Hematoma fluid and serum samples obtained from 37 patients, and samples of the dura mater and outer CSH membrane obtained from 13 patients during the operation were examined in this study. The concentrations of PGE2 in relation to COX-2 in the hematoma fluid were significantly higher than those in the serum. Immunohistochemical analyses revealed COX-2-positive cells in the outer membrane of CSHs. There was a linear and significant relationship between PGE2 concentration in hematoma fluid and the interval from trauma to initial surgery. COX-2 may play a crucial role during the development of CSHs. Our study might lead to the development of anti-COX-2 treatment options that aim to minimize repeat surgery and choose medical therapy by reducing CSH morbidity and recurrence rate in patients with CSH.

Increased expression of endothelial and neuronal nitric oxide synthase in dura and pia mater after air stress.

Stress is the leading precipitating factor for migraine attacks but the underlying mechanism is currently unknown. Nitric oxide (NO) has been implicated in migraine pathogenesis based on the ability of NO donors to induce migraine attacks. In the present study, we investigated in Wistar rats the effect of air stress on nitric oxide synthase (NOS) mRNA and protein expression in dura and pia mater using real-time polymerase chain reaction and Western blotting, respectively. Endothelial (e)NOS protein expression was significantly increased in dura and pia mater after air stress. Significantly augmented neuronal (n)NOS protein expression was detected in pia mater after air stress but not in dura mater. Inducible NOS mRNA and protein expression levels in dura and pia mater were unaffected by stress. The increased expression of eNOS in dura mater and eNOS and nNOS in pia mater seen after stress could not be antagonized by treatment with the migraine drug sumatriptan. These findings point towards the involvement of increased NO concentrations in dura and pia mater in response to air stress. However, the role of these findings in relation to migraine pathophysiology remains unclear.

Kynurenine aminotransferase in the supratentorial dura mater of the rat: effect of stimulation of the trigeminal ganglion.

Electrical stimulation of the trigeminal ganglion has been widely used as a model of nociception, characterizing migraine. This treatment is known to evoke release of neuropeptides and neurotransmitters from nerve fibers of the dura mater. On the basis of immunocytochemical investigations, we found that under normal conditions, surface membranes of Schwann cells surrounding nerve fibers in the supratentorial dura mater display kynurenine aminotransferase-immunoreaction (KAT-IR); also KAT-IR are the granules of mast cells and the cytoplasms of macrophages (histiocytes). In consequence of stimulation of the trigeminal ganglion, Schwann cells in the dura mater became conspicuously swollen while their KAT-IR decreased considerably; also KAT-IR of mast cells and macrophages decreased significantly. At the same time, nitric oxide synthase (NOS)-IR of nerve fibers in the dura mater increased, suggesting release of nitric oxide (NO), this is known to be involved in NMDA receptor activation leading to vasodilation followed by neurogenic inflammation. Because kynurenic acid (KYNA) is an antagonist of NMDA receptors, we hypothesize that KYNA and its synthesizing enzyme, KAT, may play a role in the prevention of migraine attacks.

In vivo glioma growth requires host-derived matrix metalloproteinase 2 for maintenance of angioarchitecture.

Glioma, the most common form of brain tumor, has been shown mostly by in vitro studies to utilize matrix metalloproteinase (MMP) for invasive growth through degradation of the extracellular matrix. In order to examine the in vivo role of MMP, we established a rodent model of glioma progression using C6 rat glioma cells and analyzed the effect of tissue inhibitors of metalloproteinases (TIMPs). TIMP-2 rather than TIMP-1 caused significant reduction of the tumor size accompanied by the presence of degenerated blood vessels and ischemic necrosis. Because TIMP-2 inhibits MMP-2 preferentially, we then examined glioma growth in MMP-2-deficient mice and observed essentially identical consequences. While MMP-2 activity was present in the tumor and adjacent tissues of the wild-type mice, no MMP-2 activity was detected even in the tumor of the null mice, although C6 cells are known to express MMP-2. These observations suggest that glioma induces MMP-2 and utilizes its activity in the host tissue to support angiogenesis and to maintain angioarchitecture.

Increase in meningeal blood flow by nitric oxide--interaction with calcitonin gene-related peptide receptor and prostaglandin synthesis inhibition.

This study addresses possible interactions of the vasodilators nitric oxide (NO), calcitonin gene-related peptide (CGRP) and prostaglandins, which may be implicated in the generation of vascular headaches. Local application of the NO donator diethylamine-NONOate (NONOate) to the exposed dura mater encephali of the rat caused dose-dependent increases in meningeal blood flow recorded by laser Doppler flowmetry. Pre-application of the CGRP receptor antagonist CGRP8-37 significantly attenuated the evoked blood flow increases, while the cyclooxygenase inhibitors acetylsalicylic acid and metamizol were only marginally effective. Stimulation of rat dura mater with NONOate in vitro caused increases in CGRP release. NADPH-diaphorase activity indicating NO production was restricted to the endothelium of dural arterial vessels. We conclude that increases in meningeal blood flow caused by NO depend partly on the release and vasodilatory action of CGRP from dural afferents, while prostaglandins are not significantly involved.

Nuclear factor-kappaB as a molecular target for migraine therapy.

Nitric oxide (NO) generated from inducible NO synthase (iNOS) participates in immune and inflammatory responses in many tissues. The NO donor glyceryl trinitrate (GTN) provokes delayed migraine attacks when infused into migraineurs and also causes iNOS expression and delayed inflammation within rodent dura mater. Sodium nitroprusside, an NO donor as well, also increases iNOS expression. Because inflammation and iNOS are potential therapeutic targets, we examined transcriptional regulation of iNOS following GTN infusion and the consequences of its inhibition within dura mater. We show that intravenous GTN increases NO production within macrophages. L-N(6)-(1-iminoethyl)lysine, a selective iNOS inhibitor, attenuates the NO signal, emphasizing the importance of enzymatic activity to delayed NO production. iNOS expression is preceded by significant nuclear factor kappa B (NF-kappaB) activity, as reflected by a reduction in the inhibitory protein-kappa-Balpha (IkappaBalpha) and activation of NF-kappaB after GTN infusion. IkappaBalpha degradation, NF-kappaB activation, and iNOS expression were attenuated by parthenolide (3mg/kg), the active constituent of feverfew, an anti-inflammatory drug used for migraine treatment. These findings suggest that GTN promotes NF-kappaB activity and inflammation with a time course consistent with migraine attacks in susceptible individuals. We conclude, based on results with this animal model, that blockade of NF-kappaB activity provides a novel transcriptional target for the development of anti-migraine drugs.

Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater.

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.

Increase of collagen synthesis and deposition in the arachnoid and the dura following subarachnoid hemorrhage in the rat.

Arachnoidal fibrosis following subarachnoid hemorrhage (SAH) has been suggested to play a pathogenic role in the development of late post-hemorrhagic hydrocephalus in humans. The purpose of this study was to investigate the rate of collagen synthesis in the arachnoid and the dura in the rat under normal conditions and to study the time schedule and the localization of the increased collagen synthesis following an experimental SAH. We found that the activity of prolyl 4-hydroxylase, a key enzyme in collagen synthesis, was 3-fold higher in the dura than that in the arachnoid and was similar to the activity in the skin. We then induced SAH in rats by injecting autologous arterial blood into cisterna magna. After SAH, we observed an increase in prolyl 4-hydroxylase activity of the arachnoid and the dura at 1 week. At this time point the enzyme activity in both tissues was 1.7-1.8-fold compared to that in the controls and after this time point the activities declined but remained slightly elevated at least till week 4. The rate of collagen synthesis was measured in vitro by labeling the tissues with [(3)H]proline. The rate increased to be 1.7-fold at 1 to 2 weeks after the SAH in both of the tissues. Immunohistochemically we observed a deposition of type I collagen in the meninges at 3 weeks after the SAH. SAH is followed by a transient increase in the rate of collagen synthesis in the arachnoid and, surprisingly, also the dura. Increased synthesis also resulted in an accumulation of type I collagen in the meningeal tissue, suggesting that the meninges are a potential site for fibrosis. The time schedule of these biochemical and histological events suggest that meningeal fibrosis may be involved in the pathogenesis of late post-hemorrhagic hydrocephalus.

Distribution and induction of cytochrome P450 1A1 and 1A2 in rat brain.

Cytochromes P450 1A1 and 1A2 are involved in the oxidation of a wide spectrum of endogenous compounds and xenobiotics. Although their presence has been repeatedly confirmed in brain tissue, reports regarding their distribution in the brain are often contradictory. In the present study the possibility was examined that CYP1A1 and CYP1A2 are localized and inducible in the brain-CSF barrier and regions with a leaky blood brain barrier, where they may serve as a protective metabolic barrier. CYP1A1 and CYP1A2 levels were determined in subcellular fractions of multiple brain regions, as well as tissue homogenates of circumventricular organs, and the meninges by Western blotting and catalytic activity in control male rats and rats treated with the inducer beta-naphthoflavone (BNF). In control animals CYP1A1 immunoreactive protein was undetectable in regional brain microsomes or whole tissue homogenates of the arachnoid, dura mater, choroid plexus, pineal gland, median eminence, and pituitary. However, low levels of ethoxyresorufin O-deethylase (EROD) activity were observed in homogenates of the arachnoid, dura mater, choroid plexus, pineal gland, and pituitary. Western blotting revealed only low levels of CYP1A2 immunoreactive protein in brain microsomes from the cortex, cerebellum, brainstem, thalamus, hippocampus, and striatum from control animals. Following BNF treatment, EROD activity was induced 12-42-fold in the arachnoid, choroid plexus, dura mater, pineal gland, pituitary, and median eminence. Western blot analysis revealed CYP1A1 to be induced in the arachnoid, dura mater, choroid plexus, pineal gland, and pituitary, while CYP1A2 was undetectable. No induction of CYP1A1 or CYP1A2 protein was observed in brain microsomes from the olfactory bulb, cortex, striatum, hippocampus, cerebellum, or brainstem following BNF treatment, providing that the arachnoid membranes and choroid plexus had been carefully removed prior to brain dissection. Neither CYP1A1, 1A2 protein, nor EROD activity were detected in purified brain mitochondria, regardless of treatment or region. In conclusion, catalytically active CYP1A1 is located in the meninges as well as certain circumventricular organs, is inducible by BNF, and appears to be absent or expressed constitutively at very low levels in the majority of the brain parenchyma. The localization of CYP1A1 in the blood-CSF barrier and circumventricular tissues likely plays a role in protecting the brain from xenobiotics.

Immunohistochemical localization of endothelial nitric oxide synthase in vessels of the dura mater of the Sprague-Dawley rat.

Nitric oxide (NO) and the dura mater are implicated in the pathogenesis of vascular headache. Many studies have demonstrated the participation of NO in headache; however, few studies have identified NO in the dura mater. In this study, nine Sprague-Dawley rats were examined with immunohistochemistry using two different endothelial nitric oxide synthase (eNOS) monoclonal antibodies, H32 and ECNOS. eNOS was successfully localized to the endothelium of the middle meningeal artery. To the best of our knowledge, this is the first study to report NOS immunopositive endothelial cells in the blood vessels of the rat dura mater. The authors propose that NO plays an active role in dural vasodilation, contributing to the pathogenesis of vascular headache; in the future, NO inhibitors could serve as pharmacological agents to treat vascular headache.

Nitric oxide synthase immunoreactivity in the rat dura mater.

The dura mater has been implicated as a tissue where vascular headache develops. Identification of the neural components of this tissue is a prerequisite for understanding the mechanisms of this pathological process. The nitric oxide molecule, a potent vasodilator, may contribute to the vascular headache process by dilating dural vasculature. Our immunohistochemical study using nitric oxide synthase (NOS) antibodies revealed NOS-positive nerve fibers and a prominent mast cell population in the rat dura. A majority of the immunopositive fibers were associated with the anterior meningeal artery and its branches and sparse innervation with the middle meningeal artery, its branches, and superior sagittal sinus. We propose that the NOS-positive nerve fibers and mast cells be considered as possible participants in the pathogenesis of vascular headache.

Serotonin (5-HT) fibers of the rat dura mater: 5-HT-positive, but not authentic serotoninergic, tryptophan hydroxylase-like fibers.

Serotonin (5-HT)-positive, but not tryptophan-5-hydroxylase (TPOH)-positive, authentic serotoninergic fibers were shown in the rat dura mater. 5-HT immunoreactive fibers in the dura are postulated to result from 5-HT uptake from circulating blood elements (e.g. platelets, mast cells) by perivascular sympathetic nerve fibers. A robust TPOH-immunoreactive mast cell population was identified in the dura; this result confirms the TPOH antibody specificity to cells known to synthesize 5-HT. While these results indicate that there are no authentic serotoninergic fibers in the dura mater, the mast cells, platelets and cerebrospinal fluid can serve as a source of 5-HT activating 5-HT receptors known to be present in this tissue.

[Local differences in the tissue basophils of the cerebral dura mater in the rat]. / Lokal'nye razlichiia tkanevykh bazofilov tverdoi obolochki mozga krysy.

Tissue basophils (TB) were investigated in 5 (occipital, right and left frontal and parietal) areas of the dura mater in 75 albino rats by histological and histochemical methods. Local distinctions in the structure, distribution and functional activity of cells were found. The greatest concentration of TB revealed by staining with methylene blue, cytochrome oxidation reaction and with glyoxylic acid was found in the frontal areas and the lowest concentration in the occipital areas of the mater. The amount of cells with activity of succinic dehydrogenase in the occipital area was, on the contrary, 11-13 times greater than their amount in the frontal and parietal areas of the mater. The local distinctions of TB marked with ATPase are not so considerable and those of alkaline phosphatase were not significant. The frontal areas (right and left) were found to have the greatest concentration of the degranulated forms of TB as well.

Distribution of activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase in the cranial dura mater-arachnoid interface zone of the rat.

The distribution of the activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase was studied in the encephalic dura mater-arachnoid borderline (interface) zone of albino Wistar rats. Intense clustering of electron-dense granules that indicated alkaline phosphatase activity was observed in the inner dural cells, the neurothelial cells, the outermost row of the outer arachnoidal cells and in the intercellular cleft between the latter two (the so-called electron-dense band). The remainder of the outer arachnoidal cells contained almost no reaction product. Mg-adenosine triphosphatase activity was distributed differently; a lack of reaction product was observed not only in the outer arachnoidal cells, but also in the zone occupied by the electron-dense band. The data confirm histochemically the barrier properties of the dura mater-arachnoid interface zone.

[The demonstration of the vessels of the microcirculatory bed of the dura mater by using ATPase]. / Vyiavlenie sosudov mikrotsirkuliatornogo rusla tverdoi mozgovoi obolochki pri pomoshchi ATFazy.

In order to reveal the microcirculatory bed vessels, a histochemical method is suggested, lead technique for determination of ATFase localization being taken as its base. Owing to this method, it is possible to reveal constantly and rather fully microvessels long time after death and differentiate various links of the microcirculatory bed in the connective tissue membranes.

Collagen biosynthesis in healing wounds of the spinal cord and surrounding membranes.

Kinetics of collagen synthesis and deposition were studied in the canine spinal cord, pia mater, and dura mater and in wounds of these tissues over the first 8 weeks. Little collagen is present in unwounded spinal cord compared with surrounding mesenchymal membranes. Collagen synthetic potential was found within the spinal cord, a tissue of neurectodermal origin. Rate of collagen synthesis per collagen content in the unwounded spinal cord was high. This synthetic rate was as high as that of wounds at their maximum collagen synthetic rates. Substantial deposition of collagen followed spinal cord wounding. Wounding the spinal cord, pia mater, and dura mater caused substantial elevations in rates of collagen synthesis in each tissue. These synthetic rates remained at maximum levels throughout the 8-week study, a prolonged period when compared with other wounded tissues previously studied. The role of mesenchymal tissue physiology in central nervous system wound healing is discussed. The potential value of these findings for further studies and for experimental manipulation of the healing process in spinal cord and central nervous system wounds is presented. Implications of these findings on the hypothetical relationship of scar to spinal cord and central nervous system regeneration are noted.

The influence of psychotropic drugs on the phosphodiesterase activity in the rat brain meninges.

Phosphodiesterase (PDE) is present in brain meninges. Its activity is higher in the pia than in the dura mater. Phenothiazine neuroleptics: fluphenazine, trifluoroperazine, thioproperazine, chloropromazine and thioridazine at concentration 10(-5)--10(-4) M in vitro inhibit the PDE activity in the pia and dura mater. Most potent in this respect were fluphenazine and trifluoroperazine. Much less pronounced inhibition of PDE activity in brain meninges was found after in vitro administration of tricyclic antidepressant: nortriptyline, chlorimipramine, protriptyline, desipramine and imipramine in concentrations 10(-4)--10(-3) M. Administered in vivo in a dose of 0.1 mg or 5 mg/kg ip fluphenazine inhibited the hydrolysis of 32P-cAMP injected into subarachnoid space. The results indicate that PDE present in the rat brain meninges may control the cAMP level in the cerebrospinal fluid. Treatment with phenothiazine neuroleptics which inhibit the PDE activity in meninges may significantly depress the hydrolysis of cAMP in the cerebrospinal fluid.