Impact of treatment on blood-brain barrier impairment in Wilson's disease.

INTRODUCTION: Our study assessed changes in concentrations of serum markers for brain damage and blood-brain barrier (BBB) dysfunction in untreated and treated Wilson's disease (WD) patients, and examined correlations between these changes and neurological impairment. OBJECTIVE: These results hold the potential to determine BBB impairment and neurological advancement in WD to develop the most effective treatment for patients with severe neurological deterioration. MATERIAL AND METHODS: The study groups included 171 patients with WD (77 with hepatic and 94 with neurological manifestations), treated either for up to 5 or 15 years, and 88 healthy controls. Serum concentrations of intercellular adhesion molecule 1 (ICAM1), P-selectin, matrix metallopeptidase 9 (MMP9), glial fibrillary acidic protein (GFAP), and S100 calcium-binding protein B (S100B) were measured before and during anti-copper treatment. The Unified Wilson's disease Rating Scale (UWDRS) was used to assess neurological advancement. RESULTS: ICAM1 concentrations were elevated before and during anti-copper treatment compared to controls (p < 0.01), but therapy led to substantial decreases both in patients with hepatic (p < 0.01) and in patients with neurological manifestations (p < < 0.05). P-selectin concentrations remained elevated before and during treatment (p < 0.05) regardless of the treatment duration and disease form. MMP9 concentrations before treatment were lower (p < 0.05), but reached control levels during treatment. GFAP concentrations were significantly elevated only in untreated patients with neurological symptoms in the longer-treated group compared to controls (p < 0.05). A significant reduction during treatment was observed only in the shorter-treated neurological group (p < 0.05). No substantial changes were observed in S100B. Only ICAM1 concentrations positively correlated (r = 0.27, p < 0.001) with the UWDRS. CONCLUSIONS: Our results provide evidence of endothelial activation in WD. However, inconclusive GFAP results, and no increase in S100B, do not allow us to conclude whether the reactive gliosis is not prominent or alternatively whether the BBB is disrupted. Elevated ICAM1 concentrations and their correlation with neurological advancement indicate BBB impairment. A decrease in ICAM1 during treatment suggests that the inflammatory process is reduced, and the BBB partially repaired. Decreased MMP9 concentrations may be the result of active liver fibrosis and higher copper concentrations. Elevated P-selectin concentrations indicate a systemic inflammatory process.

Blood-Brain Barrier-Associated Proteins Are Elevated in Serum of Epilepsy Patients.

Blood-brain barrier (BBB) dysfunction emerges as one of the mechanisms underlying the induction of seizures and epileptogenesis. There is growing evidence that seizures also affect BBB, yet only scarce data is available regarding serum levels of BBB-associated proteins in chronic epilepsy. In this study, we aimed to assess serum levels of molecules associated with BBB in patients with epilepsy in the interictal period. Serum levels of MMP-9, MMP-2, TIMP-1, TIMP-2, S100B, CCL-2, ICAM-1, P-selectin, and TSP-2 were examined in a group of 100 patients who were seizure-free for a minimum of seven days and analyzed by ELISA. The results were compared with an age- and sex-matched control group. Serum levels of MMP-9, MMP-2, TIMP-1, TIMP-2 and S100B were higher in patients with epilepsy in comparison to control group (p < 0.0001; <0.0001; 0.001; <0.0001; <0.0001, respectively). Levels of CCL-2, ICAM-1, P-selectin and TSP-2 did not differ between the two groups. Serum levels of MMP-9, MMP-2, TIMP-1, TIMP-2 and S100B are elevated in patients with epilepsy in the interictal period, which suggests chronic processes of BBB disruption and restoration. The pathological process initiating epilepsy, in addition to seizures, is probably the factor contributing to the elevation of serum levels of the examined molecules.

Changes in serum blood-brain barrier markers after bilateral tonic-clonic seizures.

OBJECTIVE: Seizures have been shown to increase blood-brain barrier (BBB) permeability, yet the role of this phenomenon is not fully understood. Additionally, dysfunction of the BBB leads to initiation and propagation of seizures in animal models. To demonstrate the increased permeability of the BBB in time, we investigated changes of the serum levels of BBB markers in patients with epilepsy after bilateral tonic-clonic seizures. We chose markers that might reflect endothelial activation (ICAM-1, selectins), BBB leakage (MMP-9, S100B) and mechanisms of BBB restoration (TIMP-1, thrombomodulin -TM). METHODS: We enrolled 50 consecutive patients hospitalised after bilateral tonic-clonic seizures who agreed to take part in the study and 50 participants with no history of epilepsy. Serum levels of selected markers were measured by ELISA at 1-3, 24, and 72 hours after seizures and one time in the control group. RESULTS: We found increased levels of S100B, ICAM-1, MMP-9 and P-selectin at 1-3 and 24 hours after seizures and TIMP-1 and TM at 24 and 72 hours after seizures as compared to the control group. The level of E-selectin was decreased at 72 hours after seizures. CONCLUSIONS: Our findings suggest early activation of endothelium and increased BBB permeability after seizures. While we are aware of the limitations due to the non-specificity of the tested proteins, our results might indicate the presence of prolonged BBB impairment due to seizure activity.

Serum Proteins Associated with Blood-Brain Barrier as Potential Biomarkers for Seizure Prediction.

As 30% of epileptic patients remain drug-resistant, seizure prediction is vital. Induction of epileptic seizure is a complex process that can depend on factors such as intrinsic neuronal excitability, changes in extracellular ion concentration, glial cell activity, presence of inflammation and activation of the blood−brain barrier (BBB). In this study, we aimed to assess if levels of serum proteins associated with BBB can predict seizures. Serum levels of MMP-9, MMP-2, TIMP-1, TIMP-2, S100B, CCL-2, ICAM-1, P-selectin, and TSP-2 were examined in a group of 49 patients with epilepsy who were seizure-free for a minimum of seven days and measured by ELISA. The examination was repeated after 12 months. An extensive medical history was taken, and patients were subjected to a follow-up, including a detailed history of seizures. Serum levels of MMP-2, MMP-9, TIMP-1, CCL-2, and P-selectin differed between the two time points (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0035, respectively). General linear model analyses determined the predictors of seizures. Levels of MMP-2, MMP-9, and CCL-2 were found to influence seizure count in 1, 3, 6, and 12 months of observation. Serum levels of MMP-2, MMP-9, and CCL-2 may be considered potential biomarkers for seizure prediction and may indicate BBB activation.

Autoantibodies in Wilson disease: Impact on clinical course.

Symptoms of Wilson disease (WD) vary and additional factors such as autoimmunity may play an important role in WD pathogenesis. The presence of antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies, neuronal surface antibodies, and onconeural antibodies in WD was investigated using standardized indirect immunofluorescence assays and Western Blot analysis. The presence of all studied autoantibodies was higher in WD patients in comparison to healthy subjects, but there was no statistically significant difference in autoantibodies frequency according to disease manifestation. D-penicillamine treatment was associated with a higher presence of ANA than zinc sulfate but without an increase in autoimmune diseases rate.

Ultrastructure of mitochondria and damage to small blood vessels in siblings with the same mutation in the NOTCH 3 and coexisting diseases.

We performed ultrastructural studies of mitochondria and evaluated the appearance of small blood vessels of three middle-aged siblings affected by the same mutation in the NOTCH3 gene, causing CADASIL. CADASIL pathognomonic features include granular osmiophilic material (GOM), which we observed. GOMs were located in damaged and thickened basement membranes (BM) of capillaries and arterioles. Our patients were also burdened by type II diabetes (first patient), impaired glucose metabolism (second patient), and hypertension (third patient). The ultrastructure of the capillaries in the first and second patients differed from the third patient. In diabetes/impaired glucose metabolism patients (first and second patients), we observed: pathologies of mitochondria in damaged endothelium and pericytes of capillaries; extremely thickened (BM) with visible remains of vascular cells; well-preserved GOMs anchored in the rebuilt capillary extracellular matrix. We identified degenerated or vestigial small blood vessels of skeletal muscles in the first patient. The capillary damage in the third patient (with hypertension) was milder compared to the diabetes/impaired glucose metabolism patients. We conclude that in patients with a mutation in the NOTCH3 gene, the co-occurrence of diseases such as type II diabetes/impaired glucose metabolism can cause a multiplication the damages to small blood vessels by modifying/masking the pathogenesis of CADASIL.

Kinetics of serum brain-derived neurotrophic factor (BDNF) concentration levels in epileptic patients after generalized tonic-clonic seizures.

OBJECTIVE: Epilepsy is a chronic neurological disorder characterized by the periodic and unpredictable occurrence of seizures. The serum level of brain-derived neurotrophic factor (BDNF) has been suggested to be a potential biomarker that could detect differences in epilepsy patients. Although there is considerable neurobiological evidence linking BDNF to epilepsy, only a small number of studies investigated the relationship between BDNF serum levels and epilepsy, and these studies obtained inconsistent results. The aim of this study was to elucidate BDNF serum levels in epilepsy cases. METHODS: Collectively, group of 143 patients (n = 143) were included in this study and subsequently divided into two groups consisting of individuals after singular generalized tonic-clonic seizures (n = 50) and patients with chronic epilepsy (n = 93). The samples from patients with acute epilepsy were collected 1-3 hours and 72 h after seizure, and a single collection was performed from patients with chronic epilepsy. These samples were compared to the control group (n = 48) using ELISA. RESULTS: In the present study, we observed a significant decrease of BDNF serum levels in patients after generalized tonic-clonic seizures compared to the control group. Furthermore, the observed decrease of BDNF levels in this group was sustained at 1 and 72 h after seizure insult. We did not show the relationship between BDNF levels and age, etiology of epilepsy and the duration of illness. SIGNIFICANCE: Our results and the findings of previous studies indicate that the serum BDNF level significantly decreases after seizures and should be considered when measuring BDNF in patients with chronic epilepsy. It might be also influenced by neurodegenerative processes, which may be involved in the etiopathogenesis of particular epilepsy syndromes.

Prediction of Recovery and Outcome Using Motor Evoked Potentials and Brain Derived Neurotrophic Factor in Subacute Stroke.

INTRODUCTION AND OBJECTIVES: Motor evoked potentials (MEPs) have been postulated to be useful in predicting recovery in patients with motor impairment. We aimed to investigate whether MEPs elicited by transcranial magnetic stimulation (TMS), serum brain derived neurotrophic factor (BDNF) and its genotype have prognostic value on stroke recovery in patients with hand paresis due to stroke. METHODS: This was an observational cohort study. Patients underwent TMS with MEPs from abductor digiti minimi evaluation between 2-14 (D0) and 30 days (D30) after stroke and their impact on motor function of the upper limb and general outcome was assessed after 3 months (D90). The presence of a BDNF gene polymorphism was determined and serum BDNF concentrations were measured at D0, D30 and D90. RESULTS: The presence of MEPs and their amplitude at rest and in effort significantly correlated with improvement of upper-limb paresis and general outcome after 3 months. Resting motor threshold did not have prognostic value. Central motor conduction time and MEP latency less consistently predicted stroke outcome or motor deficit improvement. Neither BDNF polymorphisms nor BDNF concentration at D0, D30 and D90 corresponded with the degree of paresis or the independence of patients 3 months after stroke. CONCLUSIONS: The presence of MEPs and their amplitude are useful predictors of upper-limb motor function recovery and general outcome after stroke. BDNF concentration and its genotype had no prognostic value. Further studies conducted on large cohorts are necessary to determine the usefulness of these methods in motor recovery and stroke outcome prediction.

Serum Brain-Derived Neurotrophic Factor is Related to Platelet Reactivity and Metformin Treatment in Adult Patients With Type 2 Diabetes Mellitus.

OBJECTIVES: The aim of this study was to investigate the association of serum brain-derived neurotrophic factor (BDNF) levels with platelet reactivity and antidiabetes treatment, as well as serum adipocytokine concentrations. METHODS: This observational, open-label study enrolled 149 patients. Serum BDNF, hematologic, biochemical parameters and platelet reactivity were measured. Blood samples were taken after the last acetylsalicylic acid dose. RESULTS: Patients with high BDNF levels were younger (65.60±8.956 vs. 68.59±8.516) and smoked cigarettes more frequently (14.6% vs. 4.1%); they were more commonly being treated by metformin (77.3% vs. 54%); had higher platelet counts (245.81±68.85 103/mm3 vs. 206.61±44.48 103/mm3); had shorter collagen-adenosine diphosphate closure time (CADP-CT) values (104.88±69.73 s vs. 140.93±86.63 s); had higher triglyceride concentrations (140.73±67.5 vs. 121.76±60.49) and had higher concentrations of serum thromboxane B2 (0.938±1.59 vs. 0.364±0.76). In univariate linear regression analyses, predictive factors for serum BDNF levels above the median were metformin treatment, current smoking, platelet count, triglyceride concentration, total cholesterol concentration and CADP-CT >74 s. In multivariate backward stepwise analysis CADP-CT >141 s; adiponectin concentration >4.22 µg/mL; total cholesterol and low-density lipoprotein levels were independently associated with serum BDNF levels above the median. CONCLUSIONS: Our results suggest that BDNF may be associated with lipid metabolism and that increased production of BDNF may be related to metformin treatment. Moreover, we showed an association between BDNF levels and platelet reactivity; we found that serum BDNF levels in patients with type 2 diabetes who had high platelet reactivity were higher than in subjects with normal platelet reactivity despite antiplatelet therapy.

Inhibition of TNF reduces mechanical orofacial hyperalgesia induced by Complete Freund's Adjuvant by a TRPV1-dependent mechanism in mice.

BACKGROUND: Inflammation in the orofacial region results in pain and is associated with many pathological states, including migraine, neuralgias and temporomandibular disorder. Although extensively studied, the mechanisms responsible for these conditions are not known and effective treatments are lacking. We reported earlier that the proinflammatory cytokine tumor necrosis factor (TNF) plays an important role in regulation of trigeminal ganglion (TG) neuron function in vitro. In the present study we investigated the role of TNF in mechanical hypersensitivity in mice. METHODS: We employed the Complete Freund's Adjuvant (CFA)-induced model of orofacial pain and evaluated the effect of blocking of soluble TNF activity by peripheral administration of the novel dominant negative TNF biologic, XPro1595. RESULTS: We show that CFA administration into the lower lip causes hyperalgesia and an increase in both expression of transient receptor potential vanilloid subfamily member 1 (TRPV1) mRNA and in the average intensity of TRPV1 protein immunoreactivity in TG neurons. We also show that intraperitoneal administration of XPro1595 prevents both CFA-induced mechanical hypersensitivity and, as shown in immunohistochemical staining - upregulation of TRPV1 protein expression in TG neurons. CONCLUSIONS: We conclude that one of the possible regulatory mechanisms of TNF in pain involves upregulation of the nociceptor TRPV1, and that peripheral treatment with a selective anti-soluble TNF biologic can prevent hyperalgesia caused by inflammation in the orofacial region. Therefore, these new findings suggest that XPro1595 may serve as a novel treatment for orofacial pain disorders.

The phosphodiesterase inhibitor, ibudilast, attenuates neuroinflammation in the MPTP model of Parkinson's disease.

BACKGROUND/AIMS: Since the degeneration of the nigrostriatal dopaminergic pathway in Parkinson's disease (PD) is associated with the inflammation process and decreased levels of cyclic nucleotides, inhibition of up-regulated cyclic nucleotide phosphodiesterases (PDEs) appears to be a promising therapeutic strategy. We used ibudilast (IBD), a non-selective PDE3,4,10,11 inhibitor, due to the abundant PDE 4 and 10 expression in the striatum. The present study for the first time examined the efficacy of IBD in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. METHODS: IBD [0, 20, 30, 40, or 50 mg/kg] was injected b.i.d. subcutaneously for nine days to three-month-old male C57Bl/10Tar mice, beginning two days prior to MPTP (60 mg/kg) intoxication. High-pressure liquid chromatography, Western blot analysis, and real time RT-PCR methods were applied. RESULTS: Our study demonstrated that chronic administration of IBD attenuated astroglial reactivity and increased glial cell-derived neurotrophic factor (GDNF) production in the striatum. Moreover, IBD reduced TNF-α, IL-6, and IL-1ß expression. CONCLUSION: IBD had a well-defined effect on astroglial activation in the mouse model of PD; however, there was no protective effect in the acute phase of injury. Diminished inflammation and an increased level of GDNF may provide a better outcome in the later stages of neurodegeneration.

Serum metalloproteinase 9 levels increase after generalized tonic-clonic seizures.

Metalloproteinase 9 (MMP9) is a member of a family of enzymes that mediate the degradation of extracellular matrix proteins, and is especially involved in blood-brain barrier maintenance. Increased levels of MMP9 have been observed in many neurological disorders, including epilepsy, suggesting it may be involved in the pathogenesis of seizures. We investigated changes in MMP9 serum levels after acute seizures in epilepsy patients. Concentrations of MMP9 in serum were measured by ELISA in 43 patients 1-3, 24, and 72h after generalized tonic-clonic seizure and once in participants of the control group. MMP9 levels were significantly increased 1-3 and 24h after seizure and decreased to control levels 72h after seizure. Our results suggest that MMP9 is released after or just before seizure; however, further studies are needed to resolve the consequences of the observed MMP9 increase.

Novel mutation of the NOTCH3 gene in a Polish family with CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small blood vessels disease caused by mutations in the gene encoding the neurogenic locus notch homolog protein 3 (NOTCH 3). We present a Polish family with a previously unreported novel mutation in exon 12 c.1851C>C/G of the NOTCH3 gene and varying disease expression. One of the two family members with the confirmed mutation presented with all the main CADASIL symptoms; while, his affected father was nearly asymptomatic. Both family members had epilepsy, coronary artery disease, and abdominal aorta aneurysm. Our observation confirms there is phenotypic variability in CADASIL not only between, but also within, families carrying the same mutation.

Serum Brain-Derived Neurotrophic Factor is Related to Platelet Reactivity but not to Genetic Polymorphisms within BDNF Encoding Gene in Patients with Type 2 Diabetes.

BACKGROUND: The aim of this study was to investigate the association between serum concentrations of the brain-derived neurotrophic factor (BDNF), platelet reactivity and inflammatory markers, as well as its association with BDNF encoding gene variants in type 2 diabetic patients (T2DM) during acetylsalicylic acid (ASA) therapy. MATERIAL/METHODS: This retrospective, open-label study enrolled 91 patients. Serum BDNF, genotype variants, hematological, biochemical, and inflammatory markers were measured. Blood samples were taken in the morning 2-3 h after the last ASA dose. The BDNF genotypes for selected variants were analyzed by use of the iPLEX Sequenom assay. RESULTS: In multivariate linear regression analysis, CADP-CT >74 sec (p<0.001) and sP-selectin concentration (p=0.03) were predictive of high serum BDNF. In multivariate logistic regression analysis, CADP-CT >74 sec (p=0.02) and IL-6 concentration (p=0.03) were risk factors for serum BDNF above the median. Non-significant differences were observed between intronic SNP rs925946, missense SNP rs6265, and intronic SNP rs4923463 allelic groups and BDNF concentrations in the investigated cohort. CONCLUSIONS: Chronic inflammatory condition and enhanced immune system are associated with the production of BDNF, which may be why the serum BDNF level in T2DM patients with high platelet reactivity was higher compared to subjects with normal platelet reactivity in this study.

Gender differences in the neurochemical response of trigeminal ganglion neurons to peripheral inflammation in mice.

It is well established that the majority of headache and other trigeminal nerve-associated disorders have higher prevalence in females than in males. However, the pathogenesis of many chronic trigeminal pain conditions, such as trigeminal neuralgia, migraine and temporo-mandibular disorders, is still not known. One of the proposed mechanisms involve calcitonin gene-related peptide (CGRP), which is considered the most important neuropeptide in the trigeminal system. In various animal models of trigeminal nerve-associated disorders concentration of CGRP has been shown to be increased in trigeminal ganglia (TG). Moreover, intraganglionic release of CGRP has been shown to modulate neuronal transmission of pain signals. In most of these models, pathological changes in the trigeminal system are accompanied by inflammation within peripheral endings of TG neurons. The aim of the present study was to investigate the relation between gender and neurochemical changes in trigeminal ganglia evoked by peripheral inflammation, induced by Complete Freund Adjuvant (CFA) administration. Our studies show significant increase in CGRP expression in female mice, comparing to male mice. Furthermore, we demonstrate, that activation of trigeminal nociceptors by peripheral inflammation causes significant increase in expression of IL-1B, IL-6, TNF and BDNF in male mice, comparing to female mice. This phenomenon may be involved in clinically observed gender-dependent differences in the frequency of both migraine and other trigeminal nerve-related facial pain disorders.

The influence of AAV2-mediated gene transfer of human IL-10 on neurodegeneration and immune response in a murine model of Parkinson's disease.

BACKGROUND: The aim of this study was to examine the effect of AAV2-hIL-10 (vector containing cDNA for human interleukin 10) on dopaminergic system activity (measured as DA levels and TH mRNA expression in mouse striata), and other monoamine and amino acid neurotransmitters concentration as well as development of inflammatory processes (measured as TGF-ß, IFN-γ and GFAP mRNA expression) in a murine MPTP neurotoxicant model of Parkinson's disease. METHODS: Male C57BL/6 mice 12 months-old were used in this study. AAV2-hIL-10 vector was bilaterally administered into striatum at 14, 21 or 28 days prior to MPTP intoxication. Animals were sacrificed at 7 days following MPTP injection. The expression of hIL-10 (human interleukin 10) was examined by ELISA. Striatal monoamine and amino acid neurotransmitters were measured by HPLC method. TH, TGF-ß, IFN-γ and GFAP mRNA expression was examined by RT-PCR method. RESULTS: MPTP treatment dramatically reduced DA levels and decreased TH mRNA expression in mouse striata, effects that were significantly impeded by AAV2-hIL-10 administration prior to MPTP intoxication. AAV2-hIL-10 infusion increased IFN-γ, TGF-ß and GFAP mRNA expression. CONCLUSIONS: Our data suggest that the transfer of AAV2-hIL-10 into the striatum may play a neuroprotective role in the mouse MPTP model of PD and these effects are mediated by the anti-inflammatory action of IL-10.