Rozprawy doktorskie na temat „Migraine with aura”

Nella tesi è descritto un modello matematico per l'aura emicranica e, più precisamente, per lo scotoma scintillante (schema di fortificazione) e per la Cortical Spreading Depression, il fenomeno neuropatofisiologico alla base dell'aura. In particolare è spiegato un modello cinematico per l'evoluzione della CSD nella corteccia visiva primaria, considerata un mezzo debolmente eccitabile, la mappa retino-corticale e il modello, tramite fibrato, dei Pinwheels di V1.

Cortical spreading depression (CSD), the underlying mechanism behind migraine aura, occurs when neurons become depolarized and lose function, while releasing signaling molecules that propagate this condition. Spreading depression occurs when abnormal flow of ions across the cell membranes of a neural cell depolarizes the cell and activates detrimental signaling pathways that cause the same disruptive ion flow to occur in nearby neural or glial cells. After depolarization, cells affected by CSD are not able to return to their normal state and remain nonfunctional for some time. This lack of function causes symptoms of migraine aura. Calcium and glutamate signaling may be primary drivers of the CSD; inhibition of either tends to prevent CSD, whereas their activation promotes it. Cerebral blood flow and oxygen consumption are considered measures of cell function during CSD and further demonstrate the significance of CSD on a larger scale. This is a review of current research about these elements of migraine aura.

Migraine is a painful neurological disease that affects at least 12% of the Australian population. It is generally characterized by recurrent head pain usually accompanied by nausea, vomiting, neurological disturbance, photo-and phonophobia. Migraine has been classified by the international headache society (IHS) into two most common types, migraine with aura (MA) and migraine without aura (MO). The underlying pathophysiology of this debilitating disease is still partially understood and there are no known diagnostic markers for these common types of migraine. Current diagnosis of migraine is based on patient reported symptoms. Studies have shown several health conditions such as epilepsy, depression and stroke to be co-morbid with migraine. Current migraine treatments work with varying efficacy and often have adverse side effects. A greater understanding of this debilitating and painful disease is thus pertinent for developing new and improved migraine treatment. Both familial clustering and twin studies have shown evidence for significant genetic mechanisms to underlie migraine pathogenesis. Migraine is thus currently defined as a complex multifactorial disorder which involves an interaction between genetic and environmental factors. We have not yet identified all migraine genes but a number of genes, causative mutations and susceptibility variants have been identified and are already of significant clinical relevance. Currently the detection of 3 rare subtypes of migraine (Familial Hemiplegic Migraine 1, 2 and 3) and several related conditions with symptom overlap (Episodic Ataxia 2, Spinocerebellar Ataxia Type 6 and Cerebral Autosomal Dominant Arteriopathy with Sub cortical Infarcts and leucoencephalopathy (CADASIL) is undertaken by sequencing, with susceptibility variants for common types of migraine detected by sequencing or genotyping.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Migraine is a serious neurological disorder that affects the central nervous system causing painful attacks of headache. Attacks of head pain vary widely in intensity, frequency and duration lasting from anywhere between 4-72 hours and are often accompanied by further symptoms of nausea, vomiting, photo- and phonophobia. In 1988, a group of world leaders in the diagnosis of migraine formed the International Headache Society (IHS) and compiled and published a consensus set of diagnostic criteria known as International Classification of Headache Disorders, ICHD-I 1988. This was the first classification system and was subsequently updated in 2004, ICHD-II 2004 and more recently a 3rd Edition beta version has been released (ICHD-3rd Ed Beta Version) and is the gold standard for diagnosing headache disorders. Migraine displays two main subtypes termed migraine with or without aura (MA and MO respectively). The two forms are distinguished from each other based on the development of aura, a period of variable and diverse neurological symptoms that precede the headache phase.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Science, Environment, Engineering and Technology
Full Text

This research was conducted to expand the understanding of the role of the X chromosome in common and familial typical migraine. The primary objective of this study was to identify new X chromosomal genetic targets that cause migraine. Overall this research has identified nine genetic targets of interest. Various obstacles were encountered throughout this study, but the knowledge gained for overcoming these obstacles are invaluable for implementation and improvement of future genetic studies investigating the X chromosome.

Migraine is a painful temporarily incapacitating disorder that affects an estimated 12% of the general population including 18% of adult women and 6% of adult men. The disorder involves two main subtypes termed migraine with or without aura (MA and MO respectively). Migraine can present with a variety of symptoms that vary between individuals and between episodes experienced by a single individual. This disorder causes significant social and economic burden and alarmingly is often poorly treated. A direct cause of this is a lack of understanding of the underlying pathology of migraine. Migraine is believed to be a neurogenic disorder that involves temporary disruption of pathways that receive and respond to sensory signals. While numerous environmental triggers may have been identified the exact mechanisms that cause the disruption are still largely unknown. However, familial aggregation of migraine suggests significant genetic contributors.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Migraine is a common, debilitating neurovascular disease charactensed by severe recurrent headache, nausea and vomiting, photophobia and phonophobia. It is clinically diagnosed based on criteria specified by the International Headache Society (IHS), defining two major classes of migraine: migraine with aura (MA) and migraine without aura (MO) MA sufferers experience neurovascular disturbances that precede the headache phase of an attack. Although migraine is partly influenced by environmental determinants, there is a significant genetic component, with disease heritability estimated to be up to 60% and mode of transmission multifactorial. The disorder is common with a large Dutch study reporting lifetime prevalence estimates of 33% in women and 13.3% in men, with an earlier study estimating 24% of women and 12% of men in the overall population. Mutations in various ion channel genes are responsible for neuromuscular and other neurological disorders. Inherited ion channel mutations or 'channelopathies' are increasingly found to be the cause of various neurological disorders in humans. In familial hemiplegic migraine (FHM), a rare subtype of migraine with aura, mutations in the CACNA1A gene (localised at C19p13) have been fbund (FHM1). This gene codes for the alphalA subunit of the neuronal voltage-dependent P/Q-type calcium channel. Recently a second gene, ATP1A2 (FHM2) (localised at C1q23), was implicated in some EHM families. The ATP1A2 ion channel gene, codes for the alpha2 subunit of the Na+, K+ ion ATPase pump. These findings of mutations in these genes have focused attention on central nervous system ionic channels and helped to better understand EHM pathophysiology, where the best genetic evidence providing molecular insight into migraine still comes flom the mutations detected in the rare form of migraine with aura; FHM. Migraine family studies, at the Genomic Research Centre (GRC), have utilised linkage analysis methods in providing results that have indicated suggestive linkage to the FHM1-CACNA1A region on l9p13, in a large multigenerational family (Migraine Family 1; MEl) affected with typical migraine. Also linkage studies conducted within the GRC have implicated an additional susceptibility region on chromosome 1q31, but still not ruling out a second susceptibility region on C1q23, with the possibility of there being two distinct loci, on the chromosome lq region. The focus of research in this thesis is on two main chromosomal regions, which were tested for migraine susceptibility on chromosome 1 and chromosome 19. The research involved a cross-disciplinary approach utilising association, linkage and mutation screening approaches. Allelic candidate gene studies can provide a suitable method for locating genes of small effect that contribute to complex genetic disorders, such as migraine. Family linkage studies are useful for detection of chromosomal susceptibility regions and association studies are powerful when a plausible candidate gene and a sequence variant with potential functional relevance is examined. Mutation screening studies can indicate a direct cause of disorders such as migraine, where possible sequence variants may alter the translation of proteins in genes, causing the disease. The first gene exanted on chromosome 19 was that of the Low Density Lipoprotein Receptor (LDLR) gene. The LDLR gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome l9pl3.2 performing an association analysis in 244 typical migraine affected patients, 151 suffering from migraine with aura, 96 with migraine without aura and 244 unaffected controls. The populations consisted of Caucasians only and controls were age and sex matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al, 2003 who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine affected genetically-isolated population. Another gene examined on chromosome l9pl3 was the insulin receptor gene (1NSR). The aim of this study was to investigate through direct sequencing the INSR gene in DNA samples from a migraine affected family previously showing linkage to chromosome l9pl3 in an attempt to detect disease associated mutations. The insulin receptor gene (INSR) on chromosome 19pl3.3-13.2 is a gene of interest since a number of SNPs located within the gene have been implicated in migraine with (MA) and without aura (MO). Six DNA samples obtained from non-founding migraine affected members of migraine family one (MF 1) were used in this study. Genomic DNA was sequenced for the 1NSR gene in exons 1-22 and the promoter region. In the six migraine family member samples, previously reported single nucleotide polymorphisms (SNP5) were detected within two exonic DNA coding regions of the INSR gene. These SNPs, in exon 13 and 17, do not alter the normal INSR polypeptide sequence. In addition, intron 7 also revealed a DNA base sequence variation. For the 5' untranslated promoter region of the gene, no mutations were detected. In conclusion, this study detected no INSR mutations in affected members of a chromosome 19 linked migraine pedigree. Hence, migraine linkage to this chromosomal region may involve other candidate genes. The NOTCH3 gene on C19p13.2-p13.l has previously been shown to be a gene involved in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and may also be implicated in migraine as there are some symptom similarities between the two disorders. The TNFSF7 gene localised on Cl9pl3 is homologous to the ligands of the TNF receptor family, including TNF-alpha and TNF-beta, genes that have both been previously associated with migraine. This study investigated the migraine susceptibility locus at Cl9p13 studying two genes that may be involved in the disorder. The NOTCH3 gene was analysed by sequencing all exons with known CADASIL mutations in a family (MF1) that has previously been shown to be linked to Cl9pl3. The sequencing results for affected members of this pedigree proved to be negative for all known sequence variants giving rise to mutation causing amino acid changes for CADASIL. The direct sequencing results displayed that of a normal coding sequence for the NOTCH3 gene F or the TNFSF7 gene, this was investigated through SNP association analysis using a matched case-control migraine diagnosed population. Chi-square results showed non-significant P values across all populations tested against controls except for the MO subgroup which displayed a weak association with the TNFSF7 SNP (genotype, allele analysis P = 0.036, P = 0 017 respectively). Our results suggest that common migraine is not caused by any known CADASIL mutations in the NOTCH3 gene of interest however, the TNFSF7 gene displayed signs of involvement in a MO affected population, but, further studies are needed to confirm these results and to further explore a TNF receptor - migraine potential interaction. A final examination on chromosome 19 involved a case report of an extremely rare and severe form of migraine. As stated earlier Familial Hemiplegic Migraine (FHM) is a severe rare sub-type of migraine and gene mutations on chromosome 19 have been identified in the calcium channel gene CACNA1A (Cl9pl3) fOr FHM. Recently a gene mutation (S218L) for a dramatic syndrome originating from FHM, commonly named 'migraine coma', has implicated exon 5 of the CACNA1A gene. The occurrence of trivial head trauma, in FHM patients, may also be complicated by severe, sometimes even fatal, cerebral edema and coma occurring after a lucid interval. Hemiplegic migraine has also been found to be sporadic in which both forms share a similar spectrum of clinical presentations and genetic heterogeneity. The case report presented in this study enhances the involvement of the S218L CACNA1A mutation in the extremely rare disorder of minor head trauma induced migraine coma. It not only proves to be a powerful diagnostic tool in detecting cases of FHM head trauma induced coma but also for sporadic hemiplegic migraine (SHM) coma subjects. We conclude from this case study that the S218L mutation, in the CACNA1A calcium channel subunit gene, is involved in sporadic hemiplegic migraine (SHM), delayed cerebral edema and coma after minor head trauma. This thesis also involved analysis of chromosome 1 for migraine susceptibility, where FHM studies provided a foundation fOr common migraine research on chromosome 1. Studies have suggested that mutations in the CACNA1A gene on chromosome l9p cause FHM in only approximately 50% of affected pedigrees. The CACNAIA gene has previously been tested, within the Genomics Research Centre, in the common forms of migraine; however no new mutations or the FHM mutations were detected in these MA/MO affected samples. A second FHM susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene have recently been implicated in two Cl-linked FHM pedigrees. As FHM is considered a rare and severe form of MA, it is possible that the chromosome 1q23 locus, and the ATP1A2 gene, may be involved in the common forms of migraine with (MA) and possibly without aura (MO). Also, we have previously reported evidence of linkage to microsatellite markers on chromosome 1q31 in a large pedigree affected predominately with MA, which suggests the possibility that there are two distinct loci for migraine susceptibility on chromosome 1. The objectives of this study were to extend our linkage analysis of chromosome lq microsatellite markers in predominantly migraine with aura pedigrees. Also, our aim was to test the novel FHM-2 ATP1A2 gene for involvement in these migraine affected pedigrees and a previous pedigree (Migraine Family 14; MF 14) showing evidence of linkage of markers to Clq31. This was performed by a chromosome 1 scan (31 markers) in 21 multiplex pedigrees affected mainly with MA. Also, the known FHM-2 ATP1A2 gene mutations were tested, by sequencing, fOr involvement in MA and MO in these pedigrees. Mutation screening by direct sequencing was also performed throughout the coding areas of the ATP1A2 gene in 3 MA individuals fiom MF14. The results of this study detected evidence for linkage in our migraine pedigrees at chromosome 1q23, to microsatellite markers spanning the ATP1A2 (FHM-2) gene. However testing of the known ATP1A2 gene mutations (for FHM) in migraine probands of pedigrees showing excess allele sharing was negative, with no mutations detected in these migraineurs. Sequencing of the entire coding areas of the gene through 3 MA affecteds from MF14, a pedigree showing significant linkage to this region, was also negative for mutations. In conclusion, this study reported that microsatellite markers on chromosome 1q23 show evidence of excess allele sharing in MA and some MO pedigrees, suggesting linkage to the common forms of migraine and the presence of a susceptibility gene in this region. The new FHM-2 (ATPIA2 gene) mutations reported by Fusco et al, 2003 do not cause migraine in probands of affected pedigrees showing excess allele sharing to markers in this genomic region. Also no mutations were detected in all exons of the ATP1A2 gene in 3 MA affected individuals from a large pedigree (MF14) showing linkage to this region. Investigation in this thesis continued on chromosome 1, with other genes being examined on C1q23, as well as the C1q31 region for a migraine susceptibility locus or gene. Previously in our laboratory, evidence for linkage was shown to migraine at C1q31 in one family predominantly affected with MA, with microsatellite markers in this region. The initial Cl study (above; ATP1A2 gene) has also provided evidence for linkage to the chromosome 1 locus 1q23, with evidence for excess allele sharing of markers in predominantly MA affected pedigrees. To further investigate both chromosome I loci, an investigation with six candidate genes that lie within the C1q23 and 1q31 regions through association analysis was undertaken. The results from this study reported non-significant chi-square results, showing P values greater than 0.05 across all SNPs (and a CA rpt) tested. An exception was the rs704326 SNP from exon 43 of the CACNA1E gene on C1q31. P values significantly less than 0.001 were obtained in the total migraine population and the MA subgroup, with similar frequency comparisons ascertained in both genotype and allele analysis. Examination through contingency table analysis of the CACNA1E flequency data indicated that the risk allele (A) was over-represented in the migraine group compared to the control group. Further comparison of the genotype data indicated a difference in frequency distributions (P less than 0 0001). Stratified analyses of migraine subtypes indicated that this association was specifically attributed to the MA subtype group. Odds ratios produced an OR of 4.14 with a 95% CI of 2.36 - 7.26 (P less than 0.0001). The positive association results obtained within the CACNA1E gene are interesting in the fact that FHM is considered to be a rare and severe form of migraine with aura (MA) and FHM-1 is caused by mutations contained within the calcium channel gene CACNA1A (localized at Cl9p13). The idea that FHM and specifically an FHM gene in the C1q31 genomic region may also contribute to susceptibility to the more common forms of migraine i e. migraine with aura, strongly supports and reinforces the idea that a common defective gene may be influencing both FHM and typical migraine. In conclusion, this thesis undertook a cross-disciplinary approach to genetic research of a complex disorder. The research involved linkage, association and mutation analysis strategies of migraine. This research implicated a specific variant on chromosome 1 and further supported the heterogeneic nature of migraine. Future directions into migraine research should involve further investigation of this specific variant and this genomic region. Such studies may aid in the development of more precise diagnosis and treatment methods for this complex disorder.

Migraine is a common, debilitating neurovascular disease charactensed by severe recurrent headache, nausea and vomiting, photophobia and phonophobia. It is clinically diagnosed based on criteria specified by the International Headache Society (IHS), defining two major classes of migraine: migraine with aura (MA) and migraine without aura (MO) MA sufferers experience neurovascular disturbances that precede the headache phase of an attack. Although migraine is partly influenced by environmental determinants, there is a significant genetic component, with disease heritability estimated to be up to 60% and mode of transmission multifactorial. The disorder is common with a large Dutch study reporting lifetime prevalence estimates of 33% in women and 13.3% in men, with an earlier study estimating 24% of women and 12% of men in the overall population. Mutations in various ion channel genes are responsible for neuromuscular and other neurological disorders. Inherited ion channel mutations or 'channelopathies' are increasingly found to be the cause of various neurological disorders in humans. In familial hemiplegic migraine (FHM), a rare subtype of migraine with aura, mutations in the CACNA1A gene (localised at C19p13) have been fbund (FHM1). This gene codes for the alphalA subunit of the neuronal voltage-dependent P/Q-type calcium channel. Recently a second gene, ATP1A2 (FHM2) (localised at C1q23), was implicated in some EHM families. The ATP1A2 ion channel gene, codes for the alpha2 subunit of the Na+, K+ ion ATPase pump. These findings of mutations in these genes have focused attention on central nervous system ionic channels and helped to better understand EHM pathophysiology, where the best genetic evidence providing molecular insight into migraine still comes flom the mutations detected in the rare form of migraine with aura; FHM. Migraine family studies, at the Genomic Research Centre (GRC), have utilised linkage analysis methods in providing results that have indicated suggestive linkage to the FHM1-CACNA1A region on l9p13, in a large multigenerational family (Migraine Family 1; MEl) affected with typical migraine. Also linkage studies conducted within the GRC have implicated an additional susceptibility region on chromosome 1q31, but still not ruling out a second susceptibility region on C1q23, with the possibility of there being two distinct loci, on the chromosome lq region. The focus of research in this thesis is on two main chromosomal regions, which were tested for migraine susceptibility on chromosome 1 and chromosome 19. The research involved a cross-disciplinary approach utilising association, linkage and mutation screening approaches. This research implicated a specific variant on chromosome 1 and further supported the heterogeneic nature of migraine. Future directions into migraine research should involve further investigation of this specific variant and this genomic region. Such studies may aid in the development of more precise diagnosis and treatment methods for this complex disorder.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

Aims: Test the putative contribution of 17-beta-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats. Main Methods: Female, Sprague-Dawley rats were intact or underwent ovariectomy followed one week later by surgery to place electrodes onto the dura to detect epidural electroencephalographic activity (dEEG). dEEG activity was recorded two days later for 12 hours after systemic administration of 17-beta-estradiol (180 mu g/kg, i.p.). A separate set of rats were observed for changes in exploratory, ambulatory, fine, and rearing behaviors; periorbital allodynia was also assessed. Key Findings: A bolus of 17-beta-estradiol significantly elevated serum estrogen levels, increased SD episodes over a 12-hour recording period and decreased rearing behaviors in ovariectomized rats. Pre-administration of ICI 182,780, an estrogen receptor antagonist, blocked 17-beta-estradiol-evoked SD events and pain behaviors; similar results were observed when the antimigraine therapeutic sumatriptan was used. Significance: These data indicate that an estrogen receptor-mediated mechanism contributes to SD events in ovariectomized rats and pain behaviors in both ovariectomized -and intact-rats. This suggests that estrogen plays a different role in each phenomenon of migraine where intense fluctuations in concentration may influence SD susceptibility. This is the first study to relate estrogen peaks to SD development and pain behaviors in awake, freely moving female rats, establishing a framework for future preclinical migraine studies.

Les gènes codant pour les canaux sodiques potentiel-dépendants (Nav) présents dans le système nerveux central sont la cible de mutations conduisant à divers phénotypes. L’objectif de mon travail de thèse est de comprendre pourquoi des mutations d’un même gène peuvent conduire à des pathologies distinctes afin d’envisager le développement de nouvelles approches thérapeutiques. Le gène SCN1A codant pour le canal Nav1.1, exprimé principalement dans les interneurones GABAergiques (IN GABA), est la cible de mutations responsables de syndromes épileptiques et de la migraine hémiplégique familiale (MHF-3), une forme rare de migraine avec aura. Il a été montré que les mutations responsables de l’épilepsie induisent une perte de fonction du canal, ce qui conduit à une hypoexcitabilité des IN GABA dont résulte une hyperexcitabilité du réseau neuronal. L’analyse fonctionnelle de mutations responsables de la MHF-3 a montré qu’elles induisent un gain de fonction du canal et une hyperexcitabilité des IN GABA pouvant être à l’origine d’une dépression corticale envahissante, un mécanisme pathologique de la migraine. En particulier, l’étude de la mutation L1649Q a montré qu’elle induit une réduction importante de la densité de courant des canaux Nav1.1 (défaut d’expression des canaux). L’analyse des propriétés biophysiques des canaux mutés après récupération de la densité de courant a mis en évidence que l’effet de la mutation correspond à un gain de fonction allant dans le sens d’une hyperexcitabilité des IN GABA (Cestèle et al.2013 PNAS). Afin d’identifier si d’autres mutations MHF-3 possèdent le même mécanisme (perte/gain de fonction), la 1ère partie de ma thèse a consisté en la caractérisation fonctionnelle d’une nouvelle mutation responsable de la MHF-3, L1670W. Cette mutation conduit à un défaut d’expression des canaux Nav1.1 (perte de fonction) cependant, après récupération de la densité de courant, la mutation induit un gain de fonction des canaux Nav1.1. Ces résultats ont permis de mettre en évidence que la mutation L1670W induit un défaut d’expression des canaux à la membrane et un gain de fonction renforçant ainsi l’hypothèse selon laquelle ce mécanisme pourrait être généralisé à d’autres mutations MHF-3. Le gène SCN2A codant pour le canal Nav1.2, exprimé principalement dans les neurones excitateurs, est la cible de mutations responsables de différentes pathologies telles que les épilepsies bénignes, les encéphalopathies épileptiques et les troubles du spectre de l’autisme (TSA). A ce jour, les mécanismes responsables de ces pathologies restent flous. Dans le but d’élucider la relation génotype/phénotype, nous avons étudié les effets fonctionnels de 23 mutants SCN2A responsables de ces différentes pathologies. Nos résultats montrent que toutes les mutations responsables de TSA induisent une perte presque totale de densité de courant tandis que pour les autres pathologies les effets sont hétérogènes. Dans le but de reproduire les conditions hétérozygotes, nous avons étudié la co-expression des canaux wild-type (WT) avec chaque canal muté. Nos résultats ont mis en évidence une réduction de la densité de courant des canaux WT uniquement en présence de canaux porteurs de mutations responsables de TSA. Par conséquent, seules les mutations responsables de TSA induisent un phénomène de dominance négative sur les canaux WT. Afin de déterminer si ce mécanisme de dominance négative est dû à l’interaction de 2 sous-unités α décrite récemment (Clatot et coll., 2018 Nat Commun), nous avons utilisé différentes stratégies pour inhiber cette interaction. Les résultats obtenus ont montré que l'effet de dominance négative des mutants responsables de TSA n’est plus observé lorsque α-α est inhibée. Par conséquent, nos résultats permettent de décrire pour la 1ère fois que les mutations des canaux Na+ responsables de TSA agissent par un mécanisme de dominance négative, lequel est médié par l’interaction entre les canaux WT et mutés
The genes encoding for the voltage-gated sodium channels (Nav) expressed in the central nervous system are the target of numerous mutations leading to various phenotypes. The aim of my work is to understand why mutations in the same gene can lead to distinct pathologies in order to consider the development of new therapeutic approaches. The SCN1A gene encoding for the Nav1.1 channels, mainly expressed in GABAergic interneurons (GABA IN), is the target of mutations responsible for epileptic syndromes and familial hemiplegic migraine (FHM-3), a rare form of migraine with aura. The mutations responsible for epilepsy have been shown to cause a loss of function, which leads to hypoexcitability of GABA IN and subsequently to the network hyperexcitability. At the opposite, the mutations responsible for MHF-3 showed a gain of function and hyperexcitability of GABA IN which can lead to the cortical spreading depression, a pathological mechanism of migarine. In particular, the functional study of the L1649Q mutation showed that the mutation leads to an important decrease of the current density (loss of function). Analysis of the biophysical properties of the mutated channels after partial recovery of the current density showed that the overall effect of the mutation is a gain of function, consistent with an hyperexcitability of GABA IN (Cestele and al. 2013 PNAS). In order to identify if other FHM-3 mutations share the same mechanism (loss / gain of function), the first part of my thesis aimed to characterize a new mutation responsible for MHF-3, L1670W. This mutation leads to a defect in the Nav1.1 channels expression at the membrane but after partial recovery of the current density, the mutation induces a clear gain of function of Nav1.1 channels. These results showed that the L1670W mutation, like the L1649Q mutation, leads to a defect in the Nav1.1 channels expression at the membrane and a gain in function, thus reinforcing the hypothesis that this mechanism could be generalized to other mutations responsible for MHF-3. The SCN2A gene encodes for the α subunit of Nav1.2 channels mainly expressed in excitatory neurons. Mutations in the SCN2A gene are responsible for different pathologies such as benign epilepsies, epileptic encephalopathies and autism spectrum disorder (ASD). To date, the detailed mechanisms responsible for these different pathologies remain unclear. In order to elucidate the genotype/phenotype relationship, we studied the functional effects of 23 SCN2A mutants responsible for these different pathologies. Our results show that all the mutations responsible for ASD induce an important decrease (almost complete) of the current density while for the other pathologies the effects are heterogeneous. In order to reproduce the heterozygous conditions, we studied the co-expression of wild-type (WT) channels with each mutated channel. Our results showed a reduction in the WT channels current density only in the presence of channels carrying mutations responsible for ASD. Consequently, only the mutations responsible for ASD induce a negative dominance on WT channels. To determine whether this negative dominance mechanism is due to the interaction of α subunits described recently (Clatot et al., 2018 Nat Commun), we used different strategies to inhibit this interaction. The results obtained showed that the negative dominance effect of the mutants responsible for ASD is no longer observed when the interaction between the α subunits is inhibited. Therefore, our results allow us to describe for the first time that mutations in Na+ channels responsible for ASD act by a negative dominance mechanism, which is mediated by the interaction between WT and mutated channels

La Dépression Corticale Envahissante (Cortical Spreading Depression) est une vague de dépolarisation neuronale qui se propage lentement à travers le cortex cérébral. Elle génère une phase rapide d’hyperactivité neuronale suivie d’une phase plus lente de silence électrique des cellules du cortex. Il est possible d’induire expérimentalement la DCE sur différents modèles animaux, in vivo ou sur tranches de néocortex, par application locale de Glutamate, d’Acétylcholine ou d’une solution hautement concentrée en potassium, ou par stimulation électrique. La DCE est largement étudiée comme mécanisme physio-pathologique de la migraine avec aura, mais aussi de l’ischémie. Les études sur la DCE expérimentale ont montré l’implication de nombreuses substances biologiques dans la génération et la propagation de cette vague, comprenant le Potassium, le Calcium, le Glutamate et d’autres neurotransmetteurs. L’approche pharmacologique permet de placer d’acteurs dans ce phénomène électrique, les canaux dépendant du potentiel, les récepteurs ionotropes du glutamate (NMDA et AMPA-kainate), les astroctyes et les pompes et échangeurs Na-K. Il existe une forme monogénique de Migraine avec Aura, la Migraine Hémiplégique Familiale, où la crise de migraine est associée à des troubles moteurs d’intensité variable, qui a permis d’identifier des mutations génétiques, donc des dysfonctions à niveau moléculaire. A ce jour trois types principaux, donc trois gènes mutés, de cette MHF sont décrits et étudiés. L’étude des modèles de souris de MHF 1 (mutation gain de fonction du Cav2.1) et de MHF 2 (mutation perte de fonction de Na-K-ATPase), confirme l’implication primordiale du Glutamate et du Potassium dans l’initiation de la DCE. Le type 3 de MHF est du à des mutations du Nav1.1, un canal sodique dépendant du potentiel, majoritairement exprimé dans les neurones GABAergiques où ils sont très importants pour leur excitabilité. Notre laboratoire a montré sur neurones transfectés en culture qu’il s’agissait d’une mutation gain de fonction, entrainant une augmentation de l’excitabilité des neurones. Cependant, le lien entre cette mutation, l’hyperexcitabilité corticale et la facilitation de la DCE – déjà observée dans les autres modèles de FHM – ou le phénotype de Migraine FHM reste inconnu. Notre hypothèse de travail, et la base de mon sujet de thèse, est que l’augmentation de l’activité des canaux Nav1.1 ou des neurones GABAergiques, déclencherait une hyperexcitabilité corticale et une DCE. Pour éprouver cette hypothèse, mes travaux de thèse ont nécessité une approche expérimentale ex vivo, sur tranches de néocortex de souris sauvages et transgéniques, associée à de l’électrophysiologie extracellulaire, imagerie IOS, pharmacologie et/ou optogénétique. L’activation des canaux Nav1.1 par une toxine activatrice sélective, ou la stimulation optogénétique des neurones GABAergiques, est capable d’induire une DCE, confirmant notre hypothèse initiale et ajoutant un nouveau modèle d’induction. Mes travaux de thèse ont permis d’identifier et de caractériser ce nouveau modèle de DCE où l’hyperexcitabilité des neurones GABAergiques conduit à une accumulation initiale de potassium dans le milieu extracellulaire, qui dépolarise et active de plus en plus de neurones excitateurs Glutamatergiques. Ceci entraine une libération soutenue de potassium jusqu’à atteindre un seuil critique pour le déclenchement de la DCE. Dans un second temps, mes travaux de thèse se sont orientés vers une modulation pharmacologique de l’activité de réseau, avec pour objectif de trouver un nouvel élément permettant de diminuer la susceptibilité à la DCE. Pour cela, par la même approche expérimentale, j’ai utilisé un agent cholinergique, le Carbachol, connu et utilisé pour moduler l’activité de réseau
Cortical Spreading Depression is a wave of neuronal depolarization that spread slowly across cerebral cortex. It generates a rapid phase of neuronal hyperactivity, followed by a slower phase of electrical silence of cortical cells. It is possible to experimentally induce CSD on several animal models, in vivo or on neocortical slices, by focal application of Glutamate, Acetylcholine or highly concentrated KCl solution, or by electrical stimulation.CSD is widely studied as the pathophysiological mechanism of migraine with aura, but also ischemia. Studies of experimental CSD have shown involvement of numerous biological substances in the wave generation and propagation, including Potassium, Calcium, Glutamate, and other neurotransmitters. The pharmacological approach allows to identify actors of this electrical phenomenon: voltage gated channel, ionotropic glutamate receptors (NMDA & AMPRA-kainate), astrocytes and Na-K pumps.Familial Hemiplegic Migraine is a monogenic form of migraine with aura; the migraine attack is associated with variable motor disorders. Genetic mutations have been described leading to molecular dysfunctions. Nowadays three main forms of this pathology caused by three mutated genes, have been described and studied. FHM type 1 (Cav2.1 gain of function) and FH% type 2 (NaK ATPase pump loss of function) mouse models studies confirmed the important involvement of Glutamate and Potassium in CSD initiation.Type 3 of FHM is caused by Nav1.1 mutations, a voltage gated sodium channel that is widely expressed in GABAergic neurons in which they are essential for excitability. Our team showed on transfected neurons in culture that he mutation is a gain of function, leading to an increased neuronal excitability. However, the link between the mutation, cortical hyperexcitability and CSD facilitation or FHM phenotype, remains unknown.Our work hypothesis and the base of my research project, is that the increasing Nav1.1 channel or GABAergic neurons’ activity, triggers a cortical hyperexcitability and CSD. To confirm this hypothesis, my work required a ex vivo experimental approach, on acute neocortical slices of wild-type and transgenic mice, associated with extracellular electrophysiology, IOS imaging, pharmacology and/or optogenetics. Nav1.1 channel activation by a selective activator (spider toxin), or GABAergic neurons stimulation by optogenetics, can trigger CSD, validating our initial hypothesis and identify a new model of CSD.My work allow us to highlight and characterised a new model of CSD by GABAergic neuron hyperexcitability leading to an initial build up of extracellular potassium, that depolarizes and activates more and more excitatory neurons. This leads to a sustained potassium release until a critical threshold of CSD triggering.In a second time, my thesis work explored a pharmacologic modulation of network excitability, to find new elements that could decrease the CSD susceptibility. To do so, with the same experimental approach, I used a cholinergic agonist, Carbachol, known for modulation the network activity. The results showed that even if Carbachol increases network excitability, it inhibits CSD induction, likely through the muscarinic pathway.In conclusion, during my thesis I identified a new mechanism of CSD induction, and a une inhibitory pathway of CSD by cholinergic modulation

Die Auslöser der Migräne sind weitgehend unbekannt. Eine Cortical Spreading Depression, das pathophysiologische Korrelat der Migräne Aura, führt im Tierexperiment zur Aktivierung des trigeminalen Schmerzsystems. Hieraus resultieren verschiedenste Veränderungen in der Dura mater, zu denen ein verzögerter Blutflussanstieg in der A. meningea media und die Extravasation von Plasmaproteinen gehören. Diese Ereignisse sind mit der Genese von Kopfschmerzen vereinbar. Die Infusion von Nitroglycerin führt zu der Entwicklung einer meningealen Inflammation, welche als Korrelat der verzögerten Migräneattacke nach Nitroglycerin Infusion in Patienten angesehen werden könnte. Die repetitive Gabe von Sumatriptan und Zolmitriptan hat keinen funktionell relevanten Einfluss in experimentellen Paradigmen zur Wirksamkeit von Triptanen. Zudem wird die Expression von Serotonin (5-HT1) Rezeptor mRNA in Geweben mit Relevanz zur Migräne nicht entscheidend verändert. Diese Untersuchungen zeigen neue Pathomechanismen in der Genese der Migräne auf und identifizieren damit bisher unbekannte Ziele, die zur Entwicklung innovativer medikamentöser Strategien zur Behandlung der Migräne dienen können.
The pathophysiological events which initiate a migraine attack are largely unknown. Cortical spreading depression (CSD) is supposed to be the pathophysiological correlate of the migraine aura. CSD is able to activate the ipsilateral trigeminal nerve system thereby leading to a series of events within meninges consistent with the notion of headache. In particular, CSD leads to a delayed increase of meningeal blood flow and the extravasation of plasma proteins. In an experimental animal model the infusion of glyceryl trinitrate causes delayed meningeal inflammation. The latter serves as the correlate of delayed migraine attacks after glyceryl trinitrate infusion in susceptible individuals. Protracted administration of sumatriptan and zolmitritpan does not alter the outcome in functional assays that are used to determine the efficacy of triptans. Moreover, the expression of serotonin (5-HT1) receptor mRNA is not significantly attenuated in tissues related to migraine pathophysiology. In summary, these studies shed light on the mechanism leading to migraine headache and thereby identify novel targets for the development of new anti-migraine drugs.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Estuda atravÃs de ensaio duplo cego, controlado, randomizado o efeito da Toxina BotulÃnica do tipo A na profilaxia de crises de migrÃnea sem aura. A migrÃnea à um tipo comum de cefalÃia primÃria, benigna, episÃdica, e recorrente que se caracteriza por dor geralmente hemicrÃnica e pulsÃtil, e que à agravada pela atividade fÃsica. Existem outros sintomas associados como nÃuseas, fotofobia, fonofobia, ou irritabilidade. Na migrÃnea com aura podem tambÃm ocorrer alteraÃÃes neurolÃgicas motoras, sensitivas, ou visuais denominadas de aura. A migrÃnea, cuja fisiopatologia ainda nÃo à perfeitamente compreendida, seria o resultado de um processo patolÃgico complexo que envolveria o tronco cerebral e levaria à inflamaÃÃo local de vasos sangÃÃneos cranianos atravÃs da liberaÃÃo de neuropeptÃdeos vasoativos como SubstÃncia P (SP), Neurocinina A (NA), e PeptÃdio Relacionado ao Gene da Calcitonina (PRGC). Apesar das vÃrias opÃÃes terapÃuticas (analgÃsicos simples, antiinflamatÃrios hormonais e nÃo hormonais, triptanos, antipsicÃticos, derivados ergotamÃnicos, e opiÃides) para tratamento da crise ou para tratamento preventivo, somente cerca de um terÃo dos pacientes fica satisfeito com o tratamento. Foi observado que pacientes utilizando toxina botulÃnica para tratamento estÃtico de rugas da face ou distonias apresentavam uma reduÃÃo na quantidade de crises de migrÃnea. A toxina botulÃnica à uma potente neurotoxina produzida pela bactÃria Clostridium botulinum. A aÃÃo da toxina à impedir a liberaÃÃo de acetilcolina nos terminais nervosos. Ela tambÃm age inibindo a liberaÃÃo de neuropeptÃdeos vasoativos. O uso da toxina botulÃnica nos faria agir exatamente no cerne do processo fisiopatolÃgico da doenÃa. Com o objetivo de testar esse possÃvel efeito analgÃsico nos pacientes portadores de migrÃnea sem aura, realizou-se um estudo duplo-cego, controlado, e randomizado. Mediu-se o nÃvel de dor atravÃs de escalas para quantificar a intensidade e o nÃmero de dias com dor na semana antes e apÃs a injeÃÃo de Toxina BotulÃnica em mÃsculos da face. O grupo controle recebeu SF como placebo. Os pacientes foram seguidos durante trÃs meses. Ao final concluiu-se que nÃo houve diferenÃa estatÃstica na intensidade nem na freqÃÃncia da dor de cabeÃa nos pacientes que usaram a toxina botulÃnica em relaÃÃo aos que usaram placebo (SF)
A randomized, double-blind, placebo-controlled study of the use of botulinum toxin type A in the prophylactic treatment of Migraine is presented. Migraine is a common type of primary, benign, episodic headache. It is characterized by pain usually unilateral and throbbing. Other associated symptoms are nausea, sensitivity to light and sound, or irritability. The pain is usually worsened by physical activity. There are also motor, sensitive, or visual neurological alterations, denominated aura. The physiopathology of migraine is not still perfectly understood but it could involve liberation of vasoactive neuropeptides as Substance P, Neurokinine A, and Calcitonin gene-related peptide, promoting an inflammation. Migraine, then, would be the result of a complex process that would involve the brainstem and induce local inflammation of cranial blood vessels. In spite of the therapeutic options (analgesics, steroidal and non-steroidal anti-inflammatory, triptans, neuroleptics, ergot derivatives, and opioids) only about one third of patients is satisfied with the treatment. The preventive treatment is appropriate for those that have frequent crises. It was observed that the patients using botulinum toxin for aesthetic treatment of wrinkles of the face, or dystonia presented a reduction in the amount of migraine crises. The botulinum toxin is a potent neurotoxin produced by the bacterium Clostridium botulinum. The action of the toxin is to inhibit the acetylcholin liberation from the nerve terminal. It acts also inhibiting the liberation of vasoactive neuropeptides. Therefore, Botulinum Toxin would act exactly in the core of the physiopathologic process of the disease. With the objective of testing possible analgesic effects of botulinum toxin in migraine without aura bearers, we performed a double-blind, controlled, and randomized study. The pain level was measured by scales and by the amount, and number of days of pain in a week, before and after botulinum toxinâs injection in muscles of the face. The placebo group received saline injection. The patients were followed for three months. At the end it was concluded that there was not statistic difference in intensity nor in frequency of the headache of the patients that used botulinum toxin in relation to the people that used placebo (saline)

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Doctors belonging to a Medical Services Cooperative in Campina Grande were interviewed with the objective to determine their diagnostic accuracy when faced with clinical cases of primary headache, and also to appreciate the amplitude of their knowledge about headache classification and procedures concerning exams prescriptions and therapeutic indications. Methodology. The method of extensive direct observation through the application of a questionnaire was used. The doctors were presented with three fictitious clinical histories which represented situations of frequent primary migraines such as: migraine with aura (MA), migraine without aura (MO) and chronic tensional-type headache (CTTH) according to criteria established on International Headache Classification, second edition (IHCD-II), 2003, by the Headache Classification Subcommittee as part of the International Headache Society (IHS). 173 out of 462 doctors members of the Cooperative were contacted and 91 of these accepted to take part in the study. Results. In the group of 91 doctors interviewed, 51 (56%) were male, 35 (38,5%) were female and five (5,5%) refused to answer. Their age varied from 27 to 70 years old (44,8 + 09 years old). Their experience as doctors varied from three to 46 years (21,4 + 8,2 years). 67 (73.6%) stated to have been in a medical internship. The doctors interviewed were doctors of several areas. About the accuracy of the diagnostic test, concerning MO, 60 participants (66%) identified the case as migraine, only two (2,2%) identified the MO subtype and two (2,2%) suggested it was a case of mere headache. For the clinical case of MA, 25 doctors (27,5%) said it was a case of migraine and only one (1,1%) identified it as MA. About the diagnostic of CTTH, 12 doctors (13,2%) acknowledged it as tensional headache and there was not any reference to the CTTH subgroup. Among other possibilities of diagnostic, the most mentioned ones were migraine 36 (39,6%), secondary headache to systemic arterial hypertension 12 (13,2%) and headaches caused by brain expansive process 06 (6,6%). For the three clinical cases, most of the doctors researched 79 (86,8%) in the example of MO, 74 (81,3%) in the cases of MA and 71 (78%) in the example of CTTH wouldn´t prescribe complementary exams. Regarding treatment 77 (84,6%) in the case of MO, 80 (87,9%) in the case of MA and 67 (73,6%) in the case of CTTH decided not to treat it. Among those who forwarded the case to a neurologist, we have 67 (73,6%) for MO, 78 (85,7%) for MA and 59 (64,8%) for CTTH. 65 (71,4%) doctors who were interviewed said to be unaware of the IHS diagnostic criteria for primary headaches. Conclusion. Migraine was the most common initial diagnosis for any kind of headache without any identification of the subgroups. The CTTH was subdiagnosed being frequently misidentified as migraine cases despite it is the most prevailing kind of primary headache. Most of the doctors interviewed (p<0,05) does not prescribe complementary exams for headache patients and prefer to forward them to a specialist, choosing not to treat them. These results show the lack of information found in the doctors interviewed about the diagnostic criteria for the several kinds of primary headaches.
Foram entrevistados médicos pertencentes a uma Cooperativa de Serviços Médicos, na cidade de Campina Grande, com o objetivo de investigar a acurácia diagnóstica desses profissionais frente a casos clínicos de cefaléia primária, além de apreciar seus conhecimentos sobre a classificação das cefaléias e condutas quanto à solicitação de exames e indicações terapêuticas. Metodologia: Utilizou-se o método de observação direta extensiva através da aplicação de questionário. Foram apresentadas a todos os médicos entrevistados três histórias clínicas, fictícias, reproduzindo quadros de cefaléias primárias freqüentes, a saber: migrânea com aura (MCA), migrânea sem aura (MSA) e cefaléia do tipo tensional crônica (CTTC), seguindo os critérios da Classificação Internacional das Cefaléias, 2ª. Edição (IHCD-II), em 2003, elaborados pelo Subcomitê de Classificação das Cefaléias da Sociedade Internacional de Cefaléia (IHS). Foram contatados 173 médicos, de um total de 462 cooperados, dos quais 91 aceitaram participar do estudo. Resultados: Eles eram 51 homens (56%) e 35 mulheres (38,5%), cinco (5,5%) não identificaram o gênero. A idade variou entre 27 e 70 anos (44,8 + 09 anos). Eles tinham entre três e 46 anos de formados (21,4 + 8,2 anos). 67 (73,6%) afirmaram ter feito residência médica. Os médicos entrevistados atuavam nas mais diversas especialidades. Em relação ao índice de acerto diagnóstico, no exemplo de MSA, 60 participantes (66,2%) identificaram o quadro como migrânea, enquanto apenas dois (2,2%) identificaram o subtipo MSA e dois (2,2 %) sugeriram tratar-se apenas de cefaléia. No caso clínico de MCA, 25 (27,5%) disseram tratar-se de quadro de migrânea e apenas um (1,1%) respondeu ser MCA. Quanto ao diagnóstico de CTTC, 12 (13,2%) reconheceram tratar-se de cefaléia tensional, não houve acerto diagnóstico no subgrupo CTTC, e dentre outras possibilidades diagnósticas as mais citadas foram: migrânea 36 (39.6%), cefaléia secundária a hipertensão arterial sistêmica 12 (13,2%) e cefaléias ocasionadas por processo expansivo cerebral seis (6,6%). Nos três casos clínicos, a maior parte dos profissionais pesquisados 79 (86,8%) no exemplo de MSA, 74 (81,3%) nos casos de MCA e 71 (78%) no exemplo de CTTC - não solicitaria exames complementares. Quanto ao tratamento: 77 médicos (84,6%) no caso de MSA, 80 (87,9%) no caso de MCA e 67 (73,6%) no caso de CTTC, optaram por não tratar. Preferiram encaminhar ao neurologista: 67 médicos (73,6%) para MSA, 78 (85,7%) para MCA e 59 (64,8%) para CTTC. 65 (71,4%) dos entrevistados afirmaram desconhecer os critérios diagnósticos da IHS para cefaléias primárias. Conclusão: O diagnóstico inicial mais freqüente foi migrânea, comumente usado para identificar qualquer tipo de cefaléia, sem, contudo identificar seus subgrupos. A cefaléia do tipo tensional foi subdiagnosticada, freqüentemente sendo confundida com quadros de migrânea, a despeito de ser o tipo mais prevalente de cefaléia primária. A maioria (p<0,05) dos médicos não solicita exames complementares para portadores de cefaléia e prefere encaminhá-los ao especialista, optando por não tratar. Esses resultados demonstram a falta de informações por parte dos médicos entrevistados sobre os critérios diagnósticos para os diversos tipos de cefaléias primárias.

Orientador: José Eduardo Tanus dos Santos
Texto em Português e Inglês
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A migrânea é uma cefaléia primária episódica com incidência mundial de 10% e alto impacto socioeconômico, afetando predominantemente as mulheres. Em 30% dos casos, as crises de migrânea são acompanhadas por sintomas neurológicos focais que caracterizam o fenômeno da aura. A fisiopatologia da migrânea continua sob investigação. Todavia, acredita-se tratar de um distúrbio neurovascular no qual a ativação de fibras aferentes do sistema trigeminovascular (TGVS) e a consequente liberação de óxido nítrico (NO) levariam à vasodilatação e neurotransmissão da dor durante a crise de migrânea. A formação de NO é atribuída à atividade de três isoformas de NO sintases (NOS): endotelial, neuronal e induzível, respectivamente eNOS, nNOS e iNOS e os genes que codificam tais enzimas exibem polimorfismos. Dessa forma, acredita-se que tais polimorfismos poderiam estar envolvidos na susceptibilidade à migrânea. No presente trabalho foi testada a hipótese de que dois polimorfismos clinicamente importantes no gene da iNOS: C-1026A (rs2779249) e G2087A (rs2297518) bem como a combinação de seus alelos em haplótipos estariam associados com a susceptibilidade à migrânea com e sem aura. Para tanto, foram estudadas 142 mulheres saudáveis (grupo controle) e 200 mulheres com migrânea, subdivididas em dois grupos: 148 pacientes com migrânea sem aura e 52 pacientes com migrânea com aura. Os genótipos e alelos foram determinados por PCR em tempo real utilizando o ensaio Taqman®. Os haplótipos foram inferidos através do programa PHASE versão 2.1. Os principais resultados do presente estudo indicam que o haplótipo H4, que combina o alelo A para os polimorfismos mencionados, pode estar associado à migrânea com aura enquanto o polimorfismo G2087A e o haplótipo H4 podem afetar a suscetibilidade à aura em pacientes com migrânea. Tais achados podem ter implicações terapêuticas ao se analisar os efeitos de possíveis inibidores seletivos da iNOS na população com esse perfil genotípico
Abstract: Migraine is an episodic primary headache with worldwide incidence of 10% and high socioeconomic impact affecting mainly women. In 30% of cases, migraine attacks are followed by focal neurological symptoms that characterize the aura phenomenon. The migraine pathophysiology continues under investigation, although it is believed to consist of a neurovascular disorder in which the activation of afferent fibers of the trigeminovascular system (TGVS), leads to an nitric oxide (NO) release which, would be responsible for vasodilation and pain during migraine attacks. The formation of NO is attributed to the activity of three isoforms of NO synthases (NOS): endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), and the genes that codify these enzymes exhibit polymorphisms. Thus, it is believed that these polymorphisms could be involved in the susceptibility to migraine. In this study, we tested the hypothesis that two clinically important iNOS gene polymorphisms: C-1026A (rs2779249) and G2087A (rs2297518), as well as the combination of their alleles in haplotypes, would be associated with migraine with or without aura. Therefore, we studied 142 healthy women (control group) and 200 women with migraine, who were subdivided into two groups: 148 with migraine without aura and 52 with migraine with aura. Genotypes and alleles were determined by real-time PCR, using the Taqman® assay. Haplotypes were inferred by the PHASE program (version 2.1). The main results of the present study indicate that H4 haplotype, which combines the A allele for both polymorphisms may be associated with migraine with aura, while G2087A polymorphism and H4 haplotype may affect the susceptibility to aura in patients with migraine. These findings may have therapeutic implications when analyzing the possible selective inhibitor effects of iNOS on the population with this genotype profile
Mestrado
Farmacologia
Mestre em Farmacologia

Background: Patent Foramen Ovale (PFO), a remnant of the foetal circulation, is emerging as a new cause of disease. It has been found to be associated with cryptogenic stroke in young adults, peripheral arterial embolism and neurological decompression sickness in divers. The detection of PFO remains a diagnostic challenge; transoesophageal echocardiogram being currently considered the ‘gold standard’. The development of a non-invasive technique is crucial for the identification of a venous-to-arterial shunt (v-aCS) which may permit paradoxical embolism. Little is known about the genetic basis of PFO and our limited knowledge is based on animal studies and gene mutations detected in patients with other cardiac septal defects. Methods: Study 1: PFO Detection and Evaluation: This study was designed to evaluate transcranial Doppler (TCD), transthoracic echocardiogram (TTE) and transoesophageal echocardiogram (TOE) with administration of contrast via arm and femoral veins. We then developed a standardized protocol for PFO detection and quantification using TCD. Study 2: PFO and Migraine: The PFO detection protocol developed from the first study formed the diagnostic technique to detect v-aCS in an adequately powered matched case control study to explore the association between PFO and migraine. Study 3: The Genetic basis of PFO: This study was designed to explore the genetic basis of a PFO using a candidate gene approach. Results: Study 1 - PFO Detection Study: When compared with TOE with femoral vein contrast injection as the ‘gold standard’, TCD with arm vein contrast was 100% sensitive and 97.4% specific for detecting a PFO. We defined a PFO positive (+ve) study on TCD as > 15 microbubbles entering the cerebral circulation, on TCD following arm vein injection and >16 microbubbles with a femoral contrast injection. A ‘major’ PFO+ve v-aCS was defined as >35 microbubbles with arm vein injection or >90 microbubbles with femoral vein injection. We then developed a new diagnostic pathway for PFO detection in clinical practice. Study 2 - PFO Migraine study: A significant difference in prevalence of v-aCS between migraine with aura M+A) and their matched controls was demonstrated with adjusted OR=3.72 (1.48-9.38) p=0.005 for a PFO+ve v-aCS, and a highly significant difference between M+A and controls for a ‘major’ PFO+ve v-aCS with adjusted OR = 6.38 (1.89 – 21.48) p = 0.003. There was significant association with APC resistance and migraine on thrombophilia screen. Study 3 - The PFO Genetics Study: This study detected mutations of GATA4 and NKX2-5 in both PFO+ve cases and PFO-ve controls. Two novel non synonymous mutations of GATA4, c.461T>A and c.994G>A were found only in PFO positive individuals and may be associated with a PFO. All the PFO+ve cases with a GATA4 gene mutation had a major PFO+ve v-aCSConclusion:TCD detects PFO with a sensitivity of 100% and specificity of 92.3% and is the most reliable non-invasive technique for PFO detection. When arm vein injections are used both cough and valsalva provocation is essential. There was a highly significant association between PFO+ve v- aCS and M+A, especially with a ‘major’ PFO+ve v-aCS. GATA 4 mutations though infrequent were found PFO+ve cases and all had major v-aCS.

Background: Migraine trigger factors are precipitating factors that can contribute to an attack by increasing the probability of a migraine occurring. For some migraineurs, the headache phase is preceded by a transient disturbance in neurological function (an aura). An aura could be visual or sensory in nature. There are medications that can be used to treat a migraine attack when it occurs (acute medication) and medication that can be used to reduce frequency and severity of migraine attacks (prophylactic medication). Objectives: The primary aim of the study was to identify if there was a relationship between migraine trigger factors, auras and treatment. Methods: The study was conducted in 2014 in Port Elizabeth and consisted of two self-administered questionnaire-based surveys, one for pharmacists and one for migraine patients. Migraine patient questionnaires were distributed to migraine patients who frequented pharmacies, physiotherapy practices and health shops. A total of 18 pharmacist questionnaires and 173 migraine patient questionnaires were analysed. Results: Experiencing an aura before a migraine attack was reported by 43.9% of respondents and only “sometimes” by 22.5% of respondents. Visual auras were experienced by 92.0% of respondents who indicated that they suffered from migraine with aura and sensory auras were experienced by 71.5% of respondents, with 62.8% of respondents experiencing both visual and sensory auras. Trigger factors were experienced by 89.0% of respondents. There was no statistical relationship between aura and trigger factors, but there was a statistical relationship between trigger factors and visual aura at the 5% level (Chi-square = 7.966, d.f. = 1, p-value = 0.005). Cramér’s V showed a small practical significance at 0.218. About 80.0% of respondents used over-the-counter (OTC) medication and only 12.6% used migraine specific medication to abort a migraine attack. There was no statistical relationship between aura (visual or sensory) and abortive medication. There was a statistical relationship between abortive medication and the presence of trigger factors (Chi-square = 8.775, d.f. = 3, p-value = 0.032). Cramér’s V showed a small practical significance at 0.244. There was no statistical relationship in the presence of trigger factors between aura and abortive medication. Conclusion: Migraine is a complex disease which affects people of all ages. There appears to be a statistical relationship between visual auras and trigger factors and between abortive medication and trigger factors. There was, however, no statistical relationship between aura and abortive medication in the presence of trigger factors. Further studies need to be conducted to substantiate these findings.

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Bachelors
Arts and Sciences
Art History

Migraine - with or without aura - is an enervating primary headache disorder that represents a heavy economic and social burden. The health-related quality of life of migraineurs is poor. The aim of this research was to investigate the health-related quality of life of migraine without aura sufferers. As the thesis was approached from a biopsychosocial perspective, potential determinants were chosen for investigation from the molecular, individual, interpersonal and wider societal levels. The research was executed in two phases: Phase 1 data (N = 341) were gathered using a survey of health-related quality of life (Short Form 6), temperament (the Tridimensional Personality Questionnaire), catastrophizing as a pain coping strategy (the four-item Pain Coping Scale) and the amount of perceived social support (the six-item Social Support Questionnaire). For phase 2, participants were requested to provide blood specimens for ELISA serum quantification of glutamate (n = 66) and gene expression analysis of the main glutamate transporter gene SLC1A2 on real-time reverse-transcription polymerase chain reaction (n = 20). Of the 341 adult residents of Gauteng Province, South Africa that participated in the survey, 94 (28%) met the criteria for migraine without aura and a further 60 (18%) suffer from possible migraine without aura, using the International Classification of Headache Diagnosis (2nd edition) criteria. This indicates that migraine without aura is a significant burden for South Africa. Health-related quality of life was significantly poorer for migraineurs versus those without migraine (p < .001), and is in fact comparable to that of liver transplant, cardiac bypass and elderly populations. This raises concerns about the severe burden of the disease on the mental and physical well-being of South African sufferers. Investigation of the predictors of health-related quality of life yielded two significant variables when controlling for sex, head and neck injury and language - Harm Avoidance and vii Catastrophizing. The regression model accounts for 29% of the variance in health-related quality of life. A reciprocal relationship likely exists between Harm Avoidance and Catastrophizing, in which a harm avoidant migraineur interprets the headache pain as a catastrophic event to be avoided – even at high cost to the self. Though there have been calls for more biopsychosocial studies of migraine, this thesis did not find added understanding of health-related quality of life through the combination of biological and psychosocial data. The implication is that the role of glutamate in migraine without aura still requires further investigation. Further study is also required with regard to which biological factors may influence the sufferer’s quality of life. The thesis indicates a key role for psychological intervention in aiding migraineurs to live a life of quality. The inclusion of interventions for the psychological aspects of migraine may yield improved outcomes for patients. However, Gauteng residents suffering from MO are potentially unaware of their diagnosis and therefore of potential management for their disorder. Awareness around migraine needs to be the first step in limiting this disorder’s devastating impact on individuals, their relationships and their potential to contribute meaningfully to society.
Psychology
D.Litt. et Phil. (Psychology)

Cloninger’s Psychobiological Theory of Personality proposes four temperament dimensions, each underpinned by a different neurotransmitter system. The serotonergic system is purportedly linked to Harm Avoidance (HA). The aim of this study was to explore the relationship between HA and serotonin in migraine without aura (MO). A second aim was to explore the personality profile of MO patients. Sixty-six participants completed an online questionnaire and donated blood samples. Results indicated no significant association between HA and serotonin and a significant relationship between MO and HA. This study indicates that both Cloninger’s Psychobiological Theory of Personality and the Tridimensional Personality Questionnaire used for its assessment have value in South African personality research. In addition, the findings of the study reveal support for personality influences on the processes involved in migraine. This not only produces worthwhile avenues of research but also an alternative perspective for clinical practice.
Psychology
M.A. (Psychology (Research Consultation))

The objective of this research was to test the effectiveness of hypnosis as a natural treatment for migraine and aura. The assumption that hypnosis is primarily psychological in nature whereas migraine and aura are psycho-neurological phenomena, determined the adoption of a psycho-neurological model for this research.

Differences in cognition between patients with migraines and healthy control subjects have been documented by several researchers, principally in the areas of attention, memory, and motor function. Patients with aura (MA) are more often reported to have interference with normal cognitive functioning and are thought to have a more severe neurological condition than do patients without aura (MO). We compared the cognitive profiles of 29 right-handed patients, recruited at the Montreal Neurological Hospital, and diagnosed with hemicranial MA and MO to that of 10 healthy control subjects (NC). Diagnosis was made according to the International Headache Society guidelines by a neurologist. From a personality perspective, higher levels of self-consciousness were documented in MA and M Left than in NC and MO (F = 2.67, p < .05). NC's response styles were more original while patients preferred more conservative ones (F = 4.65, p < .01). Examination of cognitive data from an extensive neuropsychological battery was performed. MA performed worse than NC and MO on a General Cognitive Index (F = 2.72, p< .05), Full Scale (F = 6.10, p < .001) and Verbal (F = 5.48, p < .01) IQ. Verbal IQ was also lower for MO than for NC (F = 10.69, p < .001). Compared to NC and left-hemisphere migraines (M Left ), right-hemisphere migraines (M Right ) demonstrated reduced attention (F = 3.96, p < .05), poorer constructional abilities (F = 4.38, p < .05), and lower visuo-spatial memory (F = 3.92, p < .05). M Right performed less well on executive functioning tasks (F = 2.65, p = .05). M Left was associated with lower Full Scale IQ (F = 8.34, p < .001) and Verbal IQ (F = 7.89, p < .001). MA (Right Hand: F = 3.90, p < .05; Left Hand: F = 4.10, p < .05) had poorer motor skills than NC. M Right (Left Hand: F = 3.61, p < .05) showed additional motor slowness. Thus, presence of aura and hemisphere lateralization of migraine headache can affect the cognitive and personality profiles of patients with migraine

No
Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Leão and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1¿induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 µM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ETA receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ETA receptor.

碩士
高雄醫學大學
醫學影像暨放射科學系碩士班
106
Purpose: Specific white matter alterations in migraine have been shown in many studies. Because the sensations of pain are performed by pain matrix, the integrity of white matter near these regions might be altered in migraine patients. Diffusion tensor imaging (DTI) has been used in a lot of studies to detect white matter alterations. However, because the Gaussian assumption in DTI is not a suitable model in the intracellular microenvironment, diffusion kurtosis imaging (DKI) which considers not only the Gaussian distribution but also the non-Gaussian distribution were demonstrated to better characterize white matter microstructural alterations. In addition, voxel-based morphometry (VBM) has been used in many studies to estimate the volume or density changes of gray matter in the cerebral cortex. Therefore, the aim of our study was to use DKI and VBM to comprehensively observe white matter and gray matter alterations near the regions of pain matrix in migraine. Materials and Method: Our study acquired whole brain T1 and DKI datasets from 19 Migraine without aura (MWoA) subjects (M/F=5/14, age=42±10 y/o) and13 healthy controls (M/F=5/8, age=32±9 y/o) on a Siemens 3.0T Skyra System. The DKI dataset were acquired using three b values(0, 1000 and 2000) in 20 non-collinear diffusion directions and repeated three times to improve data quality. For white matter analysis, all DKI images were post-processed with DKE tool (Diffusion Kurtosis Estimator) to obtain axial diffusivity (AD), radial diffusivity (RD), mean diffusivity (MD), Fractional Anisotropy (FA), axial kurtosis (AK), radial kurtosis (RK), and mean kurtosis (MK). For gray matter analysis, we used DARTEL VBM analysis for a series of automatic image processing, such as segmentation, modulation, and normalization. After spatial normalization, SPM8 (Statistical Parametric Mapping version 8) was performed for statistical analysis. Results: The results showed that there were more white matter alterations found by DKI than DTI. We found significantly increased diffusivity and decreased diffusion anisotropy in multiple white matter regions, especially at left side of brain near the pain matrix in migraine subjects, and also found there were more alterations in the parameters of radial direction (RD and RK). Moreover, the gray matter density were significantly decreased at the left side of brain near pain matrix in migraine subjects, but were significantly increased in cerebellum. Conclusion: The mechanism of migraine is still to be investigated. Our results demonstrated that DKI technique is helpful and more sensitive for detection of subtle white matter microstructural alterations in migraine subjects and can provide more in-depth insight into the distribution of water diffusion related to tissue microstructural alterations. Besides, DARTEL VMB is also helpful for detection of gray matter alterations in migraine subjects. Although the significance of these changes in migraine is still to be elucidated, our study preliminarily evaluated the alterations of both white matter and gray matter structures in migraine and the results may provide the direction for future research and clinical diagnosis.

M.Tech. (Chiropractic)
The object of this study was to compare two chiropractic treatment approaches to each other in the management of migraine without aura. It was hypothesised that a combination of upper cervical and upper thoracic chiropractic manipulative therapy would be more effective than upper cervical chiropractic manipulative therapy alone. Migraine without aura was diagnosed according to the criteria of the International Headache Society (1988) and based on a structured case history, physical examination as well as regional orthopaedic and neurological examinations. Forty-one (41) subjects were randomly allocated to one of the two treatment groups in this single blind, randomised trial. Thirty-three patients completed the trial. Both groups received their respective chiropractic manipulative treatments twice a week for a total period of four weeks. During this time and a period of eight weeks thereafter, each patient kept a daily headache diary, noting migraine frequency, duration, headache intensity and associated analgesic pill consumption. Statistical analysis of the collected data involved inter-group comparisons of the above mentioned variables using Mann-Whitney Rank Sum tests, and intra-group comparisons of the above. mentioned variables using Wilcoxon Signed Rank tests at a 95% level of confidence. Intra-group analysis of the data revealed statistically significant (P < 0.05) decreases in migraine frequency and headache intensity for both groups. Migraine duration followed a similar pattern but for a sudden increase in duration in the third month for the group receiving a combination of upper cervical and upper thoracic chiropractic manipulative therapy. Inter-group analysis of the data established no statistically significant differences (P > 0.05) between the two treatment groups before or during the study. Throughout the study, there was a notable difference in average analgesic pill consumption between the two groups. The results indicate that both chiropractic manipulative therapy approaches had positive effects on the frequency, duration and headache intensity of migraines without aura. The effect of chiropractic manipulative therapy on the associated analgesic pill consumption is speculative, since there was no pre-treatment assessment of analgesic pill consumption. The sudden increase in migraine duration during the third month for the group that received both upper cervical and upper thoracic manipulation may be due to this treatment being less effective than upper cervical manipulation alone. The significance of this sudden increase will need to be established by future studies. Neither one of the two chiropractic treatment protocols applied in this study fared significantly better than the other. It is suggested that future studies consider any disability associated with migraines without aura. A pre-treatment trial period would provide reliable pre-treatment statistics for the variables investigated during such a trial and larger samples would represent the overall migraineur population better. It is suggested that a third group, receiving only chiropractic manipulative therapy to the upper thoracic spine, also be included.