Utilizzo di una tossina batterica, il fattore citotossico necrotizzante 1 (CNF1), per il trattamento di glioma in un modello di topo

I glioblastomi sono un tipo di tumore cerebrale poco responsivo a tutti i trattamenti attualmente disponibili. Per questo motivo c’è necessità di sviluppare nuove terapie per questa patologia. Durante l’internato di tesi ho testato una tossina batterica per il trattamento di glioblastoma nel topo. La tossina utilizzata è il fattore citotossico necrotizzante 1 (CNF1), un’esotossina prodotta da un ceppo patogeno di Escherichia coli. CNF1 è una proteina monomerica, costituita da una porzione N-terminale che si lega alle cellule, una regione intermedia che media la traslocazione attraverso la membrana e un dominio C-terminale che possiede attività catalitica. Nel citosol la parte C-terminale catalizza la deammidazione di un residuo di glutammina delle Rho-GTPasi (RhoA, Rac1 e Cdc42), impedendo così la loro attività GTPasica e lasciandole in uno stato di costante attivazione. Questo comporta l’inibizione della citodieresi con conseguente multinucleazione nelle cellule che hanno capacità di dividersi. Inoltre è stato osservato un effetto di aumento della plasticità nei neuroni trattati, in assenza di fenomeni neurotossici. Sulla base di queste evidenze, si è deciso perciò di testare la tossina su un modello di glioma di topo, costituito dalla linea cellulare GL261. Questa è una linea di cellule di derivate da topi del ceppo C57Bl/6 in cui si è provocato l'insorgere di un astrocitoma tramite trattamenti con l'agente cancerogeno 3-metilcolantrene. Una delle loro caratteristiche è di poter essere iniettate nella corteccia di topi singenici del ceppo C57Bl/6 e dare origine a tumore senza necessità di indurre immunosoppressione. Dapprima il CNF1 è stato testato in vitro su colture di cellule GL261. Le cellule così trattate risultavano multinucleate, smettevano di proliferare e quindi andavano incontro a degenerazione. Quindi si è voluto verificare il tipo di morte cellulare cui esse vanno incontro. Circa il 65% delle cellule trattate con la tossina esprimono marker di necrosi tardiva 10 giorni dopo il trattamento, mentre si è potuta escludere la morte per apoptosi. Rimane aperta la possibilità di una morte per senescenza, suggerita anche dalla morfologia delle cellule trattate, che appaiono appiattite e con nucleo e nucleoli di dimensioni maggiori. Inoltre si è ottenuto un modello in vivo mediante l'iniezione intracorticale di cellule di glioma murino della linea GL261 in topi singenici nell’area visiva primaria. In questi topi, dopo 5 giorni dall'iniezione delle cellule, è stato poi iniettato il CNF1 in concentrazione 80 mM nella medesima area cerebrale. Nell’internato di tesi ho seguito il test di sopravvivenza di topi con glioma trattati con CNF1. La loro sopravvivenza risulta significativamente maggiore rispetto ai topi con glioma trattati con una soluzione veicolo (che muoiono circa dopo 28 giorni) e anche rispetto a topi con glioma trattati con farmaci chemioterapici classici, come il temozolomide. In effetti circa metà dei topi trattati con CNF1 è sopravvissuta per almeno 60 giorni dopo il trapianto delle cellule. Quindi ho partecipato all’analisi elettrofisiologica dell’attività corticale in un’area circostante l'iniezione delle cellule tumorali, per evidenziare differenze fra topi trattati con CNF1 e soluzione veicolo, dopo 21 giorni dall'iniezione delle cellule. L’analisi elettrofisiologica è stata eseguita tramite esperimenti acuti di inserimento di elettrodi extracellulari nella corteccia cerebrale, in cui si sono misurati i potenziali evocati da stimoli visivi (VEP). Le analisi quantitative di questi esperimenti sono tutt’ora in corso. Nei topi registrati, ho poi misurato anche il volume totale del tumore. I dati preliminari, su 6 topi di controllo e 6 topi trattati con CNF1, indicano una riduzione nel volume del tumore in seguito al trattamento con CNF1. Sto anche assistendo alla realizzazione di impianti cronici di elettrodi extracellulari nella corteccia visiva dei topi iniettati con le cellule GL261, al fine di seguire variazioni dei VEP nel corso dello sviluppo del tumore. Ho lavorato anche su due sistemi per analizzare modifiche strutturali (come le spine dendritiche) nei neuroni circostanti il tumore: la colorazione di Golgi e l'utilizzo di topi con neuroni marcati con GFP. In conclusione, i risultati ottenuti suggeriscono un effetto positivo del CNF1 sulla sopravvivenza di topi affetti da glioma e una riduzione di volume della massa tumorale, che rende questa tossina un possibile candidato nella terapia per i tumori del sistema nervoso centrale

Epileptiform activity in the mouse visual cortex interferes with cortical processing in connected areas

Epileptiform activity is associated with impairment of brain function even in absence of seizures, as demonstrated by failures in various testing paradigm in presence of hypersynchronous interictal spikes (ISs). Clinical evidence suggests that cognitive deficits might be directly caused by the anomalous activity rather than by its underlying etiology. Indeed, we seek to understand whether ISs interfere with neuronal processing in connected areas not directly participating in the hypersynchronous activity in an acute model of epilepsy. Here we cause focal ISs in the visual cortex of anesthetized mice and we determine that, even if ISs do not invade the opposite hemisphere, the local field potential is subtly disrupted with a modulation of firing probability imposed by the contralateral IS activity. Finally, we find that visual processing is altered depending on the temporal relationship between ISs and stimulus presentation. We conclude that focal ISs interact with normal cortical dynamics far from the epileptic focus, disrupting endogenous oscillatory rhythms and affecting information processing

Understanding spreading depression from headache to sudden unexpected death

Spreading depression (SD) is a neurophysiological phenomenon characterized by abrupt changes in intracellular ion gradients and sustained depolarization of neurons. It leads to loss of electrical activity, changes in the synaptic architecture, and an altered vascular response. Although SD is often described as a unique phenomenon with homogeneous characteristics, it may be strongly affected by the particular triggering event and by genetic background. Furthermore, SD may contribute differently to the pathogenesis of widely heterogeneous clinical conditions. Indeed, clinical disorders related to SD vary in their presentation and severity, ranging from benign headache conditions (migraine syndromes) to severely disabling events, such as cerebral ischemia, or even death in people with epilepsy. Although the characteristics and mechanisms of SD have been dissected using a variety of approaches, ranging from cells to human models, this phenomenon remains only partially understood because of its complexity and the difficulty of obtaining direct experimental data. Currently, clinical monitoring of SD is limited to patients who require neurosurgical interventions and the placement of subdural electrode strips. Significantly, SD events recorded in humans display electrophysiological features that are essentially the same as those observed in animal models. Further research using existing and new experimental models of SD may allow a better understanding of its core mechanisms, and of their differences in different clinical conditions, fostering opportunities to identify and develop targeted therapies for SD-related disorders and their worst consequences

Small vessel disease and biomarkers of endothelial dysfunction after ischaemic stroke

Abstract Introduction: Although pathogenesis of small vessel disease is poorly understood, increasing evidence suggests that endothelial dysfunction may have a relevant role in development and progression of small vessel disease. In this crosssectional study, we investigated the associations between imaging signs of small vessel disease and blood biomarkers of endothelial dysfunction at two different time points in a population of ischaemic stroke patients. Patients and methods: In stroke patients treated with intravenous thrombolysis, we analysed blood levels of von Willebrand factor, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and vascular endothelial growth factor. Three reviewers independently assessed small vessel disease features using computed tomography. At baseline and 90 days after the index stroke, we tested the associations between single and combined small vessel disease features and levels of blood biomarkers using linear regression analysis adjusting for age, sex, hypertension, diabetes, smoke. Results: A total of 263 patients were available for the analysis. Mean age (SD) was 69 (13) years, 154 (59%) patients were male.We did not find any relation between small vessel disease and endothelial dysfunction at baseline. At 90 days, leukoaraiosis was independently associated with intercellular adhesionmolecule-1 (b¼0.21; p¼0.016) and vascular cell adhesionmolecule- 1 (b¼0.22; p¼0.009), and lacunes were associated with vascular endothelial growth factor levels (b¼0.21; p¼0.009) whereas global small vessel disease burden was associated with vascular endothelial growth factor (b¼0.26; p¼0.006). Discussion: Leukoaraiosis and lacunes were associated with endothelial dysfunction, which could play a key role in pathogenesis of small vessel disease

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.Peer reviewe

Operationalizing mild cognitive impairment criteria in small vessel disease: The VMCI-Tuscany Study

Introduction Mild cognitive impairment (MCI) prodromic of vascular dementia is expected to have a multidomain profile. Methods In a sample of cerebral small vessel disease (SVD) patients, we assessed MCI subtypes distributions according to different operationalization of Winblad criteria and compared the neuroimaging features of single versus multidomain MCI. We applied three MCI diagnostic scenarios in which the cutoffs for objective impairment and the number of considered neuropsychological tests varied. Results Passing from a liberal to more conservative diagnostic scenarios, of 153 patients, 5% were no longer classified as MCI, amnestic multidomain frequency decreased, and nonamnestic single domain increased. Considering neuroimaging features, severe medial temporal lobe atrophy was more frequent in multidomain compared with single domain. Discussion Operationalizing MCI criteria changes the relative frequency of MCI subtypes. Nonamnestic single domain MCI may be a previously nonrecognized type of MCI associated with SVD

Unraveling alterations of excitation/inhibition balance in in vivo models of epilepsy and genetic autism

One prominent feature of brain computation is the excitation inhibition balance (E/I balance) that represents one of the main homeostatic functions of the brain. Its aim is to maintain the neural circuits in a narrow and safe range of action. Within this range, the brain network can receive and analyze sensory inputs and produce a modulated output, proportional to the stimuli intensity. Any imbalance in this equilibrium leads to abnormal responses to external stimuli and results in pathological behavior. Indeed, neurological pathologies known for featuring a deep alteration of the E\u2010I balance are epilepsy and autism, which often occur together in the same patient. Several human genetic syndromes caused by alterations of genes involved in neural development feature signs like autism and epilepsy. Thus, they represent important cases for studying and understanding the role of these single altered genes in the development and regulation of the brain balance. In return, we hope that this knowledge of these genes and more generally of human brain network can be useful in treating the patients affected by these conditions and can help us improve their quality of life. In my work, I studied the regulation of the E/I balance in mouse models of neurological diseases from three different points of view. In the first set of experiments, I studied the E/I balance in a focal model of epileptiform activity. This model is produced by the local application of bicuculline to the mouse cortex. Bicuculline is a competitive GABAA receptor antagonist that, when applied, leads to the development of persistent and periodic interictal spikes at the injection site, while activity appears to be normal in nearby areas that are not reached by bicuculline. In our experiments, we showed that, even in the apparently normal area, there is a disruption of cortical computation. Specifically, the disruption occurs whenever an interictal spike is generated in the epileptic focus. This can have important impact on our understanding of epilepsy and of its treatment since interictal spikes are a common feature not only of epileptic patients, but can also appear in non\u2010epileptic subject, apparently without any consequence. From our results, we concluded that interictal activity can actually interfere with brain operation not only in the center of the epileptiform activity, but also in the connected areas, where the E/I balance is not directly disrupted. These results provide an example of the fact that apparently non\u2010symptomatic interictal spikes can affect brain computation. The second experimental model that I studied is a mouse model for a specific human genetic disease: the Phelan\u2010McDermid syndrome. This is a developmental disease, caused by a genomic deletion at site 22q13. The main suspect for causing the disease is one gene, Shank3, which encodes for a scaffold protein localized in the post\u2010synaptic density of glutamatergic synapses. In this model, we studied the computation of visual stimuli and we found an alteration of the contrast\u2010response curve. This is a defining relationship of visual processing: it is the transfer function that converts the visual input into a neural output. This means that to each intensity of visual stimulation corresponds a certain intensity of the neural response, of the visual cortex. We determined that, in Shank3 mutant mice, this curve was altered and showed an increased response to less intense stimuli and showed also a poor modulation of responses to high\u2010contrast stimulations. An interpretation of this can be that these mice are more sensitive to low\u2010contrast stimuli, but completely lose the ability of telling apart different high\u2010contrast stimuli from each other. Therefore, the Phelan McDermid mouse becomes \u201cblinded\u201d by weak stimulations as if they were seeing strong stimulus. Finally, we studied the behavior of the chloride ion in a drug\u2010induced epileptic seizure model. Chloride ion is of pivotal importance in neurons were the activation of ionotropic GABA and glycine receptors, which increase chloride membrane conductance in response to GABA or glycine release respectively. The intracellular concentration of chloride ions decides what is the effect of GABA release. Traditionally, ionotropic GABA receptors activation was thought to be inhibitory only, but the excitatory or inhibitory nature of these receptors is determined by the intracellular concentration of chloride ions. This concentration in normal adult neurons is thought to be around 5 mM: at this concentration, the effect of the activation of GABA receptors is an inhibition of the postsynaptic element. We investigated if the chloride concentration can be varied under extreme pathologic conditions as during epileptic seizures in a drug induced mouse model. In these animals, the epileptic seizures were produced by local administration of 4\u2010aminopyridine (4\u2010AP), a potassium channel antagonist. The effect of 4\u2010AP is to cause accumulation of chloride ions in neurons and this suggests that, in epileptic crisis, the role of inhibitory neurons can actually favor excitation

Neuroinflammation: A Signature or a Cause of Epilepsy?

Epilepsy can be both a primary pathology and a secondary effect of many neurological conditions. Many papers show that neuroinflammation is a product of epilepsy, and that in pathological conditions characterized by neuroinflammation, there is a higher probability to develop epilepsy. However, the bidirectional mechanism of the reciprocal interaction between epilepsy and neuroinflammation remains to be fully understood. Here, we attempt to explore and discuss the relationship between epilepsy and inflammation in some paradigmatic neurological and systemic disorders associated with epilepsy. In particular, we have chosen one representative form of epilepsy for each one of its actual known etiologies. A better understanding of the mechanistic link between neuroinflammation and epilepsy would be important to improve subject-based therapies, both for prophylaxis and for the treatment of epilepsy

Predictors for clinical and functional outcomes in stroke patients with first-pass complete recanalization after thrombectomy

Background and purpose: The aim was to identify baseline clinical and - predictors and 24--h radiological predictors for clinical and functional outcomes in stroke patients obtaining complete recanalization in one pass of mechanical thrombectomy (MT) in an optimal baseline and procedural setting.Methods: A retrospective analysis was conducted of prospectively collected data from 924 stroke patients with anterior large vessel occlusion, Alberta Stroke Program Early Computed Tomography (ASPECT) score >= 6 and pre-stroke modified Rankin Scale score 0, who started MT <= 6 h from symptom onset and obtained first-pass complete recanalization. A first logistic regression model was performed to identify baseline clinical predictors and a second model to identify baseline-predictors. A third model including baseline clinical and - predictors was performed, and a fourth model including independent baseline predictors from the third model plus 24-h radiological variables (hemorrhagic transformation [HT] and cerebral edema [CED]).Results: In the fourth model, higher National Institutes of Health Stroke Scale (NIHSS) score (odds ratio [OR] 1.089) and higher ASPECT score (OR 1.292) were predictors of early neurological improvement (ENI) (NIHSS score <= 4 points from baseline or NIHSS score of 0 at 24 h), whereas older age (OR 0.973), longer procedure time (OR 0.990), HT (OR 0.272) and CED (OR 0.569) were inversely associated with ENI. Older age (OR 0.970), diabetes mellitus (OR 0.456), higher NIHSS score (OR 0.886), general anesthesia (OR 0.454), longer onset-to-groin time (OR 0.996), HT (OR 0.340) and CED (OR 0.361) were inversely associated with 3-month excellent functional outcome (mRS score 0-1), whereas higher ASPECT score (OR 1.294) was a predictor of excellent outcome.Conclusions: Higher NIHSS score was a predictor of ENI but inversely associated with 3-month excellent outcome. Older age, HT and CED were inversely associated with both good outcomes

Endovascular treatment beyond 24 hours from the onset of acute ischemic stroke: the Italian Registry of Endovascular Thrombectomy in Acute Stroke (IRETAS)

Background Clinical trials and observational studies have demonstrated the benefit of thrombectomy up to 16 or 24 hours after the patient was last known to be well. This study aimed to evaluate the outcome of stroke patients treated beyond 24 hours from onset. Methods We analyzed the outcome of 34 stroke patients (mean age 70.7 +/- 12.3 years; median National Institutes of Health Stroke Scale (NIHSS) score 13) treated with endovascular thrombectomy beyond 24 hours from onset who were recruited in the Italian Registry of Endovascular Thrombectomy in Acute Stroke. Selection criteria for patients were: pre-stroke modified Rankin scale (mRS) score of <= 2, non-contrast CT Alberta Stroke Program Early CT score of >= 6, good collaterals on single phase CT angiography (CTA) or multiphase CTA, and CT perfusion mismatch with an infarct core size <= 50% of the total hypoperfusion extent or involving less than one-third of the extent of the middle cerebral artery territory evaluated by visual inspection. The primary outcome measure was functional independence assessed by the mRS at 90 days after onset. Safety outcomes were 90 day mortality and the occurrence of symptomatic intracranial hemorrhage (sICH). Results Successful recanalization (Thrombolysis in Cerebral Infarction score of 2b or 3) was present in 76.5% of patients. Three month functional independence (mRS score 0-2) was observed in 41.1% of patients. The case fatality rate was 26.5%. and the incidence of sICH was 8.8%. Conclusions These findings suggest that, in a real world setting, very late endovascular therapy is feasible in appropriately selected patients