Fabio Gualtieri | Ludwig-Maximilians-Universität München (original) (raw)

Papers by Fabio Gualtieri

Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional sta... more Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional states can promote ictogenesis and epileptogenesis. So far, research has focused on the role of high-mobility group box protein 1 as an activator of TLRs. However, the development of targeting strategies might need to consider a role of additional receptor ligands. Considering the fact that heat shock protein A1 (hsp70) has been confirmed as a TLR 2 and 4 ligand, we have explored the consequences of its overexpression in a mouse kindling paradigm. The genetic modulation enhanced seizure susceptibility with lowered seizure thresholds prior to kindling. In contrast to wildtype (WT) mice, HSPA1A transgenic (TG) mice exhibited generalized seizures very early during the kindling paradigm. Along with an increased seizure severity, seizure duration proved to be prolonged in TG mice during this phase. Toward the end of the stimulation phase seizure parameters of WT mice reached comparable levels. However, a difference between genotypes was still evident when comparing seizure parameters during the postkindling threshold determination. Surprisingly, HSPA1A overexpression did not affect microglia activation in the hippocampus. In conclusion, the findings demonstrate that hsp70 can exert pro-convulsant effects promoting ictogenesis in naı¨ve animals. The pronounced impact on the response to subsequent stimulations gives first evidence that genetic HSPA1A upregulation may also contribute to epileptogenesis. Thus, strategies inhibiting hsp70 or its expression might be of interest for prevention of seizures and epilepsy. However, conclusions about a putative pro-epileptogenic effect of hsp70 require further investigations in models with development of spontaneous recurrent seizures.

Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused... more Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies. followed by pharmacoresistant subjects ( 2190/patient). Using another standardized assessment index, the international dollar purchasing power parities, candidates to neurosurgery are 25% of all cases and account for 5401,whereaspatientsaffectedbypharmacoresistanceare205401, whereas patients affected by pharmacoresistance are 20% of all cases and account for 5401,whereaspatientsaffectedbypharmacoresistanceare203010. In Italy, it has been estimated that 16% of the total direct costs for antiepileptic drugs is on newly diagnosed epilepsy, 41% on patients with seizure remission, 58% on patients with occasional seizures, and 77% on patients with pharmacoresistant epilepsy [1]. For these reasons, pharmacoresistant TLE represents large part of costs required to the health system in order to assist patients affected by epilepsy.

Bollettino - Lega Italiana contro l'Epilessia

We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metaboli... more We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metabolite desacyl-ghrelin in rats exposed to pilocarpine-induced seizures. Pilocarpine (380 mg/kg) was administered to Sprague-Dawley rats pretreated with scopolamine, followed by saline (control group) or the tested hormone (ghrelin and desacyl-ghrelin at 1.5 mg/kg). After induction of status epilepticus (SE), seizures were quelled by injecting diazepam (20 mg/kg). Four days after SE, rats were sacrificed to evaluate lesions in hippocampus. Pilocarpine did not induce seizures in 20% and SE was prevented in 60% of desacyl-ghrelin treated rats. In ghrelin treated group, all rats developed seizures and SE. Interestingly, both ghrelin and desacyl-ghrelin prevented post-SE mortality. Hippocampal lesions were larger in control rats than in ghrelin or desacyl-ghrelin treated rats (p < 0.05 unpaired Student’s t-test). We thereby conclude that desacyl-ghrelin possesses antiepileptic properties in th...

Current Medicinal Chemistry, 2014

NeuroMolecular Medicine, 2013

An early but transient decrease in oxygen availability occurs during experimentally induced seizu... more An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 to 120 min, counts of pimonidazoleimmunoreactive neurons also increased (P \ 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazoleimmunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazole-immunopositive neurons expressed remarkable immunoreactivity to hypoxiainducible factor 1a (HIF-1a). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1a and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1a, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation. equally contributed to the work.

NeuroMolecular Medicine, 2013

Plos One, 2013

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth h... more In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagoguereceptor 1a (GHS-R 1a ). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R 1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.4560.07 mm 2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P,0.05) and JMV-1843 (P,0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P,0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P,0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Plos One, 2013

Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audioge... more Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ FosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ FosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ FosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ FosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2immunopositive cells significantly decreased (-75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ FosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.

An early but transient decrease in oxygen availability occurs during experimentally induced seizu... more An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 to 120 min, counts of pimonidazole-immunoreactive neurons also increased (P < 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazole-immunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazole-immunopositive neurons expressed remarkable immunoreactivity to hypoxia-inducible factor 1α (HIF-1α). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1α and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1α, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation.

Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet not systemically... more Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet not systemically investigated astrocytic and vascular injuries. To investigate their possible association with neuronal damage, the changes in glial fibrillary acidic protein (GFAP), laminin and neuron-specific nuclear protein (NeuN) immunoreactivities were analyzed in rats treated with pilocarpine (380 mg/kg) or kainate (15 mg/ kg), and receiving diazepam (20 mg/kg) after 10 min of SE. A different group of rats was injected with endothelin-1 (ET-1) in the caudate putamen to reproduce the changes in GFAP and laminin immunoreactivities associated with ischemia. Focal loss of GFAP immunostaining was accompanied by increased laminin immunoreactivity in blood vessels, in all the examined groups. Regression analysis revealed a significant (P < 0.01) relationship between astrocytic lesion and increased laminin immunoreactivity in the piriform cortex (Pir) of both pilocarpine (R 2 = 0.88) and kainate (R 2 = 0.94) groups of treatment. A significant relationship (P < 0.01; R 2 = 0.81) was also present in the cornu Ammonis 3 (CA3) hippocampal region of pilocarpine-treated rats. At variance, neuronal and glial lesions were significantly related (P < 0.05, R 2 = 0.74) only in the substantia nigra of pilocarpine-treated rats. The ratio between areas of GFAP and laminin changes of immunoreactivity in the ET-1 group was similar to those found in pilocarpine-and kainate-treated rats in specific brain regions, such as the hippocampal CA3 subfield, Pir and the anterior olfactory nucleus. The amygdala and submedius thalamic nucleus in the pilocarpine group, and the perirhinal and entorhinal cortices in the kainate group, also presented ischemic-like changes. These results indicate that laminin immunoreactivity is upregulated in the basal lamina of blood vessels after SE induced by pilocarpine or kainate. This phenomenon is significantly associated with lesions involving more glial than neuronal cells, in specific cerebral regions.

It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this st... more It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR 1a ) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5 mg/kg), desacyl-ghrelin (1.5 mg/kg), hexarelin (330 μg/kg), EP-80317 (330 μg/kg), JMV-1843 (330 μg/kg), JMV-2959 (330 μg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic-clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P b 0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P b 0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P b 0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR 1a ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR 1a ligands display relevant anticonvulsive properties in models of limbic seizures.

We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metaboli... more We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metabolite desacyl-ghrelin in rats exposed to pilocarpine-induced seizures. Pilocarpine (380 mg/kg) was administered to Sprague-Dawley rats pretreated with scopolamine, followed by saline (control group) or the tested hormone (ghrelin and desacyl-ghrelin at 1.5 mg/kg). After induction of status epilepticus (SE), seizures were quelled by injecting diazepam (20 mg/kg). Four days after SE, rats were sacrificed to evaluate lesions in hippocampus. Pilocarpine did not induce seizures in 20% and SE was prevented in 60% of desacyl-ghrelin treated rats. In ghrelin treated group, all rats developed seizures and SE. Interestingly, both ghrelin and desacyl-ghrelin prevented post-SE mortality. Hippocampal lesions were larger in control rats than in ghrelin or desacyl-ghrelin treated rats (p < 0.05 unpaired Student's t-test). We thereby conclude that desacyl-ghrelin possesses antiepileptic properties in the pilocarpine model of temporal lobe epilepsy.

Posters by Fabio Gualtieri

Introduction: the hippocampus is a well-defined neuroanatomical structure that may not act as a u... more Introduction: the hippocampus is a well-defined neuroanatomical structure that may not act as a unit. The dorsal (Septal pole) and ventral (Temporal pole) portions of it are believed to be involved in different processes: long-term memory and hypothalamic–pituitary–adrenal (HPA) axis regulation, respectively. In addition to this, the dentate gyrus (DG) is a well-known source of adult neurogenesis. OUR AIM was to compare neurogenesis in the dorsal and ventral parts of the hippocampus in both standard and environmental enriched conditions. Methods: CD-1 female mice (n=96, age 11 weeks) underwent two experimental treatments, ENRICHED and CONTROL environmental conditions. The enrichment condition consisted of i) running wheels, ii) increased space for social activity and iii) dirty bedding coming from C57BL6 male mice. The control condition instead, consisted of standard housing in accordance with the UK’s 3Rs guidelines. Experiments lasted 8 days and brains were collected and divided a...

The hippocampus is a well-defined anatomical structure belonging to the brain’s limbic system tha... more The hippocampus is a well-defined anatomical structure belonging to the brain’s limbic system that may not act as a unitary structure, with the dorsal (Septal pole) and ventral (Temporal pole) portions of it being involved in different roles. Functionally, it has always been associated with long-term memory and spatial navigation, but it also modulates corticosteroid feedback through the type I & II glucocorticoid receptors and one of its sub regions, the dentate gyrus (DG), is a source for neurogenesis. Our aim is to compare the difference in neurogenesis within dorsal and ventral DG in both standard condition and in an environmental enrichment paradigm to identify which subdivision is more sensitive. In our experiment we used 96 eleven-week-old CD-1 female mice, running four replicate experiments in each of which 24 animals were randomly divided into 4 ENRICHED and 4 CONTROL cages. Enrichment consisted of: i) running wheels, ii) increased space for social activity and iii) murine ...

Bollettino - Lega Italiana contro l'Epilessia

Current Medicinal Chemistry, 2014

NeuroMolecular Medicine, 2013

Plos One, 2013

An early but transient decrease in oxygen availability occurs during experimentally induced seizu... more An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 to 120 min, counts of pimonidazole-immunoreactive neurons also increased (P < 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazole-immunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazole-immunopositive neurons expressed remarkable immunoreactivity to hypoxia-inducible factor 1α (HIF-1α). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1α and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1α, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation.

We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metaboli... more We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metabolite desacyl-ghrelin in rats exposed to pilocarpine-induced seizures. Pilocarpine (380 mg/kg) was administered to Sprague-Dawley rats pretreated with scopolamine, followed by saline (control group) or the tested hormone (ghrelin and desacyl-ghrelin at 1.5 mg/kg). After induction of status epilepticus (SE), seizures were quelled by injecting diazepam (20 mg/kg). Four days after SE, rats were sacrificed to evaluate lesions in hippocampus. Pilocarpine did not induce seizures in 20% and SE was prevented in 60% of desacyl-ghrelin treated rats. In ghrelin treated group, all rats developed seizures and SE. Interestingly, both ghrelin and desacyl-ghrelin prevented post-SE mortality. Hippocampal lesions were larger in control rats than in ghrelin or desacyl-ghrelin treated rats (p < 0.05 unpaired Student's t-test). We thereby conclude that desacyl-ghrelin possesses antiepileptic properties in the pilocarpine model of temporal lobe epilepsy.