Fabrizio Pizzolante

In Vivo Morphological Changes in Animal Models of Amyotrophic Lateral Sclerosis and Alzheimer's‐Like Disease: MRI Approach

Magnetic resonance imaging (MRI) is the only noninvasive technique that provides structural information on both cell loss and metabolic changes. After reviewing all the results obtained in clinical studies, reliable biomarkers in neurological diseases are still lacking. Diffusional MRI, MR spectroscopy, and the assessment of regional atrophy are promising approaches, but they cannot be simultaneously used on a single patient. Thus, for further research progress, reliable animal models are needed. To this aim, we have used the clinical MRI to assess neurodegenerative processes in the hSOD‐1G93A ALS rat model and in the trimethyltin (TMT)‐treated model of Alzheimers‐like disease. T2‐weighted (T2W) hyperintensive neurodegenerative foci were found in the brainstem of the ALS rat with apparent lateral ventricle dilation (T1W—hypointensity vs. T2W—hyperintensity). Degenerative processes in these areas were also confirmed by confocal images of GFAP‐positive astrogliosis. MRI after i.v.i. of magnetic anti‐CD4 antibodies indicated an accumulation of inflammatory cells near dilated ventricles. TMT‐treated rats also revealed the dilation of lateral ventricles. Expected deterioration in the hippocampus was not observed by clinical MRI, but immunocytochemistry could reveal significant redistribution of macro‐ and microglia in this structure. In both models, Gd‐DTPA contrast revealed a compromised blood brain barrier that may serve as the passage for inflammatory immune cells in the vicinity of dilated lateral ventricles. Moreover, in both models the midbrain region of the dorsal hippocampus was the target of BBB compromise, thus revealing a potentially vulnerable point that can be the primary target of neurodegeneration in the central nervous system. Anat Rec, 292:1882–1892, 2009.

Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration

Trimethyltin chloride (TMT) is a neurotoxicant producing neuronal degeneration and reactive astrogliosis in the mammalian central nervous system, especially the hippocampus. A previous magnetic resonance imaging investigation in TMT-treated rats evidenced dilation of lateral ventricles, also suggesting alterations in blood-brain barrier permeability and brain edema. Aquaporin 4 (AQP4), a glial water channel protein expressed mainly in the nervous system, is considered a specific marker of vascular permeability and thought to play an important role in brain edema (conditions). We studied AQP4 expression in the hippocampus and cerebral cortex of TMT-treated rats in order to explore the molecular mechanisms involved in brain edema occurring in these experimental conditions. Real-time PCR and western blotting data showed significant up-regulation of both AQP4 mRNA and protein levels starting 14 days after TMT treatment in the hippocampus and cortex. Parallel immunofluorescence studies indicated intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting 14 days after TMT intoxication. In order to study the effects of TMT on vascular integrity, double-label immunofluorescence experiments for rat immunoglobulin G (IgG) and rat endothelial cell antigen-1 (RECA-1) or neuronal nuclei (NeuN) (endothelial and neuronal markers respectively) were performed. The results indicated, at 21 and 35 days after treatment, the presence of rat IgG in paravasal parenchyma and in some neuronal cells of the hippocampus and cortex. The extravasated IgG staining was temporally correlated with over-expression of neuronal vascular endothelial growth factor (VEGF) and the active phosphorylated form of its neuronal receptor (VEGFR-2P), suggesting that these factors may cooperate in mediating vascular leakage.

Congenital extrahepatic portosystemic shunt: description of four cases and review of the literature

Congenital extrahepatic portosystemic shunt (CEPS), also known as Abernethy malformation, is a rare condition in which the splenomesenteric blood drains directly into a systemic vein, bypassing the liver through a complete or partial shunt. The diagnosis is frequently made during childhood in the setting of neonatal cholestasis, hypergalactosemia, failure to thrive, mental retardation or other congenital defects. In adulthood, CEPS is usually found incidentally during diagnostic work-up for abdominal pain, liver test abnormalities, liver nodules, portopulmonary hypertension, portopulmonary syndrome or portosystemic encephalopathy. The diagnosis depends on imaging and portal venography, but sometimes only liver biopsy can be resolutive, demonstrating the absence of venules within the portal areas. Here we report four recent cases of Abernethy malformation diagnosed in young adults, in which ultrasound (US) was the initial imaging technique and allowed to suspect the diagnosis. Furthermore, we reviewed clinical presentations, associated anomalies and treatment of the 310 cases of CEPS previously reported in the literature.SommarioLo shunt portosistemico extraepatico congenito (CEPS), anche noto come malformazione di Abernethy, è una rara condizione patologica in cui il sangue venoso refluo dal distretto spleno-mesenterico drena direttamente nel circolo venoso sistemico, bypassando il fegato attraverso un shunt completo o parziale. La diagnosi viene posta frequentemente durante l’infanzia in presenza di colestasi neonatale, ipergalattosemia, scarso accrescimento o altri difetti congeniti. Durante l’età adulta la diagnosi di CEPS è spesso incidentale nel corso di accertamenti diagnostici per dolore addominale, alterazioni degli indici di funzionalità epatica, riscontro di lesioni focali epatiche, ipertensione porto-polmonare, sindrome porto-polmonare ed encefalopatia porto-sistemica. Le tecniche di imaging o la porto-venografia consentono solitamente di porre la diagnosi, ma in alcuni casi solo la biopsia epatica risulta dirimente, documentando l’assenza di strutture venose a livello degli spazi portali. Nel presente lavoro vengono presentati 4 casi di malformazione di Abernethy diagnosticati in giovani adulti, nei quali l’ultrasonografia (US) è stata la prima tecnica di immagine utilizzata consentendo di porre il sospetto diagnostico. Inoltre, abbiamo analizzato la presentazione clinica, le anomalie associate e le opzioni terapeutiche dei 310 casi di CEPS riportati in letteratura.

Aquaporin 4 expression increases in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration

Trimethyltin (TMT) is a neurotoxicant know to produce significant and selective neuronal degeneration in the rodent CNS (for review, 1). Magnetic resonance imaging (MRI) investigation in TMT-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability .In order to explore the molecular mechanisms involved in the phenomenon we have investigated in the hippocampus and cortex of TMT-treated rats the expression of aquaporin 4 (AQP4), a glial water channel protein believed to play a role in brain oedematous conditions. AQP4 expression was tested both by real-time PCR and western blotting analysis in hippocampus and cortex homogenates. To confirm molecular results and visualize the AQP4 cell distribution double-label immunofluorescence for AQP4 and GFAP was performed. Real-time PCR and western blotting data show a significant upregulation of AQP4 starting from 14 days of TMT treatment both in the hippocampus and the cortex. Accordingly, the immunofluorescence shows an intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. In particular, AQP4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels. The study of the Rhodamine B fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of TMT-treated rats, indicating a vascular involvement in the TMT-induced neurodegenerative processes.. AQP4 over-expression and astrogliosis occurring in the brain of TMT-treated rats might putatively play a role in alterations of vascular permeability and brain oedema formation evidenced by MRI studies.

Aquaporin 4 (AQP4) expression and blood brain barrier damage in an experimental model of neurodegeneration induced by trimethyltin

Trimethyltin (TMT) is well known to produce a distinct pattern of selective neuronal degeneration in the rodent CNS. TMT intoxication is also an important factor linked to induction of brain edema. Aquaporin-4 (AQP4), a water transporting protein, is thought to be the primary route through which water moves in and out astrocytes, is over-expressed in some pathological conditions, and is considered a marker of vascular permeability. The aim of our study was to investigate the integrity of the blood brain barrier (BBB), and the expression of AQP4 in astrocytes, after TMT intoxication. The brains of adult female Wistar rats, treated and untreated with TMT, were isolated and consecutive coronal sections obtained with a vibratome were incubated with primary fluorescent antibodies AQP4-Ig goat and GFAP-Ig rabbit. Imaging of fluorescence was performed on a confocal laser scanning microscope (Zeiss). In addition, dissected hippocampi and cortex were homogenized and the proteins were separated by SDS-PAGE. Although data in literature document absence of alterations of the BBB in TMT treated rats, our preliminary published data of the MRI investigation with Gd-DTPA suggested the presence of such alterations that concur with the passage of contrast into the damaged tissue. We have analyzed the AQP4 expression 7, 14, 21 and 35 days after TMT exposure. Immunofluorescence and Western Blotting analysis have showed an upregulation of the AQP4 in astrocytes after TMT intoxication., the expression levels of which progressively increased after TMT exposure, both in hippocampus and in brain cortex. All the data suggest that the astrocytes and their AQP4 protein are involved in the brain edema formation and in the possible alteration of the vascular permeability, in this TMT model of neurodegeneration.