Effetti del litio nei nuclei motori dei nervi cranici in un modello sperimentale di sclerosi laterale amiotrofica (SLA)

RIASSUNTO Introduzione. La sclerosi laterale amiotrofica (SLA) è una malattia neurodegenerativa caratterizzata da una perdita selettiva dei motoneuroni nel sistema nervoso centrale. La degenerazione del motoneurone nella SLA coinvolge sia i motoneuroni superiori localizzati nella corteccia motoria sia quelli inferiori del midollo spinale e del tronco dell’encefalo. Vari studi sperimentali e clinici indicano che il trattamento cronico con sali di litio produce effetti promettenti, aumentando la sopravvivenza e rallentando il decorso della malattia. I topi transgenici G93A che over-esprimono forme mutate dell’enzima rame-zinco superossido-dismutasi di tipo 1 (SOD1) umano sviluppano una patologia molto simile a quella osservata nei pazienti affetti da SLA e per questo sono comunemente utilizzati come modello animale di SLA. Nella SLA umana, il coinvolgimento dei nuclei motori dei nervi encefalici contribuisce ad aggravare il quadro clinico e si trova solitamente associato a un decorso accelerato della malattia. Tuttavia, fino a questo momento, solo pochi e frammentari sono gli studi sperimentali che indagano i nuclei motori del tronco dell’encefalo nei topi G93A. Scopo dello studio. L’obiettivo primario di questo studio è stato quello di analizzare in maniera estensiva, da un punto di vista morfologico, tutti i nuclei motori del tronco dell’encefalo nel topo transgenico G93A, cercando di offrire un quadro il più possibile esaustivo che documenti il loro coinvolgimento in questo modello sperimentale di SLA. In un secondo momento l’indagine morfologica è stata rivolta ad analizzare l’effetto del trattamento cronico con sali di litio sui nuclei motori del tronco encefalico del topo G93A. Materiali e metodi. Topi transgenici G93A e i rispettivi wild-type littermate sono stati suddivisi in 4 gruppi sperimentali: wild-type+salina, wild-type+litio, G93A+salina, G93A+litio. Il trattamento con soluzione salina o carbonato di litio (corrispondente ad una dose di litio di 1 mEq/Kg) è avvenuto a giorni alterni a partire dall’insorgenza dei sintomi fino allo stadio finale della malattia (stadio tetraplegico), quando tutti gli animali sono stati sacrificati e l’encefalo prelevato e processato per l’inclusione in paraffina. Nel corso del trattamento, gli animali sono stati sottoposti ad osservazioni comportamentali, basate sull’impiego di test motori in grado di rivelare l’insorgenza e la progressione dei sintomi. Per ogni nucleo motore del tronco encefalico sono state eseguite sezioni seriate intervallate di 50 m, sulle quali sono state condotte tutte le analisi stereologiche, istochimiche e immunoistochimiche, allo scopo di valutare la presenza di cambiamenti nel numero dei motoneuroni, nella loro morfologia e nell’immunopositività per specifici marcatori. Allo scopo di rilevare il potenziale effetto neurogenetico del trattamento cronico con litio, un sottogruppo di animali è stato preventivamente trattato con il precursore modificato del DNA 5-bromo-2-deossiuridina (BrdU). In questi animali è stata valutata l’entità dell’incorporazione nucleare di tale composto all’interno dei nuclei motori del tronco encefalico. Risultati. Da questo studio è emerso che: a) I nuclei motori dei nervi trigemino, faciale, ipoglosso, il nucleo ambiguo e il nucleo motore dorsale del vago risultano significativamente compromessi nel topo G93A. La conta stereologica e l’analisi istologica e immunoistochimica dimostrano infatti una consistente perdita di motoneuroni all’interno dei suddetti nuclei e la presenza di alterazioni morfologiche nei motoneuroni superstiti nei topi G93A trattati con salina. Al contrario, i nuclei dei nervi responsabili dei movimenti oculari (oculomotore, trocleare, abducente) non appaiono interessati dal processo degenerativo che colpisce gli altri nuclei motori del topo G93A. b) Il trattamento cronico con litio dei topi G93A aumenta il numero dei motoneuroni nei nuclei lesi (ad eccezione del nucleo motore dorsale del vago) significativamente rispetto ai topi G93A trattati con salina, preserva la normale morfologia cellulare nei motoneuroni superstiti e i bottoni sinaptici ChAT-positivi sui motoneuroni del nucleo ipoglosso. Nel contempo, riduce la gliosi reattiva e induce neuronogenesi. Conclusioni. Nell’insieme, questi risultati indicano che: a) il topo G93A riproduce, anche a livello del tronco dell’encefalo, la SLA umana; b) in questo modello sperimentale il trattamento con litio sembra particolarmente protettivo per i motoneuroni somatici del tronco encefalico

High-intensity exercise training induces morphological and biochemical changes in skeletal muscles

IN THE PRESENT STUDY WE INVESTIGATED THE EFFECT OF TWO DIFFERENT EXERCISE PROTOCOLS ON FIBRE COMPOSITION AND METABOLISM OF TWO SPECIFIC MUSCLES OF MICE: the quadriceps and the gastrocnemius. Mice were run daily on a motorized treadmill, at a velocity corresponding to 60% or 90% of the maximal running velocity. Blood lactate and body weight were measured during exercise training. We found that at the end of training the body weight significantly increased in high-intensity exercise mice compared to the control group (P=0.0268), whereas it decreased in low-intensity exercise mice compared to controls (P=0.30). In contrast, the food intake was greater in both trained mice compared to controls (P < 0.0001 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively). These effects were accompanied by a progressive reduction in blood lactate levels at the end of training in both the exercised mice compared with controls (P=0.03 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively); in particular, blood lactate levels after high-intensity exercise were significantly lower than those measured in low-intensity exercise mice (P=0.0044). Immunoblotting analysis demonstrated that high-intensity exercise training produced a significant increase in the expression of mitochondrial enzymes contained within gastrocnemius and quadriceps muscles. These changes were associated with an increase in the amount of slow fibres in both these muscles of high-intensity exercise mice, as revealed by the counts of slow fibres stained with specific antibodies (P < 0.0001 for the gastrocnemius; P=0.0002 for the quadriceps). Our results demonstrate that high-intensity exercise, in addition to metabolic changes consisting of a decrease in blood lactate and body weight, induces an increase in the mitochondrial enzymes and slow fibres in different skeletal muscles of mice, which indicates an exercise-induced increase in the aerobic metabolism

High-intensity exercise training induces morphological and biochemical changes in skeletal muscles

Skeletal muscle shows an elevated plasticity and can adapt its metabolic and contractile properties in response to a variety of stimuli such as physical exercise. This implies a series of biochemical and morphological changes in the recruited muscle, in order to produce the more appropriate functional response dependent on the specific stimulation. To determine the effective role of physical exercise in the muscle plasticity, in the present study we investigated the effect of two different exercise protocols on fiber composition and metabolism of two specific muscles of mice: the quadriceps -a fast-twitch muscle- and the gastrocnemius -a typical slow-twitch muscle. Mice were run daily on a motorized treadmill for 8 weeks, at a velocity corresponding to 60% (low-intensity exercise) or 90% (high-intensity exercise) of the maximal running velocity previously determined by an incremental exercise test. We found that at the end of training the body weight was significantly increased in highintensity exercise mice (18.2 ± 1.4 %) compared to low-intensity exercise (8.7 ± 0.6 %) and control (12.7 ± 0.5 %) groups, and it was lesser in low-intensity exercise mice compared to controls. In contrast, the food intake of both exercise training mice was greater compared to control group. Whereas low-intensity exercise mice, despite consumed significantly more food compared to control mice, increased the weight lesser, the weight increase of high-intensity exercise mice, that consumed significantly more food compared to other experimental groups, was significantly greater. These effects were accompanied by a progressive reduction in blood lactate levels at the end of training in both the exercised mice compared with controls; in particular, blood lactate levels after highintensity exercise were significantly lower than those measured in low-intensity exercise mice. Moreover, in the present study we demonstrated that high-intensity exercise training produced a significant increase in the expression of mitochondrial complex enzymes (significant for the enzymes corresponding to the Complex IV, II and I of mitochondrial chain) both in gastrocnemius and quadriceps muscle, compared with controls. These changes were associated with an increase in the amount of slow fibers in both these muscle of high-intensity exercise mice. No changing in the expression of mitochondrial enzymes and in the percentage of slow fibers were found in low-intensity exercise mice

A new model of exercise training in mice: a preliminary study

Physical exercise produces a variety of effects, which might result either beneficial or negative, depending on several factors such as exercise intensity and duration. In particular, exercise training consisting of a repetition of exercises bouts over time, generally results in enhanced work capacity, mainly due to metabolic and systemic adaptations aiming to re-establish and maintain a condition of homeostasis which is disrupted during each acute exercise session. The present study, carried out in mice, shows preliminary data concerning the effect on general physical conditions and aerobic endurance capacity, namely body weight and lactate production, of two distinct training schedules both consisting of daily forced run on a treadmill for 8 weeks, but with a different intensity: a) brief sequences of intense exercise (at 90% of maximal velocity) interspersed with recovery periods (2 min running - 1 min recovery) (HIT); b) continuous exercise at moderate activity (corresponding to 60% of maximal intensity) (LOW). Distance to run was fixed to 1,000 meters for both trainings. Sedentary mice were used as control (CON). Training was preceded by a first preliminary phase consisting of a period of adaptation to running on the treadmill, followed by an incremental exercise test, in order to determine, for each mouse, the maximal running velocity. CON mice performed the first phase but were leaved in their cages during the training. At baseline conditions and during the training body weight (once a week) and plasma levels of lactate (at T1, T20, T40) were measured in exercising mice and compared with CON. For each mouse the total amount of food intake during the training was also measured. At the end of the training we found that the increase in the body weight observed in HIT mice was higher than the increase found in CON mice, suggesting that high-intensity training produces a positive effect on the body growth. In contrast, in LOW mice body weight increased significantly less than CON mice, despite LOW mice had an higher total food intake than CON. Moreover, plasma lactate was found significantly decreased in both exercising mice compared with CON, confirming the efficacy of the regular exercise in inducing muscular metabolic adaptation. This appeared more markedly in HIT mice, suggesting that a very intense exercise has a stronger impact on muscular metabolism. Studies are in progress in order to investigate whether and at which extent these changes are accompanied by morpho-functional modifications in many peripheral organs and in the nervous system, as well

Altered intercellular diffusion of misfolded proteins in neuroglia.

In the present study we performed ultrastructural dissection of cell-to-cell communication in vitro using an experimental model of glioblastoma multiforme. This experimental approach was combined with immunocytochemistry and staining for glycated end products. Evidence was provided showing an increased amount of misfolded proteins including prion protein and alpha synuclein which are released in the form of glycated compounds

Comparative ultrasonographic evaluation of the achilles paratenon in symptomatic and asymptomatic subjects: an imaging study

Medicine as well as in the common clinical practice. One of the most widely used modality to diagnose pathologies of the achilles tendon is ultrasonography (US), which is fast, repeatable, and allows dynamic assessment of the tendon gliding. However, literature studies demonstrate that only moderate correlation exists between the US appearance of the tendon and the clinical assessment of several achilles tendinopathies. For this reason we have recognized the need to consider the paratenon tissue as an integral part of the picture. In this study, sonography was used to evaluate 22 subjects complaining pain in the mid-portion of the achilles tendon and 22 healthy subjects; moreover the Victorian Institute of Sport Assessment-Achilles questionnaire, a reliable clinical index of Achilles tendinopathy severity, was administered to all participants. A significant inter-group difference was found in terms of paratenon thickness(p=0.0001). Moreover paratenon thickness was found to be positively correlated with Achilles tendinopathy severity and duration of symptoms. These findings confirm those of Harris and Leung, who found alterations in signal intensity and paratenon thickening in patients with tendinitis. In light of these results, we suggest a carefully analysis of paratenon thickness when evaluating patients with Achillodynia using ultrasound. We assume that an altered paratenon (1.27mm or above) can be not only a significant indicator of Achilles tendinophaty but, in line with the theory of Perez, it can also be a precursor sign of tendon alteration. Moreover it can be postulated that most of the symptoms are generated by the stretching of the free nerve ending of the paratenon rather than by morphological alteration of the tendon