Fabio Trimarchi

Cortical and Subcortical Connections of the Human Claustrum Revealed In Vivo by Constrained Spherical Deconvolution Tractography

The claustrum is a thin layer of gray matter that is at the center of an active scientific debate. Recently, Constrained Spherical Deconvolution (CSD) tractography has proved to be an extraordinary tool allowing to track white matter fibers from cortex to cortical and subcortical targets with subvoxel resolution. The aim of this study was to evaluate claustral connectivity in the human brain. Ten normal brains were analyzed by using the High Angular Resolution Diffusion Imaging CSD-based technique. Tractography revealed 4 groups of white matter fibers connecting the claustrum with the brain cortex: Anterior, posterior, superior, and lateral. The anterior and posterior cortico-claustral tracts connected the claustrum to prefrontal cortex and visual areas. The superior tract linked the claustrum with sensory-motor areas, while the lateral pathway connected the claustrum to the auditory cortex. In addition, we demonstrated a claustral medial pathway connecting the claustrum with the basal ganglia, specifically with caudate nucleus, putamen, and globus pallidus. An interesting and exciting new finding was the demonstration of a bilateral connection between claustrum and contralateral cortical areas and a well-represented interclaustral communication with interconnection bundles interspersed within the bulk of the trunk of the corpus callosum. The physiological and pathophysiological relevance of these findings are discussed.

Distribution and Localization of Vinculin-Talin-Integrin System and Dystrophin-Glycoprotein Complex in Human Skeletal Muscle

The vinculin-talin-integrin system and the dystrophin-glycoprotein complex (DGC) are two protein systems with structural and signaling functions, allowing interaction between muscle fibers and extracellular matrix. Although numerous studies have been conducted on these systems, their localization and distribution patterns along the nonjunctional sarcolemma are not clear. On this basis, we carried out an indirect immunofluorescence study on the vastus lateralis muscle of human adults not affected by neuromuscular diseases to better define these patterns. Our results showed that all tested proteins of the two systems have a costameric distribution; all tested proteins of the two systems colocalize with each other (about 90–95% of the cases); only α-sarcoglycan in a few cases (about 6%) does not colocalize with other proteins; in about 9–10% of the cases, dystrophin and β-dystroglycan colocalize partially with other proteins; all tested proteins can be localized in different fibers, both in the region of the sarcolemma over I or A bands. The colocalization between the vinculin-talin-integrin and DGC systems may imply their functional interaction involving the structural aspect, by providing a stronger adhesion between sarcolemma and extracellular matrix in well-defined regions of the muscle fiber. Besides, their colocalization may suggest the existence of a mechanism of mutual modulation of the transmitted signals. This reciprocal control may determine, in different conditions, the prevalence of one system over another with a consequent transmission of different messages to the sarcolemma-associated cytoskeleton.

Extensive Direct Subcortical Cerebellum-Basal Ganglia Connections in Human Brain as Revealed by Constrained Spherical Deconvolution Tractography

The connections between the cerebellum and basal ganglia were assumed to occur at the level of neocortex. However evidences from animal data have challenged this old perspective showing extensive subcortical pathways linking the cerebellum with the basal ganglia. Here we tested the hypothesis if these connections also exist between the cerebellum and basal ganglia in the human brain by using diffusion magnetic resonance imaging and tractography. Fifteen healthy subjects were analyzed by using constrained spherical deconvolution technique obtained with a 3T magnetic resonance imaging scanner. We found extensive connections running between the subthalamic nucleus and cerebellar cortex and, as novel result, we demonstrated a direct route linking the dentate nucleus to the internal globus pallidus as well as to the substantia nigra. These findings may open a new scenario on the interpretation of basal ganglia disorders.

Constrained spherical deconvolution analysis of the limbic network in human, with emphasis on a direct cerebello-limbic pathway.

The limbic system is part of an intricate network which is involved in several functions like memory and emotion. Traditionally the role of the cerebellum was considered mainly associated to motion control; however several evidences are raising about a role of the cerebellum in learning skills, emotions control, mnemonic and behavioral processes involving also connections with limbic system. In 15 normal subjects we studied limbic connections by probabilistic Constrained Spherical Deconvolution (CSD) tractography. The main result of our work was to prove for the first time in human brain the existence of a direct cerebello-limbic pathway which was previously hypothesized but never demonstrated. We also extended our analysis to the other limbic connections including cingulate fasciculus, inferior longitudinal fasciculus, uncinated fasciculus, anterior thalamic connections and fornix. Although these pathways have been already described in the tractographic literature we provided reconstruction, quantitative analysis and Fractional Anisotropy (FA) right-left symmetry comparison using probabilistic CSD tractography that is known to provide a potential improvement compared to previously used Diffusion Tensor Imaging (DTI) techniques. The demonstration of the existence of cerebello-limbic pathway could constitute an important step in the knowledge of the anatomic substrate of non-motor cerebellar functions. Finally the CSD statistical data about limbic connections in healthy subjects could be potentially useful in the diagnosis of pathological disorders damaging this system.

Culture of human skeletal muscle myoblasts: timing appearance and localization of dystrophin-glycoprotein complex and vinculin-talin-integrin complex.

The dystrophin-glycoprotein complex together with the vinculin-talin-integrin complex plays an important role in muscle function; in fact the mutations of their elements lead to diverse forms of muscular dystrophies. The relationship between the elements of dystrophin-glycoprotein complex and vinculin-talin-integrin and the time course of their formation are still not known in detail. In order to better understand this relationship we studied their expression during development in normal human skeletal muscle culture. Using a standardized muscle cell culture procedure, this study was performed to analyze the timing, appearance and the localization of some proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin complex during cellular proliferation (myoblast) and differentiation (4, 7, 15 and 21 days). The indirect immunofluorescence technique was used and cells were examined using a Meta Zeiss LSM510 confocal laser scanning inverted microscope. We examined the progressive appearance of the following proteins: α, β, γ, δ-sarcoglycans, β-dystroglycan, dystrophin, talin, vinculin and integrin isoform α7/β1. Immunofluorescence of these proteins, in satellite cells entering myogenic differentiation, revealed different patterns of localization depending on the time of culture. We showed that nondifferentiated cultures of human myoblasts expressed a perinuclear distribution of all proteins tested. During myoblast differentiation into myotubes (4 days) immunofluorescence gradually increased and was located in the whole cytoplasm. Subsequently, at day 7, a strong and homogeneous cytoplasmic labelling of all proteins was seen. At 15 days the distribution of the proteins was on the membrane. At this time some myotubes displayed a significant degree of precostameric banding pattern. As fusion proceeded at 21 days, the cytodistribution progressively changed and appeared along fibrillar longitudinal structures, and myotubes showed a clear periodic distribution (costameres). In conclusion, in normal human muscle cultures DGC and vinculin-talin-integrin proteins are first localized in the perinuclear region, then they diffuse in the cytoplasm and finally form at the plasma membrane into typical rib-like structures that are sarcolemma-associated.

Sarcoglycans in Human Skeletal Muscle and Human Cardiac Muscle: A Confocal Laser Scanning Microscope Study

Sarcoglycans are a subcomplex of transmembrane proteins which are part of the dystrophin-glycoprotein complex. They are expressed in the skeletal, cardiac and smooth muscle. Although numerous studies have been conducted on the sarcoglycan subcomplex in skeletal and cardiac muscle, the manner of the distribution and localization of these proteins along the nonjunctional sarcolemma is not clear. We therefore carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle and of healthy human atrial myocardium biopsies of patients affected by valvulopathy. Our results indicate that, in skeletal muscle, sarcoglycans have a costameric distribution and all colocalize with each other. Only in a few cases did the α-sarcoglycan not colocalize with other sarcoglycans. In addition, these glycoproteins can be localized in different fibers either in the regions of the sarcolemma over band I or band A. In cardiac muscle, our results show a costameric distribution of all proteins examined and, unlike in skeletal muscle, they show a constant colocalization of all sarcoglycans with each other, along with a consistent localization of these proteins in the region of the sarcolemma over band I. In our opinion, this situation seems to confirm the hypothesis of a correlation between the region of the sarcolemma occupied by costameric proteins and the metabolic type, fast or slow, of the muscular fibers. These data, besides opening a new line of research in understanding interactions between the sarcoglycans and other transmembrane proteins, could also be extended to skeletal and cardiac muscles affected by neuromuscular and cardiovascular pathologies to understand possible structural alterations.

Immunofluorescence distribution of actin-associated proteins in human seminiferous tubules of adolescent testes, normal and pathologic.

The aim of our study on human seminiferous tubules of adolescent testes was to study the localization of two actin-associated proteins of the adherens junctions, such as vinculin and talin, and to verify if there were modifications in their pattern in varicocele, a frequent disease of the testis in adolescent age. The study group consisted of 8 biopsies from normal testes (i.e., adolescents operated on for hydrocele or inguinal hernia) and 20 biopsies from pathological testes (i.e., adolescents operated on for idiophatic left varicocele). Biopsies were evaluated by indirect immunofluorescence using anti-human vinculin and anti-human talin antibodies. Observation was recorded with a Leica TCS 4D upright confocal microscope. In the normal testes, there was a strong positive immunoreactivity for vinculin, which was localized in the interstitial cells of Leydig, and both basal pole and lateral cell surface of Sertoli cells; the pattern of talin immunoreactivity was the same except that the lateral cell surface of Sertoli cells was not stained. In the varicocele group the pattern was different. Vinculin immunoreactivity showed small patches of fluorescence only in the cytoplasm of Sertoli cells while talin immunoreactivity showed a scanty distribution at the basal surface of Sertoli cells. These results confirm that, similarly to other tissues, vinculin is expressed at cell-cell and cell-matrix adherens junctions, while talin is present at cell-matrix adherens junctions in human seminiferous tubules of normal adolescents. Varicocele alters the patterns of these two proteins both quantitatively and qualitatively.

Volume rendering based on magnetic resonance imaging: advances in understanding the three‐dimensional anatomy of the human knee

The choice of medical imaging techniques, for the purpose of the present work aimed at studying the anatomy of the knee, derives from the increasing use of images in diagnostics, research and teaching, and the subsequent importance that these methods are gaining within the scientific community. Medical systems using virtual reality techniques also offer a good alternative to traditional methods, and are considered among the most important tools in the areas of research and teaching. In our work we have shown some possible uses of three‐dimensional imaging for the study of the morphology of the normal human knee, and its clinical applications. We used the direct volume rendering technique, and created a data set of images and animations to allow us to visualize the single structures of the human knee in three dimensions. Direct volume rendering makes use of specific algorithms to transform conventional two‐dimensional magnetic resonance imaging sets of slices into see‐through volume data set images. It is a technique which does not require the construction of intermediate geometric representations, and has the advantage of allowing the visualization of a single image of the full data set, using semi‐transparent mapping. Digital images of human structures, and in particular of the knee, offer important information about anatomical structures and their relationships, and are of great value in the planning of surgical procedures. On this basis we studied seven volunteers with an average age of 25 years, who underwent magnetic resonance imaging. After elaboration of the data through post‐processing, we analysed the structure of the knee in detail. The aim of our investigation was the three‐dimensional image, in order to comprehend better the interactions between anatomical structures. We believe that these results, applied to living subjects, widen the frontiers in the areas of teaching, diagnostics, therapy and scientific research.

Constrained Spherical Deconvolution Tractography Reveals Cerebello-Mammillary Connections in Humans.

According to the classical view, the cerebellum has long been confined to motor control physiology; however, it has now become evident that it exerts several non-somatic features other than the coordination of movement and is engaged also in the regulation of cognition and emotion. In a previous diffusion-weighted imaging-constrained spherical deconvolution (CSD) tractography study, we demonstrated the existence of a direct cerebellum-hippocampal pathway, thus reinforcing the hypothesis of the cerebellar role in non-motor domains. However, our understanding of limbic-cerebellar interconnectivity in humans is rather sparse, primarily due to the intrinsic limitation in the acquisition of in vivo tracing. Here, we provided tractographic evidences of connectivity patterns between the cerebellum and mammillary bodies by using whole-brain CSD tractography in 13 healthy subjects. We found both ipsilateral and contralateral connections between the mammillary bodies, cerebellar cortex, and dentate nucleus, in line with previous studies performed in rodents and primates. These pathways could improve our understanding of cerebellar role in several autonomic functions, visuospatial orientation, and memory and may shed new light on neurodegenerative diseases in which clinically relevant impairments in navigational skills or memory may become manifest at early stages.

Expression of muscle-specific integrins in masseter muscle fibers during malocclusion disease

Integrins are heterodimeric cell surface membrane proteins linking the extracellular matrix to actin. α7B integrin is detected in proliferating and adult myofibers, whereas α7A plays a role in regenerating muscle fibers with a minor function in mature muscle fibers. The expression levels of β1A appear to be very low, whereas β1D appears to be the predominant integrin form in mature muscle. Considering the important features of masseter muscle we have studied integrin expression in masseter muscle specimens of surgical patients with posterior right crossbite and comparing them to left side masseter muscle specimens. Our results showed that the expression of integrins was significantly lower in the crossbite side muscle. Furthermore, the most important finding is that β1A is clearly detectable in adult masseter muscle. This behavior could be due to the particular composition of masseter, since it contains hybrid fibers showing the capacity to modify the contractile properties to optimize the energy efficiency or the action of the muscle during contraction. Moreover, masseter is characterized by a high turnover of muscle fibers producing a regeneration process. This may indicate a longer time to heal, justifying the loss of β1D and the consequential increase of β1A. Thus, our data provide the first suggestion that integrins in masseter muscle play a key role regulating the functional activity of muscle and allowing the optimization of contractile forces.