Giuseppina Rizzo

Basal ganglia network by constrained spherical deconvolution: a possible cortico-pallidal pathway?

In the recent past, basal ganglia circuitry was simplified as represented by the direct and indirect pathways and by hyperdirect pathways. Based on data from animal studies, we hypothesized a fourth pathway, the cortico‐pallidal, pathway, that complements the hyperdirect pathway to the subthalamus. Ten normal brains were analyzed by using the high angular resolution diffusion imaging—constrained spherical deconvolution (CSD)‐based technique. The study was performed with a 3T magnetic resonance imaging (MRI) scanner (Achieva, Philips Healthcare, Best, Netherlands); by using a 32‐channel SENSE head coil. We showed that CSD is a powerful technique that allows a fine evaluation of both the long and small tracts between cortex and basal ganglia, including direct, indirect, and hyperdirect pathways. In addition, a pathway directly connecting the cortex to the globus pallidus was seen. Our results confirm that the CSD tractography is a valuable technique allowing a reliable reconstruction of small‐ and long‐fiber pathways in brain regions with multiple fiber orientations, such as basal ganglia. This could open a future scenario in which CSD could be used to focally target with deep brain stimulation (DBS) the small bundles within the basal ganglia loops.

Costameric proteins in human skeletal muscle during muscular inactivity.

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin–glycoprotein complex and vinculin–talin–integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive‐motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive‐motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of α7B‐integrin, β1D‐integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of α7A‐integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of α7B‐ and β1D‐integrin mRNA and a normal, or slightly higher than normal, level of α7A‐integrin mRNA in the skeletal muscle fibres of the patients with sensitive‐motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with α7B‐integrin being replaced by α7A‐integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down‐regulation of integrins and agrin in muscular dystrophies.

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.

Sarcoglycan Subcomplex Expression in Normal Human Smooth Muscle

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other members of the dystrophin–glycoprotein complex (DGC) to provide a mechanosignaling connection from the cytoskeleton to the extracellular matrix. The SGC consists of four proteins (α, β, γ, and δ). A fifth sarcoglycan subunit, ∊-sarcoglycan, shows a wider tissue distribution. Recently, a novel sarcoglycan, the ζ-sarcoglycan, has been identified. All reports about the structure of SGC showed a common assumption of a tetrameric arrangement of sarcoglycans. Addressing this issue, our immunofluorescence and molecular results showed, for the first time, that all sarcoglycans are always detectable in all observed samples. Therefore, one intriguing possibility is the existence of a pentameric or hexameric complex considering ζ-sarcoglycan of SGC, which could present a higher or lower expression of a single sarcoglycan in conformity with muscle type—skeletal, cardiac, or smooth—or also in conformity with the origin of smooth muscle. (J Histochem Cytochem 55:831–843, 2007)

Sarcoglycan and integrin localization in normal human skeletal muscle: a confocal laser scanning microscope study

Many studies have been performed on the sarcoglycan sub-complex and a7B and b1D integrins, but their distribution and localization patterns along the non-junctional sarcolemma are still not clear. We have carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle, performing double localization reactions with antibodies to sarcoglycans, integrins and sarcomeric actin. Our results indicate that the tested proteins colocalize with each other. In a few cases, a-sarcoglycan does not colocalize with the other sarcoglycans and integrins. We also demonstrated, by employing antibodies to all the tested proteins, that these proteins can be localized to regions of the sarcolemma corresponding either to the I-band or A-band. Our results seem 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 muscle fibers. On this basis, we suggest that slow fibers are characterized by localization of costameric proteins to I-bands, while fast fibers are characterized by localization of costameric proteins to A-bands. The results open a new line of research in understanding interactions between the components of the DGC and vinculin-talin-integrin complexes in the context of different fiber types. Moreover, the same results may be extended to skeletal muscle fibers affected by neuromuscular diseases to detect possible structural alterations.

Dentin Morphology of Root Canal Surface: A Quantitative Evaluation Based on a Scanning Electronic Microscopy Study

Dentin is a vital, hydrated composite tissue with structural components and properties that vary in the different topographic portions of the teeth. These variations have a significant implication for biomechanical teeth properties and for the adhesive systems utilized in conservative dentistry. The aim of this study is to analyse the root canal dentin going from coronal to apical zone to find the ratio between the intertubular dentin area and the surface occupied by dentin tubules varies. Observations were conducted on 30 healthy premolar teeth extracted for orthodontic reasons in patients aged between 10 and 14. A SEM analysis of the data obtained in different canal portions showed that, in the coronal zone, dentinal tubules had a greater diameter (4.32 μm) than the middle zone (3.74 μm) and the apical zone (1.73 μm). The average number of dentinal tubules (in an area of 1 mm2) was similar in coronal zone (46,798 ± 10,644) and apical zone (45,192 ± 10,888), while in the middle zone they were lower in number (30,940 ± 7,651). However, intertubular dentin area was bigger going from apical to coronal portion. The differences between the analysed areas must be considered for the choice of the adhesive system.

Integrins, muscle agrin and sarcoglycans during muscular inactivity conditions: an immunohistochemical study

Sarcoglycans are transmembrane proteins that seem to be functionally and pathologically as important as dystrophin. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle. It has been proposed that the dystrophin-glycoprotein complex (DGC) links the actin cytoskeleton with the extracellular matrix and the proper maintenance of this connection is thought to be crucial to the mechanical stability of the sarcolemma. The integrins are a family of heterodimeric cell surface receptors which play a crucial role in cell adhesion including cell-matrix and intracellular interactions and therefore are involved in various biological phenomena, including cell migration, and differentiation tissue repair. Sarcoglycans and integrins play a mechanical and signaling role stabilizing the systems during cycles of contraction and relaxation. Several studies suggested the possibility that integrins might play a role in muscle agrin signalling. On these basis, we performed an immunohistochemical analyzing sarcoglycans, integrins and agrin, on human skeletal muscle affected by sensitive-motor polyneuropathy, in order to better define the correlation between these proteins and neurogenic atrophy due to peripheral neuropathy. Our results showed the existence of a cascade mechanism which provoke a loss of regulatory effects of muscle activity on costameres, due to loss of muscle and neural agrin. This cascade mechanism could determine a quantitative modification of transmembrane receptors and loss of alpha7B could be replaced and reinforced by enhanced expression of the alpha7A integrin to restore muscle fiber viability. Second, it is possible that the reduced cycles of contraction and relaxation of muscle fibers, during muscular atrophy, provoke a loss of mechanical stresses transmitted over cell surface receptors that physically couple the cytoskeleton to extracellular matrix. Consequently, these mechanical changes could determine modifications of chemical signals through variations of pathway structural integrins, and alpha7A could replace alpha7B.

A Direct Cortico-Nigral Pathway as Revealed by Constrained Spherical Deconvolution Tractography in Humans.

Substantia nigra is an important neuronal structure, located in the ventral midbrain, that exerts a regulatory function within the basal ganglia circuitry through the nigro-striatal pathway. Although its subcortical connections are relatively well-known in human brain, little is known about its cortical connections. The existence of a direct cortico-nigral pathway has been demonstrated in rodents and primates but only hypothesized in humans. In this study, we aimed at evaluating cortical connections of substantia nigra in vivo in human brain by using probabilistic constrained spherical deconvolution (CSD) tractography on magnetic resonance diffusion weighted imaging data. We found that substantia nigra is connected with cerebral cortex as a whole, with the most representative connections involving prefrontal cortex, precentral and postcentral gyri and superior parietal lobule. These results may be relevant for the comprehension of the pathophysiology of several neurological disorders involving substantia nigra, such as parkinsons disease, schizophrenia, and pathological addictions.

Myofibroblasts in adolescent varicocele: an ultrastructural and immunohistochemical study

Abstract Myofibroblasts of the testes play an important role in the morphofunctional integrity of the seminiferous tubule. Previous studies in adults with varicocele have demonstrated an involvement of this cell population that tends to transform into fibroblasts. The aim of the present study was to try and verify the morphological features of myofibroblasts in the adolescent with left idiophatic varicocele. Twenty-two testicular biopsies were obtained from adolescents (aged 13–18 years, mean 15.8) and operated for left idiophatic varicocele. Biopsies were processed for electron microscopy (TEM) and immunofluorescence studies. The latter determined the level of myofibroblasts by using α-smooth muscle isoactin, a specific marker of myofibroblasts and, hence, excluding fibroblasts. TEM observations revealed a normal ultrastructure of myofibroblasts that was similar to that for the controls but an increased presence of extracellular matrix. The immunofluorescence study always demonstrated strong cell positivity to anti α-smooth muscle isoactin as also seen in the controls. This study demonstrates that adolescents with varicocele have well preserved myofibroblasts and do not show any evidence of transformation into fibroblasts, this has already been demonstrated in adult varicocele. These observations could represent an important factor for the understanding of the reversal of growth failure of the testes observed after early treatment.

A Connectomic Analysis of the Human Basal Ganglia Network

The current model of basal ganglia circuits has been introduced almost two decades ago and has settled the basis for our understanding of basal ganglia physiology and movement disorders. Although many questions are yet to be answered, several efforts have been recently made to shed new light on basal ganglia function. The traditional concept of “direct” and “indirect” pathways, obtained from axonal tracing studies in non-human primates and post-mortem fiber dissection in the human brain, still retains a remarkable appeal but is somehow obsolete. Therefore, a better comprehension of human structural basal ganglia connectivity in vivo, in humans, is of uttermost importance given the involvement of these deep brain structures in many motor and non-motor functions as well as in the pathophysiology of several movement disorders. By using diffusion magnetic resonance imaging and tractography, we have recently challenged the traditional model of basal ganglia network by showing the possible existence, in the human brain, of cortico-pallidal, cortico-nigral projections, which could be mono- or polysynaptic, and an extensive subcortical network connecting the cerebellum and basal ganglia. Herein, we aimed at reconstructing the basal ganglia connectome providing a quantitative connectivity analysis of the reconstructed pathways. The present findings reinforce the idea of an intricate, not yet unraveled, network involving the cerebral cortex, basal ganglia, and cerebellum. Our findings may pave the way for a more comprehensive and holistic pathophysiological model of basal ganglia circuits.