Francesco Speciale

Altered Cytoskeletal Structure of Smooth Muscle Cells in Ureteropelvic Junction Obstruction

PURPOSE Ureteropelvic junction obstruction is one of the most common causes of hydronephrosis in children. A malfunction of smooth muscle cells is believed to be the underlying mechanism causing obstruction. We investigated the expression of some integrins, talin and β-dystroglycan, considered the main compound of smooth muscle cell cytoskeleton, and active caspase 3 at the level of the ureteropelvic junction obstruction. MATERIALS AND METHODS Specimens were obtained at pyeloplasty in 12 children with ureteropelvic junction obstruction. Six control specimens were obtained during organ explantation. Specimens were divided into renal pelvis, ureteropelvic junction and ureter below the obstruction. Western blot analysis of active caspase 3, and immunofluorescence and polymerase chain reaction analysis were performed for α7A, β1A, α7B and β1D integrins, talin and β-dystroglycan. RESULTS Talin and β-dystroglycan were slightly impaired in ureteropelvic junction obstruction, while α7B and β1D integrins were severely reduced, and α7A, β1A and active caspase 3 were significantly enhanced compared to controls. CONCLUSIONS We demonstrated activation of apoptosis and a critical alteration of cytoskeleton that might explain the altered function and the increased apoptosis in smooth muscle cells in ureteropelvic junction obstruction. The delayed rearrangement of the cytoskeleton of smooth muscle cells in ureteropelvic junction obstruction might be linked to a postnatal splicing from α7A and β1A to α7B and β1D integrins, respectively. This relationship could explain the common clinical scenario of spontaneous improvement of hydronephrosis in children with suspected ureteropelvic junction obstruction.

Sarcoglycans in the Normal and Pathological Breast Tissue of Humans: An Immunohistochemical and Molecular Study

The sarcoglycan complex, consisting of α-, β-, γ-, δ- and ε-sarcoglycans, is a multimember transmembrane system providing a mechanosignaling connection from the cytoskeleton to the extracellular matrix. Whereas the expression of α- and γ-sarcoglycan is restricted to striated muscle, other sarcoglycans are widely expressed. Although many studies have investigated sarcoglycans in all muscle types, insufficient data are available on the distribution of the sarcoglycan complex in nonmuscle tissue. On this basis, we used immunohistochemical and RT-PCR techniques to study preliminarily the sarcoglycans in normal glandular breast tissue (which has never been studied in the literature on these proteins) to verify the effective wider distribution of this complex. Moreover, to understand the role of sarcoglycans, we also tested samples obtained from patients affected by fibrocystic mastopathy and breast fibroadenoma. Our data showed, for the first time, that all sarcoglycans are always detectable in all normal samples both in epithelial and myoepithelial cells; in pathological breast tissue, all sarcoglycans appeared severely reduced. These data demonstrated that all sarcoglycans, not only β-, δ-, and ε-sarcoglycans, have a wider distribution, implying a new unknown role for these proteins. Moreover, in breast diseases, sarcoglycans containing cadherin domain homologs could provoke a loss of strong adhesion between epithelial cells, permitting and facilitating the degeneration of these benign breast tumors into malignant tumors. Consequently, sarcoglycans could play an important and intriguing role in many breast diseases and in particular in tumor progression from benign to malignant.

Morphofunctional compensation of masseter muscles in unilateral posterior crossbite patients

Unilateral posterior crossbite is a widespread, asymmetric malocclusion characterized by an inverse relationship of the upper and lower buccal dental cusps, in the molar and premolar regions, on one side only of the dental arch. Patients with unilateral posterior crossbite exhibit an altered chewing cycles and the crossbite side masseter results to be less active with respect to the contralateral one. Few studies about morphological features of masticatory muscle in malocclusion disorders exist and most of these have been performed on animal models. The aim of the present study was to evaluate morphological and protein expression characteristics of masseter muscles in patients affected by unilateral posterior crossbite, by histological and immunofluorescence techniques. We have used antibody against PAX-7, marker of satellite cells, and against α-, β-, γ-, δ-, ε- and ζ-sarcoglycans which are transmembrane glycoproteins involved in sarcolemma stabilization. By statistical analysis we have evaluated differences in amount of myonucley between contralateral and ipsilateral side. Results have shown: i) altered fibers morphology and atrophy of ipsilateral muscle if compared to the contralateral one; ii) higher number of myonuclei and PAX-7 positive cells in contralateral side than ipsilateral one; iii) higher pattern of fluorescence for all tested sarcoglycans in contralateral side than ipsilateral one. Results show that in unilateral posterior crossbite hypertrophic response of contralateral masseter and atrophic events in ipsilateral masseter take place; by that, in unilateral posterior crossbite malocclusion masticatory muscles modify their morphology depending on the function. That could be relevant in understanding and healing of malocclusion disorders; in fact, the altered balance about structure and function between ipsilateral and contralateral muscles could, long-term, lead and/ or worsen skeletal asymmetries.

Sarcoglycan complex in masseter and sternocleidomastoid muscles of baboons: an immunohistochemical study

The sarcoglycan complex consists of a group of single-pass transmembrane glycoproteins that are essential to maintain the integrity of muscle membranes. Any mutation in each sarcoglycan gene causes a series of recessive autosomal dystrophin-positive muscular dystrophies. Negative fibres for sarcoglycans have never been found in healthy humans and animals. In this study, we have investigated whether the social ranking has an influence on the expression of sarcoglycans in the skeletal muscles of healthy baboons. Biopsies of masseter and sternocleidomastoid muscles were processed for confocal immunohistochemical detection of sarcoglycans. Our findings showed that baboons from different social rankings exhibited different sarcoglycan expression profiles. While in dominant baboons almost all muscles were stained for sarcoglycans, only 55% of muscle fibres showed a significant staining. This different expression pattern is likely to be due to the living conditions of these primates. Sarcoglycans which play a key role in muscle activity by controlling contractile forces may influence the phenotype of muscle fibres, thus determining an adaptation to functional conditions. We hypothesize that this intraspecies variation reflects an epigenetic modification of the muscular protein network that allows baboons to adapt progressively to a different social status.

Sarcoglycan Complex in Human Normal and Pathological Prostatic Tissue: An Immunohistochemical and RT‐PCR Study

The sarcoglycan complex is a trans‐membrane system playing a key role in mechano‐signaling the connection from the cytoskeleton to the extracellular matrix. While β‐, δ‐, and ε‐sarcoglycans are widely distributed, γ‐ and α‐sarcoglycans are expressed exclusively in skeletal and cardiac muscle. Insufficient data are available on the distribution of sarcoglycans in nonmuscular tissue. In the present study, we used immunohistochemical and RT‐PCR techniques to study the sarcoglycans also in normal human glandular tissue, a type of tissue never studied in relation to the sarcoglycan complex, with the aim of verifying the real wider distribution of this complex. To understand the role of sarcoglycans, we tested specimens collected from patients affected by benign prostatic hyperplasia and adenocarcinoma. For the first time, our results showed that all sarcoglycans are detectable in normal samples both in epithelial and in myoepithelial cells; in pathological prostate, sarcoglycans appeared severely reduced in number or were absent. These data demonstrated that all sarcoglycans have a wider distribution suggesting a new unknown role for these proteins. The decreased number of sarcoglycans, containing cadherin domain homologs in samples of prostate affected by hyperplasia, and the absence of proteins in prostate biopsies, in cases affected by adenocarcinoma, could be responsible for the loss of adhesion between epithelial cells, which in turn facilitates the progression of benign tumors and the invasive potential of malignant tumors. Anat Rec, 297:327–336, 2014.

Painting a global picture of basal ganglia network: from past to present!

Since the 70s it has been thought that basal ganglia integrated sensorimotor, associative and limbic inputs and then projected this information through the thalamus to the motor cortex, supplementary motor area and frontal cortex, thus playing a relevant role in planning movement. Recent literature on basal ganglia networks is going beyond the classical “dogma” of dorsal striatum as the main station for cortical inputs in basal ganglia loops and several neurophysiological studies have suggested a more segregated organization of these neural circuits. In the classical view, various tract-tracing methods combined with immunohistochemistry and in situ hybridization demonstrated that the cortical information flows through the basal ganglia via a dual-network model, based on the “direct” and “indirect” routes. However, in addition to these two major projection systems, a glutamatergic hyper-direct pathway between cerebral cortex and subthalamic nucleus has been demonstrated first in monkeys and then in humans. Furthermore, we have recently shown a i) cortico-pallidal connection; ii) a cerebello-pallidal connection; iii) a cerebello nigral connection [1, 2]. Herein, we extensively examined basal ganglia network of fifteen healthy subjects by using probabilistic constrained spherical deconvolution tractography on magnetic resonance diffusion weighted imaging data and we also performed weighted connectivity analysis for each of the subcortical nuclei. In addition, we demonstrated for the first time tractographic evidences of the existence of a direct cortico-nigral pathway in humans. 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 findings would strength the hypothesis that the cortico-basal ganglia network consists of several, parallel, segregated, and functionally distinct, but homologous loop, and may be relevant for the comprehension of the pathophysiology of several basal ganglia disorders.

Morphometric evaluation of the pedicles of the lumbar spine according to L5 lateral tilt classification

A classification of the lumbar spine according to the pedicle lateral tilt (PLT) of L5 pedicle was recently proposed [1]. In this work the sample was divided into three categories, the first or Wing Type (WT) includes people with a PLT >36° (41,8%), the second or V Type (VT) includes people with a PLT between 30° and 36° (48%) and the third or U Type (UT) includes people with a PLT <30 (10,2%). The aim of the study is to evaluate the bone morphometric values and the distance between the pedicles and the nervous structures. Similar works are present in literature [2], but some are lack or for the size of the sample or for the parameters analysed. In our work seven parameters were considered: Pedicle Width (PW), Pedicle Height, Interpedicular Distance (IPD), Pedicle-Inferior Root Distance (PIRD), Pedicle-Superior Root Distance (PSRD), Root Exit Angle (REA), Nerve Root Diameter (NRD). In this study 325 patients were evaluated, a CT and MRI scan were taken to analyse respectively bone morphometry (CT) and distance between nervous structures (MRI). Statistically significant results were observed in five out seven categories, at L5: PW has a mean value of 18,5 mm in WT, 17,2 mm in VT and 15,8 mm for UT; PH has a mean value of 13,4 mm in WT, 12,8 mm (VT) and 11,2 mm in UT; IPD has a mean value of 29,2 mm in WT, 27,3 mm in VT and 25,8 mm in UT; PSRD has a mean value of 4,9 mm in WT, 4,6 mm in VT and 4,4 mm in UT; PDSD has a mean value of 1,9 mm in WT, 1,5 mm in VT and 1,3 mm in UT; REA has a mean value of 43° in WT, 40,2° in VT and 37,8° in UT. No differences were observed for PIRD (with a mean value of 1,5 mm) and NRD (with a mean value of 4 mm). Similar results were also observed for the pedicles of L4, whereas for the proximal pedicles (L3, L2 and L1) were not observed statistically significant differences into the three categories. In conclusion, the results obtained in this paper confirms the need to adopt our proposed classification according to the anatomic differences observed into our sample; in particular VT pedicle of L5 and L4 could be considered as “complicated” in a pedicle screw fixation surgery, based on the reduced bone volume and the close distance to nervous structures.

Muscle adaptations in relation to different types of strength training: an application of diffusion tensor imaging technique

Several exercise methods are commonly used by fitness practitioners to increase muscle strength and body mass. Pre-exhaustion (PE) is a strength training method of combining two exercises, in which a single-joint exercise performed exhaustively is followed by a multi-joint exercise. The purpose of PE of the smaller muscle is to provide lower involvement of this muscle in the subsequent multi-joint exercise, thereby enabling greater participation of other muscles. Conversely, in the post-exhaustion (PO) method is reversed the sequence of the exercises: first a determined muscle is trained with a multi-joint exercise and after with a single-joint exercise (Augustsson et Al., 2003). The purpose of this study was to investigate the effects of pre-exhaustion and post-exhaustion methods on gastrocnemius muscles by Diffusion Tensor Imaging (DTI) and volumetric evaluation techniques. DTI allows for a non-invasive evaluation of water diffusion and its fractional anisotropy (FA) in tissues. Four adults men (aged 22 +/- 0.81) have been divided in two groups by two different strength trainings: pre-exhaustion method (PE) and post-exhaustion (PO) method. All subjects have been performed 3 days a week for 3 months of training. Before and after training they have been subjected to conventional T1-weighted magnetic resonance. Results, after both training protocols, have shown a growth of muscle volume differentiated in each subject, probably connected to individual variables. Moreover, we have observed that the volumetric changes are related to the FA mean and it can be assumed that this remodeling of the can’t exclude, in addition to hypertrophy, phenomena of hyperplasia.

Neural correlates of fatigue in multiple sclerosis: a diffusion tensor imaging and transcranial magnetic stimulation study

Fatigue is a common and specific symptom in neurological diseases described as an overwhelming feeling of extreme exhaustion, and it concerns the inability to sustain a force or work rate during exercise, often defined as “objective fatigue”. Many patients displaying symptoms of physical and mental fatigue have no profound weakness, persistent or progressive cognitive decline or failure of peripheral neuromuscular function. This particular type of fatigue has been termed neurasthenia, frequently reported by patients suffering from multiple sclerosis (MS). The pathological substrate of behavioral deficit in MS is not entirely understood and may reside both in grey matter involvement and white matter injury, with derangement of white matter tracts architecture [1]. In order to investigate the role of connectivity alterations in the development of fatigue and behavioral impairment in MS patients, we examined 19 MS patients combining neurophysiological paradigm by means of Transcranial Magnetic Stimulation (TMS), cognitive assessment and Magnetic Resonance Imagining (MRI) with Diffusion Tensor Imaging (DTI) based tools. DTI is a noninvasive MRI technique [2] that can be used to probe the structural integrity of white matter through quantitative parameters, such as Fractional Anysotropy (FA). We were able to reconstruct the anterior thalamic projections and the caudate-cortical loop tracts, finding lower FA values in the right hemispheres, which results in weaker connectivity in patients compared with controls. In addition, we analysed the thalamic and basal ganglia volume, showing a significant atrophy of striatum and thalamus in fatigued patients, correlated to the neurodegenerative process of the striatum-thalamus loop. We assume that fatigue and cognitive impairment could be related to micro-structural impairment of white matter tracts of the cortical-subcortical-cortical loop.

Sarcoglycan subcomplex during embryonic life of rats: an immunohistochemical study

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other member of dystrophin-glycoprotein complex (DGC) in order to provide a mechano-signaling connection from the cytoskeleton to the extracellular matrix in myocytes. Previous investigations have demonstrated that in skeletal and cardiac muscle, the SGC is a heterotetrameric unit constituted by the a-, b-, g-, and d-sarcoglycans. Other authors demonstrated that the expression of a-sarcoglycan is restricted to striated muscle cells, whereas e-sarcoglycan, is also expressed in several other tissues. Moreover, further analysis showed the presence, in vascular and visceral smooth muscle, of other sarcoglycan subcomplex, consisting of e-, b-, g-, and d-sarcoglycan, associated with sarcospan. Previous our studies have demonstrated presence of sarcoglycans also in non-muscle tissue as prostatic and breast glandular epithelial tissues in normal and pathological conditions, hypothesizing a key role for these glycoproteins in mediating the signalling between cell and extracellular matrix (1,2). Furthermore, some Authors have studied the presence of sarcoglycans also in fetal tissues demonstrating that the signals of these proteins in fetal liver are weak or absent (3), or that their immunostaining is clearly detectable (4). Although these discordant assumptions, insufficient data are present on sarcoglycans during fetal period. On this basis, in order to verify composition of sarcoglycan subcomplex during this period, we analysed fetuses of rat at 8 and 14 days using immunohistochemical techniques, observing several organs at fetal stage, as well liver, kidney and brain. Our results have shown that all sarcoglycans are constantly present in all tested embryonic organs and that their staining pattern was more detectable that that the adult life. In our opinion, these data demonstrated that, also in fetal period, sarcoglycans are present confirming a key function for these proteins in regulating of transduction signalling. In this way, sarcoglycan subcomplex could play a crucial role in cytodifferentiation processes in order to check the development of several organs during embryonic life.