Preliminary study on sarcoglycan sub-complex in rat cerebral and cerebellar cortex

The sarcoglycan sub-complex is a protein system which plays a key role in sarcolemma stabilization during muscle activity. Although numerous studies have been conducted on this system, there are few data about its localization in non-muscular tissues. On this basis we carried out an indirect immunofluorescence study on normal rat cerebral and cerebellar cortex. In particular, we carried out single localization reactions to analyze if these proteins are present in brain and double localization reactions between sarcoglycans and either SMI-32 or GFAP to verify if they are expressed both in neurons and glial cells. We found that all tested sarcoglycans are present both in cerebral and cerebellar cortex and that they are expressed both in neurons and glial cells. The typical staining pattern of all sarcoglycans is represented by “spot-like” fluorescence, with spots of 0.5-2 μm average diameter laid out mainly around the soma of the cells. The main difference about sarcoglycans expression between cerebral and cerebellar cortex is that in the cerebellar cortex the sarcoglycans positivity is detectable only in an area which is likely to correspond to Purkinje cells layer. The presence of sarcoglycans in cerebral and cerebellar cortex and their disposition mainly around the soma of the cells suggest a role of these proteins in cellular signalling and in regulating postsynaptic receptor assembly mainly in axo-somatic synapses

Sarcoglycans and GABAA Rε receptors in cerebral cortex, thalamus and hippocampus: an immunohistochemical study

The Sarcoglycans sub-complex is a protein system which plays a role in sarcolemma stabilization, protecting the fibers by any injury provoked by muscle activity. This complex is composed by six transmembrane glycoproteins, α-,β-,γ-,δ-,ε- and ξ-sarcoglycans and, although numerous studies have been conducted on this system, there are no many data about its localization in non-muscular tissues. In our previous study we have analyzed the sarcoglycans expression and localization in rat’s cerebral cortex and our results showed that all sarcoglycans are present with a staining pattern in relation to the cerebral cortex area observed. In particular we think that they could be associated with synapse sites such as inhibitory GABAA Rε receptors. In order to verify any association between sarcoglycans and GABAA Rε receptors we performed double immunolabeling to detect α-,β-,γ-,δ-,ε- and ξ-sarcoglycans and GABAA Rε receptor. Our results have shown that in cerebral cortex each sarcoglycans is equally associated with GABAA Rε receptor, showing some point of colocalization around the cellular soma. Moreover, we observed the reaction in thalamus and hippocampus where we found that all sarcoglycans are expressed with the same “spot-like” staining pattern that we observed in cerebral cortex. Instead, in the extension of the neurons the proteins present a linear staining pattern. We have also found that in these districts the fluorescence pattern of GABAA Rε receptor increase together with the sarcoglycans fluorescence pattern, supporting our previous idea about a tight correlation between sarcoglycans and GABAA Rε receptors. These results suggest again a role of sarcoglycans in cellular signalling, regulating the post-synaptic receptor assembly. On this basis it could be hypothesized that sarcoglycans could be involved in some pathologies of the brain becoming, in these districts, as important as in muscle

Use of Immunofluorescence Technique to Perform a Quantitative Analysis of Masseter Muscle Fibers in Unilateral Posterior Crossbite: A Pilot Study

Unilateral posterior crossbite is a type of malocclusion that involves morpho-functional characteristics of masticatory muscle, such as the masseter: electrophysiological data have shown that the affected side works less than the contralateral muscle, which shows a normal or increased activity, probably in order to compensate for the affected side. The aim of present work was to measure the diameter and the cross-sectional area of ipsilateral and contralateral muscle fibers to verify if hypertrophy and/or hypotrophy take place in this malocclusion. We used immunofluorescence pictures to measure, using ImageJ software, the diameter and the cross-sectional area of fibers from control and crossbite groups; after that, the data were processed to perform statistical analyses. Results show that the fiber diameters of contralateral muscle are larger than the diameters of ipsilateral and control fibers, and that this difference is statistically significant. No statistically significant difference was found between the fiber diameters of the ipsilateral and control muscles. All these data suggest that, during unilateral posterior crossbite, morphological changes take place in the contralateral masseter muscle, which undergoes hypertrophy, probably to compensate for the low activity of the affected muscle

Sarcoglycan sub-complex in the adipose organ: a molecular and immunofluorescence study

The sarcoglycan sub-complex (SGC) is made up of six glycoproteins which connect the cytoskeleton to the extracellular matrix in skeletal muscle. Recent data show that this complex is also expressed in epithelial tissue such as gingival, breast and prostatic ones [1]. The adipose organ is organized in multiple fat depots consisting of white and brown adipose cells. White adipocytes can store energy in triglycerides; brown adipocytes can dissipate energy for thermogenesis. It has been demonstrated that white adipocytes transdifferentiate to brown adipocytes after cold exposure [3]. In this study we examined the expression of sarcoglycans (SGs) in the adipose organ from two groups of mice: the first group was exposed at 25 C°, as control, and the second one was exposed at low temperature (4 C°) for 24 hours and 4 days. Fat depots from the visceral and interscapular region, but also from the mammary gland, have been examined by histological, immunofluorescence and molecular techniques. Results have shown that SGC is expressed in the adipose organ, both in brown and white adipocytes of mice exposed at 25 C°. The main results is that the expression level of all sarcoglycans increase in cold exposure experiment. For the first time the expression of all SGs in the adipose organ has been demonstrated, both at mRNA and protein levels. Since we found an increase in SGs expression after transdifferentiation from white to brown adipocytes cold exposure induced, we hypothesize that sarcoglycans could be associated with β3 adrenergic receptor; sarcoglycans associations with other receptors, as GABAr, has been already demonstrated in central nervous system. Although that, the function of these glycoproteins in the adipose organ remain still unclear and further investigation are required

Sarcoglycan sub-complex in WAG/Rij rats, a model of absence epilepsy: an immunofluorescence study

It is known that even in Central Nervous System the Dystrophin Glycoprotein Complex (DGC) exists but differs in composition from the DGC core present in muscle for the presence of several isoforms of dystrophin and for the existence of a sarcoglycan sub-complex which is made up only for ε- and ζ-sarcoglycans; for these reasons it was called “DGC-like”. Although that, in our previous studies we have found that all sarcoglycans are present in human cerebral cortex and in different regions of rat’s brain, suggesting that the composition of the brain DGC is not different from the muscle DGC but differing just for the function. In fact, our previous data showing the colocalization between sarcoglycans and GABAA Rε receptors in rat’s cerebral and cerebellar cortex, thalamus and hippocampus suggested us that in brain sarcoglycans could be associated with synaptic neurotransmission. To better understand which kind of relationship between sarcoglycans and GABAA receptors exists, we aim to investigate α-,β-,γ-,δ-,ε- and ζ-sarcoglycans and the GABAA Rε in the brain of the WAG/Rij rats, a model of absence epilepsy, using immunofluorescence techniques; we have observed cerebral cortex, thalamus and hippocampus, the structures mainly involved in absence epilepsy, comparing this to normal rat’s brain. Results show that the GABAA Rε receptors staining pattern is different from the normal rat’s brain presenting an abnormal fluorescence distribution consisting in discontinuous clusters around the cellular body. Sarcoglycans, instead, have shown the typical “spot-like” staining pattern around the cellular body, sometimes colocalizing with GABAA Rε receptor; moreover, they seem to have an higher staining pattern than normal rat’s brain. In our opinion these results, showing an alteration of GABAA Rε receptor pattern distribution and a normal or increased sarcoglycans staining pattern, allow us to hypothesize that the sarcoglycans in absence epilepsy are likely to compensate for the alteration in GABAA Rε receptor distribution. That support the opinion about the involvement of sarcoglycans in cellular signalling and receptor assembly regulating indirectly synaptic neurotransmission both in normal and in pathological condition

Sarcoglycans and mucin in epithelial tissues of digestive and respiratory tracts: an immunofluorescence study

Sarcoglycans are transmembrane glycoproteins which play a key role in maintaining sarcolemma stabilization during muscle contraction. Several studies have demonstrated that this complex is not muscle specific and that it is also expressed in epithelial tissues as gingival, breast and prostatic epithelia. In the present study we investigated sarcoglycans expression in the epithelia of digestive and respiratory tracts. We performed immunofluorescence reactions using antibody against a-, b-, g-, d-, e- and z-sarcoglycans and against mucin 4 and 16. Mucins are a superfamily of proteins which serve to protect the underlying epithelia against a wide range of injuries (bacteria, virus, parasites, toxins, pH). This protection leads to coordinate cell proliferation, differentiation and apoptosis among other cellular responses; in fact, mucins are promising biomarkers and therapeutic targets in cancer and inflammatory diseases. Our results show the expression of sarcoglycans in the basal, lateral, and apical epithelial cell’s surface; moreover, sarcoglycans show to colocalize with mucins in the cell’s apical surface of bronchi and bronchioles, stomach and intestine but no apical localization has been detected in the esophageal epithelium. These results support the role of sarcoglycans in cell-cell and cell-matrix interaction. Moreover, the colocalization between sarcoglycans and mucins at apical level of epithelia which have high mucosecretory activity suggest that sarcoglycans could interact with mucus, maybe involving in maintainig omeostasis of gastro enteric epithelia. It will be necessary to demonstrate the hypothetical correlation between sarcoglycans and the maintaining epithelial homeostasis

Mandibular bone and gingival epithelium during bisphosphonates treatment: an experimental study

Bisphosphonates (BP) are stable analogues of pyrophosphate with a P-C-P structure and 2 side chains attached to the carbon atom. Intravenous bisphosphonates are primarily used and effective in the management of cancer-related conditions in the context of solid tumors, such as breast cancer, prostate cancer, and lung cancer. Moreover bisphosphonates are subministred to patients with metabolic bone disease such as osteoporosis and Paget disease. Bisphosphonate-associated osteonecrosis of the jaws (BONJ) is a really complication of intravenous bisphosphonates therapy in patients with cancer. It is common knowledge that the jaws have a greater blood supply than other bones and a fasterbone turnover rate, related both to their daily activity and presence of teeth which mandates daily bone remodeling around the periodontal ligament; moreover bisphosphonates toxicity to epithelial cells has been well documented. On this basis, the aim of this experimental study is to evaluate the pathological changes of the mandibular bone and oral mucosa in rat treated with bisphosphonates. In details we have analyzed, by immunoistochemical and scanning electron microscopic methods, biopsy of mandibular bone and of gingival mucosa in rat treated with bisphosphonates after 7, 15, 30, 45, 60 days of assumption of drugs and after 7 and 30 days from the end of the treatment. Our results show great area demineralization bone mixed to normal bone, moreover in the demineralization bone it’s possible to observe numerous micro lacunae. In the correspondence samples of gingival epithelium we observe changes of histological structure and the disappearance of protein adhesion system cells to cells and cells to matrix. On this basis it is intriguing to speculate that the adverse effects of BP on oral epithelium may play a critical role in the initiation of BONJ an “outside-in” hypothesis

Scanning electron microscopy study on mandibular human bone and immunofluorescence study on oral mucosa in patients treated with bisphosphonates

Bisphosphonates are stable analogues of pyrophosphate with a P-C-P structure and 2 side chains attached to the carbon atom. Intravenous bisphosphonates are primarily used and effective in the management of cancer-related conditions in the context of solid tumors, such as breast cancer, prostate cancer, and lung cancer. Moreover bisphosphonates are subministrated to patients with metabolic bone diseases such as osteoporosis and Paget disease. Bisphosphonate-associated osteonecrosis of the jaws (BONJ) is a really complication of intravenous bisphosphonate therapy in patients with cancer. It is common knowledge that the jaws have a greater blood supply than other bones and a fasterbone turnover rate, related both to their daily activity and presence of teeth which mandates daily bone remodeling around the periodontal ligament; moreover bisphosphonates toxicity to epithelial cells has been well documented. On this basis, the aim of this study is to evaluate the pathological changes of the mandibular bone and oral mucosa in patients treated with bisphosphonates, during osteoporosis treatment and during chemotherapy from solid cancer. In details we have analyzed, by immunohistochemical and scanning electron microscopic methods, intrasurgical biopsy of mandibular bone and of gingival mucosa in patients treated with bisphosphonates both intravenous and oral via after 24 and 36 months from assumption of drugs. Our results show in all patients great area demineralization bone mixed to normal bone, moreover in the demineralization bone it’s possible to observe numerous micro lacunae. In the correspondence samples of gingival epithelium we observe important lesions of histological structure and the disappearance of protein adhesion system cells to cells and cells to matrix. On this basis it is intriguing to speculate that the adverse effects of BP on oral epithelium may play a critical role in the initiation of BONJ an “outside-in” hypothesis

Morpho-structural alterations of sub-chondral bone tissue in patients with osteoarthritis: a scanning electron microscopy study

Osteoarthritis focuses principally on the degeneration of articular cartilage as a primary cause of the disease. The pathophysiological process of osteoarthritis is characterized by alteration of chondrocytes and the increased bone formation by sub-chondral osteoblasts. Infiltration of macrophages and perivascular T and B lymphocytes is observed, and these infiltrates have been demonstrated in both early and advanced disease. The morphological and phenotypic characteristics of osteocytic cells attached to the normal and the osteoarthritic matrix differ from each other, suggesting that specific signalling pathways arise or are altered between matrix and cells. On this basis, we have examined biopsies of bone obtained by normal femur and by femur of subjects affected by osteoarthritis using techniques of scanning electron microscopy in order to identify the morphostructural alterations that occur in the sub-chondral bone. Our results have shown that the bone tissue of subjects not affected by any disease of bone presents a well-organized structure, while the bone tissue obtained by patients affected by osteoarthritis shows a derangement of tissue itself possibly correlated with altered function of the osteoblasts, that during the pathological process produce a less mineralized extracellular matrix with consequent loss of the normal bone structure. In our opinion, during the osteoarthritic process there would be a defective signalling between bone cells leading to the production of an irregular, amorphous extracellular matrix by osteoblasts, characteristic of the pathological condition