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The essential of bone histology for forensic applications

Objective: To test the hypothesis that some metalloproteinases (MMP-2, MMP-9) and inducible nitric oxide synthetase (iNOS) enzymes in dental pulp samples do not vary when subjected to orthodontic treatment. Materials and Methods: Human dental pulps were taken from male and female patients (N=10; age 10–14 years). A straight wire technique was used with nickel-titanium or steel archwires. The increase of pressure applied on teeth was gradual. Five patients were subjected to premolar extractions after 14 months of treatment and one after 24 months. Samples were Bouin-fixed, paraffin-embedded, and afterwards processed for immunohistochemistry using anti-MMP-2, anti-MMP-9, and anti-iNOS antibodies. Results: A reduction of MMP-2, MMP-9, and iNOS expression occurred in treated samples. This became more evident with increased treatment time. Conclusion: The hypothesis is rejected. The reduction of expression of those proteins revealed a time-dependent relationship.

Medical histology Editor / s Nadir M. Maraldi, Carlo Tacchetti Author / s Saverio Cinti, Michelangelo Cordenonsi, Simona Corso, Ottavio Cremona, Massimo De Felici, Roberta Di Pietro, Nicoletta Gagliano, Silvia Giordano, Nadir M. Maraldi, Carla Martinelli, Alessandro Moretta, Sergio Morini, Beatrice Nico, Maria Prat, Domenico Puzzolo, Domenico Ribatti, Giovanni Francesco Spatola, Carlo Tacchetti ISBN 9788870513899 Medical histology Medical histology is a modern text more oriented in the "medical" sense, in order to provide students with the tools to frame the subject in the context of preclinical disciplines. In it are presented experimentally acquired data, framed in mechanistic models whose validity is widely confirmed and which contribute to an understanding of the pathophysiological processes. The goal was to provide a model of study of a complex subject, which is also applicable to other disciplines that the student will meet in the course of his medical studies. The volume has been divided into blocks of teaching material: • text and figures - essential subjects; • in-depth analysis with figures - supplementary topics; • areas of clinical histology - physiopathological correlations; • extra content in the Virtual Campus web site - additional material available in multimedia format. Each chapter contains a paragraph concerning the Prerequisites, that is the basic knowledge necessary for a correct classification of the topics covered, a paragraph concerning the Objectives that the study of the topic is aimed at and a Summary of the topic dealt with and concludes with Concepts key to the topics covered. The topics have been developed highlighting the differentiation mechanisms that allow, in both embryonic and adult life, the exchange and integration of stem cell elements in the context of functional specialized populations. Particular attention was paid to the biomechanical properties of tissues and to the properties of the different populations of adult stem cells, also in consideration of their increasing use, also in association with biomaterials, in regenerative medicine.

Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

Heat shock proteins (Hsps) assist other proteins in their folding and drive the degradation of defective proteins. During evolution, these proteins have also acquired other roles. Hsp10 is involved in immunomodulation and tumor progression. Hsp90 stabilizes a range of "client" proteins involved in cell signaling. The present study evaluated the expression levels of Hsp10 and Hsp90 in normal mucosa and adenocarcinoma samples of human large bowel.Samples of normal mucosa and adenocarcinoma were collected and Reverse transcriptase-polymerase chain reaction RT-PCR, western blotting (WB) analyses, as well as immunohistochemistry were performed to evaluate the expression levels of Hsp10 and Hsp90.RT-PCR showed a higher gene expression of Hsp10 and Hsp90 in adenocarcinoma samples compared to healthy mucosa. WB results confirmed these findings. Immunohistochemistry revealed higher levels of Hsp10 in adenocarcinoma in both the epithelium and the lamina propria, while Hsp90 expression was higher in the adenocarcinoma samples only in the lamina propria.Hsp10 and Hsp90 may be involved in large bowel carcinogenesis.

Churg-Strauss (CSS) syndrome is rare and of unknown etiology. It is associated with vasculitis, blood eosinophilia and granulomatosis, and affects multiple organs and systems at various stages of the disease. Specific diagnostic and monitoring tests are not yet available. This study aims to assess the changes in MMP-2 and MMP-9 along with the histopathological alterations in two cases of CSS, as possible potential diagnostic and monitoring criteria. Two adult male patients were diagnosed with CSS in the otorhinolaryngology clinic in the University of Palermo, based on multiple clinical and histopathologic criteria. Biopsies of respiratory mucosa were taken after the consent of the patients, processed for routine histopathology and immunohistochemistry as well as quantitative polymerase chain reaction (qPCR). Similar biopsies were also taken from a non-CSS patient. The Assessment of MMP-2 and MMP-9 was performed using both immunohistochemistry and qPCR techniques. Histopathological alterations in the respiratory mucosa were consistent with vasculitis and granulomatous tissue formation, in addition to inflammatory cell infiltration with abundance of eosinophils. Immunohistochemistry assay performed on the samples derived from the two CSS patients showed a relative and remarkable increase of both MMP-2 and MMP-9 compared to controls. Such an increase was consistent with the qPCR results which depicted a significant increase between 20 and 30% for both MMP-2 and MMP-9, respectively. Since the secretion of MMPs is an essential step in angiogenesis, could these enzymatic factors be used as parameters to diagnose or monitor the evolution of CSS? The small number of samples analyzed in this study does not allow us to suggest a general statement correlating the increase in expression of MMP-2 and MMP-9 to the appearance or evolution of vasculitis; it is only speculative.

The aim of the study was to assess the involvement of apoptotic factors, cytokeratins and metalloproteinase- 9 in the histogenesis of both Epithelialized Gingival Lesions (EGL) and Periapical Lesions (PAL). 55 consecutive patients, 30 with PAL and 25 with EGL, were selected for the study after clinical and radiological examinations. The PAL patients had severe periapical lesions and tooth decay with exposure of the pulp chamber. All PAL and EGL biopsies were surgically extracted, fixed in 10% buffered formalin, and processed for routine light microscopy. Ten biopsies of each category were processed for immunohistochemistry (IHC). Serial paraffin sections were stained by IHC with appropriate antibodies to detect cytokeratins (CKs) 1, 5, 8, 10 and 14, caspase-3 and -9, metalloproteinase-9, and for PCNA and TUNEL assays. Both PAL and EGL showed a high expression of the cytokeratin 1, 5 and 8 with higher expression in EGL. Moreover, CK10 was markedly less intense expressed in EGL compared to PAL, while CK14 was almost three times stronger expressed in EGL. The expression of caspase-3 and -9 was stronger in PAL compared to EGL, however, the difference was only significant for caspase-9. In PAL apoptosis detected by TUNNEL method and the expression of MMP-9 were higher than in EGL, whereas PCNA was significantly more expressed in EGL. The results clearly suggest that both lesions have exclusively an epithelial origin and that epithelial proliferation was correlated with the degree of apoptosis in both entities. PAL and EGL presented mostly similar cytokeratin expression except for CK10 and CK14, though with marked differences in the distribution and intensity of IHC reactions. Finally, the degradation of extracellular matrix in both lesions could be partially attributed to the strong presence of MMP-9. (Folia Histochemica et Cytobiologica 2012, Vol. 50, No. 4, 497–503) The aim of the study was to determine by immunochemistry the expression of leptin, orexin A and orphanin FQ in the major salivary glands (parotid, submandibular and sublingual) of rat, sheep and cow. These peptides, originally synthesized in central nervous system, adipose tissue and peripheral tissues including gastrointestinal tract, play an orexigenic (orphanin and orexin) or anorexigenic (leptin) roles in the intricate neuronal network appointed to the control of nutritional homeostasis. Peptide-specific immunoreactivity was present in the studied salivary glands with various intensities in different species, in the ductal epithelium, sometimes in the acinar epithelium, and in nervous trunks spread in connective tissue stroma. The obtained data show that salivary glands present an unexpected source of orexigenic and anorexigenic peptides which with their autocrine, paracrine, and endocrine mechanisms of action may participate in the control of salivary gland function.

This research has the objective to investigate immunohistochemical expression of CB1 receptor and its probable changes in Gastroenteropancreatic system (GEP) of obese and lean Zucker rats and understand the endocannabinoid pathophysiological implications in the obesity. Male obese (fa/fa) and lean Zucker rats 6 weeks old were obtained from Harlan Italy Srl; the rats were sacrificed at 8, 12 and 16 weeks old. Normal rats also were sacrificed. Specimens of stomach, jejunum-ileum and pancreas were fixed in Bouin’s mixture and embedded in paraffin; obtained sections were processed with anti-CB1 (Biosource Europe SA) by Streptavidin-Biotin-Complex Method. The findings show that CB1 receptor is expressed not only in enteric neurons as documented by earlier studies up to now but more widely and with stronger intensity in obese animals compared with their lean counterparts by several structures of gastrointestinal tract (epithelium, glands, endocrine cells and immune cells of villi stroma). In obese Zucker rats pancreas unlike the normal rats where the CB1 receptor is essentially expressed by A-cells, the CB1 immunoreactivity even extends with higher intensity to B-cells. It is concluded that GEP System represents a new and wide peripheral target of EC action that have a direct autocrine, endocrine-paracrine and neurocrine control on many functions of GEP. In addition in GEP of obese Zucker rats in comparison with lean ones, the CB1 receptor is overexpressed and consequently the peripheral endocannabinoid system is upregulated and negatively modulated by leptin. It may contribute to increase hyperphagia, body weight and hyperglycaemia.

Objective. This study seeks to clarify the histological origin of apical lesions, through the expression of certain cytokeratins.Study design. In 3 years 30 patients were selected. After clinical and radiological exams were chosen patients who had severe apical lesions and tooth decay with exposure of the pulp chamber left untreated for very long periods (more the 12 months). Serial slides were prepared both for immunohistochemistry and Hematoxylin- Eosin. Material and methods. Twenty specimens coming from the 30 patients were used for our purpose. All periapical lesions were surgically extracted and fixed in Bouin mixture, and embedded in paraffin. Samples were processed immunohistochemically employing the instructions of the Envision Dako cytomation kit. Monoclonal antibobies against Cytokeratins (CKs) 1, 5 , 8, 10 and 14 were used. Slides were observed with Leica Laborlux-S microscope and the image were acquired with NIKON DSL2 System. Each sample has been analyzed with a “double-blind” system by two different operators. Moreover, the digital images acquired have been compared to an image analysis tools by Adobe photoshop CS5 Extended. Results. CKs 1, 5 and 8 are expressed in all samples at external epithelial layer with no statistically significant differences. CK 10 is weakly expressed in some epithelial cells lining the periapical lesion. CK 14 was negative in most samples, and little positive in some specimens. Conclusion. Our results suggest that most of the apical lesions we studied have an epithelial origin. The absence of CK14 positivity may be explained by the fact that during the extirpation of the lesion the basal epithelium remained attached to the surrounding bone., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Recent studies document that CB1 cannabinoid receptors, considered since its first identification as “brain specific”, could be expressed by peripheral tissues targets (adipose organs, enteric nervous system, striated muscle, epatocytes). This receptors were found in the GI tract of different species, including mice, rats, guinea pigs, pigs and humans. Endocannabinoids play a role in the pathophysiology of the gastrointestinal tract. They play a role in emesis, acid-related disorders, motility-related disorders, irritable bowel syndrome and diarrhea, and in inflammatory bowel disease. Endocannabinoid system might be involved in cancer differentiation, growth and cell migration. Distribution of receptors in different colonic segment is not known. We studied CB1 receptors distribution in 10 patients colonic segments; the present report shows preliminary results. Fragments of sigma, transverse and ascending human colon were obtained during endoscopic screening procedure. Materials and Methods The specimens, for immunohistochemistry (IHC), were fixed in buffered formalin and embedded in paraffin; obtained sections were processed with anti CB1 (Biosource Europe S.A.) by EnVision+System HRP (AEC) and have been studied with photomicroscope Leica DMLB. The specimens for the Reverse Transcription Polymerase Chain Reaction (RT-PCR) were frozen in liquid nitrogen and stored at -80° until use. Total RNA extraction was performed using the “illustra RNA spin Mini Kit”. RT reaction was performed using the “Enhanced avian HS RT PCR kit” The PCR was performed using the “PCR enzyme Selection Kit-High specificity”. Results and Discussion Following the endoscopic procedure was performed anatomopathological examination of specimens. The results have revealed that four of the 10 patients had a polyp the others were not suffering from any disease of the colon. Our immunohistochemical evaluation has revealed the presence of CB1 receptors expressed by regional differences in patients who had a polyp. This reactivity appeared to be lower than that shown in non-pathological patients. The PCR results instead of doubtful interpretation. In fact, despite being detected in some cases also marked a positive control, in two cases, the PCR is a totally negative compared with a positive IHC. Since the number of cases studied is not statistically significant, it is our intention to continue to evaluate additional patients both normal and suffering from polyposis in order to clarify the difference beetween immunohistochemical and molecular biology results.

The oral cavity is exposed to chronic or recurrent, physical and chemical trauma that could lead to mucosal reactions (e.g. hyperplasia, dysplasia and tumors). The objective of this study is to investigate the expression and the possible changes of the two matrix metalloproteinases MMP-2 and MMP-9 in normal and pathological human oral mucosa samples. Normal oral mucosa samples and three different types of pathological conditions (hyperplasia, dysplasia and carcinoma) were used for this study. Immunohistochemical analysis was used to evaluate protein expression for the two enzymes, while Reverse Transcription ? Polymerase Chain Reaction (RT-PCR) was used to investigate gene expression. Image analysis was used to give a quantitative evaluation of the immunohistochemical data. In control samples we identified a weak expression of both MMP-2 and MMP-9 in the epithelial layers. In hyperplasia samples MMPs expression is limited to epithelial layers but the immunoreactivity is more intense than in the control. In dysplasia and carcinoma samples the two matrix metalloproteases are expressed not only in epithelium but also in some cells of the connective tissue and in the vessel walls. Qualitative RT-PCR and image analysis confirmed the immunohistochemical data. The results obtained in this study suggest the existence of a possible relationship between the entity of morphological disorganization of the oral mucosa in different pathologies and the increase of MMP-2 and MMP-9 expression

The use of methods of image analysis has allowed us to make more reliable and reproducible results obtained by immunohistochemistry (IHC). Wider use and simplification of software allowing a colorimetric study has meant that these methods are available to everyone and made it possible to standardize the technique by reliable systems score. Moreover, the introduction in recent years of multispectral image acquisition systems methods has further refined these techniques allowing to avoid artifacts and release the evaluation of the data by the observer.

Introduction. The oral cavity contains many structures compressed in a small area. It is exposed to chronic or recurrent, physical and chemical trauma leading to mucosal reactions (e.g. hyperplasia, dysplasia and tumors). For a long time, our group has been studying the expression of some metalloproteinases (MMP-2, MMP-9) in healthy and pathological human oral mucosa through immunohistochemistry and RT-PCR to investigate the entity of the remodelling process in the different cases. Matherials and Methods. In this paper we investigate a sample of normal oral mucosa and four pathological samples (mucocele, fibroma, dysplasia and carcinoma). We identified the localization of MMP-2 and MMP-9 through immunohistochemistry and gene expression through RT-PCR analysis. Results. In control sample we identified a weak expression of both metalloproteinases (MMP-2, MMP-9) in the upper epithelial layers. The reactivity of the two enzymes is emphasized with higher intensity in pathological samples where a strong inflammatory activity is present (e.g. hyperplasia, dysplasia) or where there is a distruption of the architecture (as in tumors) while in localized lesions a functional silence is detected (mucocele). Particularly, in lesions like hyperplasia and dysplasia where (though it is present a inflammatory condition) the bio-architecture is preserved, the immunoreactivity is expressed only in the upper epithelial cell layers; while in more serious lesions (like tumors), where the architecture is fully disorganizated, the immunoreactivity is expressed also in deeper epithelial layers, in few connnective cells and in vessels wall. Qualitative RT-PCR analysis confirmed the expression of MMP-2 and MMP-9 in both normal and pathological oral mucosa samples. Conclusions. Our data suggest that there is a relationship between the increase of MMP-2 and MMP-9 expression and the entity of morphological disorganization according with the well known role of these enzymes.

We have used 12 Epulis taken during surgical operation and as control, some normal gingival specimen. The aim was to clarify the relationship between the degree of inflammation and NO production in several cases of Epulis, exhibiting different degree of inflammatory process and verifying the presence of cells involved in NO production. From the immuhistochemical data in our possession we can conclude that NO is one of the factor which module the pathogenecity degree of Epulis.

Aortic aneurysms (AA) is a degenerative vascular disease characterized by localized dilatation of the aortic wall as a result of altered matrix composition (elastin and collagen degradation). Howewer the pathogenesis of the changes is elusive and unclear. Some experimental evidences suggest that iNOS (who synthesize a large amount of NO in inflammatory processes) and the metalloproteinases (MMP) are implicated in the pathogenesis of AA but the relationship between NO and MMP to aneurismal disease is currently unknown. The aim of this study is to investigate the immunohistochemical expression of iNOS and MMP2 and MMP9 in human aneurysmal tissues.

The aim of present study is to investigate the immunohistochemical identification of cNOS-containing prostatic structures in various species and in hyperplastic human prostate. We used specimens of rat, guinea pig and hamster. Human prostatic specimens were removed from patients suffering of prostatic hyperplasia. The study was processed by ABC method. Immunohistochemical staining with anti nNOS antibody revealed the existence of strong reactivity in numerous ganglionic cell bodies and varicose nerve fibers in the fibromuscular stroma, in smooth cells around acini and in blood vessels endothelium. Surprisingly nNOS immunoreactivity appears in the glandular epithelium. In the hyperplastic prostate the nitroxidergic innervation of stroma and glandular epithelium is almost absent. These data provide a morphological evidence for a segmental differentiation of NOS-containing prostatic structures and consequently suggest that in the prostate NO plays selective neural and paraneural roles in the control of blood flow, smooth-muscle tone (relaxation) and secretory function. Further immunohistochemical studies are carrying out to elucidate the prostatic role in aging, after hormonal perturbation and pathological processes.

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