Imaging extracelluar vesicles by transmission electron microscopy: Coping with technical hurdles and morphological interpretation.

BACKGROUND: Extracellular vesicles (EVs) are cell-derived nanometric particles governing the complex interactions among cells through their bioactive cargo. Interest in EVs is rapidly increasing due to their extensive involvement in physiological and pathological conditions, their potential employment as diagnostic and therapeutic tools and their prospective use as bio-carriers of exogenous molecules. Given their nanometric size, transmission electron microscopy (TEM) provides significant contributions to assess EV presence and purity in a sample and to study morphological features. SCOPE OF REVIEW: In this review, TEM methods for EV imaging are compared with respect to their applications, benefits and drawbacks. A critical evaluation of the actual contribution of TEM to the study of EVs is also provided and the most common artifacts encountered in the literature are discussed. MAJOR CONCLUSIONS: TEM techniques are powerful tools for the investigation of EVs and have the potential to reveal sample purity, ultrastructure and molecular composition. However, technical challenges, procedural errors in sample processing or misinterpretations may result in a variety of different morphologies and artifacts. GENERAL SIGNIFICANCE: The last decades have seen exponential technological progress in EV imaging by TEM. Nevertheless, protocols have not been standardized yet and sample preparation remains a critical step. An optimized, standardized and integrated protocol of different techniques could minimize artifacts and interpretative errors that could significantly improve the quality and reliability of downstream studies.

Sympathetic Innervation and Adrenergic Receptors in Equine Deep Digital Flexor Tendinopathy: Preliminary Results.

The aim of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses with tendinopathy by immunohistochemical labelling for tyrosine hydroxylase (TH) and α-1 adrenergic receptor (α1-AR). Twelve forelimbs were obtained from 10 horses with DDFT tendinopathy and six feet obtained from six horses were used as healthy controls. Post-mortem radiographic, ultrasonographic and gross examinations were performed on the suprasesamoidean area of the DDFT to assess the presence of tendinopathy. Longitudinal sections were collected and processed. Lesions were classified as core lesions, dorsal border lesions and parasagittal oblique splits. Immunohistochemistry was performed and the degree of immunoreaction was classified as absent, mild or marked. Seven core lesions, four dorsal border lesions and one parasagittal oblique split were identified. There was no increased expression of sympathetic innervation in samples with a dorsal border lesion of the DDFT compared with healthy samples. In contrast, core lesions showed increased expression of α1-AR and reduced expression of TH, which supports the hypothesis of a compensatory imbalance between the sympathetic mediator and the sympathetic receptors as a cause or effect of structural damage. In addition, adrenergic activation could stimulate cell proliferation and differentiation within these lesions.

Effect of canine mesenchymal stromal cells loaded with paclitaxel on growth of canine glioma and human glioblastoma cell lines.

This study investigated whether canine mesenchymal stromal cells (cMSCs) are able to take up and release paclitaxel (PTX) in active form, and therefore whether they have potential as a tool for therapeutic delivery of this drug. cMSCs from bone marrow and adipose tissue were isolated, expanded and characterised phenotypically. cMSCs were loaded with PTX (cMSCs-PTX) and their capacity for release of PTX was determined by their effect on proliferation of cancer cells. cMSCs-PTX derived from bone marrow and adipose tissue were able to take up and then release active PTX. cMSCs-PTC inhibited proliferation of the canine glioma cell line J3T, and the human glioblastoma cell lines T98G and U87MG. The potential of canine cMSCs-PTX for treatment of canine gliomas should be investigated further.

Sympathetic innervation of the suprasesamoidean region of the deep digital flexor tendon in the forelimbs of horses.

The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue.

Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Immunohistochemical distribution of leptin receptor in the major salivary glands of horses.

The presence of the leptin receptor (ObR) has already been highlighted in the human major salivary glands and it has been hypothesized that leptin may act by regulating the gland's growth. No data are reported on domestic animals so, considering the important role that these glands play, not only related to food ingestion and digestion, and the important functional role hypothesized to explain the presence of ObR in humans salivary glands, the aim of the present work was to investigate the presence and the distribution of the leptin receptor in horse parotid and mandibular glands, by immunohistochemical techniques. The presence of ObR was evidenced in parotid and mandibular glands, exclusively localized in duct epithelial cells; their positivity was localized in the cytoplasm and was most evident near its apical portion. Immuno-positivity not only affects the intralobular ducts (intercalated and striated) but also the interlobular ones. Our results indicate that horse major salivary glands, like those of humans, are likely targets of leptin actions, suggesting a functional role of leptin on these glands.

Immunohistochemical detection of the orexin system in the placenta of cats.

The aim of the present study was to investigate the presence and distribution of cells containing orexin A (OXA), and orexin type 1 and 2 receptors (OX1R and OX2R, respectively) in the feline placenta by means of immunohistochemical technique. OXA was identified in several decidual and syncytiotrophoblastic cells present in the lamellar portion of the placenta. In the same placental structures, few decidual and syncytiotrophoblastic cells showed the presence of OX1R-like immunoreactivity. Characteristically, immunopositivity for OX2R, but not for OX1R, was evidenced in the cells of the glandular layer. The orexinic system was not expressed in the uterine structures that were not engaged by the chorion. Our results provide the first evidence of the presence of a placental orexinic system in a mammalian species. Orexin A and both OX1R and OX2R are unequally distributed within the cat placenta. Local OXA production and the presence of specific receptors, differentially expressed in the placental structures of the cat, suggest that the orexinic system may participate in placental growth and development as well as in the regulation of its steroidogenic capacity via endocrine, paracrine and/or autocrine mechanisms.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.

Immunohistochemical localization of CB1 receptor in canine salivary glands.

CB1 is a member of the G-protein-linked receptor superfamily that is present in the central nervous system as well as in certain peripheral neuronal and non-neuronal tissues. Recently, the presence of CB1 was found in the ductal system of the major salivary glands of laboratory animals, but no data are available for domestic mammals. Thus, in the present study, we examined the presence and distribution of CB1 in the major salivary glands of dogs using immunohistochemical techniques. CB1 was found in the parotid and mandibular glands of adult dogs; positive immunoreaction was localized to the cells of the striated ducts, with a peculiar localization on or near the apical membrane. This particular localization may be explained by the characteristics of this receptor as membrane-associated. The acinar structures were completely negative for CB1. We conclude that CB1 is involved in the control of dog salivary secretion via endogenous substances, likely endocannabinoids. The localization of CB1 highlights that endocannabinoids promote qualitative and/or quantitative changes of the primary saliva in the ductal system.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs.

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.

Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct.

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2alpha and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2alpha, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2alpha release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2alpha production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.

Apoptotic cell death in canine hair follicle.

Apoptotic cell death is an essential homeostatic mechanism involved in the control of cellular turnover in a variety of adult tissues. Cytoplasmic and nuclear condensation morphologically define this process whose biochemical hallmark is extensive DNA fragmentation into discrete oligonucleosomic units. Hair follicle growth and regression has been shown to be correlated with apoptosis in humans, mice, rats and guinea pigs. The present study was carried out to evaluate its implication in canine hair biology in order to define the spatio-temporal relationship between apoptosis and the hair cycle in dogs. As assessed by terminal deoxy-nucleotidyl transferase-mediated d-UTP nick-end-labelling (TUNEL) and by basic histological and ultrastructural assays, apoptotic cells appeared both in the growing and in the regressing follicle epithelium showing the well characterized morphological features described in the previous relevant literature.

Immunohistochemical detection of virulence-associated Rhodococcus equi antigens in pulmonary and intestinal lesions in horses.

Two horses with Rhodococcus equi infection were examined post mortem by an immunohistochemical method (peroxidase-antiperoxidase; PAP) with a monoclonal antibody (Mab 10G5) to the 15-17 kDa antigen of R. equi. One of the horses was also examined bacteriologically, R. equi being isolated in culture. Immunolabelling with this Mab was marked and widespread. On the other hand, the immunohistochemical reactivity of infected macrophages with a polyclonal antibody specific for lysozyme was slight. Thus, Mab 10G5 would appear to be a useful diagnostic reagent in R. equi infection, with or without cultural confirmation.

Outcome analysis of endoscopic III ventriculostomy.

A retrospective chart review was performed on 54 patients who had undergone endoscopic III ventriculostomy (E3V) in the past 6 years. Patient charts were reviewed to establish age at the time of operation, sex, preoperative diagnosis, preoperative shunt procedures, success or failure of the E3V, duration of success, and complications. Success of an E3V was determined by the resolution of preoperative symptoms and avoidance of a CSF shunt. The most recent clinic visit with adequate documentation of signs and symptoms of hydrocephalus was used as the last date of follow-up. The overall success rate was 74%. Children over the age of 3 years with an acquired CSF obstruction had a significantly greater probability of successful treatment (P=0.05). Younger children, especially those with hydrocephalus attributable to obstruction of the arachnoid villi, as in intraventricular hemorrhage (IVH), were less likely to benefit from E3V.