Biology subjects

Gaytan, F.

Publications and source records attributed to Gaytan, F..

6 recordsLinked to original sources

bioRxiv2023

A modified RGB trichrome (HemRGB) for improving nuclear staining: application to colorectal cancer histopathology

The RGB trichrome staining method has been used to highlight two major components of the extracellular matrix, collagen and glycosaminoglycans. While the RGB trichrome efficiently stains extracellular matrix components, it lacks a nuclear stain, limiting its application in histopathology. To address this issue, a modification of the original stain, named HemRGB trichrome, has been developed. This modification incorporates iron hematoxylin for improving nuclear staining while retaining specificity for the staining of extracellular matrix. The application of HemRGB trichrome staining to samples from both normal colonic tissues and colorectal adenocarcinomas (CRC) provides a robust nuclear staining, together with a high-contrasted staining of tumor microenvironmental components, such as infiltrating immune cells, collagen and ground substance, extracellular mucins, as well as contrasted interfaces between CRC metastases and liver parenchyma. This study underscores the potential of HemRGB trichrome as a valuable tool for histopathological studies, especially for cancer evaluation, where nuclear characteristics are particularly relevant.

pathology↗

bioRxiv2022

Staining of human skin with RGB trichrome unveils a proteoglycan-enriched zone in the hair dermal sheath

The skin is the largest organ in the body and plays several essential functions acting as a barrier that protects us from physical and chemical insults, prevents the entrance of pathogens and the loss of water, besides playing an esential role in the regulation of body temperature. The skin displays a high regenerative capacity, evidenced by its self-renewing activity and the process of wound healing, driven by the existence of several resident stem cell populations. Due to the high prevalence of skin pathology, and their anatomical accessibility, skin biopsies and their subsequent microscopic observation constitute a powerfull tool for the study of primary skin diseases, as well as cutaneous manifestations of systemic diseases. This gave rise to dermatopathology as a specific discipline that unifies dermatology and pathology. In this setting, staining with hematoxylin and eosin constitutes the gold standard method for microscopic observation and diagnosis. Yet, several additional stains are used for specific purposes, such as trichrome stains for the staining of collagens in the extracellular matrix. We have applied a recently developed stain (RGB trichrome, acronym for picrosirius Red, fast Green and alcian Blue) to human samples to assess the staining outcomes in normal skin tissues. RGB staining provides a high contrasted interface between epidermis and dermis, and a comprehensible staining of the different dermal structures such as blood vessels, nerves, and sweat and sebaceous glands. The specific staining of collagens by picrosirius red can be useful for the objective quantification of these proteins under polarized light microscopy. In hair follicles, RGB staining resulted in specific staining of the epidermal sheaths and the encasing connective tissue (i.e., dermal sheath). Interestingly, the dermal sheath show two domains in which blue predominates over red staining, thus indicating that proteoglycans prevail in these areas. These two zones are the dermal papilla and an uncharacterized zone located at the upper isthmus, that we have denominated as proteoglycan-enriched belt (PEB). While the role of the papilla in the induction and regulation of hair growth is clearly established, the possible role of PEB remains to be determined and merits additional investigation.

cell biology↗

bioRxiv2022

Application of RGB trichrome staining to the study of human parasites

Human parasitic infections are major contributors to global disease load, compromising the human life and resulting in considerable morbidity and mortality worldwide. However, many parasitic diseases have been neglected and little investigated in western medicine. Although recently developed molecular techniques have revolutionized the taxonomy of parasites and the parasitic disease diagnosis, histopathology still remains a powerfull tool for the analysis of parasitic diseases, allowing direct observation of the parasite thus providing information about the morphological features of the parasite ifself, and revealing tissue alterations at the parasite-host interface. The recently developed RGB trichrome (acronym for the primary dye components, picrosirius Red, fast Green, and alcian Blue) stains the main components of the extracellular matrix, specifically collagens and proteoglycans. We have applied the RGB tricrome staining to human tissues infected by the main three classes of human parasites: Protozoa (Leishmania donovanii and Toxoplasma gondii), helminths (Trichinella spiralis, Enterobius vermicularis, Dirofilaria spp. and Echinococcus granulosus) and ectoparasites (Demodex folliculorum and Demodex brevis). Trichrome stain results in detailed staining of the parasite microanatomical structure, and highlights host tissue alterations such as granulomatous inflammation, immune cell infiltrate, or increased amount of collagen as a sign of parasite-induced fibrosis. Yet, the use of RGB trichrome, as a complement of hematoxylin and eosin staining, provides additional valuable information to assess parasitic infection histopathology.

microbiology↗

bioRxiv2022

A comparative study of the human, mouse, and zebrafish eye after staining with RGB trichrome

Eyesight plays essential roles for the survival of most animal species, and diseases leading to blindness or partial vision loss in humans have an enormous impact in life quality. The complex structure of the eye makes it ideal for the study of structure-function relationships, and to accomplish integration of histology with other disciplines, connecting basic anatomical sciences to clinical medicine. The RGB trichrome consists of the sequential staining with three dyes, fast green (as a general protein stain), picrosirius red and alcian blue (for two main components of the extracellular matrix, collagen and glycosaminoglycans respectively). Notably, this combination of primary colors matches the physiological color detection capacity of the human trichromatic eye. Application of RGB stain to human eye samples and to the eye of two widely used animal models, the mouse and the zebrafish (Danio rerio), gives rise to brilliant staining of eye connective tissues, with a range of colors from red to magenta depending on the extracellular matrix composition, and producing highly contrasted tissue interfaces, facilitating the observation of tissue structures, as well as histomorphometric analysis. Interestingly, staining of the retina resulted in differential staining of the retinal layers in the three species analyzed. These results support the effectiveness of RGB as a reliable general staining method, complementary to routine basic hematoxylin and eosin (H&E), applicable to the study of the eye in human pathology, basic preclinical research, as well as in histo(patho)logy teaching.

cell biology↗

bioRxiv2022

Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility

Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets to reproductive control remains unfolded. To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. Mice with Tac2 cell-specific Kiss1 KO (TaKKO) displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis and locomotor activity. Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as adult metabolic homeostasis. Significance StatementNeurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders. Disclosure StatementThe authors have nothing to disclose in relation to the contents of this work.

physiology↗

bioRxiv2020

Image matters: Development of a novel staining method, the RGB-trichrome, as a reliable tool for the study of the musculoskeletal system

Morphometry and histology are essential approaches for investigation and diagnosis of musculo-skeletal disorders. Despite the advent of revolutionary methods of image analysis and high resolution three-dimensional imaging technology, basic conventional light microscopy still provides an incisive overview of the structure and tissue dynamics of the musculoskeletal system. This is crucial to both preclinical and clinical research, since several clinically relevant processes, such as bone repair, osteoarthritis, and metabolic bone diseases, display distinct, if not pathognomonic, histological features. Due to the particular characteristics of the skeletal tissues (i.e., the existence of mineralized extracellular matrices), a large number of staining methods applicable to either decalcified or undecalcified tissues are available. However, it is usually the case that several staining methods need to be sequentially applied in order to achieve the different endpoints required to fully assess skeletal tissue structure and dynamics, and to allow morphometric quantification. We describe herein a novel staining method, the RGB trichrome, amenable for application to decalcified, paraffin embedded human musculoskeletal tissues. The acronym RGB corresponds to the three primary dyes used: picrosirius Red, fast Green, and alcian Blue. Although these individual pigments are commonly used either isolated, in binary combinations, or as part of more complex polychrome staining methods, when merged in the RGB trichrome staining produce high-quality/high-contrast images, permitting not only clear identification of different tissues (i.e., the different types of cartilage, bone and fibrous connective tissue), but also discrimination between calcified and uncalcified bone and cartilage, as well as an unexpected diversity of shades of color, while displaying singular properties among polychrome staining methods, such as the unveiling of the bone osteocyte dendritic/canalicular network. Hence, we propose the RGB trichrome as simple but highly-reliable tool for the preclinical and clinical study of the musculoskeletal system.

cell biology↗