Is there still room for novelty, in histochemical papers?

Histochemistry continues to be widely applied in biomedical research, being nowadays mostly addressed to detect and locate single molecules or molecular complexes inside cells and tissues, and to relate structural organization and function at the high resolution of the more advanced microscopical techniques. In the attempt to see whether histochemical novelties may be found in the recent literature, the articles published in the European Journal of Histochemistry in the period 2014-2016 have been reviewed. In the majority of the published papers, standardized methods have been preferred by scientists to make their results reliably comparable with the data in the literature, but  many papers (approximately one fourth of the published articles) described novel histochemical methods and procedures.  It is worth noting that there is a growing interest for minimally-invasive in vivo techniques (magnetic resonance imaging, autofluorescence spectroscopy) which may parallel conventional  histochemical analyses to obtain information not only on the morphological features of living organs and tissues, but also on their functional, biophysical and molecular characteristics. Thanks to this unceasing methodological refinement, histochemistry will continue to provide innovative applications in the biomedical field.

Introduction

The impact of Histochemistry on biological and medical research is always high,[1-3] with widespread application in a large variety of topics. During the last three years, more than 65,000 articles have been published in qualified journals (according to the Web of Science and Scopus databases), and it would be interesting to know how histochemistry has been used, especially which techniques have been employed in these investigations: this would also help to answer the question “was there histochemical originality or novelty, or just instrumental application of accepted protocols?”. A survey of the whole histochemical production would have been a too difficult task; therefore, a review has been made of only the 126 articles published in the European Journal of Histochemistry from January 2014 to present. This journal traditionally accepts manuscript on functional cell and tissue biology in animals and plants, with attention to the processes of cell differentiation, development and senescence, and to the cellular basis of diseases: this should make the article sample sufficiently representative of the possible histochemical applications, though obviously limited by the small number of items considered.

For sake of simplicity, the papers have been divided into six categories, based on their main subject: Tumor biology & markers (14% of the published articles), Non-Tumors diseases (17%), Experimental medicine & Animal models (10%), Stem cells & Development (14%), Tissue Biology in Human and Animals (20%), Methods & Techniques (25%).

Tumor biology & markers, Non-Tumors diseases, Experimental Medicine & Animal Models

In most of the published papers on Tumor biology & markers,[4-21] the expression of specific protein has been investigated (in hepatocellular carcinoma,[4-6] uterine lesions,[7,8] colorectal carcinoma,[9,10] and other neoplasms[11,15,21]). The aim was either to originally define new diagnostic/prognostic markers,[4,6,8,10-12] or to identify proteins involved in tumor onset[7] or progression,[5,21] or to evaluate the effect of therapy.[9] The mechanisms of carcinogenesis have also been studied,[13-20] and it is worth noting that some new proteins have been identified as potential therapeutic targets for cancer treatment[14,16] and tailored therapy.[20,21] In most of these investigations, well-established immunohistochemical methods have been used (often in parallel with in situ hybridization, RT-PCR, Western blot assays or microarray analyses) to detect and locate single molecular species inside tissues and cells or in the extracellular matrix.

A similar situation may be found in the articles on Non-Tumors diseases.[22-42] Here again, multiple immunohistochemical assays have mostly been used to identify pathogenetic factors or to relate the changes in protein expression with tissue remodeling,[22,29-33] or the progression of the disease.[23-28,34,35] Interestingly, cartilage, bone and dentin in different pathologies were especially investigated,[36-41] which demonstrates the unique role of histochemistry in studying these tissues under normal or pathological conditions.[42]

The effect of experimental treatments or disease on the fibrous joint tissue[43,44] and cartilage[45,46] were also investigated in papers falling into the category Experimental Medicine & Animal Models.[43-54] In this article group, conventional histochemical techniques, histological examination and morphometry at light and electron microscopy were often used together with immunohistochemistry, to describe the structural organization of brain[46] or the skeletal muscle[47] in mice strains, and to investigate in rats the effects of a maternal dietary load of alpha-tocopherol on the synapse density and glial synaptic coverage in the hippocampus of adult offspring.[48] Enzyme histochemistry and immunolabeling were simultaneously applied to describe the structural and functional organization of fatty livers submitted to a preservation procedure especially suitable for organ transplantation.[49]

Cultured cell systems have also been used to analyze the effect of ozone at low concentration on cytoskeletal organization, mitochondrial activity and nuclear transcription,[52] or to elucidate the molecular pathway responsible for the increased collagen synthesis by fibroblasts in vitro after exposure to the natural flavonoid, apigenin.[53] In these articles, the use of multiple molecular and histochemical techniques at light and electron microscopy was effective to detect changes in gene expression and in the structural organization and function of subcellular organelles.

Stem cells & Development, Tissue Biology in Human and Animals

In the papers on Stem cells & Development[55-72] the expression of specific proteins was investigated during fetal development,[55] with particular reference to skin,[56] lung,[57,58] kidney,[59] pituitary and adrenal gland,[60,61] developing cartilage and bone,[62-65] and cochlea.[66,67] The presence of progenitor stem cells has been detected in the kidney,[68] cerebral cortex[69] and cerebellum[70] during human embryogenesis. Viable chondrogenic stem cells have been detected in the knee joint loose body,[71] and the ultrastructural features of mesenchymal stem cells after in vitro expansion have been reviewed.[72] Histochemistry proved to be especially suitable also for the essentially descriptive articles on Tissue Biology in Human & Animals.[73-98] Here, several proteins and protein receptors have been studied in different organs and tissues from mammals (rodents,[73-82] bat,[83] dog,[84] primates and human[85-88]), as well as from lower vertebrates[89-94] and invertebrates.[95] Few papers described the peculiar behavior of nuclear proteins involved in RNA transcription and maturation[96] or in apoptotic cell death.[97-98]

Methods & Techniques

In the great majority of the papers of all the above mentioned categories, techniques have been used to locate single proteins or nuclei acid sequences in situ (single or multiple immunohistochemistry, in situ hybridization). In these investigations, the scientists obviously preferred to use standardized methods to make their results reliably comparable with the data in the literature. As a consequence, originality resides in the investigated subjects, whereas histochemical novelty may hardly be found and, when seldom present, it is limited to the appropriate protocol adjustment to cope with special sample characteristics.

On the contrary, the papers in the section Methods & Techniques[99-129] are histochemically novel either in the proposed procedure and in its application to unusual model systems. The key role of the fixation/embedding procedures for appropriate specimen preparation was demonstrated in some studies,[99-104] and particular attention was paid to technical improvements in antigen preservation and retrieval for immunohistochemical applications,[105-110] and in multiple histochemical staining.[111-113] Imaging techniques were the subject of some articles.[114-116] Raman microspectroscopy was used to investigate stenotic aortic valve leaflets to get information on the composition and distribution of accumulated lipids, in the attempt to correlate their presence with mineralization as a precocious diagnostic marker.[114] Magnetic resonance imaging (MRI) was used to perform a longitudinal study in vivo on the spine changes occurring in an experimental rat model of ankylosing spondylitis, and the obtained results were validated at the end of the experiments by micro-computerized tomography and histological examination: this non-invasive approach may potentially be applied to follow the progress of this disease and plan therapeutic interventions in humans.[115] Advanced MRI techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, and Diffusion Tensor Imaging may be envisaged as suitable minimally-invasive techniques to investigate in vivo not only the morphological features of living organs and tissues, but also their functional, biophysical and molecular characteristics.[116] Among the non-invasive techniques, autofluorescence imaging promise to be particularly suitable and informative for diagnostic applications.[117-120] Applications of electron microscopy analyses were used for tridimensional reconstruction of apoptotic nuclei[121] and for detecting metal contaminants.[122-124] Diaminobenzidine photo-oxidation by fluorescent probes was used for detecting at electron microscopy calcium ions,[125] for visualizing endocytotic pathways[126] and for tracking nanoparticles inside the cells.[127,128] These last articles demonstrate that new varied applications in nanomedicine may be envisaged for ultrastructural histochemistry.[129]

Concluding remarks

This short survey plainly confirms that histochemical research is presently addressed to identify single molecules,[130] and to dynamically describe their intracellular location and movements; in fact, nowadays papers are rarely found where conventional histochemical techniques are used to label or quantify large macromolecular categories, such as whole proteins or nucleic acids in single cells or tissues.

Even in the small sample considered, approximately one fourth of the articles describe new or improved histochemical methods or the adaptation to electron microscopy of techniques originally designed for light microscopy; this is consistent with the recent reports on the papers published in another classical Journal of histochemistry.[1,131] Thanks to this unceasing methodological refinement, histochemistry will continue to provide novel applications in the biomedical field.

In the last decade, new technologies have been developed which made it possible to break or bypass the classical Abbe’s diffraction limit.[131,132] They are collectively known as super-resolution microscopy, and pushed the optical resolution down to the molecular level.[133] This has opened unexpected opportunities for the application of histochemistry at the nanoscale: using highly specific fluorescent labeling by immunocytochemistry, or in situ hybridization, or fluorescent protein probes, the spatial distribution and dynamics of molecules (genome sequences, RNAs or proteins) can be investigated in every subcellular organelle or structure, in living or fixed samples[134] with a resolution comparable to electron microscopy and the unique flexibility of multicolor fluorescence microscopy.

So we may surely answer the title question: “yes: there still room for novelty in histochemical papers!”.