Immunohistochemical study of the brainstem cholinergic system in the alpaca (Lama pacos) and colocalization with CGRP

Several cholinergic regions have been detected in the brainstem of mammals. In general, these regions are constant among different species, and the nuclear complement is maintained in animals belonging to the same order. The cholinergic system of the brainstem has been partially described in Cetartiodactyla, except for the medulla oblongata. In this work carried out in the alpaca, the description of the cholinergic regions in this order is completed by the immunohistochemical detection of the enzyme choline acetyltransferase (ChAT). In addition, using double immunostaining techniques, the relationship between the cholinergic system and the distribution of calcitonin gene-related peptide (CGRP) previously described is analysed. Although these two substances are found in several brainstem regions, the coexistence in the same cell bodies was observed only in the laterodorsal tegmental nucleus, the nucleus ambiguus and the reticular formation. These results suggest that the interaction between ChAT and CGRP may be important in the regulation of voluntary movements, the control of rapid eye movement sleep and states of wakefulness as well as in reward mechanisms. Comparing the present results with others previously obtained by our group regarding the catecholaminergic system in the alpaca brainstem, it seems that CGRP may be more functionally related to the latter system than to the cholinergic system.

Tumour-associated Angiogenesis and Intermediate Blood Vessels in Renal Cell Carcinoma

Background/Aim: Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry. Materials and Methods: Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells. Results: We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side. Conclusion: We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy.

Blocking Cross-Species Secondary Binding When Performing Double Immunostaining With Mouse and Rat Primary Antibodies

Immunostaining is a powerful technique and widely used to identify molecules in tissues and cells, although critical steps are necessary to block cross-reaction. Here we focused on an overlooked cross immunoreactivity issue where a secondary antibody (secondary) cross-reacts with a primary antibody (primary) from a different species. We first confirmed the previously reported cross-species binding of goat anti-mouse secondary to rat primary. This was accomplished by staining with a rat primary against glial fibrillary acidic protein (GFAP) and visualizing with goat (or donkey) anti-mouse secondary. We then further revealed the converse cross-species binding by staining with a mouse primary against neuronal nuclear protein (NeuN) and visualizing with anti-rat secondaries. We speculate that mouse and rat primaries share antigenicity, enabling either secondary to recognize either primary. To block this cross-species binding in double staining experiments, we compared three protocols using mouse anti-NeuN and rat anti-GFAP, two primaries whose antigens have non-overlapping distributions in brain tissues. Simultaneous staining resulted in cross-species astrocytic staining (anti-mouse secondary to rat anti-GFAP primary) but no cross-species neuronal staining (anti-rat secondary to mouse anti-NeuN primary). Cross-species astrocytic staining was missing after sequential same-species staining with mouse anti-NeuN primary, followed by rat anti-GFAP. However, cross-species astrocytic staining could not be diminished after sequential same-species staining with rat anti-GFAP primary, followed by mouse anti-NeuN. We thus hypothesize that a competition exists between anti-mouse and anti-rat secondaries in their binding to both primaries. Single staining for NeuN or GFAP visualized with dual secondaries at different dilution ratio supported this hypothesis.

The conditional deletion of steroidogenic factor 1 (Nr5a1) in Sox9-Cre mice compromises testis differentiation

Steroidogenic factor 1 (NR5A1) is essential for gonadal development. To study the importance of NR5A1 during early gonadal sex differentiation, we generated Sox9-Cre-Nr5a1 conditional knockout (cKO) mice: Sox9-Cre;Nr5a1flox/flox and Sox9-Cre;Nr5a1flox/− mice. Double-immunostaining for NR5A1 and AMH revealed silenced NR5A1 in Sertoli cells and reduced AMH+ cells in the gonads of XY Sox9-Cre-Nr5a1 cKO mice between embryonic days 12.5 (E12.5) and E14.5. Double-immunostaining for SOX9 and FOXL2 further indicated an early block in Sertoli cells and ectopic granulosa cell differentiation. The number of cells expressing the Leydig cell marker 3βHSD obviously reduced in the gonads of XY Sox9-Cre;Nr5a1flox/− but not Sox9-Cre;Nr5a1flox/flox mice at E15.5. The presence of STRA8+ cells indicated that germ cells entered meiosis in the gonads of XY Sox9-Cre-Nr5a1 cKO mice. The results of qRT-PCR revealed remarkably reduced and elevated levels of testis and ovary markers, respectively, in the gonads of XY Sox9-Cre-Nr5a1 cKO mice at E12.5‒E13.5. These data suggested that the loss of Nr5a1 abrogates the testicular pathway and induces the ectopic ovarian pathway, resulting in postnatal partial/complete male-to-female gonadal sex reversal. Our findings provide evidence for the critical role of NR5A1 in murine gonadal sex determination in vivo.

Adrenal Corticomedullary Mixed Tumor Associated With the FGFR4-G388R Variant

Adrenal corticomedullary mixed tumors (CMMTs) are extremely rare; with only 20 cases being reported to date, the pathogenesis has remained elusive. A 31-year-old woman developed gestational hypertension with psychiatric disturbances persistent to postpartum and was diagnosed with pheochromocytoma, for which adrenalectomy was performed. Histological findings showed mixed adrenocortical adenoma and pheochromocytoma. Double immunostaining of inhibin and INSM1 (insulinoma-associated protein 1) showed that the 2 tumor components had distinct functional properties. Exome analysis of peripheral leukocytes and tumor (singular, as anatomically it is only 1 mass) revealed a homozygous germline FGFR4-G388R variant. As a readout of the variant, serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was detected only in the nucleus of adrenocortical adenoma component but not in the pheochromocytoma component. No tyrosine phosphorylation of STAT3 was detected. We report a case of CMMT with the germline FGFR4-G388R variant. Although additional studies are required, our immunohistochemical analysis suggests that the variant may play a role in the development of the adrenocortical component within the pheochromocytoma, leading to CMMT.