3-D imaging of islets in obesity: formation of the islet-duct complex and neurovascular remodeling in young hyperphagic mice.

BACKGROUND: Obesity and insulin resistance lead to islet hyperplasia. However, how the islet remodeling influences the pancreatic environment and the associated neurovascular networks is largely unknown. The lack of information is primarily due to the difficulty of global visualization of the hyperplasic islet (>200 µm) and the neurovascular environment with high definition. METHODS: We modulated the pancreatic optical property to achieve 3-dimensional (3-D) whole-islet histology and to integrate transmitted light microscopy (which provides the ground-truth tissue information) with confocal fluorescence imaging. The new optical and imaging conditions were used to globally examine the hyperplastic islets of the young (2 months) obese db/db and ob/ob mice, which otherwise cannot be easily portrayed by the standard microtome-based histology. The voxel-based islet micrographs were digitally processed for stereo projection and qualitative and quantitative analyses of the islet tissue networks. RESULTS: Paired staining and imaging of the pancreatic islets, ducts and neurovascular networks reveal the unexpected formation of the 'neuro-insular-ductal complex' in the young obese mice. The complex consists of the peri- and/or intra-islet ducts and prominent peri-ductal sympathetic nerves; the latter contributes to a marked increase in islet sympathetic innervation. In vascular characterization, we identify a decreased perivascular density of the ob/ob islet pericytes, which adapt to ensheathing the dilated microvessels with hypertrophic processes. CONCLUSIONS: Modulation of pancreatic optical property enables 3-D panoramic examination of islets in the young hyperphagic mice to reveal the formation of the islet-duct complex and neurovascular remodeling. On the basis of the morphological proximity of the remodeled tissue networks, we propose a reactive islet microenvironment consisting of the endocrine cells, ductal epithelium and neurovascular tissues in response to the metabolic challenge that is experienced early in life.

Imaging of the islet neural network.

The islets of Langerhans receive signals from the circulation and nerves to modulate hormone secretion in response to physiological cues. Although the rich islet innervation has been documented in the literature dating as far back as Paul Langerhans' discovery of islets in the pancreas, it remains a challenging task for researchers to acquire detailed islet innervation patterns in health and disease due to the dispersed nature of the islet neurovascular network. In this article, we discuss the recent development of 3-dimensional (3D) islet neurohistology, in which transparent pancreatic specimens were prepared by optical clearing to visualize the islet microstructure, vasculature and innervation with deep-tissue microscopy. Mouse islets were used as an example to illustrate how to apply this 3D imaging approach to characterize (i) the islet parasympathetic innervation, (ii) the islet sympathetic innervation and its reinnervation after transplantation under the kidney capsule and (iii) the reactive cellular response of the Schwann cell network in islet injury. While presenting and characterizing the innervation patterns, we also discuss how to apply the signals derived from transmitted light microscopy, vessel painting and immunostaining of neural markers to verify the location and source of tissue information. In summary, the systematic development of tissue labelling, clearing and imaging methods to reveal the islet neuroanatomy offers insights to help study the neural-islet regulatory mechanisms and the role of neural tissue remodelling in the development of diabetes.

Regulation of functional muscarinic receptor expression in tracheal smooth muscle cells.

Recent studies have shown that cultured tracheal smooth muscle cells (TSMCs) do not respond to muscarinic agonists with a significant increase in intracellular Ca2+ concentration. This may be due to a downregulation of muscarinic receptors (mAChRs) in TSMCs. We report here that the individual component of growth factors or hormones at the concentration used is not sufficient to stimulate growth of TSMCs in the primary culture with 1% fetal bovine serum (FBS). In the presence of 1% FBS, TSMCs withdraw from the cell cycle and express high levels of cell surface mAChRs. Furthermore, insulin-like growth factor I (IGF-I) and insulin (Ins), alone or in combination, could stimulate the expression of mAChRs on the cultured TSMCs in 1% FBS without changing the affinity of receptors. Heparin could inhibit these stimulatory effects on mAChR expression. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and Ins than those cultured in 1% FBS. This action may be partially mediated through a cholera toxin-sensitive protein. The results conclude that IGF-I and Ins are necessary for TSMCs to express functional mAChRs.

Expression of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA in embryos and endometrial stromal cells.

The objective of the present study was to investigate the effects of insulin-like growth factor-binding protein-1 (IGFBP-1) on the interaction between mouse embryos and endometrial stromal cells in co-culture systems. To explore quantitatively the changes in expression of IGFBP-1 mRNA from endometrial stromal cells cultured alone or co-cultured with embryos, a combination of reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting followed by a densitometric analysis was used. In co-culture systems, development of embryos was significantly improved and the production of IGFBP-1 from endometrial stromal cells was stimulated by the embryos. Quantitative analysis showed that expression of IGFBP-1 mRNA in endometrial stromal cells co-cultured with embryos was higher on days 3,5 and 8 (1.6-, 4.5- and 2.3-fold respectively) than in endometrial stromal cells cultured alone. In addition, intensity of PCR products for IGFBP-1 mRNA in endometrial stromal cells co-cultured with embryos was higher on day 5 than on days 3 and 8. However, the expression of IGFBP-1 mRNA in embryos cultured alone was very low. In conclusion, co-culture of embryos with endometrial stromal cells improved the development of embryos and may be associated with the production of IGFBP-1 by the co-cultured endometrial stromal cells. A combination of RT-PCR and Southern blotting followed by a densitometric analysis appeared to be a sufficiently quantitative method to determine changes in IGFBP-1 mRNA values.