Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic, neurofilament immunoreactive myenteric neuron types of human small intestine

Abstract

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO(4) or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO(4) or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.

Quantitative Estimation and Chemical Coding of Spiny Type I Neurons in Human Intestines

“…As to the other aspect of our study, i.e. neuronal morphology, we reported earlier that we did not observe marked morphological changes of myenteric neurons in colchicine-treated versus non-treated whole mounts [Brehmer et al, 2004].…”

“…To reduce lipofuscin-induced autofluorescence, whole mounts were incubated in ammonium acetate buffer (pH 5.0) containing 1 m M CuSO 4 for 120 min followed by a short rinse in distilled H 2 O [Schnell et al, 1999;Brehmer et al, 2004]. Thereafter, both whole mounts and sections were mounted with TBS-glycerol (1: 1; pH 8.6).…”

“…One of the human myenteric neuron types described recently is co-immunoreactive for vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and neurofilaments (NF) and consistently displays, in contrast to other neuron types, spiny-shaped dendrites. This unique combination of chemical and morphological features led us to separate them as spiny from other Dogiel type I neurons [Brehmer et al, 2004Lindig et al, 2009]. Most of these spiny type I neurons have anally directed axons running within the myenteric plexus as long as they can be traced.…”

Nitrergic Myenteric Neurons are Spared in Experimental Chagasic Megacolon

“…This suggests this type of neuron is preserved in detriment of the loss of other neuron sub-types. It is known that nNOS neurons mediate most of the inhibitory responses in the gastrointestinal tract and regulate various important physiological reflexes such as the relaxation of the lower oesophageal sphincter after swallowing, receptive relaxation of the proximal stomach during feeding and descendent inhibition in response to distension [45]. Other studies, however, report a reduction in nNOS expression in the myenteric plexus during acute inflammation such as that observed in experimental models of colitis and aging [46,47] and human megacolon [34].…”

Potential conversion of adult clavicle‐derived chondrocytes into neural lineage cells in vitro

“…Anterior subventricular zone, or intestines, dissected from BrdU injected rats used as positive control tissues, and negative staining controls consisted of staining sections from naïve animals and staining of experimental tissue with omission of primary antibody. Autofluorescence, especially for explants samples, was quenched by Sudan Black B or cupric sulfate as described previously (Brehmer et al, 2004;Casella et al, 2004).…”