PRELIMINARY IMMUNOHISTOCHEMISTRY STUDY ASCERTAINED THE EXPRESSION OF FMRFAMIDE-RELATED PEPTIDES IN THE INTESTINE AND DORSAL ROOT GANGLIA IN MICE

Assiut University web-site: www.aun.edu.eg

PRELIMINARY IMMUNOHISTOCHEMISTRY STUDY ASCERTAINED THE EXPRESSION OF FMRFAMIDE-RELATED PEPTIDES IN THE INTESTINE AND DORSAL ROOT GANGLIA IN MICE

NADA ABDELLAH ABDELSAMEA ABDELLAH ¹; MADEHA AHMED HASHIM ²

AND MOHAMED-NABIL MUSTAFA ³

¹ Department of Histology, Faculty of Veterinary Medicine, Sohag University, Sohag, Egypt.

 Lecturer of Histology.

² Department of Histology, Faculty of Veterinary Medicine, Sohag University, Sohag, Egypt. Lecturer of Histology, Email address:vet_madeha@yahoo.com

³ Department of Anatomy, Embryology, and Histology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt, 71526. Professor of Histology

Received: 26 July 2020;     Accepted: 31 August 2020

INTRODUCTION

FMRFamide-related peptides are a class of amidated peptides including, Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) (Price and Greenberg 1977, Yang et al., 1985, Yang and Martin 1995 and Panula et al., 1996).

Corresponding author: Nada Abdellah Abdelsamea

E-mail address: vet_nada@yahoo.com

Present address: Department of histology, Faculty of veterinary medicine, Sohag University, Sohag, Egypt. Lecturer of Histology

They are expressed and widely distributed throughout the mammalian central nervous system and implicated in a wide range of functions (Panula et al., 1996). Although an anti-opiate effect (Tang et al., 1984 and Malin et al., 1990) and related pain modulation (Yang et al., 1985 and Gouardères et al., 1993) is the most prominent effects of FMRFamide-related peptides, they are also implicated in cardiovascular regulation (Panula et al., 1996) and neuroendocrine functions (Majane and Yang 1990, Majane et al., 1993). Interestingly, the precursor protein NPFF gene (ProNPFF), which encodes for NPFF has been cloned and shown to encode not only  NPFF, but also NPAF and NPSF in human, murine, bovine, and rat tissues (Vilim et al., 1999). Even though the biological roles of NPFF, NPAF, and related FMRFamides are well identified, their source and distribution in mammals are not well-defined. They represent the main identified family of neuropeptides in invertebrates (Peymen et al., 2014). In insects, FMRFamide-related peptides have been isolated and shown to be distributed extensively throughout the nervous system, salivary glands, accessory glands, and muscle (Orchard et al., 1997). In nematodes, they considered the chief regulators of energy balance, feeding behavior, reproduction, and sensory modulation (Peymen et al., 2014).

MATERIALS AND METHODS

Animals

Ten adult male mice BL/6  (wild black mice) were sacrificed for sample collection. Animal housing and handling procedures were conducted in accordance with the European Directive 86/609/EEC.

Tissue processing for immunohistochemistry

Male BL/6 mice were sacrificed using cervical dislocation and then dissected. Tissues of interest were fixed in 4% paraformaldehyde   solution for 2 hrs. For paraffin sections, after fixation, the tissues were placed in a cassette at the correct orientation. Next, the cassettes were placed in a Histokinette (24 hours-programm) to dehydrate the tissues in ascending series of ethanol solutions, subsequently to be cleared in methyl-benzoate and xylene solutions, and finally infiltrated with melted paraffin in order to become embedded in paraffin blocks. Next, 5-µm-thick paraffin sections were cut using a LEICA RM 2245 microtome.

Immunohistochemistry for

paraffin sections

The paraffin sections were deparaffinized using xylene then rehydrated by gradient ethanol dilutions (100%-90%-70%) and finally rinsed in distilled water. Next, microwave antigen retrieval was performed by heating the sections in 0.1M sodium citrate/0.1 M citrate buffer (pH 6.0) for 5x3 min each; subsequently, the slides were left to cool down for 45 min. Blocking of endogenous peroxidase was carried out with 0.3 % hydrogen peroxide (H₂O₂) solution for 20 min. To block unspecific binding, incubation with (1:5) normal horse serum in PBS with 1% bovine serum albumin (BSA) was performed. Subsequently, sections were incubated with primary anti-FMRFamide (1:500) (Merck-Millipore, AB15348) diluted in 0.1 M PBS (phosphate buffer solution) with 1% BSA overnight at 4°C. Sections then were incubated with secondary biotinylated donkey anti-rabbit (1:200) in PBS with 1% BSA for 2 hrs, followed by 3 washes with PBS and incubation with Extravidin peroxidase (1:200) (Sigma) in PBS with 1% BSA for 30 min. After a washing step, DAB solution (Dako K3468) was added until color developed and then the reaction was stopped by washing the section with PBS. Sections were then washed with tap water and distilled water, for 5 min each, followed by counterstaining with haematoxyline. All incubations were performed at RT (Room temperature), except for those with primary antibody which took place at 4°C.

RESULTS

We evaluated the presence of RF-amides in DRG (dorsal root ganglia) and in the ileal and colonic wall immunohistochemically. Negative controls in which the primary antibody was omitted or staining was done with IgG control did not yield any immunoreactivity. Staining of paraffin-embedded ileum, colon, and dorsal root ganglia (DRG) sections revealed the presence of FMRF-amides (Fig.1). In DRG FMRF-amide peptides were expressed within the nerve cells and nerve fibers (Fig.1A). In the ileum and colon, FMRF-amide immunoreactivity was detected in enteroendocrine cells and in intramuscular nerve fibers (Fig.1 C-F).

Fig. 1: Paraffin embedded sections, immunohistochemically stained for FMRFamide. (A) FMRFamides were expressed in nerve cells and nerve fibers in DRG (Arrows). (B) Omission of primary antibody led to elimination of immunostaining in DRG. (C) FMRFamides were detected in nerve fibers (square) and in enteroendocrine cells (arrows) in the ileum. (D-E) Enteroendocrine cells showed immunoreactive granules (square, arrow). (F) Enteroendocrine cells were also immunoreactive in the colon (square). (G-H) Staining with IgG control revealed no immunoreactivity in ileum or colon.

DISCUSSION

We used the anti-FMRFamide antibody to detect the potential sources of NPAF and NPFF in situ, which revealed the presence of FMRFamide peptides in the enteroendocrine cells and nerve fibers located in the enteric plexuses of the ileum and colon. We had proposed the cells were enteroendocrine cells according to their location and the solitary distribution. Although nothing is known before about the expression and function of RF-amides in the mammalian GI tract it is known that the FMRFamide is the most abundant neuropeptide in endocrine cells of insect alimentary tract (Oetken et al., 2004 and Haselton et al., 2008). Also, we detected the sources of NPAF and NPFF in dorsal root ganglia. Although, early studies proposed that there may be an NPFF-Iike peptide in the sympathetic ganglia and adrenal medulla and not detected NPFF in the spinal or sympathetic ganglia (Panula et al., 1987 and Lee et al., 1993). Later, they are identified to be highly condensed in the posterior pituitary, spinal cord, hypothalamus, and medulla in mouse, rat, the bovine, and human brain (Panula, Aarnisalo et al., 1996).

CONCLUSION

The current study represents the first evidence of the expression of FMRFamide peptides in the mammalian gastrointestinal tract. Also, we are suggesting that enteroendocrine cells are potential sources. Further investigations and characterizations are recommended.

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