Effects of protein diet on expression of Anoctamin1 of Cajal cell in the nervous plexus of the stomach in male mice

Abstract

Objective: To determine the association between a protein-rich diet and the expression of anoctamin 1 inInterstitial cells of Cajal within the muscle layer of the stomach wall in male mice.Method: The experimental study was conducted at the Anatomy Department of the College of Medicine, Al-Nahrain University, Iraq, from November 2020 to April 2021, and comprised male adult healthy male mice. They were divided randomly into two equal groups. Group A was fed a high protein diet, while control group B was fed a standard pellet diet. The tissue samples were harvested at day 30 post-surgery. The stomach samplings were placed in 10% neutral formalin for 24 hours to obtain paraffin sections for routine histological and immunohistochemical staining. The protein expression in stomach smooth muscle of each group was detected by immunohistochemical staining. Data was analysed using SPSS 24.Result: Of the 20 mice, 10(50%) were in each of the two groups. Group A exhibited significant weight-gaincompared to group B (p less than or equal to 0.05). There was significant elevation in muscle wall thickness in group A, compared to group B (p less than or equal to 0.05). Tunica muscularis of the stomach in group A significantly thickened compared to group B(p less than or equal to 0.05). The expression of anoctamin 1 was significantly more intense in group A compared to group B (p less than or equal to 0.01).Conclusion: Unbalanced food had a significant impact on the stomach, affecting the thickness of the muscularis layer and the expression of anoctamin 1 in cajal cells in the muscularis externa.Key Words: Anoctamin, Paraffin, Cajal, Formaldehyde

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J Pak Med Assoc (Suppl. 8) Open Access
The 16th scientific international conference S-432
RESEARCH ARTICLE
Effects of protein diet on expression of Anoctamin1 of Cajal cell in the nervous
plexus of the stomach in male mice
Khulood Ayad Majeed1, Haider Abdal-Rasool Jaafar2, Hiba Majed Rasol3
Abstract
Objective: To determine the association between a protein-rich diet and the expression of anoctamin 1 in
Interstitial cells of Cajal within the muscle layer of the stomach wall in male mice.
Method: The experimental study was conducted at the Anatomy Department of the College of Medicine, Al-
Nahrain University, Iraq, from November 2020 to April 2021, and comprised male adult healthy male mice. They
were divided randomly into two equal groups. Group A was fed a high protein diet, while control group B was fed
a standard pellet diet. The tissue samples were harvested at day 30 post-surgery. The stomach samplings were
placed in 10% neutral formalin for 24 hours to obtain paraffin sections for routine histological and
immunohistochemical staining. The protein expression in stomach smooth muscle of each group was detected by
immunohistochemical staining. Data was analysed using SPSS 24.
Result: Of the 20 mice, 10(50%) were in each of the two groups. Group A exhibited significant weight-gain
compared to group B (p≤ 0.05). There was significant elevation in muscle wall thickness in group A, compared to
group B (p≤0.05). Tunica muscularis of the stomach in group A significantly thickened compared to group B
(p≤0.05). The expression of anoctamin 1 was significantly more intense in group A compared to group B (p≤0.01).
Conclusion: Unbalanced food had a significant impact on the stomach, affecting the thickness of the muscularis
layer and the expression of anoctamin 1 in cajal cells in the muscularis externa.
Key Words: Anoctamin, Paraffin, Cajal, Formaldehyde
(JPMA 74: S432 (Supple-8); 2024) DOI: https://doi.org/10.47391/JPMA-BAGH-16-97
Introduction
Interstitial cells of Cajal (ICC) were originally identified by
Cajal, who characterised them as nerve-like cells localised
at the terminals of motor neurons within organs
innervated by peripheral nerves. He classified these cells
as primitive neurons based on distinct staining properties
using methylene blue and silver chromate. Subsequent
advancements in the field revealed that ICCs express a
receptor associated with anoctamin 1 (Ano1), a calcium-
activated chloride channel. Ano1 serves as a novel and
selective molecular marker applicable to all ICC classes
present in the gastrointestinal (GI) tracts of both humans
and mice. This discovery enabled the immunochemical
identification of ICC independently of the Kit protein. ICCs
were initially characterised using morphological criteria,
exhibiting close association with nerve varicosities and
forming numerous gap junctions among themselves and
with smooth muscle cells, creating a network across the
gut wall. The classification of ICC was multifactorial, and
there exists consensus regarding the morphology,
localisation and roles of various ICC types1. ICC are denser
in corpus/antrum than fundus. This indicates that they
likely serve as pacemaker cells regulating gastric motility.
ICC are an integral component of the enteric nervous
system, playing a pivotal role in modulating
gastrointestinal muscle movement and orchestrating
contractions facilitating food transit through the
digestive tract. Hence, the heightened concentration of
these cells in specific stomach regions may underscore
their significant involvement in governing intestinal
activity and food propulsion.
Scanning electron microscope showed complex ICC
arrangement with Auerbach's plexus in guinea-pig small
intestine. Reticular fibrils cover plexus, enabling detailed
intact nerve and tissue study2,3. ICC changes are linked to
motility issues. Abnormalities are linked to GI motility
problems and diseases4,5.
The current study was planned to determine the
association between a protein-rich diet and the
expression of Ano1 in ICCs within the muscle layer of the
stomach wall in male mice.
Materials and Methods
The experimental study was conducted at the Anatomy
Department of the College of Medicine, Al-Nahrain
1Department of Health, Iraqi Ministry of Health, Kirkuk, Iraq.2-3 Department of
Anatomy, Al-Nahrain University, Baghdad, Iraq.
Correspondence: Khulood Ayad Majeed
Email: khelowd@gmail.com

University, Iraq, from November 2020 to April 2021.
Approval was obtained from the institutional
animal ethics committee, and the study was
conducted in line with the Guidelines on Ethical
Treatment of Experimental Animals issued by the
Iraq Provincial People’s Government according to
National Institute of Health guidelines for the care
and use of laboratory animals6.
Healthy adult male albino mice aged about 8
weeks and weighing 20-30g were selected and
divided into two groups. Intervention group A was
subjected to a high protein diet, while control
group B received a standard pellet diet containing
30% protein. The allocation of protein intake
percentages was determined in the light of
literature7. The intervention lasted 30 days during
which all animals were treated according to
National Institute of Health guidelines for the care
and use of laboratory animals6. The mice were then
dissected. The stomach samplings were engaged
in 10% neutral-buffered formalin (NBF) for 24 hours
before being histologically processed for paraffin
section that included de-waxing, staining and
mounting, as well as fixation, dehydration,
clearing, impregnation, embedding and
sectioning. Tissue preparation for paraffin blocking
was done in the light of a study8.
Immunohistochemical process and monoclonal
immunoglobulin M (IgM) antibody were used
(Sigma Aldrich, Germany; Abcam, United
Kingdom). The procedures were carried out in
accordance with the instructions provided by the
manufacturers.
Data was analysed using SPSS 24. P<0.05 was considered
significant.
Results
Of the 20 mice, 10(50%) were in each of the two groups.
Group A exhibited significant weight-gain compared to
group B (p≤0.05) (Table 1). In control group B, the
stomach's distinct layers were readily distinguishable,
comprising four layers: mucosa, submucosa, muscularis
and serosa. The muscularis externa comprised two
prominent layers of smooth muscle: an inner circular layer
and an outer longitudinal layer. The myenteric plexus
(Auerbach's plexus) was positioned between these layers
of smooth muscle. In protein group A, tunica muscularis
of the stomach exhibited an increased thickness, and was
composed of multiple layers of smooth muscle fibres.
Within the interstitial spaces of the muscle cells, the
Auerbach plexus displayed hypertrophy and
degeneration, evident by the presence of vacuolated
cytoplasm (Figure 1-A-B).
The thickness of the muscularis layer displayed a more
increase in Group A compared to group B (Table 2).
The expression of Ano1 was significantly more intense in
group A compared to group B (p≤0.01) ((Table 3; Figure 1-
C-D; Figure2).
Open Access Vol. 74, No.10 (Suppl. 8), October 2024
S-433 The 16th scientific international conference
Table-1: Weight of the animals before and after using diet variation.
Group Name Mean (gm) ± SD T-test
Before After
Control Group 22.56 ±1.94 22.33 ±1.22 NS
Protein Group 21.44 ±1.67 28.44 ±1.42 1.549 **
P-value 0.110 NS 0.0001
SD: Standard deviation, NS: Non-significant.
Figure-1: (A-B) Cross-section of muscularis layer of stomach showing degenerated
myentric plexus (blue arrow), and vacuoles between smooth muscles (black arrow)
(Haemotoxylin and Eosin [H&E] X40); (C1) Intensity of the reaction at muscularis
externa, (C2) the same tissue analysed by aperio software; (D1) Intensity of the
reaction at muscularis externa; ( D2) the same tissue analysed by aperio software.

J Pak Med Assoc (Suppl. 8) Open Access
The 16th scientific international conference S-434
Discussion
The present study revealed a notable disparity in
expression, with animals on a protein-rich diet displaying
heightened aggression and quarrelsome behaviour.
Furthermore, the animals subjected to the protein diet
exhibited a noteworthy rise in the number of faecal
contents collected daily from within their cages,
accompanied by an unpleasant odour compared to the
control group. This finding is consistent with DeNapoli et
al., who observed an increase in dominance despite a
decrease in dietary protein content9. Moreover,
aggression is frequently employed in competitive
systems to secure access to limited resources within a
social context.
In the current study, the protein diet group gained
weight, which was also observed in a prior study10. The
participants on a high-protein diet had a thick muscularis
layer, which contradicts the notion that a high-fibre, low-
protein diet enhances the thickness of the muscularis
layer11.
The expression of Ano1 in the muscularis layer of protein-
fed animals exhibited a high level of expression, which is
consistent with previous research12
The protein group animals exhibited a significant increase
in Ano1 expression, which is associated with the adjacent
ganglionic neurons of the myenteric plexus, but this does
not indicate an increase in their numbers, and this is
correlated with the same conditions as malnutrition13.
Unbalanced meals can generate morpho functional
alterations in the enteric nerve plexuses, leading to
malnutrition-related diseases, such as stomach
discomfort, constipation, faecal incontinence, diarrhoea,
and malabsorption14. This explains and exhibits how a
high-protein diet induces stress by altering the muscularis
layer, resulting in a decrease in thickness and, as a result,
a significant rise in the expression of Ano1 protein in the
muscularis layer compared to the control group. This is
consistent with reports that starvation is a pathological
condition that can manifest as reversible or irreversible
organic structural alterations15. All normal metabolic
processes require protein participation, and protein-loss
affects all tissues, but the rate and type of modification
are not the same. Protein deficiency affects tissues with
high cell refurbishment rates, such as the intestinal
mucosa, while protein deficiency affects tissues with low
Figure-2: (A) Weight of the animals before and after using diet variation; (B) Comparison between difference groups in muscularis thickness; (C) Comparison between diet modality
groups in anti-anoctamin 1 (Ano1) expression.
Table-2: Comparison related to muscularis thickness.
Group Mean (μm) ± SD
Control 122.96 ±23.51
Protein Group 133.86 ±34.59
** P≤0.05
SD: Standard deviation.
Table-3: Comparison related to anti- anoctamin 1 (Ano1) expression.
Group Mean (pixels per square micron) ± SD
Control Group 0.3135 ±0.1363
Protein Group 0.5760 ±0.0953
** P≤0.01
SD: Standard deviation.

Open Access Vol. 74, No.10 (Suppl. 8), October 2024
S-435 The 16th scientific international conference
cell renovation rates, such as the myenteric nerve plexus
system.16 In malnourished rats, hypoplasia and hypo
trophic mucosa have been detected17, but other results
have also been reported12,13.
The presence of Ano1 in ICCs of the control group was
critical for maintaining a balanced peristaltic smooth
muscle activity, which guarantees coordinated stomach
motility. Ano1 regulates synchronous peristaltic motor
action. Inhibitory Ano1 protein buildup in the stomach
and digestive tract may impair normal motility. This can
result in increased relaxation and decreased contraction
force, potentially resulting in difficulty emptying the
stomach and chronic constipation. On the other hand,
symptoms like nausea, belching, bloating, heartburn,
indigestion, regurgitation, or vomiting might occur when
the nerve regulating the stomach muscles is
compromised. Gastroparesis, often known as delayed
gastric emptying, is a disorder in which the stomach
empties itself gradually. Moving partially digested food
from the stomach to the small intestine depends heavily
on the stomach's muscles.18.
In comparison to the control group, the expression of
Ano1 protein in the muscularis layer of protein diet
animals demonstrated elevated expression of Ano1. This
increased the motility of ICCs by causing them to
innervate the muscularis layer of the gut tube. This was
reported earlier as well19.
The current findings showed that dietary variability,
whether high or low in protein, affected Ano1 protein
production in the ICCs, which facilitated peristaltic
motility in the stomach. This is consistent with a prior
study20. Other studies have discovered that intraduodenal
proteins had load-dependent effect on Antro
pyloroduodenal motility as well as energy intake at a
subsequent meal in both lean and obese people,
implying that small intestinal sensitivity to protein
remains intact21. GI motor activity has a role in the
regulation of blood glucose appetite in both lean and
obese adults by decreasing gastric emptying activity and
thus appetite21.
Conclusion
An imbalanced diet significantly affected the thickness of
the muscularis externa layer and the expression of Ano1
by ICCs in the muscularis layer in the stomach. A high-
protein diet caused Ano1 levels in the stomach to rise. As
a result, gut motility was disrupted, which could result in
specific motility abnormalities that are a major
contributor to GI illnesses.
Disclaimer: The text is based on an academic thesis.
Conflict of Interest: None.
Source of Funding: None.
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