The enteric nervous system (ENS) is a highly complex structure that is considered to have the largest accumulation of neurons outside the central nervous system. It constitutes one division of the peripheral nervous system, but contains more neurotransmitters and neuromodulaters 1,2. It has been documented that ENS can be considered a migrating portion of the central nervous system that maintains communication with the latter through sympathetic and parasympathetic neurons, afferents, and efferents. The ENS has a cytoarchitecture, chemical coding, neuronal circuits, and intrinsic functional components such as sensory, motor, and interneurons. Thus, it is able to function with its own intrinsic neural circuits even when devoid of central afferent innervations 3.
The ENS consists of local nerve networks embedded in the gut wall along the entire gastrointestinal tract (GIT) 4. It controls motility, secretion, and blood flow in the GIT and is called the ‘little brain’ of the gut 2,5. Structurally, the ganglia of ENS are organized into submucosal (Meissner’s) and myenteric (Auerbach’s) plexi 6. The myenteric plexus (Auerbach’s plexus) lies between the longitudinal and circular smooth muscle layers, providing motor innervations to the two muscular layers and secremotor innervations to the mucosa. Thus, it is highly specialized both functionally and structurally 7.
In addition to neurons, the enteric ganglia contain glial cells that resemble astrocytes of the central nervous system. Unlike the other autonomic ganglia, these ganglia are compact and do not contain blood vessels or connective tissue, but contain a dense synaptic neuropil. Myenteric ganglia are surrounded by a thin layer of connective tissue, and are closely associated with a specialized cell type known as the interstitial cells of Cajal, which act as pacemaker cells, intermediary between the myenteric plexus and the smooth muscles 8.
Aging is the major risk factor for several common neurodegenerative and motor neuron diseases. It is responsible for the marked reduction in and reorganization of myenteric neurons. Up to a certain point, this results in the elimination of redundant neurons and the redistribution of work among the remaining ones 9. The digestive system is adversely affected by aging and its functions change markedly over the normal human lifespan 10. There are impairments in intestinal function, including a reduction in the gastric emptying time and the frequency and amplitude of the peristaltic wave in elderly patients. Also, there are changes in the digestion and absorption of food and a significant decrease in gastric blood flow, and certain mucosal protective mechanisms may be compromised 11.
Protein gene product (PGP 9.5) is a well-established general marker for the majority of peripheral autonomic, including enteric neurons, and has been used widely in descriptive studies. These studies have confirmed that PGP 9.5 labeling provides an accurate indication of age-related neuron loss 12. It is well established that acetylcholine is an important excitatory transmitter to the smooth muscle of the stomach 13. Thus, the neurons of the myenteric plexus of the stomach express the acetylcholine transporter, which can be determined using the neuronal marker PGP 9.5 14.
Apoptosis is a genetically regulated cell death program that occurs during neuronal development and in some neurodegenerative diseases. The Bcl-2 protein is a small intracellular nonglycosylated protein that can inhibit the apoptotic pathway. It is widely expressed in the developing central and peripheral nervous systems. Also, the Bcl-2 protein is involved in promoting the survival of enteric neurons throughout life. Thus, Bcl-2 immunostaining may be an option for neuron counting, which is important for the diagnosis of disorders in the enteric plexuses 15,16.
Many studies have reported extensive losses of 30–60% of neuronal density within the myenteric plexus in humans, rodents, guinea-pigs, and mice with aging, affecting the entire GIT 17. Nevertheless, little is known about the effect of aging on the histological structure of the myenteric plexus of the gastric fundus in rats. Therefore, the present study was designed to examine the structural changes within the myenteric plexus that may occur during normal aging in rats.
Materials and methods
Thirty male albino rats of different ages were obtained from the Animal House, Faculty of Medicine, Zagazig University. The rats were maintained under standard conditions at 21ºC under a 12 h light–dark cycle. They were supplied with water ad libitum and fed a normal standard diet. The rats were divided equally into three groups (five animals each):
Group A (adult): Rats were 4 months old.
Group B (early senile): Rats were 18 months old.
Group C (late senile): Rats were 24 months old.
At the time of sacrifice, all the animals were anesthetized with Thiopental (50▒mg/kg) intraperitoneally, and the animals were transcardially perfused, first, with warm heparinized 0.05▒mol/l PBS (pH 7.4), followed by freshly prepared 4% paraformaldehyde in 0.1▒mol/l phosphate buffer 18. The stomachs were dissected and rinsed with PBS (pH 7.4), poured into the gastric cavity with 40▒g/l paraformaldehyde (until three to four times of normal stomach volume), and placed in the same fixative for 4–6▒h (4ºC). Thereafter, the stomachs were washed with PBS 19 and opened along the greater curvature. The gastric fundui were excised and divided into two parts and processed for light and electron microscope evaluation.
For light microscope examination, specimens were fixed in 10% formol saline and processed to prepare 5▒μm thick serial paraffin sections 20. They were stained with H&E. Also, immunohistochemical staining was carried out for the pan-neuronal marker PGP 9.5 and for the detection of Bcl-2 protein 11.
An immunohistochemical reaction was carried using the avidin–biotin peroxidase system. The primary antibody used was a rabbit polyclonal antibody purchased from Sigma Laboratories (Sigma Aldrich, UK) for Bcl-2 protein and from Ultraclon for PGP 9.5. The universal kit had an avidin–biotin peroxidase system manufactured by Nova Castra Laboratories Ltd (UK). Negative control sections were treated according to the protocol, but without exposure to primary antibodies. The ganglia were identified as discrete aggregations of PGP-positive cells, located between the smooth muscle layers (myenteric plexus). Within the ganglia, neurons were considered as cells with a PGP-positive cytoplasm, whereas the positive control for Bcl-2 immunoreactions appeared as brownish coloration in the cytoplasm of lymph nodes.
For electron microscope examination, the specimens were immediately fixed in 2.5% glutaraldehyde buffered with 0.1▒mol/l phosphate buffer at pH 7.4 for 2 h and then postfixed in 1% osmium tetroxide in the same buffer for 1▒h. They were processed to prepare semithin sections and then ultrathin sections. Semithin sections (1▒μm thick) were stained with 1% toluidine blue for light microscope examination 21. Ultrathin sections were obtained using Leica ultracut UCT (Germany) and stained with uranyl acetate and lead citrate. They were examined using a JEOL JEM 1010 electron microscope (Japan) in the Electron Microscope Research Laboratory (EMRL) of Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
The image analyzer computer system Leica Qwin 500 (England) in the Histology and Cell Biology Department, Faculty of Medicine, Cairo University, Cairo, Egypt, was used to determine the mean number of PGP 9.5-positive neurons and area% of Bcl-2 protein using the interactive measure menu. The procedure was performed using immunohistochemically stained sections at a total magnification of ×400. In addition, the area of the myenteric plexus (ganglionic area) was determined in toludine blue-stained sections using the same magnification. All measurements were carried out in five fields in five sections from five rats of each group.
Data for all groups were expressed as mean ±SD (X±SD). The data obtained from the image analyzer were subjected SPSS program version 15 (Chicago, USA). Statistical analysis was carried out using the one-way analysis of variance test for comparison between different groups. P values <0.05, <0.001, and >0.05 were considered significant, highly significant, and nonsignificant, respectively. The Kruskal–Wallis test was also used for comparison of different groups in terms of measurements of the ganglionic area.
Histological and immunohistochemical results
Light microscope examination of the gastric fundus of group A (adult) showed mucosa, submucosa, muscularis externa, and serosa. The muscularis externa was composed of inner circular and outer longitudinal smooth muscles (Fig. 1). The ganglia of the myenteric plexus appeared as oval masses with multiple cells surrounded by connective tissue capsules (Fig. 2). They contained closely packed neurons (Fig. 3). Sections of the same group stained with the PGP 9.5 protein showed a strong cytoplasmic reaction in all myenteric plexus neurons (Fig. 4). Also, there was a strong reaction for Bcl-2 in neurons of this group (Fig. 5).
Ultrastructurally, the ganglia of the myenteric plexus contained glial cells, nerve cells, and neuropil and were surrounded by well-defined capsules (Fig. 6). Nerve cells had oval euchromatic nuclei and prominent nucleoli. Glial cells appeared to have irregular nuclei with peripheral condensation of heterochromatin and infolding of their nuclear envelops (Fig. 7).
In H&E-stained sections of group B (early senile), the ganglia of the myenteric plexus showed irregular outlines and a few gaps in between the neurons. Some neurons had darkly stained nuclei and a vacuolated cytoplasm (Figs. 8 and 9). In addition, there was an apparent decrease in PGP 9.5 positively stained neurons (Fig. 10) and a moderate reaction for Bcl-2 protein (Fig. 11).
Electron microscope examination of group B showed few cavities within the neuropil (Fig. 12). Glial cells had vacuoles within their cytoplasm and nerve cells appeared with peripheral condensation of heterochromatin in their nuclei (Fig. 13).
Light microscope examination of group C (late senile) showed large ganglia of the myenteric plexus with irregular outlines. Many large gaps were observed within the ganglia. The nerve cell bodies had irregular, shrunken darkly stained nuclei and cytoplasmic vacuolations (Figs. 14 and 15). PGP 9.5 immunostained sections showed a marked apparent decrease in positively stained neurons that were replaced by wide spaces (Fig. 16). Also, minimal immunoreactions for Bcl-2 protein were detected in this group (Fig. 17).
Ultrastructurally, the ganglia of this group were surrounded by many fibroblasts and collagen fibers. The nerve cells had an electron-dense cytoplasm and shrunken nuclei with peripheral chromatin condensation. Eosinophilic cellular infiltration and many large cavities were observed within the neuropil. Glial cells appeared to have electron-dense nuclei with perinuclear spaces (Figs. 18 and 19).
Morphometric and statistical results
PGP 9.5 immunostained myentric ganglia in the gastric fundus showed a decrease in PGP-positive neurons in group C (late senile) compared with groups A (adult) and B (early senile). Table 1 and Bar Chart 1 show that there was a highly significant difference in the mean number of PGP 9.5-positive neurons when group C was compared with group A or group B.
In terms of area% of immunoreactions for Bcl-2 protein, there was a decrease in area% of positive immunoreactions on comparing group A with group B or C. Table 2 and Bar Chart 2 show that there was a highly significant difference in the Bcl-2 immunoreaction (P<0.001) when group A was compared with group B or C. In addition, statistical analysis of the area% of myenteric ganglia within the wall showed a significant increase in the ganglionic area in group C compared with groups A and B (Table 3 and Bar Chart 3).
Aging involves structural and functional changes in certain aspects of GIT through its action on the ENS 22. As the ENS is responsible for coordinating and integrating the intestinal activities, the gastrointestinal disorders explained mostly by its motor dysfunctions 23,24. In addition, it has been reported that the prevalence of chronic constipation and irritable bowel syndrome in the 65–93-year-old population is as high as 24.1 and 10.9%, respectively 25. As the proportion of elderly individuals in the population is increasing rapidly, interest in this topic is growing.
In the present study, the ganglia of the myenteric plexus in group A (adult) appeared as oval masses surrounded by thin capsules and lay between the two muscular layers of muscularis externa. In groups B (early senile) and C (late senile), the outline of these ganglia appeared irregular. Ultrastructurally, the ganglia were surrounded by well-defined capsules in group A. In addition, collagen fibers appeared outside the ganglia in group B. Fibroblasts outside the ganglia and eosinophilic cellular infiltration inside the ganglia were observed in group C. The neural control of gastrointestinal function is predominantly mediated by enteric neurons that are located in small ganglia linked by nerve fiber bundles along the length of the GIT 6. Because of its location between the muscular layers, the myenteric plexus is affected by mechanical activity. Connective tissue layers and discontinuous layers of fibroblast-like cells could be observed outside the enteric ganglia. These layers envelop the ganglia, protecting them by minimizing the effect of muscular contractions on the plexus and supporting the blood vessels 26. However, connective tissue may be present within the ganglia in cats and humans during ganglia development 9.
It has been documented that the ENS has the unique ability to control most gut functions so that any damage to ENS results in digestive disorders and reduced quality of life. Experimental evidence indicates that inflammation can occur in the ENS, resulting in severe GIT motor impairment with aging. The eosinophils are specifically located around the ganglion cells, with some scattered eosinophils in the submucosa and tunica muscularis. Also, eosinophil chemoattractant has been considered as a humoral factor playing a role in gut dysmotility. Moreover, eosinophilic infiltrate of the gastrointestinal wall may occur with a predilection for the myenteric (Auerbach’s) plexus in GIT dysfunction associated with advanced age 27.
The ganglia of group A contained glial cells and nerve cells within compact neuropil. In group B, minute cavities appeared within this neuropil and in between the neurons. These cavities increased in number and size to become more obvious in group C. Some researchers have reported compact neuropil to be composed of an aggregation of cytoplasmic processes of neurons with little intercellular separation 26. These results are in agreement with those of some researchers 25. They attributed the apparent reduction in neuropil in aging ganglia to the loss of axons of the enteric neurons that underwent cell death during aging. They added that the effect of aging was not limited to pruning away of some neurons but may have compromised even those that survived.
The neurons of group A appeared closely packed, with vesicular nuclei. They had euchromatic nuclei with prominent nucleoli. In group B, some neurons had darkly stained nuclei with peripheral condensation of heterochromatin. Cytoplasmic vacuolations appeared in nerve cells of this group and increased in group C. Nerve cells of the group C contained interstitial cell of Cajal (ICC), shrunken darkly stained nuclei. The cellular mechanisms of neural injury in aging included cell necrosis, apoptosis, and production of reactive oxygen species (ROS). These ROS is implicated in age-related nonpathological neurodegeneration and neuronal death 28,29. It is known that neurons are considered particularly susceptible to free radical damage because of their large size, high level of metabolic activity, and relatively poor antioxidant defense 12. Also, age-related vulnerability of enteric neurons can be attributed to calcium dyshomeostasis 30 and altered mitochondrial ROS generation in old age 31. Furthermore, some authors 30 have posited that neuron loss in the myenteric plexus is characteristic of a degenerative process that would eventually result in a decline in the digestive system function in aging.
In the current work, glial cells of group A had irregular nuclei that had peripheral condensation of heterochromatin and infolding of their nuclear envelops. These cells changed with age, and cytoplasmic vacuoles were observed in group B and electron-dense nuclei with perinuclear spaces in group C. The gut function is controlled by a dynamic interaction between different cell types, including epithelia that form the mucosal barrier, immune cells, smooth muscle, neurons, glial, and interstitial cell of Cajal (ICC) 32. These enteric glial cells originate from the neural crest to provide support for neuronal elements and are believed to act as intermediaries in enteric neurotransmission 33. It has been reported that the glial cells in myenteric ganglia appear in larger numbers than nerve cells. They were small, with few cytoplasms and scarce organelles 34. With aging, the glial cell populations undergo marked changes that result in tissue inflammation 35–37. Also, their decrease might further weaken the already precarious neuroenteric balance because of the decrease in neuronal elements and ICC 33.
In this research, there was a significant increase in this research, there was a significant increase in the area% of myenteric ganglia with advancing age. This ganglionic expansion may be responsible for the increased proportion of cavities within the ganglia. Throughout life, there is an increase in the surface area of the colon, and in parallel, an increase in the ganglionic area. Furthermore, the process of stretching of the ganglia requires the moving apart of neurons, which might be accompanied by the breakage of synaptic contacts. The greater distances between neurons may increase the conduction times between neurons and may impair information processing within the ganglia. Finally, these events might be associated with disturbed motility 25. In contrast, other workers have reported that the changes in the ganglionic area are caused by two mechanisms: a loss of neurons and a collapse upon itself or involution of the ganglia. Thus, it seems that both occur independently or concurrently to result in a decrease in the ganglion area with age 10.
In the present study, PGP 9.5-stained sections in group B showed an apparent decrease in the number of positively stained neurons. This decrease in neurons progressed to become more obvious in group C. In this group, the lost neurons were replaced by many gaps. Although it has been reported that only 80% of myenteric neurons are stained by PGP 9.5, this is considered the marker of choice for the study of neuronal numbers during aging. It provides a more accurate and complete measure of neuronal numbers particularly in the aged ENS. The authors attributed the decreased number of positively stained neurons to the presence of unstained cells. These cells lost their ability to stain because of alterations occurring as a result of old age 38.
Bcl-2 immunoreaction appeared to show a gradual decrease from group A to group C. Thus, the reaction varied from strong (in group A) to moderate (in group B), to minimal (in group C). These results indicated that the neuronal death increased with advanced age. Over a normal lifespan, a subpopulation of myenteric neurons in the stomach decreases. This loss could be one possible mechanism responsible for the disruptions in gastrointestinal function observed in the elderly. Also, the deterioration in the myenteric innervation of the gut does not appear to be an indication of the normal development of the ENS; rather, it appears to be a process of gradual deterioration that eventually results in a decrease in function 12.
The science of enteric neurodegeneration in aging is considered an area of interest and may potentially impact future therapies for gastrointestinal motility disorders, including the restoration of function in the elderly. This could be approached by maintenance of the normal structure of ENS throughout the life. From the results of this study, we concluded that aging is associated with significant histological changes in the ganglia of myenteric plexus in rat stomach fundus. We recommend further detailed analysis of the nature and mechanisms of age-associated changes in the ENS that will provide insights into how ‘normal’ aging impacts upon physiological functions in the elderly and how such changes can be prevented.
Conflicts of interest
There is no conflict of interest to declare.
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