Can severe reflux symptoms without endoscopic evidence of esophagitis be due to irritation of the nerve of the submucosa?
Y. Hamanaka, Y. Hirose, H. Hayashi, T. Murakami (Yamaguchi)
There have been many studies regarding the effects of reflux content, such as hydrochloric acid, pepsin, bile and pancreatic juice, on the esophageal mucosa. Each has been shown to play an important role in causing reflux esophagitis [1]. However,
clinically and potentially, the relative contributions of each factor of the refluxate to the reflux esophagitis have not been elucidated. Although the pH of the refluxate in the esophagus and/or the stomach have been recently elucidated using 24-h pH monitoring, the clinical relationship between the above mentioned barrier breakers and pH remains unclear. There are many questions to be answered, such as:
- Why can we not find early esophagitis by endoscopy in patients with severe reflux symptoms?
- Microscopically, where are the initial foci of the esophagitis?
Experimental study using canine esophagus [2]
Under general anesthesia, right thoracotomy was performed and a double lumen tube was inserted orally into the esophagus, the esophagogastric junction was then occluded with rubber tape. The perfusion system incorporated a reservoir, a water bath and a microtube pump. Esophageal perfusion was performed for 2 h with 200 ml of perfusate. Perfusate solutions consisted of 100 mM HC1 and various concentrations of pepsin (0, 1,2 g/l) and sodium taurocholate (Na-Tc) (0, 10, 20 mM) in different combinations.
Histochemical examination
After 2-h perfusion, the esophageal segment was removed and opened longitudinally. Macroscopic findings were then assessed. The full thickness of the mucosa was fixed in 10% phosphate buffered formalin, embedded in paraffin and sectioned. The sections were stained with hematoxylineosin, alcian blue (pH 2.5) and high iron diamine [3] to detect mucins and submucosal glands.
On gross inspection, pepsin-perfused segments of the esophagus showed fine unevenness compared to marked mucosal edema in Na-Tc perfused segments of the esophagus. Pepsin injury was characterized by substantial mucosal erosion and inflammatory cell infiltrations in the lamina propria, while the esophageal glands in the submucosal layer were not involved (Fig. 1). Submucosal edema, destruction of esophageal glands and leakage of mucins were pathognomonic in the Na-Tc perfused esophageal mucosa (Fig. 2). These characteristic findings (i.e., mucosal erosion and destruction of esophageal glands) were graded according to the following scale: 0 = none; 1 = slight; 2 = moderate; 3 = severe (Table 1). Also, it is conceivable that bile induced reflux esophagitis initially develops not only in the mucosa but also in the submucosal layer. This hypothesis, if correct, might explain pathogenesis of reflux esophagitis in patients with severe reflux symptom but without endoscopic findings of esophagitis.
Selection of experimental animals
Human esophagi have a few but obvious esophageal glands [2,4] which may serve as protection against several injurious substances. Although many investigators have
Figure 1..
Figure 2..
Table 1. .Histochemical findings in pepsin (P) and/or sodium taurocholate (Na-Tc) perfused canine esophagus (n = 18)
|
Na-Tc 0 mM |
Na-Tc 10 mM |
Na-Tc 20 mM |
||||||
|
|
erosion |
destruction |
erosion |
destruction |
erosion |
destruction |
||
|
P 0 g/l |
0.1 |
0.0 |
0.0 |
1.3 |
0.0 |
2.3 |
||
|
P 1 g/l |
2.2 |
0.1 |
0.1 |
0.0 |
1.1 |
1.1 |
||
|
P 2 g/l |
3.3 |
0.1 |
0.0 |
2.2 |
0.2 |
0.3 |
||
Note: 0 = none; 1 = slight; 2 = moderate; 3 = severe.
used rabbit esophagi for a model of esophagitis, there are no esophageal glands in this animal. In contrast, an opossum or a dog have many esophageal glands in the submucosal layer [5]. It is important and of interest to clarify whether the submucosal gland and its duct or orifice are relevant to the development of reflux esophagitis as a site of origin.
Detailed examination of each barrier breaker
Under general anesthesia, a canine esophagus was opened longitudinally and fixed horizontally by stay sutures. Several chambers (made of Teflon, content volume 2 ml, weight 5 g) were placed on the esophageal mucosa. Saline used as a control and several combinations of barrier breakers (i.e., HC1, pepsin, Na-Tc, bile, pancreatic juice) were added to the chamber for 30 min.
Figure 3 shows that 100 mM HC1 plus pepsin 2 g and 100 mM HC1 plus Na-Tc 20 mM was added to the chamber. After 30 min, the resected esophagus revealed fine unevenness in the case of pepsin and mucosal edema in Na-Tc (Fig. 4). The specimen was fixed in formalin for microscopic analyses and 2% glutaraldehyde for scanning electron microscopy (SEM). Figure 5 shows SEM findings of normal canine esophageal mucosa after being treated with saline. A fine microridge was seen on the surface of the mucosa. After being treated with canine bile, the microridge had disappeared and squamous cell exfoliation was seen (Fig. 6). Na-Tc caused more severe exfoliation of the superficial squamous cell layer (Fig. 7). Pepsin induced complete desquamation, exposing collagen fibers and fibrine (Fig. 8). This procedure has the following advantages:
- It is easy to change the combination of barrier breakers.
- It is easy to change the contact time.
- Several breakers can be applied at the same time in various parts of the same esophagus in vivo.
- Both the surface and deeper layers of the esophagus can be examined using light and electron microscope.
Figure 3..
Figure 4..
Figure 5..
Figure 6..
Figure 7..
Figure 8..
Summary
Based on our experimental findings, Na-Tc acted on deeper layers of the canine esophagus while pepsin acted superficially. Therefore, we hypothesized that the early stage of bile reflux esophagitis may develop in the orifices of the esophageal gland and submucosal glands.
References