Materials and methods
The study was approved by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects (Approve No. 20-084). The profiles of human autopsy cases in this study are as follows: totally, 47 cases (male:female is 2:1) were studied for mandibular and bronchial glands, in which 37 cases were studied for both glands, whereas 5 cases were studied for either one of the glands. The ages range from 0 to 95 years old. The postmortem intervals range from 0.5 to 7.5 days. The causes of death are 3 cases of cardiac diseases, 5 cases of respiratory disease, 5 cases of trauma, 4 cases of asphyxia, 6 cases of drowning, 4 cases of burning, 3 cases of intoxication, 13 cases of other causes, 4 cases of unknown causes. None of the cases had the history of COVID-19 infection, and all cases were negative for COVID-19 PCR at the time of autopy.
Human autopsy samples were fixed in 16% formalin, embedded in paraffin, and sectioned. The immunohistochemistry was performed by the BOND RX automated immunostainer (Leica, Germany) using BOND polymer refine detection system [5]. The antibody used was 500 times diluted anti-ACE2 (rabbit polyclonal, ab15348, Abcam, UK). Diaminobenzidine (DAB) was used for the staining. The immunohistochemical positivity was measured using cellSence software (Olympus, Tokyo, Japan). The range of interest was set on the mandibular salivary glands and tracheal glands, and the brown color of DAB was selected. The percentages of the positive areas in the glands were calculated using the cellSense software (Olympus, Japan).
Statistical analysis
Linear regression test was used for statistical analyses, and graphs were made by Prism 7 (GraphPad Software, San Diego, USA). The p values less than 0.05 were considered statistically significant.
Results and discussion
Our study clearly proves the presence of ACE2 immunoreactivity in salivary and tracheal glands (Fig. 1A and B for 0 year old, and Fig. 2A and B for 69 years old). Our results demonstrate that ACE2 is immunohistochemically expressed in salivary glands and tracheal gland cells through all ages studied (Fig. 2A, B). COVID-19 has a characteristic in age distribution. That is, the susceptibility to infection in individuals under 20 years of age is approximately half that of adults aged over 20 years [6]. The ACE2 prevalence in children compared with adults would offer the important information as to the reason why the susceptibility is different in terms of age. Previously, the expression of ACE2 gene in nasal epithelium has been reported lower in young children compared with adults [4]. Since saliva is an important source of SARS-CoV-2 infection [7], we investigated the immunohistochemical expression of ACE2 in submandibular salivary glands as well as tracheal glands as the possible infection sites. Contrary to the above nasal result, our results reveal the novel finding that the ACE2 protein immunoreactivity in mandibular salivary glands (Fig. 2A) and tracheal glands (Fig. 2B) shows the statistically significant decrease with the increase of age. Thus, our results indicate that immunohistochemical ACE2 expressions may inversely correlate with the age. This indicates that the susceptibility of aged individuals to SARS-CoV-2 may be due to various factors including but not limited to ACE2 protein expressions.
Limitations
Since this is an immunohistochemical study, the functional aspects of ACE2 in the glands are not covered.
