Histopathology assessment.

Histopathology assessments were performed on Hematoxylin and Eosin-stained 4-micron tissue sections from heart, kidney, liver, pancreas, muscle, lung, and brain as previously described [10]. Age-related lesion severity levels were determined by a scoring system from 0 (no lesion present) to a range of 1 to 4 (lesion presence and severity). The absence or presence and severity of age-related lesions were then used to determine organ morbidity defined as the presence of two-level 1 lesions or one lesion with a score of 2 or greater. The body organ disease number (BODN) was then calculated as the number of organ systems with morbidity as a proxy of multimorbidity and a counterpart of clinically determined BODN in humans. With the premise that different pathology entity levels incorporate into BODN heterogeneously, all organ pathologies in a model were used to predict BODN for each mouse to quantify PathoClock independent of chronological age.