Reduced DNA Synthesis in Primary Cultures of Hepatocytes From Old Mice is Restored by Thymus Grafts

Abstract

We previously observed in vivo that a neonatal thymus grafted into old mice can correct age-related changes such as occurrence of hepatocyte tetraploid nuclei and impaired isoproterenol-induced DNA synthesis in submandibular glands. The aim of the present paper was to study the influence of age and thymus on basal and beta-adrenergic-stimulated DNA synthesis using primary cultures of mouse hepatocytes. In the absence of any adrenergic agents, cells from young mice show peak DNA synthesis between 36 and 48 h; old mice show a similar time course, but the peak is significantly reduced statistically. The main result is represented by the behavior of hepatocytes from old thymus-grafted mice, which recover the levels of [3H]-thymidine incorporation toward young-like values. Grafted animals also show a correction of total DNA content that is increased in old mice. The addition of isoproterenol does not modify the DNA synthetic pattern, whereas the antagonist propranolol causes a slight but statistically significant decrease.

Aging Reversibility: from Thymus Graft to Vegetable Extract Treatment – Application to Cure an Age-associated Pathology

“…The intriguing hypothesis of an unblocking action on the division process of polyploid liver cells cannot be excluded. These results are similar to those obtained with the neonatal thymus graft in old mice (Basso et al 1998). The hepatocyte culture model is interesting because it allows to reduce drastically the number of animals killed in each experiment.…”

“…Hepatocytes were isolated as previously reported by liver perfusion with collagenase (Seglen 1976;Basso et al 1998). Mice were killed by cervical dislocation and the liver was first perfused with EGTA solution containing 0.5 mM EGTA, 5.5 mM glucose, 142 mM NaCl, 6.7 mM KCl, 10 mM HEPES (pH 7.6), 100 nM insulin, 0.1 mg/ml kanamicin and then with collagenase solution containing collagenase, 5 mM CaCl2, 117 mM NaCl, 6.7 mM KCl, 30 mM HEPES (pH 7.4), 5.5 mM glucose, 0.005% trypsin inhibitor, 0.1 mg/ml kanamycin, 1% BSA at 5.5 ml/ min.…”

“…Age-related alterations have also been shown in vitro experiments. DNA synthesis of mouse and rat primary cultures of hepatocytes decreases with aging (Ishigami et al 1993); neonatal thymus graft in old mice recovers the levels of DNA synthesis toward young-like value (Basso et al 1998). Other liver age-related modifications, such as the increased volume and number of tetraploid nuclei, can be corrected by grafting a neonatal thymus into old mice (Pieri et al 1980;Pieri et al 1982).…”

Insulin receptors in mouse brain: Reversibility of age-related impairments by a thymic extract

“…Thus, thymic gland is needed for both development and maintenance of physiological functions of the 13-adrenergic system, but it is also able to recover physiologically relevant age-related alterations. The regulatory influence of thymus has been observed on different tissues, such as submandibular glands, brain cortex and liver, and on different neuronal systems, such as c~-and B-adrenergic ones (21,23,(55)(56)(57)(58). The corrective action of the thymus, in addition, does not seem to act in a nonspecific stimulatory way.…”

“…It is worth noting that the restorative action of the thymus was previously observed also on age-related modifications of several biological functions at level of different tissues. Examples are represented by DNA synthesis in hepatocytes (21), blood levels of insulin and thyroid hormones (22), and 13-adrenergic functions in brain cortex and submandibular glands (23)(24)(25)(26).…”