ROCK inhibitor(Devgen)

Male Sprague Dawley rats were used for mature hepatocytes (HEP) isolation to induce CLiP using 3 small molecules cocktail (ROCK-inhibitor, TGF beta-1 inhibitor, GSK3 inhibitor). Harvested rat CLiP (1 × 10⁶) were transplanted to the posterior lobe of the liver (30 % of whole liver) of Nagase analbuminemic rats (NAR) through portal vein injection. One week later, portal venous branch to non-transplanted lobe was ligated to induce liver regeneration in CLiP bearing liver lobe (portal branch ligation: PBL). Only CLiP Tx or HEP Tx of same dose were used as control groups. Serial measurement of serum albumin level was measured by ELISA method. All NARs were subject to immunosuppression therapy using tacrolimus s.q. 3 days per week.

After HEP Tx especially with PBL, serum albumin level was significantly elevated. However, even with PBL, CLiP Tx group did not show significant increase in serum albumin. CLiP were surrounded by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) positive cells in portal veins suggesting that CLiP were exposed to the risk of innate immunity.

Although CLiP respond to growth factors and proliferate better than HEP in vitro, their behavior in vivo in response to liver regenerative stimulus were not similar to in vitro probably because of less immaturity of receptors and/or high immunogenicity as compared to HEP.

JOURNAL/nrgr/04.03/01300535-202509000-00026/figure1/v/2024-12-31T000210Z/r/image-tiff Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer’s disease. The accumulation of amyloid-β peptides, a key hallmark of Alzheimer’s disease, is believed to induce neuritic abnormalities, including reduced growth, extension, and abnormal growth cone morphology, all of which contribute to decreased connectivity. However, the precise cellular and molecular mechanisms governing this response remain unknown. In this study, we used an innovative approach to demonstrate the effect of amyloid-β on neurite dynamics in both two-dimensional and three-dimensional culture systems, in order to provide more physiologically relevant culture geometry. We utilized various methodologies, including the addition of exogenous amyloid-β peptides to the culture medium, growth substrate coating, and the utilization of human-induced pluripotent stem cell technology, to investigate the effect of endogenous amyloid-β secretion on neurite outgrowth, thus paving the way for potential future applications in personalized medicine. Additionally, we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition. We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway, achieved through modulation with Y-27632 (a ROCK inhibitor) and Ibuprofen (a Rho A inhibitor), respectively, can restore and even enhance neuronal connectivity in the presence of amyloid-β. In summary, this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition, but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-β peptides, along with potential intervention points to restore neurite growth. Thereby, we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical, predictive outcomes of drugs and their ability to promote neurite outgrowth, both generally and in a patient-specific manner.