Increasing the inspiratory time and I:E ratio increased the production of proinflammatory cytokines in VILI. Mice were mechanically ventilated for 4 hours with either low tidal volume (LVT 9 ml/kg) or high tidal volume (HVT 34 ml/kg) and an inspiratory/expiratory ratio of 1:2 or 1:1, respectively. An alternative end point was defined as decreasing of mean arterial blood pressure below 40 mm Hg, which predicts death with certainty in this model. Controls (ctr) were subjected to LVT 1:2 ventilation only during operation and were killed before the 4-hour ventilation protocol started. (A) mRNA levels of interleukin (IL)-1β, IL-6, macrophage chemotactic protein (MCP)-1, and keratinocyte-derived cytokine (KC) were measured with quantitative reverse transcription polymerase chain reaction in lung homogenates and normalized to GAPDH levels. (B) Protein levels of IL-1 β , IL-6, MCP-1, and KC were determined in lung homogenates by multiplex ELISA technique. n = 6 to 8 each group; *P < 0.05, **P < 0.01, ***P < 0.001.