The effects of rapid stretch injury on rat neocortical cultures

Several key biological mechanisms of traumatic injury to axons have been elucidated using in vitro stretch injury models. These models, however, are based on the experimentation of single cultures keeping productivity slow. Indeed, low yield has hindered important and well founded investigations requiring high throughput methods such as proteomic analyses. To meet this need, a multi-well high throughput injury device is engineered to accelerate and accommodate the next generation of traumatic brain injury research. This modular system stretch-injures neuronal cultures in either a 24-well culture plate format or six individual wells simultaneously. Custom software control allows the user to accurately program the pressure pulse parameters to achieve the desired substrate deformation and injury parameters. Classically, in vitro research in TBI has shown increases in [Ca2+]i levels following injury. The Ca2+ sensitive fluorescent dye, Fluo-4AM, is used to observe the effects of strain rate on the changes in [Ca2+]i levels following injury. Neuronal cultures are injured at three strain levels: 20%, 40% and 60% strain. At each of these strain levels, two strain rates are applied; 30s-1 (slow) and 70s-1 (rapid). At each strain level, the data show that neurons injured at 70s-1 experience larger maximum F/F0 and longer sustained Ca2+ fluorescence than neurons injured at 30s-1. It is also shown that at high strain rates TTx no longer blocks increases in [Ca2+]i levels after injury. Traumatic injury to the brain is known to cause dysfunction in surviving neurons. The effects of simulated traumatic injury of rat neocortical neurons cultures are investigated. These neurons are subjected to a stretch injury of 60% strain over 20 ms using a custom in vitro injury device. Spontaneous and stimulated electrical properties are measured 20-60 minutes after stretch using current and voltage clamp techniques. The same measurements are performed in non-stretched neurons. All neurons display TTX-inhibitable action potentials when ...