Document Type


Date of Award

Spring 5-31-2013

Degree Name

Doctor of Philosophy in Materials Science and Engineering - (Ph.D.)


Committee for the Interdisciplinary Program in Materials Science and Engineering

First Advisor

Bryan J. Pfister

Second Advisor

Joshua R. Berlin

Third Advisor

Michael Jaffe

Fourth Advisor

Raquel Perez-Castillejos

Fifth Advisor

Camelia Prodan

Sixth Advisor

Andrew Hill


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 basal membrane potential was set at -60 mV, and many display bursting behavior in response to depolarizing current injection. No differences in resting membrane potential (-40 ± 1 mV [n=20]) or input resistance (1.0 ± 0.1 GΩ [n=20]) are observed in injured and non-injured neurons. Interestingly, stretch injury reduces the frequency of spontaneous action potentials (33 ± 6 min-1 [ n=13] and 11 ± 3 min-1 [n=16] in non-injured and injured neurons, respectively) and decreases spontaneous bursting activity by almost 90%. ADP50 and action potential amplitude are unchanged. However, A D P90 is significantly prolonged in injured neurons and characterized by a less pronounced action potential after-hyperpolarization. These data are consistent with an alteration in kinetics of K+ currents in injured neurons. Since spontaneous action potentials are blocked by 20 µM bicuculline and 3 mM kyneuri nic acid, the frequency of subthreshold depolarizations is measured to estimate overall neuronal network activity. The frequency of spontaneous subthreshold depolarizations is not significantly different in injured and non-injured neurons. These data show that spontaneous electrical signaling is acutely altered and suggest that action potential initiation is reduced by in vitro stretch in neuronal cell cultures.



To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.