Seeking New Molecular Targets for Treating Traumatic Brain Injury

Travis C. Jackson, PhD, University of Pittsburgh
Travis Jackson

Travis C. Jackson, PhD, is an Assistant Professor in the Department of Critical Care Medicine at the University of Pittsburgh. He is also an Associate Director at the Safar Center for Resuscitation Research (SCRR). Travis earned a BSc degree from the University of Pittsburgh in 2005 and a PhD in Biomedical Science in 2010 from the University of Florida in the Department of Neuroscience. He returned to the University of Pittsburgh in 2010 for his postdoctoral work at the SCRR, prior to joining the faculty in 2012.

Dr. Jackson's laboratory utilizes lentivirus, transgenic knockouts, pharmacological tools, and in vitro as well as in vivo models of brain injury to investigate cell signaling mechanisms contributing to neuronal or axonal injury. His work has been consistently funded by the American Heart Association and by the National Institutes of Health.


I think that one of the greatest strengths of the scientific process is a commitment to periodically re-evaluate what is “optimal” (in all circumstances)... [BrainPhys™] is a different medium vs. other classic growth support media commonly used to study neurotoxicity and neuroprotection. Different is good. Different is one of the fundamental ingredients in the recipe of advancement. In my opinion, BrainPhys™ is an exciting and interesting new tool to explore.

Travis C. Jackson, PhD, University of Pittsburgh

Travis’ Story: Modern Day Treasure Hunting

What made you choose a career in science?
I have always been captivated by stories of explorers on the hunt for treasure, or of the unearthing of secrets hidden throughout the world. In my opinion scientific research is one of the last bastions for modern day treasure hunting. The job requirements share common elements—you need a map (hypothesis), a brave crew (great colleagues), and a steady ship (laboratory). Also, environmental factors like the trade winds (research funding) strongly impact where you plan to go vs. where you end up. Thus, I was drawn into the sciences, in part, in the spirit of adventure, discovery, and exploration. Furthermore, I think that biomedical scientists have unearthed some of the greatest treasures on the planet (e.g. penicillin, ibuprofen, the polio vaccine, anti-hypertensive drugs, MRI imaging). These priceless artefacts are worth more than gold or jewels because unlike the latter they hold the promise of a healthier future for everyone, and knowing that it is possible to make such amazing finds, ignites my optimism to stay the course and keep digging.

Who/What inspires you?
I am inspired by family, friends, and colleagues, each gifted with amazing and unique skills. Their incredible talents serve as a mirror for self-reflection to recognize my own limitations but most importantly motivate me to learn more and attempt even greater heights. As to what inspires me—quite literally, the human brain.

What is your current research focus?
Brain research is my current focus. Specifically, my lab investigates the intracellular mechanisms that are activated by acute brain injury. We use this information to explore novel approaches to improving brain recovery, by either targeting novel pathways, or by finding methods that enhance the effectiveness of current neuroprotective strategies (e.g. therapeutic hypothermia). My studies utilize in vitro and in vivo models of traumatic brain injury (TBI), and also models of cardiac arrest to investigate global brain ischemia.

The Paper: BrainPhys® Increases Neurofilament Levels in CNS Cultures, and Facilitates Investigation of Axonal Damage After a Mechanical Stretch-Injury In Vitro1

What is the significance of studying traumatic brain injury and mechanical-stretch injury?
TBI is a leading cause of death and disability in the young. However, the epidemiology is changing, reflecting a growing aged population, and a concomitant increase in the incidence of TBIs among the elderly (mostly due to accidental falls). In terms of the scope of the problem, statistics provided by the CDC’s Blue Book on TBI2 indicate that there are ~1.7 million TBIs each year in the US and others estimate that the societal cost (direct and indirect) is a staggering ~$86 billion annually. The modern management of severe TBI involves clinical maneuvers that limit secondary evolving injury. However, no therapies are clinically available to directly target neuroprotection. Thus, there is a pressing need to learn more about the molecular sequelae of a TBI, and to rapidly screen drugs for neuroprotective potential. The mechanical stretch-injury model is an in vitro tool which helps to achieve both of these goals. Specifically, it models the mechanical-injury component of a TBI on isolated cells from the brain.

Can you briefly describe the main findings in your paper?1
First, we compared the effect of growth medium formulations (Neurobasal®/B-27™ medium vs. NeuroCult™/BrainPhys™/SM1) on the levels of several molecular protein targets in primary neuron cultures. Among the striking differences, we observed that the levels of neurofilament (NF) proteins were robustly increased in NeuroCult™/BrainPhys™/SM1 cultures. The significance of this finding is that NF light (NFL) and NF heavy (NFH) are commonly measured biomarkers in the TBI literature for assessing the extent of axonal injury. Second, we also showed that NFL levels dramatically decrease after a mechanical stretch-injury in NeuroCult™/BrainPhys™/SM1 cultures. Thus, we think that our reported stretch-injury protocol may be useful to expedite the investigation of axonal-injury targeting therapies, which is relevant across the spectrum of TBI severity.

What is the clinical relevance to your research/paper?
Therapies for the treatment of TBI are lacking. Pre-clinical models that accurately describe injury processes in the human brain might have the greatest chance of identifying therapeutic breakthroughs faster. Germane to in vitro work, the extracellular neuronal culture environment strongly influences background gene expression. Thus, the choice of medium alters the experimental outcome. It remains to be determined if any medium can truly provide a bona fide “clinically relevant” system to test drugs. However, we found quantifiable differences in the expression levels of clinically relevant molecular targets comparing BrainPhys™ vs. other medium. Thus, we conclude that: (a) additional medium-dependent differences are likely present but need to be brought to light, (b) more studies are merited to further compare BrainPhys™ vs. alternative formulations and elucidate extracellular factors contributing to alterations in neuronal properties that affect neuronal signaling fidelity vs. the intact brain, and (c) that the choice of medium should be a carefully weighed factor during the design stage of neuronal culture experiments. It is worth noting that our work was done in rodent primary neurons, which is a limitation in terms of the clinical relevance of our findings.

How Travis Used BrainPhys™

Why did you decide to use BrainPhys™ in your experiment?
The mechanical stretch-injury model uses special culture plates that incorporate a Silastic® membrane which neurons are grown on top of. In early pilot experiments we used standard Neurobasal medium to maintain neuronal growth. It proved quite difficult. The neurons did not adhere well to the Silastic® membrane, and were easily dislodged during regular media swaps for maintenance (prior to a stretch-injury). We hypothesized that adjusting various culture factors (cell adhesion substrates and the culture media) might improve the overall adherence. This led us to test NeuroCult™/SM1. In our hands NeuroCult™ improved the adherence of neurons to the Silastic® membrane, which was further augmented by additional changes to the coating substrates. Independent of this troubleshooting (but around the same time) we came across an article from Dr. Fred Gage’s group at the Salk Institute in California, published in PNAS,3 reporting the development of a new neuronal culture formulation that uniquely supported neuronal electrophysiology (i.e. BrainPhys™). Thus, it was a logical decision to further modify our protocol by replacing/diluting the NeuroCult™ over culture days with BrainPhys™, and testing the effect it had on stretch-injury outcomes.

Were you surprised with any of the results?
Yes and No. Before our stretch-injury studies began, for an unrelated project, we attempted to replicate a protocol (4AP/bicuculline treatment) to chemically activate synaptic NMDAR receptors and increase pERK activation in neuron cultures. These experiments failed. We did not really understand why they failed but decided to move on to more fruitful directions. After reading the rigorous work concerning the development of BrainPhys™, and in particular confirmation that neurons were far more responsive to depolarizing stimuli with the new medium, we were fairly optimistic that 4AP/bicuculline treatment in our experiments would have the desired effect to increase pERK under conditions of BrainPhys™. In contrast, we were genuinely surprised at the extent of differences in the protein levels of various targets between culture medium.

What is it about BrainPhys™ that makes it a suitable medium to study neuronal injury?
I think that one of the greatest strengths of the scientific process is a commitment to periodically re-evaluate what is “optimal” (in all circumstances). Brainphys™ certainly needs additional study. It is quite evident that it is altering fundamental properties of CNS cells in an enormous way. The full implications of these findings in the context of a neuronal injury are not yet clear. It is a different medium vs. other classic growth supports commonly used to study neurotoxicity and neuroprotection. Different is good. Different is one of the fundamental ingredients in the recipe of advancement. In my opinion, BrainPhys™ is an exciting and interesting new tool to explore. However, it is up to the scientific community to unleash their creativity upon this new resource and see where it leads. A more direct answer to your question is that the enhanced electrophysiological parameters really standout.

References

  1. Jackson TC et al. (2018) BrainPhys® increases neurofilament levels in CNS cultures, and facilitates investigation of axonal damage after a mechanical stretch-injury in vitro. Exp Neurol. 300:232-46.
  2. Faul M et al. (2010) Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations and Deaths 2002 – 2006. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Injury Prevention and Control.
  3. Bardy C et al. (2015) Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc Natl Acad Sci. 112 (20) E2725-4.