Travis C Jackson1, Shawn E Kotermanski2, Edwin K Jackson2, Patrick M Kochanek3. 1. University of Pittsburgh School of Medicine, Safar Center for Resuscitation Research, Children's Hospital of Pittsburgh of UPMC, John G. Rangos Research Center, 6th Floor, 4401 Penn Avenue, Pittsburgh, PA 15224, United States; University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Scaife Hall, 3550 Terrace Street, United States. Electronic address: jacksontc@upmc.edu. 2. University of Pittsburgh School of Medicine, Department of Pharmacology and Chemical Biology, Bridgeside Point Building 1, 100 Technology Drive, United States. 3. University of Pittsburgh School of Medicine, Safar Center for Resuscitation Research, Children's Hospital of Pittsburgh of UPMC, John G. Rangos Research Center, 6th Floor, 4401 Penn Avenue, Pittsburgh, PA 15224, United States; University of Pittsburgh School of Medicine, Department of Critical Care Medicine, Scaife Hall, 3550 Terrace Street, United States.
Abstract
Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. METHODS/ RESULTS: Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as "BrainPhys® maintained neurons"). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau. CONCLUSION: BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro.
Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. METHODS/ RESULTS: Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as "BrainPhys® maintained neurons"). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau. CONCLUSION:BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro.
Authors: Travis C Jackson; Lina Du; Keri Janesko-Feldman; Vincent A Vagni; Cameron Dezfulian; Samuel M Poloyac; Edwin K Jackson; Robert S B Clark; Patrick M Kochanek Journal: J Cereb Blood Flow Metab Date: 2015-03-31 Impact factor: 6.200
Authors: Patrick M Kochanek; Travis C Jackson; Ruchira M Jha; Robert S B Clark; David O Okonkwo; Hülya Bayır; Samuel M Poloyac; Amy K Wagner; Philip E Empey; Yvette P Conley; Michael J Bell; Anthony E Kline; Corina O Bondi; Dennis W Simon; Shaun W Carlson; Ava M Puccio; Christopher M Horvat; Alicia K Au; Jonathan Elmer; Amery Treble-Barna; Milos D Ikonomovic; Lori A Shutter; D Lansing Taylor; Andrew M Stern; Steven H Graham; Valerian E Kagan; Edwin K Jackson; Stephen R Wisniewski; C Edward Dixon Journal: J Neurotrauma Date: 2019-02-01 Impact factor: 5.269
Authors: Jeffrey Farooq; Kara Snyder; Keri Janesko-Feldman; Kiersten Gorse; Vincent A Vagni; Patrick M Kochanek; Travis C Jackson Journal: J Neurotrauma Date: 2022-03-01 Impact factor: 5.269
Authors: Stefania Mondello; Viktor Sandner; Mona Goli; Endre Czeiter; Krisztina Amrein; Patrick M Kochanek; Sakshi Gautam; Byeong Gwan Cho; Ryan Morgan; Ali Nehme; Giacomo Fiumara; Ali H Eid; Chloe Barsa; Muhammad Ali Haidar; Andras Buki; Firas H Kobeissy; Yehia Mechref Journal: EClinicalMedicine Date: 2022-06-17
Authors: Travis C Jackson; Keri Janesko-Feldman; Kiersten Gorse; Vincent A Vagni; Edwin K Jackson; Patrick M Kochanek Journal: Neuroscience Date: 2020-04-24 Impact factor: 3.590
Authors: Travis C Jackson; C Edward Dixon; Keri Janesko-Feldman; Vincent Vagni; Shawn E Kotermanski; Edwin K Jackson; Patrick M Kochanek Journal: Sci Rep Date: 2018-05-08 Impact factor: 4.379