SCCM Resource Library-Reduction in Reaction Oxygen Species With Novel Intervention Following TBI in a Large Animal Model

Video Transcription

looking at therapeutics in a pig model for traumatic brain injury. Specifically, I'll be  presenting data from our randomized, blinded, placebo-controlled preclinical trial using a large  animal model for TBI. Listed below are my co-authors, most notable, our lab's primary  investigator, Dr. Todd Kilbaugh. So to start off, let's discuss the impact of TBI on individuals  and society. It affects many people in the U.S. each year, and for those who survive,  even those with mild to moderate injuries, there are costs, including quality of life and financial  from post-injury therapy and lost school or work time. The other problem with TBI is that as  intensivists, short of stabilizing the patients who end up in our ICU, we don't have a lot of  therapeutic options to improve how their brain heals. A big reason for this is the lack of a  reliable animal model. Much of the TBI research comes from rodents, who not only have smaller brains than humans, but also significantly different brain structure, including white  matter distribution that simply isn't translatable to our human patients. Well, our lab, led by Dr.  Todd Kilbaugh, hopes to overcome some of these challenges. For this study, we utilized a controlled  cortical impact injury model in Yorkshire pigs to examine the effects of TBI. Our three groups  included placebo treatment and sham animals, and the treatment intervention was a novel therapeutic  called CMX2043. CMX2043 is an alpha lipoic acid analog that reduces reactive oxygen species.  This study was 24-hour survival. To understand how CMX2043 can affect mitochondrial function, we have to understand the harmful cycle that starts at mitochondria following TBI.  TBI causes excitotoxicity, leading to glutamate release, which increases intracellular calcium, hindering oxidative phosphorylation by injuring complex one in the ETC.  With oxidative phosphorylation not working as well, the mitochondria can't create as many ATP, and there's an increase in reactive oxygen species, which cause more mitochondrial  dysfunction and less ATP to be produced, impeding the ability of other organelles to sequester calcium, furthering the damage calcium can cause to the electron transport chain.  Why does this matter? Well, after enough repetitions of this harmful cycle, injured mitochondria play a role in signaling to the cell that it's time for apoptosis.  We hypothesized that if we could intervene at this part of the cycle with an alpha lipoic acid analog that can decrease reactive oxygen species, both by increasing glutathione and  increasing transcription of antioxidants, we could improve mitochondrial function.  Here we have the results of respirometry using the Ouroboros machine to assess oxidative phosphorylation. This chart shows the differences in the respiratory control ratio among  groups. For those of you who are unfamiliar, the Ouroboros provides a way for us to examine  mitochondrial function. We can add substrates or inhibitors depending on what parts of the  ETC we want to investigate. The RCR tells us about oxygen consumption. To derive this information,  we give the mitochondria oxygen, ADP, and inorganic phosphate, with the idea being that  if everything is humming well in the ETC, oxygen consumption should be high as the ingredients  needed at the end are in maximal supply. So a high RCR means mitochondria are working well, while a low one tells you that even with all the ingredients needed to make ATP,  oxygen utilization is low, meaning there's damage somewhere along the ETC upstream from ATP  synthase. Here we see the treatment group had significantly better oxidative phosphorylation  capabilities compared to the placebo. This graph shows the differences in reactive oxygen species.  In addition, it shows results of injury from the contralateral side. Initially, I thought it would  be worthwhile to see if we could use the contralateral side of the brain as a control to better account for differences among animals. However, as you can see, not only were there  significantly less reactive oxygen species in the treatment animals compared to the placebo animals  shown in maroon, but when looking at the striped bars that represent the contralateral side of the  pigs, the focal controlled cortical impact injury also caused an increase in reactive oxygen species  compared to sham on the contralateral side of the brain as well, which is important to note when we  think about the widespread effects that the brain sustains from even a focal injury. When looking at  mitochondrial reactive oxygen species using the Ouroboros, we're limited to hydrogen peroxide and  superoxide. For that reason, we used ELISA and Western blot to look at other indices of reactive  oxygen species, including protein carbonyl and 4-HNA. Protein carbonyl tells us about protein  carboxylation as a result of reactive oxygen species, while 4-HNA builds up as a result of  lipid peroxidation. Again, what we see here is the animals who received treatment had a significant  reduction in these markers of reactive oxygen species compared to placebo. In conclusion, we were able to show that this therapeutic intervention reduced mitochondrial reactive  oxygen species and improved oxidative phosphorylation. Most importantly, though, this  data comes from a large animal model cohort that is reproducible and can help us better understand  TBI in humans, as well as the role for potential therapeutic interventions. Thank you.

Video Summary

In this video, the presenter discusses the impact of traumatic brain injury (TBI) on individuals and society. They highlight the lack of effective therapeutic options for improving brain healing in TBI patients due to the lack of reliable animal models. The presenter then introduces a study using a pig model to examine the effects of TBI and test a novel therapeutic called CMX2043, which reduces reactive oxygen species. The results show that CMX2043 improved mitochondrial function, reducing reactive oxygen species and enhancing oxidative phosphorylation. This study provides valuable insights into TBI and potential therapeutic interventions.

Asset Subtitle

Neuroscience, Trauma, 2022

Asset Caption

INTRODUCTION: Traumatic brain injury [TBI] is a significant source of morbidity and mortality in the United States, affecting 2.8 million people per year. Lifetime costs following TBI have been estimated as high as $76.5 billion dollars. Despite high costs to society, there are no approved therapeutics. One significant hurdle is the lack of translational large animal platforms in TBI. Generation of mitochondrial reactive oxygen species [mtROS] following TBI impairs complexes in the electron transport chain, in a cycle that further harms mitochondrial function, increasing mtROS. mtROS can influence apoptosis. We examined a novel therapeutic, CMX-2043 - an alpha lipoic acid [ALA] analogue - to see if it could reduce mtROS following TBI in a swine model. METHODS: Yorkshire piglets, n=16, 4-weeks old, were randomized and blinded into sham [5], placebo [6] and treatment [5] groups. Sham animals had surgical incision and anesthesia similar to injured animals. Placebo and treatment pigs received a moderate-severe controlled cortical impact [CCI] injury. Treatment group received 18mg/kg CMX-2043 at 1-hour and 13-hours following injury. 24-hours after injury, animals were sacrificed. Mitochondrial respirometry function was analyzed utilizing ex-vivo brain cortical homogenates. Mitochondria were exposed to a sequence of substrate, uncoupler, inhibitor titrations that interrogate the mitochondrial oxidative phosphorylation system. Respirometry control ratios [RCR] were used as an overall measure of mitochondrial health. Measurements of mtROS were assessed simultaneously with respirometry measurements by an O2k-Fluorescence LED2-Module (Oroboros) utilizing an Amplex UltraRed assay. ELISA was performed to measure 4-HNE, a downstream marker of lipid peroxidation. RESULTS: Markers of mtROS and lipid peroxidation were significantly decreased in animals treated with CMX-2043 as compared to placebo (mtROS: 8.28+/-1.47 vs. 18.77+/-1.58, p < 0.05; 4-HNE 0.287+/-0.051 vs. 1.432+/-0.59, p < 0.05). CMX-2043 animals demonstrated significantly higher mitochondrial respiration compared to placebo; RCR (11.61+/-2.284 vs. 6.006+/-0.2678, p < 0.05). CONCLUSIONS: Treatment with CMX-2043 demonstrated a statistically significant reduction in mtROS, lipid peroxidation and improved mitochondrial function at 24-hours post-TBI in a large animal model.

Meta Tag

Content Type Presentation

Knowledge Area Neuroscience

Knowledge Area Trauma

Knowledge Level Advanced

Membership Level Select

Tag Traumatic Brain Injury TBI

Tag Trauma

Year 2022

Keywords

traumatic brain injury

TBI

brain healing

animal models

CMX2043

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