Data Analysis: Exogenous mitochondrial transplantation improves survival and neurological outcomes after resuscitation from cardiac arrest

In this study, the authors aim to demonstrate that mitochondrial transplantation (MTx) can improve outcomes after cardiac arrest (CA) resuscitation. They test the hypothesis that MTx can mitigate the severe effects of ischemia-reperfusion injury and improve survival and neurological recovery. Specifically, the study seeks to establish several key claims(Hayashida et al., 2023):

1. That exogenous mitochondria can be taken up and enter neural cells in vitro, with higher ATP content compared to frozen mitochondria.
2. That freshly isolated mitochondria, but not frozen-thawed ones, significantly improve 72-hour survival rates in a rat model of CA.
3. That MTx leads to improvements in critical physiological parameters, including arterial lactate and glucose levels, cerebral microcirculation, lung edema, and neurological function.
4. That the transplanted mitochondria persist in vital organs for at least 24 hours following resuscitation.

In this paper, I will analyze the data and figures presented in the study to assess whether the results support these claims and to evaluate the overall efficacy of MTx as a potential therapeutic strategy for improving outcomes after CA.

Methods

In this study, exogenous mitochondria were isolated from rat brain or muscle tissue, stained with MitoTracker Deep Red, and cocultured with primary neural cells stained with MitoTracker Green for 24 hours to visualize mitochondrial transfer using confocal microscopy. Mitochondria were isolated through tissue homogenization in a mitochondrial isolation buffer, followed by centrifugation and resuspension in prewarmed PBS for transfer. ATP content was measured with a luminescent assay, and mitochondrial membrane potential (ΔψM) was assessed by flow cytometry using the JC1 assay. In vivo, rats were subjected to a controlled cardiac arrest (CA) model, anesthetized, mechanically ventilated, and paralyzed with vecuronium bromide to induce cardiac arrest. After 10 minutes of CA, ventilation was resumed, manual chest compressions were performed, and epinephrine was administered. Post-resuscitation, the rats remained on mechanical ventilation for 10 minutes before being weaned off over a 2-hour period, with pain medications administered throughout the procedure. Mitochondrial infusion was used to assess the functional impact of mitochondrial transfer(Hayashida et al., 2023).

Analysis

In Figure 1 DAPI is used as a nuclear stain, which highlights the cell nuclei, while Actin staining visualizes the cytoskeleton of the neural cells (Hayashida et al., 2023). These stains help in understanding the cell structure alongside the mitochondrial labeling, providing context to the co-localization of exogenous and endogenous mitochondria within the cells. The DAPI and Actin stains assist in confirming the location and structural integrity of the cells during the mitochondrial uptake process. In Figure 1, the study demonstrates that isolated brain- and muscle-derived mitochondria are successfully taken up by neural cells in vitro. In Figure 1A the exogenous mitochondria, labeled with MitoTracker Deep Red, were co-cultured with neural cells, whose mitochondria were stained with MitoTracker Green. After 24 hours, exogenous mitochondria were observed inside the neural cells, co-localizing with the cells’ endogenous mitochondria, as shown by the merged yellow staining. This indicates efficient mitochondrial transfer from both brain and muscle tissues into neural cells. In Figure 1B, the study also demonstrates that muscle-derived mitochondria, similarly stained, are transferred into neural cells. This further supports the claim that both brain- and muscle-derived mitochondria can be efficiently transferred and integrated into neural cells.

In Figure 2, the study assesses mitochondrial functionality through two primary methods: ATP content (Panel A) and mitochondrial membrane potential (ΔψM) (Panel B).

• Panel 2A: ATP content in freshly isolated mitochondria was approximately four times higher than that in frozen-thawed mitochondria, indicating that freezing significantly diminishes mitochondrial energy production.
• Panel 2B: The mitochondrial membrane potential (ΔψM) was significantly higher in freshly isolated mitochondria, as measured by flow cytometry using JC-1 dye. JC-1 is a color-switching dye that emits red fluorescence in healthy mitochondria (with high membrane potential) and green fluorescence in unhealthy mitochondria (with low membrane potential). In the fresh mitochondria group, the red-to-green ratio was much higher than in the frozen-thawed group, indicating intact, functional mitochondria. The freezing process disrupted mitochondrial membrane potential, as evidenced by the significantly lower red-to-green ratio in the frozen-thawed mitochondria group.

These results demonstrate that fresh mitochondria maintain better functionality, with both higher ATP production and membrane potential, compared to their frozen counterparts.

Figure 3 illustrates the effects of mitochondrial transplantation on survival, neurological function, and body weight following cardiac arrest (CA) and resuscitation in rats. Panel A shows survival rates during the first 72 hours post-CA, where rats receiving fresh mitochondria had a significantly higher survival rate (90.9%) compared to the vehicle (54.5%) and frozen-thawed-mito (54.5%) groups. The p-values of 0.048 (vs. vehicle) and 0.038 (vs. frozen-thawed-mito) indicate that the differences are statistically significant, suggesting a beneficial effect of fresh mitochondria on survival.

Neurological function significantly deteriorates following cardiac arrest (CA) due to ischemia and reperfusion injury, which causes widespread damage to brain cells. This damage impairs cellular bioenergetics, disrupts mitochondrial membrane potential, and triggers inflammation and apoptosis, leading to neurological deficits. In the study, these deficits were quantified using the Neurological Functional Score (NFS), where lower scores indicate more severe impairment (Hayashida et al., 2023).

The paper demonstrates that mitochondrial transplantation, particularly with freshly isolated mitochondria, helps restore neurological function after CA. Rats receiving fresh mitochondria had significantly higher NFS values at 72 hours post-CA compared to those in the vehicle or frozen-thawed mitochondria groups  (P=0.047) . This indicates better preservation and recovery of neurological function. Fresh mitochondria likely contribute to this recovery by integrating into host neural cells, restoring ATP production, and mitigating oxidative stress and inflammatory responses, thereby reducing neuronal damage and promoting functional recovery. These data findings suggest that mitochondrial transplantation could be a promising therapeutic approach for improving neurological outcomes after CA. In Panel C, the body weight analysis shows that the fresh-mito group had significantly higher body weights at 72 hours post-resuscitation compared to the vehicle group (P=0.044), indicating better physical recovery. These findings, supported by statistical tests like the Gehan–Breslow–Wilcoxon test, Kruskal-Wallis test, and mixed-effects model, demonstrate the significant improvement in survival, neurological function, and weight maintenance after fresh mitochondrial transplantation.

Lactate Levels (Fig. 4A)
Lactate is a biomarker for tissue hypoxia, and elevated levels indicate impaired oxygen delivery and utilization. Following CA, lactate levels spiked in all groups due to ischemia and metabolic stress. However, rats treated with freshly isolated mitochondria showed a significantly faster reduction in arterial lactate levels within 15 minutes post-resuscitation compared to vehicle and frozen-thawed mitochondria groups. These findings suggest that freshly isolated mitochondria enhance metabolic recovery by restoring cellular energy production and reducing anaerobic glycolysis, bringing lactate levels closer to baseline more rapidly.

Lung Edema (Fig. 4B)
Lung edema, a hallmark of reperfusion injury, was assessed by measuring lung water content at 72 hours post-resuscitation. Rats treated with freshly isolated mitochondria exhibited significantly lower lung water content (W/D ratio 4.23 ± 0.85) compared to the vehicle (5.70 ± 0.75, P = 0.027) and frozen-thawed mitochondria groups (6.21 ± 1.40, P = 0.003). This reduction indicates that fresh mitochondria mitigate pulmonary inflammation and vascular leakage, likely by decreasing oxidative stress and inflammatory signaling during reperfusion.

Heart Function and Hemodynamics (Fig. 4C, 4D)
The study observed reduced left ventricular ejection fraction (LVEF) at 2 hours post-resuscitation in all groups, reflecting acute myocardial dysfunction caused by CA. However, there were no significant differences in LVEF or other hemodynamic parameters (e.g., arterial pressure and heart rate) between groups at this early time point. This suggests that while fresh mitochondria improved lactate clearance and lung injury, their effects on cardiac function were not evident within the 2-hour monitoring window (Hayashida et al., 2023).

In conclusion, freshly isolated mitochondria effectively improve early outcomes by accelerating lactate normalization and reducing lung edema, highlighting their potential to alleviate systemic and pulmonary complications following CA(Hayashida et al., 2023).

Figure 5 highlights how mitochondrial transplantation (MTx) affects glucose levels, blood pH, and arterial partial pressure of carbon dioxide (PaCO₂) following cardiac arrest (CA). Post-CA, glucose levels typically rise due to stress-induced hyperglycemia. In this study, glucose levels were initially elevated in all groups but were significantly lower in the fresh-mitochondria (fresh-mito) group compared to the vehicle and frozen-thawed-mito groups at 15 minutes post-resuscitation. By 2 hours, glucose levels in all groups had returned to baseline, indicating that MTx accelerated the normalization of glucose levels. Blood pH, which drops during ischemia and acidosis, showed faster recovery in the fresh-mito group (7.35 ± 0.03) compared to the vehicle group (7.21 ± 0.02) and the frozen-thawed group at 15 minutes post-resuscitation, reflecting improved acid-base homeostasis. Similarly, PaCO₂, which rises during impaired ventilation and perfusion, decreased more effectively in the fresh-mito group (40 ± 3 mmHg) than in the vehicle (51 ± 5 mmHg) and frozen-thawed groups (46 ± 4 mmHg). These findings suggest that MTx with fresh mitochondria facilitates rapid metabolic recovery, including glucose normalization and improved respiratory efficiency, following CA​. 

Exogenous mitochondrial transplantation with freshly isolated mitochondria facilitates rapid metabolic recovery, including glucose normalization and improved respiratory efficiency, following cardiac arrest (CA) by restoring mitochondrial function and enhancing cellular energy production. During CA, ischemia and reperfusion injuries lead to mitochondrial dysfunction, impairing ATP synthesis and causing metabolic disturbances like hyperglycemia. Fresh mitochondria are able to integrate into damaged cells and restore mitochondrial bioenergetics, which improves ATP production and helps normalize glucose metabolism. This is particularly important in reducing stress-induced hyperglycemia. Additionally, fresh mitochondria help restore mitochondrial membrane potential and oxidative phosphorylation, promoting more efficient oxygen consumption and better gas exchange, which contributes to improved respiratory function and reduced levels of carbon dioxide. These processes underline the potential of mitochondrial transplantation to mitigate metabolic dysfunctions and accelerate recovery following CA (Bhatti , 2016). 

Figure 7 of the paper presents a detailed analysis of cerebral blood flow (CBF) following cardiac arrest (CA) and mitochondrial transplantation (MTx). The figure shows a map of brain blood vessels, where red indicates areas with the highest blood flow density and blue indicates the least. The graph summarizes the recovery of cerebral blood flow (rCBF) in the three experimental groups—vehicle, frozen-thawed mitochondria (frozen-thawed-mito), and freshly isolated mitochondria (fresh-mito)—at 2 hours post-CA. The results show a marked improvement in rCBF in the fresh-mito group, with a value of 107.7% ± 5.6% of baseline CBF at 2 hours, compared to 77.5% ± 4.5% in the vehicle group and 81.3% ± 15.9% in the frozen-thawed-mito group. This significant improvement in CBF, especially in the fresh-mito group (P < 0.0001 vs. vehicle, P = 0.024 vs. frozen-thawed-mito), suggests that the transplantation of fresh mitochondria accelerates the recovery of cerebral perfusion after CA. This enhancement in blood flow is crucial for improving neuronal recovery and function, demonstrating the beneficial effects of mitochondrial transplantation on cerebral microcirculation. Transplantation of fresh mitochondria accelerates the recovery of cerebral perfusion after cardiac arrest (CA) by restoring mitochondrial function and improving endothelial cell function, which are crucial for the maintenance and restoration of blood flow. After CA, ischemic damage leads to mitochondrial dysfunction, impairing the ability of cells to generate ATP and regulate vascular tone. Fresh mitochondria, being intact and functional, can integrate into damaged cells, improving mitochondrial bioenergetics and cellular function. This, in turn, enhances the ability of endothelial cells to produce nitric oxide and other vasodilators, leading to improved vasodilation and increased cerebral blood flow (CBF). Studies have shown that mitochondrial transplantation can reduce oxidative stress, enhance energy metabolism, and improve microcirculatory function, which accelerates the recovery of CBF after ischemic events like CA. This mechanism is particularly important for improving neuronal survival and function after reperfusion injury, as seen in studies of mitochondrial therapies following cardiac arrest and stroke (Park et al., 2021).

In Figure 8, the persistence of transplanted mitochondria in the brain, kidney, and spleen at 1 and 24 hours post-cardiac arrest (CA) is analyzed. Confocal fluorescence imaging using MitoTracker Deep Red labeled the donor mitochondria, and these were observed in the targeted organs at both time points. At 24 hours post-CA, transplanted mitochondria were clearly visible in the brain, kidney, and spleen, indicating their successful integration and retention within these organs. Specifically, mitochondria were evident in the brain and kidney (red staining), while no significant presence was observed in the heart, liver, or lung, as seen in additional data (Additional file 1: Fig. S3). The images clearly demonstrate that exogenous mitochondria were still present and functional in the brain, kidney, and spleen at 24 hours after transplantation. These findings suggest that freshly isolated mitochondria can persist in critical tissues for at least 24 hours post-CA, offering insight into their potential role in therapeutic applications for post-resuscitation recovery. The limited persistence of transplanted mitochondria in tissues beyond 24 hours is influenced by several factors, including the body’s immune response, mitochondrial integration into host cells, and the specific tissue environment. After mitochondrial transplantation, the foreign mitochondria can be taken up by cells, but their long-term retention depends on their ability to integrate into the host cell’s metabolic machinery. The body may also recognize the transplanted mitochondria as foreign, triggering immune responses that promote their clearance. Additionally, transplanted mitochondria may undergo oxidative damage or dysfunction over time, leading to their degradation by the host cell’s mitophagic machinery. Furthermore, the tissue-specific conditions, such as metabolic needs and vascularization, can impact mitochondrial persistence, with certain organs like the brain and kidney providing a more favorable environment than others. These factors collectively contribute to the relatively short persistence of transplanted mitochondria in tissues (Park et al., 2021).

Based on the data from the attached study, fresh mitochondria are significantly more effective than frozen-thawed mitochondria in improving outcomes after cardiac arrest (CA). The study shows that freshly isolated mitochondria led to a marked improvement in survival, neurological function, and metabolic recovery, including better lactate clearance and lung edema reduction, compared to frozen-thawed mitochondria. The results from Figure 2 indicate that fresh mitochondria had substantially higher ATP content and mitochondrial membrane potential (ΔψM) than frozen-thawed mitochondria, which had reduced functionality. Additionally, Figure 4 showed that the fresh-mito group had improved metabolic parameters such as faster normalization of lactate levels, blood pH, and CO₂ levels, which were not as effectively normalized in the frozen-thawed group (Hayashida et al., 2023).

Frozen mitochondria undergo damage during the freezing and thawing process, which can lead to impaired membrane integrity, loss of cytochrome c from the intermembrane space, and reduced respiratory competence. As a result, frozen mitochondria cannot provide the same level of functional support to the recipient cells as fresh mitochondria can. This compromised functionality likely contributes to the less favorable outcomes observed with frozen mitochondria, including lower survival rates and poorer metabolic recovery (Hayashida et al., 2023).

In conclusion, the study demonstrates that fresh mitochondrial transplantation significantly improves survival, neurological function, and metabolic recovery following cardiac arrest (CA), whereas frozen-thawed mitochondria show reduced functionality and less favorable outcomes. Fresh mitochondria are more effective in restoring mitochondrial bioenergetics, improving ATP production, and reducing lactate levels, lung edema, and oxidative stress. These benefits are particularly evident in the normalization of blood pH, CO₂ levels, and glucose metabolism, which are crucial for recovery after ischemia and reperfusion injury. Furthermore, freshly isolated mitochondria persist in critical organs like the brain, kidney, and spleen for at least 24 hours, highlighting their potential for therapeutic use. In contrast, frozen-thawed mitochondria exhibit impaired mitochondrial membrane potential and reduced efficacy in these processes. This study underscores the importance of using fresh mitochondria for optimal therapeutic outcomes, suggesting that mitochondrial transplantation could be a promising strategy for improving recovery after cardiac arrest (Hayashida et al., 2023).

References

Bhatti , J. S. (2016, November 9). Mitochondrial dysfunction and oxidative stress in metabolic disorders – a step towards mitochondria based therapeutic strategies. Biochimica et biophysica acta. Molecular basis of disease. https://pubmed.ncbi.nlm.nih.gov/27836629/

Hayashida, K., Takegawa, R., Endo, Y., Yin, T., Choudhary, R. C., Aoki, T., Nishikimi, M., Murao, A., Nakamura, E., Shoaib, M., Kuschner, C., Miyara, S. J., Kim, J., Shinozaki, K., Wang, P., & Becker, L. B. (2023, March 16). Exogenous mitochondrial transplantation improves survival and neurological outcomes after resuscitation from cardiac arrest – BMC medicine. BioMed Central. https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-023-02759-0

Park, A., Oh, M., Lee, S. J., Oh, K.-J., Lee, E.-W., Lee, S. C., Bae, K.-H., Han, B. S., & Kim, W. K. (2021, April 30). Mitochondrial transplantation as a novel therapeutic strategy for mitochondrial diseases. International journal of molecular sciences. https://pmc.ncbi.nlm.nih.gov/articles/PMC8124982/