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Published in final edited form as: Stroke Vasc Interv Neurol. 2023 Mar 2;3(3):e000644. doi: 10.1161/svin.122.000644

Intraarterial Transplantation of Mitochondria After Ischemic Stroke Reduces Cerebral Infarction

Pedro Norat 1, Jennifer D Sokolowski 1, Catherine M Gorick 2, Sauson Soldozy 1, Jeyan S Kumar 1, Youngrok Chae 1, Kaan Yagmurlu 1, Joelle Nilak 1, Khadijeh A Sharifi 1,3, Melanie Walker 6, Michael R Levitt 6, Alexander L Klibanov 5, Zhen Yan 4, Richard J Price 2, Petr Tvrdik 1,3,*, M Yashar S Kalani 7,*
PMCID: PMC10399028  NIHMSID: NIHMS1868004  PMID: 37545759

Abstract

Background-

Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by ischemia and reperfusion.

Methods-

Using murine stroke models of middle cerebral artery occlusion, we sought to evaluate feasibility of delivery of viable mitochondria to ischemic brain parenchyma. We evaluated the effects of concurrent focused ultrasound activation of microbubbles, which serves to open the blood-brain barrier, on efficacy of delivery of mitochondria.

Results-

Following intra-arterial delivery, mitochondria distribute through the stroked hemisphere and integrate into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, the transplanted mitochondria elevate concentration of adenosine triphosphate in the stroked hemisphere, reduce infarct volume and increase cell viability. Additional of focused ultrasound leads to improved blood brain barrier opening without hemorrhagic complications.

Conclusions-

Our results have implications for the development of interventional strategies after ischemic stroke and suggest a novel potential modality of therapy after mechanical thrombectomy.

Graphical Abstract

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INTRODUCTION

Stroke is a leading cause of death and severe disability in the United States1. Thus far, clinical trials of neuroprotective agents have been unsuccessful, and studies preventing stroke-related injury in animal models have not translated well into clinical settings. Current treatment options for stroke patients are limited to restoring blood flow via intravenous administration of thrombolytics (tissue plasminogen activator, tPA) or endovascular thrombectomy 2, 3. However, reperfusion intervention itself has been shown to trigger secondary injury pathways in the ischemic tissue4. Therefore, new therapeutic strategies addressing the ischemic and reperfusion-induced injury are needed to improve post-stroke outcomes.

Mitochondrial function is fundamental for metabolic homeostasis in all multicellular eukaryotes. In the nervous system, mitochondria-generated adenosine triphosphate (ATP) is essential for establishing electrochemical gradients across the plasma membrane and reliable synaptic transmission. Within minutes after arterial occlusion, brain mitochondria begin to lose electrochemical proton gradients, causing cessation of ATP synthesis and overproduction of reactive oxygen species (ROS). Prolonged depletion of ATP leads to plasma membrane depolarization and eventually apoptotic cell death5, 6.

Mitochondrial transplantation as a therapeutic approach was first explored in cardiovascular disease. Pioneered by McCully et al.7–9, mitochondria were harvested from autologous skeletal muscle and transplanted through coronary vasculature into the ischemic zone of a rabbit’s heart, improving post-ischemic myocardial functional recovery 8. In a clinical trial, Emani et al. transplanted mitochondria into human pediatric patients who sustained a myocardial ischemic event following cardiac surgery and found promising preliminary results 10. Recent studies have also suggested that neurons can incorporate mitochondria released in the extracellular space by other cells, such as astrocytes 11. Mitochondrial re-uptake can preserve neuronal viability and improve recovery after stroke 11–13. Exploiting this mechanism, Zhang et al. demonstrated that transplantation of muscle-derived autologous mitochondria in the lateral ventricle of the stroked brain reduced infarct size 14. In a more recent study, placenta-derived mitochondria delivered intravenously were shown to decrease infarct size after transient focal ischemia in mice 13.

While the evidence for beneficial effects of mitochondrial supplementation after ischemia is mounting, optimal routes of delivery to affected brain tissue have not been investigated in detail.

METHODS

Details of the methods used in this manuscript can be found in the supplemental materials available online.

Mouse model establishment

All experiments were approved by the Institutional Animal Care and Use Committee of the University of Virginia. Adult male or female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). All mice were housed in a controlled environment on 12 hours light/dark cycles and fed a standard chow. Only adult animals (8 to 10 weeks) were used in this study and animals from different cages were selected for each experimental group to ensure randomization. Littermates of the same sex were randomly assigned in a 1:1 ratio to experimental groups. All analysis was pooled, and sex was not used for sub-analysis of data.

Mitochondrial isolation and staining

Mitochondrial isolation was performed as previously described 18, and summarized in the supplemental methods.

Intra-arterial delivery of mitochondria

One hour after MCA occlusion, the mice were anesthetized and set up for reperfusion as described previously. Immediately prior to the withdrawal of the occluding filament, a micro-clip was placed on the ICA artery to avoid retrograde bleeding. The existing arteriotomy in the CCA was then used to insert an intra-arterial catheter (SAI infusion technologies, REF: MAC-01) filled with mitochondria or buffer solution. The catheter was secured in the CCA with a silk tie. Using a connected Hamilton syringe, slow infusion of 200 μl of the fresh mitochondrial suspension was performed at a rate of 100 μl per minute over two minutes. The catheter was then removed from the ICA and the CCA was permanently tied. The skin was closed, and analgesics were provided as above.

Stereotactic delivery of mitochondria

Mice were anaesthetized with isoflurane; the scalp was shaved and the animal was positioned in a stereotaxic frame (Kopf Instruments). Using aseptic techniques, a midline incision was made with small surgical scissors. The skin was removed and bregma and lambda were exposed. A small burr hole was drilled over the target area with a hand-held drill. A 5 μl Hamilton syringe filled with mitochondrial suspension was attached to the stereotaxic apparatus moved to the stereotactic target in the striatum. Two μl of mitochondria suspension were slowly deposited using Hamilton syringe. The skin was sutured, the animal was treated with analgesics and allowed to recover in a heated cage.

RESULTS

Rapid isolation of viable mitochondria

Mitochondria were isolated from the gastrocnemius muscle of C57Bl/6 mice using the method of Preble et al. 18, and their morphology and viability was assessed with electron microscopy, flow cytometry and bioluminometric ATP assays. Transmission electron microscopy confirmed organelle integrity, presence of cristae in the matrix and the mean area of isolated mitochondria (Figure 1A). Next, isolated mitochondria were labeled with fluorescent dye (MitoTracker Red CMXRos, Thermo Fisher), and their size was analyzed with flow cytometry. The peak size of isolated mitochondria was smaller than the 6 μm-diameter reference beads (Figure 1B), in line with previously reported dimensions and with the measurements we performed for our isolated mitochondria (Figure 1A) 21. In addition, MitoTracker CMXRos accumulation is dependent upon membrane potential 22, therefore bright fluorescence observed in our preparations indicated sustained electrochemical proton gradients in these mitochondria (Figure 1C). To follow the fate of isolated mitochondria in the ischemic brain independent of synthetic dye labeling, in a subset of experiments we took advantage of a transgenic line in which mitochondria are genetically labelled with a DsRed variant termed MitoTimer 17. Using fluorescence microscopy, mitochondria isolated from these mice appeared bright red, with a similar size distribution as MitoTracker-labelled organelles (Figure 1D). Finally, we evaluated the ATP content in isolated mitochondria to further confirm they were metabolically active. The mean ATP concentration in the fresh mitochondrial suspension was 0.20 ± 0.04 pM, while no ATP was detectable in solution buffer alone (Figure 1E).

Figure 1. Morphological and functional properties of skeletal muscle-isolated mitochondria.

Figure 1.

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Mitochondria were isolated from the murine gastrocnemius muscle with gentleMACS Octo Dissociator. A, Electron microscopy image showing representative isolated mitochondria from 94 different specimens from three batches of isolates with preserved membranes and organized matrix. Mitochondrial area was analyzed using the imageJ software and the area is reported in the graph in μm2. Scale bar, 500 nm. B, Flow cytometry analysis. Isolated mitochondria were labeled with MitoTracker Red CMRox, analyzed with flow cytometry and compared with reference beads. The size of the isolated mitochondria was found to be below the size of the reference (6 μm). C, Fluorescence microscopy image showing mitochondria labeled with MitoTracker Red CMXRox, indicating viable mitochondria with a preserved membrane potential. Scale bar, 10 μm. D, Fluorescence image or mitochondria genetically labeled with MitoTimer. Scale bar, 10 μm. E, A freshly isolated mitochondrial suspension, or the suspension buffer, were analyzed with the CellTiter-Glo ATP Assay. In 5 independent mitochondrial preparations, the mean ATP concentration was 0.20 ± 0.04 pM, while no ATP was detected in the buffer. **, p<0.01.

Intra-arterially infused mitochondria can permeate a disrupted blood-brain barrier

To determine how mitochondria distribute in the brain following intra-arterial infusion, with or without ultrasound treatment, we performed a series of experiments in healthy and ischemic brains. C57Bl/6 mice were subjected to transient proximal middle cerebral artery occlusion (tpMCAo) by inserting an occluding filament in the common carotid artery (CCA), as previously described 23. After 60 min, the ischemic hemisphere was re-perfused by withdrawing the occluding filament, and fresh mitochondrial suspension was infused through the same arteriotomy in the CCA, while the control mice received mitochondrial infusion after exposing the CCA without occlusion. To test whether we could increase permeability of the blood-brain barrier, we sonicated intra-arterially injected MBs with pulsed focused ultrasound in the ischemic brain immediately prior to mitochondrial infusion. Finally, a direct stereotactic injection in the ischemic brain was also performed as a control group. In all cases, the animals were sacrificed 2 hours after mitochondrial transplantation and brain sections were analyzed with confocal microscopy.

To measure delivery of mitochondria, we quantified the density of mitochondria signal in the vessels and in the parenchyma of comparable areas in dorsolateral striatum (Figure 2). We used CD31 staining to identify vessels to calculate intravascular density and used the area outside the vessels to define parenchymal density (Figure 2A, 2B, 2C, 2D). A parenchymal-to-intravascular signal density ratio was calculated as a measure of mitochondrial extravasation (Figure 2E). Mitochondrial infusion in the normal brains with intact blood-brain barrier resulted in low extravasation, and the ratio of parenchymal to intravascular mitochondrial signal was 0.33 ± 0.09 (Figure 2A). In the brains subjected to tpMCAo, this ratio increased to 6.96 ± 4.50, consistent with partial disruption of the blood-brain barrier (Figure 2C). However, this increase was not statistically significant. In another set of mice subjected to tpMCAo, blood-brain barrier permeability was further increased by stereotactic FUS (see Methods). In these animals, infused mitochondria reached a much higher concentration in the extravascular parenchyma; the parenchymal to intravascular ratio was 20.20 ± 7.57 (Figure 2E). Finally, for comparison, we performed direct stereotactic injections of mitochondrial suspension in the striatum. This resulted in robust delivery of mitochondria along the site of delivery but showed limited diffusion beyond the injection perimeter (Figure 2G).

Figure 2. Intra-arterially infused mitochondria permeate ischemic blood-brain barrier.

Figure 2.

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A, Intra-arterial delivery of mitochondria in the normal brain results in a limited diffusion of mitochondria in the parenchyma. Scale bar, 700 μm. High Magnification image demonstrating details of a CD31-labeled blood vessel containing fluorescently labeled mitochondria with limited extravasation. Scale bar, 15 μm. B, Intra-arterial injection of mitochondria into acute ischemic stroke. The DAPI-stained whole hemisphere image shows the highest density of extravasating mitochondria in the infarct core. Scale bar, 700 μm. In the high magnification image, mitochondrial signal was detected inside the blood vessels as well as outside of the vessels in the brain parenchyma. Scale bar, 15 μm. C, After intra-arterial delivery of mitochondria into acute ischemic stroke with subsequent FUS + MBs, an increased permeation of mitochondria in the stroked hemisphere was observed, including in the surrounding penumbra. Scale bar, 700 μm. Furthermore, in the magnified image, mitochondrial deposits in the blood vessels were infrequent. Scale bar, 15 μm. D, For comparison, direct stereotactic injections into the stroked hemisphere were performed. This route resulted in a robust loading with limited diffusion. Scale bar, 700 μm. High power image shows a fluctuating density of mitochondria in the parenchyma and absence of mitochondrial signal in the vessels. Scale bar, 20 μm. E, Plot summarizing the ratios of signals shown in the extra vascular and intra vascular space. NS + Mito IA, intra-arterial mitochondrial infusion in the control brains; S + Mito IA, intra-arterial mitochondrial infusion in the stroked brains; S + Mito IA + FUS, intra-arterial mitochondrial infusion in the stroked brains preceded by focused ultrasound. Bars represent mean. *, p<0.05; **, p<0.01, one-way ANOVA, n = 3.

To further characterize the synergistic effects of ischemic stroke and FUS on permeabilization of the blood-brain barrier, we performed intra-arterial injections of Evans blue dye for each experimental paradigm. The animals were sacrificed 4 hours after injection and the brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC) to delineate infarcted tissue (Figure S1A–S1D). Evans blue signal was quantified using a flatbed scanner, analyzed in ImageJ and expressed volumetrically. In the control brains, the mean volume of Evans blue extravasation was 0.048 ± 0.083 mm3. Application of stereotactic FUS to normal brains significantly increased Evans blue extravasation (7.914 ± 1.888 mm3). As expected, tpMCAo also substantially elevated the volume of Evans blue extravasation, to 10.64 ± 4.729 mm3. However, when we applied stereotactically guided FUS to brains post-tpMCAo, the volume of extravasated dye increased dramatically to 38.45 ± 16.30 mm3, significantly higher than either of the treatments alone (p < 0.05) (Figure S1E). This data reiterates that ischemic injury weakens the blood-brain barrier and that additional permeabilization can be achieved by activated MBs with pulsed FUS in the ischemic region.

Limited FUS does not cause hemorrhage in post-stroke brain

Given the significant improvement in delivery of mitochondria with the use of FUS, we asked if the use of FUS during acute ischemia could cause any additional injury, such as hemorrhage. First, we obtained T1- and T2-weighted Gd-DTPA-enhanced MRI scans in 4 mice undergoing acute phase of ischemic tpMCAo stroke. Next, we applied MRI-guided FUS delivery to 3 different focal points distributed over cortical and striatal areas in the ischemic side of the brain (Figure 3A–3C). Following FUS exposure, all animals were re-imaged using an analogous Gd-DTPA MRI sequence. Also, after FUS we acquired gradient-echo (GRE) T2*-weighted MRI sequences to detect brain hemorrhage 20. Evaluation of pre- and post-FUS T1 MRI sequences demonstrated the anticipated enhancement of mean grayscale intensity value over the areas targeted with FUS (pre-FUS, 72.30 ± 17.19; post-FUS, 112.5 ± 35.35; p = 0.0229) (Figure 3D). Hence, the results of MRI imaging afford additional support for synergistic effects of ischemia and focused ultrasound on focal permeabilization of the blood-brain barrier. Also, the GRE sequences did not detect any cerebral macro-hemorrhages in these mice (Figure 3E). Finally, histological staining was used to inspect for microhemorrhages. We serially sectioned infarcted regions of all brains receiving 3 targeted FUS deliveries and found no indication of vascular damage or bleeding (Figure 3F). Thus, limited use of focused ultrasound can be a safe and effective enhancement for mitochondrial delivery in the stroked brain.

Figure 3. Limited FUS delivery to the ischemic brain tissue does not cause hemorrhage.

Figure 3.

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A-C, Mice undergoing acute ischemic stroke following tpMCAo were imaged with Gd-DTPA-enhanced MRI, exposed to focused ultrasound (FUS) and re-imaged with the Gd-DTPA protocol. A, A T2 sequence showing the stroke area and three selected targets for FUS (red circles). B, A T1-weighted sequence confirming modest Gd-DTPA enhancement in the ischemic stroke area before FUS. C, T1-weighted sequence showing a strong Gd-DTPA enhancement over the targeted areas after FUS. D, A plot showing significant increase of the mean Gd-DTPA MRI grayscale pixel value over the selected areas after FUS. This data demonstrates a substantial blood-brain barrier opening in the ischemic brain tissue with limited FUS exposure. *, p = 0.02, n= 4. E, GRE sequence prior (left) and after (right) to FUS sonication indicating absence of detectable hemorrhagic complications. F, The absence of microhemorrhages was confirmed with histological staining. No microbleeds were found with H&E staining in serial sections thought the exposed brain regions. Scale bar: 300 μm (250 μm in the inset).

Neurons and other cells in the brain incorporate infused mitochondria

To follow the fate of mitochondria after extravasation, we studied coronal sections from mitochondria-infused brains with immunohistochemistry and confocal imaging. Specific antibodies for neurons (anti-NeuN, anti-MAP2), astrocytes (anti-GFAP) and microglia (anti-Iba1) were used to colocalize mitochondrial fluorescence signal with these cell types in the brain parenchyma. Mitochondria were seen in the extracellular space and vascular endothelia. However, under all tested conditions with compromised blood-brain barrier, transplanted mitochondria were also found in close apposition, as well as incorporated into all investigated cell types (Figure 4). We did not notice any preferred integration among the cell types investigated. To further investigate the mitochondria incorporation to cells, we analyzed 728 neurons in the penumbra area of 4 stroked brains using Z stacks and quantified the percentage of neuron’s co-localization with mitochondria (Figure S2A). We found that 85% of neurons, in the penumbra area, incorporated the transplanted mitochondria. To strengthen the evidence of intracellular localization, we also performed 3D volume rendering of the confocal Z-stacks with the Imaris software package. We have identified fluorescent puncta, consistent with mitochondria, which had been internalized inside the cytoplasm of the brain cells, including neurons (Figure S2B and C).

Figure 4. Brain cells can incorporate exogenous mitochondria.

Figure 4.

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High power confocal images show that multiple cell types in the brain can integrate exogenous mitochondria after FUS-assisted mitochondrial delivery. A, GFAP-positive astrocytes, B, Iba1-positive microglia, C, MAP2-positive neurons have been found to contain MitoTracker-labeled exogenous mitochondria (white arrows). Scale bar (a-c), 5µm.

Because MitoTracker is a synthetic dye which can possibly dissociate from mitochondria under certain conditions, and inadvertently label endogenous mitochondria, we also used genetically labeled mitochondria expressing a DsRed variant MitoTimer to corroborate our results. Unlike MitoTracker, which is a membrane-bound synthetic dye that remains significantly fluorescent even in fragmented and degraded mitochondria, MitoTimer is a soluble mitochondrial matrix protein. If MitoTimer leaks out from the damaged mitochondria, the protein becomes diluted in the cytosol and degraded by the proteasome. Alternatively, if a mitochondrion is degraded via mitophagy, the MitoTimer protein is completely degraded by the lysosomal enzymes. We have previously shown that all MitoTimer signals are Cox4 positive, i.e. of mitochondrial origin 16, supporting the claim that MitoTimer is a reliable marker of intact mitochondria (Figure S3).

Transplanted mitochondria increase ATP concentration in the stroked hemisphere

We evaluated for metabolic rescue by testing whether mitochondrial infusion augmented ATP concentrations in the brain (Figure 5). Using the paradigm of tpMCAo, we induced stroke in C57Bl/6 mice and delivered freshly isolated mitochondria. Two hours later, we flash-froze the brains and determined total ATP concentrations per hemisphere with bioluminometry. A predicted drop in endogenous ATP levels was measured in the ischemic hemi-dissected brains (from 10 ± 6 pmol/kg to 3 ± 2 pmol/kg) (Figure 5A). FUS-enhanced intra-arterial infusion as well as direct stereotactic injection of mitochondrial suspension increased total ATP to control levels. Surprisingly, the highest total ATP levels were reached after intra-arterial infusion without FUS facilitation (40 ± 6 pmol/kg), significantly greater than other groups (Figure 5A). Thus, mitochondrial transfusions appear to improve baseline ATP content in the infarcted brain, providing exogenous energy support to the ischemic tissue.

Figure 5. Mitochondrial transplantation improves ischemic stroke outcome.

Figure 5.

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A, ATP levels in the brains undergoing acute ischemic stroke. The ATP levels were decreased in the ipsilateral hemisphere after stroke and restored after intraarterial mitochondrial infusions with FUS (Mito IA + FUS), as well as after direct stereotactic injections of mitochondria (Mito STI). Intraarterial mitochondrial infusions without FUS facilitation (Mito IA) delivered significantly higher ATP levels than other procedures and exceeded even the normal control levels. B,C, Flow cytometry was used to assess cell viability in the stroke hemisphere following mitochondrial transplantations. B, Gating strategy used to separate live and dead cell populations. C, Plot showing the percentage of live cells in the stroked hemisphere. A significant decrease in cell viability was measured in the stroked hemisphere after tpMCAo, using buffer infusion as control (Buffer IA). Intra-arterial infusion of mitochondria (Mito IA) significantly rescued cell viability in the ischemic tissue. FUS-facilitated infusion of mitochondria (Mito IA + FUS) did not improve cell viability in this assay. D,E, Infarct size decreases after intraarterial mitochondrial infusion. Distal MCA coagulation was used to induce stroke. After 1 hour of ischemia, mitochondria or buffer were injected intra-arterially. At 24 hours, the brains were harvested and stained for infarct with TTC. D, Representative TTC-stained brains from a pdMCAo mouse (Stroke) and E, Mitochondria-infused mouse (Stroke + Mito). F, Evaluation of the infarct size between the group of mice treated intraarterially with buffer (Buffer IA) or mitochondria (Mitochondria IA). The mean infarct size in the Mitochondria IA group was reduced by 34% (p = 0.012, n = 6). *, p<0.05; **, p<0.01, one-way ANOVA, n = 3.

Mitochondrial transplantation improves cell viability and reduces infarct size

To address whether mitochondrial transplantation decreases infarct size, we used flow cytometry to investigate the basis of infarct size reduction at a single-cell level. Mice received tpMCAo and subsequent intraarterial infusion of freshly isolated mitochondria. Two hours after the procedures, the brains were dissociated to single-cell suspensions, stained for cell viability using Live/Dead-Aqua (Invitrogen) and analyzed with flow cytometry. The gating strategy and robustness of viability-based discrimination is shown in Figure 5B. We found that the percentage of viable cells in cell suspensions from ischemic brains decreased from 68.40 ± 1.70% to 32.47 ± 13.95% (p = 0.04, n = 3). Intra-arterial infusion mitochondria alone significantly rescued cell viability in the ischemic brains, (56.63 ± 1.30%), although remaining below the normal brain values (Figure 5C).

Finally, we used a permanent distal middle cerebral artery occlusion (pdMCAo) stroke model, which creates a more reproducible ischemic injury than transient proximal occlusion. After distal MCA electrocoagulation (see Methods), fresh mitochondrial suspension was infused through catheterized ipsilateral common carotid artery in the experimental mice, while the control group received the suspending buffer only. Twenty-four hours after infusion, the brains were extracted, sliced, and stained for infarct evaluation (Figure 5D and 5E). Importantly, mitochondrial infusions brought about a 34% reduction of the infarct volume (9.919 ± 2.154 mm3 to 6.523 ± 1.659 mm3, p = 0.012, n = 6) (Figure 5F).

DISCUSSION

We provide evidence that mitochondrial transplantation may be beneficial as therapeutic intervention for ischemic stroke. First, we show that healthy muscle-derived mitochondria can be isolated in a clinically relevant timescale. Second, we demonstrate that intra-arterial injections of mitochondria result in perfusion of extravascular spaces in a minimally invasive manner, and that this outflow can we further enhanced by FUS. Third, mitochondria from the interstitial spaces become integrated by multiple cell types in the ischemic region. Fourth, mitochondrial transplantations elevate ATP concentrations in the brain parenchyma which might provide a basis for a partial metabolic rescue of ischemic tissue. Finally, cerebral infarction volume is reduced, and cell survival is increased after mitochondrial infusions.

Mitochondrial dysfunction is a major contributor to neurological disease 24, 25. Mitochondria are particularly critical to neuronal survival during ischemic stroke and supplementing mitochondrial function could reverse cell death and protect the ischemic penumbra from further injury. Given the narrow treatment window for ischemic stroke has few medical options, autologous mitochondrial transplantation offers an attractive paradigm for therapeutic intervention in cerebral infarction.

With respect to cerebral ischemic injury, Zhang et al. transplanted mitochondria by stereotactic injections in the lateral ventricle of stroked rat brains 14. Their data revealed reduction in brain infarct volume, increased neurogenesis and reversed neurological deficits. The study also found reduced astrogliosis, cellular oxidative stress and apoptosis. While these results are very encouraging, direct intracerebroventricular injections into lateral ventricles are inherently invasive, which would add additional peri-procedural time and risks to stroke patients. Furthermore, lateral ventricles occupy only about 1% of the whole adult human brain 29, rendering this route of delivery less favorable from a clinical perspective. Furthermore, we have previously noted that stereotactic intraparenchymal injection is unlikely to be useful in the setting of stroke as the range of delivery is limited compared to intra-arterial delivery 25.

Our results favor the intra-arterial approach to mitochondrial transplantations. Whereas the normal blood-brain barrier is not highly permeable to infused mitochondria, the endothelial barrier coincidentally opens during the acute phase of ischemic stroke (Figure 2, Figure 4, Figure S1, S2, S3.). These results are also supported by a recent study that showed benefit with intravenous delivery of mitochondria after stroke 13. The advantage of arterial delivery over the intravenous approach includes potentially higher concentrations of mitochondria delivered specifically to the reperfused brain, and less non-specific delivery and diffusion into other tissues. Either way, delivery via blood vessels ensures homogeneous distribution of labeled mitochondria in the parenchyma, clearly superior to stereotactic injections (Figure 2). The intra-arterial route also robustly supplements brain tissue with exogenous ATP (Figure 5A) which may assist with metabolic recovery in the ischemic areas.

Our data also indicate that even greater permeabilization of the ischemic blood-brain barrier can be attained with judicious use of FUS. In the laboratory setting, blood-brain barrier permeability is consistently increased through mechanical effects of FUS on injected microbubbles 31–34. Utilizing FUS, we were able to achieve enhanced delivery of macromolecules to the distal ischemic penumbra (Figure 2, Figure S1). Achieving enhanced delivery through use of focused ultrasound seems appealing and we show that effective, yet safe and hemorrhage-free conditions can be established in an animal stroke model (Figure 3). However, we acknowledge that our functional tests yielded lower overall ATP levels and cell viability after FUS-assisted intra-arterial infusions (Figure 5A and 5C); ultimately, further investigation of this inconsistency is warranted, but we speculate that the sonication associated with the FUS-assisted delivery may result in the decrease in ATP levels and cellular viability.

Molecular mechanisms responsible for incorporating extracellular mitochondria in the surviving cells of ischemic penumbra are unclear. Several processes have been proposed to mediate the internalization, including endocytosis, formation of nanotubes, gap junctions or connexin bridges 35. A recent study found that mitochondrial transfer from healthy cells to injured neurons occurs naturally in vivo 11. Ischemia-induced release of mitochondria from astrocytes involves CD38-cADPR pathway and mitochondrial transfer into neurons appear to be dependent on integrin-mediated Src/Syk signaling 11. However, other cell types in the central nervous system have been shown to be able to integrate mitochondria from the extracellular space, including astrocytes 11, 36, microglia and endothelial cells 37. With two different mitochondrial labeling strategies, using synthetic and genetic fluorophores, we have observed mitochondria integrating in several different cell types with no obvious preference (Figure 4 and Figure S3).

The precise mechanism by which infusion of mitochondria mediates a benefit is not definitively addressed by our study. There could be other paracrine effects of mitochondria that are contributing. For example, mitochondrial constituents could be modulating inflammation or other protective cascades 38. It is noteworthy, however, that Ramirez-Barbieri and co-authors investigated the immune and damage-associated molecular patterns (DAMPs)-associated response after injections of allogeneic mitochondria in a transplant rejection system of fully MHC-mismatched skin allografts, and found no direct or indirect, acute or chronic alloreactivity or DAMPs associated reactions to single or serial injections of allogeneic mitochondria 39. The interested reader is pointed to our recent review (Norat et al. Ref 25) on potential mechanisms of mitochondrial-based neuroprotection. There remain main outstanding questions as to how these cellular powerhouses are responsible for the phenotypic changes noted after injury.

This study, as many others have done in the past, pooled results from male and female subjects. The role of sex differences in recovery from injury is an area of intense discourse, with notable examples of differences after traumatic brain injury, for example. The pooling of results is a potential limitation of this study.

We propose that autologous mitochondrial transplantation is extremely well-poised to complement and augment the revascularization treatment for maximum benefit to stroke patients. While the role for FUS in enhancing delivery of mitochondria in this context remains unclear, we do show that safe and hemorrhage-free conditions for FUS can be established in an animal stroke model.

Supplementary Material

supinfo

Clinical Perspective.

  • The authors present a novel strategy of autologous mitochondrial harvest and transplantation after thrombectomy for large vessel occlusion using a murine model of cerebral ischemia and reperfusion that mimics current clinical paradigms.

  • They demonstrate that mitochondria can be harvested in a clinically viable time and that they can be delivered to the brain and integrate into the parenchyma. Upon integration, the mitochondria elevate concentration of ATP, reduce infarct volume, and increase cell viability.

  • This novel strategy lends itself to clinical translation because the cellular components are autologously harvested and minimally modified.

ACKNOWLEDGEMENTS

We gratefully acknowledge resources at the Center for Brain Immunology and Glia (BIG) at the University of Virginia School of Medicine. We thank G. Wilson Miller and William Garrison for assistance with MRI imaging and Stacey Criswell for help with transmission electron microscopy. We also thank Kalil Alves de Lima and Aminata Coulibaly for advice and helpful suggestions on flow cytometry. Image analysis support by Daniel Miranda (Bitplane) is acknowledged. This study was supported by National Institutes of Health (Grants R01EB020147 and R21EB024323 to R.J.P.). C.M.G. is supported by the American Heart Association Fellowship (18PRE34030022). M.R.L. is supported by R01NS105692 and P.T. is supported by R21NS116431.

Abbreviations:

FUS

Focused Ultrasound

MB

Microbubbles

TTC

2,3,5-Triphenyltetrazolium chloride

MCA

Middle Cerebral Artery

tpMCAo

Transient proximal middle cerebral artery occlusion

pdMCAo

Permanent distal middle cerebral artery occlusion

Footnotes

DECLARATION OF INTERESTS AND DISCLOSURES

The authors declare no competing financial interests or disclosures.

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