# Mitochondria dysfunction, a potential cytoprotection target against ischemia-reperfusion injury in a mouse stroke model

Elodie Ong, Paul Clottes, Christelle Leon, hala guedouari, Noelle Gallo-Bona, Megane Lo Grasso, Lucas Motter, Radu Bolbos, Michel Ovize, Norbert Nighogossian, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Elodie Ong, Paul Clottes, Christelle Leon, hala guedouari, Noelle Gallo-Bona, et al. Mitochondria dysfunction, a potential cytoprotection target against ischemia-reperfusion injury in a mouse stroke model. Neurotherapeutics, 2025, 22 (2), pp.e00549. 10.1016/j.neurot.2025.e00549. hal-04956501

HAL Id: hal-04956501

https://hal.science/hal-04956501v1

Submitted on 19 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
Original Article

# Mitochondria dysfunction, a potential cytoprotection target against ischemia-reperfusion injury in a mouse stroke model

Elodie Ong<sup>a,b,1</sup>, Paul Clottes<sup>a,b,1</sup>, Christelle Leon<sup>b</sup>, Hala Guedouari<sup>b</sup>, Noelle Gallo-Bona<sup>b</sup>, Megane Lo Grasso<sup>b</sup>, Lucas Motter<sup>b</sup>, Radu Bolbos<sup>c</sup>, Michel Ovize<sup>a,b</sup>, Norbert Nighogossian<sup>a,b</sup>, Marlene Wiart<sup>b,d,2</sup>, Melanie Paillard<sup>b,*,2</sup>

ARTICLE INFO

Keywords:

Cerebral ischemia-reperfusion

PTP

Mitochondrial respiration

Therapeutic

ABSTRACT

More than 50 % of patients undergoing mechanical thrombectomy (MT) for ischemic stroke have a poor func-
tional outcome despite timely and successful angiographic reperfusion, highlighting the need for adjunctive
treatments to reperfusion therapy. Mitochondria are key regulators of cell fate, by controlling cell bioenergetics
via oxidative phosphorylation (OXPHOS) and cell death through the mitochondrial permeability transition pore
(mPTP). Whether these two main mitochondrial functions are altered by reperfusion and could represent a new
cytoprotective approach remains to be elucidated in mice. Swiss male mice underwent either permanent or
transient middle cerebral artery occlusion (pMCAO or tMCAO), with neuroscore evaluation and multimodal
imaging. The area at risk of necrosis was evaluated by per-occlusion dynamic contrast-enhanced ultrasound. Final
infarct size was assessed at day 1 by MRI. Cortical mitochondrial isolation was subsequently performed to assess
mPTP sensitivity by calcium retention capacity (CRC) and OXPHOS. A cytoprotective treatment targeting mito-
chondria, ciclosporine A (CsA), was tested in tMCAO, to mimick the clinical situation of patients treated with MT.
Reperfusion after 60 min of ischemia improves neuroscores but does not significantly reduce infarct size or
mitochondrial dysfunction compared to permanent ischemia. CsA treatment at reperfusion mitigates stroke
outcome, decreases final infarct size and improves mitochondrial CRC and OXPHOS. Mitochondrial dysfunctions,
i.e. reduced mPTP sensitivity and decreased oxygen consumption rates, were observed in pMCAO and tMCAO
regardless of the reperfusion status. CsA improved mitochondrial functions when injected at reperfusion. These
suggest that both mPTP opening and OXPHOS alterations are thus early but reversible hallmarks of cerebral
ischemia/reperfusion.

Introduction

Stroke is the second most common cause of death and a leading cause
of disability worldwide: the 30-year projection forecasts an increase of
around 30 % of its burden in the European Union [1,2]. The target of
acute ischemic stroke (AIS) treatment is based on early reperfusion to
salvage ischemic penumbra (i.e. the severely hypoperfused, electrically
silent, at-risk brain tissue) in order to avoid irreversible brain damage
and subsequent neurological disability. Multimodal stroke magnetic

resonance imaging (MRI) allows an individual assessment of the initial
infarct size, of the site of occlusion and of the hypoperfused region,
namely the area at risk of necrosis (AAR). This so-called “ischemic pen-
umbra” is the target of AIS therapies [3]. Intravenous thrombolysis with
recombinant tissue plasminogen activator (rt-PA) and mechanical
thrombectomy (MT) for selected patients with large vessel occlusion,
have demonstrated their efficacy [4]. Nevertheless, more than 50 % of
patients undergoing MT have a poor functional outcome despite timely
and successful angiographic reperfusion [5]. The term “futile

<footnote><sup>a</sup> Stroke Department, Hospices Civils de Lyon, 69500 Bron, France</footnote>

<footnote><sup>b</sup> Laboratoire CarMeN - IRIS Team, INSERM, INRA, Université Claude Bernard Lyon-1, Univ-Lyon, 69500 Bron, France</footnote>

<footnote><sup>c</sup> CERMEP-Imagerie du Vivant, 69500 Bron, France</footnote>

<footnote><sup>d</sup> CNRS, 69100 Villeurbanne, France</footnote>

<footnote>* Corresponding author.
*E-mail address: melanie.paillard@univ-lyon1.fr (M. Paillard).</footnote>

<footnote n="1">¹ These authors share the first authorship.</footnote>

<footnote n="2">² These authors share the last authorship.</footnote>
reperfusion" is used to describe this condition encompassing the components of the ischemia/reperfusion injury (IRI) including infarct growth and post-treatment complications such as haemorrhagic transformation, malignant edema, and ultimately neurological disability [6]. In fact, sudden reperfusion following thrombectomy promotes ischemic brain damage [7,8]. Although cytoprotective strategies for ischemic stroke have failed to demonstrate efficacy in several randomized clinical trials, the advent of successful reperfusion provides the opportunity to reconsider therapeutic approaches [9]. In the context of MT, IRI remains an important therapeutic issue [9]. To assess candidate cytoprotective therapies against IRI in preclinical studies, the intraluminal transient middle cerebral artery occlusion (tMCAO) murine model is commonly used, since it replicates the prompt recanalization obtained by MT in stroke patients [10,11].

Mitochondria, as the « powerhouse of the cell », contributes to the cell fate notably by maintaining the bioenergetics through oxidative phosphorylation. Accordingly, mitochondria may play a key role in IRI [12]. The mitochondrial permeability transition pore (mPTP), a non-selective pore located in the inner mitochondrial membrane, is a Ca²⁺-activated channel involved in mitochondrial injury and cell death upon stress [13]. During reperfusion after a brain ischemia, a combination of Ca²⁺ overload, oxidative stress and adenosine tri-phosphate lead to mPTP opening and necrotic death [14,15]. Cyclophilin D (CypD) appears to be an essential regulator of the mPTP: a significant reduced infarct size in tMCAO is observed in CypD-deficient mice in which inhibition of mPTP opening has been described [15]. Targeting mPTP opening could be a potential strategy to limit IRI [14]. Cyclosporine A (CsA), a potent in vitro mPTP inhibitor via CypD binding, led to a decreased infarct size in patients with proximal artery occlusion and successful recanalization [16,17].

Whether the two main mitochondrial functions, i.e. oxidative phosphorylation (OXPHOS) and mPTP opening (assessed by calcium retention capacity, CRC, which reflects maximum Ca²⁺ overload that mitochondria can support before mPTP opening [18]), are altered specifically by reperfusion, and could potentially be modulated by a cytoprotective strategy, has not been assessed so far in a mouse model of MCAO to the best of our knowledge.

In this context, the aims of this study were:

(i) To evaluate mitochondrial dysfunctions (using CRC and OXPHOS analysis) in a mouse model of MCAO (transient and permanent) using multimodal imaging (per-occlusion dynamic contrast-enhanced ultrasound (CEUS) assessment of the AAR and MRI at day 1 to evaluate infarct size) and neuroscore evaluation;

(ii) To study CsA as a cytoprotective treatment potentially targeting mitochondria in the tMCAO model combining imaging approach, neuroscores assessment and mitochondrial function analyses (mPTP sensitivity to Ca²⁺ and OXPHOS).

## Materials and methods

### Ethics and animals

All animal experiments were approved by the local review board in Lyon ("Comité d'éthique pour l'Expérimentation Animale Neurosciences Lyon": CELYNE; CNREEA no. 42; APAFIS agreement no. #24275) and authorized by the French ministry of higher education, research, and innovation. Experiments were performed on 6-week-old male mice (Envigo CD-1®, France), weighting between 25 and 36 g on the day of surgery. Mice were acclimated to the animal facility conditions for at least 7 days, housed in a controlled temperature (22 ± 2 °C) with a 12/12-h light/dark cycle and ad libitum access to pelleted food and water. Humane endpoints were: no reaction to stimuli, prolonged inactivity (>6 h), distress signs (stereotypies) and weight loss >15 %. These signs were monitored at 1 h, 5 h and 24 h post-surgery. Results are reported according to the ARRIVE guidelines 2.0 [19]. A total of 52 adult male mice were included in this study.

### Study design

Fig. 1A presents the study design. The study adhered to RIGOR guidelines for stroke research [20]. Randomization was performed by block of 3 animals per day (https://www.graphpad.com/quickcalculs/randomize1.cfm). Cofounders such as animal/cage location were not controlled. All experimenters were blinded to the allocation group.

Sample size was calculated a priori using G\*power (Heinrich Heine Düsserldorf Universität, Germany). An alpha level of 0.05 and a power of 0.8 were defined. The primary endpoint was infarct volume measured on T2-weighted MRI at 24 h normalized by the area-at-risk of infarction measured on per-occlusion CEUS. We hypothesized a 40 % reduction in infarct volume in treatment groups and a variability of 40 % according to our previous studies (effect size = 1.21, Mann-Whitney test) [21,22]. The minimal number of animals to be included was determined as 10 mice per group. A 30 % exclusion rate (including mortality and exclusion from study or analysis according to a priori-defined criteria as defined below) was anticipated, therefore 52 mice underwent randomization in total.

### Transient and permanent ischemic stroke models

For analgesia, buprenorphine was injected (0.075 mg/kg body-weight; Vetergesic 0.3 mg/mL, Alcyon, France) subcutaneously. For induction, mice were anesthetized with 8 % sevoflurane. During all procedures, the core temperature of the animals was kept constant at 37 °C, using a homothermic blanket heating system. The intraluminal filament model of focal ischemia was used [22,23]. Briefly, under 4 % sevoflurane maintenance anesthesia, a size-matched silicon-coated monofilament (Doccol Corporation, USA) was advanced through the right internal carotid artery toward the middle cerebral artery (MCA) junction until resistance was felt (~8 mm). For tMCAO animals, the filament was withdrawn after 60 min of occlusion, while it remained in position for pMCAO animals. For Sham animals, the filament was introduced into the MCA but remained not occlusive. CsA (10 mg/kg, Sandimmun 50 mg/mL diluted in NaCl) was injected in the caudal vein as a bolus (5 µl/g body weight) in tMCAO+CsA mice 5 min before reperfusion. After suture of the carotid artery access, mice were returned to their respective cage for 24 h and were monitored with continuous analgesia (buprenorphine at 0.075 mg/kg sc after surgery and on the next morning) until their euthanasia on the next day (cervical dislocation under 2 % isoflurane anesthesia).

### Per-occlusion echography imaging

Echography imaging was used per-occlusion to ascertain ischemia (and thus include only animals that were correctly occluded) and to provide a quantitative biomarker of the area at risk of infarction using perfusion imaging. Several acquisitions modes were performed on a Vevo3100 system: B-mode, color doppler and contrast imaging. Per-occlusion dynamic contrast-enhanced ultrasound (CEUS) was performed at 4 slice levels during the infusion of 250 µL microbubbles at 50 µL/min using the burst-replenishment method (Vevo® 3100 LAZR-X, MX201 probe, Vevo MicroMarker, Visual Sonics) [23].

### Functional status evaluation

Neuroscores were performed 24 h after the onset of reperfusion using a 0 to 5 scale score, as previously described [24]. Three points were assessed: parachute reflex, lateral resistance and circling. Mice with no spontaneous motion were assigned a grade of 0. Mice with symmetrical forelimbs extension toward the floor without neurological impairments were assigned a grade of 5. According to the score, we defined three outcome categories: mild (4–5), moderate (2–3) and severe (0–1).
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /><figcaption>**Fig. 1. Description of the study.** A. Study design. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) using the monofilament model. Mice underwent permanent or transient MCAO (60 min) or Sham surgery. Ciclosporin A (CsA) was given in a subgroup of mice with tMCAO. Per-occlusion ultrasound imaging was performed to verify: (i) arterial occlusion using color Doppler mode and (ii) hypoperfusion of the MCA territory using contrast (microbubbles)-enhanced ultrasound. Neuroscores and MRI were obtained 24 h post-surgery. Brains were sampled and mitochondria extracted for functional analysis (CRC and OXPHOS). B. Study Flow Chart. A total of 52 animals were randomized. Inclusion/exclusion criteria were defined a priori. MCAO mice that did not have occlusion and/or perfusion defect on per-occlusion ultrasound were excluded. C. Imaging protocol: the yellow arrow shows the right MCA occlusion on Doppler and the perfusion defect appears in blue on CEUS. The yellow dotted line on the MRI delineates the infarct.</figcaption></figure>
## Magnetic resonance imaging

The MRI protocol was performed on mice using a 7T horizontal-bore Bruker BioSpec Avance I preclinical imaging system (Bruker Biospin MRI GbmH Bruker, Germany). This MRI was equipped with a set of gradients of 440 mT/m maximum amplitude and controlled via a Bruker workstation interfaced with ParaVision5.1 software for data acquisition. A Bruker birdcage volume coil (inner diameter = 72 mm and outer diameter = 112 mm) was used for the signal transmission, and a Bruker single loop surface coil (25 mm diameter) was used for signal reception and positioned on the head of the animals to target the brain. Anesthesia was performed with a dedicated system (TEM Sega Lormont, France), induced with a mixture of 3.5 % isoflurane gas and air and delivered at a rate of 600 mL/min. The animals were placed in supine position in an MRI-compatible mouse cradle provided with a stereotactic holder (Bruker Biospec Animal Handling Systems) and maintained during the entire MRI protocol under anesthesia at 1.5 % isoflurane delivered via a cone mask. The respiratory rate was carefully monitored using a pressure sensor linked to a monitoring device (ECG Trigger Unit HR V2.0, RAPID Biomedical, Rimpar, Germany), and the physiological body temperature was maintained by means of a thermo-regulated water circuit.

The MRI protocol was performed 24 h post-MCAO experimental model. To visualize and quantify the post-stroke cerebral vasogenic edema, 2D T2-weighted RARE spin-echo MR images were obtained with the following parameters: field of view (FOV) = 30 × 30 mm², matrix size = 256 × 256 pixels, leading to an in-plane spatial resolution of 117 × 117 µm², effective echo time (eff-TE) = 75 ms, repetition time (TR) = 5000 ms, RARE factor = 8, number of averages (NA) = 2, bandwidth = 35 kHz. A total of 9 adjacent slices of 1 mm thickness were acquired in axial orientation for a 4 min scan time, covering the mouse brain from olfactory bulb to cerebellum. Finally, in order to ascertain the middle cerebral artery in pMCAO and recanalization in tMCAO, a 2D time of flight (TOF) with flow compensation angiography images was obtained with the following parameters: field of view (FOV) = 20 × 20 mm², matrix size = 192 × 192 pixels, leading to an in-plane spatial resolution of 104 × 104 µm², effective echo time (eff-TE) = 3.6 ms, repetition time (TR) = 23 ms, flip angle: 80 deg, number of averages (NA) = 4, bandwidth = 75 kHz. A total of 29 adjacent slices of 0.40 mm thickness and 0.25 mm interslice distance were acquired in coronal orientation for a 6.24 min scan time.

## Inclusion and exclusion criteria

Mice were excluded from the study if they did not have a vascular occlusion on per-occlusion Doppler imaging and/or a perfusion defect as visually assessed on dynamic contrast-enhanced ultrasound. Mice were excluded from analysis if they did not have a complete longitudinal follow-up, if they did not have an infarct in the MCA territory or if they had an infarct lesion superior to the AAR.

## Lesion volume assessment

**CEUS analysis:** Dynamic images were imported from VevoLab and processed using an in-house Matlab code. Dynamic curves were fitted to the burst-replenishment model to generate perfusion maps. The median value in the contralateral hemisphere was used to normalize perfusion maps on a voxel-by-voxel basis. The AAR was defined automatically as the voxels with hypoperfusion <26 % of contralateral hemisphere and its volume is presented as a percentage of the hemisphere. This threshold was defined based on the hypoperfused area that evolved to infarction on T2WI in pMCAO [23].

**MRI analysis:** For assessment of lesion size, T2WI data were analyzed by a blinded examiner using ImageJ software (National Institute of Health, USA imagej.nih.gov/ij/) by manually contouring the lesion, the ipsilateral and the contralateral hemispheres. Lesion volume was corrected for edema using Gerriets correction and presented as a percentage of the hemisphere, %HLVc [25,26].

## Mitochondrial function study

### Cortical mitochondrial isolation

The brain was extracted quickly after the sacrifice of the mouse and the two hemispheres were separated. The cerebellum, brain stem and underlying midbrain structures were removed from the forebrain. After mechanical homogenization in an isolation buffer (Mannitol 225 mM, Sucrose 75 mM, Hepes 20 mM, EGTA 0.1 mM, BSA 0.5 %, pH 7.4), a first centrifugation was performed at 500 g for 5 min at 4 °C to cast out the unwanted membranes and organelles, and then a second centrifugation at 9000 g for 10 min at 4 °C to pellet mitochondria. Mitochondria were finally suspended in a storage buffer (Mannitol 225 mM, Sucrose 75 mM, Hepes 10 mM, pH 7.4) and kept on ice for a maximum of 4 h. Final protein content was assessed with the Lowry method before subsequent analyses.

### Calcium retention capacity

For CRC, we used two Hitachi Research Fluorescence Spectrophotometer (F-2500 and F-7000). 500 µg of isolated cortical mitochondria from each hemisphere were suspended in 2 mL of buffer at 25 °C (150 mM Saccharose, 50 mM KCL, 2 mM KH₂PO₄, 5 mM Succinate in 20 mM Tris/HCl, pH 7.4) with 0.5 µM Calcium Green in the spectrophotometer cuvette to follow the extramitochondrial Ca²⁺ level. After a 2-min phase of stabilization, we performed repetitive pulses (every minute) of 10 µL CaCl₂ at 500 µM until the burst of Ca²⁺ by mitochondria, corresponding to mPTP opening. The results were expressed in nmol of Ca²⁺ per mg of mitochondria.

### Oxidative phosphorylation (OXPHOS)

To determine the oxygen consumption rates (OCR) through the different complexes, a successive substrate-inhibitor titration protocol was performed on a high-resolution oxygraph (Oxygraph-2k; Oroboros, Innsbruck, Austria) at 25 °C. 500 µg of isolated mitochondria from each hemisphere were added in 2 mL of respiration buffer (100 mM KCL, 50 mM Mops, 1 mM EGTA, 5 mM Kpi, pH 7.4 with 1 mg/mL of bovine serum albumine without fatty acid) in each cuve. Addition of glutamate/pyruvate/malate (final concentration 3 mM) followed by ADP (2 mM) led to the measurement of OCR after complex I stimulation. Successive additions of rotenone (complex I inhibitor, 0.5 µM), succinate (complex II substrate, 10 mM), TTFA (complex II inhibitor, 40 µM), TMPD/ascorbate (complex IV substrate, 0.25/2.5 mM) and azide (complex IV inhibitor, 1.5 mM) allowed determination of sensitive rates of oxidative phosphorylation after stimulation of complexes II and IV. Data were analyzed with the Oroboros DatLab4 software and expressed as nanomoles of oxygen per minute per milligram of proteins.

## Statistical analysis

GraphPad Prism 4 (Graph Pad Software Inc, La Jolla, CA) was used for statistical analysis. Due to the effective per group, data were analyzed by Kruskal-Wallis and Mann-Whitney tests. Data are presented as Median [interquartile range 25 %; 75 %]. A p value < 0.05 was considered significant. Treatment-effect modification on neuroscores was evaluated in subgroups of mice (mild, moderate, and severe): the chi-square test of proportions (with a two-sided alpha level of 5 %) was used to determine the between-group differences.

## Results

Study flow chart is displayed on Fig. 1B and imaging protocol on Fig. 1C. Fifty-two mice were randomized (n = 12 Sham, n = 14 tMCAO, n = 12 tMCAO + CsA, n = 14 pMCAO). Ten mice were excluded from analysis: 6 mice did not meet the inclusion criteria and 3 did not have a complete follow-up because they died before MRI at 24 h (2 in pMCAO and 1 in tMCAO groups).
Reperfusion after 60 min of ischemia improves neuroscores but does not significantly reduce infarct size compared to permanent ischemia

After reperfusion, tMCAO mice displayed significantly improved neuroscores in comparison with pMCAO mice (p = 0.0193) (Fig. 2A). Importantly, the AAR was similar in tMCAO and pMCAO groups (48 % [34%; 61 %] vs 52 % [41%; 56 %] p = 0.45) (Fig. 2B). The primary endpoint (%HLVc/AAR) revealed no significant difference between tMCAO and pMCAO groups (83 % [45; 104]) vs 92 % [75; 137] p = 0.14) (Fig. 2C). For a given AAR, the lesion size was not statistically different between tMCAO and pMCAO model (Fig. 2D) (p = 0.1107).

Sixty minutes of ischemia with 24h of reperfusion adversely affects mitochondrial CRC and OXPHOS

We next investigated the impact of reperfusion on mPTP opening and oxidative phosphorylation. Mitochondria were extracted in both groups from the left and right hemispheres. On mitochondria isolated from the right ischemic hemisphere, we observed a significantly decreased CRC in pMCAO and tMCAO compared to Sham mitochondria (pMCAO: 60 [25; 65] and tMCAO: 70 [50; 90] versus Sham: 105 [80; 118] nmol Ca²⁺/mg protein) (Fig. 3A): no further difference was measured in CRC between pMCAO and tMCAO ischemic mitochondria. In the left non-ischemic hemisphere, CRC reached the same level between groups, averaging 100 nmol Ca²⁺/mg protein (Fig. 3C). While no difference was observed in CRC between the right and left hemisphere in the Sham group, CRC was lower in mitochondria isolated from the right ischemic hemisphere compared to their respective left non-ischemic hemisphere in the tMCAO group (p < 0.0059) and in the pMCAO group (p < 0.0039).

Concerning the OXPHOS analysis, we quantified the oxygen consumption rates induced by the stimulation of the different electron transport chain complexes. We found a significant difference between Sham and pMCAO groups respectively for complex I (24 [21.1; 29.5] vs 11 [4.8; 16.4] nmol O₂/min/mg, p = 0.0002), II (15.9 [14.7; 19.3] vs 6.93 [4.2; 9.2] nmol O₂/min/mg, p < 0.0001) and IV (24.8 [23.2; 32.9] vs 15.1 [12.1; 21.4] nmol O₂/min/mg, p = 0.0011) respiratory rates in mitochondria from the right ischemic hemisphere (Fig. 2B). Oxygen consumption rates were significantly decreased in the pMCAO ischemic mitochondria compared to the Sham ones, whatever the substrates used. In the tMCAO group, respiratory rates in the right ischemic mitochondria hemisphere were significantly decreased compared to the Sham group after addition of substrates of either complex I (tMCAO: 13.4 [11.1; 16.2] vs Sham: 24 [21.1; 29.5] nmol O₂/min/mg, p = 0.0015), II (tMCAO: 15.9 [14.7; 19.3] nmol O₂/min/mg, vs Sham: 11.5 [6.7; 12.7] nmol O₂/min/mg, p = 0.0142) or IV (tMCAO: 24.8 [23.2; 32.9] vs Sham: 22.99 [18.6; 23.5] nmol O₂/min/mg, p = 0.0441). However, no significant difference in oxygen consumption rates was observed between tMCAO and pMCAO ischemic mitochondria. Moreover, no differences were observed between groups regarding respiratory rates for each complex in mitochondria isolated from the non-ischemic left hemisphere.

In short, our data showed that similar anomalies, i.e. increase of mPTP sensitivity to Ca²⁺ and alteration of OXPHOS in the right ischemic hemisphere, were induced in both pMCAO and tMCAO models.

CsA treatment at reperfusion improves stroke outcome and decreases final infarct size

To evaluate the potential reversibility of mitochondrial dysfunction induced by tMCAO, we chose a well-known cytoprotective drug that modulates in vitro mitochondrial function, CsA. tMCAO mice received a single bolus of 10 mg/kg CsA 5 min before reperfusion. Neuroscores tended to improve in the tMCAO+CsA vs tMCAO mice (tMCAO+CsA: 4 [1; 4] vs tMCAO: 2 [1; 3], p = 0.0836) (Fig. 4A). Fig. 4B presents representative infarct lesions from tMCAO+CsA and tMCAO groups. No differences were observed in AAR between groups (tMCAO: 48 %

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Fig. 2.** Comparison between transient middle cerebral artery (tMCAO) and permanent MCAO (pMCAO) mice. A. Neuroscores in the two groups, tMCAO and pMCAO. Three outcome categories were defined based on the Bederson score (0–5): mild (4–5), moderate (2–3) and severe (0–1). One mouse from the tMCAO group and 2 mice from the Sham group did not get a neuroscore because of neurobehavior experimenter unexpected absence. Neuroscores were significantly improved in tMCAO compared to pMCAO. Chi-square test. B. Quantification of the area at risk (AAR) in tMCAO and pMCAO groups. The AAR was defined as the hypoperfused region on per-occlusion contrast-enhanced ultrasound. As expected, AAR were not statistically different between tMCAO and pMCAO. Mann-Whitney test, Median [interquartile range 25%; 75%]. C. Quantification of the infarct size calculated as the ratio of lesion volume measured on T2-weitghed MRI over AAR (%HLVc/%AAR) in tMCAO and pMCAO groups. Infarct sizes were not different between groups. Mann-Whitney test, Median [interquartile range 25%; 75%]. D. Lesion volume presented as percentage of the hemisphere (%HVLc) relative to the AAR, showing that for a given AAR, infarct size was lower for tMCAO mice, although this was not significant (p = 0.11).
<figure><img src="image_9.png" /><figcaption>Fig. 3. Comparison of mitochondrial functions between sham, transient middle cerebral artery (tMCAO) and permanent MCAO (pMCAO) mice. Upper figures: right ischemic hemisphere. A. Calcium Retention Capacity (CRC), B. OXPHOS after stimulation of complex I, II or IV. Bottom figures: left non-ischemic hemisphere. C. CRC, D. OXPHOS after stimulation of complex I, II or IV. Data are expressed as Median [interquartile range 25%; 75%]; Kruskal-Wallis test. As expected, no statistical difference was found between groups in the non-ischemic hemisphere. In contrast, CRC and OXPHOS were decreased in the ischemic hemisphere in MCAO groups vs Sham, with no statistically significant difference between permanent and transient MCAO.</figcaption></figure>

[34%; 61 %] vs tMCAO+CsA: 46 [39%; 50 %]) (Fig. 4C). tMCAO+CsA mice displayed a significantly decreased infarct size (tMCAO+CsA: 51% [29%; 72 %]) vs tMCAO: 83% [45%; 104 %] % HLVc/AAR, p = 0.02) (Fig. 4D). For the same AAR, the lesion size in the tMCAO + CsA group lies below the regression line of the tMCAO mice, suggesting significantly smaller lesion size compared to tMCAO mice (Fig. 4E, p = 0.0494).

### CsA treatment at reperfusion improves mitochondrial CRC and OXPHOS

A significant increase of CRC in the tMCAO+CsA group vs tMCAO mice was observed for the ischemic mitochondria (tMCAO + CsA: 90 [70; 110] vs tMCAO: 70 [50; 90] nmol $Ca^{2+}$/mg, p = 0.0412) (Fig. 5A). Concerning OXPHOS, we measured in tMCAO+CsA ischemic mitochondria when compared to tMCAO group a significant improvement of respiration with complex I substrate (18.8 [15.2; 24.1] vs 13.4 [11.2; 16.2] nmol $O_2$/min/mg, p = 0.0079) (Fig. 5B). This was also true for complex IV substrate, albeit to a lesser extent (23.8 [22.2; 28.2] vs 23.0 [18.6; 23.5] nmol $O_2$/min/mg, p = 0.044) (Fig. 5B). There was no difference of either CRC or OCR in the left hemisphere mitochondria regardless of the group (Fig. 5C–D), except a trend toward an improved complex I-linked respiration by CsA (p = 0.0665, Fig. 5D). We finally investigated if the lesion size could correlate with the mPTP sensitivity. In the right ischemic hemisphere, there was a significantly

negative correlation between lesion size and CRC, i.e. CRC in ischemic mitochondria decreases as lesion size increases (Fig. 6B). On the other hand, no correlation was observed between CRC and lesion size in the left hemisphere (Fig. 6A).

### Discussion

In the present study, we investigated the impact of cerebral ischemia-reperfusion on the two main mitochondrial functions involved in both cell fate and bioenergetics (mPTP sensitivity to $Ca^{2+}$ overload and OXPHOS analysis) in a mouse model of thrombectomy. Mitochondrial dysfunctions have been well addressed in ischemia-reperfusion models in heart and kidney [27,28], but has been less explored in ischemic stroke, notably in mouse models. Our study describes mitochondrial alterations observed in a mouse model of focal cerebral ischemia. The characterization of the two models, pMCAO versus tMCAO with MT, using a novel imaging approach combining ultrasound and MRI methods, is also valuable for the stroke research community with the ambition of improving the quality of preclinical experiments. The study of mitochondrial functions in parallel sheds new light on the physiopathology of IRI damage.

Here, we observed that in comparison to Sham-operated mice, pMCAO mice had an increased mPTP sensitivity to $Ca^{2+}$ and an OXPHOS
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Effect of Cyclosporine (CsA) treatment in mice with transient middle cerebral artery (tMCAO). A. Neuroscores in tMCAO and tMCAO+CsA groups. Neurofunctional outcome was improved in CsA-treated mice although statistical significance was not reached. B. Representative infarct lesions in tMCAO and tMCAO + CsA groups (4 central slices on T2-weighted MRI). C. Quantification of the area at risk (AAR) in tMCAO and tMCAO + CsA groups. As expected, AAR were not statistically different between tMCAO and pMCAO. Mann-Whitney test, Median [interquartile range 25%; 75%]. D. Quantification of the infarct size calculated as the ratio of lesion volume over AAR (%HVLC/%AAR) in tMCAO and tMCAO + CsA groups. The primary endpoint was statistically lower in CsA-treated animals. Mann-Whitney test, Median [interquartile range 25%; 75%]. E. Lesion volume presented as percentage of the hemisphere (%HVLC) relative to the area at risk (%AAR). For a given AAR, infarct size was significantly lower for CsA-treated mice (p = 0.0494).</figcaption></figure>

dysfunction (after stimulation of either complex I, II or IV), as expected. Surprisingly, reperfusion did not significantly improve these markers of mitochondrial dysfunction. In line with these results, final infarct sizes of tMCAO mice did not statistically differ from that of pMCAO. In contrast, neuroscores were statistically improved in tMCAO vs pMCAO groups. Altogether, these results suggest that despite a neurofunctional benefit, reperfusion could at the same time promote mitochondrial injuries. This is concordant with previous studies that used different experimental protocols or mitochondrial endpoints [29–31]. We thus hypothesized that we could prevent post-reperfusion mitochondrial injuries and consequently improve stroke outcome.

Several therapeutic strategies at reperfusion, called post-conditioning, have been evaluated in ischemic stroke. Post-conditioning with interruptions of reperfusion in a rat model reduces infarct size [32]. Local post-conditioning directly in a brain artery is compromised in clinical practice because it is unsafe and operationally difficult. However, pharmacological post-conditioning is easier to use in clinical routine. CsA is known to inhibit mPTP opening [17,22]. A clinical study assessing CsA in stroke showed a significant decrease in cerebral infarct size in a subgroup of patients who received thrombolysis with successful recanalization, as this trial was performed before the onset of MT [16]. This promising study motivated us to focus on mitoprotection by CsA to elucidate the mechanism of action before going back from bench to bedside. In the present study, CsA improved mPTP sensitivity and OXPHOS, decreased infarct size and further improved neuroscores at day 1. Recently, CsA was shown to reduce cerebral inflammation in a non-human primate model of stroke with MT [33]. Altogether, these data suggest that preventing mitochondrial dysfunction is a relevant therapeutic target in cerebral ischemia-reperfusion and supports the launching of a new clinical trial assessing CsA protective effect in MT-treated ischemic stroke patients.

To address methodological flaws commonly identified in preclinical stroke studies, we implemented a robust methodology that included a priori sample size calculation, randomization, blinding and longitudinal imaging. Multimodal echography (Doppler and perfusion) was performed during the occlusion: (i) to select animal with effective MCAO and (ii) to account for individual variability in arterial collaterality by normalizing the final infarct size to the initial hypoperfused area, each animal being its own control. A previous study showed that MRI may be used to replace 2,3,5-triphenyltetrazolium chloride (TTC) post-mortem staining for assessing infarct size at day 1 [21]. By using MRI to assess final infarct size, we were able to study mitochondrial function in animals for which infarct size was controlled. This improves the results' robustness and has the advantage to reduce the number of animals. Interestingly, thanks to this approach, we were able to demonstrate a significant correlation between the PTP sensitivity in the ischemic hemisphere and final infarct size. We complemented our approach by a neurofunctional evaluation using neuroscores at day 1. This methodology is original and may become the gold standard to study cytoprotective drugs targeting mitochondria.

Nevertheless, our study presents some limitations. Surgery was performed on male young mice, without comorbidities. Since mitochondria were isolated from the whole hemisphere, we cannot address if they belong to grey matter or white matter, as these tissues have a different tolerance according to the severity of ischemia [34]. While we did not perform a dose-response study of CsA effect, we chose a validated dosage of CsA in post-conditioning as previously reported [27]. The duration of 60 min of ischemia and 24 h of reperfusion was chosen to study acute secondary ischemic injury only [35]. It would be important in a future work to evaluate whether CsA benefits are sustained on the long term. Finally, we cannot assert if CsA action was acting directly on the mPTP or via its known effect on calcineurin [36,37]. Some previous studies also mentioned that CypD could regulate OXPHOS activity notably by modifying respirasome assembly [38].
<figure><img src="image_14.png" /><figcaption>Fig. 5. Effect of CsA treatment on mitochondrial function in mice with transient middle cerebral artery (tMCAO). Upper figures: right ischemic hemisphere. A. Calcium Retention Capacity (CRC), B. OXPHOS after stimulation of complex I, II or IV. Bottom figures: left non-ischemic hemisphere. C. CRC, D. OXPHOS after stimulation of complex I, II or IV. Data are expressed as Median [interquartile range 25%; 75%]; Mann-Whitney test. As expected, no statistical difference was found between groups in the non-ischemic hemisphere. In contrast, CRC and OXPHOS were improved in the ischemic hemisphere of CsA-treated mice.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>Fig. 6. Correlation between CRC and %HVLc in the left non-ischemic hemisphere (A) and right ischemic hemisphere (B). There was no relationship between CRC and infarct size in the non-ischemic hemisphere. In contrast, CRC was negatively correlated to infarct size in the ischemic hemisphere.</figcaption></figure>

<figure><img src="image_16.png" /></figure>

In conclusion, mitochondrial dysfunctions represented by early mPTP opening and OXPHOS alterations were observed in pMCAO and tMCAO mice regardless of the reperfusion status. CsA improved mitochondrial functions when injected at reperfusion. Therefore, these mitochondrial dysfunctions are partially reversible using a cytoprotective therapeutic approach targeting mitochondria. As time windows for the treatment of AIS have widened, drug and mechanical reperfusion therapies could be combined with cerebral cytoprotective techniques in the future, a promising approach to prevent ischemic damage associated with ischemia-reperfusion injury.

Author Contributions

Conceptualization: MW, MP.

Data curation: LM, EO, PC, MW, MP.

Formal analysis: LM, EO, PC.

Funding acquisition: MO, NN, MW, MP.

Investigation: EO, PC, HG, NGB, CL, MLG, RB.

Methodology: MO, NN, MW, MP.

Project administration: MW, MP.

Resources: HG, NGB, CL, MLG, RB.
Software: LM.

Supervision: MW, MP.

Validation: MO, NN, MW, MP.

Visualization: LM, EO, PC.

Writing – original draft: EO, MW, MP.

Writing – review & editing: All authors.

## Funding

This project was funded by the OPERa IHU research program (ANR-10-IBHU-0004) to MO, the Leducq Transatlantic Network of Excellence "Targeting mitochondria to treat heart disease" (MitoCardia, 16CVD04) to MP and MO, a grant from the Agence Nationale de la Recherche (ANR-20-CE14-0013-01) to MP, and a grant from the Agence Nationale de la Recherche (ANR-18-CE19-0003) to MW.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Melanie Paillard reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We would like to thank the iXplora platform (Celphedia) and CERMEP imaging platform for their in vivo expertise.

## Data availability

The processed data required to reproduce these findings and perform the statistical analyses are available for download at the figshare repository: https://figshare.com/s/59226fa3098ae31726da.

## References

[1] Campbell BCV, De Silva DA, Macleod MR, Coutts SB, Schwamm LH, Davis SM, et al. Ischaemic stroke. Nat Rev Dis Prim 2019;5(1):70.

[2] Wafa HA, Wolfe CDA, Emmett E, Roth GA, Johnson CO, Wang Y. Burden of stroke in Europe: thirty-year projections of incidence, prevalence, deaths, and disability-adjusted life years. Stroke 2020;51(8):2418-27.

[3] Kidwell CS, Wintermark M. The role of CT and MRI in the emergency evaluation of persons with suspected stroke. Curr Neurol Neurosci Rep 2010;10(1):21-8.

[4] Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, et al. Guidelines for the early management of patients with acute ischemic stroke: 2019 update to the 2018 guidelines for the early management of acute ischemic stroke: a guideline for healthcare professionals from the American heart association/American stroke association. Stroke 2019;50(12):e344-418.

[5] Hussein HM, Saleem MA, Qureshi AI. Rates and predictors of futile recanalization in patients undergoing endovascular treatment in a multicenter clinical trial. Neuroradiology 2018;60(5):557-63.

[6] Rabinstein AA, Albers GW, Brinjikji W, Koch S. Factors that may contribute to poor outcome despite good reperfusion after acute endovascular stroke therapy. Int J Stroke 2019;14(1):23-31.

[7] Aronowski J, Strong R, Grotta JC. Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats. J Cerebr Blood Flow Metabol 1997;17(10):1048-56.

[8] Xu WW, Zhang YY, Su J, Liu AF, Wang K, Li C, et al. Ischemia reperfusion injury after gradual versus rapid flow restoration for middle cerebral artery occlusion rats. Sci Rep 2018;8(1):1638.

[9] Savitz SI, Baron JC, Fisher M, Consortium SX. Stroke treatment academic industry roundtable X: brain cytoprotection therapies in the reperfusion era. Stroke 2019; 50(4):1026-31.

[10] Lyden PD. Cerebroprotection for acute ischemic stroke: looking ahead. Stroke 2021; 52(9):3033-44.

[11] Savitz SI, Baron JC, Yenari MA, Sanossian N, Fisher M. Reconsidering neuroprotection in the reperfusion era. Stroke 2017;48(12):3413-9.

[12] An H, Zhou B, Ji X. Mitochondrial quality control in acute ischemic stroke. J Cerebr Blood Flow Metabol 2021;41(12):3157-70.

[13] Rasola A, Bernardi P. The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis 2007;12(5): 815-33.

[14] Li Y, Sun J, Wu R, Bai J, Hou Y, Zeng Y, et al. Mitochondrial MPTP: a novel target of ethnomedicine for stroke treatment by apoptosis inhibition. Front Pharmacol 2020;11: 352.

[15] Schinzel AC, Takeuchi O, Huang Z, Fisher JK, Zhou Z, Rubens J, et al. Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl Acad Sci USA 2005;102(34): 12005-10.

[16] Nighoghossian N, Berthezene Y, Mechtouff L, Derex L, Cho TH, Ritzenthaler T, et al. Cyclosporine in acute ischemic stroke. Neurology 2015;84(22):2216-23.

[17] Nighoghossian N, Ovize M, Mewton N, Ong E, Cho TH. Cyclosporine A, a potential therapy of ischemic reperfusion injury. A common history for heart and brain. Cerebrovasc Dis 2016;42(5-6):309-18.

[18] Ichas F, Jouaville LS, Mazat JP. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 1997;89(7): 1145-53.

[19] Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol 2020;18(7):e3000410.

[20] Lapchak PA, Zhang JH, Noble-Haeusslein LJ. RIGOR guidelines: escalating STAIR and STEPS for effective translational research. Transl Stroke Res 2013;4(3):279-85.

[21] Basalay MV, Wiart M, Chauveau F, Dumot C, Leon C, Amaz C, et al. Neuroprotection by remote ischemic conditioning in the setting of acute ischemic stroke: a preclinical two-centre study. Sci Rep 2020;10(1):16874.

[22] Cho TH, Aguettaz P, Campuzano O, Charriaut-Marlangue C, Riou A, Berthezene Y, et al. Pre- and post-treatment with cyclosporine A in a rat model of transient focal cerebral ischaemia with multimodal MRI screening. Int J Stroke 2013;8(8):669-74.

[23] Ong E, Costes M, Dumot C, Leon C, Paillard M, Canet-Soulas E, et al. Per Occlusion dynamic contrast-enhanced ultrasound may replace perfusion MRI for assessing the area at risk of infarction in a mouse model of acute ischemic stroke. 18th annual meeting of the European society for molecular imaging (ESMI) – European molecular imaging meeting (EMIM). Salzburg (Austria); 2023. 14-17 March 2023.

[24] Henaut L, Grissi M, Brazier F, Assem M, Poirot-Leclercq S, Lenglet G, et al. Cellular and molecular mechanisms associated with ischemic stroke severity in female mice with chronic kidney disease. Sci Rep 2019;9(1):6432.

[25] Gerrietts T, Stolz E, Walberer M, Muller C, Kluge A, Bachmann A, et al. Noninvasive quantification of brain edema and the space-occupying effect in rat stroke models using magnetic resonance imaging. Stroke 2004;35(2):566-71.

[26] Koch S, Mueller S, Foddis M, Bienert T, von Elverfeldt D, Knab F, et al. Atlas registration for edema-corrected MRI lesion volume in mouse stroke models. J Cerebr Blood Flow Metabol 2019;39(2):313-23.

[27] Gomez L, Thibault H, Gharib A, Dumont JM, Vuagniaux G, Scalfaro P, et al. Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am J Physiol Heart Circ Physiol 2007;293(3):H1654-61.

[28] Schleef M, Gonton F, Pillot B, Leon C, Chanon S, Vieille-Marchiset A, et al. Mild therapeutic hypothermia protects from acute and chronic renal ischemia-reperfusion injury in mice by mitigated mitochondrial dysfunction and modulation of local and systemic inflammation. Int J Mol Sci 2022;23(16).

[29] Liu W, Tian F, Kurata T, Morimoto N, Abe K. Dynamic changes of mitochondrial fusion and fission proteins after transient cerebral ischemia in mice. J Neurosci Res 2012;90(6):1183-9.

[30] Rutkai I, Merdzo I, Wunnava S, McNulty C, Chandra PK, Katakam PV, et al. Detrimental effects of transient cerebral ischemia on middle cerebral artery mitochondria in female rats. Am J Physiol Heart Circ Physiol 2022;323(6): H1343-51.

[31] Wu X, Zheng Y, Liu M, Li Y, Ma S, Tang W, et al. BNIP3L/NIX degradation leads to mitophagy deficiency in ischemic brains. Autophagy 2021;17(8):1934-46.

[32] Zhao H, Sapolsky RM, Steinberg GK. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cerebr Blood Flow Metabol 2006;26(9):1114-21.

[33] Becker G, Debatisse J, Riviere M, Crola Da Silva C, Beaudoin-Gobert M, Eker O, et al. Spatio-temporal characterization of brain inflammation in a non-human primate stroke model mimicking endovascular thrombectomy. Neurotherapeutics 2023;20(3):789-802.

[34] Ong E, Mewton N, Bouvier J, Chauveau F, Ritzenthaler T, Mechtouff L, et al. Effect of cyclosporine on lesion growth and infarct size within the white and gray matter. Front Neurol 2017;8:151.

[35] Neumann-Haeefelin T, Kastrup A, de Crespigny A, Yenari MA, Ringer T, Sun GH, et al. Serial MRI after transient focal cerebral ischemia in rats: dynamics of tissue injury, blood-brain barrier damage, and edema formation. Stroke 2000;31(8): 1965-72. discussion 72-3.

[36] Hefler J, Marfil-Garza BA, Campbell S, Freed DH, Shapiro AMJ. Preclinical systematic review & meta-analysis of cyclosporine for the treatment of myocardial ischemia-reperfusion injury. Ann Transl Med 2022;10(18):954.

[37] Azzi JR, Sayegh MH, Mallat SG. Calcineurin inhibitors: 40 years later, can't live without. J Immunol 2013;191(12):5785-91.

[38] Porter Jr GA, Beutner G, Cyclophilin D. Somehow a master regulator of mitochondrial function. Biomolecules 2018;8(4).