Ameliorative Role of Mitochondrial Therapy in Cognitive Function of Vascular Dementia Mice

Abstract

Background:

Mitochondrial dysfunction plays a vital role in the progression of vascular dementia (VaD). We hypothesized that transfer of exogenous mitochondria might be a beneficial strategy for VaD treatment.

Objective:

The study was aimed to investigate the role of mitochondrial therapy in cognitive function of VaD.

Methods:

The activity and integrity of isolated mitochondria were detected using MitoTracker and Janus Green B staining assays. After VaD mice were intravenously injected with exogenous mitochondria, Morris water maze and passive avoidance tests were used to detect cognitive function of VaD mice. Haematoxylin and eosin, Nissl, TUNEL, and Golgi staining assays were utilized to measure neuronal and synaptic injury in the hippocampus of VaD mice. Detection kits were performed to detect mitochondrial membrane potential (ΔΨ), SOD activity and the levels of ATP, ROS, and MDA in the brains of VaD mice.

Results:

The results showed that isolated mitochondria were intact and active. Mitochondrial therapy could ameliorate cognitive performance of VaD mice. Additionally, mitochondrial administration could attenuate hippocampal neuronal and synaptic injury, improve mitochondrial ΔΨ, ATP level and SOD activity, and reduce ROS and MDA levels in the brains of VaD mice.

Conclusions:

The study reports profitable effect of mitochondrial therapy against cognitive impairment of VaD, making mitochondrial treatment become a promising therapeutic strategy for VaD.

Keywords

Alzheimer’s disease cognitive function dementia mitochondrial therapy oxidative stress vascular dementia

INTRODUCTION

Vascular dementia (VaD), the most commonly diagnosed dementia in the elderly after Alzheimer’s disease (AD), is characterized by cognitive and memory impairments [1]. In North America and Europe, VaD accounts for approximately 15–20% of dementia cases [2, 3]. The estimated prevalence of VaD is about 30% in Asia and developing countries [3 –5]. However, no licensed therapies are available for VaD [1]. Therefore, developing novel therapy is urgently needed.

Mitochondria, the primary energy source of eukaryotic cells, play a crucial role in the maintenance of cellular homeostasis and function [6]. Apart from maintaining energy, mitochondria produce reactive oxygen species (ROS) as a byproduct of oxidative phosphorylation [6, 7]. Dysfunction of mitochondria causes oxidative stress due to excessive ROS generation, leading to cell injury [6]. Mitochondrial dysfunction has been reported to be implicated in neurological disorders, including VaD [8]. It has been demonstrated that increased oxidative stress and reduced mitochondrial respiration have been found in a VaD rat model, indicating mitochondrial bioenergetic deficits in the development of VaD [9, 10]. Restoring mitochondrial function either by acupuncture or by overexpression of PGC-1α in neurons reduced ROS production and improved the cognitive deficits in VaD models [11]. Collectively, these studies indicate that improving mitochondrial function may be beneficial to VaD treatment.

Mitochondrial therapy refers to the transfer of functional exogenous mitochondria into cells with mitochondrial defects so as to prevent the progress of diseases by recovering cellular activity [12, 13]. It has been documented that mitochondria administrated locally or intravenously can enter into brain cells and improve mitochondrial function in mitochondria-deficient neurological diseases. For example, Nitzan et al. found that intravenous administration of human isolated mitochondria could decrease hippocampal neuronal loss and ameliorate cognitive deficits by improving cerebral mitochondrial function in AD mice [14]. Additionally, mitochondria-targeted therapeutic strategies have also been used for PD. The authors found that mitochondrial transfer through medial forebrain bundle of PD rats could reduce dopaminergic neuron loss and improve locomotive activity of PD rats [15]. Furthermore, Wang, et al. found that mitochondrial transplantation via intravenous injection could inhibit neuroinflammation, increase BDNF expression and attenuate depression-like behaviors of mice induced by lipopolysaccharide via improving cerebral mitochondrial function [16].

Based on the above studies, we hypothesized that mitochondrial therapy might ameliorate cognitive impairments of VaD. In the present study, we aimed to investigate whether mitochondrial therapy contributes to the improvement of cognitive impairments via enhancing mitochondrial function in the brains of VaD mice. This study provides a novel insight for mitochondrial therapy in the treatment of VaD.

MATERIALS AND METHODS

Development of VaD mouse model

Male healthy Kunming strain mice (25–28 g), which were obtained from Xuzhou Medical University, were used to develop VaD mouse model according to previous studies with modifications [17, 18]. Briefly, mice were anesthetized and fixed on operation table. The bilateral common carotid arteries were separated from the vague nerve. Then the left common carotid artery was ligated with an artery clamp for 20 min and then released for 20 min. The same ligation regimen was performed on the right common carotid artery. The above procedure was repeated three times. The mice in the sham group underwent the same procedure except for the ligation of common carotid artery. After the procedure, mice were housed at room temperature with 12:12 h light-dark cycle and intraperitoneally administrated with carprofen to relieve pain and penicillin to prevent infection. Animal care and experimental procedures were approved by the animal welfare committee of Xuzhou Medical University (NO. 202110A023). After behavior tests, mice were euthanized with carbon dioxide and brain samples were prepared for staining assays, and the detection of mitochondrial membrane potential (ΔΨ), SOD activity and the levels of ATP, ROS, and MDA.

Mitochondrial isolation

Mitochondria were isolated from the brains of male healthy Kunming strain mice according to previous studies with modifications [12, 16]. Briefly, after mice were euthanized, the whole brain was dissected out, washed in cold PBS and cut into pieces. Then brain samples were homogenized in cold SE buffer (8.56 g sucrose, 0.36 g Tris, 0.37 g Na₂-EDTA and 0.055 g CaCl₂ in 100 mL ddH₂O, pH 7.6) and centrifuged at 3000 rmp for 5 min at 4°C. The supernatant was collected and recentrifuged at 3000 rmp for 5 min at 4°C followed by centrifugation at 12000 rmp for 15 min at 4°C. The precipitate was suspended in cold normal saline and recentrifuged at 12000 rmp for 15 min at 4°C. The mitochondrial precipitate was resuspended and washed with normal saline. The concentration of mitochondria was measured using Coomassie brilliant blue (CBB) staining assay according to the protocol of manufacture (Nanjing Jiancheng Bioengineering institute, Nanjing, China; catalog NO. A045-2).

Mitochondrial therapy

7 days after the procedure, VaD mice were divided into model and mitochondrial groups. Mice in the mitochondrial group were slowly injected with mitochondria (40μg per mouse) through tail veins once a day for consecutive 7 days. Mitochondria were injected immediately after isolation. Mice in the model and sham groups were injected the same volume of normal saline.

Morris water maze test

The ability of spatial learning and memory of mice was measured by Morris water maze (MWM) test according to a previous study [19]. Briefly, the mice were trained to locate the hidden platform for four days. On day 5, the ability of spatial memory was detected by recording the number of platform entry and time spent in the platform and target quadrant.

Passive avoidance test

The memory retention of mice was detected using passive avoidance test as previously described [19]. In brief, mice were allowed to enter the dark compartment where mice were given 2-s foot-shock (0.2 mA) on training day. After 24 h, step-through latency and memory errors were recorded.

Mitochondrial ΔΨdetection

Mitochondrial ΔΨ was measured using JC-1 assay kit according to the protocol of manufacture (Dalian Meilun Biotechnology Co. Ltd., Dalian, China; catalog NO. MA0338). Isolated mitochondria were mixed with JC-1 solution at room temperature. The fluorescence intensity of the mixture was detected at the wavelength of 490 nm excitation and 530 nm emission for JC-1 monomers, and the wavelength of 525 nm excitation and 590 nm emission for JC-1 aggregates, by a microplate reader (Varioskan lux, Thermo Fisher Scientific Inc., MA, USA). The ΔΨ of mitochondria was calculated by the ratio of fluorescence intensity of JC-1 aggregates and monomers.

Hematoxylin and eosin (H&E) and Nissl staining assays

The H&E and Nissl staining assays were performed using H&E and Nissl staining kits (Beyotime Biotech. Inc., Jiangsu, China; catalog NO. C0105S and C0117, respectively) in accordance with the manufacture’s protocols. Briefly, paraffin-embedded brain sections were deparaffinized and stained with hematoxylin and eosin and Nissl staining solution, respectively. The percentage of Nissl body in the hippocampal CA1 region of each section was determined after the images were photographed by a microscope (Olympus Corporation, Tokyo, Japan).

TUNEL staining assay

Neuronal apoptosis in the hippocampal CA1 region was measured using TUNEL staining assay according to the protocol of manufacture (Dalian Meilun Biotechnology Co. Ltd., Dalian, China; catalog NO. MA0224). Microtubule-associated protein 2 (MAP2) (ABclonal Technology Co.,Ltd., Wuhan, China; catalog NO. A22206), a neuronal marker, was detected for identification of neurons in the CA1 region of hippocampus. The apoptotic neurons were detected by a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The apoptotic rate of neuron was calculated by counting the number of apoptotic and total neurons.

MitoTracker Red CMXRos and Janus Green B staining assays

The activities of mitochondria were determined using MitoTracker Red CMXRos (Dalian Meilun Biotechnology Co. Ltd., Dalian, China; catalog NO. MB6046) and Janus Green B staining (Solarbio Life Science, Beijing, China; catalog NO. J8020) assays in accordance with the manufacture’s protocols. For MitoTracker Red CMXRos staining, mitochondria were stained with 500 nM MitoTracker at 37°C for 30 min. Then, mitochondria were collected, washed, and resuspended in PBS, and subsequently photographed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). For Janus Green B staining, mitochondria were stained with Janus Green B solution at room temperature for 1 min, and then were photographed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Golgi staining assay

Golgi staining assay was conducted using FD Rapid GolgiStaintrademark Kit (FD NeuroTechnologies, Inc, Columbia, USA; catalog NO. PK401A) according to the instruction of manufacture. Briefly, brains were excised, washed with cold PBS and immersed in solution A and B for 2 weeks followed by solution C for 3 days. Then, brains were sliced and stained with staining solution for 10 min at room temperature. Synaptic morphology in the hippocampus of mice was photographed by a microscope (Olympus Corporation, Tokyo, Japan). Branch length and dendritic spine of neurons in the hippocampus were analyzed using Image J software.

2,3,5-Triphenyltetrazolium chloride (TTC) staining assay

The infarction area was determined using TTC staining assay according to the protocol of manufacture (Solarbio Life Sciences, Beijing, China; catalog NO. T8170). Briefly, brains were excised and frozen at –20°C. 2mm-thick coronal sections were prepared and stained in TTC solution at 37°C for 30 min in the dark. The sections were then immersed in 4% paraformaldehyde. Then the images of brain sections were photographed by a microscope (Olympus Corporation, Tokyo, Japan) and the areas of cerebral infarction were calculated by Image-Pro Plus software.

ROS, ATP, and MDA detection

ROS, ATP, and MDA levels in the brain were detected according to the instructions of ROS, ATP, and MDA assay kits (Nanjing Jiancheng Bioengineering institute, Nanjing, China; catalog NO. E004-1-1, A095-1-1 and A003-1, respectively). For ROS detection, brain tissues were cut into pieces, digested by 0.25% trypsin at 37°C for 20 min and filtrated through 200 meshes net followed by centrifugation. The cell suspension was collected and washed with PBS. Then, 5-(and-6)-carboxy-2^′,7^′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to the suspension followed by the incubation at 37°C for 30 min. The fluorescence intensity of ROS was detected at 500 nm excitation wavelength and 525 nm emission wavelength using a microplate reader (Varioskan lux, Thermo Fisher Scientific Inc., MA, USA). For ATP and MDA detection, brain tissues were homogenized, centrifugated for supernatant collection and mixed with action buffers according to the protocol of manufactures, followed by the detection of OD values of ATP (636 nm) and MDA (532 nm) using a microplate reader (Varioskan lux, Thermo Fisher Scientific Inc., MA, USA).

SOD activity detection

The activity of SOD was measured according to the instructions of SOD activity detection kit (Nanjing Jiancheng Bioengineering institute, Nanjing, China; catalog NO. A001-3). In brief, brain tissues were cut into pieces, homogenized and centrifugated for collecting supernatants. Then, the supernatants were mixed with reaction mixture at 37°C for 20 min followed by the measurement of OD value at 450 nm using a microplate reader (Varioskan lux, Thermo Fisher Scientific Inc., MA, USA).

Statistical analysis

The data of each group were expressed as mean±SEM. One-way ANOVA or t test was utilized to analyze the variance using SPSS software 20.0. p < 0.05 was considered to be statistically significant.

RESULTS

Detection of exogenous mitochondrial activity and integrity

Mitochondria were isolated from the brains of healthy mice. The activity and integrity of mitochondria were detected using MitoTracker and Janus Green B staining assays. The results showed that the isolated mitochondria were stained with MitoTracker and exhibited high red fluorescence (Fig. 1A). Additionally, the mitochondria stained with Janus Green B showed green or dark green (Fig. 1B), indicating that isolated mitochondria are intact and active. The activity and integrity of isolated mitochondria were further determined by detecting mitochondrial ΔΨ using JC-1 staining assay, the results of which showed that isolated mitochondria exhibited normal ΔΨ, while mitochondrial ΔΨ was significantly reduced after mitochondria were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial ΔΨ disrupter (Fig. 1C), suggesting that isolated mitochondria have integrity and normal activity. Collectively, these results indicate that exogenous mitochondria are functional.

Fig. 1

Detection of exogenous mitochondrial activity and integrity. A, B) Isolated mitochondria were stained by Mitotracker red CMXRos and Janus Green B, respectively. C) ΔΨ of isolated mitochondria. ΔΨ of mitochondria was detected using JC-1 staining assay. CCCP was used as positive control. Data represent mean±SEM. n = 3 mice. Statistically significant differences were calculated by t test using SPSS 20.0 software. **p < 0.01.

Mitochondrial therapy ameliorated the cognitive deficits of VaD mice

MWM test

The effect of mitochondrial therapy on spatial learning and memory of VaD mice was investigated using MWM test. Number of platform entry as well as platform and quadrant time on day 5 were recorded. The results showed that, as compared to the sham mice, the number of platform entry of VaD mice was significantly reduced, and the platform and quadrant time of VaD mice were remarkably shortened (Fig. 2A-D), indicating the impairment of spatial learning and memory of VaD mice. Notably, mitochondrial therapy could increase the number of platform entry, and prolonged the platform and quadrant time of VaD mice (Fig. 2A-D), suggesting mitochondrial therapy improves the spatial learning and memory of VaD mice.

Fig. 2

Effect of mitochondrial therapy on cognitive function of VaD mice. After VaD mice were treated with mitochondria, MWM and passive avoidance tests were used to detect cognitive function of mice. A-D) MWM test. A) Track diagrams of MWM. Quadrant time (B), platform time (C), and platform entries (D) on day 5 were recorded. Data represent mean±SEM. n = 11–12 mice. E, F) Passive avoidance test. Retention latency (E) and memory errors (F) were recorded. Data represent mean±SEM. n = 11–12 mice. Statistically significant differences were calculated by one-way ANOVA using SPSS 20.0 software. *p < 0.05, **p < 0.01.

Passive avoidance test

The effect of mitochondrial therapy on retention memory was evaluated using passive avoidance test. As illustrated in Fig. 2E and F, VaD mice exhibited shorter retention latency and higher memory errors as compared to the sham mice, suggesting retention memory deficit of VaD mice. Importantly, mitochondrial therapy could prolong the retention latency and decrease the memory errors of VaD mice, implying that mitochondrial therapy ameliorates the retention memory impairment of VaD mice.

Mitochondrial therapy attenuated the cerebral infarction of VaD mice

VaD is often accompanied with cerebral infarction [20]. Thus, in the study, cerebral infarction was evaluated using TTC staining assay. As shown in Fig. 3A and B, no infarction area was observed in the sham mice. The infarction area in the VaD mice was significantly increased as compared to the sham mice. It should be noted that mitochondrial therapy could reduce the infarction area of VaD mice, indicating that mitochondrial therapy attenuates the cerebral infarction of VaD mice.

Fig. 3

Effect of mitochondrial therapy on cerebral infarction and neuronal injury in the hippocampus of VaD mice were evaluated by TTC, H&E, and Nissl staining assays, respectively. A, B) TTC staining. A) Representative images of TTC staining. B) Infarction area. Data represent mean±SEM. n = 3 mice. C-E) H&E and Nissl staining. C, D) Representative images of H&E and Nissl staining, respectively. E) Percentage of Nissl body. Data represent mean±SEM. n = 3 mice. Statistically significant differences were calculated by one-way ANOVA using SPSS 20.0 software. *p < 0.05, **p < 0.01.

Mitochondrial therapy alleviated the neuronal injury in the hippocampus of VaD mice

Because hippocampus is one of the most vulnerable regions in the brain and involved in learning and memory formation [21, 22], we then investigated whether mitochondrial therapy has protective effect on the neuronal injury in the hippocampal CA1 region of VaD mice. As shown in Fig. 3C, the hippocampal neurons in CA1 region of the sham mice exhibited organized arrangements with clear boundaries, whereas the hippocampal neurons of VaD mice were arranged irregularly and loosely. After mitochondrial therapy, the hippocampal neurons showed regular and dense arrangements. The result of Nissl staining assay showed that the percentage of Nissl body in the hippocampal CA1 region of VaD mice were significantly decreased as compared with the sham mice, while mitochondrial therapy attenuated the reduction in the percentage of Nissl body of VaD mice (Fig. 3D, E). Additionally, neuronal apoptosis in the hippocampal CA1 region was evaluated using TUNEL assay. The results showed that the apoptotic rate of VaD mice was remarkably higher than that of the sham mice, while mitochondrial therapy reduced the apoptotic rate of VaD mice (Fig. 4A, B). These results indicate that mitochondrial therapy alleviates neuronal injury in the hippocampus of VaD mice.

Fig. 4

Effect of mitochondrial therapy on neuronal apoptosis and injury of synaptic structure in the hippocampus of VaD mice were evaluated by TUNEL and Golgi staining assays. A, B) TUNEL staining. A) Representative images of TUNEL staining. B) Apoptotic rate of neurons in hippocampal CA1 region. Data represent mean±SEM. n = 3 mice. C-F) Golgi staining. C, E) Representative images of Golgi staining. D, F) Branch length and density of dendritic spine. Data represent mean±SEM. n = 3 mice. Statistically significant differences were calculated by one-way ANOVA using SPSS 20.0 software. *p < 0.05, **p < 0.01.

Mitochondrial therapy ameliorated the injury of synaptic structure in the hippocampus of VaD mice

It has been demonstrated that the cognitive deficits of VaD is associated with damaged synaptic structure [17]. Therefore, the hippocampal synaptic structure was measured using Golgi staining assay. The results showed that the branch length and density of dendritic spine in the hippocampus of VaD mice were significantly reduced as compared to the sham mice, and mitochondrial therapy restored the hippocampal synaptic structure of VaD mice (Fig. 4C-F), suggesting the ameliorative role of mitochondrial therapy in the injury of synaptic structure in the hippocampus of VaD mice.

Mitochondrial therapy improved mitochondrial ΔΨ and ATP level in the brains of VaD mice

Since mitochondrial impairments are implicated in the neuronal injury and cognitive dysfunction of VaD [9], we investigated whether mitochondrial therapy ameliorates mitochondrial impairments in the brains of VaD mice by detecting ΔΨ of mitochondria. As shown in Fig. 5A, a significant reduction in mitochondrial ΔΨ in the brains of VaD mice was observed as compared to the sham mice, and the reduction in mitochondrial ΔΨ in the brains of VaD mice was reversed by mitochondrial therapy. Mitochondria are the main source of energy and mitochondrial dysfunction causes a decrease of energy supply. We then detected the effect of mitochondrial therapy on the energy supply of mitochondria in the brains of VaD mice. The results showed that ATP level in the brains of VaD mice were remarkably reduced as compared to the sham mice (Fig. 5B). Notably, mitochondrial therapy significantly increased cerebral ATP level of VaD mice (Fig. 5B), implying that mitochondrial therapy recovers mitochondrial function in the energy supply of VaD mice.

Fig. 5

Effect of mitochondrial therapy on mitochondrial ΔΨ, the levels of ATP, ROS, and MDA, and SOD activity in the brains of VaD mice. (A) ΔΨ of mitochondria. Mitochondrial ΔΨ was detected using JC-1 staining assay. Data represent mean±SEM. n = 4–5 mice. B-E) ATP, ROS, and MDA levels, and SOD activity. Cerebral levels of ATP (B), ROS (C), MDA (D), and SOD activity (E) were detected using ATP, ROS, MDA, and SOD activity detection kits, respectively. Data represent mean±SEM. n = 3–5 mice. Statistically significant differences were calculated by one-way ANOVA using SPSS 20.0 software. *p < 0.05, **p < 0.01.

Mitochondrial therapy inhibited MDA level and ROS production and elevated SOD activity in the brains of VaD mice

Mitochondria are not only the main source of energy but also the primary producers of ROS [7]. Mitochondrial dysfunction in VaD animals may cause excessive ROS production, subsequently leading to neuronal injury [9, 10]. Therefore, the levels of cerebral ROS and MDA, a peroxidation product of lipid, were detected in the study. The results showed that the levels of ROS and MDA in the brains of VaD mice were significantly higher than that in the sham mice (Fig. 5C, D), whereas mitochondrial therapy suppressed cerebral ROS and MDA levels of VaD mice (Fig. 5C, D). Furthermore, the activity of antioxidant enzyme SOD in the brains of VaD mice has been detected after mitochondrial therapy. The results demonstrated that the activity of SOD was reduced in the brains of VaD mice as compared to the sham mice, while mitochondrial therapy could elevate cerebral SOD activity of VaD mice (Fig. 5E).

DISCUSSION

VaD is a very common dementia affecting millions of people each year. However, no effective treatments are available for the disease. Studies have proven that mitochondrial dysfunction is closely related to VaD [9 –11]. Therefore, exploring strategies targeting mitochondria may be profitable to VaD therapy. The current study demonstrated for the first time that therapy based on mitochondrial transfer alleviated neuronal and synaptic injury and ameliorated cognitive performance in VaD mice (Figs. 2–4). Furthermore, mitochondrial therapy improved mitochondrial ΔΨ, ATP level and SOD activity, and reduced ROS and MDA levels in the brains of VaD mice (Fig. 5). Hence, mitochondrial therapy may be a potential therapeutic strategy for VaD treatment.

Natural transfer of mitochondria between different cell types occurs under both physiological and pathological conditions. For example, the damaged mitochondria can be released from neurons and transferred to astrocytes for recycling and disposal [23]. Additionally, functional mitochondria in astrocytes can transfer into adjacent neurons to amplify cell survival signals in focal cerebral ischemia [24]. Therefore, transferring functional exogenous mitochondria could be used to correct mitochondrial deficits in mitochondria-deficient neurological diseases. Indeed, local or systemic injection of exogenous mitochondria could improve cerebral mitochondrial function, thereby considering as alternative strategies for the treatment of neurological diseases including neurodegenerative diseases, such as AD and PD, as well as cerebral ischemia and depression [14–16 , 25]. In the study, we show that transfer of intact mitochondria via intravenous injection produces therapeutic efficacy in VaD.

Although the data of distribution of exogenous mitochondria to the brain were lacked in the study, it has been proven that exogenous mitochondria can entrance into the brain. One study showed that exogenous mitochondria could distribute to the brain, liver, lung, kidney and muscle after intravenous injection for 2 h [26]. Similar results were observed in another study which showed that the signals of exogenous mitochondria labeled with Mitotracker Deep Red were detected in the brain sections after intravenous injection [27]. Additionally, Huang and colleagues found that 5-bromo-2’-deoxyuridine (BrdU) signals were detected in neurons, microglia and astrocytes after brain-ischemic rats were administrated with mitochondria pre-labeled with BrdU via intra-arterial injection, indicating the entrance of exogenous mitochondria into the brain [28]. These studies indicate the distribution of mitochondria in the brain. Therefore, the beneficial effect of mitochondria therapy in the study may be associated with the entrance of exogenous mitochondria into the brain. It has been demonstrated that intravenously injected mitochondria could accumulate in the liver [14], which may affect brain functions due to cross talk between the liver and brain [29 –31]. Thus, we cannot rule out the possibility that other organs, such as the liver may mediate the effect of mitochondrial therapy. Future studies should be designed to elucidate the mechanisms.

Mitochondrial dysfunction causes a reduction of ATP supply and an excessive ROS generation, which leads to the damages of cell components, resulting in neuronal injury and synaptic dysfunction, subsequently causing cognitive impairments of VaD [9, 10]. Thus, the supplement of exogenous mitochondria might recover neuronal function of VaD by increasing ATP supply and decreasing ROS production through restoring mitochondrial function. In the study, we found that mitochondrial therapy could elevate ATP level and reduce ROS and MDA levels in the brains of VaD mice (Fig. 5C, D), indicating that the ameliorative role of mitochondrial therapy in the neuronal and cognitive impairments of VaD may be associated with the increment of ATP supply and inhibition of oxidative stress by mitochondrial therapy. Mitochondrial enzymatic and non-enzymatic antioxidant systems, such as SOD and GSH, rapidly scavenge ROS [6, 7]. Several reports have shown that exogenous mitochondria reduced ROS production and elevated SOD and GSH expressions [12, 32]. In the current study, increased SOD activity by mitochondrial therapy was found (Fig. 5E), implying that reduced levels of ROS and MDA by mitochondrial therapy may be associated with the increment of SOD activity.

Some limitations should be interpreted. Firstly, cerebral mitochondrial dysfunction is proven to be associated with neuroinflammation [33, 34], which contributes to the pathology of VaD [35, 36]. Further research is needed to investigate whether mitochondrial therapy alleviates neuroinflammation in VaD mice. Secondly, although the safety of systematic delivery of mitochondria has been confirmed in a previous study [14], the safety of mitochondrial therapy in the study needs to be addressed in additional studies due to different dosing regimen. Thirdly, aberrant mitochondrial quality control, including mitochondrial dynamics, mitophagy, mitochondrial biogenesis, antioxidant defense, etc., could lead to mitochondrial destruction and play vital roles in neurological diseases [37, 38]. It would be interesting to investigate the role of mitochondrial therapy in mitochondrial quality control and its mechanisms in further studies. Fourthly, although the beneficial effect of mitochondrial therapy on VaD was observed in the study, the mechanisms are still elusive. Further studies in this respect should be designed.

Taken together, our study demonstrates that mitochondrial therapy holds the potential to ameliorate neuronal injury and cognitive deficits in VaD mice. The study reports profitable effect of mitochondrial therapy against cognitive impairments of VaD, making mitochondrial therapy become a promising therapeutic strategy for VaD.

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This project was supported by the National Natural Science Foundation of China under grants 82003718 and 82160681, the Natural Science Foundation of Jiangsu Province under grant BK20231179.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

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