Platelets: Emerging as a Cutting-Edge Source for Mitochondrial Transplantation in Rodent Models of Traumatic Brain Injury

Platelets: Emerging as a Cutting-Edge Source for Mitochondrial Transplantation in Rodent Models of Traumatic Brain Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Platelets: Emerging as a Cutting-Edge Source for Mitochondrial Transplantation in Rodent Models of Traumatic Brain Injury Chia Bamshad, Zahra Pourmohammadi-Bejarpasi, Fatemeh Amiri, Nima Najafi-Ghalehlou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6356471/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Traumatic brain injury (TBI) remains a significant clinical challenge due to the absence of effective therapies for mitigating oxidative damage and neuronal dysfunction. Given the brain’s high metabolic demand, mitochondrial transplantation has emerged as a promising therapeutic strategy. However, identifying an optimal, functional source of mitochondria remains a major obstacle. In this study, we explore the therapeutic potential of mitochondria isolated from senescent platelets as a readily available, GMP-compliant source for promoting recovery in TBI models. TBI was induced in rats using a weight-drop model, with confirmation through histopathological analysis. The animals were then divided into four experimental groups: healthy control, TBI, TBI + RB (receiving respiration buffer), and TBI + Mito (receiving mitochondrial transplantation). Neurobehavioural recovery was assessed using a series of sensorimotor tests, including the beam walk test, horizontal bars test, grid-walking test, and cylinder test. Our results show that mitochondrial transplantation significantly improved neurobehavioural function in TBI animals, with performance in the TBI + Mito group comparable to that of the healthy control group. Histological examination further revealed that brain tissue morphology in the TBI + Mito group closely resembled that of the control group. These findings provide compelling evidence for the therapeutic potential of platelet-derived mitochondrial transplantation as an accessible and effective treatment for TBI. However, further investigation is needed to determine the long-term efficacy and underlying mechanisms of this novel approach. Traumatic brain injury Mitochondria Mitochondrial transplantation Platelets Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION TBI affects as many as 69 million individuals worldwide and is reputed to be a concerning challenge in medicine (Dewan et al. 2019 ), which pursuant to the World Health Organisation includes half of all trauma-wise mortalities (Iaccarino et al. 2018 ). TBI is classified into three types mild, moderate and severe contingent on the Glasgow Coma Scale (GCS), with the first one being characterised by headache, nausea, vomiting, fatigue or lethargy, depression, and anxiety (Marshall et al. 2012 ). Moderate TBI manifests more acutely in blurred vision, confusion, temporary loss of consciousness, and focal neurologic problems (Malec et al. 2007 ), while the severe type is associated with delusion, hallucination, euphoria, aberrant motor behaviour, and death (Ciurli et al. 2011 ). In addition, TBI could be pathologically categorised into primary and secondary brain injuries, with the former disrupting the brain tissue integrity within the initial hours of injury. The subsequent death of local brain cells and the leakage of pernicious chemical content and apoptotic factors leads to secondary injury in the adjacent non-traumatic area (Greve and Zink 2009 ). Reactive oxygen species (ROS) are among the hazardous molecules released after injury and the imbalance between ROS generation and mitochondria-mediated ROS scavenging leads to oxidative stress, damage to the cell membrane of neighbouring cells. and the expansion of the injury site (Abdul-Muneer et al. 2015 ). Meticulously, ROS clearance is primarily mediated by mitochondrial permeability transition pores (mPTPs), which are located in the inner mitochondria membrane and are responsible for transporting ions and chemicals in response to Ca 2+ accumulation in the mitochondrial matrix, as well as phosphate levels elevation, adenine nucleotide depletion, and, most importantly, oxidative stress. Therefore, excessive ROS-triggered mPTPs opening leads to ROS-induced ROS release (RIRR) into the cytosol and apoptotic cell death (Bopassa et al. 2005 ; Cheng et al. 2012 ; Zorov et al. 2014 ). As the current surgical procedures and rehabilitation methods for treating TBI patients fail to effectively demote secondary brain injury or promote damaged tissue recovery (Chang et al. 2016 ; Jabbari et al. 2020 ; Pourmohammadi-Bejarpasi et al. 2020 ), one practical solution to counteract the lethal effects of ROS surplus and restore its physiological balance in the injured tissue involves augmenting healthy and functional mitochondria through mitochondrial transplantation (Emani et al. 2017 ). This approach has demonstrated therapeutic potential in various conditions, including acute kidney injury (Jabbari et al. 2020 ), Parkinson’s disease (Chang et al. 2016 ), brain ischaemia (Pourmohammadi-Bejarpasi et al. 2020 ), spinal cord injury, peripheral neuropathy (Gollihue et al. 2017 ; Kuo et al. 2017 ), and human breast cancer by fostering chemosensitivity (Elliott et al. 2012 ). In this regard, we conducted the present study to evaluate the therapeutic efficacy of platelet-derived mitochondrial transplantation in rat models of TBI. Platelets, traditionally known for their role in haemostasis and clotting, were selected for isolating healthy and functional mitochondria because they serve as reservoirs of bioactive molecules, including growth factors that induce tissue repair and regeneration (Locatelli et al. 2021 ). In addition, an advantage of platelets in therapeutic applications relates to their short lifespan, such that platelet concentrates are stored for up to 5–7 days prior to transfusion (Aubron et al. 2018 ). Therefore, expired or unused platelets are considered a readily available biowaste that can be repurposed for mitochondrial isolation and transplantation (Najafi-Ghalehlou et al. 2022 ). In this study, we evaluated the potential of platelet-derived mitochondria for TBI treatment, introducing a cost-effective solution for advancing regenerative medicine as well as emphasising their feasibility as a GMP-compliant source for mitochondrial isolation that would otherwise be discarded. STUDY DESIGN AND METHODS Animal Ethics All animal studies were undertaken after being reviewed and approved by Iran National Committee for Ethics in Biomedical Research responsible for ethical clearance (Approval Code: IR.NIMAD.REC.1400.003). Mitochondria Isolation from Platelets Figure 1 illustrates a succinct overview of the study, beginning with harvesting mitochondria from the platelets provided by Guilan Blood Transfusion Department conforming to Pourmohammadi-Bejarpasi et al . (Pourmohammadi-Bejarpasi et al. 2020). Briefly, each platelet storage bag with a volume of 50-70 mL and a pH of 6.2 contained at least 6×10 10 platelets. The samples were centrifuged at 3,731 g for 4 minutes and the plasma was discarded, followed by resuspending the remaining pellet in phosphate-buffered saline (PBS) and re-centrifuging to remove the remaining plasma. Afterwards, the platelets were resuspended in 2 mL of homogenisation buffer (HB; 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 2 mM EGTA, 0.1% BSA, and 250 mM sucrose) harbouring 1% of protease inhibitor (Sigma-Aldrich, USA). The cell suspension was then incubated on ice for 10 minutes and for 35-40 times on a cell homogeniser. Next, it was centrifuged at 2,000 g and 4℃ for 10 minutes, with the supernatant carefully transferred to another microtube and centrifuged at 10,000 g and 4℃ for another 10 minutes. After discarding the supernatant, the mitochondria pellet was resuspended in 10 µL of respiration buffer (RB, 0.5 mM EGTA, 2 mM KH 2 PO 4 , 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , and 250 mM sucrose), incubated on ice, and sent to operating theatre for transplantation. Moreover, mitochondrial quality control and mitochondrial membrane potential (ΔΨm) were evaluated by JC-1 staining (Cayman Chemical, USA) as instructed by the manufacturer, observing the red and green fluorescence under the fluorescent microscope (Nikon, Japan), and merging the corresponding images. Animal Models of TBI TBI establishment in male Wistar rats weighing 300 g was achieved as described in Feeney’s study (Feeney et al. 1981) and the animals were randomly allocated to four groups, including the control group, TBI group, TBI+RB group (TBI models receiving intracerebroventricular (ICV) injection of RB), and TBI+Mito group (TBI models receiving ICV injection of healthy mitochondria). All experimental units were housed under a 12-hour light/dark cycle at 20-22℃ and 52% humidity with food and water supplied ad libitum . Afterwards, they were fully anaesthetised by intraperitoneal injection of ketamine (100 mg/Kg), xylazine (10 mg/Kg), and acepromazine (2.5 mg/kg). the animals were then mounting them on a stereotaxic device by a head holder on the skull. A 2-cm-wide midline scalp incision was made to expose the skull, followed by a 5-mm-wide craniectomy, 1 mm lateral to the sagittal suture between the bregma and lambda, to assess the brain surface without damaging the dura. In accordance with Feeney et al. (Feeney et al. 1981), TBI induction was carried out by dropping an 80-gram weight through a guide tube, intuitively ascertained by observing brain tissue inflammation and paleness in the exposed region. The injury site was then covered with cold-cure acrylic (Acropars, Iran) and the animals were returned to the animal room for recovery. Sensorimotor and histopathological studies were subsequently performed to further confirm TBI induction. Mitochondrial Transplantation Mitochondria were isolated from 3×10 10 platelets and transplanted into the brain tissue of TBI rats. ICV injection was performed using a 10 µL Hamilton syringe (Hamilton, Cat. No.8929A70, USA) at stereotaxic coordinates of −0.8 mm posterior, −1.5 mm lateral, and −4.0 mm ventral from Bregma (Zhang et al. 2019). The respiratory rate, heart rate, and body temperature were constantly monitored during the surgery and post-surgery recovery in the animal room. Blood Serum Analysis Prior to animal sacrifice, blood samples were obtained from the animals for measuring serum creatine phosphokinase (CPK) levels. Histopathological Studies Subsequent to anaesthetising the animals as mentioned earlier, animal perfusion fixation was carried out by flushing 4% paraformaldehyde solution through the circulatory system. The brain tissues were then fixed in 10% formaldehyde and 5-µm-thick paraffin-embedded sections were acquired for haematoxylin and eosin (H&E; Merck, Germany) staining. In situ Cell Death Detection The TUNEL assay in situ apoptosis detection kit (Takara, Japan) was used to estimate the number of apoptotic cells in tissue samples pursuant to the manufacturer’s instruction. Deparaffinisation was performed by incubating paraffin-fixed tissue sections in xylene for 30 minutes at 60℃, followed by rehydration with descending concentrations of ethanol and antigen retrieval using proteinase K (Thermo Fisher Scientific, USA). Afterwards, the tissue sections were incubated with 50 µL of TUNEL labelling mixture for 70 minutes at 37℃, washed with PBS, counterstained with DAPI, and observed under a fluorescent microscope (Nikon, Japan). Immunohistochemistry First, 3-µm-thick brain sections were deparaffinised and soaked in 1X tris-buffered saline (TBS) solution (Sigma-Aldrich, T5912). Subsequently, microwave tissue processing was performed until the sections reached the boiling point and were allowed to cool for 20 minutes. In the next step, the sections were washed three times with PBS for 5 minutes each and were incubated in H 2 O 2 mixed with methanol in a 1:9 ratio for 10 minutes. After another PBS wash, the sections were incubated with anti-glial fibrillary acidic protein (-GFAP), -ionised calcium-binding adapter molecule 1 (-Iba1), or -Nestin antibodies for 1 hour at room temperature, followed by three PBS washes for 5 minutes each. Next, the samples were incubated in 100 µL linker (Diagnostic BioSystems-PVP1000D) for 15 minutes, washed three times with PBS, and incubated in 100 µL polymer solution (Diagnostic BioSystems-PVP1000D) for half an hour. Following another, PBS wash, the sections were incubated in 100 µL DAB solution (ScyTek-ACV999) for 5 minutes. In the final step, the sections were rinsed with water, counterstained with haematoxylin for 10 seconds, and examined under a microscope (Labomed, US) to observe GFAP + , Iba1 + , or Nestin + cells. Animal Sensorimotor Skills Assessment Sensorimotor skills of the animals were minutely assessed by a series of four tests. (a) in the beam walk test animals were required to travers a 1×2.5×4 cm 3 wooden beam placed 1 metre above the bench surface and forelimb and hind limb placement errors were recorded to assess balance (Hausser et al. 2018). (b) the horizontal bars test measured the motor coordination and strength of forelimbs. Animals were placed at the centre of a 60-cm-long and 6-mm-wide rod placed in the notches of two support columns. Performance was scored based on their ability to grasp the bar for a minimum of 30 seconds or reaching either of the support columns (Deacon 2013). (c) the grid-walking test assessed tactile sensitivity and spatial awareness by allowing the animals to navigate freely for 5 minutes over a 46×92 cm 2 wire grid comprising 25×25 mm 2 cells 30 cm above the bench surface. The number of foot slips was recorded for scoring as a measure of motor impairment (Russell et al. 2011). (d) the cylinder test, otherwise the spontaneous forelimb elevation test, related to placing the animals inside a glass cylinder (21 cm in diameter and 34 cm in height) and allowing them to rear freely for 5 minutes. The total number of whole-palm contacts with the surrounding wall was recorded, especially for the left forelimb, since the experimental units were undergone TBI induction in the cortex of the right hemisphere of the brain (Cenci and Lundblad 2005). Statistical Analysis Statistical analysis was conducted by GraphPad Prism software version 8 (La Jolla, CA, USA), with One-way ANOVA used for comparing groups and p-values of 0.05 at the maximum were deemed statistically significant. RESULTS Exogenous Mitochondria Quality Control Figure 2.a-c and Figure 2.d-f illustrate ΔΨm evaluation by JC-1 staining of the platelets and the isolated mitochondria, respectively, such that the dominance of J-aggregates (red fluorescence) over the J-monomers (green fluorescence) in both cases marks a strong ΔΨm and thus affirms the high quality of the mitochondria for transplantation. Neuroprotection against Secondary Brain Injury Figure 3I and Figure 3Ib show low magnification images of brain tissue from the TBI and control groups. In the control group, no significant alterations in brain tissue were observed, with clear visibility of cellular nuclei and cytoplasm (Figure 3I.c). In contrast, the TBI group displayed evidence of secondary brain injury and tissue destruction at the injury site, characterized by paler surrounding tissue (Figure 3I.d), loss of tissue integrity, and vacuolation (Figure 3I.e-f), as well as bleeding and pyknosis (Figure 3I.g-h). A similar pattern was observed in the TBI+RB group. However, in the TBI+Mito group, mitochondrial transplantation was associated with a significant reduction in brain tissue damage and preservation of neuronal cell structure (Figure 3I.i-j). Figure 3II presents the plasma levels of the brain trauma biomarker creatine phosphokinase (CPK) across the four experimental groups. The control group exhibited the lowest CPK levels, while the TBI group had significantly higher CPK levels compared to controls (p < 0.0001). Additionally, CPK levels were significantly higher in the TBI+RB group compared to the TBI+Mito group (p < 0.001). Notably, no significant difference in CPK levels was found between the control and TBI+Mito groups, suggesting that mitochondrial transplantation restored normal plasma CPK levels and effectively mitigated secondary brain injury. Brain Cells Apoptosis Reduction The apoptosis rate in brain tissue of the animals was evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay four days post-surgery, disclosing a statistically significant difference between the TBI+Mito and the TBI groups (Figure 4; p < 0.0001) and no statistically significant difference between the control and the TBI+Mito groups. Additionally, the TBI and TBI+RB groups displayed similar apoptosis rates, with no significant difference between them. These findings suggest a robust association between mitochondrial transplantation and post-TBI apoptosis progress obstruction. Astrogliosis Reduction The evaluation of astrogliosis-specific biomarker GFAP across the four groups revealed that unlike the TBI and TBI+RB groups (Figure 5.b-c), which exhibited elevated GFAP expression, the TBI+Mito group possessed a notably lower GFAP level (Figure 5.d; p < 0.0001) compared to TBI and TBI+R groups. In other words, astrogliosis had remarkably decline in the TBI+Mito group and was closely similar to that of the control group, which suggests that mitochondrial transplantation effectively mitigates astrogliosis. Microglial Activation Decrease The expression of microglial activation biomarker Iba1 was significantly high in the TBI group compared to TBI+Mito group (p < 0.001; Figure 6). In addition, a similar trend was observed in the TBI+RB group (P<0.01); however, no significant difference was found between the TBI and TBI+RB groups (Figure 6.b-c). In contrast, the TBI+Mito group exhibited a dramatic reduction in Iba1 level (Figure 6.d), closely analogous to that of the control group (Figure 6.a). Therefore, Iba1 levels in the TBI+Mito group were significantly different from those in the TBI and TBI+RB groups (p < 0.001 and p < 0.01, respectively), indicating that mitochondrial transplantation effectively attenuates microglial activation following TBI. Nestin Upregulation Nestin designated as being the neurogenesis biomarker present in neuroepithelial stem cells becomes elevated post-TBI brain injuries (Sahin Kaya et al. 1999). In this study, no significant difference in Nestin levels was observed between the TBI and TBI+RB groups (Figure 7.b-c). However, Nestin levels in the TBI+Mito were significantly higher than the TBI and TBI+RB groups (p <0.0001 and P<0.001 respectively) highlighting the role of mitochondrial transplantation in promoting neurogenesis. Additionally, Nestin expression was significantly upregulated in the TBI+Mito group compared to the control group (p < 0.0001). Sensorimotor Skills Revival The results of the sensorimotor skills tests following mitochondrial transplantation corroborate a significant restoration of motor function in the experimental units receiving mitochondria. The recovery levels in the TBI+Mito group was comparable to the control group, highlighting the therapeutic potential of mitochondrial transplantation. In detail, the beam walk test results depicted in Figure 8.a exhibit a significantly faster recovery in the TBI+Mito group compared to the TBI+RB and TBI groups, with a considerable difference on day 7 (p < 0.001), insofar as the TBI+Mito group was not statistically different than the control group on days 14 and 21, indicating effective mitigation of secondary brain injury. As for the horizontal bars test results illustrated in Figure 8.b, mitochondrial transplantation was associated with motor coordination and forelimb strength restoration with no significant difference was recorded between the TBI+Mito group and the control group by the end of week 1. In contrast, the control group remained significantly different from the TBI and TBI+RB groups (p <0.01 and 0P<0.001 respectively). The grid-walking test results, based on the frequency of foot slips, proved to be highly sensitive in detecting sensorimotor deficits in TBI models compared to the other tests. In line with the previous tests, the difference between the control and the TBI+Mito group began to become statistically indistinguishable by day 7 (Figure 8.c). Conversely, both the TBI and the TBI+RB groups showed negligible rate of sensorimotor skills recovery, with a statistically conspicuous difference compared to the control group (p < 0.001). Consistently, the cylinder test revealed that the TBI+Mito group scores greatly resembled that of the control group and thus were not statistically different from week 2 onward (Figure 8.d). In contrast, both the TBI and TBI+RB groups continued to show significant impairments compared to the control group (p < 0.001 on day 7, p < 0.001 on day 14, and p < 0.01 and p < 0.05, respectively, on day 21). DISCUSSION Mitochondria are credited with playing a crucial role in cellular physiology and survival, especially in highly dynamic neurons, by constantly regulating adenosine triphosphate (ATP) generation, proliferation, homeostasis, ROS-mediated apoptosis, and autophagy (Reddy 2007 ; de Castro et al. 2010 ; Norat et al. 2020 ). Consequently, mitochondrial transplantation has struck as an advanced and promising therapeutic approach for treating various disorders associated with mitochondrial dysfunction and disturbance in ATP production, such as myocardial ischaemia, acute kidney injury (AKI), and acute ischaemic stroke (AIS) (McCully et al. 2009 ; Hayakawa et al. 2016 ; Tang 2019 ; Zhang et al. 2019 ; Huang et al. 2020 ; Youn et al. 2020 ; Zhang et al. 2020 ). This study builds on our previous endeavours to investigate the efficacy of transplanting mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in treating rat models of traumatic brain injury (TBI) (Bamshad et al. 2023 ; Baharvand et al. 2024 ), which demonstrated consistent results in terms of accelerated neurogenesis, rescued sensorimotor skills, reduced apoptosis rate, and decreased microglial activation (Bamshad et al. 2023 ). Similar results were noted in ischaemic heart disease (Baharvand et al. 2024 ), AIS (Pourmohammadi-Bejarpasi et al. 2020 ), and AKI (Jabbari et al. 2020 ), along with prostate cancer (PC-3) cell line (Nikoo et al. 2023 ) that was observed to be interconnected with promoted proliferation and demoted cisplatin sensitivity, but not erastin sensitivity, in PC-3 cells. However, the therapeutic applications of hUC-MSCs-derived mitochondria are largely hindered by the relatively long population doubling time, low yield, survival in culture, isolation complexity, and high cost. In order to address these challenges, we explored platelet-derived healthy and exogenous mitochondria as an alternative source, highlighting their feasibility and therapeutic potential in animal models of TBI. Our findings indicate that transplanting the mitochondria derived from expired platelets is meaningfully linked with mitigating and ameliorating secondary brain injury by reducing apoptosis and astrogliosis, suppressing microglial activation, restoring homeostasis, and fostering neurogenesis (Singh et al. 2006 ; Fischer et al. 2016 ; Kheirandish-Rostami et al. 2020 ), reflected in motor functions recovery. In line with our study, Zhao et al . reported that transplanting exogenous mitochondria derived from allogeneic liver and/or autogenic muscle reduced neuronal apoptosis, improved motor functions, alleviated anxiety, and upregulated astrocytic brain-derived neurotrophic factor (BDNF) (Zhao et al. 2021 ). Consistently, Zhang et al. investigating the therapeutic effects of exogenous brain-derived mitochondria in mouse models of TBI concluded that administering exogenous mitochondria in mice enhanced mitochondrial respiratory control ratio, upregulated synaptic plasticity-related tight junction (TJ) protein expression, demoted apoptosis, promoted angiogenesis, and alleviated brain oedema and blood-brain barrier (BBB) leakage (Zhang et al. 2020 ). The neurovascular unit (NVU), comprising neuronal cells, astrocytes, BBB endothelial cells, pericytes, myocytes, and extracellular matrix (ECM) components, plays a pivotal role in central nervous system (CNS) disorders. Astrocytes, in particular, have been found to contribute to neuroprotection and repair by releasing and transferring mitochondria into damaged neurons via the calcium signalling enzyme CD38 (38, 46). Thus, NVU disruption is a fundamental factor in CNS pathologies, highlighting the importance of mitochondrial homeostasis in brain injury recovery (Hayakawa et al. 2016 ; Nakamura et al. 2020 ). One of the major drawbacks of mitochondrial transplantation pertains to identifying an optimal source. Platelets present a potentially desirable and ideal alternative for harvesting healthy mitochondria in basic and translational research due to their accessibility, high mitochondrial content, non-invasive isolation, compliance with GMP standards, high biocompatibility, and low pathogenicity. Despite the short shelf life of platelets (5 days), storage requirement and efficacy control at 20–24℃ prior to transfusion (Humbrecht et al. 2018 ), our study demonstrated that mitochondria harvested from old platelets remained viable and effective. Furthermore, the route of mitochondrial administration requires further investigation. Our results support intracerebroventricular (ICV) administration of mitochondria as a suitable and effective method for targeting the brain cells. In this manner, Huang et al . reported that both the intracerebral and intra-arterial administration of mitochondria, isolated from baby hamster kidney (BHK-21) cells, to a rat model of middle cerebral artery occlusion (MCAO) was interrelated with improved motor functions, decreased apoptosis rate, and shrunk infarct area (Hayakawa et al. 2016 ). Hence, future studies are encouraged to focus on optimising delivery routes for different pathological conditions. CONCLUSIONS In conclusion, our findings suggest that transplanting mitochondria harvested from aged platelets into rat models of TBI holds significant therapeutic potential, not only by slowing the progression from primary to secondary brain injury but also by aiding the recovery of sensorimotor function. While mitochondrial transplantation shows promise as a reliable treatment, several challenges remain, as with any therapeutic approach. These include determining the optimal route of administration for different pathological conditions, establishing an effective dosing regimen, addressing immune system overreaction, and mitigating potential side effects. Future research is needed to overcome these hurdles and further enhance the efficacy of this treatment. Declarations ACKNOWLEDGMENTS This work was supported by the National Institute for Medical Research and Development (NIMAD) under grant number 996448. The authors would like to dedicate this paper to the cherished memory of Chia Bamshad, who tragically passed away during the submission process. His passion for science, unwavering dedication, and invaluable contributions were the driving forces behind this work. While his absence leaves a profound void, his legacy will continue to inspire us. We honor his memory and the lasting impact he has made on this field. CONFLICT OF INTEREST The authors report there are no competing interests to declare. Author Contributions Statement CB, NNG and MHR : Wrote the main manuscript text. CB , ZPB and NNG prepared figures, FA, ZPB, KT, YK, NNG, TS and MHR ; Writing – review & editing, MHR : supervision and Funding acquisition. All authors reviewed the manuscript DATA AVAILABILITY All data generated or analysed during this study are included in this published article References Abdul-Muneer PM, Chandra N, Haorah J (2015) Interactions of oxidative stress and neurovascular inflammation in the pathogenesis of traumatic brain injury. Mol Neurobiol 51:966-979 doi: 10.1007/s12035-014-8752-3 Aubron C, Flint AWJ, Ozier Y, McQuilten Z (2018) Platelet storage duration and its clinical and transfusion outcomes: a systematic review. Crit Care 22:185 doi: 10.1186/s13054-018-2114-x Baharvand F, Habibi Roudkenar M, Pourmohammadi-Bejarpasi Z, et al. 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Journal of Neurotrauma 24:1417-1424 doi: 10.1089/neu.2006.0245 Marshall S, Bayley M, McCullagh S, Velikonja D, Berrigan L (2012) Clinical practice guidelines for mild traumatic brain injury and persistent symptoms. Canadian Family Physician 58:257 McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H, Levitsky S (2009) Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Physiol Heart Circ Physiol 296:H94-h105 doi: 10.1152/ajpheart.00567.2008 Najafi-Ghalehlou N, Feizkhah A, Mobayen M, Pourmohammadi-Bejarpasi Z, Shekarchi S, Roushandeh AM, Roudkenar MH (2022) Plumping up a Cushion of Human Biowaste in Regenerative Medicine: Novel Insights into a State-of-the-Art Reserve Arsenal. Stem Cell Rev Rep 18:2709-2739 doi: 10.1007/s12015-022-10383-3 Nakamura Y, Park J-H, Hayakawa K (2020) Therapeutic use of extracellular mitochondria in CNS injury and disease. Experimental Neurology 324:113114 doi: https://doi.org/10.1016/j.expneurol.2019.113114 Nikoo A, Roudkenar MH, Sato T, et al. (2023) Mitochondrial transfer in PC-3 cells fingerprinted in ferroptosis sensitivity: a brand new approach targeting cancer metabolism. Human Cell doi: 10.1007/s13577-023-00896-5 Norat P, Soldozy S, Sokolowski JD, et al. (2020) Mitochondrial dysfunction in neurological disorders: Exploring mitochondrial transplantation. NPJ Regen Med 5:22 doi: 10.1038/s41536-020-00107-x Pourmohammadi-Bejarpasi Z, Roushandeh AM, Saberi A, et al. (2020) Mesenchymal stem cells-derived mitochondria transplantation mitigates I/R-induced injury, abolishes I/R-induced apoptosis, and restores motor function in acute ischemia stroke rat model. Brain Research Bulletin 165:70-80 Reddy PH (2007) Mitochondrial dysfunction in aging and Alzheimer's disease: strategies to protect neurons. Antioxid Redox Signal 9:1647-1658 doi: 10.1089/ars.2007.1754 Russell KL, Kutchko KM, Fowler SC, Berman NEJ, Levant B (2011) Sensorimotor behavioral tests for use in a juvenile rat model of traumatic brain injury: assessment of sex differences. Journal of neuroscience methods 199:214-222 Sahin Kaya S, Mahmood A, Li Y, Yavuz E, Chopp M (1999) Expression of nestin after traumatic brain injury in rat brain. Brain Res 840:153-157 doi: 10.1016/s0006-8993(99)01757-6 Singh IN, Sullivan PG, Deng Y, Mbye LH, Hall ED (2006) Time course of post-traumatic mitochondrial oxidative damage and dysfunction in a mouse model of focal traumatic brain injury: implications for neuroprotective therapy. J Cereb Blood Flow Metab 26:1407-1418 doi: 10.1038/sj.jcbfm.9600297 Tang WHW (2019) The New Promise of Mitochondrial Transplantation for Myocardial Recovery∗. JACC: Basic to Translational Science 4:889-890 doi: https://doi.org/10.1016/j.jacbts.2019.11.009 Youn DH, Kim BJ, Kim Y, Jeon JP (2020) Extracellular Mitochondrial Dysfunction in Cerebrospinal Fluid of Patients with Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage. Neurocritical Care 33:422-428 doi: 10.1007/s12028-019-00895-1 Zhang B, Gao Y, Li Q, et al. (2020) Effects of brain-derived mitochondria on the function of neuron and vascular endothelial cell after traumatic brain injury. World Neurosurgery 138:e1-e9 Zhang Z, Ma Z, Yan C, et al. (2019) Muscle-derived autologous mitochondrial transplantation: A novel strategy for treating cerebral ischemic injury. Behav Brain Res 356:322-331 doi: 10.1016/j.bbr.2018.09.005 Zhao J, Qu D, Xi Z, et al. (2021) Mitochondria transplantation protects traumatic brain injury via promoting neuronal survival and astrocytic BDNF. Translational Research 235:102-114 Zorov DB, Juhaszova M, Sollott SJ (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiological reviews 94:909-950 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6356471","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446642099,"identity":"329ed85e-6060-4ea7-8f49-5341c8be311c","order_by":0,"name":"Chia Bamshad","email":"","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chia","middleName":"","lastName":"Bamshad","suffix":""},{"id":446642101,"identity":"a54f5249-90e2-48a3-a01e-7d44be1f2f0a","order_by":1,"name":"Zahra Pourmohammadi-Bejarpasi","email":"","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Pourmohammadi-Bejarpasi","suffix":""},{"id":446642103,"identity":"60c6728d-258e-46c3-91cb-17d0e8852052","order_by":2,"name":"Fatemeh Amiri","email":"","orcid":"","institution":"Hamadan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Amiri","suffix":""},{"id":446642104,"identity":"9750fa8a-34e5-4eb5-a285-919d844165ff","order_by":3,"name":"Nima Najafi-Ghalehlou","email":"","orcid":"","institution":"Dartmouth College","correspondingAuthor":false,"prefix":"","firstName":"Nima","middleName":"","lastName":"Najafi-Ghalehlou","suffix":""},{"id":446642108,"identity":"77aace9a-a3c3-4c82-906d-bc135a3f7ed2","order_by":4,"name":"Kazuo Tomita","email":"","orcid":"","institution":"Kagoshima University, Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Kazuo","middleName":"","lastName":"Tomita","suffix":""},{"id":446642109,"identity":"d4d8085f-abfc-4b67-8b18-ec3758e101ae","order_by":5,"name":"Yoshikazu Kuwahara","email":"","orcid":"","institution":"Tohoku Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yoshikazu","middleName":"","lastName":"Kuwahara","suffix":""},{"id":446642111,"identity":"80bfc63c-15cf-4d2d-8fb1-770281609af4","order_by":6,"name":"Tomoaki Sato","email":"","orcid":"","institution":"Kagoshima University, Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Tomoaki","middleName":"","lastName":"Sato","suffix":""},{"id":446642112,"identity":"c22811f7-77e3-4f0a-9166-e0439a5d6f81","order_by":7,"name":"Mehryar Habibi Roudkenar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYDACCQbmx38qbEAM4rWwGfCcSSNNC4MEb8thErTozu59YCDZcD6xf3bzwQcMNTbRBLWY3Tlu8MBwx+3EGXeOJRswHEvLbSCo5UYag0HimduJDTdyzCQYGw4Tp0XiYNu5xPkkaZFsbDuQuIEULWzGDGeSjTfeSEs2SCDSL8yPGSrsZOfdSD744EONDWEtMOAIVplArHIQsCdF8SgYBaNgFIwwAAD3/UQMIku+bwAAAABJRU5ErkJggg==","orcid":"","institution":"Guilan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mehryar","middleName":"Habibi","lastName":"Roudkenar","suffix":""}],"badges":[],"createdAt":"2025-04-02 01:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6356471/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6356471/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81380745,"identity":"e8fd8403-9fff-4d7e-abb3-846fe7450d2f","added_by":"auto","created_at":"2025-04-25 12:40:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2022702,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flowchart. All models were pre-trained for sensorimotor tests prior to TBI induction, with mitochondrial transplantation performed on day 0 and plasma CPK level evaluated 72 hours post-surgery. Apoptosis assessment and immunohistochemistry tests were performed on day 4 and 7, respectively, with sensorimotor tests performed on days 0, 1, 4, 7, 14, and 21. CPK; creatine phosphokinase, IHC; Immunohistochemistry, Mito; mitochondria, TBI; traumatic brain injury\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/5103fa59ff8f346679e2a270.png"},{"id":81380746,"identity":"7bef2dac-a5ec-48da-a566-c222dd3d24af","added_by":"auto","created_at":"2025-04-25 12:40:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2086918,"visible":true,"origin":"","legend":"\u003cp\u003eJC-1 staining of both the platelets and the isolated mitochondria for ΔΨm evaluation. a) Platelets harbouring J-aggregates (red fluorescence). b) Platelets harbouring J-monomers (green fluorescence). c) Figures a and b merged. The preponderance of the red fluorescence over the green fluorescence indicates a strong ΔΨm prior to isolation from the platelets. d) J-aggregates after isolation of mitochondria (red fluorescence). e) J-monomers subsequent to isolation (green fluorescence). f) Figures d and e merged. The preponderance of the red fluorescence over the green fluorescence and thus a high ΔΨm of the isolated mitochondria ascertain their suitability for transplantation. Scale bar = 100 µm\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/508562ac120a297e965ded9e.png"},{"id":81381098,"identity":"ce196036-bbf9-4fe6-8176-f2e3c9fcd25b","added_by":"auto","created_at":"2025-04-25 12:48:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9623685,"visible":true,"origin":"","legend":"\u003cp\u003eHistological configuration of the brain tissue and plasma CPK levels in the four groups. I) Brain tissue staining with haematoxylin and eosin. a) The coronal section of brain tissue in TBI group in low magnification (Red circle shows the damaged area). b) shows the coronal section of brain tissue in control group in low magnification. c) micrograph of brain tissue in control group with normal nucleus and cytoplasm. d-e) The brain tissue of the TBI group with pale and loss of integrity configuration. f-g) The brain tissue of the TBI group with vacuoles and bleeding (black arrows). h) The brain tissue of the TBI group with a high number of cells with dense nuclei (black arrow). i-j) The brain tissue of the TBI+Mito group with nearly normal cytoarchitecture and neuronal morphology. II) The plasma CPK levels in the four groups. Ctrl; control, TBI; traumatic brain injury, Mito; mitochondria, CPK; creatine phosphokinase, RB; respiration buffer. Scale bar = 1mm, 200 and 100 µm (ns; non-significant, * P\u0026lt;0.05, *** p \u0026lt; 0.001, **** P\u0026lt;0.0001, Number of replicates = 3)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/bb6ab5a20a7a91739581963c.png"},{"id":81382039,"identity":"ff3e4625-44aa-436e-95cc-abaf897fa715","added_by":"auto","created_at":"2025-04-25 12:56:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2978325,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent imaging of apoptotic cells and cell nuclei by TUNEL and DAPI staining, respectively. Statistical analysis of the apoptosis rate indicated an analogy between the TBI+Mito and the control groups, confirming the efficiency of mitochondrial transplantation. TBI; traumatic brain injury, Ctrl; control, RB; respiration buffer, Mito; mitochondria. Scale bar = 100 µm (ns; non-significant, **** p \u0026lt; 0.0001, Mean ± SD, Number of replicates = 3).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/fc80887377e2d634ee1f9b54.png"},{"id":81381097,"identity":"0f94e131-93ed-4359-addb-4ae3d715f7f4","added_by":"auto","created_at":"2025-04-25 12:48:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4254085,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry analysis of the astrogliosis-specific biomarker GFAP expression 7 days following mitochondrial transplantation, with the brown cells being positive for GFAP and the number of GFAP\u003csup\u003e+\u003c/sup\u003e cells/field shown in the chart. a) GFAP expression in the control group. b-c) GFAP expression in the TBI and the TBI+RB groups with positive cells in abundance. d) GFAP expression in the TBI+Mito group with relatively fewer positive cells. GFAP; glial fibrillary acidic protein, Ctrl; control, TBI; traumatic brain injury, RB; respiration buffer, Mito; mitochondria. Scale bar = 20µm (ns; non-significant, **** p \u0026lt; 0.0001, Number of replicates = 4)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/6ccdf68e109dde1268964b51.png"},{"id":81380756,"identity":"354bf63f-8813-4d0e-b3dc-18e39854064a","added_by":"auto","created_at":"2025-04-25 12:40:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4028260,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry analysis of the microglial activation biomarker Iba1 expression 7 days following mitochondrial transplantation, with the brown cells being positive for Iba1 and the number of Iba1\u003csup\u003e+\u003c/sup\u003e cells/field shown in the diagram. a) Iba1 expression in the control group with roughly no positive cells. b-c) Iba1 expression in the TBI and the TBI+RB groups with positive cells in abundance. d) Iba1 expression in the TBI+Mito group with relatively fewer positive cells. Ctrl; control, TBI; traumatic brain injury, RB; respiration buffer, Mito mitochondria. Scale bar = 20 µm (Mean ± SD, Number of replicates = 4, ** P\u0026lt;0.01 and *** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/b95bc4b19e519e0db570cdc9.png"},{"id":81380751,"identity":"f7487f74-9bea-49f5-84b9-5f9e4f5dac47","added_by":"auto","created_at":"2025-04-25 12:40:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4292914,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry analysis of the neurogenesis biomarker Nestin expression with the Nestin expression level of the four groups shown in the chart. a) Normal Nestin expression in the brain tissue of the control group. b) Relatively high Nestin expression in the brain tissue of the TBI group. c) TBI group-alike Nestin expression in the brain tissue of the TBI+RB group. d) Significantly high Nestin expression in the brain tissue of the TBI+Mito group. Ctrl; control, TBI; traumatic brain injury, RB; respiration buffer, Mito; mitochondria. Scale bar = 20µm (ns; non-significant, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, Number of replicates = 4)\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/d7ae78e5fbaa1d2bcb742f5a.png"},{"id":81380755,"identity":"8d2b2799-036d-491b-9d48-681c04a56436","added_by":"auto","created_at":"2025-04-25 12:40:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1651548,"visible":true,"origin":"","legend":"\u003cp\u003eSensorimotor tests results. a) The beam walk test. b) The horizontal bars test. c) The grid-walking test. d) The cylinder test. TBI; traumatic brain injury, Ctrl; control, RB; respiration buffer, Mito; mitochondria. (ns; non-significant, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/0f82cb60e1be8ac71474f0dc.png"},{"id":82695906,"identity":"711fc74a-630d-4d99-8236-809071f99c0c","added_by":"auto","created_at":"2025-05-14 08:47:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29419971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6356471/v1/ee0e2134-a687-4e7c-b2d6-6dfa5c0255c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Platelets: Emerging as a Cutting-Edge Source for Mitochondrial Transplantation in Rodent Models of Traumatic Brain Injury","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTBI affects as many as 69\u0026nbsp;million individuals worldwide and is reputed to be a concerning challenge in medicine (Dewan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which pursuant to the World Health Organisation includes half of all trauma-wise mortalities (Iaccarino et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). TBI is classified into three types mild, moderate and severe contingent on the Glasgow Coma Scale (GCS), with the first one being characterised by headache, nausea, vomiting, fatigue or lethargy, depression, and anxiety (Marshall et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moderate TBI manifests more acutely in blurred vision, confusion, temporary loss of consciousness, and focal neurologic problems (Malec et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), while the severe type is associated with delusion, hallucination, euphoria, aberrant motor behaviour, and death (Ciurli et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In addition, TBI could be pathologically categorised into primary and secondary brain injuries, with the former disrupting the brain tissue integrity within the initial hours of injury. The subsequent death of local brain cells and the leakage of pernicious chemical content and apoptotic factors leads to secondary injury in the adjacent non-traumatic area (Greve and Zink \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Reactive oxygen species (ROS) are among the hazardous molecules released after injury and the imbalance between ROS generation and mitochondria-mediated ROS scavenging leads to oxidative stress, damage to the cell membrane of neighbouring cells. and the expansion of the injury site (Abdul-Muneer et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Meticulously, ROS clearance is primarily mediated by mitochondrial permeability transition pores (mPTPs), which are located in the inner mitochondria membrane and are responsible for transporting ions and chemicals in response to Ca\u003csup\u003e2+\u003c/sup\u003e accumulation in the mitochondrial matrix, as well as phosphate levels elevation, adenine nucleotide depletion, and, most importantly, oxidative stress. Therefore, excessive ROS-triggered mPTPs opening leads to ROS-induced ROS release (RIRR) into the cytosol and apoptotic cell death (Bopassa et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zorov et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the current surgical procedures and rehabilitation methods for treating TBI patients fail to effectively demote secondary brain injury or promote damaged tissue recovery (Chang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jabbari et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pourmohammadi-Bejarpasi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), one practical solution to counteract the lethal effects of ROS surplus and restore its physiological balance in the injured tissue involves augmenting healthy and functional mitochondria through mitochondrial transplantation (Emani et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This approach has demonstrated therapeutic potential in various conditions, including acute kidney injury (Jabbari et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Parkinson\u0026rsquo;s disease (Chang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), brain ischaemia (Pourmohammadi-Bejarpasi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), spinal cord injury, peripheral neuropathy (Gollihue et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kuo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and human breast cancer by fostering chemosensitivity (Elliott et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this regard, we conducted the present study to evaluate the therapeutic efficacy of platelet-derived mitochondrial transplantation in rat models of TBI. Platelets, traditionally known for their role in haemostasis and clotting, were selected for isolating healthy and functional mitochondria because they serve as reservoirs of bioactive molecules, including growth factors that induce tissue repair and regeneration (Locatelli et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, an advantage of platelets in therapeutic applications relates to their short lifespan, such that platelet concentrates are stored for up to 5\u0026ndash;7 days prior to transfusion (Aubron et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, expired or unused platelets are considered a readily available biowaste that can be repurposed for mitochondrial isolation and transplantation (Najafi-Ghalehlou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we evaluated the potential of platelet-derived mitochondria for TBI treatment, introducing a cost-effective solution for advancing regenerative medicine as well as emphasising their feasibility as a GMP-compliant source for mitochondrial isolation that would otherwise be discarded.\u003c/p\u003e"},{"header":"STUDY DESIGN AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal Ethics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were undertaken after being reviewed and approved by Iran National Committee for Ethics in Biomedical Research responsible for ethical clearance (Approval Code: IR.NIMAD.REC.1400.003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMitochondria Isolation from Platelets\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1 illustrates a succinct overview of the study, beginning with harvesting mitochondria from the platelets provided by Guilan Blood Transfusion Department conforming to Pourmohammadi-Bejarpasi \u003cem\u003eet al\u003c/em\u003e. (Pourmohammadi-Bejarpasi et al. 2020). Briefly, each platelet storage bag with a volume of 50-70 mL and a pH of 6.2 contained at least 6\u0026times;10\u003csup\u003e10\u003c/sup\u003e platelets. The samples were centrifuged at 3,731 g for 4 minutes and the plasma was discarded, followed by resuspending the remaining pellet in phosphate-buffered saline (PBS) and re-centrifuging to remove the remaining plasma. Afterwards, the platelets were resuspended in 2 mL of homogenisation buffer (HB; 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM EGTA, 0.1% BSA, and 250 mM sucrose) harbouring 1% of protease inhibitor (Sigma-Aldrich, USA). The cell suspension was then incubated on ice for 10 minutes and for 35-40 times on a cell homogeniser. Next, it was centrifuged at 2,000 g and 4℃ for 10 minutes, with the supernatant carefully transferred to another microtube and centrifuged at 10,000 g and 4℃ for another 10 minutes. After discarding the supernatant, the mitochondria pellet was resuspended in 10 \u0026micro;L of respiration buffer (RB, 0.5 mM EGTA, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 20 mM HEPES (pH 7.4), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 250 mM sucrose), incubated on ice, and sent to operating theatre for transplantation. Moreover, mitochondrial quality control and mitochondrial membrane potential (\u0026Delta;\u0026Psi;m) were evaluated by JC-1 staining (Cayman Chemical, USA) as instructed by the manufacturer, observing the red and green fluorescence under the fluorescent microscope (Nikon, Japan), and merging the corresponding images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal Models of TBI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTBI establishment in male Wistar rats weighing 300 g was achieved as described in Feeney\u0026rsquo;s study (Feeney et al. 1981) and the animals were randomly allocated to four groups, including the control group, TBI group, TBI+RB group (TBI models receiving intracerebroventricular (ICV) injection of RB), and TBI+Mito group (TBI models receiving ICV injection of healthy mitochondria). All experimental units were housed under a 12-hour light/dark cycle at 20-22℃ and 52% humidity with food and water supplied \u003cem\u003ead libitum\u003c/em\u003e. Afterwards, they were fully anaesthetised by intraperitoneal injection of ketamine (100 mg/Kg), xylazine (10 mg/Kg), and acepromazine (2.5 mg/kg). the animals were then mounting them on a stereotaxic device by a head holder on the skull. A 2-cm-wide midline scalp incision was made to expose the skull, followed by a 5-mm-wide craniectomy, 1 mm lateral to the sagittal suture between the bregma and lambda, to assess the brain surface without damaging the dura. In accordance with Feeney \u003cem\u003eet al.\u003c/em\u003e (Feeney et al. 1981), TBI induction was carried out by dropping an 80-gram weight through a guide tube, intuitively ascertained by observing brain tissue inflammation and paleness in the exposed region. The injury site was then covered with cold-cure acrylic (Acropars, Iran) and the animals were returned to the animal room for recovery. Sensorimotor and histopathological studies were subsequently performed to further confirm TBI induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMitochondrial Transplantation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondria were isolated from 3\u0026times;10\u003csup\u003e10\u003c/sup\u003e platelets and transplanted into the brain tissue of TBI rats. ICV injection was performed using a 10 \u0026micro;L Hamilton syringe (Hamilton, Cat. No.8929A70, USA) at stereotaxic coordinates of \u0026minus;0.8 mm posterior, \u0026minus;1.5 mm lateral, and \u0026minus;4.0 mm ventral from Bregma (Zhang et al. 2019). The respiratory rate, heart rate, and body temperature were constantly monitored during the surgery and post-surgery recovery in the animal room.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlood Serum Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to animal sacrifice, blood samples were obtained from the animals for measuring serum creatine phosphokinase (CPK) levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistopathological Studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequent to anaesthetising the animals as mentioned earlier, animal perfusion fixation was carried out by flushing 4% paraformaldehyde solution through the circulatory system. The brain tissues were then fixed in 10% formaldehyde and 5-\u0026micro;m-thick paraffin-embedded sections were acquired for haematoxylin and eosin (H\u0026amp;E; Merck, Germany) staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn situ Cell Death Detection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TUNEL assay\u003cem\u003e in situ\u003c/em\u003e apoptosis detection kit (Takara, Japan) was used to estimate the number of apoptotic cells in tissue samples pursuant to the manufacturer\u0026rsquo;s instruction. Deparaffinisation was performed by incubating paraffin-fixed tissue sections in xylene for 30 minutes at 60℃, followed by rehydration with descending concentrations of ethanol and antigen retrieval using proteinase K (Thermo Fisher Scientific, USA). Afterwards, the tissue sections were incubated with 50 \u0026micro;L of TUNEL labelling mixture for 70 minutes at 37℃, washed with PBS, counterstained with DAPI, and observed under a fluorescent microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, 3-\u0026micro;m-thick brain sections were deparaffinised and soaked in 1X tris-buffered saline (TBS) solution (Sigma-Aldrich, T5912). Subsequently, microwave tissue processing was performed until the sections reached the boiling point and were allowed to cool for 20 minutes. In the next step, the sections were washed three times with PBS for 5 minutes each and were incubated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mixed with methanol in a 1:9 ratio for 10 minutes. After another PBS wash, the sections were incubated with anti-glial fibrillary acidic protein (-GFAP), -ionised calcium-binding adapter molecule 1 (-Iba1), or -Nestin antibodies for 1 hour at room temperature, followed by three PBS washes for 5 minutes each. Next, the samples were incubated in 100 \u0026micro;L linker (Diagnostic BioSystems-PVP1000D) for 15 minutes, washed three times with PBS, and incubated in 100 \u0026micro;L polymer solution (Diagnostic BioSystems-PVP1000D) for half an hour. Following another, PBS wash, the sections were incubated in 100 \u0026micro;L DAB solution (ScyTek-ACV999) for 5 minutes. In the final step, the sections were rinsed with water, counterstained with haematoxylin for 10 seconds, and examined under a microscope (Labomed, US) to observe GFAP\u003csup\u003e+\u003c/sup\u003e, Iba1\u003csup\u003e+\u003c/sup\u003e, or Nestin\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal Sensorimotor Skills Assessment \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSensorimotor skills of the animals were minutely assessed by a series of four tests. (a) in the beam walk test animals were required to travers a 1\u0026times;2.5\u0026times;4 cm\u003csup\u003e3\u003c/sup\u003e wooden beam placed 1 metre above the bench surface and forelimb and hind limb placement errors were recorded to assess balance (Hausser et al. 2018). (b) the horizontal bars test measured the motor coordination and strength of forelimbs. Animals were placed at the centre of a 60-cm-long and 6-mm-wide rod placed in the notches of two support columns. Performance was scored based on their ability to grasp the bar for a minimum of 30 seconds or reaching either of the support columns (Deacon 2013). (c) the grid-walking test assessed tactile sensitivity and spatial awareness by allowing the animals to navigate freely for 5 minutes over a 46\u0026times;92 cm\u003csup\u003e2\u003c/sup\u003e wire grid comprising 25\u0026times;25 mm\u003csup\u003e2\u003c/sup\u003e cells 30 cm above the bench surface. The number of foot slips was recorded for scoring as a measure of motor impairment (Russell et al. 2011). (d) the cylinder test, otherwise the spontaneous forelimb elevation test, related to placing the animals inside a glass cylinder (21 cm in diameter and 34 cm in height) and allowing them to rear freely for 5 minutes. The total number of whole-palm contacts with the surrounding wall was recorded, especially for the left forelimb, since the experimental units were undergone TBI induction in the cortex of the right hemisphere of the brain (Cenci and Lundblad 2005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was conducted by GraphPad Prism software version 8 (La Jolla, CA, USA), with One-way ANOVA used for comparing groups and p-values of 0.05 at the maximum were deemed statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExogenous Mitochondria Quality Control\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2.a-c and Figure 2.d-f illustrate \u0026Delta;\u0026Psi;m evaluation by JC-1 staining of the platelets and the isolated mitochondria, respectively, such that the dominance of J-aggregates (red fluorescence) over the J-monomers (green fluorescence) in both cases marks a strong \u0026Delta;\u0026Psi;m and thus affirms the high quality of the mitochondria for transplantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Neuroprotection against Secondary Brain Injury\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3I and Figure 3Ib show low magnification images of brain tissue from the TBI and control groups. In the control group, no significant alterations in brain tissue were observed, with clear visibility of cellular nuclei and cytoplasm (Figure 3I.c). In contrast, the TBI group displayed evidence of secondary brain injury and tissue destruction at the injury site, characterized by paler surrounding tissue (Figure 3I.d), loss of tissue integrity, and vacuolation (Figure 3I.e-f), as well as bleeding and pyknosis (Figure 3I.g-h). A similar pattern was observed in the TBI+RB group. However, in the TBI+Mito group, mitochondrial transplantation was associated with a significant reduction in brain tissue damage and preservation of neuronal cell structure (Figure 3I.i-j).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3II\u003c/strong\u003e presents the plasma levels of the brain trauma biomarker creatine phosphokinase (CPK) across the four experimental groups. The control group exhibited the lowest CPK levels, while the TBI group had significantly higher CPK levels compared to controls (p \u0026lt; 0.0001). Additionally, CPK levels were significantly higher in the TBI+RB group compared to the TBI+Mito group (p \u0026lt; 0.001). Notably, no significant difference in CPK levels was found between the control and TBI+Mito groups, suggesting that mitochondrial transplantation restored normal plasma CPK levels and effectively mitigated secondary brain injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBrain Cells Apoptosis Reduction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptosis rate in brain tissue of the animals was evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay four days post-surgery, disclosing a statistically significant difference between the TBI+Mito and the TBI groups (Figure 4; p \u0026lt; 0.0001) and no statistically significant difference between the control and the TBI+Mito groups. Additionally, the TBI and TBI+RB groups displayed similar apoptosis rates, with no significant difference between them. These findings suggest a robust association between mitochondrial transplantation and post-TBI apoptosis progress obstruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAstrogliosis Reduction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of astrogliosis-specific biomarker GFAP across the four groups revealed that unlike the TBI and TBI+RB groups (Figure 5.b-c), which exhibited elevated GFAP expression, the TBI+Mito group possessed a notably lower GFAP level (Figure 5.d; p \u0026lt; 0.0001) compared to TBI and TBI+R groups. In other words, astrogliosis had remarkably decline in the TBI+Mito group and was closely similar to that of the control group, which suggests that mitochondrial transplantation effectively mitigates astrogliosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicroglial Activation Decrease\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of microglial activation biomarker Iba1 was significantly high in the TBI group compared to TBI+Mito group (p \u0026lt; 0.001; Figure 6). In addition,\u0026nbsp;a similar trend was observed in the TBI+RB group (P\u0026lt;0.01); however, no significant difference was found between the TBI and TBI+RB groups (Figure 6.b-c). In contrast, the TBI+Mito group exhibited a dramatic reduction in Iba1 level (Figure 6.d), closely analogous to that of the control group (Figure 6.a). \u0026nbsp;Therefore, Iba1 levels in the TBI+Mito group were significantly different from those in the TBI and TBI+RB groups (p \u0026lt; 0.001 and p \u0026lt; 0.01, respectively), indicating that mitochondrial transplantation effectively attenuates microglial activation following TBI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNestin Upregulation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNestin designated as being the neurogenesis biomarker present in neuroepithelial stem cells becomes elevated post-TBI brain injuries (Sahin Kaya et al. 1999). In this study, no significant difference in Nestin levels was observed between the TBI and TBI+RB groups (Figure 7.b-c). However, Nestin levels in the TBI+Mito were significantly higher than the TBI and TBI+RB groups (p \u0026lt;0.0001 and P\u0026lt;0.001 respectively) highlighting the role of mitochondrial transplantation in promoting neurogenesis. Additionally, Nestin expression was significantly upregulated in the TBI+Mito group compared to the control group (p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSensorimotor Skills Revival\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the sensorimotor skills tests following mitochondrial transplantation corroborate a significant restoration of motor function in the experimental units receiving mitochondria. The recovery levels in the TBI+Mito group was comparable to the control group, highlighting the therapeutic potential of mitochondrial transplantation. In detail, the beam walk test results depicted in Figure 8.a exhibit a significantly faster recovery in the TBI+Mito group compared to the TBI+RB and TBI groups, with a considerable difference on day 7 (p \u0026lt; 0.001), insofar as the TBI+Mito group was not statistically different than the control group on days 14 and 21, indicating effective mitigation of secondary brain injury. As for the horizontal bars test results illustrated in Figure 8.b, mitochondrial transplantation was associated with motor coordination and forelimb strength restoration with no significant difference was recorded between the TBI+Mito group and the control group by the end of week 1. In contrast, the control group remained significantly different from the TBI and TBI+RB groups (p \u0026lt;0.01 and 0P\u0026lt;0.001 respectively). The grid-walking test results, based on the frequency of foot slips, proved to be highly sensitive in detecting sensorimotor deficits in TBI models compared to the other tests. In line with the previous tests, the difference between the control and the TBI+Mito group began to become statistically indistinguishable by day 7 (Figure 8.c). Conversely, both the TBI and the TBI+RB groups showed negligible rate of sensorimotor skills recovery, with a statistically conspicuous difference compared to the control group (p \u0026lt; 0.001). Consistently, the cylinder test revealed that the TBI+Mito group scores greatly resembled that of the control group and thus were not statistically different from week 2 onward (Figure 8.d). In contrast, both the TBI and TBI+RB groups continued to show significant impairments compared to the control group (p \u0026lt; 0.001 on day 7, p \u0026lt; 0.001 on day 14, and p \u0026lt; 0.01 and p \u0026lt; 0.05, respectively, on day 21).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eMitochondria are credited with playing a crucial role in cellular physiology and survival, especially in highly dynamic neurons, by constantly regulating adenosine triphosphate (ATP) generation, proliferation, homeostasis, ROS-mediated apoptosis, and autophagy (Reddy \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; de Castro et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Norat et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, mitochondrial transplantation has struck as an advanced and promising therapeutic approach for treating various disorders associated with mitochondrial dysfunction and disturbance in ATP production, such as myocardial ischaemia, acute kidney injury (AKI), and acute ischaemic stroke (AIS) (McCully et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hayakawa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tang \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Youn et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This study builds on our previous endeavours to investigate the efficacy of transplanting mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in treating rat models of traumatic brain injury (TBI) (Bamshad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Baharvand et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which demonstrated consistent results in terms of accelerated neurogenesis, rescued sensorimotor skills, reduced apoptosis rate, and decreased microglial activation (Bamshad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similar results were noted in ischaemic heart disease (Baharvand et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), AIS (Pourmohammadi-Bejarpasi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and AKI (Jabbari et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), along with prostate cancer (PC-3) cell line (Nikoo et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) that was observed to be interconnected with promoted proliferation and demoted cisplatin sensitivity, but not erastin sensitivity, in PC-3 cells.\u003c/p\u003e \u003cp\u003eHowever, the therapeutic applications of hUC-MSCs-derived mitochondria are largely hindered by the relatively long population doubling time, low yield, survival in culture, isolation complexity, and high cost. In order to address these challenges, we explored platelet-derived healthy and exogenous mitochondria as an alternative source, highlighting their feasibility and therapeutic potential in animal models of TBI. Our findings indicate that transplanting the mitochondria derived from expired platelets is meaningfully linked with mitigating and ameliorating secondary brain injury by reducing apoptosis and astrogliosis, suppressing microglial activation, restoring homeostasis, and fostering neurogenesis (Singh et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fischer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kheirandish-Rostami et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), reflected in motor functions recovery. In line with our study, Zhao \u003cem\u003eet al\u003c/em\u003e. reported that transplanting exogenous mitochondria derived from allogeneic liver and/or autogenic muscle reduced neuronal apoptosis, improved motor functions, alleviated anxiety, and upregulated astrocytic brain-derived neurotrophic factor (BDNF) (Zhao et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consistently, Zhang \u003cem\u003eet al.\u003c/em\u003e investigating the therapeutic effects of exogenous brain-derived mitochondria in mouse models of TBI concluded that administering exogenous mitochondria in mice enhanced mitochondrial respiratory control ratio, upregulated synaptic plasticity-related tight junction (TJ) protein expression, demoted apoptosis, promoted angiogenesis, and alleviated brain oedema and blood-brain barrier (BBB) leakage (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe neurovascular unit (NVU), comprising neuronal cells, astrocytes, BBB endothelial cells, pericytes, myocytes, and extracellular matrix (ECM) components, plays a pivotal role in central nervous system (CNS) disorders. Astrocytes, in particular, have been found to contribute to neuroprotection and repair by releasing and transferring mitochondria into damaged neurons via the calcium signalling enzyme CD38 (38, 46). Thus, NVU disruption is a fundamental factor in CNS pathologies, highlighting the importance of mitochondrial homeostasis in brain injury recovery (Hayakawa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nakamura et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the major drawbacks of mitochondrial transplantation pertains to identifying an optimal source. Platelets present a potentially desirable and ideal alternative for harvesting healthy mitochondria in basic and translational research due to their accessibility, high mitochondrial content, non-invasive isolation, compliance with GMP standards, high biocompatibility, and low pathogenicity. Despite the short shelf life of platelets (5 days), storage requirement and efficacy control at 20\u0026ndash;24℃ prior to transfusion (Humbrecht et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), our study demonstrated that mitochondria harvested from old platelets remained viable and effective.\u003c/p\u003e \u003cp\u003eFurthermore, the route of mitochondrial administration requires further investigation. Our results support intracerebroventricular (ICV) administration of mitochondria as a suitable and effective method for targeting the brain cells. In this manner, Huang \u003cem\u003eet al\u003c/em\u003e. reported that both the intracerebral and intra-arterial administration of mitochondria, isolated from baby hamster kidney (BHK-21) cells, to a rat model of middle cerebral artery occlusion (MCAO) was interrelated with improved motor functions, decreased apoptosis rate, and shrunk infarct area (Hayakawa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hence, future studies are encouraged to focus on optimising delivery routes for different pathological conditions.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, our findings suggest that transplanting mitochondria harvested from aged platelets into rat models of TBI holds significant therapeutic potential, not only by slowing the progression from primary to secondary brain injury but also by aiding the recovery of sensorimotor function. While mitochondrial transplantation shows promise as a reliable treatment, several challenges remain, as with any therapeutic approach. These include determining the optimal route of administration for different pathological conditions, establishing an effective dosing regimen, addressing immune system overreaction, and mitigating potential side effects. Future research is needed to overcome these hurdles and further enhance the efficacy of this treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institute for Medical Research and Development (NIMAD) under grant number 996448. The authors would like to dedicate this paper to the cherished memory of Chia Bamshad, who tragically passed away during the submission process. His passion for science, unwavering dedication, and invaluable contributions were the driving forces behind this work. While his absence leaves a profound void, his legacy will continue to inspire us. We honor his memory and the lasting impact he has made on this field.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCB, NNG\u003c/strong\u003e and \u003cstrong\u003eMHR\u003c/strong\u003e: Wrote the main manuscript text. \u003cstrong\u003eCB\u003c/strong\u003e, \u003cstrong\u003eZPB\u003c/strong\u003e and \u003cstrong\u003eNNG\u003c/strong\u003e prepared figures, FA, ZPB, \u003cstrong\u003eKT, YK, NNG, TS\u003c/strong\u003e and \u003cstrong\u003eMHR\u003c/strong\u003e ; Writing \u0026ndash; review \u0026amp; editing, \u0026nbsp;\u003cstrong\u003eMHR\u003c/strong\u003e: supervision and Funding acquisition. All authors reviewed the manuscript\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdul-Muneer PM, Chandra N, Haorah J (2015) Interactions of oxidative stress and neurovascular inflammation in the pathogenesis of traumatic brain injury. Mol Neurobiol 51:966-979 doi: 10.1007/s12035-014-8752-3\u003c/li\u003e\n\u003cli\u003eAubron C, Flint AWJ, Ozier Y, McQuilten Z (2018) Platelet storage duration and its clinical and transfusion outcomes: a systematic review. Crit Care 22:185 doi: 10.1186/s13054-018-2114-x\u003c/li\u003e\n\u003cli\u003eBaharvand F, Habibi Roudkenar M, Pourmohammadi-Bejarpasi Z, et al. (2024) Safety and efficacy of platelet-derived mitochondrial transplantation in ischaemic heart disease. 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(2021) Mitochondria transplantation protects traumatic brain injury via promoting neuronal survival and astrocytic BDNF. Translational Research 235:102-114 \u003c/li\u003e\n\u003cli\u003eZorov DB, Juhaszova M, Sollott SJ (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiological reviews 94:909-950 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Traumatic brain injury, Mitochondria, Mitochondrial transplantation, Platelets","lastPublishedDoi":"10.21203/rs.3.rs-6356471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6356471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraumatic brain injury (TBI) remains a significant clinical challenge due to the absence of effective therapies for mitigating oxidative damage and neuronal dysfunction. Given the brain\u0026rsquo;s high metabolic demand, mitochondrial transplantation has emerged as a promising therapeutic strategy. However, identifying an optimal, functional source of mitochondria remains a major obstacle. In this study, we explore the therapeutic potential of mitochondria isolated from senescent platelets as a readily available, GMP-compliant source for promoting recovery in TBI models. TBI was induced in rats using a weight-drop model, with confirmation through histopathological analysis. The animals were then divided into four experimental groups: healthy control, TBI, TBI\u0026thinsp;+\u0026thinsp;RB (receiving respiration buffer), and TBI\u0026thinsp;+\u0026thinsp;Mito (receiving mitochondrial transplantation). Neurobehavioural recovery was assessed using a series of sensorimotor tests, including the beam walk test, horizontal bars test, grid-walking test, and cylinder test. Our results show that mitochondrial transplantation significantly improved neurobehavioural function in TBI animals, with performance in the TBI\u0026thinsp;+\u0026thinsp;Mito group comparable to that of the healthy control group. Histological examination further revealed that brain tissue morphology in the TBI\u0026thinsp;+\u0026thinsp;Mito group closely resembled that of the control group.\u003c/p\u003e \u003cp\u003eThese findings provide compelling evidence for the therapeutic potential of platelet-derived mitochondrial transplantation as an accessible and effective treatment for TBI. However, further investigation is needed to determine the long-term efficacy and underlying mechanisms of this novel approach.\u003c/p\u003e","manuscriptTitle":"Platelets: Emerging as a Cutting-Edge Source for Mitochondrial Transplantation in Rodent Models of Traumatic Brain Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 12:39:58","doi":"10.21203/rs.3.rs-6356471/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22d18a63-0252-4442-8a99-b0ebd733dcb2","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-14T08:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-25 12:39:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6356471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6356471","identity":"rs-6356471","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}