Ketone Esters partially and selectively rescue mitochondrial bioenergetics after acute cervical spinal cord injury in rats: A time-course

Ketone Esters partially and selectively rescue mitochondrial bioenergetics after acute cervical spinal cord injury in rats: A time-course | 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 Ketone Esters partially and selectively rescue mitochondrial bioenergetics after acute cervical spinal cord injury in rats: A time-course Oscar Seira, HyoJoon Park, Jie Liu, Michelle Poovathukaran, Kieran Clarke, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3751380/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 Spinal cord injury (SCI) pathology and pathophysiology can be attributed to both primary physical injury and secondary injury cascades. Secondary injury cascades involve dysregulated metabolism and energetic deficits, which are directly linked to compromised mitochondrial bioenergetics. Rescuing mitochondrial function and reducing oxidative stress are associated with neuroprotection. In this regard, ketosis after traumatic brain injury (TBI), or after SCI, improves secondary neuropathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics. Here, we follow up on our previous study and have used an exogenous ketone monoester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE), as an alternative to a ketogenic diet, focusing on mitochondrial function between 1 and 14 days after injury. Starting 3 hours following a C5 hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) combined with KE administered orally (OKE). We found that mitochondrial function was reduced after SCI at all times post-SCI, accompanied by reduced expression of most of the components of the electron transport chain (ETC). The KE rescued some of the bioenergetic parameters 24 hours after SCI when BHB concentrations were ~ 2 mM, but most of the beneficial effects were observed at 2 weeks after injury with BHB concentrations reaching values of 4–6 mM. To our knowledge, this is the first report of beneficial effects of KE in rescuing mitochondrial function after SCI and demonstrates the suitability of KE to ameliorate the metabolic dysregulation that occurs after traumatic SCI without requiring a restrictive dietary regime. Ketone monoester electron transport chain spinal cord injury mitochondria temporal changes Figures Figure 1 Figure 2 Figure 3 Introduction Secondary injury cascades after traumatic spinal cord injury (SCI) result in significant pathophysiological effects (Alizadeh, Dyck et al. 2019 ). A better understanding of the injury mechanisms following SCI is essential for the development of therapies to increase neuroprotection, restore metabolic function, and promote functional recovery. It is known that metabolism and cellular energetics are severely affected due to mitochondrial dysfunction after SCI (Rabchevsky, Michael et al. 2020 ), for review); targeting mitochondrial function might have an important role in promoting recovery after SCI (McEwen, Sullivan et al. 2011 ; Slater, Dominguez-Romero et al. 2022 ; Cheng, Cai et al. 2023 ; Patel, Michael et al. 2023 ). For example, reduced oxidative stress is associated with improved cellular bioenergetics providing neuroprotection (Ferdous, Burnett et al. 2022 ). In this regard, ketosis after traumatic brain injury (TBI), or after traumatic spinal cord injury (SCI) improves secondary pathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics (Almeida-Suhett, Namboodiri et al. 2022 )(Koh, Dupuis et al. 2020 ; Tan, Jiang et al. 2020 ; Gough, Casella et al. 2021 ; Kong, Liu et al. 2021 ; Seira, Kolehmainen et al. 2021 ; Yarar-Fisher, Li et al. 2021 ). In this study, we follow up on our previous work showing that ketosis induced by a ketogenic diet (KD) was able to mitigate mitochondrial oxidative damage and dysfunction 1 week post SCI in rats (Seira, Kolehmainen et al. 2021 ). However, the KD is highly restrictive and may not be suitable for many people with SCI. The ketone body, d -β-hydroxybutyrate (BHB), has multiple mechanisms of action ranging from metabolite for mitochondrial energy production to anti-inflammatory receptor ligand, signaling molecule, and epigenetic modifier (Clarke, Tchabanenko et al. 2012 ; Newman and Verdin 2017 ; Soto-Mota, Norwitz et al. 2020 ; Yarar-Fisher, Li et al. 2021 ). Circulating BHB can also be increased using exogenous ketone esters (KE). In particular, the (R)-3-hydroxybutyl (R)-3-hydroxybutyrate ketone monoester (∆G®) can elevate BHB for extended periods in humans at higher levels than those seen with a KD, without side effects and without sodium overload (Clarke, Tchabanenko et al. 2012 ; Soto-Mota, Vansant et al. 2019 ). To assess the use of an exogenous ketone as an alternative to a KD, and to evaluate effects on mitochondrial function in a temporal manner, we focused on the acute-subacute phases of SCI (24 hours post-injury, HPI; to 2 weeks post-injury, WPI) during which the spinal cord undergoes the most dramatic metabolic changes (Ahuja, Wilson et al. 2017 ). Starting 3 hours following a C5 hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) which is a standard chow containing KE (12.5% w/w). The KED was combined with KE administered orally by gavage to achieve robust BHB concentrations of 2–6 mM. As expected, mitochondrial function was lower after SCI at all timepoints post-SCI, and this was accompanied by a reduced expression of most of the components of the electron transport chain (ETC) for most timepoints. Although KE rescued some bioenergetic parameters at early timepoints, most of the beneficial effects were observed 2 weeks after treatment. To our knowledge, this is the first study to investigate the suitability of an exogenous ketone to counteract the metabolic dysregulation and mitochondrial dysfunction at multiple timepoints during the acute and subacute phases of traumatic SCI. Our results highlight the beneficial role of KE immediately, and for at least up to 2 weeks after trauma. Material and Methods Experimental design, SCI and Treatment All procedures were conducted according to the guidelines of the Canadian Council for Animal Care and overseen by the University of British Columbia Animal Care Committee. We used 44 adult male Sprague-Dawley Harlan Breeding Laboratory), group-housed (21°C; 12 h:12 h light:dark cycle) and given ad libitum access to standard rodent diet prior to the injury (Tekland 2020X, Envigo). The injury was performed as we described previously in JoVE (Lee, Streijger et al. 2012 ). After a unilateral C5 laminectomy, the dorsal processes of C4-C6 were held with a clamp and fixed in a frame tilted at a 22.5° angle. The contusion force was set to 150 Kdynes using the Infinite Horizon impactor (Precision Systems and Instrumentation, LCC, Brimstone, Virginia, USA). KE administration The ketone ester (KE) was provided by TdeltaS Global Inc. (∆G® www.deltaGketone.com ). The KED and control diet (CD) were prepared by Dyets Inc. (Bethlehem, Pennsylvania, USA) using the formulation from the Veech lab as previously described (Kashiwaya et al. 2013). The KE content was 0.125g/1g food and, as our rats ate around 15–20 g per day, the KE intake was approximately 1.9 to 2.5 g or 1.8 to 2.3 mL (KE density = 1.0731g/mL at 22ºC) (Soto-Mota et al. 2019 ). For the oral ketone ester (OKE), rats were gavaged with either 1 mL of KE or water (controls) mixed 1:1 with Boost® Nutritional Supplement (total volume per gavage = 2 mL). Boost® was used to help mitigate weight loss following injury and improve overall animal health without masking the effects of the KE. (For detailed nutritional information of Boost® visit BOOST Chocolate Meal Replacement | madewithnestle.ca). Blood BHB levels Ketone levels were measured from blood obtained by tail poking. Precision Xtra Blood Ketone Test Strips (Diabetes Express) were used with the Precision Xtra Blood and Ketone Meter (Abbott) according to manufacturer guidelines. Mitochondrial Respiration, Protein extraction, and Western Blots Rats were euthanized at various times post-injury with an overdose of chloral hydrate injected intraperitoneally. Rats were perfused with cold PBS and the injury epicenter (approximately half the cord, 5 mm in length) was harvested. Mitochondrial respiration in permeabilized spinal cord segments was measured using high-resolution respirometry (Oroboros Instruments, Innsbruck, Austria). Spinal cord tissue was permeabilized with saponin at 5 ug/ml for 20’ in the shaker with gentle agitation. Tissue was washed for 10’ with respiration media. Respirometry experiments were performed at 37°C in respiration medium containing EGTA (0.5 mM), MgCl 2 (3 mM), K-lactobionate (60 mM), taurine (20 mM), KH 2 PO 4 (10 mM), HEPES (20 mM), sucrose (110 mM), and BSA (1 g/l), with the addition of mitochondrial substrates and inhibitors to measure coupled and uncoupled respiration, flux through complex I and II, and maximal respiration of the electron transport chain ETC. Mitochondrial respiration was expressed as weight-specific oxygen flux (pmolO 2 ·s − 1 ·mg − 1 ). Chamber oxygen levels were maintained between 240 and 400 nmol·ml − 1 to avoid O 2 limitation. As previously described in our lab (Seira, Kolehmainen et al. 2021 ), basal respiration was first measured, then malate (2mM), pyruvate (5mM), and glutamate (10 mM) were added to measure LEAK respiration (respiration due to proton leakage and the circuit of electrons and cations that is not dependent on ATP synthase activity). ADP (2.5 mM) was then added to stimulate NADH-dependent coupled respiration through complex I, followed by oligomycin (2.5 µM) to measure LEAK respiration at high membrane potential during inhibition of ATP synthase. Complete, non-physiological uncoupled respiration of the ETC was measured with the titration of carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (CCCP) (0.5 + 0.5 µM). Succinate (10 mM) was added to measure reduced flavin adenine dinucleotide (FADH 2 )-dependent complex II respiration followed by complex I inhibition with rotenone (0.5 µM). Residual oxygen consumption (ROX) attributed to non-mitochondrial respiration was measured with addition of Antimycin A (2.5 µM) to inhibit complex III of the ETC. Finally, ascorbate (2 mM) and N , N , N ′, N ′-tetramethyl- p -phenylenediamine dihydrochloride (TMPD) (0.5 mM) was added to measure the isolated respiratory rate of cytochrome oxidase (complex IV respiration). Protein lysate was prepared using plastic Dounce homogenizers to disrupt tissue in Tris-EDTA SDS lysis buffer (0.01M Tris-HCl (pH 8, VWR)), 1mM EDTA (Ambion, ThermoFisher Scientific), 0.1% SDS (VWR), cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Mississauga, ON), 100mM NaF (ThermoFisher Scientific), 25mM β-glycerophosphate (MilliporeSigma), 10mM Pyrophosphate (ThermoFisher Scientific), and 200µM Orthovanadate (ThermoFisher Scientific)). Samples were spun at 14000rpm and an aliquot of supernatant was taken for Western blotting. Statistical analysis Data are presented as mean values, and error bars indicate ± SEM, as noted. All statistical analysis were performed using the Prism software ( http://www.graphpad.com ). Information about the tests used can be found in the Figure Legends. Results A Ketone Ester Diet (KED) in combination with orally administered Ketone Esters (OKE) induces ketosis acutely after SCI in rats. We examined the effects of the ketone ester (KE) in raising BHB levels at 24HPI, 48HPI, 7DPI and 14DPI (Fig. 1 A). Blood samples were collected before injury and before starting with the dietary treatments for all timepoints. We found that baseline levels are fairly consistent throughout all timepoints (Fig. 1 B). As seen in Fig. 1 B, BHB levels were significantly higher in the KE treated groups compared to the control group both, 24HPI and 48HPI. Note the higher BHB levels at 48HPI compared to 24HPI. The BHB levels for these two timepoints were analyzed before euthanasia around 8 Hours Post Oral Gavage (HPOG) (See treatment paradigm schematic for clarification, Fig. 1 A). Figure 1 C shows BHB levels taken throughout the treatment of the 7 day survival cohort. Measurements during the first 3 days post injury were taken 8HPOG. Note the increase in BHB levels over the first 3 days. BHB levels were then analyzed at 24HPOG at 6DPI (as an arbitrary half way checkpoint control BHB measurement) and before euthanasia (12HPOG) at 7DPI. Surprisingly, we did not find any significant increase in BHB levels at 7DPI measured 12HPOG. The 14 days survival group shows significant BHB increases over time during the first 7DPI in the KE group compared to the Control group (Fig. 1 D). At 24HPOG-10DPI, another half way through checkpoint, an increase in BHB levels in the treatment group was still observed. Finally, at 14DPI one last measurement before euthanasia was taken 24HPOG, showing a significant increase in BHB levels in the KE treated group compared to the control group as well (Fig. 1 D). Ketone Esters selectively improve mitochondrial respiration at 24 hours and 2 weeks post-SCI. Mitochondrial respirometry is a commonly used method to evaluate mitochondrial function and oxidative phosphorylation (OXPHOS) in mitochondria, cells, and tissues (Hoeks, Hesselink et al. 2012 ). This methodology enables assessment of the overall function of mitochondria in a specific tissue, and discrete functional components and characteristics of the electron transport chain (ETC) (Fig. 2 A) that may be dysfunctional as a result of trauma or pathological disorders. To evaluate the possible beneficial effects of ketone esters (KE) in modulating the metabolic distress observed acutely after spinal cord trauma, we performed a respirometry study of nervous tissue located at the epicenter of the injured cervical spinal cords at 24HPI, 48HPI, 1WPI and 2WPI. We followed the same titration protocol we previously used in our ketogenic diet study (Seira, Kolehmainen et al. 2021 ) (See Material and Methods). Basal mitochondrial respiration (oxygen consumption at baseline) was consistent for the different timepoints. Overall, no significant differences were observed when comparing the uninjured cords to the injured-treated spinal cords (Fig. 2 B-E), however, we detected a significant increase in basal respiration in the injured cords compared to the uninjured cords at 48H (Fig. 2 D). No differences in respiration between the KE and the control treated group were observed at baseline for any time point studied. In addition, an overall pattern of a decline in mitochondrial respiration across all timepoints was observed (Fig. 2 B-E). Yet, at 2 weeks post injury, while there was a trend towards a lower maximum respiration via CI and CII, respiration via these 2 complexes was not significantly decreased when compared to the uninjured spinal cords (Fig. 2 E). The respirometry data from all separate timepoints show that uncoupled mitochondrial respiration with Complex I (CIu) and Complex II (CIIu) substrates was reduced at 24H following SCI (Fig. 2 B). LEAK respiration measured after oligomycin addition to block ATP-synthase activity was significantly decreased after injury in the control group. The decrease in LEAK after injury was rescued with the KE treatment (Fig. 2 B). In the KE group, mitochondrial respiration with electron supply through Complex I was significantly higher compared to the control group. No differences in maximum mitochondrial respiration via Complex II and or the isolated respiration rate of cytochrome c oxidase (Complex IV) were seen (Fig. 2 B). Next, we looked at the oxygen consumption at 48H and 7 weeks post-SCI, and did not find any differences in mitochondrial respiration between the KE treated group and the control group after injury (Fig. 2 C-D). However, we noticed a trend towards an increase in respiration with Complex II substrate supply at 48H (Fig. 2 C). Lastly, we analyzed the 2 weeks post-SCI data. Overall, and as seen in Fig. 2 E, respirometry data at this timepoint show the most significant differences between KE treatment and the controls compared to the results from earlier timepoints studied. Specifically, the treatment of the injured group with KE showed a significant increase of ADP-stimulated OXPHOS for Complex I compared to the injured control group. Furthermore, LEAK, Complex I (CIu) and Complex II (CIIu) uncoupled respiration also showed a significant increase in the injured KE group compared to the injured control group (Fig. 2 E). Similar to earlier timepoints, no significant differences in maximum mitochondrial respiration of Complex IV were detected. Interestingly, the treatment with KE led to an overall increase in mitochondrial respiration that not only rescued the mitochondrial bioenergetic dysfunction associated with the injury, but it was also able to significantly increase the oxygen consumption to levels that were higher than the ones observed in the uninjured group (Fig. 2 E). Ketone Esters selectively modify the expression of OXPHOS complexes of the Electron Transport Chain (ETC). Changes in mitochondrial gene expression have been previously linked to changes in mitochondrial function after neurotrauma in the CNS (Harris, Black et al. 2001 ). In fact, we demonstrated that the treatment with a KD led to an increase in protein expression for some of the OXPHOS complexes that are part of the electron transport chain (ETC) in the spinal cord after injury (Seira, Kolehmainen et al. 2021 ). To further elucidate if the use of KE might have similar effects to those observed with a KD, and whether the data from our mitochondrial respirometry could be associated with changes in protein expression of those complexes, we performed individual western blotting analysis on tissue homogenates from the epicenter of the injury for each of the OXPHOS complexes (Fig. 3 ). Figure 3 illustrates the western blots corresponding to the protein subunits of the OXPHOS complexes for the four different timepoints after SCI. Similar to our mitochondrial respirometry data, a single uninjured group was used as non-injured tissue control, and to normalize all quantification data (see uninjured blot in Fig. 3 D). The quantification of the bands revealed that at 24H post SCI, there was a general and significant decrease in protein expression of all complexes after injury compared to the uninjured spinal cords. However, the treatment with KE did not reverse the injury effect (Fig. 3 A). Similar to the 24H post-SCI, quantification of the 48 hour post-SCI blots, showed significantly less expression of Complex I, III and IV after injury, while Complex II and V did not appear to change at this timepoint. It was interesting that Complex II expression significantly increased after injury with the KE resulting in an even larger increase in expression compared to the control group. A trend towards an increase in Complex V with KE treatment was also found (Fig. 3 B). The band quantifications at 1 week post-SCI revealed a significant decrease in protein expression of Complex II, III and V (Fig. 3 C) after injury. Interestingly, the expression of Complex I and IV remained at the same levels than the uninjured cords. Regarding the treatment effects, and surprisingly to us, we found a significant decrease in protein expression after KE treatment of Complexes I, III and V. Overall, KE caused a decrease in expression at 1 week post-SCI (Fig. 3 C). Lastly, Fig. 3 D illustrates the quantification for the 2 weeks post-SCI timepoint. Here, we also found a generalized decrease in protein expression for the OXPHOS complexes I, II, III and IV after injury, with the exception of Complex V (Fig. 3 D). At this timepoint, the KE changes in expression were similar to those at 1 week post-SCI. Thus, Complex III and V expression were significantly reduced after treatment compared to the control group (and also to the uninjured group regarding Complex V). No significant differences were found in the levels of expression between KE and controls for Complexes I, II and IV. Discussion Mitochondria play an essential role in creating energy to drive cellular function and biological processes. Dysregulation of mitochondrial function has been closely linked to numerous neurological diseases and disorders (Cabral-Costa and Kowaltowski 2020 ; Norat, Soldozy et al. 2020 ). Secondary injury occurs after initial damage of the CNS, and leads to systemic as well as tissue metabolic changes (Scholpa and Schnellmann 2017 ; Rabchevsky, Michael et al. 2020 ; Slater, Dominguez-Romero et al. 2022 ). The secondary injury propagates for weeks-to-months following the primary insult, and contributes to the lack of recovery in SCI patients. Over the last few years, progress has been made in studying and understanding the role and use of dietary interventions to treat the metabolic dysregulation in neurological diseases and disorders, including neurotrauma events ((Carneiro and Pellerin 2021 ; Chelluboina and Vemuganti 2021 ; Yarar-Fisher, Li et al. 2021 ; Field, Field et al. 2022 ; Yassine, Self et al. 2022 ). Furthermore, improving mitochondrial function after SCI has been already been shown to lead neuroprotection and functional recovery (Patel, Sullivan et al. 2012 ; Patel, Cox et al. 2017 ). In this study, we sought to evaluate the use of KE on restoring energy metabolism following SCI. We studied mitochondrial respiratory function and oxidative phosphorylation (OXPHOS) on injured cervical spinal cord segments from rats during the acute and subacute phases of the injury. Additionally, we also wanted to investigate whether KE might potentially mimic some of the beneficial effects on mitochondrial function that we found using a ketogenic diet (KD) (Seira, Kolehmainen et al. 2021 ). To induce ketosis in rats that underwent C5 hemi-contusion, we delivered the KE orally (OKE), and incorporated them in the diet (KED). After SCI, animals tend to lose their appetite for the first 24H-48H. Based on previous work from our lab (unpublished data), we knew that in order to increase β-hydroxybutyrate (BHB) levels in the bloodstream of the animals during those initial hours, an oral supplementation of KE was required. Our treatment paradigm was designed with that consideration in mind, and combines the use of KED and oral administration of KE (OKE). Based on previous literature, we knew that those initial days after injury are critical in regards to cell bioenergetics dysregulation (Sullivan, Krishnamurthy et al. 2007 ; McEwen, Sullivan et al. 2011 ), so an intensive treatment regime was designed (Fig. 1 A). Using this treatment paradigm, we were able to induce ketosis in the animals at all timepoints studied. We found a certain degree of variability between the different cohorts regarding the BHB levels that were reached. For example, the levels 8 hours post oral gavage (HPOG) after 2 days of treatment were singly lower in the 1 week post–SCI timepoint compared to the others. BHB levels usually peak between 3 and 7 days post-treatment, and are followed by a trend towards a decrease after that. In addition, the differences between control and treated groups becomes smaller. We believe that this time adaptation to the treatment could be associated with cell regulation of some receptors such as the Monocarboxylate transporters (MCTs) (Leino, Gerhart et al. 2001 ). MCTs have been identified as transporters of lactate, pyruvate, and ketone bodies (Perez-Escuredo, Van Hee et al. 2016 ). Some of these transporters are expressed not only in the central nervous system (MCT1, MCT2 and MCT4) (Pierre and Pellerin 2005 ), but in other organs and tissues such as skeletal muscle, liver, and fat tissue (Bonen, Heynen et al. 2006 ). MCTs can be regulated in tissues in response to changes in production or availability of their targeted metabolites (Hajduch, Heyes et al. 2000 ; Pierre, Parent et al. 2007 ). For example, diet-induced ketosis has been shown to increase MCT1 levels in rat brain (Leino, Gerhart et al. 2001 ), and in the injured spinal cord (Streijger, Plunet et al. 2013 ). Hence, we hypothesized that there may be an adaptive mechanism that explains the reduction in BHB levels in the bloodstream over time, in which an increase in circulating BHB can increase the expression of MCTs in some tissues, leading to a rapid increase in BHB uptake by the cells. Our mitochondrial respirometry results showed that KE selectively increases mitochondrial ETC activity in the spinal cord after SCI; increases were only observed at 24H and 2 weeks post–SCI. In particular, KE led to changes of Complex I and LEAK respiration at 24H; and changes of Complex I and II, OXPHOS + CI , and LEAK respiration at 2 weeks post-SCI. Simil arly to other studies, mitochondrial function assessment was performed in tissue homogenates. Although this approach is still very relevant to assess the overall energetic status of the cord after trauma, the spinal cord contains a variety of different cell types (i.e. astrocytes, oligodendrocytes and neurons). Determining whether KE rescues mitochondrial function in all cell types or is targeting mostly a specific cell type would be of great value in order to develop more targeted therapies. Interestingly, and in that regard, a recent work by Koppel et al. demonstrated that BHB preferentially enhances neuron over astrocyte respiration in a naïve state (Koppel, Wilkins et al. 2023 ). Temporally, mitochondrial dysfunction has been shown to start as early as 2h after SCI and to continue until 24H after SCI (Sullivan, Krishnamurthy et al. 2007 ). Our measurements obtained beyond the 24H mark show that, in fact, mitochondrial dysregulation continues for at least 2 weeks after SCI. This suggests that a feasible therapeutic window not only needs to start early, as previously suggested (Sullivan, Krishnamurthy et al. 2007 ), but may also need to be extended for at least 2 weeks following injury. Contrary to what we observed using a KD (Seira, Kolehmainen et al. 2021 ), the treatment with KE did not show significant benefits at 1 week after SCI, however similar effects were seen at 2 weeks after SCI. While no differences between the injury parameters were found between groups (see Supplementary Fig. 1), inter-individual variability amongst animals, cohorts (van der Goot, Kooij et al. 2021 ), and most likely differences in the biochemical composition of the treatments might be some of the factors leading to those differences at that specific timepoint. Indeed, treatment with KE only supplies one ketone body, β-hydroxybutyrate (BHB), whereas when using a KD, the fats from the diet are broken down in the liver to the three ketone bodies: β-hydroxybutyrate (BHB), acetoacetate (AcAC), and acetone. AcAc can be further broken down into BHB (Dhillon and Gupta 2023 ). Additionally, AcAc has also been shown to act as signaling metabolite to promote muscle cell growth (Rahman, Muhammad et al. 2014 ; Zou, Meng et al. 2016 ; Zhong, Miao et al. 2021 ), increase mitochondrial function in kidney cells in vitro (Denoon, Sunilkumar et al. 2020 ), protect against glutamate toxicity in neurons (D'Agostino, Pilla et al. 2013 ), and improve motor coordination and cognition in mice with Angelman syndrome (Ciarlone, Grieco et al. 2016 ). Whether AcAc might activate signaling pathways in the CNS that could contribute to the early beneficial effects of the KDs after neurotrauma still needs to be further investigated. Differences in glycemic control between KD and KE treatments might also be relevant to explain the differences in mitochondrial function rescue observed in this study when compared to our previous KD study. For example, KD has been proven to have a therapeutic effect on glucose levels after TBI (Ritter, Robertson et al. 1996 ). Indeed, high-glucose levels have been associated with induction of mitochondrial dysfunction in cardiac models, retina, and neurons (Russell, Golovoy et al. 2002 ; Dassanayaka, Readnower et al. 2015 ; Fiorello, Treweeke et al. 2020 ; Lam, Cheung et al. 2022 ). Contrary to KD, in our experience, KE treatment alone does not change glucose levels to the same extent that KD does (data not shown). Although glucose levels decrease initially, it quickly recovers to baseline after KE. Moreover, in a comparative study, Modica et al., described that the supplementation with ketone diester had no effect on glucose when compared to KD (Modica, Flores-Felix et al. 2021 ). This may suggest that in our study, the inability of KE to decrease glucose might be accountable for the less positive effects on mitochondrial bioenergetics observed at 1 week post SCI. Nonetheless, the 2 weeks post-SCI data indicate that KE can still confer relevant bioenergetic benefits, but that these may be slightly different and delayed. Similar to our previous KD study, we investigated the protein expression levels of all the ETC complexes. Interestingly, general changes in protein expression were found at 48H, 1 and 2 weeks. Specifically KE seemed to significantly reduce the protein expression of some of the subunits of the complexes when compared to the control treated group. Only the 48H timepoint was an exception, in which we saw an increase in protein expression for Complex II after treatment (Fig. 3 ). In fact, at this timepoint, we also saw an increase in expression in the control treated group for Complex II and Complex V. The enhanced expression of some of the complexes after injury may reflect a compensatory mechanism to sustain cellular basal oxygen consumption after an increase in cellular energy demand in the injured spinal cord. Furthermore, although not in a time dependent manner, variability in the protein expression of the ETC complexes (up- or down-regulation) in different regions of the brain have been previously seen in a model of repeated stress in mild traumatic brain injury (mTBI) (Xing, Barry et al. 2013 ). Thus, it would seem reasonable to think that variability in protein expression of the ETC complexes in the spinal cord exists at different timepoints as well. Even though KE led to changes in protein expression, the increase in activity through Complexes I and II was not correlated with an increase or change in protein expression of the targeted subunits (NDUFB8 and SDH respectively). This lack of correlation between activity and protein expression levels has been previously described in cardiac tissue; in a pressure-overload hypertrophy model in rabbits, in human atrial fibrillation (Griffiths, Friehs et al. 2010 ; Emelyanova, Ashary et al. 2016 ), and in response to exercise training (Jacobs and Lundby 2013 ; Montero, Cathomen et al. 2015 ). Proposed mechanisms for the lack of synergy between subunit protein expression and respiratory function are post-translational modifications of subunits as a result of changes in oxidation. Unfortunately, we did not measure reactive oxygen production, carbonylation, nor mutations of mitochondrial DNA in this study (Ryan, Backos et al. 2012 ; Alexeyev, Shokolenko et al. 2013 ; Emelyanova, Ashary et al. 2016 ; Ngo, Sverdlov et al. 2019 ). Moreover, the formation of enzymatic supercomplexes between some of the mitochondrial complexes might also be contributing to the lack of correlation between individual complex activity and protein content. In fact, it’s been described that these supercomplexes might compensate complex dysfunction by stabilizing individual complexes such as CI to changes in the environment. Interestingly, the regulation of these supercomplexes has been also been linked to neurological disorders (Nesci, Trombetti et al. 2021 ). Whether some of these mechanisms occur in the CNS, or are involved in traumatic events is still unknown. Lastly, the understanding of the mechanisms underlying the different outcomes in regards of mitochondrial activity between KE and KD treatments at 1 week after SCI, will require further research. In summary, our study provides evidence that KE partially mitigate mitochondrial dysfunction in the acute and subacute phases after SCI. The rescue of mitochondrial function predominantly affects Complexes I and II. Furthermore, our data show that this improvement might not be fully correlated with changes in protein expression of the different ETC complexes. Overall, the work presented here provides support for the beneficial use of KE as an alternative to KD to treat acute metabolic dysfunction that occurs after SCI. Declarations Ethics approval and consent to participate All procedures were conducted according to the guidelines of the Canadian Council for Animal Care and overseen by the University of British Columbia Animal Care Committee. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests Professor Kieran Clarke is a non-executive director of TdeltaS Ltd, a spin out company of the University of Oxford, which owns the intellectual property rights to the D-3-β-hydroxybutyrate-1,3-butanediol ketone monoester. The rest of authors declare that they have no competing interests. Funding Funding for this project was granted by the Canadian Health Reserarch Funding (CIHR). M.P. was partially funded by the UBC Work Learn program. W.T. holded the John and Penny Ryan British Columbia Leadership chair in spinal cord injury in part supported by the Rick Hansen Foundation. Authors' contributions O.S., and W.T., conceived the study. O.S., and W.T., wrote the manuscript. O.S. designed experiments. K.C., and R.B., gave input and suggestios. O.S., H.P., J.L., and M.P., performed experiments and analyzed the data. O.S. designed all the drawings using BioRender. Acknowledgements We would like to thank all laboratory members for their help and suggestions. References Ahuja CS, Wilson JR, et al. Traumatic spinal cord injury. Nat Rev Dis Primers. 2017;3:17018. Alexeyev M, Shokolenko I, et al. The maintenance of mitochondrial DNA integrity–critical analysis and update. Cold Spring Harb Perspect Biol. 2013;5(5):a012641. Alizadeh A, Dyck SM, et al. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. Front Neurol. 2019;10:282. Almeida-Suhett C, Namboodiri AM, et al. The ketone ester, 3-hydroxybutyl-3-hydroxybutyrate, attenuates neurobehavioral deficits and improves neuropathology following controlled cortical impact in male rats. Nutr Neurosci. 2022;25(6):1287–99. Bonen A, Heynen M, et al. Distribution of monocarboxylate transporters MCT1-MCT8 in rat tissues and human skeletal muscle. Appl Physiol Nutr Metab. 2006;31(1):31–9. Cabral-Costa JV, Kowaltowski AJ. Neurol disorders mitochondria Mol Aspects Med. 2020;71:100826. Carneiro L, Pellerin L. Nutritional Impact on Metabolic Homeostasis and Brain Health. Front Neurosci. 2021;15:767405. Chelluboina B, Vemuganti R. Therapeutic potential of nutraceuticals to protect brain after stroke. Neurochem Int. 2021;142:104908. Cheng L, Cai B, et al. The role of mitochondrial energy metabolism in neuroprotection and axonal regeneration after spinal cord injury. Mitochondrion. 2023;69:57–63. Ciarlone SL, Grieco JC, et al. Ketone ester supplementation attenuates seizure activity, and improves behavior and hippocampal synaptic plasticity in an Angelman syndrome mouse model. Neurobiol Dis. 2016;96:38–46. Clarke K, Tchabanenko K, et al. Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects. Regul Toxicol Pharmacol. 2012;63(3):401–8. D'Agostino DP, Pilla R, et al. Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats. Am J Physiol Regul Integr Comp Physiol. 2013;304(10):R829–836. Dassanayaka S, Readnower RD, et al. High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation. Biochem J. 2015;467(1):115–26. Denoon T, Sunilkumar S, et al. Acetoacetate enhances oxidative metabolism and response to toxicants of cultured kidney cells. Toxicol Lett. 2020;323:19–24. Dhillon KK, Gupta S. (2023). Biochemistry, Ketogenesis. Emelyanova L, Ashary Z, et al. Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation. Am J Physiol Heart Circ Physiol. 2016;311(1):H54–63. Ferdous KA, Burnett G, et al. Neuroprotective Function of Fucoxanthin in Oxidative Stress-Mediated Mitochondrial Dysfunction. Curr Developments Nutr. 2022;6(Supplement1):787–7. Field R, Field T, et al. Ketogenic diets and the nervous system: a scoping review of neurological outcomes from nutritional ketosis in animal studies. Nutr Res Rev. 2022;35(2):268–81. Fiorello ML, Treweeke AT, et al. The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes. Sci Rep. 2020;10(1):19547. Gough SM, Casella A, et al. Neuroprotection by the Ketogenic Diet: Evidence and Controversies Front Nutr. 2021;8:782657. Griffiths ER, Friehs I, et al. Electron transport chain dysfunction in neonatal pressure-overload hypertrophy precedes cardiomyocyte apoptosis independent of oxidative stress. J Thorac Cardiovasc Surg. 2010;139(6):1609–17. Hajduch E, Heyes RR, et al. Lactate transport in rat adipocytes: identification of monocarboxylate transporter 1 (MCT1) and its modulation during streptozotocin-induced diabetes. FEBS Lett. 2000;479(3):89–92. Harris LK, Black RT, et al. Traumatic brain injury-induced changes in gene expression and functional activity of mitochondrial cytochrome C oxidase. J Neurotrauma. 2001;18(10):993–1009. Hoeks J, Hesselink M, et al. Mitochondrial Respiration. Encyclopedia of Exercise Medicine in Health and Disease. F. C. Mooren. Berlin, Heidelberg: Springer Berlin Heidelberg; 2012. pp. 587–90. Jacobs RA, Lundby C. (2013). Mitochondria express enhanced quality as well as quantity in association with aerobic fitness across recreationally active individuals up to elite athletes. J Appl Physiol (1985) 114(3): 344–350. Koh S, Dupuis N, et al. Ketogenic diet and Neuroinflammation Epilepsy Res. 2020;167:106454. Kong G, Liu J, et al. Ketone Metabolite beta-Hydroxybutyrate Ameliorates Inflammation After Spinal Cord Injury by Inhibiting the NLRP3 Inflammasome. Neurochem Res. 2021;46(2):213–29. Koppel SJ, Wilkins HM, et al. beta-Hydroxybutyrate preferentially enhances neuron over astrocyte respiration while signaling cellular quiescence. Mitochondrion. 2023;68:125–37. Lam CH, Cheung JK et al. (2022). Proteomic Profiling Revealed Mitochondrial Dysfunction in Photoreceptor Cells under Hyperglycemia. Int J Mol Sci 23(21). Lee JH, Streijger F et al. (2012). A contusive model of unilateral cervical spinal cord injury using the infinite horizon impactor. J Vis Exp(65). Leino RL, Gerhart DZ, et al. Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain. Neurochem Int. 2001;38(6):519–27. McEwen ML, Sullivan PG, et al. Targeting mitochondrial function for the treatment of acute spinal cord injury. Neurotherapeutics. 2011;8(2):168–79. Modica LCM, Flores-Felix K, et al. Impact of ketogenic diet and ketone diester supplementation on body weight, blood glucose, and ketones in Sprague Dawley rats fed over two weeks. Food Chem (Oxf). 2021;3:100029. Montero D, Cathomen A, et al. Haematological rather than skeletal muscle adaptations contribute to the increase in peak oxygen uptake induced by moderate endurance training. J Physiol. 2015;593(20):4677–88. Nesci S, Trombetti F et al. (2021). Molecular and Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System: Implications for Pathology. Life (Basel) 11(3). Newman JC, Verdin E. beta-Hydroxybutyrate: A Signaling Metabolite. Annu Rev Nutr. 2017;37:51–76. Ngo DTM, Sverdlov AL, et al. Oxidative modifications of mitochondrial complex II are associated with insulin resistance of visceral fat in obesity. Am J Physiol Endocrinol Metab. 2019;316(2):E168–77. Norat P, Soldozy S, et al. Mitochondrial dysfunction in neurological disorders: Exploring mitochondrial transplantation. NPJ Regen Med. 2020;5(1):22. Patel SP, Cox DH, et al. Pioglitazone treatment following spinal cord injury maintains acute mitochondrial integrity and increases chronic tissue sparing and functional recovery. Exp Neurol. 2017;293:74–82. Patel SP, Michael FM, et al. Delivery of mitoceuticals or respiratory competent mitochondria to sites of neurotrauma. Mitochondrion. 2023;68:10–4. Patel SP, Sullivan PG, et al. Acetyl-L-carnitine treatment following spinal cord injury improves mitochondrial function correlated with remarkable tissue sparing and functional recovery. Neuroscience. 2012;210:296–307. Perez-Escuredo J, Van Hee VF, et al. Monocarboxylate transporters in the brain and in cancer. Biochim Biophys Acta. 2016;1863(10):2481–97. Pierre K, Parent A, et al. Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice. J Physiol. 2007;583(Pt 2):469–86. Pierre K, Pellerin L. Monocarboxylate transporters in the central nervous system: distribution, regulation and function. J Neurochem. 2005;94(1):1–14. Rabchevsky AG, Michael FM, et al. Mitochondria focused neurotherapeutics for spinal cord injury. Exp Neurol. 2020;330:113332. Rahman M, Muhammad S, et al. The beta-hydroxybutyrate receptor HCA2 activates a neuroprotective subset of macrophages. Nat Commun. 2014;5:3944. Ritter AM, Robertson CS, et al. Evaluation of a carbohydrate-free diet for patients with severe head injury. J Neurotrauma. 1996;13(8):473–85. Russell JW, Golovoy D, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 2002;16(13):1738–48. Ryan K, Backos DS, et al. Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis. J Neurosci. 2012;32(33):11250–8. Scholpa NE, Schnellmann RG. Mitochondrial-Based Therapeutics for the Treatment of Spinal Cord Injury: Mitochondrial Biogenesis as a Potential Pharmacological Target. J Pharmacol Exp Ther. 2017;363(3):303–13. Seira O, Kolehmainen K, et al. Ketogenesis controls mitochondrial gene expression and rescues mitochondrial bioenergetics after cervical spinal cord injury in rats. Sci Rep. 2021;11(1):16359. Slater PG, Dominguez-Romero ME, et al. Mitochondrial function in spinal cord injury and regeneration. Cell Mol Life Sci. 2022;79(5):239. Soto-Mota A, Norwitz NG, et al. Why a d-beta-hydroxybutyrate monoester? Biochem Soc Trans. 2020;48(1):51–9. Soto-Mota A, Vansant H, et al. Safety and tolerability of sustained exogenous ketosis using ketone monoester drinks for 28 days in healthy adults. Regul Toxicol Pharmacol. 2019;109:104506. Streijger F, Plunet WT, et al. Ketogenic diet improves forelimb motor function after spinal cord injury in rodents. PLoS ONE. 2013;8(11):e78765. Sullivan PG, Krishnamurthy S, et al. Temporal characterization of mitochondrial bioenergetics after spinal cord injury. J Neurotrauma. 2007;24(6):991–9. Tan BT, Jiang H, et al. Neuroprotective effects of a ketogenic diet in combination with exogenous ketone salts following acute spinal cord injury. Neural Regen Res. 2020;15(10):1912–9. van der Goot MH, Kooij M, et al. Incorporating inter-individual variability in experimental design improves the quality of results of animal experiments. PLoS ONE. 2021;16(8):e0255521. Xing G, Barry ES, et al. Impact of repeated stress on traumatic brain injury-induced mitochondrial electron transport chain expression and behavioral responses in rats. Front Neurol. 2013;4:196. Yarar-Fisher C, Li J, et al. Ketogenic regimens for acute neurotraumatic events. Curr Opin Biotechnol. 2021;70:68–74. Yassine HN, Self W, et al. Nutritional metabolism and cerebral bioenergetics in Alzheimer's disease and related dementias. Alzheimers Dement; 2022. Zhong R, Miao R, et al. Acetoacetate promotes muscle cell proliferation via the miR-133b/SRF axis through the Mek-Erk-MEF2 pathway. Acta Biochim Biophys Sin (Shanghai). 2021;53(8):1009–16. Zou X, Meng J, et al. Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice. J Biol Chem. 2016;291(5):2181–95. Supplementary Files SupplementaryFigure1.docx 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-3751380","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264437985,"identity":"15f12979-dbd6-4e2a-a785-b5d2907f066e","order_by":0,"name":"Oscar Seira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYJCCAwwMNjwghgQpWtJI1AIEhxmI18LPf/jg4Yqa8zL87b0PbzBU3LNrIKRFckZawsEzx27zSJw5bmzBcKY4maAWgxs8Bgcb2G7zGEiksUkwtiUkE3SYwfnzHw42/DtHipYDOQwHG9sOwLXYEdQC9IvBwca+ZKBfjjFbJJxJSCCoBRhijz82fLOz529vY7zxoSLBnqAWVAC0IrGBRD0MDKTaMgpGwSgYBSMAAAAq1To0/4muzAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0002-0753-9669","institution":"UBC: The University of British Columbia","correspondingAuthor":true,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Seira","suffix":""},{"id":264437986,"identity":"52d02226-7160-4004-a330-9342bde47558","order_by":1,"name":"HyoJoon Park","email":"","orcid":"","institution":"UBC: The University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"HyoJoon","middleName":"","lastName":"Park","suffix":""},{"id":264437987,"identity":"ba661bdc-799f-407c-9576-fe0eebbd48b7","order_by":2,"name":"Jie Liu","email":"","orcid":"","institution":"UBC: The University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Liu","suffix":""},{"id":264437988,"identity":"ab6ff16f-883d-472e-ae45-bb2cb69a0fe5","order_by":3,"name":"Michelle Poovathukaran","email":"","orcid":"","institution":"UBC: The University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Poovathukaran","suffix":""},{"id":264437989,"identity":"8330a7a9-dec6-4ab4-b08b-416ab38486b0","order_by":4,"name":"Kieran Clarke","email":"","orcid":"","institution":": University of Oxford Department of Physiology Anatomy and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Kieran","middleName":"","lastName":"Clarke","suffix":""},{"id":264437990,"identity":"e356ade5-afc1-4565-9c05-ddcdf659a8b1","order_by":5,"name":"Robert Boushel","email":"","orcid":"","institution":"UBC: The University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Boushel","suffix":""},{"id":264437991,"identity":"434d37cc-1c98-4614-8eb7-b2bd12711367","order_by":6,"name":"Wolfram Tetzlaff","email":"","orcid":"","institution":"UBC: The University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Wolfram","middleName":"","lastName":"Tetzlaff","suffix":""}],"badges":[],"createdAt":"2023-12-14 04:36:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3751380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3751380/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49117010,"identity":"58608dfa-b397-4e53-addf-cbe1ab89320e","added_by":"auto","created_at":"2024-01-03 11:31:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental plan and BHB levels. A)\u003c/strong\u003e Experimental timeline showing the final outcome measurements of the study: BHB Levels, mitochondrial function and western blot analysis. Created with BioRender.com\u003cstrong\u003e. B-D)\u003c/strong\u003e BHB levels measured at 24H, 48H, and at several hours post oral gavage (HPOG) throughout the 1 and 2 weeks post SCI cohorts (see Experimental timeline in (A) for details.). One-way ANOVA, Tukey’s Post-hoc test. All data are mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3751380/v1/0acd35c81128fc5c80b0f0ef.jpeg"},{"id":49117009,"identity":"45a21627-aa3c-44d8-8425-d1930a580433","added_by":"auto","created_at":"2024-01-03 11:31:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":836667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial respiration.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003eSchematic representation of the mitochondrial Electron Transport Chain (ETC). Adapted from “Electron Transport Chain”, by BioRender.com. \u003cstrong\u003eB-E) \u003c/strong\u003eQuantification of mitochondrial respiration by high-resolution respirometry showing Oxygen flux values of the respiratory states. OXPHOS: Representing the maximum oxidative phosphorylation in the presence of metabolic substrates after the addition of ADP; LEAK respiration (proton leak): Mitochondrial respiration rate in the presence of an ATP synthase inhibitor (Oligomycin); Complex I (CI) Maximum respiration: Maximum respiration measured after addition of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) causing a depolarization of the cell membrane and leading to an increase in respiration; Complex II (CII) Maximum respiration: Maximum respiration measured after inhibition of CI with Rotenone and the subsequent addition of its substrate, Succinate; Complex IV (CIV) Maximum respiration: Maximum respiration estimated after inhibition of Complex III with Antimycin A, and the subsequent addition of ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as proton donor substrates. Finally, NaN3 (Sodium Azide) was added to block the respiratory chain by blocking ATPase and leaving the fraction of total respiration that is non-mitochondrial, this value is subtracted from the Complex CV Maximum respiration in order to estimate the real mitochondrial CIV respiration. One-way ANOVA, Fisher’s LSD Post-hoc test. All data are mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3751380/v1/fe1e40f2fa68faeb0de99cae.jpeg"},{"id":49117008,"identity":"59db2f9d-a746-4602-9442-f95aa4327cda","added_by":"auto","created_at":"2024-01-03 11:31:08","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":782192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blotting analysis of protein levels of the 5 ETS complexes at 24H, 48H and 1 and 2 weeks post-SCI.\u003c/strong\u003e \u003cstrong\u003eA-D)\u003c/strong\u003e Western blots of total OXPHOS proteins and subsequent quantifications showing an overall decrease in protein expression for most complexes, and for all timepoints analyzed with the exception of CII and CIV at 48H, CI and CIV at 1 week, and CV at 2 weeks. Note the increase in the protein levels for CII, CIV at 48H. As well as, decreases in expression in CI, CIII, CIV and CV at 1 week, and CIII and CV at 2 weeks in the KE versus Controls. \u0026nbsp;One-way ANOVA, Fisher’s LSD Post-hoc test (per complex). All data are mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3751380/v1/f59a9f2c0e44ffd7c90e1bde.jpeg"},{"id":49939400,"identity":"bd1d9be4-1d5b-4ed3-a463-4fbeb256f0e6","added_by":"auto","created_at":"2024-01-22 00:41:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":739326,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3751380/v1/d9b2ca5b-643e-4c89-a71a-de671791b1c0.pdf"},{"id":49117007,"identity":"10ffee4d-8f59-4a0b-aeca-4456f21bc82b","added_by":"auto","created_at":"2024-01-03 11:31:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":427464,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3751380/v1/28348e8e99a9508676df4f28.docx"}],"financialInterests":"","formattedTitle":"Ketone Esters partially and selectively rescue mitochondrial bioenergetics after acute cervical spinal cord injury in rats: A time-course","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSecondary injury cascades after traumatic spinal cord injury (SCI) result in significant pathophysiological effects (Alizadeh, Dyck et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A better understanding of the injury mechanisms following SCI is essential for the development of therapies to increase neuroprotection, restore metabolic function, and promote functional recovery.\u003c/p\u003e \u003cp\u003eIt is known that metabolism and cellular energetics are severely affected due to mitochondrial dysfunction after SCI (Rabchevsky, Michael et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), for review); targeting mitochondrial function might have an important role in promoting recovery after SCI (McEwen, Sullivan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Slater, Dominguez-Romero et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cheng, Cai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Patel, Michael et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, reduced oxidative stress is associated with improved cellular bioenergetics providing neuroprotection (Ferdous, Burnett et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this regard, ketosis after traumatic brain injury (TBI), or after traumatic spinal cord injury (SCI) improves secondary pathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics (Almeida-Suhett, Namboodiri et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)(Koh, Dupuis et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tan, Jiang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gough, Casella et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kong, Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yarar-Fisher, Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we follow up on our previous work showing that ketosis induced by a ketogenic diet (KD) was able to mitigate mitochondrial oxidative damage and dysfunction 1 week post SCI in rats (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the KD is highly restrictive and may not be suitable for many people with SCI. The ketone body, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-β-hydroxybutyrate (BHB), has multiple mechanisms of action ranging from metabolite for mitochondrial energy production to anti-inflammatory receptor ligand, signaling molecule, and epigenetic modifier (Clarke, Tchabanenko et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Newman and Verdin \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Soto-Mota, Norwitz et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yarar-Fisher, Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Circulating BHB can also be increased using exogenous ketone esters (KE). In particular, the (R)-3-hydroxybutyl (R)-3-hydroxybutyrate ketone monoester (∆G\u0026reg;) can elevate BHB for extended periods in humans at higher levels than those seen with a KD, without side effects and without sodium overload (Clarke, Tchabanenko et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Soto-Mota, Vansant et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess the use of an exogenous ketone as an alternative to a KD, and to evaluate effects on mitochondrial function in a temporal manner, we focused on the acute-subacute phases of SCI (24 hours post-injury, HPI; to 2 weeks post-injury, WPI) during which the spinal cord undergoes the most dramatic metabolic changes (Ahuja, Wilson et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Starting 3 hours following a C5 hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) which is a standard chow containing KE (12.5% w/w). The KED was combined with KE administered orally by gavage to achieve robust BHB concentrations of 2\u0026ndash;6 mM. As expected, mitochondrial function was lower after SCI at all timepoints post-SCI, and this was accompanied by a reduced expression of most of the components of the electron transport chain (ETC) for most timepoints. Although KE rescued some bioenergetic parameters at early timepoints, most of the beneficial effects were observed 2 weeks after treatment.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first study to investigate the suitability of an exogenous ketone to counteract the metabolic dysregulation and mitochondrial dysfunction at multiple timepoints during the acute and subacute phases of traumatic SCI. Our results highlight the beneficial role of KE immediately, and for at least up to 2 weeks after trauma.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design, SCI and Treatment\u003c/h2\u003e \u003cp\u003e All procedures were conducted according to the guidelines of the Canadian Council for Animal Care and overseen by the University of British Columbia Animal Care Committee. We used 44 adult male Sprague-Dawley Harlan Breeding Laboratory), group-housed (21\u0026deg;C; 12 h:12 h light:dark cycle) and given \u003cem\u003ead libitum\u003c/em\u003e access to standard rodent diet prior to the injury (Tekland 2020X, Envigo). The injury was performed as we described previously in JoVE (Lee, Streijger et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). After a unilateral C5 laminectomy, the dorsal processes of C4-C6 were held with a clamp and fixed in a frame tilted at a 22.5\u0026deg; angle. The contusion force was set to 150 Kdynes using the Infinite Horizon impactor (Precision Systems and Instrumentation, LCC, Brimstone, Virginia, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKE administration\u003c/h2\u003e \u003cp\u003eThe ketone ester (KE) was provided by TdeltaS Global Inc. (∆G\u0026reg; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.deltaGketone.com\" target=\"_blank\"\u003ewww.deltaGketone.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.deltaGketone.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The KED and control diet (CD) were prepared by Dyets Inc. (Bethlehem, Pennsylvania, USA) using the formulation from the Veech lab as previously described (Kashiwaya et al. 2013). The KE content was 0.125g/1g food and, as our rats ate around 15\u0026ndash;20 g per day, the KE intake was approximately 1.9 to 2.5 g or 1.8 to 2.3 mL (KE density\u0026thinsp;=\u0026thinsp;1.0731g/mL at 22\u0026ordm;C) (Soto-Mota et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For the oral ketone ester (OKE), rats were gavaged with either 1 mL of KE or water (controls) mixed 1:1 with Boost\u0026reg; Nutritional Supplement (total volume per gavage\u0026thinsp;=\u0026thinsp;2 mL). Boost\u0026reg; was used to help mitigate weight loss following injury and improve overall animal health without masking the effects of the KE. (For detailed nutritional information of Boost\u0026reg; visit BOOST Chocolate Meal Replacement | madewithnestle.ca).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBlood BHB levels\u003c/h2\u003e \u003cp\u003eKetone levels were measured from blood obtained by tail poking. Precision Xtra Blood Ketone Test Strips (Diabetes Express) were used with the Precision Xtra Blood and Ketone Meter (Abbott) according to manufacturer guidelines.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial Respiration, Protein extraction, and Western Blots\u003c/h2\u003e \u003cp\u003eRats were euthanized at various times post-injury with an overdose of chloral hydrate injected intraperitoneally. Rats were perfused with cold PBS and the injury epicenter (approximately half the cord, 5 mm in length) was harvested.\u003c/p\u003e \u003cp\u003eMitochondrial respiration in permeabilized spinal cord segments was measured using high-resolution respirometry (Oroboros Instruments, Innsbruck, Austria). Spinal cord tissue was permeabilized with saponin at 5 ug/ml for 20\u0026rsquo; in the shaker with gentle agitation. Tissue was washed for 10\u0026rsquo; with respiration media. Respirometry experiments were performed at 37\u0026deg;C in respiration medium containing EGTA (0.5 mM), MgCl\u003csub\u003e2\u003c/sub\u003e (3 mM), K-lactobionate (60 mM), taurine (20 mM), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (10 mM), HEPES (20 mM), sucrose (110 mM), and BSA (1 g/l), with the addition of mitochondrial substrates and inhibitors to measure coupled and uncoupled respiration, flux through complex I and II, and maximal respiration of the electron transport chain ETC. Mitochondrial respiration was expressed as weight-specific oxygen flux (pmolO\u003csub\u003e2\u003c/sub\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Chamber oxygen levels were maintained between 240 and 400 nmol\u0026middot;ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to avoid O\u003csub\u003e2\u003c/sub\u003e limitation. As previously described in our lab (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), basal respiration was first measured, then malate (2mM), pyruvate (5mM), and glutamate (10 mM) were added to measure LEAK respiration (respiration due to proton leakage and the circuit of electrons and cations that is not dependent on ATP synthase activity). ADP (2.5 mM) was then added to stimulate NADH-dependent coupled respiration through complex I, followed by oligomycin (2.5 \u0026micro;M) to measure LEAK respiration at high membrane potential during inhibition of ATP synthase. Complete, non-physiological uncoupled respiration of the ETC was measured with the titration of carbonyl cyanide-\u003cem\u003ep\u003c/em\u003e-trifluoromethoxyphenylhydrazone (CCCP) (0.5\u0026thinsp;+\u0026thinsp;0.5 \u0026micro;M). Succinate (10 mM) was added to measure reduced flavin adenine dinucleotide (FADH\u003csub\u003e2\u003c/sub\u003e)-dependent complex II respiration followed by complex I inhibition with rotenone (0.5 \u0026micro;M). Residual oxygen consumption (ROX) attributed to non-mitochondrial respiration was measured with addition of Antimycin A (2.5 \u0026micro;M) to inhibit complex III of the ETC. Finally, ascorbate (2 mM) and \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026prime;,\u003cem\u003eN\u003c/em\u003e\u0026prime;-tetramethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine dihydrochloride (TMPD) (0.5 mM) was added to measure the isolated respiratory rate of cytochrome oxidase (complex IV respiration).\u003c/p\u003e \u003cp\u003eProtein lysate was prepared using plastic Dounce homogenizers to disrupt tissue in Tris-EDTA SDS lysis buffer (0.01M Tris-HCl (pH 8, VWR)), 1mM EDTA (Ambion, ThermoFisher Scientific), 0.1% SDS (VWR), cOmplete\u0026trade;, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Mississauga, ON), 100mM NaF (ThermoFisher Scientific), 25mM β-glycerophosphate (MilliporeSigma), 10mM Pyrophosphate (ThermoFisher Scientific), and 200\u0026micro;M Orthovanadate (ThermoFisher Scientific)). Samples were spun at 14000rpm and an aliquot of supernatant was taken for Western blotting.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean values, and error bars indicate\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, as noted. All statistical analysis were performed using the Prism software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.graphpad.com\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Information about the tests used can be found in the Figure Legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eA Ketone Ester Diet (KED) in combination with orally administered Ketone Esters (OKE) induces ketosis acutely after SCI in rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe examined the effects of the ketone ester (KE) in raising BHB levels at 24HPI, 48HPI, 7DPI and 14DPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Blood samples were collected before injury and before starting with the dietary treatments for all timepoints. We found that baseline levels are fairly consistent throughout all timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, BHB levels were significantly higher in the KE treated groups compared to the control group both, 24HPI and 48HPI. Note the higher BHB levels at 48HPI compared to 24HPI. The BHB levels for these two timepoints were analyzed before euthanasia around 8 Hours Post Oral Gavage (HPOG) (See treatment paradigm schematic for clarification, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows BHB levels taken throughout the treatment of the 7 day survival cohort. Measurements during the first 3 days post injury were taken 8HPOG. Note the increase in BHB levels over the first 3 days. BHB levels were then analyzed at 24HPOG at 6DPI (as an arbitrary half way checkpoint control BHB measurement) and before euthanasia (12HPOG) at 7DPI. Surprisingly, we did not find any significant increase in BHB levels at 7DPI measured 12HPOG. The 14 days survival group shows significant BHB increases over time during the first 7DPI in the KE group compared to the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). At 24HPOG-10DPI, another half way through checkpoint, an increase in BHB levels in the treatment group was still observed. Finally, at 14DPI one last measurement before euthanasia was taken 24HPOG, showing a significant increase in BHB levels in the KE treated group compared to the control group as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKetone Esters selectively improve mitochondrial respiration at 24 hours and 2 weeks post-SCI.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMitochondrial respirometry is a commonly used method to evaluate mitochondrial function and oxidative phosphorylation (OXPHOS) in mitochondria, cells, and tissues (Hoeks, Hesselink et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This methodology enables assessment of the overall function of mitochondria in a specific tissue, and discrete functional components and characteristics of the electron transport chain (ETC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) that may be dysfunctional as a result of trauma or pathological disorders.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the possible beneficial effects of ketone esters (KE) in modulating the metabolic distress observed acutely after spinal cord trauma, we performed a respirometry study of nervous tissue located at the epicenter of the injured cervical spinal cords at 24HPI, 48HPI, 1WPI and 2WPI. We followed the same titration protocol we previously used in our ketogenic diet study (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (See Material and Methods). Basal mitochondrial respiration (oxygen consumption at baseline) was consistent for the different timepoints. Overall, no significant differences were observed when comparing the uninjured cords to the injured-treated spinal cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E), however, we detected a significant increase in basal respiration in the injured cords compared to the uninjured cords at 48H (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). No differences in respiration between the KE and the control treated group were observed at baseline for any time point studied. In addition, an overall pattern of a decline in mitochondrial respiration across all timepoints was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). Yet, at 2 weeks post injury, while there was a trend towards a lower maximum respiration via CI and CII, respiration via these 2 complexes was not significantly decreased when compared to the uninjured spinal cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe respirometry data from all separate timepoints show that uncoupled mitochondrial respiration with Complex I (CIu) and Complex II (CIIu) substrates was reduced at 24H following SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). LEAK respiration measured after oligomycin addition to block ATP-synthase activity was significantly decreased after injury in the control group. The decrease in LEAK after injury was rescued with the KE treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the KE group, mitochondrial respiration with electron supply through Complex I was significantly higher compared to the control group. No differences in maximum mitochondrial respiration via Complex II and or the isolated respiration rate of cytochrome c oxidase (Complex IV) were seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we looked at the oxygen consumption at 48H and 7 weeks post-SCI, and did not find any differences in mitochondrial respiration between the KE treated group and the control group after injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). However, we noticed a trend towards an increase in respiration with Complex II substrate supply at 48H (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Lastly, we analyzed the 2 weeks post-SCI data. Overall, and as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, respirometry data at this timepoint show the most significant differences between KE treatment and the controls compared to the results from earlier timepoints studied. Specifically, the treatment of the injured group with KE showed a significant increase of ADP-stimulated OXPHOS for Complex I compared to the injured control group. Furthermore, LEAK, Complex I (CIu) and Complex II (CIIu) uncoupled respiration also showed a significant increase in the injured KE group compared to the injured control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Similar to earlier timepoints, no significant differences in maximum mitochondrial respiration of Complex IV were detected. Interestingly, the treatment with KE led to an overall increase in mitochondrial respiration that not only rescued the mitochondrial bioenergetic dysfunction associated with the injury, but it was also able to significantly increase the oxygen consumption to levels that were higher than the ones observed in the uninjured group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKetone Esters selectively modify the expression of OXPHOS complexes of the Electron Transport Chain (ETC).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eChanges in mitochondrial gene expression have been previously linked to changes in mitochondrial function after neurotrauma in the CNS (Harris, Black et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In fact, we demonstrated that the treatment with a KD led to an increase in protein expression for some of the OXPHOS complexes that are part of the electron transport chain (ETC) in the spinal cord after injury (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further elucidate if the use of KE might have similar effects to those observed with a KD, and whether the data from our mitochondrial respirometry could be associated with changes in protein expression of those complexes, we performed individual western blotting analysis on tissue homogenates from the epicenter of the injury for each of the OXPHOS complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the western blots corresponding to the protein subunits of the OXPHOS complexes for the four different timepoints after SCI. Similar to our mitochondrial respirometry data, a single uninjured group was used as non-injured tissue control, and to normalize all quantification data (see uninjured blot in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe quantification of the bands revealed that at 24H post SCI, there was a general and significant decrease in protein expression of all complexes after injury compared to the uninjured spinal cords. However, the treatment with KE did not reverse the injury effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similar to the 24H post-SCI, quantification of the 48 hour post-SCI blots, showed significantly less expression of Complex I, III and IV after injury, while Complex II and V did not appear to change at this timepoint. It was interesting that Complex II expression significantly increased after injury with the KE resulting in an even larger increase in expression compared to the control group. A trend towards an increase in Complex V with KE treatment was also found (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The band quantifications at 1 week post-SCI revealed a significant decrease in protein expression of Complex II, III and V (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) after injury. Interestingly, the expression of Complex I and IV remained at the same levels than the uninjured cords. Regarding the treatment effects, and surprisingly to us, we found a significant decrease in protein expression after KE treatment of Complexes I, III and V. Overall, KE caused a decrease in expression at 1 week post-SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Lastly, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD illustrates the quantification for the 2 weeks post-SCI timepoint. Here, we also found a generalized decrease in protein expression for the OXPHOS complexes I, II, III and IV after injury, with the exception of Complex V (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At this timepoint, the KE changes in expression were similar to those at 1 week post-SCI. Thus, Complex III and V expression were significantly reduced after treatment compared to the control group (and also to the uninjured group regarding Complex V). No significant differences were found in the levels of expression between KE and controls for Complexes I, II and IV.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMitochondria play an essential role in creating energy to drive cellular function and biological processes. Dysregulation of mitochondrial function has been closely linked to numerous neurological diseases and disorders (Cabral-Costa and Kowaltowski \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Norat, Soldozy et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Secondary injury occurs after initial damage of the CNS, and leads to systemic as well as tissue metabolic changes (Scholpa and Schnellmann \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rabchevsky, Michael et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Slater, Dominguez-Romero et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The secondary injury propagates for weeks-to-months following the primary insult, and contributes to the lack of recovery in SCI patients. Over the last few years, progress has been made in studying and understanding the role and use of dietary interventions to treat the metabolic dysregulation in neurological diseases and disorders, including neurotrauma events ((Carneiro and Pellerin \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chelluboina and Vemuganti \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yarar-Fisher, Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Field, Field et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yassine, Self et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, improving mitochondrial function after SCI has been already been shown to lead neuroprotection and functional recovery (Patel, Sullivan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Patel, Cox et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we sought to evaluate the use of KE on restoring energy metabolism following SCI. We studied mitochondrial respiratory function and oxidative phosphorylation (OXPHOS) on injured cervical spinal cord segments from rats during the acute and subacute phases of the injury. Additionally, we also wanted to investigate whether KE might potentially mimic some of the beneficial effects on mitochondrial function that we found using a ketogenic diet (KD) (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To induce ketosis in rats that underwent C5 hemi-contusion, we delivered the KE orally (OKE), and incorporated them in the diet (KED). After SCI, animals tend to lose their appetite for the first 24H-48H. Based on previous work from our lab (unpublished data), we knew that in order to increase β-hydroxybutyrate (BHB) levels in the bloodstream of the animals during those initial hours, an oral supplementation of KE was required. Our treatment paradigm was designed with that consideration in mind, and combines the use of KED and oral administration of KE (OKE). Based on previous literature, we knew that those initial days after injury are critical in regards to cell bioenergetics dysregulation (Sullivan, Krishnamurthy et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; McEwen, Sullivan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), so an intensive treatment regime was designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using this treatment paradigm, we were able to induce ketosis in the animals at all timepoints studied. We found a certain degree of variability between the different cohorts regarding the BHB levels that were reached. For example, the levels 8 hours post oral gavage (HPOG) after 2 days of treatment were singly lower in the 1 week post\u0026ndash;SCI timepoint compared to the others. BHB levels usually peak between 3 and 7 days post-treatment, and are followed by a trend towards a decrease after that. In addition, the differences between control and treated groups becomes smaller. We believe that this time adaptation to the treatment could be associated with cell regulation of some receptors such as the Monocarboxylate transporters (MCTs) (Leino, Gerhart et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). MCTs have been identified as transporters of lactate, pyruvate, and ketone bodies (Perez-Escuredo, Van Hee et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Some of these transporters are expressed not only in the central nervous system (MCT1, MCT2 and MCT4) (Pierre and Pellerin \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), but in other organs and tissues such as skeletal muscle, liver, and fat tissue (Bonen, Heynen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). MCTs can be regulated in tissues in response to changes in production or availability of their targeted metabolites (Hajduch, Heyes et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pierre, Parent et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). For example, diet-induced ketosis has been shown to increase MCT1 levels in rat brain (Leino, Gerhart et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and in the injured spinal cord (Streijger, Plunet et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Hence, we hypothesized that there may be an adaptive mechanism that explains the reduction in BHB levels in the bloodstream over time, in which an increase in circulating BHB can increase the expression of MCTs in some tissues, leading to a rapid increase in BHB uptake by the cells.\u003c/p\u003e \u003cp\u003eOur mitochondrial respirometry results showed that KE selectively increases mitochondrial ETC activity in the spinal cord after SCI; increases were only observed at 24H and 2 weeks post\u0026ndash;SCI. In particular, KE led to changes of Complex I and LEAK respiration at 24H; and changes of Complex I and II, OXPHOS\u003csub\u003e+\u0026thinsp;CI\u003c/sub\u003e, and LEAK respiration at 2 weeks post-SCI. Simil arly to other studies, mitochondrial function assessment was performed in tissue homogenates. Although this approach is still very relevant to assess the overall energetic status of the cord after trauma, the spinal cord contains a variety of different cell types (i.e. astrocytes, oligodendrocytes and neurons). Determining whether KE rescues mitochondrial function in all cell types or is targeting mostly a specific cell type would be of great value in order to develop more targeted therapies. Interestingly, and in that regard, a recent work by Koppel et al. demonstrated that BHB preferentially enhances neuron over astrocyte respiration in a na\u0026iuml;ve state (Koppel, Wilkins et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTemporally, mitochondrial dysfunction has been shown to start as early as 2h after SCI and to continue until 24H after SCI (Sullivan, Krishnamurthy et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Our measurements obtained beyond the 24H mark show that, in fact, mitochondrial dysregulation continues for at least 2 weeks after SCI. This suggests that a feasible therapeutic window not only needs to start early, as previously suggested (Sullivan, Krishnamurthy et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), but may also need to be extended for at least 2 weeks following injury. Contrary to what we observed using a KD (Seira, Kolehmainen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the treatment with KE did not show significant benefits at 1 week after SCI, however similar effects were seen at 2 weeks after SCI. While no differences between the injury parameters were found between groups (see Supplementary Fig.\u0026nbsp;1), inter-individual variability amongst animals, cohorts (van der Goot, Kooij et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and most likely differences in the biochemical composition of the treatments might be some of the factors leading to those differences at that specific timepoint. Indeed, treatment with KE only supplies one ketone body, β-hydroxybutyrate (BHB), whereas when using a KD, the fats from the diet are broken down in the liver to the three ketone bodies: β-hydroxybutyrate (BHB), acetoacetate (AcAC), and acetone. AcAc can be further broken down into BHB (Dhillon and Gupta \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, AcAc has also been shown to act as signaling metabolite to promote muscle cell growth (Rahman, Muhammad et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zou, Meng et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhong, Miao et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), increase mitochondrial function in kidney cells \u003cem\u003ein vitro\u003c/em\u003e (Denoon, Sunilkumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), protect against glutamate toxicity in neurons (D'Agostino, Pilla et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and improve motor coordination and cognition in mice with Angelman syndrome (Ciarlone, Grieco et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Whether AcAc might activate signaling pathways in the CNS that could contribute to the early beneficial effects of the KDs after neurotrauma still needs to be further investigated.\u003c/p\u003e \u003cp\u003eDifferences in glycemic control between KD and KE treatments might also be relevant to explain the differences in mitochondrial function rescue observed in this study when compared to our previous KD study. For example, KD has been proven to have a therapeutic effect on glucose levels after TBI (Ritter, Robertson et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Indeed, high-glucose levels have been associated with induction of mitochondrial dysfunction in cardiac models, retina, and neurons (Russell, Golovoy et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Dassanayaka, Readnower et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fiorello, Treweeke et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lam, Cheung et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Contrary to KD, in our experience, KE treatment alone does not change glucose levels to the same extent that KD does (data not shown). Although glucose levels decrease initially, it quickly recovers to baseline after KE. Moreover, in a comparative study, Modica et al., described that the supplementation with ketone diester had no effect on glucose when compared to KD (Modica, Flores-Felix et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This may suggest that in our study, the inability of KE to decrease glucose might be accountable for the less positive effects on mitochondrial bioenergetics observed at 1 week post SCI. Nonetheless, the 2 weeks post-SCI data indicate that KE can still confer relevant bioenergetic benefits, but that these may be slightly different and delayed.\u003c/p\u003e \u003cp\u003eSimilar to our previous KD study, we investigated the protein expression levels of all the ETC complexes. Interestingly, general changes in protein expression were found at 48H, 1 and 2 weeks. Specifically KE seemed to significantly reduce the protein expression of some of the subunits of the complexes when compared to the control treated group. Only the 48H timepoint was an exception, in which we saw an increase in protein expression for Complex II after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In fact, at this timepoint, we also saw an increase in expression in the control treated group for Complex II and Complex V. The enhanced expression of some of the complexes after injury may reflect a compensatory mechanism to sustain cellular basal oxygen consumption after an increase in cellular energy demand in the injured spinal cord. Furthermore, although not in a time dependent manner, variability in the protein expression of the ETC complexes (up- or down-regulation) in different regions of the brain have been previously seen in a model of repeated stress in mild traumatic brain injury (mTBI) (Xing, Barry et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, it would seem reasonable to think that variability in protein expression of the ETC complexes in the spinal cord exists at different timepoints as well.\u003c/p\u003e \u003cp\u003eEven though KE led to changes in protein expression, the increase in activity through Complexes I and II was not correlated with an increase or change in protein expression of the targeted subunits (NDUFB8 and SDH respectively). This lack of correlation between activity and protein expression levels has been previously described in cardiac tissue; in a pressure-overload hypertrophy model in rabbits, in human atrial fibrillation (Griffiths, Friehs et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Emelyanova, Ashary et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and in response to exercise training (Jacobs and Lundby \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Montero, Cathomen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Proposed mechanisms for the lack of synergy between subunit protein expression and respiratory function are post-translational modifications of subunits as a result of changes in oxidation. Unfortunately, we did not measure reactive oxygen production, carbonylation, nor mutations of mitochondrial DNA in this study (Ryan, Backos et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Alexeyev, Shokolenko et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Emelyanova, Ashary et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ngo, Sverdlov et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, the formation of enzymatic supercomplexes between some of the mitochondrial complexes might also be contributing to the lack of correlation between individual complex activity and protein content. In fact, it\u0026rsquo;s been described that these supercomplexes might compensate complex dysfunction by stabilizing individual complexes such as CI to changes in the environment. Interestingly, the regulation of these supercomplexes has been also been linked to neurological disorders (Nesci, Trombetti et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Whether some of these mechanisms occur in the CNS, or are involved in traumatic events is still unknown. Lastly, the understanding of the mechanisms underlying the different outcomes in regards of mitochondrial activity between KE and KD treatments at 1 week after SCI, will require further research.\u003c/p\u003e \u003cp\u003eIn summary, our study provides evidence that KE partially mitigate mitochondrial dysfunction in the acute and subacute phases after SCI. The rescue of mitochondrial function predominantly affects Complexes I and II. Furthermore, our data show that this improvement might not be fully correlated with changes in protein expression of the different ETC complexes. Overall, the work presented here provides support for the beneficial use of KE as an alternative to KD to treat acute metabolic dysfunction that occurs after SCI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were conducted according to the guidelines of the Canadian Council for Animal Care and overseen by the University of British Columbia Animal Care Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProfessor Kieran Clarke is a non-executive director of TdeltaS Ltd, a spin out company of the University of Oxford, which owns the intellectual property rights to the D-3-\u0026beta;-hydroxybutyrate-1,3-butanediol ketone monoester. The rest of authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this project was granted by the Canadian Health Reserarch Funding (CIHR). M.P. was partially funded by the UBC Work Learn program. W.T. holded the John and Penny Ryan British Columbia Leadership chair in spinal cord injury in part supported by the Rick Hansen Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eO.S., and W.T., conceived the study. O.S., and W.T., wrote the manuscript. O.S. designed experiments. K.C., and R.B., gave input and suggestios. O.S., H.P., J.L., and M.P., performed experiments and analyzed the data. O.S. designed all the drawings using BioRender.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all laboratory members for their help and suggestions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhuja CS, Wilson JR, et al. Traumatic spinal cord injury. Nat Rev Dis Primers. 2017;3:17018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexeyev M, Shokolenko I, et al. The maintenance of mitochondrial DNA integrity\u0026ndash;critical analysis and update. Cold Spring Harb Perspect Biol. 2013;5(5):a012641.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlizadeh A, Dyck SM, et al. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. Front Neurol. 2019;10:282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmeida-Suhett C, Namboodiri AM, et al. The ketone ester, 3-hydroxybutyl-3-hydroxybutyrate, attenuates neurobehavioral deficits and improves neuropathology following controlled cortical impact in male rats. Nutr Neurosci. 2022;25(6):1287\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonen A, Heynen M, et al. Distribution of monocarboxylate transporters MCT1-MCT8 in rat tissues and human skeletal muscle. Appl Physiol Nutr Metab. 2006;31(1):31\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabral-Costa JV, Kowaltowski AJ. Neurol disorders mitochondria Mol Aspects Med. 2020;71:100826.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarneiro L, Pellerin L. Nutritional Impact on Metabolic Homeostasis and Brain Health. Front Neurosci. 2021;15:767405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChelluboina B, Vemuganti R. Therapeutic potential of nutraceuticals to protect brain after stroke. Neurochem Int. 2021;142:104908.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng L, Cai B, et al. The role of mitochondrial energy metabolism in neuroprotection and axonal regeneration after spinal cord injury. Mitochondrion. 2023;69:57\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiarlone SL, Grieco JC, et al. Ketone ester supplementation attenuates seizure activity, and improves behavior and hippocampal synaptic plasticity in an Angelman syndrome mouse model. Neurobiol Dis. 2016;96:38\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarke K, Tchabanenko K, et al. Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects. Regul Toxicol Pharmacol. 2012;63(3):401\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Agostino DP, Pilla R, et al. Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats. Am J Physiol Regul Integr Comp Physiol. 2013;304(10):R829\u0026ndash;836.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDassanayaka S, Readnower RD, et al. High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation. Biochem J. 2015;467(1):115\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenoon T, Sunilkumar S, et al. Acetoacetate enhances oxidative metabolism and response to toxicants of cultured kidney cells. Toxicol Lett. 2020;323:19\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhillon KK, Gupta S. (2023). Biochemistry, Ketogenesis.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmelyanova L, Ashary Z, et al. Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation. Am J Physiol Heart Circ Physiol. 2016;311(1):H54\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerdous KA, Burnett G, et al. Neuroprotective Function of Fucoxanthin in Oxidative Stress-Mediated Mitochondrial Dysfunction. Curr Developments Nutr. 2022;6(Supplement1):787\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eField R, Field T, et al. Ketogenic diets and the nervous system: a scoping review of neurological outcomes from nutritional ketosis in animal studies. Nutr Res Rev. 2022;35(2):268\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFiorello ML, Treweeke AT, et al. The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes. Sci Rep. 2020;10(1):19547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGough SM, Casella A, et al. Neuroprotection by the Ketogenic Diet: Evidence and Controversies Front Nutr. 2021;8:782657.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffiths ER, Friehs I, et al. Electron transport chain dysfunction in neonatal pressure-overload hypertrophy precedes cardiomyocyte apoptosis independent of oxidative stress. J Thorac Cardiovasc Surg. 2010;139(6):1609\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHajduch E, Heyes RR, et al. Lactate transport in rat adipocytes: identification of monocarboxylate transporter 1 (MCT1) and its modulation during streptozotocin-induced diabetes. FEBS Lett. 2000;479(3):89\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris LK, Black RT, et al. Traumatic brain injury-induced changes in gene expression and functional activity of mitochondrial cytochrome C oxidase. J Neurotrauma. 2001;18(10):993\u0026ndash;1009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoeks J, Hesselink M, et al. Mitochondrial Respiration. Encyclopedia of Exercise Medicine in Health and Disease. F. C. Mooren. Berlin, Heidelberg: Springer Berlin Heidelberg; 2012. pp. 587\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacobs RA, Lundby C. (2013). Mitochondria express enhanced quality as well as quantity in association with aerobic fitness across recreationally active individuals up to elite athletes. J Appl Physiol (1985) 114(3): 344\u0026ndash;350.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoh S, Dupuis N, et al. Ketogenic diet and Neuroinflammation Epilepsy Res. 2020;167:106454.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong G, Liu J, et al. Ketone Metabolite beta-Hydroxybutyrate Ameliorates Inflammation After Spinal Cord Injury by Inhibiting the NLRP3 Inflammasome. Neurochem Res. 2021;46(2):213\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoppel SJ, Wilkins HM, et al. beta-Hydroxybutyrate preferentially enhances neuron over astrocyte respiration while signaling cellular quiescence. Mitochondrion. 2023;68:125\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLam CH, Cheung JK et al. (2022). Proteomic Profiling Revealed Mitochondrial Dysfunction in Photoreceptor Cells under Hyperglycemia. Int J Mol Sci 23(21).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JH, Streijger F et al. (2012). A contusive model of unilateral cervical spinal cord injury using the infinite horizon impactor. J Vis Exp(65).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeino RL, Gerhart DZ, et al. Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain. Neurochem Int. 2001;38(6):519\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcEwen ML, Sullivan PG, et al. Targeting mitochondrial function for the treatment of acute spinal cord injury. Neurotherapeutics. 2011;8(2):168\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eModica LCM, Flores-Felix K, et al. Impact of ketogenic diet and ketone diester supplementation on body weight, blood glucose, and ketones in Sprague Dawley rats fed over two weeks. Food Chem (Oxf). 2021;3:100029.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero D, Cathomen A, et al. Haematological rather than skeletal muscle adaptations contribute to the increase in peak oxygen uptake induced by moderate endurance training. J Physiol. 2015;593(20):4677\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNesci S, Trombetti F et al. (2021). Molecular and Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System: Implications for Pathology. Life (Basel) 11(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewman JC, Verdin E. beta-Hydroxybutyrate: A Signaling Metabolite. Annu Rev Nutr. 2017;37:51\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgo DTM, Sverdlov AL, et al. Oxidative modifications of mitochondrial complex II are associated with insulin resistance of visceral fat in obesity. Am J Physiol Endocrinol Metab. 2019;316(2):E168\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorat P, Soldozy S, et al. Mitochondrial dysfunction in neurological disorders: Exploring mitochondrial transplantation. NPJ Regen Med. 2020;5(1):22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel SP, Cox DH, et al. Pioglitazone treatment following spinal cord injury maintains acute mitochondrial integrity and increases chronic tissue sparing and functional recovery. Exp Neurol. 2017;293:74\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel SP, Michael FM, et al. Delivery of mitoceuticals or respiratory competent mitochondria to sites of neurotrauma. Mitochondrion. 2023;68:10\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel SP, Sullivan PG, et al. Acetyl-L-carnitine treatment following spinal cord injury improves mitochondrial function correlated with remarkable tissue sparing and functional recovery. Neuroscience. 2012;210:296\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Escuredo J, Van Hee VF, et al. Monocarboxylate transporters in the brain and in cancer. Biochim Biophys Acta. 2016;1863(10):2481\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierre K, Parent A, et al. Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice. J Physiol. 2007;583(Pt 2):469\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierre K, Pellerin L. Monocarboxylate transporters in the central nervous system: distribution, regulation and function. J Neurochem. 2005;94(1):1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabchevsky AG, Michael FM, et al. Mitochondria focused neurotherapeutics for spinal cord injury. Exp Neurol. 2020;330:113332.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman M, Muhammad S, et al. The beta-hydroxybutyrate receptor HCA2 activates a neuroprotective subset of macrophages. Nat Commun. 2014;5:3944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitter AM, Robertson CS, et al. Evaluation of a carbohydrate-free diet for patients with severe head injury. J Neurotrauma. 1996;13(8):473\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRussell JW, Golovoy D, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 2002;16(13):1738\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyan K, Backos DS, et al. Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis. J Neurosci. 2012;32(33):11250\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScholpa NE, Schnellmann RG. Mitochondrial-Based Therapeutics for the Treatment of Spinal Cord Injury: Mitochondrial Biogenesis as a Potential Pharmacological Target. J Pharmacol Exp Ther. 2017;363(3):303\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeira O, Kolehmainen K, et al. Ketogenesis controls mitochondrial gene expression and rescues mitochondrial bioenergetics after cervical spinal cord injury in rats. Sci Rep. 2021;11(1):16359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlater PG, Dominguez-Romero ME, et al. Mitochondrial function in spinal cord injury and regeneration. Cell Mol Life Sci. 2022;79(5):239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto-Mota A, Norwitz NG, et al. Why a d-beta-hydroxybutyrate monoester? Biochem Soc Trans. 2020;48(1):51\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto-Mota A, Vansant H, et al. Safety and tolerability of sustained exogenous ketosis using ketone monoester drinks for 28 days in healthy adults. Regul Toxicol Pharmacol. 2019;109:104506.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStreijger F, Plunet WT, et al. Ketogenic diet improves forelimb motor function after spinal cord injury in rodents. PLoS ONE. 2013;8(11):e78765.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSullivan PG, Krishnamurthy S, et al. Temporal characterization of mitochondrial bioenergetics after spinal cord injury. J Neurotrauma. 2007;24(6):991\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan BT, Jiang H, et al. Neuroprotective effects of a ketogenic diet in combination with exogenous ketone salts following acute spinal cord injury. Neural Regen Res. 2020;15(10):1912\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Goot MH, Kooij M, et al. Incorporating inter-individual variability in experimental design improves the quality of results of animal experiments. PLoS ONE. 2021;16(8):e0255521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXing G, Barry ES, et al. Impact of repeated stress on traumatic brain injury-induced mitochondrial electron transport chain expression and behavioral responses in rats. Front Neurol. 2013;4:196.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarar-Fisher C, Li J, et al. Ketogenic regimens for acute neurotraumatic events. Curr Opin Biotechnol. 2021;70:68\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYassine HN, Self W, et al. Nutritional metabolism and cerebral bioenergetics in Alzheimer's disease and related dementias. Alzheimers Dement; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong R, Miao R, et al. Acetoacetate promotes muscle cell proliferation via the miR-133b/SRF axis through the Mek-Erk-MEF2 pathway. Acta Biochim Biophys Sin (Shanghai). 2021;53(8):1009\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou X, Meng J, et al. Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice. J Biol Chem. 2016;291(5):2181\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\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":"Ketone monoester, electron transport chain, spinal cord injury, mitochondria, temporal changes","lastPublishedDoi":"10.21203/rs.3.rs-3751380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3751380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) pathology and pathophysiology can be attributed to both primary physical injury and secondary injury cascades. Secondary injury cascades involve dysregulated metabolism and energetic deficits, which are directly linked to compromised mitochondrial bioenergetics. Rescuing mitochondrial function and reducing oxidative stress are associated with neuroprotection. In this regard, ketosis after traumatic brain injury (TBI), or after SCI, improves secondary neuropathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics. Here, we follow up on our previous study and have used an exogenous ketone monoester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE), as an alternative to a ketogenic diet, focusing on mitochondrial function between 1 and 14 days after injury. Starting 3 hours following a C5 hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) combined with KE administered orally (OKE). We found that mitochondrial function was reduced after SCI at all times post-SCI, accompanied by reduced expression of most of the components of the electron transport chain (ETC). The KE rescued some of the bioenergetic parameters 24 hours after SCI when BHB concentrations were ~\u0026thinsp;2 mM, but most of the beneficial effects were observed at 2 weeks after injury with BHB concentrations reaching values of 4\u0026ndash;6 mM. To our knowledge, this is the first report of beneficial effects of KE in rescuing mitochondrial function after SCI and demonstrates the suitability of KE to ameliorate the metabolic dysregulation that occurs after traumatic SCI without requiring a restrictive dietary regime.\u003c/p\u003e","manuscriptTitle":"Ketone Esters partially and selectively rescue mitochondrial bioenergetics after acute cervical spinal cord injury in rats: A time-course","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 11:31:03","doi":"10.21203/rs.3.rs-3751380/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":"da2bb649-acd8-4276-ba00-eacaf2554655","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T00:33:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 11:31:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3751380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3751380","identity":"rs-3751380","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}