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A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice

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Abstract

Cerebral blood flow (CBF) is uniquely regulated by the anatomical design of the cerebral vasculature as well as through neurovascular coupling. The process of directing the CBF to meet the energy demands of neuronal activity is referred to as neurovascular coupling. Microvasculature in the brain constitutes the critical component of the neurovascular coupling. Mitochondria provide the majority of ATP to meet the high-energy demand of the brain. Impairment of mitochondrial function plays a central role in several age-related diseases such as hypertension, ischemic brain injury, Alzheimer’s disease, and Parkinson disease. Interestingly, microvessels and small arteries of the brain have been the focus of the studies implicating the vascular mechanisms in several age-related neurological diseases. However, the role of microvascular mitochondrial dysfunction in age-related diseases remains unexplored. To date, high-throughput assay for measuring mitochondrial respiration in microvessels is lacking. The current study presents a novel method to measure mitochondrial respiratory parameters in freshly isolated microvessels from mouse brain ex vivo using Seahorse XFe24 Analyzer. We validated the method by demonstrating impairments of mitochondrial respiration in cerebral microvessels isolated from old mice compared to the young mice. Thus, application of mitochondrial respiration studies in microvessels will help identify novel vascular mechanisms underlying a variety of age-related neurological diseases.

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References

  • Belanger M, Allaman I, Magistretti PJ (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab 14:724–738

    Article  PubMed  CAS  Google Scholar 

  • Castellani R, Hirai K, Aliev G, Drew KL, Nunomura A, Takeda A, Cash AD, Obrenovich ME, Perry G, Smith MA (2002) Role of mitochondrial dysfunction in Alzheimer’s disease. J Neurosci Res 70:357–360

    Article  PubMed  CAS  Google Scholar 

  • Chan PH (2005) Mitochondrial dysfunction and oxidative stress as determinants of cell death/survival in stroke. Ann N Y Acad Sci 1042:203–209

    Article  PubMed  CAS  Google Scholar 

  • Clark LC (1956) Monitor and control of blood and issue oxygen tensions. Trans Am Soc Artif Intern Organs 2:41–48

    Google Scholar 

  • Clark LC Jr, Sachs G (1968) Bioelectrodes for tissue metabolism. Ann N Y Acad Sci 148:133–153

    Article  PubMed  CAS  Google Scholar 

  • Csiszar A, Tarantini S, Fulop GA et al (2017) Hypertension impairs neurovascular coupling and promotes microvascular injury: role in exacerbation of Alzheimer’s disease. Geroscience 39:359–372

    Article  PubMed Central  PubMed  Google Scholar 

  • Dai DF, Rabinovitch PS, Ungvari Z (2012) Mitochondria and cardiovascular aging. Circ Res 110:1109–1124

    Article  PubMed  CAS  Google Scholar 

  • de la Torre JC (1994) Impaired brain microcirculation may trigger Alzheimer’s disease. Neurosci Biobehav Rev 18:397–401

    Article  PubMed  Google Scholar 

  • Deepa SS, Bhaskaran S, Espinoza S, Brooks SV, McArdle A, Jackson MJ, van Remmen H, Richardson A (2017) A new mouse model of frailty: the Cu/Zn superoxide dismutase knockout mouse. Geroscience 39:187–198

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Devi L, Prabhu BM, Galati DF, Avadhani NG, Anandatheerthavarada HK (2006) Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer’s disease brain is associated with mitochondrial dysfunction. J Neurosci 26:9057–9068

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Farkas E, Luiten PG (2001) Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog Neurobiol 64:575–611

    Article  PubMed  CAS  Google Scholar 

  • Ferrick DA, Neilson A, Beeson C (2008) Advances in measuring cellular bioenergetics using extracellular flux. Drug Discov Today 13:268–274

    Article  PubMed  CAS  Google Scholar 

  • Girouard H, Iadecola C (2006) Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease. J Appl Physiol (1985) 100:328–335

    Article  CAS  Google Scholar 

  • Grassi B, Poole DC, Richardson RS, Knight DR, Erickson BK, Wagner PD (1996) Muscle O2 uptake kinetics in humans: implications for metabolic control. J Appl Physiol (1985) 80:988–998

    Article  CAS  Google Scholar 

  • Grimmig B, Kim SH, Nash K, Bickford PC, Douglas SR (2017) Neuroprotective mechanisms of astaxanthin: a potential therapeutic role in preserving cognitive function in age and neurodegeneration. Geroscience 39:19–32

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Horan MP, Pichaud N, Ballard JW (2012) Review: quantifying mitochondrial dysfunction in complex diseases of aging. J Gerontol A Biol Sci Med Sci 67:1022–1035

    Article  PubMed  CAS  Google Scholar 

  • Iadecola C (1993) Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci 16:206–214

    Article  PubMed  CAS  Google Scholar 

  • Izzo C, Carrizzo A, Alfano A, Virtuoso N, Capunzo M, Calabrese M, de Simone E, Sciarretta S, Frati G, Oliveti M, Damato A, Ambrosio M, de Caro F, Remondelli P, Vecchione C (2018) The impact of aging on cardio and cerebrovascular diseases. Int J Mol Sci 19

  • Jung SK, Gorski W, Aspinwall CA, Kauri LM, Kennedy RT (1999) Oxygen microsensor and its application to single cells and mouse pancreatic islets. Anal Chem 71:3642–3649

    Article  PubMed  CAS  Google Scholar 

  • Katakam PV, Gordon AO, Sure VN, Rutkai I, Busija DW (2014) Diversity of mitochondria-dependent dilator mechanisms in vascular smooth muscle of cerebral arteries from normal and insulin-resistant rats. Am J Physiol Heart Circ Physiol 307:H493–H503

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Katakam PV, Dutta S, Sure VN et al (2016) Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide. Am J Physiol Heart Circ Physiol 310:H1097–H1106

    Article  PubMed  PubMed Central  Google Scholar 

  • Kunz A, Iadecola C (2009) Cerebral vascular dysregulation in the ischemic brain. Handb Clin Neurol 92:283–305

    Article  PubMed  PubMed Central  Google Scholar 

  • Marzetti E, Csiszar A, Dutta D, Balagopal G, Calvani R, Leeuwenburgh C (2013) Role of mitochondrial dysfunction and altered autophagy in cardiovascular aging and disease: from mechanisms to therapeutics. Am J Physiol Heart Circ Physiol 305:H459–H476

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Merdzo I, Rutkai I, Tokes T, Sure VN, Katakam PV, Busija DW (2016) The mitochondrial function of the cerebral vasculature in insulin-resistant Zucker obese rats. Am J Physiol Heart Circ Physiol 310:H830–H838

    Article  PubMed  PubMed Central  Google Scholar 

  • Merdzo I, Rutkai I, Sure VN, McNulty CA, Katakam PV, Busija DW (2017) Impaired mitochondrial respiration in large cerebral arteries of rats with type 2 diabetes. J Vasc Res 54:1–12

    Article  PubMed  CAS  Google Scholar 

  • Navarro G, Allard C, Morford JJ, Xu W, Liu S, Molinas AJR, Butcher SM, Fine NHF, Blandino-Rosano M, Sure VN, Yu S, Zhang R, Münzberg H, Jacobson DA, Katakam PV, Hodson DJ, Bernal-Mizrachi E, Zsombok A, Mauvais-Jarvis F (2018) Androgen excess in pancreatic beta cells and neurons predisposes female mice to type 2 diabetes. JCI Insight 3

  • Nehlig A (2004) Brain uptake and metabolism of ketone bodies in animal models. Prostaglandins Leukot Essent Fat Acids 70:265–275

    Article  CAS  Google Scholar 

  • Pasarica M, Sereda OR, Redman LM, Albarado DC, Hymel DT, Roan LE, Rood JC, Burk DH, Smith SR (2009) Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes 58:718–725

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Payne BA, Chinnery PF (1847) Mitochondrial dysfunction in aging: much progress but many unresolved questions. Biochim Biophys Acta 2015:1347–1353

    Google Scholar 

  • Reddy PH, Beal MF (2008) Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer’s disease. Trends Mol Med 14:45–53

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sakamuri S, Sperling JA, Sure VN et al (2018) Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research. Geroscience 40:347–356

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Silbergeld DL, Ali-Osman F (1991) Isolation and characterization of microvessels from normal brain and brain tumors. J Neuro-Oncol 11:49–55

    Article  CAS  Google Scholar 

  • Sonntag WE, Lynch CD, Cooney PT, Hutchins PM (1997) Decreases in cerebral microvasculature with age are associated with the decline in growth hormone and insulin-like growth factor 1. Endocrinology 138:3515–3520

    Article  PubMed  CAS  Google Scholar 

  • Springo Z, Tarantini S, Toth P, Tucsek Z, Koller A, Sonntag WE, Csiszar A, Ungvari Z (2015) Aging exacerbates pressure-induced mitochondrial oxidative stress in mouse cerebral arteries. J Gerontol A Biol Sci Med Sci 70:1355–1359

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Starkov AA, Chinopoulos C, Fiskum G (2004) Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury. Cell Calcium 36:257–264

    Article  PubMed  CAS  Google Scholar 

  • Stirone C, Duckles SP, Krause DN, Procaccio V (2005) Estrogen increases mitochondrial efficiency and reduces oxidative stress in cerebral blood vessels. Mol Pharmacol 68:959–965

    Article  PubMed  CAS  Google Scholar 

  • Sure VN, Katakam PV (2016) Janus face of thrombospondin-4: impairs small artery vasodilation but protects against cardiac hypertrophy and aortic aneurysm formation. Am J Physiol Heart Circ Physiol 310:H1383–H1384

    Article  PubMed  Google Scholar 

  • Tarantini S, Fulop GA, Kiss T, Farkas E, Zölei-Szénási D, Galvan V, Toth P, Csiszar A, Ungvari Z, Yabluchanskiy A (2017a) Demonstration of impaired neurovascular coupling responses in TG2576 mouse model of Alzheimer’s disease using functional laser speckle contrast imaging. Geroscience 39:465–473

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tarantini S, Yabluchanksiy A, Fulop GA et al (2017b) Pharmacologically induced impairment of neurovascular coupling responses alters gait coordination in mice. Geroscience 39:601–614

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tarantini S, Valcarcel-Ares NM, Yabluchanskiy A, Fulop GA, Hertelendy P, Gautam T, Farkas E, Perz A, Rabinovitch PS, Sonntag WE, Csiszar A, Ungvari Z (2018) Treatment with the mitochondrial-targeted antioxidant peptide SS-31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice. Aging Cell 17

  • Tucsek Z, Noa Valcarcel-Ares M, Tarantini S, Yabluchanskiy A, Fülöp G, Gautam T, Orock A, Csiszar A, Deak F, Ungvari Z (2017) Hypertension-induced synapse loss and impairment in synaptic plasticity in the mouse hippocampus mimics the aging phenotype: implications for the pathogenesis of vascular cognitive impairment. Geroscience 39:385–406

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ungvari Z, Kaley G, de Cabo R, Sonntag WE, Csiszar A (2010a) Mechanisms of vascular aging: new perspectives. J Gerontol A Biol Sci Med Sci 65:1028–1041

    Article  PubMed  Google Scholar 

  • Ungvari Z, Sonntag WE, Csiszar A (2010b) Mitochondria and aging in the vascular system. J Mol Med (Berl) 88:1021–1027

    Article  CAS  Google Scholar 

  • van Hall G, Stromstad M, Rasmussen P et al (2009) Blood lactate is an important energy source for the human brain. J Cereb Blood Flow Metab 29:1121–1129

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Ying Z, Bosy-Westphal A, Zhang J, Schautz B, Later W, Heymsfield SB, Müller MJ (2010) Specific metabolic rates of major organs and tissues across adulthood: evaluation by mechanistic model of resting energy expenditure. Am J Clin Nutr 92:1369–1377

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Williams LR, Leggett RW (1989) Reference values for resting blood flow to organs of man. Clin Phys Physiol Meas 10:187–217

    Article  PubMed  CAS  Google Scholar 

  • Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA (2007) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292:C125–C136

    Article  PubMed  CAS  Google Scholar 

  • Yousif S, Marie-Claire C, Roux F, Scherrmann JM, Decleves X (2007) Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy. Brain Res 1134:1–11

    Article  PubMed  CAS  Google Scholar 

  • Yu H, Yang C, Chen S, Huang Y, Liu C, Liu J, Yin W (2017) Comparison of the glycopattern alterations of mitochondrial proteins in cerebral cortex between rat Alzheimer’s disease and the cerebral ischemia model. Sci Rep 7:39948

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhang J, Nuebel E, Wisidagama DR et al (2012) Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells. Nat Protoc 7:1068–1085

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Ms. Sufen Zheng for her technical help for the studies.

Funding

This research project was supported by the National Institutes of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (NS094834—PV Katakam), National Heart, Lung and Blood Institute (HL-077731 and HL093554—DW Busija), National Institute on Aging (R01AG047296—R Mostany, R01AG049821—WL Murfee); American Heart Association (National Center Scientist Development Grant, 14SDG20490359—PV Katakam, Greatersoutheast Affiliate Predoctoral Fellowship Grant,16PRE27790122—V.N. Sure), Louisiana Board of Regents grants (Endowed Chairs for Eminent Scholars program; DW Busija, RCS, LEQSF(2016-19)-RD-A-24—R Mostany), and COBRE on Aging and Regenerative Medicine (P20GM103629—R Mostany). The content does not necessarily represent the official views of the NIH and is solely the responsibility of the authors.

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Authors and Affiliations

  1. Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA, 70112, USA

    Venkata N. Sure, Siva S. V. P. Sakamuri, Jared A. Sperling, Wesley R. Evans, Ivan Merdzo, Ricardo Mostany, David W. Busija & Prasad V. G. Katakam

  2. Tulane Brain Institute, Tulane University, 1430 Tulane Avenue; Room 3554C, 8683, New Orleans, LA, 70112, USA

    Wesley R. Evans, Ricardo Mostany, David W. Busija & Prasad V. G. Katakam

  3. Department of Pharmacology, University of Mostar School of Medicine, Mostar, Bosnia and Herzegovina

    Ivan Merdzo

  4. J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, 32611, USA

    Walter L. Murfee

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  1. Venkata N. Sure
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  2. Siva S. V. P. Sakamuri
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  3. Jared A. Sperling
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  5. Ivan Merdzo
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  6. Ricardo Mostany
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  8. David W. Busija
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  9. Prasad V. G. Katakam
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Corresponding author

Correspondence to Prasad V. G. Katakam.

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All experimental protocols were approved by the Institution of Animal Care and Use Committee (IACUC) of Tulane University School of Medicine in accordance with the National institute of Health (NIH) guidelines for the animal care and use.

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The authors declare that they have no conflict of interest.

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Sure, V.N., Sakamuri, S.S.V.P., Sperling, J.A. et al. A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice. GeroScience 40, 365–375 (2018). https://doi.org/10.1007/s11357-018-0037-8

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