Skip to main content
Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Beta1-receptor blockade attenuates atherosclerosis progression following traumatic brain injury in apolipoprotein E deficient mice

  • Jintao Wang ,

    Roles Conceptualization, Formal analysis, Investigation, Writing – original draft

    ‡ JW and JV are co-first authors as each contributed equally to this work.

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Jessica Venugopal ,

    Roles Formal analysis, Investigation, Writing – original draft

    ‡ JW and JV are co-first authors as each contributed equally to this work.

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Paul Silaghi,

    Roles Formal analysis, Investigation

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Enming J. Su,

    Roles Formal analysis, Investigation

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Chiao Guo,

    Roles Formal analysis, Investigation

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Daniel A. Lawrence,

    Roles Conceptualization, Formal analysis, Investigation

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America

  • Daniel T. Eitzman

    Roles Conceptualization, Funding acquisition, Supervision, Writing – original draft

    Affiliation Department of Internal Medicine, Cardiovascular Research Center, University of Michigan, Ann Arbor, Michigan, United States of America


Traumatic brain injury (TBI) is associated with cardiovascular mortality in humans. Enhanced sympathetic activity following TBI may contribute to accelerated atherosclerosis. The effect of beta1-adrenergic receptor blockade on atherosclerosis progression induced by TBI was studied in apolipoprotein E deficient mice. Mice were treated with metoprolol or vehicle following TBI or sham operation. Mice treated with metoprolol experienced a reduced heart rate with no difference in blood pressure. Six weeks following TBI, mice were sacrificed for analysis of atherosclerosis. Total surface area and lesion thickness, analyzed at the level of the aortic valve, was found to be increased in mice receiving TBI with vehicle treatment but this effect was ameliorated in TBI mice receiving metoprolol. No effect of metoprolol on atherosclerosis was observed in mice receiving only sham operation. In conclusion, accelerated atherosclerosis following TBI is reduced with beta-adrenergic receptor antagonism. Beta blockers may be useful to reduce vascular risk associated with TBI.


Traumatic brain injury (TBI) is a common cause of morbidity and mortality worldwide [1]. In addition to neurocognitive deficits, TBI is associated with other comorbidities, including cardiovascular complications. In the acute setting, cardiovascular effects of TBI include stress-related cardiomyopathy [2], arrhythmias [3], and ECG changes [4], all of which are associated with increases in catecholamines. Chronic cardiovascular effects, including increased coronary artery calcification and cardiovascular mortality have also been demonstrated [5]. Preclinical studies in mice support a causal relationship between TBI and atherosclerosis [6] which may be related to enhanced sympathetic activity. Chronic increases in sympathetic activity following TBI have been identified in studies of both rodents and humans [79]. Therapeutic blockade of beta-adrenergic receptors with beta-blockers have been shown to improve neurological and functional outcomes in humans following TBI [10, 11]. Beta-blockers may also be useful in reducing other comorbidities that may be associated with TBI. The current preclinical study was designed to determine whether the beta-receptor antagonist, metoprolol, would be effective in preventing the increased atherosclerosis observed following TBI in hyperlipidemic mice.

Materials and methods


Male apolipoprotein E–deficient (ApoE−/−) on the C57BL6/J strain background were purchased from Jackson Laboratory (Bar Harbor, Maine) at 8 weeks of age. Mice were housed under specific pathogen-free conditions in static microisolator cages with tap water ad libitum in a temperature-controlled room with a 12:12-hour light/dark cycle. At 10 weeks of age mice were started on a Western diet (TD88137, Harlan, WI) and at 14 weeks of age, mice underwent the CCI or sham procedures, with or without metoprolol. Mice receiving metoprolol were grouped in same cage as drug was supplied in water source. Metoprolol or vehicle control was administered via the drinking water at a concentration of 2 mg/mL for 6 weeks following TBI.

Ethics statement

All animal use protocols complied with the Principle of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use and Care of Animals. This study is reported in accordance with ARRIVE guidelines as set by the National Centre for the Replacement Refinement and Reduction of Animals in Research [12].

Model of TBI

To induce TBI, male ApoE−/− mice were anesthetized with 2% isoflurane and placed in a stereotactic frame (Kopf, Tujunga, CA, USA) as previously described [13, 14]. Briefly, a 5 mm circular craniotomy, centered between the bregma and lambda, was made and then a controlled cortical impact (CCI) was delivered to the midline at an impact speed of 3.00 m/s, tissue displacement of 1.1 mm, and impact duration of 50 ms. Following impact, the circular bone fragment from the craniotomy was glued back to the cranial window. The sham procedure was identical except for craniotomy and delivery of the CCI.

Blood pressure measurement

Blood pressure and pulse rate were measured 3 weeks after CCI or sham operation in non-anesthetized, trained mice by tail plethysmography using the BP-2000 Blood Pressure Analysis System (Visitech System, Apex, NC) as previously described [15].

Histological analysis

Quantification of atherosclerosis, the primary outcome, was performed as previously described [15, 16]. Briefly, mice were euthanized under pentobarbital anesthesia (i.p., 100 mg/kg), and arterial trees were perfused at physiological pressure and fixed in 10% zinc formalin. Paraffin-embedded hearts, which included aortic valves, were sectioned for lesion analysis. A series of 5 μm sections were obtained at the level of the aortic sinus and 4 cross sections were analyzed from each mouse. Sections were stained with hematoxylin and eosin for quantification of lesion area normalized by adjacent medial area of aorta to control for possible tangential sectioning [15, 17]. The lesion area was defined as the area between the endothelial cell layer and internal elastic lamina.

For plaque composition analysis, macrophages were quantified with an antibody to MAC3 (1:100, BD Biosciences, San Jose, CA) followed by detection of the biotin-conjugated secondary goat anti-rat IgG (BD Biosciences, San Jose, CA) with AEC substrate kit (Vector Laboratories, Newark, CA). Negative controls consisted of tissues handled identically to experimental samples except that the primary antibody was omitted. The detection system was streptavidin-HRP and endogenous peroxidase was quenched with hydrogen peroxide. Sections were counterstained with hematoxylin. Positive staining area was analyzed from three fields in each section and expressed as percentage of the total area. All images were analyzed by automated detection of positive stained area using Nikon MetaMorph software with observer blinded to treatment allocation.

Measurements of plasma samples

Plasma samples were collected via terminal heart puncture in 3.2% sodium citrate (50μL/mL of blood) 6 weeks after TBI or sham operation. Total cholesterol was measured using Infinity cholesterol enzymatic-colorimetric kit (Thermo Fisher, #TR13421).

Statistical analysis

All data are presented as mean ± standard deviation. Statistical analysis was carried out using GraphPad Prism. Shapiro-Wilk normality test was used for normal distribution testing. Results were analyzed using ANOVA with Dunnett’s post-hoc testing. Sample size was determined by power calculation based on variability of atherosclerosis previously established in this model [6]. No animals were excluded from analysis.


Effect of TBI and metoprolol on baseline parameters in ApoE−/− mice

At 10 weeks of age, ApoE−/− mice were started on a western diet to induce hyperlipidemia and accelerate the development of atherosclerosis. Four weeks later mice underwent a TBI or sham control procedure. Mice recovered quickly from the procedure and demonstrated grossly normal activity and eating behavior. Twenty-four hours following injury, mice were given metoprolol (M) or placebo (P) in the drinking water (TBI + M, n = 6; TBI +P, n = 6; Sham + M, n = 8; Sham +P, n = 9). Body weights were not different between the TBI ± M or sham groups ± M of mice 6 weeks following the procedure (Fig 1A) indicating the TBI procedure did not impair feeding or cause illness. There were also no significant differences in total cholesterol between the groups of mice (Fig 1B). Tail-cuff plethysmography was used to measure blood pressure and pulse 3 weeks following injury in non-anesthetized mice (n = 4 mice per group for this analysis). To ensure reliable and stable blood pressure measurements, mice were first trained for seven consecutive days and all blood pressure measurements were performed in the morning. No differences in pulse rate or blood pressure were observed between the groups (Fig 1C and 1D).

Fig 1. Effect of TBI and metoprolol on baseline parameters.

(A) body weight, (B) total cholesterol, (C) heart rate, and (D) systolic blood pressure.

Effect of TBI and metoprolol on atherosclerosis in ApoE−/− mice

Total surface area quantitation of atherosclerosis confined to the aorta or total tree was increased in mice subjected to TBI compared to sham-operated mice (Fig 2A and 2B). However, this TBI-induced increase in atherosclerosis was attenuated in mice treated with metoprolol (Fig 2A and 2B). No effect of metoprolol on atherosclerosis was observed in sham-operated mice (Fig 2A and 2B).

Fig 2. Surface area atherosclerosis involving aorta and major branches.

(A) Representative photographs of aortic trees 6 weeks following TBI or sham operation with (+M) or without (-M) metoprolol (2 mg/mL) for 6 weeks following the surgeries. (B) Quantification of oil-red-O staining plaque area of aortic trees confined to the aorta or total tree 6 weeks following TBI or sham operation (*p < 0.05).

Similarly, quantitation of lesion thickness performed at the level of the aortic sinuses revealed increased I/M (intima/media) ratio in mice subjected to TBI compared to sham-operated mice (Fig 3A and 3B). This TBI-induced increase in atherosclerosis was attenuated in mice treated with metoprolol (Fig 3A and 3B) while no effect of metoprolol on atherosclerosis was observed in sham-operated mice.

Fig 3. Plaque area in response to TBI and metoprolol.

A) Representative images of sections of the aortic root stained with H and E for ApoE-/- mice subjected to Sham or TBI surgeries with (+M) or without (-M) metoprolol (2 mg/mL) for 6 weeks following the surgeries. B) Quantification of intima:media ratio. All data are presented as mean ± standard deviation. Results were analyzed using ANOVA with Dunnetts post-hoc testing (* = p<0.05, ** = p<0.01).

Plaque area occupied by MAC3 positive staining cells was not different in the TBI-treated mice compared to sham-treated mice and not different in TBI mice treated with metoprolol (Fig 4A and 4B).

Fig 4. MAC3 immunostaining in response to TBI and metoprolol.

A) Representative images of sections of the aortic root stained with anti-MAC3 antibody for ApoE-/- mice subjected to Sham or TBI surgeries with (+M) or without (-M) metoprolol (2 mg/mL) for 6 weeks following the surgeries. B) Quantification of MAC3 staining as percent of the plaque area. Scale bar: 50μm. All data are presented as mean ± standard deviation.


Cardiovascular complications are increased following brain injury [5, 18] and these effects may be mediated by catecholamine surges as chronic and paroxysmal sympathetic hyperactivity are common after traumatic brain injury [19]. Sympathoadrenal activation following TBI has been shown to induce a coagulopathy and endotheliopathy as evidenced by biomarkers and these effects have been associated with a poor prognosis [20]. In a preclinical model, TBI was shown to accelerate atherosclerosis in hyperlipidemic mice [6].

Pharmacologic blockade of adrenergic receptors, which mediate effects of sympathetic hyperactivation, with beta blockers have been shown to improve neurologic outcome following TBI [10, 11] and a beneficial effect on survival has been demonstrated with metoprolol following severe TBI that was independent of heart rate [21]. Since a murine model demonstrating effects of TBI on a relevant vascular endpoint has been previously established [6], the current study was designed to determine whether therapy with a beta blocker might be effective in preventing TBI-induced accelerated atherosclerosis.

The CCI model has been commonly used in mice to explore pathways involved in post-traumatic brain injury [22]. As in humans, cascades are activated subsequent to injury that lead to chronic systemic effects involving apoptosis [23], inflammation [24] and oxidative stress [25, 26]. The mortality rate associated with the CCI model in rodents is low, so long term survival studies are feasible. Pathophysiological changes have been shown to occur even 1 year after CCI, including ongoing neurodegeneration, microglial activation [27], and neurologic compensatory responses [28]. The model may therefore be useful to analyze effects of TBI on vascular disease processes.

Atherosclerotic-prone rodents are widely employed to genetic and environmental factors involved in atherosclerosis [29]. Vascular endpoints are accelerated with a western diet [15, 2931], allowing the current study to focus on a timepoint 6 weeks following the TBI.

Consistent with a previous study [6], atherosclerosis was increased in mice subjected to TBI. This effect was not associated with increases in blood pressure or pulse. Previous studies have demonstrated effects of TBI on leukocyte activation and biomarkers of endothelial adhesiveness [6], although the precise mechanism(s) for acceleration of atherosclerosis remains to be proven. In the current study, therapy with the β1-adrenergic antagonist, metoprolol, prevented the increase in atherosclerosis following TBI. This was not associated with hemodynamic effects as no difference between the groups were noted related to pulse or blood pressure. A previous study demonstrated acceleration of atherosclerosis following stroke in ApoE deficient mice suggesting common proatherogenic pathways may be activated following acute cerebrovascular events [32]. While further mechanistic studies are warranted to define the specific downstream mediators responsible for vascular effects of TBI, these findings are consistent with an important role of the central nervous system and catecholamine signaling in promoting atherogenesis. In addition to systemic catecholamine effects on circulating monocytes or endothelial cells following brain injury, efferent sympathetic peripheral nervous system axons may be stimulated to produce adrenaline locally in disease-prone arteries leading to enhanced inflammatory plaque activity [16]. The current study was limited by the use of a single, selective beta1 receptor antagonist. Additional studies are therefore necessary to also delineate the contributions of various beta receptor pools and downstream mechanism(s) responsible for beta1-blocker-mediated atheroprotection following TBI. Measurement of plasma steroid levels and catecholamines could also be revealing, in addition to detailed analysis of circulating leukocytes and plaque characteristics. However given benefits in neuroprotection and possible vascular protection, the threshold may be lowered for using this relatively benign class of drugs in patients following TBI.


  1. 1. Maas AI, Stocchetti N, Bullock R. Moderate and severe traumatic brain injury in adults. The Lancet Neurology. 2008;7(8):728–41. pmid:18635021.
  2. 2. Riera M, Llompart-Pou JA, Carrillo A, Blanco C. Head injury and inverted Takotsubo cardiomyopathy. The Journal of trauma. 2010;68(1):E13–5. pmid:19065115.
  3. 3. Bourdages M, Bigras JL, Farrell CA, Hutchison JS, Lacroix J, Canadian Critical Care Trials G. Cardiac arrhythmias associated with severe traumatic brain injury and hypothermia therapy. Pediatric critical care medicine: a journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies. 2010;11(3):408–14. pmid:20464781.
  4. 4. Krishnamoorthy V, Prathep S, Sharma D, Gibbons E, Vavilala MS. Association between electrocardiographic findings and cardiac dysfunction in adult isolated traumatic brain injury. Indian journal of critical care medicine: peer-reviewed, official publication of Indian Society of Critical Care Medicine. 2014;18(9):570–4. pmid:25249741; PubMed Central PMCID: PMC4166872.
  5. 5. Ahmadi N, Hajsadeghi F, Yehuda R, Anderson N, Garfield D, Ludmer C, et al. Traumatic brain injury, coronary atherosclerosis and cardiovascular mortality. Brain injury. 2015;29(13–14):1635–41. pmid:26399477.
  6. 6. Wang J, Su E, Wang H, Guo C, Lawrence DA, Eitzman DT. Traumatic Brain Injury Leads to Accelerated Atherosclerosis in Apolipoprotein E Deficient Mice. Sci Rep. 2018;8(1):5639. Epub 2018/04/06. pmid:29618740; PubMed Central PMCID: PMC5884790.
  7. 7. Zhu K, Zhu Y, Hou X, Chen W, Qu X, Zhang Y, et al. NETs Lead to Sympathetic Hyperactivity After Traumatic Brain Injury Through the LL37-Hippo/MST1 Pathway. Front Neurosci. 2021;15:621477. Epub 2021/05/18. pmid:33994918; PubMed Central PMCID: PMC8116628.
  8. 8. Meyfroidt G, Baguley IJ, Menon DK. Paroxysmal sympathetic hyperactivity: the storm after acute brain injury. The Lancet Neurology. 2017;16(9):721–9. Epub 2017/08/18. pmid:28816118.
  9. 9. Hilz MJ, Wang R, Markus J, Ammon F, Hosl KM, Flanagan SR, et al. Severity of traumatic brain injury correlates with long-term cardiovascular autonomic dysfunction. J Neurol. 2017;264(9):1956–67. Epub 2017/08/05. pmid:28770375; PubMed Central PMCID: PMC5587629.
  10. 10. Florez-Perdomo WA, Laiseca Torres EF, Serrato SA, Janjua T, Joaquim AF, Moscote-Salazar LR. A Systematic Review and Meta-Analysis on Effect of Beta-Blockers in Severe Traumatic Brain Injury. Neurol Res. 2021;43(8):609–15. Epub 2021/01/23. pmid:33478359.
  11. 11. Khalili H, Ahl R, Paydar S, Sjolin G, Cao Y, Abdolrahimzadeh Fard H, et al. Beta-Blocker Therapy in Severe Traumatic Brain Injury: A Prospective Randomized Controlled Trial. World J Surg. 2020;44(6):1844–53. Epub 2020/02/01. pmid:32002583.
  12. 12. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020;18(7):e3000410. Epub 2020/07/15. pmid:32663219; PubMed Central PMCID: PMC7360023
  13. 13. Su EJ, Fredriksson L, Kanzawa M, Moore S, Folestad E, Stevenson TK, et al. Imatinib treatment reduces brain injury in a murine model of traumatic brain injury. Frontiers in cellular neuroscience. 2015;9:385. pmid:26500491; PubMed Central PMCID: PMC4596067.
  14. 14. Liu NK, Zhang YP, O’Connor J, Gianaris A, Oakes E, Lu QB, et al. A bilateral head injury that shows graded brain damage and behavioral deficits in adultmice. Brain research. 2013;1499:121–8. pmid:23276498.
  15. 15. Wang H, Wang J, Guo C, Luo W, Kleiman K, Eitzman DT. Renal denervation attenuates progression of atherosclerosis in apolipoprotein E-deficient mice independent of blood pressure lowering. Hypertension. 2015;65(4):758–65. pmid:25646301; PubMed Central PMCID: PMC4776645.
  16. 16. Mohanta SK, Peng L, Li Y, Lu S, Sun T, Carnevale L, et al. Neuroimmune cardiovascular interfaces control atherosclerosis. Nature. 2022;605(7908):152–9. Epub 2022/04/29. pmid:35477759.
  17. 17. Stubbendorff M, Hua X, Deuse T, Ali Z, Reichenspurner H, Maegdefessel L, et al. Inducing myointimal hyperplasia versus atherosclerosis in mice: an introduction of two valid models. Journal of visualized experiments: JoVE. 2014;(87). pmid:24893977; PubMed Central PMCID: PMC4186352.
  18. 18. Larson BE, Stockwell DW, Boas S, Andrews T, Wellman GC, Lockette W, et al. Cardiac reactive oxygen species after traumatic brain injury. The Journal of surgical research. 2012;173(2):e73–81. pmid:22172132; PubMed Central PMCID: PMC3299814.
  19. 19. Choi HA, Jeon SB, Samuel S, Allison T, Lee K. Paroxysmal sympathetic hyperactivity after acute brain injury. Current neurology and neuroscience reports. 2013;13(8):370. pmid:23780802.
  20. 20. Di Battista AP, Rizoli SB, Lejnieks B, Min A, Shiu MY, Peng HT, et al. Sympathoadrenal Activation is Associated with Acute Traumatic Coagulopathy and Endotheliopathy in Isolated Brain Injury. Shock. 2016;46(3 Suppl 1):96–103. pmid:27206278; PubMed Central PMCID: PMC4978599.
  21. 21. Zangbar B, Khalil M, Rhee P, Joseph B, Kulvatunyou N, Tang A, et al. Metoprolol improves survival in severe traumatic brain injury independent of heart rate control. The Journal of surgical research. 2016;200(2):586–92. Epub 2015/09/15. pmid:26365164.
  22. 22. Osier ND, Dixon CE. The Controlled Cortical Impact Model: Applications, Considerations for Researchers, and Future Directions. Frontiers in neurology. 2016;7:134. pmid:27582726; PubMed Central PMCID: PMC4987613.
  23. 23. Schaible EV, Steinstrasser A, Jahn-Eimermacher A, Luh C, Sebastiani A, Kornes F, et al. Single administration of tripeptide alpha-MSH(11–13) attenuates brain damage by reduced inflammation and apoptosis after experimental traumatic brain injury in mice. PloS one. 2013;8(8):e71056. pmid:23940690; PubMed Central PMCID: PMC3733710.
  24. 24. Haber M, Abdel Baki SG, Grin’kina NM, Irizarry R, Ershova A, Orsi S, et al. Minocycline plus N-acetylcysteine synergize to modulate inflammation and prevent cognitive and memory deficits in a rat model of mild traumatic brain injury. Experimental neurology. 2013;249:169–77. pmid:24036416.
  25. 25. Miller DM, Singh IN, Wang JA, Hall ED. Nrf2-ARE activator carnosic acid decreases mitochondrial dysfunction, oxidative damage and neuronal cytoskeletal degradation following traumatic brain injury in mice. Experimental neurology. 2015;264:103–10. pmid:25432068; PubMed Central PMCID: PMC4323924.
  26. 26. Lewen A, Fujimura M, Sugawara T, Matz P, Copin JC, Chan PH. Oxidative stress-dependent release of mitochondrial cytochrome c after traumatic brain injury. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2001;21(8):914–20. pmid:11487726.
  27. 27. Loane DJ, Kumar A, Stoica BA, Cabatbat R, Faden AI. Progressive neurodegeneration after experimental brain trauma: association with chronic microglial activation. Journal of neuropathology and experimental neurology. 2014;73(1):14–29. pmid:24335533; PubMed Central PMCID: PMC4267248.
  28. 28. Dixon CE, Kochanek PM, Yan HQ, Schiding JK, Griffith RG, Baum E, et al. One-year study of spatial memory performance, brain morphology, and cholinergic markers after moderate controlled cortical impact in rats. Journal of neurotrauma. 1999;16(2):109–22. pmid:10098956.
  29. 29. Getz GS, Reardon CA. Animal models of atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology. 2012;32(5):1104–15. pmid:22383700; PubMed Central PMCID: PMC3331926.
  30. 30. Luo W, Wang H, Ohman MK, Guo C, Shi K, Wang J, et al. P-selectin glycoprotein ligand-1 deficiency leads to cytokine resistance and protection against atherosclerosis in apolipoprotein E deficient mice. Atherosclerosis. 2012;220(1):110–7. pmid:22041028; PubMed Central PMCID: PMC3246103.
  31. 31. Ohman MK, Luo W, Wang H, Guo C, Abdallah W, Russo HM, et al. Perivascular visceral adipose tissue induces atherosclerosis in apolipoprotein E deficient mice. Atherosclerosis. 2011;219(1):33–9. pmid:21835408; PubMed Central PMCID: PMC3206153.
  32. 32. Dutta P, Courties G, Wei Y, Leuschner F, Gorbatov R, Robbins CS, et al. Myocardial infarction accelerates atherosclerosis. Nature. 2012;487(7407):325–9. Epub 2012/07/06. pmid:22763456; PubMed Central PMCID: PMC3401326.