A Multimodal, Regenerative Approach to Traumatic Brain Injury

1. AMultimodal, RegenerativeApproach toTraumaticBrain Injury Dr. John C. Hughes, D.O. OMED – VIRTUAL October 2020

2. Disclosure The content of this presentation has been peer reviewed for fair balance and evidence-based medicine. Dr. John Hughes, DO has no relevant financial relationships to disclose.

3. Dr.JohnHughes,DO  Doctor of Osteopathy  From Georgia  Arizona College of Osteopathic Medicine – 2007  Aspen Integrative Medicine – 2009  TBI Therapy – 2014

4. LearningObjectives Define Define the clinical, biochemical and metabolic effects from TBI Understand Understand the specific hormonal reactions associated with TBI Treatment An osteopathic, integrative, regenerative approach to treating TBI

5. Clinical Symptoms from TBI Cognitive Memory decline / loss Slow reaction time Inability to pay attention Executive dysfunction Slow learning Interrupted speech Difficulty understanding Unable to concentrate Confusion Difficulty communicating thoughts Unable to plan, reason, problem-solve Physical Headache Fatigue Sleep disorders Vertigo or dizziness Tinnitus or hyperacusis Photosensitivity Anomia Reduced tolerance to psychotropic medications Disorientation Loss of mobility Seizures Loss of smell Psychological Irritability Easy frustration Tension Anxiety Affective lability Personality changes Disinhibition Apathy Suspiciousness Suicidality Depression PTSD

6. Neurobiochemical CascadeinTBI Mechanical stress Disruption to cellular membranes Neuronal depolarization, firing, and release of neurotransmitters Increased extracellular glutamate Efflux of K+ with Ca2+ going intracellular Hyperglycolysis Kawa, 2017

7. Increased intracellular Ca2+ concentration gets sequestered by mitochondria Oxidative metabolism gets inhibited Energy demands must be met by glycolysis = lactate accumulation = decreased ATP production Cell enters phase of metabolic suppression and widespread depression Intracellular Ca2+ causes enzyme activation and initiation of apoptotic pathways Immediate decreases in Mg 2+ slows down recovery Kawa, 2017 Neurobiochemical CascadeinTBI

8. Neurobiochemical Response to TBI Kawa, 2017

9. Neurobiochemical Cascadein TBI: TakeHome Disruptions in the neurobioochemical cascade of TBI lead directly to the patient’s persistent emotional, cognitive, and somatic symptoms Further support for this conclusion: Pre-treatment of animals with magnesium results in improved post-traumatic outcomes Kawa, 2017

10. Biochemical and Physiological Responses fromTBI  Disproportional proinflammatory cytokine production and release  Increased counterregulatory hormones work against the action of insulin  Hypermetabolic and catabolic states  Severely impaired nitrogen homeostasis  Oxidative Stress

11. Oxidative Stress From TBI Impairs cerebral vascular function Impairs circulation Impairs the energy metabolism Damages mitochondria and DNA

12. SerotoninTransporterProtein (SERT)LevelsDecreasedinTBI  Decreased SERT immunoreactivity in neuronal fibers  Decreased SERT mRNA and protein expression  Decreased SERT expression in the cerebral cortex  Take Home: Decreased serotonin neurotransmission means increased depression with TBI and PTSD patients Abe et al., 2016

13. Serotonin(5HT) Originsand Functions  Origins and rate limiting steps:  The raphe nuclei neuron cell groups, B1-B9 are the principal neurons that give rise to spinal and extensive serotonergic forebrain projections  These nuclei in the brain express the rate-limiting enzyme tryptophan hydroxylase 2 for 5-HT synthesis  Functions: 5-HT regulates sleep, appetite, pain and mood. Abe et al., 2016; Gao et al., 2008; Maes et al., 1995; McAllister, 2011

14. Effectsof Decreased Serotonin  5-HT levels are reduced in following conditions:  Chronic stress: reduces 5-HT found in the plasma and CSF (Gao et al., 2008)  Depression: Decreased 5-HT transporter binding in post- mortem brains of depressed patients (Maes et al., 1995)  “5-HT plays a role in stress and PTSD—possibly modulating the “fight-or-flight” response.” (McAllister, 2011)  Bottom Line: Lower serotonin is found in patients with chronic stress, depression, and PTSD Abe et al., 2016; Gao et al., 2008; Maes et al., 1995; McAllister, 2011

15. Neurotransmitter SystemsPost-TBI  TBI increases noradrenaline levels  Increasing inflammation in the forebrain  Increasing anxiety-like behavior  TBI decreases the serotonin metabolite 5-HIAA in pre- frontal cortex  Bottom Line: ↑ catecholamines + ↓ 5HT metabolism = PTSD (internal anxiety with poor ability for patient to manage) Kawa et al., 2015

16. Neurotransmitters GoingAwryPost- TBI  Cholinergic excess:  Amplifies destructive effects of excitatory amino acid excesses  Cerebral monoaminergic excesses:  Initially induced elevations of certain cerebral monoamines  Leads to decreased cerebral glucose use  Results in a metabolic crisis that characterizes TBI McAllister, 2011

17. What Happens Metabolically withaTBI? “The brain is in a metabolic crisis with concussion… potassium ion from inside the cell going extracellularly, calcium ions going intracellularly, neurotransmitters widely released in a chaotic manner. It takes energy to pump that potassium back, put the neurotransmitters back on so the cell can function.” Dr Robert Cantu, MD, 2013

18. What Happens Metabolically withaTBI? An Energy Crisis

19. Hormonesand TBI  Head injury can damage the pituitary gland and reduce production of two important hormones.  Growth hormone releasing hormone  Reduced production of GH causes symptoms of fatigue, reduced interest in sex, reduced stamina, anxiety, and depression.  18 percent of people with brain injuries, most of whom had moderate to severe TBI.

20. Hormonesand TBI  Part of the pituitary gland that makes antiduretic hormone (ADH) is also vulnerable to injury.  Reduced production of ADH can cause diabetes insipidus, which causes excessive urination and extreme thirst. (Unlike diabetes mellitus, diabetes insipidus does NOT raise blood sugar levels and is NOT treated with insulin.) Sometimes the resulting dehydration causes a sensation of fatigue.  Diabetes insipidus occurs in about 25 percent of the people with brain injury, most of whom have had severe TBI

21. Hormonesand TBI  Treatments–  1) heal brain with Multimodal protocols  2) Testosterone boosting supplements; HGH boosting aminos  3) Glandulars:  hypothalamus/pituitary/adrenals/orchex/thyrotrophin

22. Mainstream Treatments • Occupational and physical rehabilitation • Speech therapy • Pharmaceutical drugs • Cognitive maintenance exercises • Patients simply cope with their condition

23. Alternative Treatments • Do not seek to regenerate but rather simply treat symptoms • Do not combine regenerative treatments in a multimodal manner in order to maximize patient benefit

24. It is hypothesized that the practical, effective combination of multiple regenerative TBI therapies can produce synergistic benefits to the patient that exceed the use of one particular TBI treatment. A Multimodal, Regenerative Approach to TBI

25. A Multimodal, Regenerative Approach to TBI I. Hyperbaric Oxygen Therapy II. Intranasal Therapies III. IV Nutrition IV. Cranial Osteopathy V. Diet and Nutrition

26. Hyperbaric Oxygen Therapy (HBOT) for TBI Part I

27. Hyperbaric Oxygen Therapy (HBOT) • Allows the body to absorb about 10-15 times its normal supply of oxygen • Stimulates the growth of tissue, bone and blood vessels, and reduces inflammation Thom, et al., 2006

28. VolumerenderedBrain SPECTperfusion mapsof a51-year-old woman sufferingfrommTBIthat hadoccurred2yearsprior toinclusion inthestudy Boussi-Gross et al., 2013

29. HBOT for TBI • Induces neuroplasticity • Increases tissue oxygenation • Generates new capillary networks • Restores blood supply • Increases stem cells in the blood

30. HBOT andStemCells • 2 hours of HBOT triples the patients own circulating stem cells • 20 sessions of HBOT increases circulating stem cells to 8 fold (800%) Thom, et al., 2006

31. MeanCD34+populationinbloodofhumansbeforeandafterHBO2treatments. Data are the fraction of CD34+ cells within the gated population using leukocytes obtained from 26 patients before and after their 1st, 10th, and 20th HBO2 treatment. Thom, et al., 2006

32. InhaledOxygen Increases Serotonin  Participants: Six healthy participants (3 male, 3 female) breathed a 15% or 60% oxygen mixture 15 min before injection of tracer and during acquisition period  Observations: Two sets of PET images were acquired  Before and after each of the oxygen mixtures and after reconstruction  All images were converted into brain functional images illustrating the brain trapping constant K(*) (microL/g/min).  Results: Highly significant increases (50% on average) in brain serotonin synthesis (K(*) values) at high (mean value of 223+/- 41 mmHg) relative to low (mean value 77.1+/-7.7 mmHg) blood oxygen levels Nishikawa et al., 2005

33. Serotonin Increasedby Oxygen: Conclusions  Increasing blood oxygen increases 5-HT synthesis in brain  Tryptophan hydroxylase 2 is not saturated with oxygen in brain  Take Home: Inhaled oxygen (60%) over a short period of time rapidly increases serotonin levels in comparison to 15% inhaled oxygen  Other studies: Support that hyperbaric medicine increases serotonin (Silliphant, 2017) Nishikawa et al., 2005; Silliphant, 2017

34. “[Hyperbaric oxygen therapy] is the safest way clinically to increase stem cell circulation, far safer than any of the pharmaceutical options.” STEPHEN THOM, MD, PH.D. (2006)

35. IntranasalTherapies (Insulin,PRP, and StemCells) forTBI Part II

36. JourneyThroughtheNose Through the olfactory nerves Bypasses the blood-brain barrier Into the CSF within 10 minutes

37. Solidarrowsrepresent thepathsofmigration ofcellsintothebrain, dashedarrowsreflect possiblehypothetical routesofcelldelivery Danielyan, et al., 2014 MouseBrain

38. Intranasal InsulinforTBI  Improves brain ATP production  Decreases CSF cortisol  Improves neuronal viability in the hippocampus  Increases the expression of anti- inflammatory microglia  Reduces beta-amyloid and tau protein deposition

39. Improved neuronal viability in the hippocampus of the insulin treated rats. Intranasal insulin increases the expression of anti- inflammatory microglia in the hippocampus Brabazon, Khayrullina, Frey, & Byrnes, 2014

40.  Autologous plasma contains growth factors and cytokines to aid the injured brain: VEGF, EGF increases angiogenesis PDGF, TGF-p enhance collagen growth IGF-1 stimulates protein synthesis PlateletRichPlasma(PRP)

41. PlateletRichPlasma(PRP) The infusion of concentrated platelets results in an exponential increase in numerous growth factors at the sight of infusion Plasma cytokines control inflammatory mediators cox1, cox2 and guide stem cells to areas of injury

42. Intranasal Platelet RichPlasma (PRP)forTBI • “Basic fibroblast growth factor infusion enhances injury- induced cell proliferation in the dentate gyrus and improves cognitive function in rats following fluid percussive injury.” • “Other studies have found that infusion of S100β or VEGF can also enhance neurogenesis in the hippocampus and improve the functional recovery of animals following TBI.” Sun, 2014

43. Peripheral BloodBased AdultStemCells Recently discovered in peripheral blood PLURIPOTENT adult stem cells Behave like embryonic stem cells Give rise to all the cell types Long lifespan Work in combination with PRP

44. Intranasal PeripheralBlood StemCells forTBI Have regenerative and reparative properties Adult stem cells from BMA have been used to treat ischemic brain damage by reducing gray and white matter loss (Danielyan, et al., 2014). Downregulate neuroinflammatory cytokines

45. Intranasal Nutrients for TBI IN glutathione has been used to reduce oxidative stress and enhance cellular detoxification in Parkinson’s disease patients (Mischley, et al., 2016). IN methylcobalamin has been shown to improve QEEG Theta activity in ADHD and autism patients (Kurtz, 2008).

46. Intravenous Nutrition forTBI Part III

47. IVNutrition forTBI  PRP  Adult peripheral blood stem cells  NAD+  Myer’s cocktail with potassium, magnesium, calcium, B-complex, B5, B6, and B12, ascorbate, and glutathione

48. CranialOsteopathyforTBI Part IV

49. Cranial Osteopathy for TBI • Manual manipulation of the cranial bones and membranes to allow the cerebral spinal fluid to flow properly • The central nervous system, including the brain and spinal cord, has a subtle, rhythmic pulsation

50. Cranial Osteopathy for TBI • This rhythmic pulsation can be blocked in brain injuries – impedes CSF and blood flow • Effective at treating vertigo and headaches associated with TBIs

51.  Time shift between peaks of TCD and В-Imp is determined by the replacement of some portion of CSF out from (or into) zone of В- Imp electrodes.  This time interval represents the mobility of CSF inside the cranium during the pulse cycle. Moskalenko, Frymann, Kravchenko, & Weinstein, 2003

52. Nutrition forTBI Part V

53. KetogenicDiet forTBI High-fat Adequate- protein Low- carbohydrate

54. Glucose/Carbohydrates = Kindling Ketones/Fats = Logs Which burns more even?

55. Ketonesarelike diesel fuel(Glucoseislikegasoline) • Diesel fuel has a high flash point than gasoline • Harder to oxidize – Less flammable (excitable) • The brain works like a diesel engine • Burns more efficiently – lasts longer

56. Whatelsedo ketonesdo? Increases GABA Decreases Depression, Fear, Anxiety Decreases Glutamate Decreases Oxidative Stress Increases Neuroprotection Increases Calming

57.  Possible anticonvulsant effects of ketone bodies on the brain  Increased GABA synthesis  Decreased glutamate release by competitive inhibition of vesicular glutamate transporters. McNally & Hartman, 2012

58.  Increased membrane potential hyperpolarization via KATP channels  Decreased reactive oxygen species production from glutamate exposure  Electron transport chain subunit transcription McNally & Hartman, 2012

59. Neuroprotective Actionsofthe Ketogenic Diet Upregulates energy metabolism genes Stimulates of mitochondrial biogenesis Promotes synthesis of ATP Limits glutamate toxicity

60. TreatingTBIand PTSD with TryptophanDiet  Serotonin levels enhanced by carb ingestion  Insulin release accelerates the serum removal of competing valine, leucine, and isoleucine  Increased protein in diet slows serotonin elevation so a strict Keto paleo diet is not the best  Tryptophan hydroxylase is the rate-limiting enzyme for serotonin production so serotonin levels are directly on bioavailable tryptophan  Converts Trp to 5-HTP  Trp plentiful in chocolate, oats, bananas, dried dates, milk, cottage cheese, meat, fish, turkey, and peanuts.  Take Home: Along with turkey ingestion, a diet with some healthy carbs can improve oral tryptophan assimilation Thorne Research. Retrieved October 12, 2019 http://www.altmedrev.com/archive/publications/11/1/52.pdf

61. OralIngestion ofTryptophan forTBI/PTSD  Daily nutritional requirement for L-tryptophan (Trp) = 5 mg/kg  Most adults consume much more, up to 4–5g/d (60–70 mg/kg)  Ingesting L-Trp raises brain tryptophan levels and stimulates its conversion to serotonin in neurons  Side effects at higher doses (70–200 mg/kg), include tremor, nausea, and dizziness, with a drug that enhances serotonin function (e.g., antidepressants)  Tryptophan can be taken as supplement (used for 50 years now)  Risks: “serotonin syndrome” occurs – too much serotonin stimulation when Trp combined with serotonin drugs  Symptoms include delirium, myoclonus, hyperthermia, and coma  Tryptophan supplement dosing: Should be individualized for each patient Fernstrom, 2012

62. OralTryptophan (Trp)CanIncrease SerotonininBrain  Variations in Trp concentrations in the brain found to modify the rate of 5HT synthesis in and release by neurons  5HT synthesis falls when brain Trp declines  Trp is a large neutral amino acids (LNAA) and competes with other LNAA for a shared, competitive transporter across the blood-brain barrier  Raising plasma levels of the LNAA (other than Trp) reduces Trp transport into the brain and lowers brain (and CSF) Trp concentrations Fernstrom, 2012

63. Serotonininthe Gut  Microbes can also alter availability of tryptophan – amino acid building block required for serotonin production  After synthesis in intestinal enterochromaffin cells, serotonin is stored in platelets and released upon stimulation  Estimated that 90% of the body’s serotonin is made in the digestive tract  Beneficial microbes produce short-chain fatty acids like butyrate that influence production of serotonin in enterochromaffin cells  Take Home: A healthy gut microbiome leads to increased serotonin levels Banskota, Ghia, and Khan, 2019

64. The Multimodal Regenerative Protocol

65. Cranial osteopathy HBOT IV PRP + Nutrition IN PRP + Insulin Day 1: IV pluripotent stem cells (VESLs) from the blood + NAD IN pluripotent stem cells (VESLs) from the blood HBOT Day 2: 2-Day Program

66. HBOTProtocol Medical Grade HBOT 10 – 20 before and after treatment Home HBOT Chamber 5 – 7 days/wk 1 month before treatment 5 – 7 days/wk 2 – 9 months after treatment

67. CaseReport1:46year-oldmale Before Treatment: • Memory loss • Depression and anxiety • Emotionally unstable • Headaches daily • Inability to carry on conversation • Inability to do math or read • Light and sound sensitivity • Could not drive • Insomnia After Treatment: • “Memory download” • “An awakening” • Mood and personality improvements • Improvements intellectually, physiologically, and psychologically • Improved ability to read • Able to turn on lights /electronics • Able to drive • Sleep normalized

68. TBITherapy: CaseReport1 “It was like a stream of information had been let loose… I felt for the first time in a year that I had some clarity. I was excited and able to read more than 2-3 sentences without triggering a migraine… The ability to think and plan returned.”

69. CaseReport2:30year-oldfemale Before Treatment: • Insomnia • Mood swings • Depression • Unable to work • Head pressure • Sound and light sensitivity After Treatment: • Able to travel and work • Light and sound sensitivity decreased • Improved mood • Less fatigued • Relief from anxiety

70. TBITherapy: CaseReport2 “I felt well enough that I started saying yes again. TBI Therapy has turned me into a TBI THRIVER, not just a survivor. I’m happy. I enjoy life again, can travel and am doing work in the world that’s more aligned with myself than ever.”

71. CaseReport3:48year-oldfemale Before Treatment: • Anger • Depression • Suicidal ideation • Anosmia • Extreme mental fatigue • PTSD After Treatment: • Calm • No longer “reactive” and irritable • Confident • No thoughts of suicide • Feeling of less inflammation • Improved memory • Improved sense of smell

72. TBITherapy: CaseReport3 “The results for me have been are nothing short of MIRACULOUS! Popeye may have his spinach but I have stem cells and PRP! Yes, my brain is strong!”

73. Outof100patientstreated,mostreport: More mental clarity Improved memory Improved executive function/decision making More stable emotions and less stress Better ability to cope with pain More physical and mental energy

74. Outof100patientstreated,somereport: Less sound and light sensitivity Improved eyesight Improved sleep and libido Improved motor function (ability to open a clenched fist, ability to walk) Less muscle spasticity

75. Conclusion: The Multimodal, Regenerative Approach is a Superior Way to Treat TBI The practical, effective combination of multiple regenerative TBI therapies can produce synergistic benefits to the patient superior to mainstream TBI or single modality TBI treatments.

76. References Boussi-Gross, R., Golan, H., Fishlev, G., Bechor, Y., Volkov, O., et al. (2013) Hyperbaric Oxygen Therapy Can Improve Post Concussion Syndrome Years after Mild Traumatic Brain Injury – Randomized Prospective Trial. PLoS ONE 8(11): e79995. doi: 10.1371/journal.pone.0079995. Brabazon, F. P., Khayrullina, G. I., Frey, W. H., & Byrnes, K. R. (2014, June). INTRANASAL INSULIN TREATMENT OF TRAUMATIC BRAIN INJURY. In JOURNAL OF NEUROTRAUMA (Vol. 31, No. 12, pp. A106-A106). 140 HUGUENOT STREET, 3RD FL, NEW ROCHELLE, NY 10801 USA: MARY ANN LIEBERT, INC. Cantu, R. (August, 2013). What Physical and Cognitive Rest Really Mean After a Concussion. Retrieved from https://www.brainline.org/video/what-physical-and-cognitive-rest-really-mean-after-concussion. Danielyan, L., Beer-Hammer, S., Stolzing, A., Schäfer, R., Siegel, G., Fabian, C., … & Novakovic, A. (2014). Intranasal delivery of bone marrow-derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer’s and Parkinson’s disease. Cell transplantation,23(1), S123-S139. European Society of Endocrinology. (2010). Vitamin D deficiency associated with chronic fatigue in brain injured patients. ScienceDaily. Retrieved August 15, 2016 from www.sciencedaily.com/releases/2010/04/100427182609.htm Gladstone Institutes. (2008). Collagen May Help Protect Brain Against Alzheimer’s Disease. ScienceDaily. Retrieved August 15, 2016 from www.sciencedaily.com/releases/2008/12/081210150713.htm Gunther, N. & Queen, E. (2013). What Physical and Cognitive Rest Really Mean After a Concussion. Brainline. Retrieved from http://www.brainline.org/content/multimedia.php?id=9022 Haller, H., Cramer, H., Werner, M., & Dobos, G. (2015). Treating the sequelae of postoperative meningioma and traumatic brain injury: a case of implementation of craniosacral therapy in integrative inpatient care. The Journal of Alternative and Complementary Medicine, 21(2), 110-112. Huskisson, E., Maggini, S., & Ruf, M. (2007). The role of vitamins and minerals in energy metabolism and well-being. Journal of international medical research, 35(3), 277-289. Kurtz, S. (2008). U.S. Patent Application No. 12/077,296. Retrieved August 15, 2016 from https://www.google.com/patents/US20090012039 McNally, M. A., & Hartman, A. L. (2012). Ketone bodies in epilepsy. Journal of neurochemistry, 121(1), 28-35. Mischley, L. K., Conley, K. E., Shankland, E. G., Kavanagh, T. J., Rosenfeld, M. E., Duda, J. E., … & Padowski, J. M. (2016). Central nervous system uptake of intranasal glutathione in Parkinson’s disease. npj Parkinson’s Disease, 2, 16002. Moskalenko, Y., Frymann, V., Kravchenko, T., & Weinstein, G. (2003). Physiological background of the Cranial Rhythmic Impulse and the Primary respiratory Mechanism. Am Acad Osteopath J, 13(2), 21-33. Rho, J. M., & Stafstrom, C. E. (2012). The ketogenic diet as a treatment paradigm for diverse neurological disorders. Frontiers in pharmacology, 3, 59. Sun, D. (2014). The potential of endogenous neurogenesis for brain repair and regeneration following traumatic brain injury. Neural regeneration research, 9(7), 688.). Thom, S. R., Bhopale, V. M., Velazquez, O. C., Goldstein, L. J., Thom, L. H., & Buerk, D. G. (2006). Stem cell mobilization by hyperbaric oxygen. American Journal of Physiology-Heart and Circulatory Physiology, 290(4), H1378-H1386. Tithon Biotech (n.d.). Retrieved from http://tithonbiotech.com/index/ UHN Staff. (2015). Vitamins for Memory Loss and Stroke Prevention – These 3 Are Critical. University Health News Daily. Retrieved August 15, 2016 from http://universityhealthnews.com/daily/memory/vitamins-for-memory-loss-and-stroke-prevention-these-3-are-critical/ Van Velthoven, C. T., Kavelaars, A., van Bel, F., & Heijnen, C. J. (2010). Nasal administration of stem cells: a promising novel route to treat neonatal ischemic brain damage. Pediatric research, 68, 419-422.