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1. A Multimodal, Regenerative Approach to Traumatic Brain Injury Dr. John C. Hughes, D.O. OMED 2018 – San Diego, CA October 8th, 2018

2. Disclaimer I have no relevant financial relationships with any commercial interests to disclose. The content of this presentation has been peer reviewed for fair balance and evidence based medicine.

3. LearningObjectives Define Define the clinical, biochemical and metabolic effects from TBI Identify Identify mainstream and alternative treatments for TBI Understand Understand the regenerative model of TBI treatment

4. 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

5. 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

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

7. “The brain is in a metabolic crisis in a concussion, potassium ion from inside the cell going extracellular, calcium ions going intracellular, 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 An Energy Crisis What Happens Metabolically withaTBI?

8. Mainstream Treatments • Occupational and physical rehabilitation • Speech therapy • Pharmaceutical drugs • Cognitive maintenance exercises • Patients resign to simply cope with their condition as they reach a plateau of overall treatment benefit.

9. 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 • Singular treatments can be prohibitive for patients and their families, both in cost and time

10. A Multimodal, Regenerative Approach to TBI 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.

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

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

13. 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, 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.

14. VolumerenderedBrain SPECTperfusionmapsofa 51-year-oldwoman sufferingfrommTBIthat hadoccurred2yearsprior toinclusioninthestudy 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

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

16. HBOT MobilizesStem Cells • 2 hours of HBOT triples the patients own circulating stem cells • 20 sessions of HBOT increases circulating stem cells to 8 fold (800%) 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.

17. MeanCD34+population inbloodofhumans beforeandafterHBO2 treatments.  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, 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.

18. “[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. (2005)

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

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

21. Solidarrowsrepresentthepathsofmigrationofcellsintothebrain, dashedarrowsreflectpossiblehypotheticalroutesofcelldelivery 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.

22. 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

23. NeuN, an immunohistochemical marker of neurons, was used to examine the effect of intranasal insulin on neurons after injury. Qualitative assessment of histology showed improved neuronal viability in the hippocampus of the insulin treated rats. Intranasal insulin increases the expression of anti- inflammatory microglia in the hippocampus. 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: MARYANN LIEBERT, INC.

24. PlateletRich Plasma(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

25. IntranasalPlatelet RichPlasma(PRP) forTBI  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  Enhanced collagen IV in neurons of the brain has been shown to have a neuroprotective effect and reduce amyloid-beta proteins.

26. IntranasalPlatelet RichPlasma(PRP) forTBI • “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.” (Kleindienst et al., 2005; Lee and Agoston, 2010; Thau-Zuchman et al., 2010 cited from Sun, 2014). • “Basic fibroblast growth factor infusion enhances injury-induced cell proliferation in the dentate gyrus and improves cognitive function in rats following fluid percussive injury.”

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

28. Intranasal PeripheralBlood StemCells forTBI  Plasma contains millions of these cells per mL  Have regenerative and reparative properties  Have been used to treat ischemic brain damage by reducing gray and white matter loss  Downregulate neuroinflammatory cytokines

29. Intranasal Nutrients forTBI IN glutathione has been used to reduce oxidative stress and enhance cellular detoxificaton in Parkinson’s disease patients. IN methylcobalamin has been shown to improve QEEG Theta activity in ADHD and autism patients.

30. IntravenousNutrition (B-vitamins,Minerals, VitaminC,Glutathione andothernutrients) forTBI Part III

31. IVNutritionfor TBI  Includes PRP, stem cells, NAD+, Myer’s cocktail with potassium, magnesium, calcium, B-complex, B5, B6, and B12, ascorbate, and glutathione  B vitamin supplementation improves memory, mood, and energy levels

32. CranialOsteopathyforTBI Part IV

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

34.  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 оf CSF inside the cranium during the pulse cycle. At this period no active processes could operate.  Investigations under different conditions have shown that “t” reflects CSF mobility. 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. The difference of “t” between inspiratory and expiratory phases of the secondary respiration in a Healthy person, Athlete trained in diving and Patient after head injury

35. MCTOils andtheKetogenic Diet forTBI Part V

36. Ketogenic Diet for TBI DO NOT EAT Grains – wheat, corn, rice, cereal, etc. Sugar – honey, agave, maple syrup, etc. Fruit – apples, bananas, oranges, etc. Tubers – potato, yams, etc. DO EAT Meats Leafy Greens Above ground vegetables High Fat Dairy Nuts and seeds Avocado and berries Other fats – avocado oil, coconut oil, grass-fed ghee, high-fat salad dressing, saturated fats, etc. • High-fat • Adequate-protein • Low-carbohydrate

37. How doesthebodycreateenergy? Glucose Glycogen/Blood Sugar/Insulin Decreases Body uses fat for energy If we cut out carbohydrates and sugar =Beta-oxidation / Ketosis / Decrease Oxidative Stress

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

39. 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

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

41.  Possible anticonvulsant effects of ketone bodies on the brain.  Increased GABA synthesis through alteration of glutamate cycling in glutamate-glutamine cycle or altered neuronal responsiveness to GABA at GABAA receptors.  Decreased glutamate release by competitive inhibition of vesicular glutamate transporters.  Other neurotransmitters, including norepinephrine and adenosine.  Increased membrane potential hyperpolarization via KATP channels possibly mediated by GABAB receptor signaling.  Decreased reactive oxygen species production from glutamate exposure.  Electron transport chain subunit transcription. McNally, M. A., & Hartman, A. L. (2012). Ketone bodies in epilepsy. Journal of neurochemistry, 121(1), 28-35.

42. Neuroprotective Actionsofthe KetogenicDiet  Increases resistance to metabolic stress  Increases resilience to neuronal loss  Upregulates energy metabolism genes  Stimulates of mitochondrial biogenesis  Enhances alternative energy substrates  Promotes synthesis of ATP  Interferes with glutamate toxicity  Bypasses the inhibition of complex I in the mitochondrial respiratory chain (Pillsbury, Oria, & Erdman, 2011)

43. KetogenicDietforTBI • Proven treatment for patients suffering from epileptic seizures • Produce cortical sparing and less apoptotic neuro-degeneration • Overall improvements in cognitive and motor functioning • Increase the available calming neurotransmitter GABA • With less glutamate, there is less oxidative stress and improved neuroprotection • MCT oils are a rich source of ketone bodies

44. The TBI Therapy Protocol

45. TheTBITherapyProtocol I. HBOT: at 1.3 ATA to 1.75 ATA from 10 to 40 sessions II. Intranasal therapies: utilized 1 to 4 x during HBOT treatment series (IN plasma, insulin, glutathione, B12) administered first followed by IN platelet-derived, pluripotent stem cells within 7 days of IN plasma  Patients are also sent home with 10 days IN insulin to self administer III.Cranial osteopathy: administered throughout HBOT treatment series IV.IV nutrition: administered 1-4 x during HBOT treatment series V. Ketogenic Diet, MCT Oils and Supplementation  Blueberries, Vitamin D3, and elk antler recommended daily 3 weeks before and after treatment  Ketogenic dietary counseling and MCT oils are begun on day 1 of HBOT series and continued for 3 months after treatment

46. TBITherapyHBOTProtocol Medical Grade HBOT 10 – 20 before stem cell infusion 10 – 20 after stem cell infusion Home HBOT Chamber 5 – 7 days/wk 1 month before stem cell infusion 5 – 7 days/wk 2 – 9 months after stem cell infusion

47. Consultation HBOT Cranial therapy IV therapy Intranasal (IN) PRP and insulin Day 1: IV and IN NAD+ IV and IN pluripotent stem cells (VESLs) from the blood HBOT Day 2: TBI Therapy 2-Day Program

48. CaseReport 46year-oldmalefromBoulder,CO Before Treatment:  Light and sound sensitivity  Could not drive  Emotionally unstable  Headaches daily  Inability to carry on conversation  Inability to do math or read  Loss of libido  Depression and anxiety  Insomnia  Memory loss After Treatment:  “Memory download”  “An awakening”  Mood and personality improvements  Improvements intellectually, physiologically, and psychologically  Improved ability to read  Able to turn on lights and get on computer / TV  Able to drive  Sleep normalized

49. TBITherapy:Case Report “It was like a stream of information had been let loose like a dam that had busted. 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. I found that I was able to get back on the computer and learn more about my trauma and recent treatments. Within the following days it was like an awakening. It seemed like a light switch was turned back on inside my head. The ability to think and plan returned.”

50. TBITherapy:Case Report “I felt well enough that I started saying yes again to facilitating events and speaking gigs. I also experienced relief from anxiety. With the stem cell procedures, the results were never immediate but 8-12 weeks post procedure I experienced a noticeable jump in my healing. 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.”

51. TBITherapy:Case Report “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!”

52. TBITherapy:ClinicalResults Out of 100 patients treated, nearly every patient reports:  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

53. TBITherapy:ClinicalResults Out of 100 patients treated, some patients report:  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

54. 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

55. 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. 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.

56. Treats TBI patients by combining regenerative therapies: HBOT, stem cells, PRP, and nutritional therapies. tbitherapy.com Treats chronic pain and major medical problems using modern and natural medicine. aspenintegrativemedicine.com

57. HBOT and Peripheral Blood Stem Cells: An Essential Component for Regenerative Treatment Dr. John C Hughes, DO OMED 2018 – San Diego, CA October 8th, 2018

58. Disclaimer  I have no relevant financial relationships with any commercial interests to disclose.  The content of this presentation has been peer reviewed for fair balance and evidence based medicine.

59. AdvancedEvidenceBasedMedicine=CreativeExpertise The Novice Stage: Learns the basic rules and applies them mechanically with no attention to context. Second and Third Stages: Increasing depth of knowledge and sensitivity to context when applying rules. Fourth and Fifth Stages: Rule following gives way to expert judgments – characterized by rapid, intuitive reasoning informed by imagination, common sense, and judiciously selected research evidence.

60. AdvancedEvidenceBasedMedicine=Creative Expertise CreativePeople[CreativeBrains]havean“opennesstonewexperiencethatpermitsthemtoobservethingsthan otherscannot…[this]opennessisaccompaniedbyatoleranceforambiguity.Creativepeopledonotcravethe absolutismofablackandwhiteworld;theyarequitecomfortablewithshadesofgray. Infact,theyenjoylivingin aworldwithunansweredquestionsandblurryboundaries.” Nancy Andreasen, The Creative Brain: The Science of Genius, p. 31

61. HBOT: An Essential Component for Regenerative Treatment  Introduction to HBOT  HBOT: Mechanisms for Addressing Chronic Pain  HBOT: Adjunctive Treatment for Sports Injuries  HBOT: Upregulates Pluripotent Peripheral Blood Adult Stem Cells  VSELs over MSCS: Regenerative Treatments with Pluripotent Stem Cells for Sports Injuries and Arthritis

62. Introduction to HBOT: Physics  Henry’s Law of Gas Solubility: The solubility of a gas in a liquid is directly proportional to the partial pressure of the gas above the liquid.  Increasing the atmospheric pressure increases the amount of gas that is dissolved into a fluid.  Oxygen → Blood Plasma A B

63. IntroductiontoHBOT:Physiology  What Gets Hyper-Oxygenated?  Blood Plasma  Cerebrospinal Fluid  Lymph Fluid  Clinical Hyperbaric Pressures  7 – 22 psi  10 – 15 normal amount of oxygen  Bypasses body’s normal system of transporting oxygen

64. IntroductiontoHBOT:MechanismofAction Limits ischemic damage, cell death, inflammation Promotes collagen synthesis (fibroblast stimulation) Decreases lactate production and tissue acidosis Aids in oxygen dependent killing of bacteria – WBC Limits leukocyte adhesion and degranulation Decreases tissue edema

65. HBOT: Mechanisms for Addressing Chronic Pain  Decreases inflammation, reduces hypoxia, and improves microcirculation  For neuropathic pain, analgesic and antinociceptive effects are due to cellular modulation  Autophagy in the mitochondria of microglia (mitophagy) (Han et al., 2017)

66. HBOT: Mechanismsfor Addressing ChronicPain  Mitochondria are the primary source of ROS  ROS can:  Induce mutations in mtDNA causing protein deficiencies  Restrict ability to self-repair, leaving cells more vulnerable to ROS attack  Damage mitochondrial proteins and lipids by inducing oxidative stress (Nie et al., 2015; Koirala et al., 2013; Lupfer et al., 2013)

67. Latent mitochondria arelikecampfires leftburning all night

68.  HBOT modulates cellular autophagy (mitochondria of microglia) and directly reduces pain  Appropriate clearance of mitochondria is important for maintaining homeostasis in cells HBOT:Addressing Chronic Pain withMitophagy

69. HBOT:Addressing ChronicPainwith Mitophagy  20 rats were given a CCI (chronic constriction injury); 20 rats got CCI+ HBOT  20 rats were sham CCI and 20 rats were controls  All 80 rats were given CSI (a mitophagy inhibitor) before testing  MMP was used to measure mitophagy (lower MMP observed with more mitophagy) (Han et al., 2017)

70. HBOT:AddressingChronicPainwithMitophagy  HBOT improved mitochondrial permeability via transitive pores on the mitochondrial membrane  More permeability results in more mitophagy (see as lowered MMP) which reduces ROS calming neuro- inflammation and pain Control & Sham – minimal to no mitophagy (no change in MMP) MMP: Mitochondrial membrane potential CCI: Chronic constriction injury (Han et al., 2017)

71. Mitophagy is putting the mitochondrial fires out by involuting the ashes and soil upon the remaining embers. Without mitophagy, wildfires (of pain) get out of control. July 4th, 2018 Basalt, CO (Courtesy of Pete McBride)

72. Whatelse encouragescellular autophagy (includingneuronal autophagy)? Intermittent Fasting!  Dr. Yoshinori Ohsumi Wins Nobel Prize for this discovery  https://www.garmaonhealth.com/intermittent-fasting- cellular-autophagy/ Fun Fact

73. HBOT:OtherMechanisms forAddressingChronicPain  Suppresses pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-alpha and simultaneous releases anti-cytokines  Suppresses astrocyte activation and inflammatory responses (stopping gliosis) by:  Decreasing TNF-α  Decreasing Kindlin-1 and Wnt- 10a in the dorsal root ganglia (DRG), spinal cord, and hippocampus of rats (Zhao, B., Pan, Y., Xu, H., & Song, X., 2017)

74. HBOT: Mechanismsfor ChronicPain: CaseStudy  40 year old spinal cord injury (C4 burst fx from mtn biking accident) paraplegic patient with chronic spasticity and pain in lower extremities  Reports almost immediate reduction in neuroplasticity, inflammation, and pain when treated in a HBOT chamber at 2.4 ATA

75. HBOTforSports Injuries  Reduces swelling  Blunts the inflammatory process  Improves range of motion earlier/ PT  Increases and enhances tissue growth  Fibroblast and osteoblast proliferation  Improves bone regeneration-faster and stronger fracture repair

76. CaseStudy  Injured on January 5th 2009  Shearing fracture, surgically repaired  High risk for Non-Union  Started HBO January 7th 2009  30 tx over 6 week period  Cleared to ski March 3rd 2009

77. HBOT:UpregulatesPluripotentAdult StemCells(akaVSELs-verysmall embryonic-likestemcells)intheblood

78. Adult StemCells • Derived from bone, adipose, or blood • Require physician expertise and quality control • Mostly used for regenerative and cosmetic purposes • Readily available • Less expensive • Autologous use is permitted in US (with restrictions)

79. Peripheral Blood-Based AdultStemCells • Originate in bone marrow • Present in peripheral blood • Dr. Young (2004) • Forms cells from the three primary germ-layer lineages • Also known as very small embryonic-like stem cells (VSELs) or blastomere-like stem cells • Have a long lifespan (can double more than 70 times) • *Not derived from umbilical cord blood (mesenchymal) (Pluripotent / Embryonic-Like)

80. Peripheral Blood-Based AdultStemCells • Understanding lineage uncommitted pluripotent stem cells requires an understanding of the germ layers • Lineage uncommitted pluripotent stem cells can produce all types of cells in the germ later (Young & Black, 2004) (Pluripotent / Embryonic-Like)

81. Peripheral Blood-Based AdultStemCells Clinical indications: • Regenerative in their applications unlike mesenchymal • Actually develop into new target tissue such as organs, cartilage, neurons, muscle, skin, etc. • Conditions treated: traumatic brain injury, chronic pain, ligament / tendon injuries, diabetes, osteoarthritis, osteoporosis, Alzheimer’s disease, fertility, aging, etc. (Pluripotent / Embryonic-Like)

82. Mean CD34+ population (hematopoetic and pluripotent cells) in blood of humans before and after HBO2 treatments. 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, 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.

83. PeripheralBlood Pluripotent(VSELs) vs.Multipotent (Mesenchymal- MSCs) • Many stem cell clinics are focused on the use of mesenchymal stem cells (MSCs) • MSCs are derived from bone marrow, umbilical, or fat • MSCs have merit for homologous use (bone marrow to bone marrow or fat to fat transplantation) • MSCs do not actually transform, in vivo, to new tissues

84. Pluripotent (VSELs) Multipotent (Mesenchymal) Recently discovered in peripheral blood From bone marrow, fat, and cord blood Also known as very small embryonic-like stem cells (VSELs) Mesenchymal stem cells (MSCs) Does not have a specialized trajectory of development On a development trajectory Give rise to all the cell types Specialization potential limited to one or more cell lines Lineage uncommitted Lineage committed Long lifespan Short-lived Not restricted by FDA Increased FDA restriction for non- homologous tissue use Best for regeneration Best for homologous use

85. Stemcellsand prpwork together  Stem cells = seeds  Growth factors = soil/water/fertilizer/sunlight  Without growth factors, the seed cannot mature and grow Stem Cells and Growth Factors

86. StemCellsand GrowthFactors (PRP) • Signaling molecules between cells • Cytokines and hormones that bind to specific receptors • Promotes cell differentiation and maturation • Designed to improve metabolism of nutrients • Stimulate growth of collagen: cartilage, bone, ligaments, tendons, blood vessels, and neurons • Guide stem cells to area of injury • Nurture stem cells to maturity

87. Pluripotent Stem Cells (VSELs) Displaced (5mm) C-7 proximal spinal fracture failed to heal 9 months post trauma Pre-Treatment Post-Treatment 4 months post-treatment of peripheral blood-based stem cells – the fracture is fully healed

88. ArthritisCase Report  80 year old with tricompartmental arthritis x 10 years, confirmed by xray, worse in R knee  Treated with VSELs in Bilat Knee joints, menisci, and associated ligaments on 2/9/2018  Reports on 4/13/2018 that her left knee does not hurt  Reports improvements in walking with less R knee pain on 6/7/2018. Patient provided booster PRP injection into R knee joint and IT band at 6/7/2018  “The only consistent symptom I have is that it is always uncomfortable when I stand up from a sitting position and when I first get up in the morning. Usually just a few steps and the discomfort is gone.”

89. Conclusion  The scientific mechanisms and effects of HBOT used in combination with PRP- PBSC (Platelet Rich Plasma and Peripheral Blood Stem Cells) provide a solid basis for use in the treatment of pain, inflammation, tissue damage, and degeneration associated with TBI, sports injuries, and arthritic conditions.

90. Treats TBI patients by combining regenerative therapies: HBOT, stem cells, PRP, and nutritional therapies. tbitherapy.com Treats chronic pain and major medical problems using the best of modern and natural medicine. aspenintegrativemedicine.com