This document summarizes the potential benefits of low-level laser therapy (LLLT) for anti-aging and healing applications. It discusses how LLLT may work by increasing ATP production and reducing oxidative stress at the mitochondrial level. It also reviews evidence that LLLT can reduce inflammation, promote wound healing, and help with conditions like arthritis, brain injuries, and hair regrowth through photobiomodulation effects on cell signaling and gene expression. The document examines key parameters for effective LLLT dosing and provides examples of clinical studies supporting its use.

1. Photobiomodulation in Anti-Aging: Harnessing the Power of Low Level Laser Therapy (LLLT) for Healing and Regeneration Michael R Hamblin PhD

2. Michael R Hamblin PhD A4M 2011 THOR LLLT Knowledge Matrix Safe mixed acceptance.health economics Introduction (context) Phototherapy is not new Not like laser surgery History & future Large evidence base Mechanism Primary Absorption / molecular rotations and vibrations Secondary NO ATP ROS membrane permeability Tertiary Transduction Gene transcription Secretion Signalling Quaternary Amplification / systemic Clinical Benefits Repair Inflammation Analgesia Function muscle, lymph Physics Photons Electromagnetic spectrum Wavelength Laser vs other Coherence polarisation spectral width Propagation Depth of target, RATS Dosimetry Wavelength absorption Irradiance dose rate Pulse Period on, period off Peak & average power Tx time How many times Interval Where Injury Lymph Nerve Ganglia, trigger points Blood ? Glands ? Preconditioning ? Other Clinical Safety Contraindications Regulations Reimbursement Increasing ATP, Reduces oxidative stress and ??? when LLLT does not work .... Safe, credible Many applications What is light How does it behave Define medicine Time is the dose Clinical targets

3. Michael R Hamblin PhD A4M 2011 Dentistry, pain Brain, stroke, TBI Non-union fractures Lateral epicondylitis Tinnitus Skin rejuvenation Reduction of heart attack Carpal tunnel syndrome Arthritis Laser acupuncture Wound healing Laser lipolysis Hair regrowth Mucositis Achilles tendonitis Muscle fatigue Neck pain Temporomandibular joint disorder

4. History 1967 - Prof. Mester - Budapest - discovers laser biostimulation 1983 - UK Physios first use low power laser for sports injuries treatment 1998 - NASA conducted LED therapy wound healing study for space 1960 - Theodore “Ted” Maiman built the first working laser 1988 - James Carroll raised £ for laser research at Guys Hospital London 1999 - US Military nerve regeneration research with THOR lasers 1916 - Einstein first proposed the phenomenon of stimulated emission 1993 - THOR formed 2003 - First FDA clearance for THOR 2005 - Spinal cord regeneration 2006 - LLLT published in Nature and Pain 2008 - WHO Bone & Joint Task Force recommend on neck pain (Spine) 2009 - Lancet review neck pain 2010 - APTA recommends LLLT for Achilles Tendinopathies 2010 - BMJ “strong evidence” for LLLT on frozen shoulder 2010 - Intl Assoc for the Study of Pain, “strong evidence“ Chronic Myofascial Pain 2011 - ???

5. Low Level Laser Therapy (LLLT) Published in the worlds top scientific journals Over 200 (RCT) clinical trials Over 2000 laboratory studies

6. Michael R Hamblin PhD A4M 2011 Treatment

7. Complex I Complex II Mitochondrial ATP ADP Complex III Complex IV Michael R Hamblin PhD A4M 2011 H + H + H + H + H +

8. Michael R Hamblin PhD A4M 2011 Subunit II Subunit I 4 cyt c (ox) 4 cyt c (red) 4 e - Cu A Cu B Heme a Heme a3 O 2 4 H 2 H 2 O

9. Michael R Hamblin PhD A4M 2011 Respiration↑ ATP↑ Red or NIR light Subunit II Subunit I 4 cyt c (ox) 4 cyt c (red) 4 e - Cu A Cu B Heme a Heme a3 4 H 2 H 2 O e- e- e-

10. Michael R Hamblin PhD WALT 2010 Bergen Norway Study mechanisms of 810-nm laser in mouse cultured primary cortical neurons Look at biphasic dose response

11. Intracellular calcium post 810-nm laser Biphasic dose response

12. Mitochondrial membrane potential post 810-nm laser Biphasic dose response .

13. ATP levels post 810 nm laser. Biphasic dose response

14. Intracellular ROS post 810nm laser Triphasic dose response

15. NO levels post 810-nm laser Triphasic dose response

17. Myoglobin Hemoglobin Cytochrome c OX

19. Reactive oxygen species ( ROS) sensors and signaling ROS Heat shock factor - HSF-1 Nuclear factor kappa B - NF-kB Activator protein 1 - AP1 Nuclear factor erythroid 2 related factor 2 - Nrf2 Protein kinase D PKD Cellular analog of Rous sarcoma virus c-SRC kinase Thioredoxin - Trx apoptosis signal regulating kinase 1 ASK1 Glutathione - GSH c-Jun N-terminal kinase JNK

20. near infrared light extracellular matrix deposition growth factor production cell proliferation & motility anti-apoptosis and pro-survival Gene transcription signaling

21. LLLT activates NF-kB P50 NEMO IκBα IκBβ RelA IκB P50 RelA IκB P Phosphorylation P50 RelA Transcription Proteasomal degradation IκB Nucleus Cytoplasm LLLT O - , OH - ,NO - PKD Ub ROS

22. Submitted Used MEFs from transgenic NF-kB-luc reporter mice

23. NF-kB Anti-apoptotic c-IAP1, c-IAP2, survivin, Bcl-2, Bcl-xL, Bcl-xS, Bfl-1/A1, XIAP, c-FLIP, E2F3A, NR13, IEX-1, GADD45  , TRAF1, TRAF2 Anti-oxidant Mn-SOD, heme oxygenase 1, Glutathione peroxidase Ferritin heavy chain, NQO1  -glutamylcysteine synthetase Cytokines & chemokines IL1, IL2, IL6, IL2-R, IL8, IL9, IL11, GRO, IP10, MIP1, MCP, RANTES, eotaxin Pro-inflammatory iNOS, TNF-  , COX2, LTA, LTB, phospholipase A, Pro-proliferation MCSF, GCSF, GMCSF, c-myc, VEGF-C, PDGFB, BMP2, c-myb, cyclin (D1, E) Adhesion molecules E-selectin, ICAM1, VCAM1 ELAM, MADCAM1 Adaptive immunity MHC-I, MHC-II, IgG k light chain, IGHG3, CD3  , CD105, TAP1, CD69, Acute phase response C reactive protein, serum amyloid A, angiotensin, tissue factor, MMPs, complement (B, C4)

24. PKC Transcription AP1 activation Does LLLT activate AP-1? ROS GDP+P ↑ GTP ELK-1 RAF1 NEKK1 MEK JNNK1 JNK1 ERK C-FOS c-JUN CK-II SRF

25. Laser abrogates glutamate excitotoxicity

26. Conclusion 810-nm laser is anti inflammatory

28. Do you need a laser? What about coherence? Laser speckles Low energy High energy NIR

30. epidermis dermis subcutaneous fat muscle tendon bone CW 100 ms 10 ms 1 ms 100 µs 10 µs 100 ns 1 µs

31. Cell proliferation Cell adhesion Cell apoptosis LLLT Cell migration MCSF,Cyclin(D1,E), CDC42, L-actin E-selectin , ICAM1, VCAM1,IL8 Bcl-2,c-IAP1,Survivin

32. LLLT Increases Growth Factors and Cytokines TGF-  bFGF, HGF and SCF IL-1  IL-8 VEGF LLLT collagen synthesis Fibroblasts M yofibloblasts initial inflammatory phase Neo-vascularization Fibroblast proliferation and migration 

33. Mitochondrion NO cytC ox respir Mitochondrion NO cytC ox O 2 Red or NIR light Mitochondrion cytC ox Mitochondrion cytC ox respir O 2 O 2 Healthy cell High pO 2 HIF1  Hypoxic cell respir low pO 2 HIF1  VEGF New blood vessels Hypoxic cell with NO med pO 2 HIF1  low pO 2 HIF1  VEGF New blood vessels VEGF respir X The hypoxic cell that doesn ’t know it is hypoxic

34. Biphasic dose response? Response Dose

35. Stimulation/Inhibition of Wound Healing in Mice

36. C57/BL6 Balb/c SKH1 hairless Green:  -smooth muscle actin, blue: DAPI nuclei Red = phalloidin (all actin)

37. Low level light reduces inflammation and swelling in arthritis in rats Control (Zymosan 4 mg/kg) Zymosan dexamethasone Zymosan 3 J/cm2 @ 50 mW/cm2 1 minute Zymosan 3 J/cm2 @ 5 mW/cm2 10 minutes Zymosan 30 J/cm2 @ 50 mW/cm2 10 minutes Zymosan 30 J/cm2 @ 5 mW/cm2 100 minutes Treatments 1X day for 5 days 810-nm laser

38. 3 out of 4 laser regimens work well Non effective Effective

39. Traumatic brain injury in military and civilian medicine Civilian Blunt trauma MVAs, sports, assaults Military US: 2003-2007 - 43779 cases 28% of patients at Walter-Reed >$100,000,000 Explosive blast injury, Overpressure, Penetrating injury, Diffuse axonal injury 1,400,000? Total 1,111,000 Emergency Room Visits 235,000 Hospitalizations 50,000 Deaths 80,000 Disabilities

40. Clinical trials of pharmacological and physical therapies for stroke/TBI Therapeutic approaches for stroke/TBI QuickTime™ and a decompressor are needed to see this picture. Antioxidants Ebselen NXY-059 a nitrone spin-trap agent Tirilazad Edaravone Iron chelator Traditional Chinese medicine Anti-inflammatory Anti- neutrophil adhesion molecule Nitric oxide signal transduction down-regulator: lubeluzole Corticosteroid Interleukin-1 receptor antagonist Circulation Volume expansion Flow enhancer Vasodilator Hemodilution Blood pressure-related strategy Oxygen Hyperbaric oxygen Oxygenated fluorocarbon Oxygen supplementation Physical intervention Hypothermia (brain cooling) Hemicraniectomy Osmotic agent Excitotoxicity Potassium channel opener Sodium channel blocker Calcium chelator Magnesium GABA agonist Glutamate/AMPA antagonist NMDA receptor/polyamine blocker Metabolism Ganglioside , Astrocyte modulator Beta blocker,CNS stimulant Phosphatidylcholine precursor Fibroblast growth factor Opioid antagonist Prostanoid , Statin Neurochemical Serotonin antagonist Serotonin receptor agonist Serotonin uptake inhibitor STROKE TBI

41. Michael R Hamblin PhD MGH Sports Medicine

42. NEST-1

43. NEST-2

45. 665-nm laser 810-nm laser 980-nm laser Four different wavelength lasers 732nm Laser

46. Transcranial LLLT for TBI in mouse model (IACUC approved) closed head weight drop method based on Marmarou (1994)

47. Neurological performance testing Neurological Severity Score(NSS) for Brain-Injured Mice Presence of mono- or hemiparesis 1 Inability to walk on a 3-cm-wide beam 1 Inability to walk on a 2-cm-wide beam 1 Inability to walk on a 1-cm-wide beam 1 Inability to balance on a 1-cm-wide beam 1 Inability to balance on a round stick (0.5 cm wide) 1 Failure to exit a 30-cm-diameter circle (for 2 min) 1 Inability to walk straight 1 Loss of startle behavior 1 Loss of seeking behavior 1 Maximum total 10 One point is awarded for failure to perform a task.

48. Transcranial LLLT for TBI in mouse model Mice were performance tested 1 hour post-TBI Mice received a single exposure to laser on top of head at 4 hours post-TBI Fluence was 36 J/cm 2 delivered at 150 mW/cm 2 over 4 min with spot size 1-cm diameter Estimated light penetration to dura is 3% Subsequent performance testing for 4 weeks

49. Transcranial LLLT for TBI in mouse model

50. Controlled cortical impact TBI in mice Amscien Instruments

51. Biphasic dose response in LLLT for TBI Figure 4. (A) NSS scores from mice with CCI TBI treated with 1 or 14 exposures to 810-nm laser (36 J/cm2 at 50 mW/cm2) commencing 4h post-TBI. (B) Mice were treated with a single exposure to 810-nm laser at different fluences and irradiances. (C) Mice were treated with a single exposure to 810-nm laser (36 J/cm2 at 50 mW/cm2) that was either CW or pulsed at 10 or 100 Hz and a 50% duty cycle. (D) Mean lesion area measured at 4 weeks from mice in Fig ?A.

54. LLLT for Depression and PTSD

55. MedX LED cluster 870-nm & 633-nm Case1. 59 yo F, 7 yr. post-MVA after 8 weekly Tx. ’s, ability to do computer work had improved 10-fold, obtained home unit and has used daily for 5 years. Case 2. 52 yo F, multiple concussions and PTSD, Tx. ’d daily with home unit, memory and “executive function” tests improved >2 SD, after 9 months. Off “Medical Disability” status after 4 months of home treatments; returned to full-time work.

57. Low-level laser therapy for hair regrowth

59. Acknowledgments Michael J Whalen MD Sulbha K Sharma PhD Margaret Naeser PhD Frederic Schiffer MD Gitika B Kharkwal PhD Qiuhe Wu, MD, PhD Tatiana N Demidova-Rice PhD Ana P Castano MD Tianhong Dai, PhD Takahiro Ando MS Tao Xu PhD Weijun Xuan MD, PhD Aaron C-H Chen Ying-Ying Huang MD