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This video was created to support their published research. The authors did research using several lasers and slices of a sheep’s brain to try and determine the best parameter for treating TBI (Traumatic Brain Injury) with a desired fluency of 0.9 to 15 joules/cm2 at a depth of 2 cm. They state that getting the energy through the skull is especially difficult so they test multiple options so test the transfer rate. They started out using a continuous output split 980/810nm system (the only company that makes that type of split system, 80% of the power at 980nm and 20% of the power at 810nm, is LiteCure with their LightForce series). The result was less than 1/2% of the energy reached a depth of 2cm. Then they switched to pulsing and got an increase in the energy transfer. When they switched to a 810nm-only 15 watt system with pulsing the transfer rate increased to 16% of the output energy reached the target depth.

 Here are some rough numbers to review the feasibility of using this system for treatment. If the duty cycle is 70%, the system will deliver 1.68 joules per second at a depth 2cm (15wattS*70%*16%). To get 5 joules/cm2 over 15 x 15 cm treatment area would require a total of 1125 joules at depth. This would take 23 minutes.

This research shows that only class 4 systems can delivery the level of power needed for this kind of therapy in a typical rushed doctor's office. A class 3b system with 1 watt would take 4 - 5 hours per treatment to get the same dosage.

The original research publication is titled " Treatments for traumatic brain injury with emphasis on transcranial near-infrared laser phototherapy"

video length: (9:18)

 1Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

2Department of Cell Biology and Immunology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

Email: *oronu@post.tau.ac.il

Received 27 May 2013; revised 29 June 2013; accepted 16 July 2013

ABSTRACT

In this study, we investigated the hypothesis that photo- biostimulation by low-energy laser therapy (LLLT) applied to the bone marrow (BM) of myocardial in- farcted rats may attenuate the scarring processes that follow myocardial infarction (MI). Wistar rats under- went experimental MI. LLLT (Ga-Al-As diode laser) was applied to the BM of the exposed tibia at differ- ent time intervals post-MI (4 hrs, 48 hrs and 5 days). Sham-operated infarcted rats served as control. In- farct size was significantly reduced (55%) in the la- ser-treated rats as compared to the control non-treat- ed rats, at 2 weeks post-MI. A significant 3-fold in- crease was observed in the density of desmin immu- nopositive stained cells 14 days post-MI in the infarc- ted area of the laser-treated rats as compared to the non-laser-treated controls. The electron microscopy from the control infarcted rat hearts revealed a typi- cal interphase area between the intact myocardium and the infarcted area, with conspicuous fibroblasts with collagen deposition dispersed among them. In rats that were laser treated (to BM), the interphase zone demonstrated cells with different intracellular struc- tures. There was also a significant increase in the per- centage of c-kit positive cells and macrophages in the circulating blood of the laser treated rats as compar- ed to control non treated ones. In the majority of the cells clusters of myofibrils anchored to well-developed Z-lines and structures resembling the morphological characteristics of mature intact cardiomyocytes were evident. In conclusion, LLLT to the BM of rats post- MI induces cardiogenesis mainly at the borders of the infarcted area in the heart.

Keywords: Low-Level Laser Therapy; Myocardial Infarction; Macrophage; Desmin; Ultrastructure; c-Kit Positive Cells

1. INTRODUCTION

Regenerative capacity and mitotic activity in the heart are confined mainly to the lower vertebrates [1]. Amputation of ~20% of the zebrafish’s ventricular myocardium re- sulted in full regeneration without scarring [2]. In am- phibians, heart injury was associated with increased cell proliferation of myocytes and enhanced regeneration [3]. The adult mammalian heart was traditionally considered to be a post-mitotic organ with terminally differentiated cardiac myocytes. However, this dogma has recently been challenged by several studies and reviews [4-8]. These studies have suggested that cardiac myocytes are replaced throughout the lifespan even in the human heart, and that myocytes can regenerate from resident cardiac progenitor cells (CPC) as well as from bone marrow (BM). Studies in human infarcted hearts have shown evidence of cytoki- nesis of cells in the heart and evidence of cardiac stem cells that are activated in response to ischemic injury. This growth response is attenuated in chronic heart fail- ure [9]. Some studies have reported that cardiac myocyt- es can be derived from BM; specifically, side population precursor cells following induction of myocardial infarc- tion (MI) by left anterior descending artery (LAD) liga- tion [10-12]. Contradicting these findings, other laborato- ries using genetic markers have reported that lineage ne- gative, c-kit+ BM cells did not differentiate into cardio- myocytes [13]. It was also suggested that BM-derived stem cells may stimulate the small population of stem cells in the ischemic heart to proliferate and differentiate to enhance cardiac repair post-MI [14]. In a recent study transient regenerative potential in the mouse heart was demonstrated during the neonatal period [15].

Low-level laser therapy (LLLT) has been found to modulate various biological processes [16,17], such as increasing mitochondrial respiration and ATP synthesis [18], facilitating wound healing and promoting the proc- ess of skeletal muscle regeneration and angiogenesis [19- 21]. In an experimental model of the infarcted heart in rats and dogs, it was demonstrated that LLLT application directly to the infarcted area in the heart at optimal power parameters significantly reduced scar tissue formation [22-24]. This phenomenon was partially attributed to a significant elevation in ATP content, heat shock proteins, vascular endothelial growth factor (VEGF), inducible ni- tric oxide (NO) synthase, and angiogenesis in the ischemic zone of the laser-irradiated rats, as compared to non- irradiated rats [25].

The effect of photobiostimulation on stem cells or pro- genitor cells has not been extensively studied. LLLT ap- plication to normal human neural progenitor cells signi- ficantly increases ATP production in these cells [26]. LLLT delivery to MSCs and cardiac stem cells in vitro caused a significant enhancement in their proliferation rate [27,28]. LLLT has also been shown to increase the proliferation rate of adipose-derived stem cells in vitro [29]. Recently, we demonstrated that LLLT application to autologous BM could induce mesenchymal stem cells (MSCs) in the BM to proliferate and cause their recruit- ment and specific homing in on the infarcted rat heart and not on other organs [30,31]. The laser treatment to the BM also caused a marked and statistically significant reduction of 79% in the scarring and ventricular dilata- tion followed MI as compared to infarcted non-laser- treated rats. The aim of the present study was to investi- gate the possibility that induction of stem cells in the BM of rats by LLLT could also affect cardiogenesis in the in- farcted rat heart.

2. MATERIALS AND METHODS

2.1. Experimental Procedures

A total of 21 Wistar male rats, weighing 200 - 250 gr, that underwent ligation of the LAD artery to induce MI, were used as described by us previously [23]. All the ex- perimental procedures were approved by the animal care committee of Tel-Aviv University. Briefly, rats were anes- thetized with Avertin (1 ml/100 g body weight I.P.) and the lungs were ventilated. Thoractomy was performed by invasion of the intercostals muscles between the 5th and 6th rib to expose the heart. The LAD artery was occluded 2 mm from the origin with 5-0 polypropylene thread (Ethicon Inc., Cincinnati, OH). Following LAD artery occlusion the chest muscles and skin were sutured and the rats were ventilated until they woke up. The infarcted rats were divided randomly into two groups. In one group LLLT was applied directly to the BM 4 hrs, 48 hrs and 5 days post-MI (see below). The second group was non-laser-treated (the rat’s bone was exposed for the same duration as the laser-treated group but the laser was not turned on). Food and water were supplied ad libitum. Rats were sacrificed 14 days post-MI.

2.2. Laser Application

After induction of MI rats were randomly assigned to a laser-treated or control non-laser-treated group. A diode (Ga-Al-As) laser, wavelength 804 nm with a tunable po- wer output of maximum of 400 mW (Lasotronic Inc., Zug, Switzerland) for application to the BM was used. The laser device was equipped with a metal-backed glass fiber optic (1.5 mm diameter). An infrared viewer (Laso- tronic Inc. Zug, Switzerland) and infrared-sensitive de-tecting card (Newport, Inc., Irvine, CA) were used to de- termine the infrared irradiation area. Laser application was done by a 10 mm longitudinal cut in the skin above the medial aspect, and further delicate cleaning of the bone surface was carried out. The tip of the fiber optic (1.5 mm diameter) was placed perpendicularly to the center of the exposed medial aspect of the tibia and power den- sity of 10 mW/cm2 was applied to the BM. The laser was applied for a duration of 100 sec (energy density 1.0 J/cm2). Left or right exposed tibias were chosen at random for LLLT application. In sham-operated infarcted rats that served as control the tibias were exposed and the fi- ber optic was placed as described above but the laser beam was not turned on.

2.3. Histology and Electron Microscopy

A defined cross-section sample (2 mm thick) from the central part of the infarcted area was taken from all hearts for histology. Eight micron paraffin sections were pre- pared from the tissue samples of each heart. Infarct size was determined using Masson’s trichrome staining as described by us previously [23]. Three observers, blinded to control or laser-treated rats, analyzed infarct size. Six microscopic slides from the infarcted area of each heart were chosen at random for determination of infarct size. Infarct size was expressed as the percentage of the total infarcted area relative to the total area of the left ventri- cle (LV) in each section, using image analysis software Sigma Scan Pro (Sigma, St. Louis, MO).

For electron microscopy three tissue samples from each of the control and laser-irradiated rat hearts were taken from the interphase zone between the infarcted and non-infarcted tissue by macroscopic examination. Fixa- tion was performed in 3.5% glutaraldehyde in 0.1 M ca- codylate buffer for 24 hrs followed by embedment in Epon-812. Semi-thin sections (1 micron) were prepared in order to localize the interphase zone. Thin sections were then prepared and stained with uranyl acetate and lead citrate followed by examination with a Jeol electron microscope.

2.4. Immunohistochemistry

The total number of cells immunostained for desmin (bone marrow cells or newly formed) in the infarcted area were determined using a desmin kit (Zytomed Laboratory, Ber- lin, Germany). The procedure was performed at room temperature with anti-mouse (dilution 1:25 - 1:50) primary antibody for 60 min. Following washing, slides were in- cubated with HRP secondary antibody for mouse for 30 min followed by DAB Chromogen system (Covance Inc., Dedham). Slides were rinsed again in wash buffer, stain- ed in Hematoxylin for nuclei detection, mounted and viewed using a Zeiss microscope equipped with a camera and video screen. The total number of desmin immuno- stained cells within the infarcted area was counted and their density expressed as the percentage of the total area of the infarct using SigmaPro software.

2.5. Flow Cytometry Analysis

Blood samples were taken 2 and 7 days post-IR injury for fluorescence-activated cell sorting (FACS) analysis. 100 μl of blood were mixed with different antibodies: anti-mouse CD117 (c-kit) PE (eBioscience San Diego, USA) and rat IgG2b isotype control PE (eBioscience San Diego, USA) and anti-rat macrophage marker PE (eBio- science San Diego, USA) and mouse IgG2a K isotype control PE (eBioscience San Diego, USA), were used for the FACS analysis according to the manufacturer’s guide- lines. Forty five min post incubation of the whole fresh blood with the relevant antibodies, 2 ml of Fix/Lyse so- lution (eBioscience, San Diego, USA) was added. After mixture the suspended cells were left for 60 min in the dark at room temperature. Centrifugation was performed for 10 min, supernatant was removed and washing of the pellet was performed with 2 ml of Flow Cytometry Stain- ing Buffer Solution (eBioscience, San Diego, California, USA). After another centrifugation for 10 minutes the supernatant was decanted. The pellet containing mono- nucleated cells was resuspended in 200 μl of flow stain buffer for FACS analysis.

2.6. Statistical Analysis

The SigmaStat 2.0 (Sigma, St. Luis, USA) software was used for statistical analysis. Tests were performed first for normality distribution, followed by parametric (stu- dent’s t-test) test.

3. RESULTS

Application of LLLT to the infarcted heart caused a sig- nificant (p = 0.049) reduction of 55% in infarct size as compared to control. The present of macrophages and c- kit positive cells in the blood was determined by FACS analysis (Figure 1). It was found that at 5 days post MI there was a statistical significant 2-fold higher concentra- tion of macrophages and significant 1.4-fold higher c-kit positive cells (mesenchymal cells) in the laser treated rats as compared to the infarcted non laser treated rats. Des- min immunostaining of histological sections of the in- farcted zone from laser-treated rats demonstrated a higher density of positively stained cells than in the non laser-treated ones (Figures 2-4). In the interphase zone, cells extending from the myocardium towards the in 

Figure 1. Percent (out of total mononucleated cells) of macro- phages and c-kit positive cells in blood of control and laser treated rats (to the bone marrow) 5 days post MI as revealed by FACS analysis. The results are mean ± S.E.M of 15 rats at each group. Statistical significance *p < 0.05; **p < 0.01.

Figure 2. Representative desmin immunostained light micro- graphs of the infarcted zone of non-laser-treated rats (a, c) and laser-treated rats (to the bone marrow at 4 and 48 hrs and 5 days) (b, d) taken 2 weeks post-MI. Note that the zone in the control non-laser-treated rats contains mainly collageneous mate- rial with a few desmin immunopositive cells in the infarcted area (a, c); while in the laser-treated rats the zone displays posi- tive desmin staining in extended outgrowths (arrow) from the myocardium (MC) in (b), and in the cytoplasm of many cells in the infarcted area in (d). IF, Infarcted area. Bar = 50 μm.

farcted area showed higher immunostaining for desmin in the laser-treated rat hearts as compared to the control non-treated ones (Figure 2). The cell density of desmin immune-positive cells was also determined quantitatively in histological sections of both the infarcted laser-treated rats and infarcted non-laser-treated rats. The cell density was significantly (p < 0.01) 3-fold higher in the infarcted area of the laser-treated rats as compared to the non-la- ser-treated controls (Figure 4).

The electron micrographs of all samples taken from the control non-laser-treated infarcted rat hearts revealed a typical interphase area between intact and infarcted heart (Figure 5(a)). Adjacent to the non-ischemic intact myocardium there were conspicuous fibroblasts with col- lagen deposition dispersed among them (Figure 5(a)). In all samples taken from the laser-irradiated hearts the in- terphase zone between intact and infarcted area demon- strated different characteristics to those of the non-laser- treated infarcted rat hearts. Cells with newly-formed or- ganized contractile myofilaments dispersed in the cyto- plasm were detected in groups of several cells (Figure 5(b)). In these cells numerous mitochondria, clusters of ribosomes, and conspicuous clusters of contractile pro- teins were evident in the cytoplasm (Figures 6-8). Some cells contained dispersed contractile myofilaments in the cytoplasm that were still in an early stage of organization (Figure 6). The organization of newly-formed contractile myofilaments in the cytoplasm was observed in various

Figure 3. Representative desmin immunostained light micro- graphs of the interphase of the infarcted zone of laser-treated rats. Note that desmin positively stained cross-sections of myo- fibers (arrows) intermingled in the infarcted zone in (a). In (b) immunopositively stained cross-sections of myofibers (arrow) are visible in the infarcted area (IF). In (c) newly-formed car- diomyocytes (NC) are seen, with the desmin immunostaining mainly confined to the Z-line. Bar = 50 μm.

Figure 4. Density of desmin positively stained area (relative to total area) in the infarcted areas of control (non-laser-treated) and laser-treated (to the bone marrow) rats at 14 days post-MI. Results are mean+ S.E.M from 6 - 8 rats in each group. **p < 0.01.

Figure 5. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of control non-laser- treated (a) and laser-treated (b) to bone marrow rats. Note intact myocardium (MY) and adjacent fibroblast (FB) in the infarcted area surrounded by collagen (CL) deposition in (a). In (b) sev- eral newly-formed cardiomyocytes (marked with asterix) with conspicuous well-organized myofilaments (MF) in their cyto- plasm are evident adjacent to blood capillaries (CA). EN, En- dothelial cell.

degrees of maturation in those cells. In some cells the myofilaments were dispersed in the cytoplasm and in others they were organized in clusters anchored to well- developed Z-lines (Figure 7(a)). In certain cells the myo- filaments were organized parallel to the longitudinal di- rection of the cells, resembling the morphological char- acteristics of mature intact cardiomyocytes (Figure 7(b)). Some of the cells were also seen in a process of forma- tion of typical intercalated disc between them (Figure 9).

4. DISCUSSION AND CONCLUSION

The most significant outcome of this study was the ap- pearance of newly-formed cardiomyocytes following laser treatment to the BM, as indicated by light and electron microscopy. There was a 3-fold increase in the density of

Figure 6. Electron micrographs of most probably newly-formed cardiomyocytes at an early stage of organization of contractile myofilaments. Note myofilaments (MF) in the cytoplasm. M, Mitochondrion. Bar = 1 μm.

Figure 7. Electron micrographs of most probably newly-formed cardiomyocytes with early (a) and late (b) stages of the organi- zation of the contractile myofilaments in the cytoplasm. Note contractile myofilaments that are dispersed (DMF) in the cyto- plasm with a few organized in clusters anchored to Z-lines (Z) in (a). In (b) myofilaments (MF) are organized in parallel to the longitudinal axis of the cardiomyocyte, resembling their orga- nization in mature cardiomyocyte. N, Nucleus. Bar = 1 μm.

desmin immunostained cells in the infarcted rat hearts that had been laser treated. Desmin is a protein found in the cytoplasm of developing myocytes and cardiomyo- cytes [32]. The significantly higher occurrence of des- min-positive cells in the infarcted area of the laser- treated hearts may indicate the synthesis of new contrac- tile proteins in the developing new cardiomyocytes, re- sembling the process that takes place during embryonic development. The ultrastructural features of the cells in the interphase between the intact myocardium and the

Figure 8. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of laser-treated infarcted rat heart. Note numerous mitochondria (M) in the cytoplasm of the cardiomyocytes in (a) and (b). Also note organized contrac- tile myofilament with well-developed Z-lines (Z), some dis- persed myofilaments and clusters of ribosomes (R). Bar = 1 μm.

Figure 9. Electron micrographs of typical intercalated disk formation in the interphase region of the infarcted heart of la- ser-treated rats. Formation of intercalated disks (ID) between cells (marked with asterix) is evident. Note that the most proba- bly newly-formed cardiomyocytes contain clusters of myofila- ments (MF) in the cytoplasm that are conspicuous in their obli- que or cross-sections (arrows). Bar = 1 μm.

infarcted myocardium of the laser-treated rats, as shown in this study, clearly resemble the characteristics of car- diomyocytes during embryonic development of the heart [33]. Furthermore, the clusters of ribosomes and the nu- merous clusters of mitochondria in the cytoplasm of these cells may characterize cells that are active in the synthe- sis of proteins. It was previously demonstrated that direct LLLT to the infarcted hearts of rats, dogs and pigs caus- ed a significant reduction of scarring post-MI [23,24]. It was suggested that part of this reduction could be ex- plained by the regenerative response that takes place in the interphase zone [24].

The results of the present study indicate that the LLLT

applied to autologous BM attenuates the concentration of macrophages and MSC in the circulating blood. We have previously shown that LLLT application to the BM of infarcted rats caused a 2 fold enhancement in the rate of proliferation of MSC in the BM [30]. Those cells that most probably leave the BM to the circulating blood in- deed show a significant elevation of their concentration (as reveled by the FACS analysis in the present paper) at 5 days post MI. Consequently these cells probably home in on the infarcted heart, and even migrate specifically to the infarcted area [30]. These cells may induce cardiac stem cells to differentiate to newly-formed cardiomyo- cytes, as suggested previously by Hatzistergos et al. [14]. Indeed, it was found that endogenous c-kit+ cardiac stem cells were increased by 20-fold in the rat infarcted heart compared to control, following transcardial injection of BM-derived MSCs [14]. Such induction may be enabled due to paracrine secretion of various growth factors by the laser-stimulated MSC that originated from the BM. The possibility that paracrine secretion occurs in im- planted stem cells during cell therapy to the heart post- MI has been suggested previously [34]. Another mecha- nism that may take place after homing of stem cells to the infarcted heart of the laser-stimulated rats is that these cells continue to proliferate in the appropriate mi-lieu of the interphase zone in the infarcted heart and then differentiate to cardiomyocytes [30].

Another possible mechanism that maybe associates with the reduction of infarct size is the significant increase in the concentration of macrophages in the circulation fol- lowing LLLT to the BM as revealed from the FACS analysis in the present study. These findings corroborate with studies indicating that macrophages activity in the infarcted area at early stages post MI cause reduction of scarring post MI [35,36]. Thus, it could be postulated that more macrophages that will eventually home in the infarcted area from the circulating blood in the laser treated rats will also contribute to the reduction of scar- ring.

Although the findings of the present study do not in- dicate the extent of regenerative capacity of the rat in- farcted heart post-laser-irradiation, they do reveal a shift from practically no cardiomyocytes in the tissue samples taken from the non-laser-treated hearts, to the presence of newly-formed cardiomyocytes in all the electron mi- croscope sections taken from the hearts of rats that are laser-treated to the BM.

In conclusion, to the best of our knowledge, this is the first study to demonstrate the appearance of newly-form- ed cardiomyocytes in the infarcted area following LLLT to autologous BM in the infarcted rat heart. The mecha- nisms associated with this phenomenon remain to be elu- cidated in further studies.

5. ACKNOWLEDGEMENTS

This study was partially supported by the Elizabeth and Nicholas Shle- zak Super-center for Cardiac Research and Medical Engineering. The authors wish to acknowledge N. Paz for editing the manuscript and V. Wexler for helping with preparation of the figures.

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Oron, U. (2011) Light therapy and stem cells: A thera- peutic intervention of the future. Journal of Interventio- nal Cardiology, 3, 627-629.

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Toma, C., Pittenger, M.F., Cahill, K.S., Byrne, B.J. and Kessler, P.D. (2002) Human mesenchymal stem cells dif- ferentiate to a cardiomyocyte phenotype in the adult mu- rine heart. Circulation, 105, 93-98. doi:10.1161/hc0102.101442

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Mummery, C.L., Davis, R.P. and Krieger, J.E. (2010) Challenges in using stem cells for cardiac repair. Science Translational Medicine, 14, 1-5.

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Dr. Hamblin discusses the use of low level laser therapy for all type of brain injuries. He is an expert in all type of light healing (see below). He has performed much of his research on rats. He claims several key points:

• 10 Hertz is the preferred pulsing frequency for the brain
• 810nm is the preferred wavelength for cell interaction.
• The best treatment period is about 7 sessions but  less than 14 days of  treatments. Going to longer term treatments seems to reduce the effectiveness.

This video is mainly about TBI but the principals are universal. Dr Hamblin is associated with Thor laser so there is some potential for bias but he is also assocated with Harvard and the Wellman Centre. Introduction to Low Level Laser Therapy (LLLT) for Traumatic Brain Injury (TBI) by Mike Hamblin. Wellman Centre for Photomedicine, Harvard Medical School.

video length: (6:18)

This video is restricted for minors.

In this video Dr. Michael Hamblin is interviewed by youtuber Selfhacked. Dr. Hamblin discusses the use of red and near infrared light in low level light therapy, he talks about the different possibilities of LLLT as well as some of his personal experiences with LLLT. He explains the reasons behind some of the effects from LLLT noticed in patients over the years, including it's effect on calcium in cells, ion channels, and infrared light vs. ultraviolet light. Dr. Hamblin also notes the differences between LLLT, bright light therapy, and light from the sun that is used for theraputic purposes. The majority of the video is spent discussing the effect of LLLT on the brain specifically.

video length: (52:08) 

This video features Dr. Juanita Anders who is a pioneer and expert in the field of PBM. This is a great overview of the science and concepts in PBM. While the talk does get pretty technical and involved in places, a full understanding of all of the biological mechanisms that Dr. Anders discusses isn't necessary to get a high level understanding of the science and benefits of light therapy.

This link is an audio interview with Dr. Michael Hamblin (an associate professor at Harvard Medical School) and is an informative podcast type of interview regarding the latest in PBM research.

Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster Rana Begum 1 , Karin Calaza 2 , Jaimie Hoh Kam 1 , Thomas E. Salt 1 , Chris Hogg 3 and Glen Jeffery Institute of Ophthalmology, University College London, London EC1V 9EL, UK 2 Program of Neuroscience, Institute de Biologia, Universidade Federal Fluminense, Rio de Janeiro 24210130, Brazil 3 Moorfields Eye Hospital, London EC1V 2PD, UK Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflam- mation with age. Critically, there was a significant increase in average lifespan: 100–175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age. 1. Introduction Mitochondria provide cellular energy via adenosine triphosphate (ATP). But, their DNA (mtDNA) suffers from progressive mutations resulting in reduced ATP production, which is thought to run concomitantly with an increase in pro-inflammatory reactive oxygen species (ROS) [1,2]. Hence, hallmarks of ageing are reduced cellular energy and progressive systemic inflammation. Meta- bolic demand also plays a role as tissues and organisms with high metabolic rates generally suffer from rapid ageing [3,4]. The retina has the greatest metabolic demand in the body [5], but ATP decline in the central nervous system can be significantly improved by near-infrared/infrared light (NIR/IR, [6]). Specific wavelengths in this range are absorbed by cytochrome c oxidase in mitochondrial respiration, improving its efficiency [7–10]. These wavelengths improve mito- chondrial membrane potentials, significantly reduce inflammation and reduce macrophage numbers with brief exposures of around 60–90 s repeated over approximately a week [11,12]. NIR/IR also reduces experimental pathology when insult impacts on mitochondrial function, as in experimental Parkinson’s disease, where NIR significantly reduces cell death in the substantia nigra [13]. However, NIR/IR studies have largely used light for short periods and their impact on lifespan has not been assessed [7,11,12]. If NIR improves mitochondrial function we predict it may extend life. The fly has been used here because of its relatively short life [14]. Hence, we ask if long-term exposure to 670 nm in Drosophila melanogaster can increase lifespan and improve function in old age. 2. Material and methods Drosophila melanogaster were used. Hatched male flies were housed on 12/12 light cycle at 258C within a season. Half were exposed to 670 nm for 20 min per day at & 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from 40 mW cm 22 in clear plastic 50 cm 3 (28 mm wide) containers, illuminating flies from either side, which were counted weekly. Room illumination was 2 mW cm 22 . 670 nm energies were approximately 100 times lower than indirect sunlight, consistent with earlier studies [7]. Light devices were built by C. H. Elec- tronics UK and contained 50 670 nm LEDS over 20 cm 2 . Six independent replicates were used in lifespan experiments (n ¼ 620 flies). ATP, inflammation and mobility were assessed at seven weeks, when ATP and mobility are known to decline [15]. ATP was measured by luciferin–luciferase assay (Enliten w ATP Assay System, Promega). Flies were killed with liquid nitrogen, transferred to 2.5% trichoroacetic acid (TCA), then homogenized at 48C. Supernatant was collected and the TCA was neutralized with 1 M Tris–acetate buffer (pH 7.75, final TCA concentration 0.0625%); 10 ml of neutralized solution was added to 100 ml of luci- ferin–luciferase in fresh buffer. ATP was measured using an Orion microplate luminometer (Berthold Detection Systems GmbH) and data normalized to fly numbers. Tissues were homogenized in 2% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail for Western blot (Roche Diagnos- tics), and centrifuged; the supernatant was pipetted out, separated with 10% SDS–PAGE and electrophoretically transferred onto nylon membranes. Immunoblotting was undertaken for complement component C3 (Cappel, MP Biomedicals), which is highly conserved [16]. Protein was quantified by densitometric X-ray scanning and values were normalized to a-tubulin. Fly mobility assessment was as Bjedov et al. [14]. Flies were placed in 100 ml clear cylinders (seven flies per trial), tapped to the bottom and then videoed, the last two steps repeated three times. Using the videos, the number of flies above the 50 ml mark (9 cm from the bottom) was counted after 1 min. Individual flies were traced, with absolute distance travelled measured. Data w er e analysed with GraphPad P RISM v. 5 and sta t is ti cal analysis was undertaken using Mann –Whitne y U non-parametric and log-rank tests. 3. Results (a) ATP levels are elevated and systemic inflammation reduced Whole body ATP declines with age only after appr o xima tely seven weeks [14], when ATP was measured here. A TP concen- trations wer e significantly grea t er, by approxima tely 80%, in 670 nm exposed animals compared with unexposed (figure 1a, Mann–Whitney test p ¼ 0.028). At seven weeks, W estern blots wer e undertaken for inflammatory marker complement com- ponent C3. This was reduced in 670 nm exposed flies compared with controls (figure 1b). Hence, 670 nm radiation elevates ATP and reduces inflammation. (b) Lifespan increases Fly numbers in experimental and control groups were similar in the two weeks post-hatching. From week 3, fly deaths were greater in controls than 670 nm exposed flies and they remained so at each time point until week 11–12, when all flies were dead in both groups. This difference was significant (figure 2, log-rank test p ¼ 0.008). The progressive mean percentage increase in 670 nm flies alive over controls is given in figure 2b. Group differences accelerated from week 4, when 10% extra 670 nm treated flies were alive compared with controls, to approximately 50% extra when the control population had halved. By the time the control population was reduced by 80%, at week 8, more than 100% extra 670 nm treated flies remained alive. Subsequently, group differences reached almost 180% before declining to zero in both groups at week 11–12. Hence, 670 nm did not extend absolute lifespan. (c) Aged mobility increases Mobility of 670 nm treated and control flies was measured at seven weeks. Significantly more 670 nm treated flies climbed above the 50 ml level (9 cm) and significantly more travelled a greater distance than controls (Mann–Whitney test p ¼ 0.028, p ¼ 0.014, respectively). Twice as many 670 nm flies climbed above 50 ml (9 cm) compared with controls and these travelled twice the distance in 1 min compared with controls (figure 2c,d). Hence, 670 nm exposure significantly improves both lifespan and mobility. 4. Discussion Drosophila melanogaster has been widely used in lifespan studies as they are short lived and their genomic sequence is relatively well understood [14,17], hence their adoption experimentally here to extend lifespan. Our results reveal [ATP] (nM)/fly ATP level(a) C3 expression (Western blot)(b) control absolute intensity 670 control 670 control C3 ~ 110 kDa a-tubulin 55 kDa 670 * 0 0 500 1000 200 400 600 Figure 1. Exposur e to 670 nm radiation increases ATP in aged flies and reduces inflammati on. (a) Sev en week old flies exposed to 670 nm had a significant incr ease in whole body ATP compared with controls, p ¼ 0.028. n ¼ 25 flies per group. (b) Whole body inflammation (C3) was measured in seven week flies using W estern blot. This was reduced in 670 nm exposed flies by approxima tely 15%. Here, flies were pooled within groups as C3 protein levels were lo w in individuals. Hence there are no error bars. n ¼ 15 flies per group. (Online version in colour.) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 2 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from that when flies are exposed to 670 nm radiation they have reduced inflammation, improved ATP, improved mobility and extended average lifespans. These data are consistent with the majority of studies undertaken using 670 nm on mammals, showing reduced inflammation in experimental models and in ageing, and improved ATP levels [6,7]. How- ever, it would be difficult to undertake lifespan experiments in mice as the light would not penetrate the entire body as it does in flies and hence its influence would not be systemic. There are many factors and pathways in ageing, and nine candidate hallmarks have been suggested, which may be separate, but also are likely to have interactions [1]. Mito- chondrial function is one. Previously, mitochondrial function and ageing were viewed within a framework of pro- gressive mtDNA mutations/deletions resulting in reduced ATP and increased ROS. The balance of these factors was seen as a driver in the mitochondrial theory of ageing [18]. However, evidence has undermined the role of ROS in ageing [19,20]. Hence, some mutant mice have reduced life- span as a result of mtDNA mutations/deletions not associated with increased ROS [21,22]. Further, increased ROS can prolong lifespan in yeast and Caenorhabditis elegans [22,23], and in mammals it does not accelerate ageing [20]. These data are reviewed by Lopez-Otin et al. [1], who argue that low ROS may activate compensatory mechanisms and not directly contribute to ageing. Such data may undermine the ROS element in Harman’s mitochondrial theory [18]. If correct, it places greater potential emphasis on ATP in ageing. NIR has been successful in treating induced pathology [7] and ageing, particularly in the retina, where progressive age- related inflammation is marked owing to high metabolic rate [11,12]. These wavelengths penetrate deeply and 670 nm trans-illuminated our flies at 40 mW cm 22 . In relation to this, it may be significant that, while old domestic incandescent light- ing contained significant NIR elements, none is present in modern strip lighting or energy-saving domestic lighting [12]. The absence of these wavelengths from artificial lighting may have long-term consequences. As longer wavelengths penetrate deeply, this may be of significance not only for the ageing eye, but also potentially for other tissues. Ethics statement. Fly research is free of legal ethical constraint. Data accessibility. All data are presented in the manuscript. Acknowledgement. We thank Iris Salecker, Giovanna Vinti and Tobi Weinrrich for technical assistance. Author contributions. G.J. designed experiments and wrote the manu- script. All authors undertook the experiments and approved the final version of the manuscript. R.B. analysed the data. Funding statement. Supported by the Rosetrees Trust UK. K.C. was a research fellow from CAPES Brazil (proc. 18134/12-2). Competing interests. We have no competing interests. References 1. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. 2013 The hallmarks of aging. Cell 153, 1194–1217. (doi:10.1016/ j.cell.2013.05.039) 2. Balaban RS, Nemoto S, Finkel T. 2005 Mitochondrial, oxidants and aging. Cell 120, 483–495. (doi:10.1016/j.cell.2005.02.001) 3. Speakman JR. 2005 Body size, energy, metabolism and lifespan. J. Exp. Biol. 208, 1717–1730. (doi:10. 1242/jeb.01556) 4. Wang Z, Ying Z, Bosy-Westphal A, Zhang J, Schautz B, Later W, Heymsfield SB, Mu¨ller MJ. 2010 Specific metabolic rates of major organs and tissues across adulthood: evolution by mechanistic model of resting expenditure. Am. J. Clin. Nutr. 92, 1369– 1377. (doi:10.3945/ajcn.2010.29885) 5. Yu DY, Cringle SJ. 2001 Oxygen distribution and consumption within the retina in vascularized and avascular retinas and in animal models of disease. Prog. Retin. Eye Res. 20, 175–208. (doi:10.1016/ S1350-9462(00)00027-6) 0 0 10 20 30 40 50 60 70 80 100 (a) (c) (d ) (b) % survival over controls n = 620 flies fly survival curves for 670 nm (–) and control (–) 90 123456 time (weeks) % survival 78910 0 0 100 200 300 123456 no. weeks % increase over control control distance travelled in 1 min 670 control 670 * 0 20 60 40 80 100 distance (mm) fly climbing (>90 mm) * 0 20 60 40 80 % >90 mm in 1 min 78910 11 12 Figure 2. Lifespan and mobility. (a) Fly numbers at progressive weeks in groups exposed to 670 nm supplemented light each day (red line) and controls (black line). Curves are averages for six independent experiments with a minimum of 40 flies per group in each experiment. Fly death rates separated between three and six weeks with fewer flies dying in 670 nm exposed animals. Reduction in the two population followed similar patterns from six weeks but with the 670 nm exposed group having greater numbers at any point until week 12. In all replicates, there was no indication that 670 nm increased absolute lifespan beyond weeks 11 – 12. Differences between the two groups were statistically significant ( p ¼ 0.008). (b) Inset: percentage increase of 670 nm exposed flies alive at pro- gressive weeks. (c) Seven week old 670 nm exposed flies were more active than controls. (d) Mobility measures the percentage of flies that climbed above 90 mm in a clear 100 ml cylinder. (d) This was filmed and then the distance travelled by each fly was measured in each group. In both cases, the 670 nm exposed flies where significantly more mobile. There were 21 flies in each group in each condition. (Online verion in colour.) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 3 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from 6. Gkotsi D, Begum R, Salt T, Lascaratos G, Hogg C, Chau KY, Schapira AH, Jeffery G. 2014 Recharging mitochondrial batteries in old eyes. Near infra-red increases ATP. Exp. Eye Res. 122, 50 –53. (doi:10. 1016/j.exer.2014.02.023) 7. Fitzgerald M et al . 2013 Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders. Rev. Neurosci. 24, 205–226. (doi:10.1515/revneuro-2012-0086) 8. Wilson M, Greenwood C. 1970 The long-wavelength absortion band of cytochrome c oxidase. Biochem. J. 116, 17 –18. 9. Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI. 2005 Absorption measurements of cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation. J. Photochem. Photobiol. 81, 98– 106. (doi:10.1016/j.jphotobiol.2005.07.002) 10. Cooper CE, Springett R. 1997 Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy. Phil. Trans. R. Soc. Lond. B 352, 669– 676. (doi:10.1098/rstb.1997.0048) 11. Kokkinopoulos I, Colman A, Hogg C, Heckenlively J, Jeffery G. 2013 Age-related inflammation is reduced by 670 nm light via increased mitochondrial membrane potential. Neurobiol. Aging 34, 602– 609. (doi:10.1016/j.neurobiolaging.2012.04.014) 12. Begum R, Powner MB, Hudson N, Hogg C, Jeffery G. 2013 Treatment with 670 nm up regulates cytochrome C oxidase expression and reduces inflammation in an age-related macular degeneration model. PLoS ONE 8, e57828. (doi:10. 1371/journal.pone.0057828) 13. Purushothuman S, Nandasena C, Johnstone DM, Stone J, Mitrofanis J. 2013 The impact of near- infrared light on dopaminergic cell survival in a transgenic mouse model of parkinsonism. Brain Res. 1535, 61 –70. (doi:10.1016/j.brainres.2013.08.047) 14. Bjedov I, Toivonen JM, Kerr F, Slack C, Foley A, Partridge L. 2010 Mechanisms of life span extension by rampamycin in the fruit fly Drosophila melanogaster. Cell Metab. 11, 35–46. (doi:10.1016/ j.cmet.2009.11.010) 15. Vernace VA, Arnaud L, Schmidt-Glenewinkel T, Figueiredo-Pereira ME. 2007 Aging perturbs 26S proteasome assembly in Drosophila melanogaster . FASEB J. 21, 2672–2682. (doi:10.1096/fj.06- 6751com) 16. Nonaka M, Kimura A. 2006 Genomic view of the evolution of the complement system. Immunogenetics 58, 701– 713. (doi:10.1007/ s00251-006-0142-1) 17. Celniker SE, Rubin GM. 2003 The Drosophila melanogaster genome. Annu. Rev. Genomics Hum. Genet. 4, 89–117. (doi:10.1146/annurev.genom.4. 070802.110323) 18. Harman D. 1981 The ageing process. Proc. Natl Acad. Sci. USA 78, 7124 –7128. (doi:10.1073/pnas. 78.11.7124) 19. Edgar D et al. 2009 Random point mutations with major effects on protein coding genes are the driving force behind premature aging in mtDNA mutator mice. Cell Metab. 10, 131–138. (doi:10. 1016/j.cmet.2009.06.010) 20. Hiona A et al. 2010 Mitcohondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial mutator mice. PLoS ONE 5, e11468. (doi:10.1371/ journal.pone.0011468) 21. Doonan R, McElwee JJ, Matthijssens F, Walker GA, Houthoofd K, Back P, Matscheski A, Vanfleteren JR, Gems D. 2008 Against the oxidative damage theory of aging: superoxide disumatases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 22, 3236–3241. (doi:10.1101/gad. 504808) 22. Mesquita A et al. 2010 Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H 2 O 2 and superoxide dismutase activity. Proc. Natl Acad. Sci. USA 107, 15 123–15 128. (doi:10.1073/pnas.1004432107) 23. Zhang Y et al. 2009 Mice deficient in both Mn superoxide dismutase and glutathione peroxidase-1 have increased oxidative damage and a greater incidence of pathology but no reduction in longevity. J. Gerontol. A Biol. Sci. Med. Sci. 64, 1212– 1220. (doi:10.1093/gerona/glp132) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 4 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from

This video shows before and after treatment footage of a patients with advanced Parkinsons. Treatment lasts about 2 weeks. Dr Riner is using the brain and neurostim setting on the brain, C5 Nerve Root and the Ulnar nerve in the elbow.

The TheraLazr is the prototype for the Avant LZ30 series of lasers.

video length: (2:03)

LLLT being used to treat a patient with TMJ. She experiences pain in her cheeks and neck particularly, and afterward claims to have no pain or discomfort. The doctor states that the LLLT has reduced inflammation, and relaxed the muscles that were causing the spasms.

 video length: (2:43)

The laser info starts around 30 minutes into the presentation.

The presentation includes research done on rats for the following conditions:

• Traumatic Brain Injury (TBI)
• Spinal Cord Injury
• Nerve regeneration

The research was supported by Thor so it could be biased but their research indicates that 810nm provides better stimulation of the cells.

video length: (17:09)

Dr. Burke shares some of his quite impressive results in patients who recieved LLLT, incuding:

• a woman with severe ulcers on her hand
• a horse with a leg wound
• an 88 year old woman with diabetes and an infected ulcer on her leg, soon to be amputated
• a 68 year old woman with a non-healing knee surgury wound
• a man with a 40 year old venous ulcer
• a woman with blood clots in her feet that led to gangreen, soon to be amputated
• a 12 year old girl with post herpetic Bell's Palsy
• a man with very bad leg fracture
• a woman with a jones fracture

video length: (13:46) 

News report about the use of low level light therapy (LLLT) on a surgical wound that wouldn't heal after nearly a year in a patient with diabetes.

video length: (2:17) 

Young man with mucositis recieved a bone marrow transplant and was given LLLT both pre-, and post-op. He was expected to have serious mouth sores, but thanks to the LLLT he had minimal sores that quickly went away.

video length: (1:35) 

Chad Davis, DVM, gives a demonstration of the effecacy of LLLT on a horse.

 video length: (4:39)

This short video shows 3 before and after videos of paralized dogs who recieved LLLT and were able to walk again.

video length: (1:25) 

This video discusses the basics of Low Level Laser Therapy. You will learn a little bit about lasers and laser history, and what makes a cold laser a cold laser. It also talks about the difference between lasers and LED's and why the latter may be less effective for medical therapy.

You'll find information on the treatment parameters of LLLT which are:

• wavelength
• power
• duty cycle (continueous or pulsed)
• energy density (dosage)
• treatment duration

It also goes over what indications the FDA has approved LLLT and infrared light for.

 video length (12:13)

 Dr. Michael Hamblin Talks about the differences between photodynamic therapy (PDT) and low level laser therapy (LLLT), and explains the basics of LLLT.

video length: (4:35) 

In this video, you will see one of the better graphic representations of the photobiomodulation process including the release of ATP, NO, ROS.

This video was created by LightCure so at the end of the video, they try to make the point that class 4 laser are better than class 3 system. With the release of high-power class 3b (with power levels over 15 watts) system, this part of the video out of date.

video length: (3:08)

Class IV K Laser is an excellent new treatment for helping muscle pulls, sprains, strains, and joint injuries. Cold Laser is a great treatment for speeding the recovery of knee pain, chondromalacia patella, patella tendonitis, and knee sprains. Lasers help increase cellular ATP, which is the cells energy source. Cells use the increased energy for healing and repair. Lasers are also excellent at increasing metabolic and repair process within the tissue. They decrease inflammation and pain around muscles, tendons, and joints. In addition, the speed recovery and healing of nerves, especially pain nerves.

video length: (1:51) 

 Dr. Adam Zuckerman talks about his use of LLLT in patients with diabetes.

video length: (1:46) 

Veterinarian Ron Hirschberg tells of how he first experienced laser therapy on his own arthritis, and decided to invest in lasers for his practice. Since he started using laser therapy on the pets he works with he has seen many positive results, he explains that gross profits from NSAIDs decreased from 0.71% to 0.3%, and laser profit now makes up 4% of the practice's income. He will treat between 3 and as many as 13 patients with LLLT, 95% of which he says improve noticably faster than those without LLLT.

viedo length: (13:37) 

In this video Spokane Chiropractor Dr. Patrick Dougherty gives a quick demonstration of how cold laser therapy is used as a chiropractic treatment to help with range of motion by affecting the nervous system. This can be used as an effective adjunct to chiropractic adjustments when the brain is having a difficult time holding on to the input that the adjustments provides to the brain.

video length: (4:34) 

This short animation from gives a simple description of how LLLT effects individual cells and things like:

• cytochrome c oxidase
• oxygen
• NADH
• Nitric oxide
• ATP 

video length: (1:21) 

Dr. Larry Lytle discusses LLLT, particularly for pain in an interview.

video length: (27:37) 

Cold Laser Therapy can be very effective for those suffering from pain caused by auto accidents. Cold Laser Therapy is equally effective for those suffering from pain caused by work-related accidents. Athletes get great relief from sports-related injuries using Cold Laser Therapy. Cold Laser Therapy is a powerful therapy in the battle to relieve back pain, neck pain and joint pain. 

Importantly, studies to date indicate that Cold Laser Therapy has no serious side effects when used by a trained healthcare professional. It is a non-invasive procedure requiring no surgical incision. There is no recovery time after a treatment. You do not have to take any medications relating to Cold Laser Therapy.

 video length: (5:49)

Using LLLT to treat neck pain, procedure without wavelength or power, or any other laser parameters given.

video length: (1:14) 

Dr. Fritz teaches Obstetrics & Gynecology at the Academy for Oriental Medicine in Austin.

After getting undergraduate and graduate degrees in Biology from the University of Virginia, Dr. Vanessa Fritz graduated from the National College of Natural Medicine in Portland, Oregon, with a doctorate in Naturopathic Medicine (ND) as well as a Master of Science in Oriental Medicine (MSOM). 

video length: (9:32)

video length: (11:05) 

This video gives a somewhat in-depth list of dental issues that LLLT can be used to treat and how to treat them, along with a simple explaination of what LLLT is. At (16:48) there is a demonstration on how to use a Zolar laser for LLLT.

video length: (19:49) 

This video shows how red laser light interacts with blood and it talks in detail about the chemical process.

K-Laser Mechanism of Action, Animal from K-LaserUSA on Vimeo

LLLT is known to dramatically improve conditions associated with soft tissue inflammations, not only by reducing pain, but also by providing a significant therapeutic advantage resulting in inflammation reduction, as well as expediting the healing process.

• Plantar fasciitis is an inflammation of the band of tissue (the plantar fascia) that extends from the heel to the toes. In this condition, the fascia first becomes irritated and then inflamed, resulting in heel pain. The most common cause of plantar fasciitis relates to faulty structure of the foot. Wearing non-supportive footwear on hard, flat surfaces puts abnormal strain on the plantar fascia and can also lead to plantar fasciitis. 
• Achilles tendinitis is an overuse injury of the Achilles tendon, the band of tissue that connects calf muscles at the back of the lower leg to your heel bone. The structure of the Achilles tendon also weakens with age, which can make it more susceptible to injury — particularly in people who may participate in sports only on the weekends or who have suddenly increased the intensity of their running programs
• A neuroma is a thickening of nerve tissue that may develop in various parts of the body. The most common neuroma in the foot is a Morton’s neuroma, which occurs between the third and fourth toes. It is sometimes referred to as an intermetatarsal neuroma. Morton's neuroma seems to occur in response to irritation, pressure or injury to one of the nerves that lead to the toes.

video length: (3:25) 

A man experiencing tooth sensitivity atests to pain reilief from LLLT.

video length: (0:54)

Dr. Bernard Filner MD Discusses his use of LLLT in pain management.

video length: (2:48) 

12.1. INTRODUCTION

Significant changes in blood flow or in the integrity of cerebral vessels are believed to cause cerebrovascular disease (CVD) and to contribute to dementias including Alzheimer’s disease [1]. Stroke, the most serious form of CVD, is one of the leading causes of death and adult disability worldwide. Acute treatments for stroke, however, are severely limited. Neuroprotective drugs under development show promise at halting the ischemic cascade, but as yet, no such compound has received federal approval in the United States. One of the biggest limitations to this development is the lack of understanding of the mechanisms by which cerebral vessels react to factors such as ischemia, inflammation, blood pressure changes, metabolic demands, and trauma [2]. In order to address these fundamental questions, functional brain imaging techniques such as fMRI and intrinsic signal optical imaging (ISOI) have emerged as tools to visualize and quantify cerebral hemodynamics.

In the neuroscience community, ISOI has long been used to study the organization and functional architecture of different cortical regions in animals and humans [3–5] (see other chapters in this book). Three sources of ISOI signals that affect the intensity of diffusely reflected light derive from characteristic physiologic changes in the cortex. For functional neuronal activation, these have been observed to occur over a range of timescales, including (1) light scattering changes, both fast (over 10 s of milliseconds) and slow (i.e., > ~0.5 s) (2) early (~0.5–2.5 s) absorption changes from alterations in chromophore redox status, i.e., the oxy/deoxy-hemoglobin ratio (known as the “initial dip” period), and (3), slower (~2–10 s) absorption changes due to blood volume increase (correlated with the fMRI BOLD signal). Light scattering changes have been attributed to interstitial volume changes resulting from cellular swelling, organelle swelling due to ion and water movement, capillary expansion, and neurotransmitter release [6,7]. The slower absorption factors have been demonstrated to correlate with the changes in metabolic demand and subsequent hemodynamic cascades following neuronal activation [4,8,9].

Using animal models of acute and chronic brain injury, ISOI has been used to quantify the acute hemodynamic events in response to stroke, including focal ischemia and cortical spreading depression (CSD) [10–21]. Researchers have also used ISOI to locate and quantify the spatial extent of the stroke injury, including ischemic core, penumbra, and healthy tissue zones [18,22]. CSD also plays a key role in migraine headache, and recent laser speckle imaging studies have revealed the neurovascular coupling mechanism to the transmission of headache pain [23,24].

To fully understand the underlying mechanisms in vascular changes associated with cerebrovascular diseases such as stroke, an optical imaging technique that has the capability to rapidly separate absorption from scattering effects can enhance the information content of traditional ISOI, enabling (1) more accurate quantitation of hemodynamic function, (2) isolation of the electro-chemical changes characterized by light scattering, and (3) longitudinal chronic injury studies of function where structural reorganization due to neovascularization can cause significant alterations in scattering [25,26].

Quantitative diffuse optical methods [27] such as spatially-resolved reflectance, diffuse optical spectroscopy (DOS), and tomography (DOT), and diffuse correlation spectroscopy (DCS) possess exquisite sensitivity to these functional and structural alterations associated with brain injury, and have been applied to the study of CSD [11,15,28]. DOS and DOT utilize the near-infrared spectral region (600–1000 nm) to separate and quantify the multispectral absorption (μ_a) and reduced scattering coefficients (μ_s′), providing quantitative determination of several important biological chromophores such as deoxy-hemoglobin (HbR), oxy-hemoglobin (HbO₂), water (H₂O), and lipids. Concentrations of these chromophores represent the direct metrics of tissue function such as blood volume fraction, tissue oxygenation, and edema. Additionally, the scattering coefficient contains important structural information about the size and density of scatterers and can be used to assess tissue composition (exctracellular matrix proteins, cell nuclei, mitochondria) as well as follow the process of tissue remodeling (wound healing, cancer progression). DOS utilizes a limited number of source-detector positions, e.g., 1–2, but often employs broadband content in temporal and spectral domains [29]. In contrast, DOT typically utilizes a limited number of optical wavelengths (e.g., 2–6) and a narrow temporal bandwidth, but forms higher resolution images of subsurface structures by sampling a large number of source-detector “views.” To achieve maximal spatial resolution, the ideal DOT design would employ thousands of source-detector pairs and wavelengths. However, several engineering considerations including measurement time and instrument complexity currently limit the practicality of this approach.

In this chapter we present the basic principles of a new, noncontact quantitative optical imaging technology, modulated imaging (MI) [30–32], and provide examples of MI performance in 2 rat models of brain injury, cortical spreading depression (CSD) and stroke. MI enables both DOS and DOT concepts with high spatial (<1 mm) and temporal resolution (<1 s) in a simple, scan-free platform. MI is capable of both separating and spatially-resolving optical absorption and scattering parameters, allowing wide-field quantitative mapping of tissue optical properties. While compatible with time-modulation methods, MI alternatively uses spatially modulated illumination for imaging of tissue constituents. Periodic illumination patterns of various spatial frequencies are projected over a large area of a sample. The diffusely reflected image is modified from the illumination pattern due to the turbidity of the sample. Typically, sine-wave illumination patterns are used. The demodulation of these spatially modulated waves characterizes the modulation transfer function (MTF) of the material, and embodies the sample optical property information.

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12.2. METHODS AND INSTRUMENTATION

12.2.1. Modulated Imaging Spectroscopy

The MI instrument platform was introduced originally by Cuccia et al. [30] Based on this design, we have developed a custom multispectral near-infrared (NIR) MI spectroscopy system capable of imaging between 650 and 1000 nm. A diagram of this system is shown in Figure 12.1.

FIGURE 12.1

Modulated imaging platform. QTH—quartz tungsten halogen; L1—aspheric condenser; H—hybrid hot mirror; DMD—digital micromirror device; L2—projection lens; L3—camera lens; LCTF—liquid crystal tunable (more...)

Broadband NIR illumination is provided by an intensity-stabilized 250 W quartz-tungsten-halogen (QTH) lamp (Oriel QTH Source with Light Intensity Controller, Newport Corporation-Oriel Instruments, Stratford, Connecticut). Light is collimated and refocused with a pair of aspheric F/#0.7 optical lens systems (Oriel Aspherab). A custom-sized 3.5 in square hybrid hot mirror (Reynard Corporation, i.e., R00670-00) was placed between the lenses to limit the illumination to wavelengths below 1000 nm. Light engine optics taken from a digital projector (NEC HT1000) serve to homogenize and direct the light onto a 0.7 in digital micromirror device (DMD Discovery™ 1100 with ALP Accessory Package, ViALUX, Germany). Grayscale spatial sinusoid patterns are projected at 400 Hz using the ViALUX software development toolkit, which generates the necessary pulse-width modulation of binary sub-frames to produce a specified grayscale bit-depth (1–8 bits). Finally, a fixed focal length (f = 100 mm) projection lens illuminates the tissue at a slight angle from normal with a 15 × 25 mm illumination field. Detection was performed at normal incidence using a CRI Nuance™ camera system, which combines a 12-bit CCD camera and a liquid crystal tunable filter (LCTF; λ = 650–1100 nm, Δλ = 10 nm). To avoid specular reflection, crossed linear polarizers are used in the illumination and detection arms. For this system, the former is a 1.5 in diameter NIR linear polarizer (Meadowlark Optics, VLM-200-IR-R) placed immediately after the projection lens, and the first stage of the Nuance LCTF serves as the latter. The DMD, CCD, and LCTF are controlled via USB by a laptop computer, and synchronized using LabVIEW software (LabVIEW 8, National Instruments), enabling fast acquisition of a series of patterns with various spatial frequencies.

12.2.2. SFD Measurement, Calibration, and Modeling

A detailed description of SFD measurement, calibration, and diffusion modeling is provided by Cuccia [32]. In this work, we modeled diffuse reflectance using a transport-based White Monte Carlo (WMC) method [33,34]. Previously, we have found that compared with Monte Carlo, (1) diffusion predictions over- and underestimate low- and high-frequency diffuse reflectance, respectively, and (2) the quantitative accuracy of diffusion degrades with decreasing albedo [32]. Due to the moderate albedo of brain tissue (μ_s′/μ_a ~ 10–20), we chose to analyze all brain data with the WMC approach. This homogeneous tissue model is a significant simplification of the multilayered rat brain, and more work is necessary to accurately model this complex system. We discuss further the consequences of our simple model in Section 12.2.5.

12.2.3. Optical Property Inversion Methods

In this chapter, we use two inversion methods to calculate the absorption and reduced scattering from measurements of diffuse reflectance. When high measurement precision is desired, we use a “sweep” in spatial frequency space, producing an overdetermined set of diffuse reflectance measurements, which can be fitted to our WMC forward model predictions using least-squares minimization. This method is performed for all spatially averaged region analysis of optical properties and chromophores. When increased acquisition and/or processing speed is desired, we alternatively use a rapid two-frequency lookup table method based on cubic spline interpolation [32]. This data can be achieved with a minimal 3-phase, single frequency image set (by demodulating and averaging the images to obtain AC and DC amplitude maps, respectively). On typical personal computers this approach is capable of millions of inverse lookup calculations per second, and is therefore used to calculate all high-resolution images including time sequences. The signal-to-noise ratio (and thus the measurement precision) of either approach is limited by the data sampling, with the two-frequency method having a lower precision with the tradeoff of higher acquisition and processing speed.

12.2.4. Spectral Analysis-Chromophore Calculation

The quantitative absorption coefficient is assumed to be a linear (Beer’s law) summation of individual chromophore absorption contributions:

μa(λ)=2.303∑i=13ci?i(λ),

12.1

where c_i and ?_i(λ) represent chromophore concentrations and molar extinction coefficients, respectively. Using reported extinction coefficients of HbO₂/HbR35 and H₂O,36 we can invert Equation 12.1 and calculate tissue chromophore concentration separately at each pixel by linear least-squares fitting to the multispectral absorption images. Total hemoglobin (HbT) and oxygen saturation (S_tO₂) can then be calculated as HbT = HbR + HbO₂ and S_tO₂= HbO ₂/(HbR + HbO₂) * 100, respectively.

12.2.5. Optical Property Mapping: Resolution Versus Quantitation

On a pixel-by-pixel basis, diffuse reflectance versus spatial frequency is fitted to the WMC forward model to extract the local absorption and reduced scattering optical property contrast. This process is repeated for each wavelength, resulting in multi-spectral absorption and scattering spectra at each pixel. The measured contrast from discrete absorbers and scatterers on millimeter and submillimeter spatial scales, however, will possess partial volume effects in all three spatial dimensions. This is due to the physical light transport length scales in tissue, limiting the true x-y resolution of optical property contrast to many detector pixels [32]. This phenomenon is not unique to MI, but present in all planar reflectance imaging measurements of turbid media. Absorption and scattering are calculated using a homogeneous reflectance model, extracting a locally averaged sampling of optical property contrast. Based on simulations of the tissue MTF for varying optical properties [32], we expect the resulting image resolution to scale directly with the transport length, l* = (μ_a + μ_s′)^{− 1}, and the spatial frequency of illumination. In this chapter, we place quantitative emphasis on average optical properties and chromophores measured over a field of view that is greater than l*. Spatial maps and videos of these parameters are displayed and referred to as “contrast maps,” with the caveat that high resolution features will exhibit degraded quantitative accuracy.

12.2.6. In Vivo Rat CSD Experiments

12.2.6.1. Animal Preparation

MI spectroscopy measurements were performed on an in vivo Wistar rat model with a thinned-skull preparation. All procedures were performed in accordance with approved IACUC protocol guidelines. The animals were anesthetized, placed in a stereotaxic frame, their skulls thinned and glass coverslip applied. This preparation is described in detail by Masino et al. [37] The resulting thinned skulls allowed direct imaging of the cortex over a 5 × 7 mm field-of-view (whisker barrel cortex, centered at the C2 location). In order to investigate the sensitivity of MI toward studying acute cortical injury, we induced cortical spreading depression (CSD) by applying 1 M KCl solution to the surface of the cortex through a perforated section of skull and dura, located approximately 3 mm above the camera’s imaging field.

12.2.6.2. MI Measurement Protocol

For each of three animals, our MI measurement protocol was twofold. Prior to CSD induction, baseline spatial modulation data were acquired at 6 spatial frequencies (3-phase projections each) from 0 to 0.26 mm⁻¹, at 10 nm intervals over the entire range between 650 and 980 nm. Depending on the wavelength, image acquisition times ranged from 200 ms to 4 s, with total spectral imaging time of approximately 30 s per spatial pattern. The entire measurement (34 wavelengths, 3 phases, 6 frequencies) was repeated three times for statistical averaging yielding an entire measurement time of approximately 30 min.

Next, rapid dynamic measurements were performed, beginning 1 min prior to K⁺Cl⁻ administration. Here, a significantly reduced data set was chosen in order to achieve high temporal resolution. Two spatial frequencies (0 and 0.26 mm⁻¹) were acquired with three phase projection images, as described in Section 12.2.2, at each of four wavelengths (680, 730, 780, and 830 nm). The resulting 12 images took in total 6 s, permitting a repetition rate of 10 measurements per minute. The animals were followed for a period of 10 min for rats 1 and 2, and a period of 30 min for rat 3.

All images in this study were smoothed by 2D convolution with a Gaussian filter function (FWHM = 3 pixels), and baseline repetitions were averaged prior to data processing. Additionally, time-series data were post-processed by smoothing slightly in time (Gaussian FWHM of 2 timepoints = 12 s).

12.2.6.3. Spatial Frequency Sensitivity Analysis

Because of the differential absorption sensitivity at low and high frequencies, optimal optical property separation is achieved when a large range of frequencies is used [31]. In Figure 12.2a, we depict this differential sensitivity using diffuse reflectance (MTF) predictions versus frequency, increasing μ_a by 100% from 0.02 (black line) to 0.04mm⁻¹ (gray line). This is done for two values of μ_s′, 0.6 (solid lines) to 1.2mm⁻¹ (dashed lines), simulating a 100% change in scattering. Notice that the low frequencies have a significant reflectance change due to absorption, while high frequency reflectance remains nearly unchanged. Conversely, reflectance changes due to scattering are observed at all spatial frequencies. In Figure 12.2b, we further visualize this by plotting the reflectance sensitivity to 1% changes in absorption and scattering. Whereas DC reflectance is equivalently sensitive to a fractional change in either absorption or scattering, at high spatial frequencies absorption contrast is lost while scattering contrast is retained. For instance, notice that at our maximum measurement frequency of 0.26 mm⁻¹ the reflectance is roughly 24 times more sensitive to scattering compared to absorption (ΔR_d = 0.56 μ_s′ versus 0.024 * 10⁻³ for μ_a). This plays an important role in Section 12.3.2 during our discussion of dynamic scattering measurement.

FIGURE 12.2

(a) Reflectance contrast in absorption and scattering covering a typical range of brain optical properties. (b) The frequency-dependent sensitivity to absorption (black line) and scattering (gray line), respectively. Reflectance at fx = 0.26 mm⁻¹ (more...)

In realistic heterogeneous tissues, a tradeoff exists between maximizing the frequency range for optical property accuracy and obtaining similar sampling volumes. As tissue is a low-pass spatial filter, high frequencies are attenuated quickly with depth. Using diffusion-based forward modeling, we have estimated mean sampling depths at 650 nm using measured average background optical properties of brain tissue. This was done by predicting the depth sensitivity to contrast from a planar perturbation in absorption, given a background fluence profile from spatial frequencies 0 and 0.26 mm⁻¹. Based on these results, we observe qualitatively similar depth sampling, with mean depth sampling ranging between 2.5 mm and 1.2 mm (for f_x = 0 and 0.26 mm⁻¹, respectively). In all cases maximal sensitivity was found in the first 1–2 mm, where cortical hemodynamic changes occur.

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12.3. RESULTS AND DISCUSSION

12.3.1. Baseline MI Spectroscopy

In Figure 12.3a we show a grayscale planar reflectance image of the cortical region of rat 1 at 650 nm. A dotted-line box denotes the region-of-interest (ROI) used for analysis, selected for its uniform illumination and the absence of cerebral bruising. The Monte Carlo-model fitting of spatial frequency data allows calculation of the absorption and reduced scattering coefficients. In Figure 12.3b we show the spatially averaged diffuse reflectance at 650 nm and the corresponding multi-frequency fit. Excellent agreement is observed between measurement data and the model-based fit, with derived μ_a and μ_s′ coefficients of 0.033 and 0.70 mm⁻¹, respectively.

FIGURE 12.3

(a) Reflectance map for rat 1, showing the 3.8 × 5.9 mm region chosen for quantitative analysis. (b) Sample MTF reflectance data (squares) and fit (solid line) at 650 nm. (c) Recovered optical property maps (above) and corresponding image histogram (more...)

Analysis of multifrequency reflectance data separately at each pixel results in spatial maps of absorption and reduced scattering contrast. In Figure 12.3c, we plot the μa and μs′ maps recovered at 650 nm for rat 1. Note the strong absorption in the vein region, due to a large absorption by HbR at this wavelength. Below the images, we show histogram distributions of the corresponding quantitative maps above, indicating the degree of spatial variation in recovered optical properties. The mean and standard deviation for the pixel-wise μa and μs ′ were 0.030 ± 0.007 mm⁻¹ and 0.63 ± 0.13 mm⁻¹, respectively. These statistical results are in good agreement with the spatially averaged reflectance fit from Figure 12.3b, suggesting that our simple pixel-wise fitting approach yields optical properties similar to that calculated using a global analysis.

By mapping the absorption coefficient at multiple wavelengths, we can perform quantitative spectral imaging of tissue. In Figure 12.4, we summarize the baseline spectroscopy results for all three animals. In Figure 12.4a we show the μ_a (left) and μ_s′ (right) coefficients versus wavelength (circles) recovered from spatially averaged fitting. Data for rat 1 is shown in black (rat 2 in dark gray; rat 3 in light gray). Note the distinct spectral features in absorption, resulting from oxy- and deoxy-hemoglobin (HbO₂, HbR), and water (H₂O) absorption. The calculated scattering coefficient generally decays with increasing wavelength, and the results from a power law (μ_s ′ = A·λ(nm) ^−b, solid lines) fit are shown. A small residual coupling is observed between measured scattering and absorption spectral features. In particular, the scattering at the shortest and longest wavelengths appears to be underestimated by 5–10%, occurring where the corresponding absorption is highest (due to HbR and H₂O, absorption features, respectively). Based on our experiments in layered tissue phantoms [38], we believe this effect is primarily due to frequency-dependent probing volumes in the presence of depth-heterogeneous structures.

FIGURE 12.4

(a) Average μ_a (left) and μ_s′(right) spectra over entire ROI (circles). HbO ₂, HbR, and H₂O concentrations are determined by subsequent least-squares fitting (solid lines) of molar extinction coefficients to the absorption. Data (more...)

Simultaneous linear fitting of the absorption to known extinction coefficients yields measures of chromophore concentration. Shown in Figure 12.4a, multispectral fitting (solid line) for rat 1 yields HbO₂, HbR, H₂O, HbT and S_tO₂ values of 56.3 μM, 33.2 μM, 63.9%, 89.6 μM, and 56.3%, respectively. Tabulated results of chromophore values for all three animals are shown in Figure 12.4b. Lipid absorption near 930 nm was not apparent in the μ_a spectrum, and when included in the spectral analysis was not found to significantly affect the results. The small absorption “bump” at 900–910 nm is an artifact of imperfect phantom calibration due to the presence of a sharp, strong silicone absorption peak that is present in the phantom.

We note that the solution for chromophore concentration is well-determined when the number of wavelengths is at least equal to the number of chromophores. Therefore, as few as two wavelengths can be used to separate HbO₂ and HbR (if a constant value of H₂O is assumed). Repeating the above analysis with 780 and 830 nm only (assuming H₂O = 65%) yields results for HbO₂ and HbR within 10% of those from full spectral fitting. Repeating the above analyses using a simple diffusion-based model provided qualitatively similar results for absorption and scattering spectra, but in general was found to overestimate the absorption coefficient by 10–25%.

Absorption spectra at each pixel can be separately analyzed to yield spatial maps of local HbO₂, HbR, and H₂O distribution, shown in Figure 12.5. Notice the high concentration of HbR over the large superficial draining vessel (venous) regions, also reflected in the S_tO₂ image, highlighting the effect of tissue oxygen extraction. Conversely, notice that the high albedo regions with less structural detail are highly oxygenated, with S_tO₂ levels between 60 and 70%. Lastly, the H₂O map reveals a relatively homogeneous distribution of water.

FIGURE 12.5

Chromophore fits to absorption spectra at each pixel yield maps of local HbO₂, HbR, and H₂O concentration (left). Total hemoglobin (HbT) and oxygen saturation (S_tO₂) maps can then be calculated from HbO₂ and HbR.

12.3.2. Dynamic MI Spectroscopy of CSD

We performed measurements of CSD in each of the three rats, as described in Section 12.2.3. The results are presented as follows. We first present data for a single animal, choosing rat 3 for its long observation period of 30 minutes. Three ROIs are selected for analysis, and baseline MI spectroscopy results are reported for each of these regions. Next, the observed dynamic time courses of diffuse reflectance, optical properties, and chromophore concentrations are shown for each ROI. We then present the full spatio-temporal dynamic contrast data for rat 3 (2D + time) in the form of “snapshot” images.

Figure 12.6 summarizes the baseline spectroscopy measurements for rat 3. In Figure 12.6a, we show three regions of interest superimposed on the DC reflectance map, chosen to highlight three different characteristic temporal profiles observed within the field of view. In Figure 12.6b we show the baseline spectral fits for each of these regions, and in Figure 12.6c we tabulate the resulting calculated chromophore concentrations. In general, Region A (black) is a high albedo region lacking any large blood vessels, whereas Regions B (dark gray) and C (light gray) include high-absorption blood vessels and mild cerebral bruising from surgery. These differences are apparent in their recovered absorption spectra and fits, with on average 27% higher HbT, and 32% lower saturation in the vascular regions. Also, 7% higher H₂O is found in Regions B and C, which may indicate increased edema due to bruising.

FIGURE 12.6

Regionwise spectral analysis of rat 1 baseline data including the respective (A) ROIs, (B) spectral absorption data (circles) and fit (lines), and (C) tabulated recovered chromophore data for each region

In Figures 12.7–12.9 (for regions A–C, respectively), we present the temporal dynamics of CSD in each ROI of rat 3 as measured by MI. In part (a) of each figure, we plot the multispectral diffuse reflectance changes at f_x = 0 mm⁻¹ (DC, top) and f_x = 0.26 mm⁻¹ (AC, bottom). In part (b), we plot the recovered Δμ_a (top) and Δμ_s′ (bottom) optical properties at each wavelength. While absolute values of diffuse reflectance and optical properties are measured separately at each time point, for visualization purposes all data are displayed as a change from that prior to KCl administration. Absolute optical property values at t = 0 (not shown) demonstrate excellent agreement (~5–10%) with full multifrequency baseline data.

FIGURE 12.7

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx =.26 mm⁻¹, bottom) for Region A of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) coefficients. (more...)

FIGURE 12.8

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx = 0.26 mm⁻¹, bottom) for Region B of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) (more...)

FIGURE 12.9

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx = 0.26 mm⁻¹, bottom) for Region C of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) (more...)

Looking first at the reflectance time courses of Figure 12.7a (Region A), we see in general a series of three CSD events over the 30 minutes, with each transient event occurring for approximately 4.3 minutes. The first event occurs at minute 2.9 after KCl application, indicating an initial latency between the insult and the first resulting spreading depression wave. Reflectance contrast is present in both DC and AC frequency components, but with markedly different signatures. Generally, the DC time course shows a slow, gradual decay, punctuated by sharp, wavelength-dependent spikes/dips (for short/long wavelengths, respectively). Alternatively, the AC signature contains three sets of transient dips consistent across all wavelengths, with final values leveling off progressively lower than baseline. Discussed in detail in the following paragraph, we believe these AC changes are due primarily a result of optical scattering and may be related to neuronal depolarization. The corresponding derived optical properties in Figure 12.7b reflects this, with μ_s′ trends tracking directly with the measured AC reflectance. As expected, μ_a trends reveal similar wavelength-dependence of the DC reflectance (with opposite polarity), reflecting changes in HbO₂ and HbR.

In Section 12.2.3.3 we noted that the diffuse reflectance at f_x = 0.26 mm⁻¹ is 23 times more sensitive to scattering changes compared to absorption. In this context, we propose that the observed magnitude of the CSD-induced AC reflectance changes can only be explained by changes in optical scattering. To concretely illustrate this point, we pick as an example the observed 780 nm AC diffuse reflectance dip in Figure 12.7a at t = 3.7 min of -0.003. Here, the corresponding change in reduced scattering in Figure 12.7b, Δμ_s′, is calculated to be −0.03 mm⁻¹. In order for this change to instead be due to an absorption-only event, μ_a would need to increase by 121% from baseline (from 0.038 to 0.084 mm⁻¹). This increase would also need to be accompanied by a drop in R_d (f_x = 0 mm⁻¹) of 0.12 (33%), whereas the actual observed DC reflectance only drops by 0.008 (<1%) and thus cannot explain the change. Secondly, we note that the three sets of AC reflectance dips occur consistently across all four wavelengths. While an approximate 120% increase in HbT could induce this decrease at high frequency, it would also require a large broad-wavelength decrease in the DC reflectance. We instead observe during these events that the DC increases at short wavelengths while the DC decreases at long wavelengths, suggesting primarily an exchange between HbO₂ and HbR volume fractions, as opposed to a dramatic HbT change.

Regions A–C (Figures 12.7–12.9) were chosen to highlight three different time signatures observed in the field of view during the CSD dynamics. The most contrasting feature between all three regions is the measured AC reflectance and the derived scattering coefficient. In Region B (Figure 12.8), each CSD event appears to cause a biphasic scattering change, with a sharp increase and then decrease, whereas a monophasic dip was observed in Region A (Figure 12.7). Region C (Figure 12.9) appears even more complex with a triphasic rise-dip-rise temporal profile. We observe that Regions A to C are located with increasing proximity to the CSD induction point (3 mm above the imaging field).

Because fractional changes in scattering and absorption have an equal (and opposite) effect on DC reflectance (see Section 12.2.3.3), any scattering (i.e., pathlength) changes measured here could be misinterpreted as absorption events with traditional ISOI analyses (i.e., DC reflectance only). In our observations, the measured scattering change of up to −0.05 mm⁻¹ would be interpreted as an increase in absorption of up to +0.005 mm⁻¹, more than the maximum measured absorption change for wavelengths 730, 780, or 830 nm in any of the three regions. In order to account for differential pathlength changes, Kohl et al. proposed a multispectral model [39], which they used to differentiate dynamic scattering and absorption changes using ISOI. This approach improves ISOI accuracy, and has been generally adopted as the method of choice for quantitative functional imaging. For dynamic measurements, we see MI as an improvement over this approach as it alternatively uses frequency domain measurements at a single wavelength to derive absolute scattering and absorption coefficients. This potentially provides a simplified single-wavelength measurement apparatus for detection of scattering, and also avoids potential mis-estimation of background optical properties.

Light scattering changes induced by spreading depression have been reported previously, and a comprehensive review is provided by Somjen. With in vivo spatially resolved reflectance measurements, Kohl et al. [11] separated absorption from scattering and observed a biphasic scattering response similar to that of Region A. With simultaneous laser scattering and electrophysiological measurements, both Jarvis et al. and Tao et al. found a strong correlation between electrical and optical scattering changes [12,13,40]. Tao et al. noted spatial heterogeneity in the dynamic spreading depression (SD) waveform related to the proximity to the SD induction site, similar to our results.

Using linear spectral analysis of absorption at all four wavelengths, we calculated the time-dependent chromophore concentration for Regions A, B, and C, presented in Figure 12.10A,B,C, respectively. In each region, the calculated baseline concentrations of H₂O were assumed to be constant. All three regions exhibit remarkably similar trends in HbR, HbO₂, HbT, and S_tO₂. This similarity is not clear in the DC traces of Figures 12.7–12.9, further highlighting the benefit of accurate separation of μa and μs′. Focusing on the first CSD event, there is a very consistent signature of: (1) a 2-minute latency post-KCl administration, (2) a 30-second period of decreasing S_tO₂ (3) a dramatic spike in both S_tO₂ (3–10%) and HbT (2–4 μM) with rise and decay times of approximately 1 minute each. For each region, the final S_tO₂ is approximately 5–10% lower than baseline, while the HbT restores to baseline values. This process repeats again twice more, except that the phase (2) desaturation appears to be absent. Additionally, in the “vessel” Region 3, we observe a gradual increase in HbT over the 30 minutes, indicating chronic blood pooling.

FIGURE 12.10

Recovered HbR, HbO₂, HbT, and STO2, for ROIs A, B, and C (top, middle, and bottom), recovered by analysis of the multispectral absorption coefficients from Figures 12.7–12.9b (top).

We show in Figure 12.11 the spatio-temporal evolution of both chromophore concentration and scattering changes from the first SD wave in rat 3. These are depicted in the form of a time derivative, i.e., (C(tn + 1) − C(tn))/(tn + 1 − tn), where C represents concentration/saturation/scattering values and tn represents time of acquisition for data point n. This visualization is appealing as it highlights the changes with high contrast [18]. From left to right, we show HbO₂, HbR, HbT, S_tO₂, and μ_s′. Notice the wave in scattering which propagates from top right to bottom left, at a rate of approximately 3 mm/min. An increase, or “spike” in scattering is observed initially in the top right hand corner, in close proximity to the location of KCl administration. Note the large spikes in HbT and S_tO₂ due to vascular activity from depression wave propagation through the measurement field. We observe a transient increase in saturation and blood volume. Over the longer time periods, however, we observe a slow, sustained trend toward hypoxia in the vein regions.

FIGURE 12.11

Spatio-temporal evolution of the hemodynamic and neural scattering response during a single spontaneous CSD event in rat 3. For visualization, a time derivative of the image sequence is displayed to highlight changes.

The spatio-temporal evolution of the scattering coefficient in Figure 12.11 reveals a spatially defined scattering wave (reduction in μs′) that precedes hemodynamic changes. The scattering drop is presumed to be a consequence of neuronal depolarization accompanying CSD. This observed wave pattern has been shown previously with reflectance ISOI and attributed to blood volume changes [18]. Interestingly, the scattering depolarization wave is clearly followed in space and time by the increase in deoxyhemoglobin (HbR), decrease in saturation (S_tO₂), and drop in oxyhemoglobin (HbO₂); changes that are consistent with depolarization-induced neural tissue oxygen consumption.

12.3.3. Dynamic MI Spectroscopy of Stroke

In order to assess the sensitivity of MI to stroke, we conducted preliminary studies in a rat middle cerebral artery occlusion (MCAo) model, the most commonly involved artery in ischemic strokes. The left MCA was surgically cauterized using monopolar cautery or ligated to produce a permanent stroke. Figure 12.12 shows pre-versus post-MCAo results for a representative animal. Data were acquired at 5 wavelengt