Lasers in Surgery and Medicine

Quantitative Analysis of Transcranial andIntraparenchymal Light Penetration in Human CadaverBrain Tissue

Clark E. Tedford,1 Scott DeLapp,1 Steven Jacques,2 and Juanita Anders3�1LumiThera, Inc., Poulsbo, Washington 983702Oregon Health and Science University, Portland, Oregon3Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda,Maryland 20814

BackgroundandObjective:Photobiomodulation (PBM)also known as low-level light therapy has been usedsuccessfully for the treatment of injury and disease of thenervous system. The use of PBM to treat injury anddiseases of the brain requires an in-depth understanding oflight propagation through tissues including scalp, skull,meninges, and brain. This study investigated the lightpenetration gradients in the human cadaver brain using aTranscranial Laser Systemwith a 30mmdiameter beamof808nm wavelength light. In addition, the wavelength-dependence of light scatter and absorbance in intra-parenchymal brain tissue using 660, 808, and 940nmwavelengths was investigated.Study Design/Material and Methods: Intact humancadaver heads (n¼8) were obtained for measurement oflight propagation through the scalp/skull/meninges andinto brain tissue. The cadaver heads were sectioned ineither the transverse or mid-sagittal. The sectioned headwas mounted into a cranial fixture with an 808nmwavelength laser system illuminating the head frombeneath with either pulsed-wave (PW) or continuous-wave (CW) laser light. A linear array of nine isotropicoptical fibers on a 5mm pitch was inserted into the braintissue along the optical axis of the beam. Light collectedfrom each fiber was delivered to a multichannel powermeter. As the array was lowered into the tissue, the powerfrom each probewas recorded at 5mm increments until theinner aspect of the dura mater was reached. Intrapar-enchymal light penetration measurements were made bydelivering a series of wavelengths (660, 808, and 940nm)through a separate optical fiber within the array, whichwas offset from the array line by 5mm. Local lightpenetration was determined and compared across theselected wavelengths.Results: Unfixed cadaver brains provide good anatomicallocalization and reliablemeasurements of light scatter andpenetration in theCNS tissues. Transcranial application of808nm wavelength light penetrated the scalp, skull,meninges, and brain to a depth of approximately 40mmwith an effective attenuation coefficient for the system of2.22 cm�1. No differences were observed in the resultsbetween the PW and CW laser light. The intraparenchy-

mal studies demonstrated less absorption and scatteringfor the 808nm wavelength light compared to the 660 or940nm wavelengths.Conclusions: Transcranial light measurements of un-fixed human cadaver brains allowed for determinations oflight penetration variables. While unfixed human cadaverstudies do not reflect all the conditions seen in the livingcondition, comparisons of light scatter and penetration andestimates of fluence levels can be used to establish furtherclinical dosing. The 808nm wavelength light demonstrat-ed superior CNS tissue penetration. Lasers Surg. Med.� 2015 Wiley Periodicals, Inc.

Key words: central nervous system; isotropic opticalfibers; low level light therapy; photobiomodulation;transcranial light therapy; 808 nm wavelength

INTRODUCTION

Photobiomodulation (PBM), also referred to as low-levellight therapy, uses non-thermal red andnear infrared light(600–1000nm) to alter cellular and tissue function by theinteraction of photonswithoneormore chromophores [1,2].Recent reviews summarized the data supporting themechanisms that underlie PBM [3,4] including theabsorption of near-infrared (NIR) light by the terminalenzyme in the mitochondrial respiratory chain, cyto-chrome C oxidase. The energy absorbed by cytochrome Coxidase leads to increased enzyme activity and productionof ATP. Critical downstream signaling cascades are alsoactivated [5–7]. NIR light causes increased ATP andreactive oxygen species production, nitric oxide release,

Contract grant sponsor: CRADA between USUHS and Photo-thera, Inc.; Contract grant number: # G1709V.

�Correspondence to: Juanita J. Anders, PhD, Department ofAnatomy, Physiology and Genetics, Uniformed Services Uni-versity of the Health Sciences, 4301 Jones Bridge Road,Bethesda, MD 20814. E-mail: juanita.anders@usuhs.edu

Accepted 5 January 2015Published online in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/lsm.22343

� 2015 Wiley Periodicals, Inc.

and cyclic AMP production, all of which can contribute tobeneficial effects in tissues with compromised func-tion [4]. In vitro and in vivo studies have shown thatPBM can directly modify markers of inflammation andoxidative stress [8–12], reduce pain [13], and prevent celldeath [7,14] in a number of tissue types. More recently, agrowing number of preclinical and clinical publishedreports have shown positive outcomes for PBM indiseases and injuries related to the central nervoussystem (CNS) [11,15–29].

Non-invasive delivery of photons from the light sourceto the brain for the treatment of brain injuries anddiseases is termed transcranial light therapy (TLT). TLTholds promise as a treatment for acute conditionsincluding ischemic stroke and traumatic brain injury(TBI), as well as global ischemia following cardiacarrest. It also has the potential to improve cellularfunction in age-related neurodegenerative CNS diseasesthat include Alzheimer’s Disease, Parkinson’s Disease,and Huntington’s Disease and to restore normal meta-bolic activity in psychiatric illnesses including MajorDepressive Disorders.

We recently analyzed transcranial light penetration inanesthetized rats, mice, and rabbits. In these animals, asingle NIR light application site on the scalp irradiated theentire brain. Positive effects of TLT have been reported inanimal models of acute TBI [15–17], Alzheimer’s Dis-ease [20], depression [18], and cerebral ischemia [24–28].In humans, TLT improved cognition in two chronic TBIcases [19] and increased regional cerebral blood flow andimproved the psychological status of patients withdepression [23]. No adverse side effects were noted inthese studies. In fact, safety of TLT for human use wasdemonstrated in a clinical trial for treatment of ischemicstroke [29,30]. Based on the preclinical work and cadavermeasurements done to support the ischemic stroke clinictrial, it was determined that delivery of energy density of7–10mW/cm2 was an effective dose for photobiomodula-tion of the injured cortex (personal communicationand [31]).

The expansion of PBM as an effective, non-invasive, andinnovative non-pharmaceutical approach to treat humanbrain injuries and diseases requires an in-depth under-standing of light propagation through the scalp, skull,meninges, and brain. Human cadaver studies provide oneapproach to determine optimal light treatment parametersto target global or specific brain structures. Human scalp,skull, and brain tissue have been examined for absorptionand scattering properties of light [32] and for NIR lighttransmission and attenuation [33] in vivo during brainsurgery. Transmittance and diffuse reflectance of ex vivosamples of human cadaver skull [34] and skull and scalpand brain have also been measured [35–37].

The objective of this study was to characterize thetransmission of 808nm wavelength light through non-fixed, intact human scalp, skull, meninges, and brain atvarious depths and locations. Additionally, the attenuationthrough the intraparenchymal brain tissue was character-ized locally at wavelengths of 660, 808, and 940nm.

METHODS

Cadaver Laser System

The Laser System was a research device specificallydesigned to provide non-invasive TLT to cadavers (Fig. 1).The instrument provided high power capacity and atemperature controlled primary laser affixed to a stereo-tactic apparatus. The system included a Primary Laserand three Reference Lasers of different wavelengths. Thebasic characteristics of these lasers are presented inTable 1.The Laser System consisted of a console (location of

control electronics), a laptop computer, a primary laserassembly (location of the laser source and interface to thecadaver tissue), a reference laser system with threewavelengths for brain parenchyma measurements, apower verification unit, a cart assembly, a cranial fixture,and data collection equipment. The treatment modalitywas a predetermined sequence that was preprogrammedinto the Research Device Console and data acquisition wascontrolled and stored by a laptop computer. A treatmentregimen consisted of either treating a user-determinedlocation on the cadaver scalp with the primary laser ortreating locally in the brain with the reference laserprobes. ThePrimaryLaserResearchDevice utilized a laserdiode source and had a maximal output power of 50Wcontinuous wave (CW) or 70W pulsed wave (PW) at awavelength of 808nm and nominal beam diameter of30mm applied to the surface of the head of cadavers. CWand PW were examined to determine if pulsing alteredfluence rate.

Power Measurements

Measurement of the optical power within the tissue wasaccomplished by sampling the light with an array of nine400um fibers each with an isotropic detector (MedlightIP85). Each fiber was bonded into a stainless steelhypodermic tubing at the collection end to providemechanical rigidity (Fig. 2). The isotropic probes were

Fig. 1. Photograph of the cadaver laser system and the cranialfixture.

2 TEDFORD ET AL.

used with a distance of 5mm between the probes. Eachfiber was connected to a separate channel of a multi-channel power meter (OptoTest OP710-Si), which wascapable of a dynamic measurement range of þ6decibel-milliwatts (dBm) (4.0mW) to �80dBm (1.0� 10–8mW).An additional 1,000mm core optical fiber was run fromanother channel of the power meter to a sampling portwithin the laser assembly for continuous monitoring oflaser output power. The total output power of the primarylaser was measured with a thermal-based power detector(Ophir LD40–150) and meter (Ophir Nova), which con-tained a large enough collection area to capture the full30mm primary beam. Power from the emitting probes wasmeasured using a single channel optical power andwavelength detector (ILX Lightwave OHM-6722B) con-nected to its associated meter (ILX Lightwave OMM-6810B). Characterization of the primary laser output inpulsedmode was performed using an amplified photodiode(Thorlabs PDA10A) and an oscilloscope (TectronicsTDS2024B).

Instrument Calibration

Due to the multitude of sensors used to measure opticalpower in the study (i.e., 12 channels in the OP710, the ILXsystem, and theOphirmeter and detector), calibration of allthe detectors to a single standard was required to reducemeasurement error. The chosen standard was the ILX,whichwasNISTtraceable.Thecalibrationof theOP710andassociated collection fibers to the ILX was undertaken by alengthy procedure that involved the following abbreviatedsteps: (1) the probes and power meter channels wereserialized such that a given probe would always beconnected/reconnected to the same channel; (2) a spatiallyuniform beam (<2.5% variation) of 808nm wavelengthlaser light was generated, and the fluence rate withinthe beam was measured using the ILX; (3) the angularresponse of each probe-channel was measured at thesame point within the beam. This process was repeatedwith theprobe tips submerged inwater; and (4)a calibrationfactor was calculated for each probe-channel pair. This

TABLE 1. Laser System Overview: Basic Characteristics of the Primary and Three Reference Lasers

Laser

808nm

Primary

660nm

Reference

808nm

Reference

940nm

Reference

Power supply 120V AC 3.5–5V DC 3.5–5V DC 3.5–5V DC

Emission power 70W max 50mW max 50mW max 50mW max

Laser class IV IIIb IIIb IIIb

Laser diode 4.6�4.6 VCSEL Array Single mode diode Single mode diode Single mode diode

Beam type Flat Top, <0.1NA Gaussian, 0.39NA Gaussian, 0.39NA Gaussian, 0.39NA

Wavelength 808� 5 nm 660� 3nm 808�3nm 940� 6nm

Fig. 2. Diagram of the cranial tissue fixture including the optical sensor probe array and laser.

TRANSCRANIAL AND INTRAPARENCHYMAL LIGHT PENETRATION 3

factor was used to convert the indicated power on themultichannel power meter (mW) to the actual fluencerate at the probe tip (mW/cm2). The Ophir detector wascalibrated directly to the ILX by exposing each to the samelaser source and calculating a percentage offset betweenthe two.

Cadaver Tissue

Cadaver heads (n¼ 8) were used with permission of theUSUHS Anatomical Materials Review Committee afterreview and approval of the submitted protocol. The eightcadavers ranged in age between 66 and 97. Unfixedcadaver tissue was utilized and the average number ofdays postmortem was 25. The extended post-mortemperiod was required to allow blood testing for infectiousdisease. The cause of death was reviewed and no CNSdiseases or pathologies were included in the cadaverselections. Standard refrigerated conditions at USUHSAnatomical Teaching Laboratories were used for main-taining cadaver specimens. The morphology of the brainwas very good with recognizable gray and white matterand defined nuclei.

Experimental Procedure

Multiple sites on each head were selected for lightpenetration analysis under different parameter sets andat multiple depths. A total of 20 different sites wereexamined across the eight heads. Four of the eight headswere sectioned transversely. These heads were sectionedjust dorsal to the top of the ear resulting in the upperportion of the calvarium with its intact scalp, skull,meninges, and brain portion. The remaining four headswere sectioned in the mid-sagittal plane. At this level, thecut passed through the occipital, parietal, and frontallobes of the cerebral cortices. A transfer plate was placedon the cut side of the head to allow inversion andmounting of the sectioned head into the removablefixation ring. The head was secured by four positionalscrews into a cranial fixation ring. Once secured, thehead was inverted again wherein the cut surface ofthe brain was dorsal and the top of the skull and scalpwere in the ventral position. The removable fixation ringwas placed into position in the cranial fixture and securedwith the primary laser positioned below (Fig. 3). In thisposition, the optical sensor probe array was lowered forentry into the brain tissue. The probe array wasconstrained to move along the optical axis of the laser.The coordinates were recorded and established the laserlight illumination path through the head at a fixedlocation and angle. Once the coordinates were secure, theprimary laser was raised until the output lens was flushwith the scalp, applying a light pressure (�<5 lbs). Thesensor probe array was lowered to the cut surface of thebrain and distance from the lens surface was recorded.The sensor probe array was then lowered into the braintissue at 5mm increments until the inner aspect of thedura mater was reached. At each 5mm increment, thefluence rate at each probe was recorded.

Typically, a series of measurements was taken at eachposition within the tissue, corresponding to a differentlaser (primary laser 808nm wavelength, reference lasers660, 808, and 940nm wavelengths) or modality (PW vs.CW). The general sequence at each depth was lower theprobe array into the tissue and record probe depth; activateprimary laser, 5W CW and collect data; activate primarylaser, pulsed at 10Hz, 20% duty cycle (DC), 5W peakpower; collect data; activate primary laser, pulsed at100Hz, 20%DC, 5Wpeak power; collect data; sequentiallyactivate the reference lasers, 50mW CW; collect data foreach reference laser; lower the probe array 5mm andrepeat. This processwas continueduntil the inner aspect ofthe dura mater was reached. A similar procedure wasconducted for themid-sagittal cuts. At each data collection,the collection time ranged from 2 to 6 seconds, with thepower meter streaming all channels to the PC at a rate of7 times per second. This resulted in 14–42 measurementsof the fluence rate at each probe tip for every collectionwindow.

Data Analysis

The raw data collected from the multichannel powermeter were converted to measured fluence rates using thepreviously calculated calibration factor for each probe. Thedata from each site were then normalized to an input laserpower of 1W on the surface of the scalp. For the primarylaser, this normalization was performed on each acquisi-tion using the power recorded from the laser powermeasurement fiber at the same instance, thus eliminatingany potential errors due to laser power drifting over thecourse of sequential acquisitions. For the reference lasers,

Fig. 3. Illustration of the cranial fixture with the optical sensorprobe array (above) and laser delivery system (below).

4 TEDFORD ET AL.

normalization was performed using the reference laserpowers measured at the start of each test. An additionalcorrection to the data from the reference lasers was madeto account for the wavelength-dependent sensitivity of thepower meter. Raw data that fell below an establishedminimum detection level were discarded. The minimumdetectable signal (also known as the noise floor) is theresult of a number of factors including basic equipmentresponsiveness, electrical noise, stray light, fiber collectionefficiency, etc. For this system, the level was established bytaking a series of identical measurements sequentiallythroughout the tissue at 5 and at 15W of primary laserpower. Once normalized, the 5 and 15W measurementswould be expected to produce nearly identical results.Thus, the point at which these results begin to differsignificantly can be taken as the point at which the noisebegins to swamp the signal in the 5Wcase. For this system,a percentage difference of <4% between the 5 and 15Wcases was selected as the threshold for minimum detect-able signal. This difference was seen at approximately the55–60mm distance from the scalp. All raw data corre-sponding to values above this threshold were discarded.Because of the multiple anatomical brain substructuresand unique set of angles of light penetration studied,comparative group data were generated but should berecognized as containing confounding variables. Con-founding variables included subject variability, brainsize, shape, age, which provided unique cadaver datasets and thus sample pooling is somewhat limited whengroup analysis was conducted.

RESULTS

The measured fluence rate within the cadaver brainacross the nine isotropic probes for a typical coronal sectionis presented in Fig. 4a. The external surface of the scalp,where the laser made contact, was defined to be themeasurement origin. Themaximal probe distance from theprimary laser was the exposed surface of the dissectedbrain tissue, where the isotropic probes entered. Allexperiments were carried out in the dark to minimizenoise caused by ambient light. This maximal probedistance varied from subject to subject depending on thelevel of the dissection. At the distance farthest away fromthe primary laser beam where the probes entered thetissue, typically depths of 60mm or more, backgroundminimal fluence rateswere established. As the probeswerelowered into the tissue and the distance to the internalcortical surface decreased, an increase in fluence rates wasseen. This generally resulted in an exponential increase inthe measured fluence rate for a given linear decrease inprobe depth. The distance of the probe depth from theinternal cortical surface to the external surface of the scalpwas typically 5–15mm. However, curvature of the skulland angle of the probe insertion impacted how close to thecortical surface each individual probe reached. A contourplot of the above data was generated to graphicallyillustrate the resultant light distribution within the tissue(Fig. 4b). Near the cortical surface, the center and adjacentprobes showed the highest fluence rates, which generally

corresponds to the 30mm beam diameter of the laser.Measurements taken beyond the lateral extent of the beamindicated lower fluence rates. Measurable power wasobserved at cortical depths of greater than 40mm.

The data from all probes over all examined siteswere collapsed into a single data set, and a curve of theform F(x)¼Foe

�ux was fit using a least-squares method(Fig. 5a). The resulting value of u can be interpreted as theeffective attenuation coefficient for the system, and wasfound tobe2.22 cm�1. It isworthnoting thata largedriverofdeviation in the data was the initial transmission throughthe scalp and the skull. Normalizing the data from eachprobe to 1mW/cm2 at the inner surface of the skull removesthe effect of the skull and scalp variability, and illustratesthe optical variability in the brain tissue itself (Fig. 5b). Theresting value ofwas found tobe2.43 cm�1whennormalized.

The fluence rate wasmeasured by a given probe with theprimary laser operating sequentially in CW mode, pulsedat 10Hz, 20% duty cycle, and pulsed at 100Hz, 20% dutycycle. No differencewas observed in the normalized fluencerates for any of the probes at any of the sites.

The results of the intraparenchymal brain tissue meas-urements using the internal emitter probe to launchreference laserwavelengths of660,808,and940nmdirectlyinto the brain tissue are presented in Figure 6. As with thedata generated with the primary laser, any data beneaththe established minimum detectable signal was discarded,and the results have been normalized to remove the powerdifferences between each laser. The typical power emittedfrom each reference laser was �40mW. There is clearly ahigher attenuation of the 660nm wavelength than the940nm wavelength and of the 940nm wavelength com-pared to the 808nm wavelength. In most instances, the660nmwavelength failed to produce a response in the outertwo probes that was above the noise threshold.

The experimental values of meff [cm�1] for cadaver brain

at 660, 808, 940nm wavelengths were compared toliterature on brain tissues [38]. This comparison is showninFigure 7. The experimental data are shownasmeff [cm

�1]versus wavelength (l). Also shown is the least-squares fitto the data using diffusion theory, which specifies the bloodvolume fraction (B), the hemoglobin oxygen saturation (S),and the reduced scattering coefficient at 500nm wave-length (ms’500nm). Literature cited in Jacques, 2013 [38]provided spectra for comparison, and the median value ofthe scattering power (b). The water content was assumedto be 0.75. The fitting was conducted on all the data at 660,808, and 940nm simultaneously, using the equation:

mef f ¼1

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3maðma þ ms

0Þp

wherema¼BSma.oxy(l)þB(1-S)ma.deoxy(l)þWma.water(l)b¼1.41 (median of literature values for brain tissues)ms’¼ms’500nm (l/500nm)�b

D¼ 1/(3(maþms’))meff¼ sqrt(ma/D)

TRANSCRANIAL AND INTRAPARENCHYMAL LIGHT PENETRATION 5

Fig. 4. NIR light fluencemeasurements in the human brain for a typical coronal section. (a) depictsthe measured fluence rate at each of the nine individual isotropic detector probes. Measurementswere serially collected at 5mm depth of CNS tissue. Each data point represents a mean of datacollected over several secondswhile the primary laserwas activated and the light intensity datawascollected at a rate of 7 points/seconds. The datawere normalized to 1Wat the external surface of thescalp. The fluence rates are provided in mW/cm2. (b) depicts the same dataset as (a), plotted as alogarithmic contour plot to illustrate the spatial distribution of the fluence rate within the tissue.

6 TEDFORD ET AL.

Fig. 5. (a) shows a composite plot of the fluence rates measured by all probes at all 20 sitesexamined (eight independent cadavers, various coronal, and sagittal dissections). Data below theestablished noise floor were discarded, and data from each site were normalized at each site to 1Wdelivered to the surface of the scalp. The fluence rates are provided in mW/cm2. A curve of the formF(x)¼Foe

�ux was fit to the data using a least-squares method. (b) represents the same dataset,normalized at each site to 1W at the cortical surface, rather than at the surface of the scalp. Thisremoves the variability of the transmission through the scalp and skull and illustrates thevariability in just the brain tissue.

TRANSCRANIAL AND INTRAPARENCHYMAL LIGHT PENETRATION 7

andma.oxy(l) is the absorption coefficient of oxygenatedwhole

bloodma.deoxy(l) is the absorption coefficient of deoxygenated

blood (whole blood defined ashaving 150g hemoglobin/liter)ma.water(l) is the absorption coefficient of water.Table 2 lists the optical properties, ma and ms’, of the

cadaver brain for the three wavelengths.

DISCUSSION

Acritical component for establishingPBMasan effectivetherapy in CNS disorders is identifying and deliveringthe appropriate dose to alter aberrant cellular functions.While in vitro cellular and in vivo animal studies canprovide clues to what light parameters and fluence ratesare beneficial, scaling to the human condition is qualita-tive. Several animal studies have suggested the potentialuse of PBM in acute TBI [15–17], Alzheimer’s Disease [20],depression [18], and cerebral ischemia [24–28]. In humans,TLT improved cognition in two chronic TBI cases [19]and increased regional cerebral blood flow and improvedthe psychological status of patients with depression [23].

The need to reach deep brain structures in many of thesediseases and disorders is critical for establishing efficacy inlong and costly human clinical trials. Recent failures in theuse of TLT in stroke trials (Personal communication, ClarkTedford) may reflect the absence of consideration of thedosing parameters ofNIRwavelengths in complex diseasesor conditions. In this study, determination of transmissionof 808nm wavelength light through non-fixed, intacthuman scalp, skull, meninges, and brain at various depthsand locations was undertaken to establish light penetra-tionmaps. Thesemaps will help researchers and cliniciansto better understand the potential dosing parametersrequired to provide an effective non-invasive treatment forCNS disorders. In addition, the absorption and scatteringproperties of the intraparenchymal brain tissue at wave-lengths of 660, 808, and 940nmwere established to furtheraid in optimizing PBM parameters.Human scalp, skull, and brain tissue have been

examined for absorption and scattering properties oflight [32] and for NIR light transmission and attenua-tion [33] in vivo during brain surgery. Transmittance anddiffuse reflectance of ex vivo samples of human cadaver

Fig. 6. Results of the intraparenchymal CNS fluence rate measurements at 660, 808, and 940nmwavelengths for all probes at all sites examined. Data were normalized to 1W from the emitterprobe. The fluence rates are provided in mW/cm2.

8 TEDFORD ET AL.

skull [34] and skull, scalp and brain have also beenmeasured [35–37]. Those studies involved transmission,scattering, and temperature measurements on excisednon-preserved human skull and brain tissue as well asformalin preserved tissue. A number of these papers werereferenced in a recent review of optical properties ofbiological tissues which compared the wavelength-depen-dent behavior of scattering and absorption of brain tissueto other tissues in the body [38].Several limitations to the current study were identified

during the course of data collection. Themost obvious is theuse of cadaver tissue, which may not mimic in vivoconditions. However, the instrument and detector probecapabilities were initially tested in fresh bovine brain andlight scatter and penetration parameters were similar tothose measured in the cadaver brains (unpublishedobservations). The non-fixed cadavers were kept refriger-ated and the skulls were cut on the testing dayimmediately prior to evaluation. The anatomical land-marks and overall integrity of the CNS tissue was verygood. The laser system was capable of collecting extensiveand detailed information and was only limited byanatomical considerations. The laser beam was fixed inline with the array probe, which meant that the laserposition and probe angle of entrywas dependent on the sizeand shape of the skull. The study attempted to evaluate thesame number of placements around the skull, butmaintaining a flat laser contact with the scalp and skull

was themost critical and variable aspect which limited theability to definitively collect same site data from subject tosubject. This observation implies that a flexible apparatuswould be most useful in the clinical instrument setting toallow for specific regional targeting.

Our results clearly demonstrate the penetration ofmeasurable 808nm wavelength light through the scalp,skull, and meninges to a brain depth of 40–50mm. Thesedata were used to create color-coded fluence maps of theNIR light as it penetrated the brain and scattered thusestablishing a gradient of light energy as the photons wereabsorbed and distributed within the brain tissue. It isessential to understand these light fluence gradientswithin the brain to adjust the dose of light to reach thetarget tissues and provide a therapeutic range for CNSapplications

In a previous study, post-mortem, human brain tissuewas manually cut at non-specified thicknesses to estab-lish gross penetration values [36]. In this study, fourdifferent wavelengths were tested: 632.8, 657, 780, and835nm. The optical penetration depth reported for eachwavelength was 0.92�0.08, 1.38� 0.13, 3.46� 0.23, and2.52� 0.19mm, respectively. In another recent study,preserved cadaver skulls and heads that had beenpreviously dissected or sectioned were used to measuretranscranial red and NIR light transmission [37].The light source consisted of LEDs emitting at peakwavelengths of either 633 or 830 nm with output powersin the mW/cm2 range (33.3mW/cm2) for the 830 nmwavelength and 67.5mW/cm2 for the 633nm wavelength.The relative percentages of light penetration weredetermined for various skull regions. For the occipitalregion which consisted of skull and some intactbrain tissue (approximately 10mm thick), it was 11.7%for the 830 nm wavelength and 0.7% of the 633 nmwavelength. Although the optical penetration depthsreported earlier [36] were much less than we measuredin our system, our intraparenchymal comparisons oflight penetration of 660, 808, and 940nm wavelengthsconfirmed that the NIR wavelength of 808 nm wassuperior in brain tissue penetration. Scattering andabsorption of light is wavelength dependent [38] and isreflected in our wavelength-dependent transcranial lightpenetration data. This conclusion is also supportedtheoretically by Monte Carlo simulations that predictednear infrared light propagation in realistic head mod-els [39–42]. Recognition of this wavelength-dependenttranscranial and intraparenchymal light penetration isvery important in establishing the potential use of PBMto treat CNS disorders. It appears highly unlikely thatcertain wavelengths would provide the transcranialdepth of penetration needed for use in treating braintissue. Continued research is needed to establish theoptimal parameters for treatment of specific CNSdiseases or disorders. In spite of the early stage ofresearch into PBM treatment of CNS disorders, there isgood evidence that NIR wavelengths will penetrate thescalp and skull and provide an opportunity for a non-invasive treatment.

Fig. 7. Comparison of experimental values of ueff [cm�1] forcadaver brain at 660, 808, 940nmwavelengths (red circles), versusliterature on brain tissues (magenta lines, cited in review byJacques 2013 [38]). The diffusion theory fit to the cadaver braindata is shown as green line, with fitting parameters listed infigure. The diffusion theory predictions of fully oxygenated (redline) and fully deoxygenated (blue line) blood are shown.

TABLE 2. Optical Properties of Cadaver Brain

l [nm] ma [cm�1] ms’ [cm�1] meff [cm

�1]

808 0.22 9.5 22.2

940 0.47 7.7 21.8

660 0.29 12.7 21.5

TRANSCRANIAL AND INTRAPARENCHYMAL LIGHT PENETRATION 9

ACKNOWLEDGMENTS

The opinions and assertions contained herein are theprivate ones of the authors and are not to be construed asofficial or reflecting the views of theDepartment of Defenseor the Uniformed Services University of the HealthSciences. The authors thank Mr. Ronald Rivenburgh,Anatomical Curator, for his help and expertise in themaintenance and preparation of the cadaver specimens.We want to thank Stephen Fry and Andreas Rose for theirwork on the calibration of the optical probes.

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