Non‐invasive treatment of ischemia/reperfusion injury: Effective transmission of therapeutic near‐infrared light into the human brain through soft skin‐conforming silicone waveguides

Abstract Noninvasive delivery of near‐infrared light (IRL) to human tissues has been researched as a treatment for several acute and chronic disease conditions. We recently showed that use of specific IRL wavelengths, which inhibit the mitochondrial enzyme cytochrome c oxidase (COX), leads to robust neuroprotection in animal models of focal and global brain ischemia/reperfusion injury. These life‐threatening conditions can be caused by an ischemic stroke or cardiac arrest, respectively, two leading causes of death. To translate IRL therapy into the clinic an effective technology must be developed that allows efficient delivery of IRL to the brain while addressing potential safety concerns. Here, we introduce IRL delivery waveguides (IDWs) which meet these demands. We employ a low‐durometer silicone that comfortably conforms to the shape of the head, avoiding pressure points. Furthermore, instead of using focal IRL delivery points via fiberoptic cables, lasers, or light‐emitting diodes, the distribution of the IRL across the entire area of the IDW allows uniform IRL delivery through the skin and into the brain, preventing “hot spots” and thus skin burns. The IRL delivery waveguides have unique design features, including optimized IRL extraction step numbers and angles and a protective housing. The design can be scaled to fit various treatment areas, providing a novel IRL delivery interface platform. Using fresh (unfixed) human cadavers and isolated cadaver tissues, we tested transmission of IRL via IDWs in comparison to laser beam application with fiberoptic cables. Using the same IRL output energies IDWs performed superior in comparison to the fiberoptic delivery, leading to an up to 95% and 81% increased IRL transmission for 750 and 940 nm IRL, respectively, analyzed at a depth of 4 cm into the human head. We discuss the unique safety features and potential further improvements of the IDWs for future clinical implementation.

transmission for 750 and 940 nm IRL, respectively, analyzed at a depth of 4 cm into the human head. We discuss the unique safety features and potential further improvements of the IDWs for future clinical implementation.

K E Y W O R D S
cadaver, infrared light, ischemia/reperfusion, light penetration, mitochondria, stroke, waveguide

| INTRODUCTION
Stroke is one of the leading causes of death globally, with more than 15 million people affected each year. 1,2 With the blood flow to the tissue blocked due to the stroke, the affected area quickly becomes ischemic. During ischemia, energy is rapidly depleted due to the lack of oxygen preventing oxidative phosphorylation by the mitochondrial electron transport chain (ETC). 3 Disrupted mitochondrial calcium levels and the low ATP concentration result in activation of the various mitochondrial complexes in a futile attempt to meet the energy demand. 4,5 The restoration of blood flow is necessary to prevent total death of the affected area, but also results in transient mitochondrial hyperpolarization and excessive reactive oxygen species (ROS) production, which causes reperfusion injury. 6,7 Clinical trials to treat stroke pharmacologically, such as with ROS scavengers, have failed to deliver clinically in human patients despite success in the laboratory environment with animal models. 8,9 These failures may be due, in part, to difficulties in delivering relevant concentrations to the specific subcellular targets during the early period of reperfusion, when most reactive oxygen species (ROS) are generated. The spectrum of near-infrared light (IRL), ranging from 700 to 1000 nm, has been evaluated as a possible noninvasive treatment for stroke. 10 IRL offers many benefits over conventional treatments. IRL is nonpharmacological and therefore can be noninvasively applied locally and does not affect the body systemically. Additionally, IRL does not depend on blood flow, which is compromised during stroke, to be delivered to the target tissue.
Cytochrome c oxidase (COX), also known as complex IV in the mitochondrial ETC, has been identified as the primary target of IRL due to its two enzymatic copper centers, which broadly absorb IRL between 700 and 1000 nm as can be seen with the COX absorption spectrum. [11][12][13] Although the precise mechanism of IRL action on COX is not fully clear, it should be noted that there are two copper atoms located at the cytochrome c binding site (electron acceptor site) and a third copper atom is located near heme a 3 and binds the COX substrate, oxygen. Therefore, all three coppers are located at the catalytic sites of COX. Since infrared light adds vibrational energy when absorbed, a straightforward hypothesis would be that additional movement at the catalytic site(s) alters cytochrome c and/or oxygen binding. Generally, IRL is believed to be stimulatory to COX and the mitochondria, particularly the commonly used 810 nm. 14 Treatment strategies with 810 nm focus on increasing mitochondrial activity in areas affected by stroke to increase ATP levels and improve cognitive function. 10 However, we have previously established that certain wavelengths of IRL, specifically 750 nm and 940/950 nm, are inhibitory to COX and the mitochondria. 15 This provides a unique treatment opportunity for reperfusion injury following ischemia. By applying COX-inhibitory IRL during early reperfusion, ETC flux is reduced in the hyperpolarized mitochondria, which normalizes the mitochondrial membrane potential and prevents the generation of excessive ROS. We have demonstrated inhibition using both purified COX protein and intact cells. Additionally, applying 750 and 950 nm during early reperfusion showed robust neuroprotection in rat models of focal stroke and global brain ischemia. 15,16 The IRL spectrum is able to penetrate through biological tissues via an "optical window" which occurs due to the low absorbance of hemoglobin above 650 nm and water below 1000 nm. 17 Despite this, most IRL is lost within the first millimeter of the skin. 18 This highlights the need for the development of IRL delivery technologies that can enhance transmission of IRL through biological tissues to reach therapeutic amounts of IRL at the deepest structures of the brain. Here, we describe the development and testing of our IRL delivery waveguides (IDWs) which facilitate the transmission of IRL into the brain. Our IDWs consist of low-durometer silicone waveguides, which have good optical transparency in the near IRL range. 19 Additionally, silicone waveguides have previously been used to efficiently transmit IRL for spectroscopic purposes and biomarker detection. [20][21][22] Similar technology has been found to enhance delivery of light to a target tissue. 23 Our IDWs offer several advantages over traditional IRL delivery methods. The IDWs are placed in direct contact with the scalp, conforming to the patient's head to prevent pressure points. IRL is transmitted through fiberoptic cables from a laser source to the IDWs, allowing higher IRL outputs without the heat production associated with light-emitting diodes (LEDs). 24 Extract steps within the IDWs allow uniform distribution of IRL toward the scalp, preventing "hot spots" which could burn the skin. Additionally, the rear surface of the IDW is coated with a reflective material to capture IRL that reflects off the skin and redirect it back toward the body. Furthermore, this solution is scalable, providing a novel platform for IRL delivery into any desired tissue type. We have previously reported that COX-inhibitory IRL can penetrate up to 4 cm into the human head using fresh, unfixed cadavers. 25 Here, using the same wavelengths and IRL output intensities, we compare IRL penetration via fiberoptic delivery of IRL versus IRL delivered by our IDW into the human head and isolated human skin samples.

| IDW assembly and system integration
The IRL delivery unit clamshell housing was CNC milled from poly- Two SMA-to-SMA mating sleeves purchased from ThorLabs (Newton, NJ) were used to connect each laser diode to a splitter aligning the laser diode fiber guide ferrule and splitter input fiber guide ferrule apertures tip-to-tip. A custom IDW-splitter fixture assembly was 3D-printed by ProtoLabs from acrylonitrile butadiene styrene (ABS)-like gray photopolymer resin using a stereolithography printer. Each IDW assembly received three of the fibers from the splitter connected to the 750 nm laser diode assembly from Akela Laser (Jamesburg, NJ) and three fibers from the splitter connected to the 940 nm laser diode assembly from BWT Beijing Ltd.
(Fengtai, Beijing, China) leaving a null fiber (center fiber) disconnected from each splitter for monitoring output stability from each laser diode when necessary.

| IDW performance verification
A performance verification study was conducted to evaluate the optical delivery performance of the IDWs (n = 5 IDWs). Each IDW assembly was evaluated for extraction efficiency and uniformity of IRL power across the emitting surface.

| Extraction efficiency
The waveguide emitting surface contributing output power from each laser diode was measured using a 5 cm diameter Thermopile Sensor and an Optical Laser Power Meter, model 843-R, from Newport (Avenue Irvine, CA). To determine the power contribution from each wavelength separately, the corresponding wavelength was selected in the handheld optical meter menu. Power (W) from the splitter fibers connected to the 750 nm laser diode P 750in ð Þand 940 nm laser diode P 940in ð Þ was measured by first offsetting the ambient light with the meter's "Zero" function before deploying the laser. The laser diode's power supply was incrementally adjusted until the desired output from the three contributing fibers was achieved for each wavelength contributing power measurement. The sum of the contributing power inputs from each wavelength was the total power input (T in ) to one IDW: This process was repeated using the IDW as an extended IRL delivery source after placing three output ferrules from the 750 nm splitter and three ferrules from the 940 nm splitter into each of the IDW housing's six input dowel holes in alternating sequence. Before deploying the laser diode, the waveguide emitting surface was placed over the 5 cm sensing aperture of the thermopile and the ambient light was offset with the "Zero" function. The sum of the contributing power outputs from each wavelength was the total power output (T out ) of one IDW: The IDW extraction efficiency was measured for each wavelength by dividing the total power output from the waveguide emitting surface (T out ) by the total power input of IRL from the splitter (T in ).

| Uniformity pixel analysis and surface percentage uniformity
To quantitatively verify the distribution of output IRL power across the waveguide's emitting surface, six inputs ferrules from the splitter connected to the 940 nm IRL source were connected to the IDW. The output power from the 940 nm IRL source was incrementally adjusted until the total measured output from the emitting surface was 2 W using the methods described above. A photomask was 3D-printed from Nylon by ProtoLabs using fused deposition modeling (FDM) printing. The photomask dimensions were designed to fit the dimen-

| Emitting surface imaging
The emitting surfaces of IDWs (n = 5) were imaged using a D5500 Nikon DSLR camera that was modified to capture IRL. Six inputs ferrules from the splitter connected to the 940 nm IRL source were connected to the IDW. The output power from the 940 nm IRL source was incrementally adjusted until the total measured output from the emitting surface was 2 W using the methods described above. The Profile" tool was used to plot the combined average gray value intensity of pixels in the Y-axis against its location along the X-axis. The image was rotated 90 degrees and this process was repeated to plot the intensity along the perpendicular axis.

| Surgical dissection
Surgical dissection of the cadaver proceeded as previously described. 25 After shaving the head, skin and soft tissues of the scalp were excised to expose the skull. An arch-shaped window into the brain was generated via craniotomy of the skull using a Stryker 810 Autopsy Saw (Kalamazoo, MI). Lastly, the ThorLabs PM160 optical power meter (Newton, NJ) was inserted into the brain after performing a micro-durotomy at the site, and IRL transmission measurements were taken. After IRL transmission measurements on the intact head were complete, an isolated skin sample was taken from the top of the scalp.

| IRL transmission measurements through cadaver head
After the dissection, the optical power meter was sequentially inserted into occipital, parietal, frontal, and temporal lobes of the

| RESULTS
The goal of this study was to generate IDWs with improved IRL delivery characteristics into the human body that (1) consider safety concerns by separating the IRL generation source and thus heat source from the human interface to limit unspecific heating at the IRL delivery point, (2) provide a more uniformly distributed IRL delivery over  Table 1. The simulations also revealed that the waveguide maintained an extraction efficiency of $80% covering the full emitting surface along the proximalto-distal axis using an extraction feature step angle of 58 even when the emitting surface was curved to a 50 mm radial curve (Figure 2a).
The ray traces showed a striated pattern of IRL output reflecting the patterning of steps along the IRL extracting surface (Figure 2b).

| IDW form
The final IDW assembly form is represented in Figure 1

| Uniformity pixel analysis
The distribution of output IRL power density from the emitting surface was measured through 4 Â 4 mm "pixels" using a thermopile with photomask to determine the proportional disparity of IRL density from one location to the next across the emitting surface of the IDW (see Section 2). The emitting surfaces of five IDWs were measured pixel-by-pixel for a total of 25 locations per surface spanning the entire area. Table 3

| Emitting surface uniformity imaging
To show the uniformity of IRL emission from the IDW, the emitting surfaces of five IDWs were imaged using a Nikon camera converted to image IRL (Figure 4a). Each of the captured images were converted to 8-bit gray-scaled images using ImageJ. Next, the image scale was converted from pixels to millimeters. Pixel intensities were averaged along the left-to-right axis and plotted against the position along the distal-to-proximal axis (Figure 4b). The image was rotated by 90 and the process was repeated to average all of the pixel intensities along distal-to-proximal axis and plotted against the position along the leftto-right axis ( Figure 4C). lobes of the brain were evaluated as an IRL delivery point in order to compare the difference in IRL intensity delivered to 4 cm in the brain by the fiberoptic cable versus the waveguide (Figure 5a). For the frontal lobe, the IDW delivered 51% and 65% more IRL than the fiberoptic for 750 and 940 nm IRL, respectively. The parietal lobe saw similar levels, with the IDW delivering 51% and 42% more IRL than the laser for 750 and 940 nm IRL, respectively. The occipital lobe experienced almost a doubling in the detected power of IRL with 95% and 81% more IRL than the laser for 750 and 940 nm IRL, respectively.  The IDW was in direct contact with the skin, whereas the fiberoptic cable was positioned at 2 and 3 cm distance from the skin in a and b, respectively. Statistical significance was determined using a two-tailed Student's t test. N = 5; *p < 0.05. Error bars represent SD.

| Cadaver testing of the IDWs reveals improved IRL delivery efficiency
Meanwhile, the temporal lobe experienced a smaller increase in IRL delivered with the IDW over the fiberoptic with a 30% and 28% increase for 750 and 940 nm IRL, respectively. Overall, for each lobe of the brain, the waveguide delivered significantly more IRL into the brain than the fiberoptic cable.
IRL has been proposed as a therapeutic for conditions outside of the brain. No matter the biological target, IRL will first need to penetrate the skin. Therefore, skin samples were taken from the cadaver in order to compare IRL transmission by the fiberoptic cable to the IDW.
For the isolated skin transmission measurements ( Figure 5b)

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
In accordance with the Uniform Anatomical Gift Act of Michigan (Act. No. 368, Public Acts of 1978, Article 10), donor consent for educational and research purposes was obtained prior to death.