Optical Modalities for Research, Diagnosis, and Treatment of Stroke and the Consequent Brain Injuries

Abstract

Stroke is the second most common cause of death and third most common cause of disability worldwide. Therefore, it is an important disease from a medical standpoint. For this reason, various studies have developed diagnostic and therapeutic techniques for stroke. Among them, developments and applications of optical modalities are being extensively studied. In this article, we explored three important optical modalities for research, diagnostic, and therapeutics for stroke and the brain injuries related to it: (1) photochemical thrombosis to investigate stroke animal models; (2) optical imaging techniques for in vivo preclinical studies on stroke; and (3) optical neurostimulation based therapy for stroke. We believe that an exploration and an analysis of previous studies will help us proceed from research to clinical applications of optical modalities for research, diagnosis, and treatment of stroke.

1. Introduction

A stroke occurs when partial blood supply to brain suddenly stops or when the blood vessels of the brain burst, consequently spilling blood into a space surrounding the brain cells which die when they no longer receive oxygen and nutrients from the blood or when there is sudden bleeding in or around the brain. Stroke has two forms: (1) ischemic blockage in blood vessels of the brain, and (2) hemorrhagic stroke that causes bleeding in the brain. Globally, stroke is the second-largest cause of death and third-largest cause of disability [1]. In 2019, a number of global stroke patients reached 115 million. A number of ischemic stroke patients was 77.2 million, a number of hemorrhagic stroke patients was 20.7 million, and a number of stroke patients due to subarachnoid hemorrhage was 8.4 million [2]. Also, 3.3 million people worldwide died from stroke in 2019.

To look a little further at the progression of stroke in the cell, tissue, and brain function, first, clogging or bursting blood vessels results in irreversible damage to the center of the brain tissue directly associated with it. After that, brain damage and penumbra occur in the surrounding brain tissues. In neuronal cells of tissues damaged by stroke, glutamate is released from ion channel collapse and excessive inflow of calcium occurs [3,4]. In addition, energy production decreases due to a damage of DNA (deoxyribonucleic acid) [5,6]. As a result, both necrosis and apoptosis of neuronal cells occur in brain tissues damaged by stroke. Also, blood corpuscles are accumulated by factors such as interleukin 8 (IL-8) expressed in damaged brain tissue by stroke [7]. It enhances an inflammatory response of the brain tissue. In such damaged brain tissues by stroke, neurons and blood vessels are regenerated and recovery proceeds for weeks to months [8]. Studies to understand anatomical and functional brain damages related to complex connectivity and study functional recovery mechanisms due to neuroplasticity after stroke have been actively conducted [9,10,11]. Research, development, and medical applications in the diagnosis, prevention, treatment immediately after stroke, and rehabilitation/therapeutic techniques for post-stroke patients have extensively progressed for high incidence and mortality of stroke and studies on mechanism of stroke, recovery processes, and therapeutic techniques developments.

Biomedical optics is a research field that studies both the principle of light-biological specimen interactions and development of medical applications [12,13]. Research and diagnosis techniques using light have advantages such as lower invasiveness and the ability to obtain varied biological and medical information compared to diagnostic modalities using radiation and other energies. Additionally, benefits of light-based therapeutic methods have fewer side effects and can be combined with conventional treatments such as chemical drugs. In particular, neurophotonics has been extensively used in studies using light for research, diagnostic, and therapeutic modalities in brain and nervous system diseases [14,15].

In this article, we explored optical techniques for research, diagnostics, and therapeutic modalities for stroke and related brain injuries. Specifically, three detailed techniques are addressed: (1) photochemical thrombosis to establish stroke animal models with localized brain lesions; (2) optical imaging techniques for in vivo preclinical studies for understanding stroke mechanism and developing diagnostic or treatment modalities; and (3) optical neurostimulation based therapy of stroke and preclinical and clinical assays related to it; and. An exploration, an identification, and an analysis of previous studies of optical modalities for stroke are confident that they will help with the processes from studies to clinical applications of optical methods for research, diagnosis, and treatment of stroke.

2. Investigating In Vivo Stroke Model Using Photochemical Thrombosis

2.1. Photochemical Thrombosis-Based Stroke Animal Model Establishments

Each of research, diagnosis and treatment modalities require verification, especially preclinical and clinical tests, which are mandatory for diagnosis and treatment devices that are to be employed for medical purposes. Development of a stroke-induced animal model for preclinical tests and modalities to build such an animal model are being extensively investigated [16,17,18,19]. A typical stroke animal model investigation is achieved either through surgical blocking of specific vessels [20,21,22] or through a biochemical method [23,24]. However, stroke in local brain regions is investigated using photochemical thrombosis. An investigation of a photochemical stroke animal model is based on a generation of blood clots using rose bengal [25,26,27,28]. Rose bengal reacts with a green light irradiation to produce reactive oxygen species, which then activate factors that are involved in a coagulation of blood and production of thrombosis. To use this characteristic of rose bengal, an animal’s blood vessel is first injected with rose bengal, and after waiting for it to spread to all of the blood vessels, a green light is irradiated in a desired brain region, which causes photochemical thrombosis and stroke in the desired area. This section describes several studies focused on photochemical thrombosis investigation modalities for performance enhancements such as an improvement of location accuracy of brain damages and an inhibition of excessive brain damage.

A. Sigler et al. investigated a photothrombosis inducing module that was integrated with an upright microscope to generate a photothrombotic stroke on a single brain arteriole [29]. To be specific, a 532-nm laser diode module and an optical cage system were applied for a construction of the photothrombosis inducing module, which was connected to a filter slot of an upright microscope. Moreover, this module provides a function that can form blood clots in certain cerebral blood vessels while observing them under a microscope. When laser irradiation on a single brain vessel in mice was maintained for 120 s, a simultaneous blockage of a targeted brain vessel was verified using microscopic images (Figure 1a). In addition, laser speckle imaging was utilized to confirm the vascular obstruction caused by photothrombotic stroke. For a similar purpose, M. Alaverdashvili et al. developed a reconfigured laser system to reduce a deviation in induced sizes of brain damages caused by photothrombosis [30]. In the laser system that they proposed, a polarizer or a variable neutral density filter were employed to maintain a constant output irradiation power of laser, which was verified using an optical power meter. An in vivo study on photothrombosis inducing in rats’ brains and 2,3,5-Triphenyltetrazolium chloride (TTC) staining used for estimating locations and depths of photothrombotic strokes confirmed that the system with a maintained optical irradiation power is useful for inducing a stroke and the consequent brain damage of a certain size. U.I. Tuor et al. investigated a minor photothrombotic stroke animal model using a fiber-optic laser irradiation probe to generate Rose Bengal photoactivation [31]. In detail, a probe with a halogen lamp consisting of an optical fiber with a 13-mm diameter, a heat filter, and collimator was constructed to induce a photothrombotic stroke. According to magnetic resonance images described in Figure 1b, an investigation of a small animal model with minor stroke was achieved using a fiber-optic photothrombosis inducing probe. A photothrombosis inducing device integrated with a stereotaxic frame was also developed to produce brain damage at a certain position [32,33]. The stereotaxic frame, which was applied in this study, was magnetic resonance compatible (i.e., it was configured to move with an animal’s location to acquire magnetic resonance images of the brain). Additionally, it provided a function to link indicators of the stereotaxic frame and coil to provide support for image acquisitions, so that it could be held firmly, ensuring the least possible movement.

Various optical imaging modalities were utilized to analyze stroke and its resultant brain damages in cell tissue. The preclinical animal studies were discussed in a previous section. Relevant studies have also been conducted extensively in which these optical imaging techniques are applied to methods used for verifying stroke-inducing animal models or for analyzing changes in lesions or treatments. Q. Liu et al. used a real-time and high-resolution laser speckle brain imaging system to confirm a blockage of blood vessels and vascular changes in the brain of a rodent model using photochemical thrombosis [34]. In this study, changes in a specific vessel over time were quantitatively explored through high-speed imaging of brain vessels. S. Zhang and T.H. Murphy employed in vivo two-photon fluorescence microscopy to observe changes in dendrites as well as a distribution of blood vessels in a photothrombotic animal (mice) model [35]. In addition, it was confirmed that changes in neurons and biomolecules could also be observed using in vivo two-photon imaging, and precise measurements were achieved for damage and changes in dendrites through photochemical thrombosis, as illustrated in Figure 2. H. Lu et al. investigated a miniaturized and lightened infrared (780 nm) vessel imaging device, which was equipped with a combination of fiber-coupled blue and green lasers. The device is designed to generate photochemical thrombosis and acquire a brain vessel image of a freely moving animal [36]. The miniaturized imaging device is significant in that it can immediately monitor effects of photochemical thrombosis and changes in cerebral blood vessels without a need for location-limited noninvasive brain imaging techniques such as an MRI.

2.2. Summary of In Vivo Stroke Model Investigations

In this section, we explored several studies and techniques to establish photothrombosis animal models. Compared to surgical stroke animal model establishment methods, the photothrombosis has advantages of being relatively easy to construct stroke animal models, less likely to die from important vascular obstruction, and easier to control a location of localized stroke. When an establishment of standardized experimental devices and protocols is further carried out by follow-up studies, it is expected that the photothrombosis can be applied to secure a certain stroke animal model and use it for research of various diagnostic and treatment techniques.

3. Optical Imaging Modalities to Develop and Verify Diagnosis/Treatment Techniques of Stroke

3.1. Fluorescence Imaging

Fluorescence imaging is a modality that measures biological, chemical, or biophysical information and its time-varying changes by imaging fluorescence indicators that emit fluorescent signals including required information. Moreover, it has an advantage of non-invasive acquisition of functional and specific bio-information without destroying any biomaterials, for instance, cells [37,38,39,40,41]. For this reason, fluorescence imaging is utilized in both in vitro and in vivo preclinical assays for understanding their causes and verifying diagnostic/therapeutic techniques of stroke by imaging the fluorescent markers that display biochemical and biophysical information related to strokes and its consequent brain damages. In particular, structural neuroplasticity [42,43], axonal regrowth and sprouting [44,45], a regeneration of dendrites [46] in recovery and rehabilitation after stroke were understand by high-resolution fluorescence imaging techniques. In this section, several studies on in vivo animal studies on stroke using fluorescence imaging were explored.

Confocal and two-photon fluorescence microscopy are some of the advanced fluorescence imaging techniques that overcome limits of penetration depth in general fluorescence imaging with a higher resolution [47,48,49,50]. Therefore, these have been employed to analyze stroke-induced in vivo animal models and conduct preclinical tests of stroke-related medical devices. N. Nishimura et al. identified blood clots, which were generated by photothrombosis, in arteries penetrating the longitudinal axis, and changes in blood clots and surrounding blood vessels by observing fluorescein/dextran labeled plasmas using two-photon microscopy [51]. L.T. Watts et al. obtained fluorescence images of blood clots formed inside a cerebral blood vessel beyond a thinned skull of mice [52]. In this study, thrombosis in cerebral blood vessels was confirmed by obtaining fluorescence images of injected fluorescein/dextran in the vessels using a confocal fluorescence microscope. Brain damages from a generation of thrombosis were also verified by 2,3,5-triphenyltetrazolium chloride (TTC) staining after an extraction of the brain. C.J. Schrandt et al. conducted a study to analyze a transient progression of a distribution of the cerebral blood vessels and blood flows using optical speckle and two-photon fluorescence microscopes [53]. The multi-exposure optical speckle imaging used in this study showed a possibility of a long-term analysis of cerebral blood vessels in stroke-induced animal models through its comparison with the two-photon fluorescence imaging.

Studies that analyze strokes and a resultant brain damage using fluorescence imaging of in vivo brain of animal models mainly employed near-infrared probes, which correspond to one of the highest transmission windows [54]. G. Hong et al. developed an optical imaging system and technique to acquire a near-infrared image of a distribution of blood vessels in a mouse brain without removing a skull using a carbon nanotube indicator that emits functionalized optical signals between 1300 and 1400 nm [55]. This imaging technique can obtain arterial and venous veins in the mouse brain with minimal invasiveness up to a depth of approximately 2 mm. Moreover, an experiment to observe vascular loss in a local brain region, which is caused by middle cerebral artery occlusion (MCAO) induced stroke, confirmed its applicability as a stroke research tool. Y. Zhang et al. applied a near-infrared fluorescence indicator to noninvasive acquisitions of fibrin protein deposition in stroke-induced mice as illustrated in Figure 3a [56]. A comparative study of damaged brain areas for ex vivo brain slices confirmed that the fluorescence indicator and noninvasive near-infrared imaging based on it has sufficient correlation to indicate lesions caused by a stroke.

3.2. Functional Near-Infrared Spectroscopy

Both ischemic and hemorrhagic strokes are diseases caused by changes and abnormalities in a distribution of blood in cerebral blood vessels. Therefore, methods to extract information on cerebral blood flows and hemoglobin distributions in cerebral blood vessels, for instance, Doppler ultrasound imaging and vascular magnetic resonance imaging (MRI), can be employed as a noninvasive diagnostic method for stroke. The optical imaging technology, defined as a functional near-infrared spectroscopy (fNIRS), provides an ability to obtain distribution information of oxy-/deoxy-hemoglobin and total blood concentrations using infrared light sources [57,58,59,60]. fNIRS does not use specific indicators, unlike fluorescence imaging. Therefore, it has been applied to preclinical tests as well as to brain function analysis and a verification of stroke rehabilitation at a clinical stage.

Y. Shang et al. investigated a diffuse optical oxy-/total hemoglobin monitoring system using infrared light sources with two peak wavelengths of 785 nm and 854 nm [61]. The system, which consists of two light sources and detectors, can measure hemoglobin concentrations and blood flows in left and right sides of a mouse brain. Moreover, it demonstrated high correlation in a comparative study using a laser Doppler flowmeter and a mouse model with repeated ischemia. C.H. Han et al. applied fNIRS to confirm cerebral hemodynamic responses derived using transcranial electrical stimulation, which is one of the neurostimulation-based therapeutic techniques for brain diseases including stroke [62]. Two light sources with central wavelengths of 690 nm and 830 nm, and detectors associated with fiber-optic probes, were installed in left and right sides of a rat brain. Further, hemodynamic responses were measured for direct current simulation by an electrode, which was placed between the fiber-optic probes of the light sources and the detector. Z.J. Lin et al. applied an infrared diffusion tomography to establish three-dimensional hemodynamic changes to a rat model that performed ischemic stroke (derived by middle cerebral artery to create a permanent occlusion (MCAO)) and its treatment (reperfusion), as illustrated in Figure 3b [63].

fNIRS is also applied to a diagnosis of stroke and to monitoring an effectiveness of rehabilitation and treatment in cerebrovascular stroke patients in a clinical stage. M.J.H. Aries et al. conducted a pilot study of fNIRS to detect acute ischemic stroke and a good responsiveness in stroke-derived changes was confirmed in a small clinical research study [64]. G. Giacalone et al. studied a diagnostic feature extraction of acute ischemic stroke for oxy-/deoxy-/total hemoglobin and tissue oxygen saturation that can be measured by fNIRS (with detailed probe distribution described in Figure 3c) in a clinical stage [65]. Several research groups analyzed a validity and performance of rehabilitation applications and gait training for stroke through a noninvasive observation of cortical activations using fNIRS [66,67,68,69,70]. P.-Y. Lin et al. studied typical cortical activations during a cycle rehabilitation exercise of stroke patients [71]. The study found that despite the existing differences in a degree of activation, both passive and active cycle rehabilitation exercises contribute to premotor cortex activations. Studies using fNIRS as a clinical validation tool for stroke treatments based on various physical stimulations are also being conducted extensively. A. Dutta et al. measured electroencephalographic signals and oxy-hemoglobin during transcranial direct current electrostimulation therapies in stroke patients using a multimodal neuro-signal measurements probe linked to electroencephalography (EEG) and fNIRS [72,73]. From the measurements, variations in cerebrovascular coupling due to the electrical brain stimulation were observed and a potential for development of devices capable of brain stimulation and simultaneous monitoring of multimodal neural signals for treatment efficiency verification was identified. G. Litscher et al. conducted a study using fNIRS to explore brain activities and its responses when a laser stimulation and traditional acupuncture treatments were applied in parallel to stroke patients [74]. Although precisions and evidences should be reinforced through follow-up clinical studies, variations in the brain activity in local areas were noninvasively observed using the multimodal stimulations by fNIRS.

fNIRS was commercialized as a cerebral hematoma diagnostic device at an emergency site due to an advantage that the location of a hematoma can be searched quickly and noninvasively [75,76]. Typically, a clinical study was conducted to diagnose bleeding and the hematoma in the brain, using a commercialized fNIRS-based intracranial hematoma diagnostic device (Infrascanner 2000, InfraScan, Inc., Philadelphia, PA, USA) at helicopter emergency medical services in The Netherlands [77].

Figure 3. (a) Near-infrared images of in vivo fluorescence using fibrin deposition in stroke-induced mice at 6, 8, 20, and 96 h after stroke was triggered. Reprinted with permission from Ref. [56]. Copyright 2012 Public Library of Science. (b) Near-infrared optical diffusion tomographic rat brain images of oxygenate/deoxygenate/total hemoglobin concentrations 30 min after an ischemic stroke. Reprinted with permission from Ref. [63]. Copyright 2014 Elsevier B.V. (c) fNIRS probe arrays designed for an analysis of acute ischemic stroke patients in clinical tests and computed tomographic images used to determine a measurement possibility for areas with and without brain lesions. Reprinted with permission from Ref. [65]. Copyright 2019 International Society for Optics and Photonics (SPIE).

Figure 3. (a) Near-infrared images of in vivo fluorescence using fibrin deposition in stroke-induced mice at 6, 8, 20, and 96 h after stroke was triggered. Reprinted with permission from Ref. [56]. Copyright 2012 Public Library of Science. (b) Near-infrared optical diffusion tomographic rat brain images of oxygenate/deoxygenate/total hemoglobin concentrations 30 min after an ischemic stroke. Reprinted with permission from Ref. [63]. Copyright 2014 Elsevier B.V. (c) fNIRS probe arrays designed for an analysis of acute ischemic stroke patients in clinical tests and computed tomographic images used to determine a measurement possibility for areas with and without brain lesions. Reprinted with permission from Ref. [65]. Copyright 2019 International Society for Optics and Photonics (SPIE).

3.3. Optical Coherence Tomography

Optical coherence tomography (OCT) is an imaging technique that employs an infrared interferometer to obtain optical interference information in depth using light scattering materials and scans it to acquire two- or three-dimensional images of optical coherence information [78,79,80]. An imaging resolution of OCT is typically in micron units, and OCT is generally used as a tool and a diagnostic device for obtaining biomedical information such as eyes (retina) and skin [81,82,83,84]. A more advanced form of OCT, which can acquire bio-information in deeper areas through light sources with a longer wavelength and can be used for acquiring functional information such as vascular distributions through high-speed signal measurements and post-processing techniques, has been developed, which increases a scope of its applications [85,86,87,88]. In this section, a series of studies that conducted studies of cerebrovascular stroke by analyzing information about blood vessels and blood flow rates using advanced OCT instruments are discussed.

L. Yu et al. investigated a Doppler OCT to measure blood circulation in ischemic stroke-induced mice [89]. The study employed a 532 nm LASER to induce an ischemic stroke and changes in blood flow in a localized area, where stroke was generated, were observed using spectral Doppler OCT. E. Osiac et al. analyzed structural brain damages induced by artificial stroke [90]. The rat brain was extracted after different time periods after stroke was induced, and areas of brain damages were imaged using OCT. The results suggested a possibility of OCT as a pathological analysis equipment for ex vivo brain tissues with an acute or chronic stroke. As a follow-up study, an application of an image analysis based on artificial neural networks was additionally applied to classifications of stroke injuries in ex vivo brain tissues [91]. V.J. Srinivasan et al. employed a depth-resolved OCT angiographic imaging, which was integrated in a conventional upright microscope, to analyze acute cerebral ischemic stroke, which was induced by distal MCAO, and a chronic restoration of blood vessels over time in in vivo small animal studies, as illustrated in Figure 4 [92]. S. Chen et al. applied depth-coded visible OCT angiography for making accurate analyses of microvascular blood flow variations when a localized ischemic stroke was induced through photothrombosis [93,94]. In this study, the difference in the blood oxygen saturation caused by a localized ischemic stroke induction was also observed simultaneously by calculating oxygen saturations using the spectroscopic images from visible OCT angiography.

3.4. Photoacoustic Imaging and Tomography

Photoacoustic imaging is a label-free bio-imaging technique in which minute thermal expansion and its consequent pressure changes, which are caused by excitations of pulsed light to a specific biomaterial, are measured in a range of acoustic or ultrasonic waves, and are scanned in multiple dimensions to obtain information on a distribution of a particular biomaterial [95,96]. In detail, photoacoustic imaging consists largely of five steps to obtain images: pulsed light irradiation to excite a specific material, excitation → relaxation, increasing a pressure, measurement of the increased pressure, and finally, image reconstruction [97]. This technique is widely employed in in vivo studies due to its advantages such as deeper imaging depth compared to that of optical imaging, which gives and receives optical signals, and it is label-free [98,99,100,101]. It also exhibits potential for application in minimally invasive clinical diagnosis of several diseases such as breast cancer and tumors [102,103]. As a research tool for stroke, several studies were conducted to analyze photoacoustic signals generated by oxy-/deoxy-hemoglobin for understanding processes of stroke and verifying diagnostic/therapeutic techniques.

J. Laufer et al. developed a high-resolution photoacoustic imaging system that allows precise, three-dimensional observation of blood vessels in mice brains [104]. The system consists of a nanosecond pulsed LASER with excitation wavelength ranges between 590 nm and 889 nm, and an interferometric detection film as a photoacoustic signal sensor. Moreover, it provides images of brain blood vessels with a high sensitivity, precision and greater image acquisition depth. L. Lin et al. investigated a high-speed photoacoustic imaging system to acquire time-varying distributions of blood vessels in mice brains [105]. The photoacoustic microscopy measured a loss of vessels caused by micro-hematoma and laser irradiation using precise photoacoustic brain blood vessels image acquisition, and an analysis of shrinkage response of cerebral blood vessels, which was carried out by injection of phenylephrine, was also performed using photoacoustic imaging. M. Kneipp et al. employed a multispectral optoacoustic imaging to acquire oxy-/deoxy-hemoglobin distributions of mice brains when an artificial stroke was induced by a MCAO, as illustrated in Figure 5 [106]. The imaging method using this proposed optoacoustic imaging system is meaningful in that it provides functional hemodynamic images with enhanced depth ranges while ensuring noninvasiveness. Z. Deng et al. monitored responses of blood vessel distributions and blood oxygen saturations in a rat brain after an induction of acute stroke using a multimodal imaging system, which consisted of laser speckle and photoacoustic imaging [107]. D. Wu et al. developed a multispectral photoacoustic imaging system for a noninvasive analysis of intracerebral hematoma locations induced by collagenase [108]. Images computed using photoacoustic signals at 532 nm, 750 nm, and 875 nm as excitation wavelengths offered location and size information of a lesion in hematoma and perihematomal regions. R. Ni et al. applied multispectral photoacoustic imaging to acquire noninvasive measurements of a matrix-metalloproteinase, a unique group of enzymes associated with acute stroke and brain damages, in in vivo mouse model of an ischemic stroke [109].

3.5. Summary of Optical Imaging Modalities

In this section, we looked at four optical imaging techniques (fluorescence imaging, fNIRS, OCT, and photoacoustic imaging) to study and diagnosis stroke. Optical imaging employed in clinical applications is fNIRS. Fluorescence imaging could be limited in use in clinical sites due to limitations in clinical compatibility of fluorescence staining markers, and it is expected to be actively used in cellular and preclinical studies of stroke. Compared with ultrasound and magnetic resonance imaging, OCT and photoacoustic imaging clearly have limitations in penetrations depth, but are expected to be used in clinical applications through further researches to improve the performance. In addition, OCT and photoacoustic imaging can be employed in combination with fluorescence imaging for cellular and preclinical studies. An application of image post-processing and artificial intelligence-based data analysis techniques will help each optical imaging technique provide more meaningful information for research and diagnosis of stroke.

4. Light Therapy for Mitigation/Treatment of Stroke

4.1. Light Therapy for Stroke Based on Photobiomodulation

Photobiomodulation means a use of light in selected wavelengths for purposes of healing tissues, enhancing a recovery speed, alleviating pain, or restoring function [110,111,112,113]. Among several types of photobiomodulation, a method of irradiating light to the brain for performing functions of treatments, recovery and symptom mitigation is called transcranial photobiomodulation [114,115,116,117,118], and light with spectral bands of red or infrared is generally employed in this process due to its relatively high penetration efficiency. Several research groups have studied transcranial photobiomodulation for brain diseases in three categories: stroke/ischemia, degenerative diseases (such as Alzheimer’s disease), and psychiatric diseases (for instance, bipolar disorder). This section explores the latest progress and important points regarding the studies focused on transcranial photobiomodulation to stroke and its consequent brain injuries.

Several research groups conducted preclinical studies on small and middle-sized animals to identify effects of therapeutic optical stimulation on treatment of stroke and symptoms related to it. L. DeTaboada et al. explored a possibility of recovering neurological deficits caused by a stroke using low-level laser therapy with a peak wavelength of 808 nm in a rat stroke model, which was established using a middle cerebral artery to create a permanent occlusion (MCAO) [119]. The quantitative estimation of neurological deficits from six behavioral tests confirmed that the therapeutic photostimulation on both sides (i.e., a stroke-induced side and on an opposite side improved neurological deficits after stroke). An immunohistochemical analysis of neurons using Bromodeoxyuridine and Tubulin isotype III also confirmed a decrease in neurological damages using therapeutic photostimulation with an 808 nm laser at 7.5 mW/cm² [120]. Preclinical tests using a rabbit stroke model, where stroke was caused by artificially injecting blood clot particles, also indicated that pulsed near-infrared laser irradiations aid healing of brain damages [121]. In this study, a significant impact was observed on a treatment of pulsed photostimulation under two different conditions (1000 Hz with a duty cycle of 30% and 100 Hz with a duty cycle of 20%, with a power density = 7.5 mW/cm²) after 6 h of stroke. However, no therapeutic effect of continuous laser irradiation could be observed when compared statistically with control results, as illustrated in Figure 6. H.I. Lee et al. employed a light emitting diode (LED), with 610 nm as a peak wavelength, in a preclinical study of photostimulation-based stroke therapies [122]. Furthermore, light irradiation using the LED-integrated medical device prototype was applied to a photothrombotic mouse stroke model, and a possibility of using the LED-integrated medical device to treat stroke and mitigate resultant symptoms was confirmed through a comparison of a volume of ischemic brain damages with a control model that was untreated. Moreover, a functional recovery from treatments through the LED-integrated medical device was verified using immunofluorescence assays (Bromodeoxyuridine and an astrocyte marker) [123]. B. Argibary et al. examined preclinical tests to apply infrared (830 nm) LEDs to treatments based on photobiomodulation after an ischemic stroke [124]. Specifically, these researchers applied the therapeutic photostimulation to rats that were subjected to stroke by a transient MCAO, and employed functional MRI (fMRI) to explore functional recoveries of the brain area where ischemic stroke was induced. Although the study could not identify a recovery of stroke-induced lesions and functional reduction due to stroke by LED-based optical neurostimulation, it confirmed that fMRI is a sufficient modality to validate an efficiency of therapeutic photostimulation, both structurally and functionally. Additionally, it was suggested that an application of efficient light irradiation protocols and therapeutic photostimulation for stroke damages in smaller areas should be further studied using fMRI.

Clinical studies for stroke patients and a possibility of noninvasive treatments based on photobiomodulation have been progressed for a treatment of stroke. Y. Lampl et al. reported that therapeutic infrared photobiomodulations had successful outcomes in clinical trial studies with 120 ischemic stroke patients [125,126]. This clinical trial dealt with infrared laser treatments immediately after stroke or within 24 h from the occurrence of stroke, and researchers confirmed that treatments conducted using photo-therapy within 24 h had a satisfactory effect. Moreover, clinical trial studies and validity analyses of transcranial photostimulation to treat an acute ischemic stroke were later conducted on a larger scale, covering 780 patients in 5 countries [127]. H.L. Casalechi et al. conducted clinical studies on an effectiveness of a therapy combining photobiomodulation with magnetic stimulations to a muscle and a nerve for restoring functional mobilities of stroke patients [128]. It has been confirmed that the magnetic stimulation (30 J per site) with light therapy using four infrared diodes (905 nm), pulsed laser diodes, four infrared (with 875 nm as their peak wavelength) LEDs, and four red (640 nm as a peak wavelength) LEDs can significantly recover the functional mobility of stroke patients.

4.2. Optogenetic Neurostimulation to Treat Stroke

Meanwhile, several researchers have developed a technique to treat stroke using optogenetic-based neurostimulation at a preclinical stage and secured a basis for stroke treatment by optogenetic brain stimulations. Optogenetics is a biological technique that genetically ion channels of neurons so that they can be opened or closed in response to light [129,130,131]. So, the neurons modified by optogenetic techniques can be activated or inhibited by light. M.Y. Cheng et al. suggested an optogenetic stimulation as a method to promote functional recovery [132]. The result of this study indicated that when channelrhodopsin-2 was expressed to neurons in an ipsilesional brain region of mice that caused stroke and optical stimulation was induced, blood flow and a neurovascular response increased. M. Song et al. introduced that optogenetic stimulations promoted a neurogenesis of stroke mice [133]. A.M. Shah et al. conducted a preclinical study that optical stimulations of neurons in lateral cerebellar nucleus can contribute to brain function recovery after stroke [134]. A follow-up study by the same research group confirmed that optogenetic neurostimulations reduced neuronal nitric oxide synthase, one of the regulators of neurovascular responses in stroke [135]. It was confirmed that this decrease in neuronal nitric oxide synthase causes recovery of functional degradation by stroke. A preclinical in vivo study was also conducted that both optogenetic neurostimulations and rehabilitation training. E. Conti and co-researchers confirmed that brain function degradation caused by stroke was be more recovered when optogenetic neuronal activations to a motor cortex and foreleg exercise training were applied together to mice with an induction of stroke and an expression of channelrhodopsin-2 [136].

4.3. Summary of Optical Treatments for Stroke

In this section, we explored two types of optical therapeutic techniques (photobiomodulation-based treatment and optogenetic neurostimulation) for stroke. Photobiomodulation-based stroke treatment is highly likely to be employed as a non-drug therapeutic tool for brain diseases, and can be applied in parallel with conventional drugs. Based on the merits of non-causing unexpected problems by spreading through a body such as conventional drugs and low-cost, photobiomodulation-based treatments for brain diseases are expected to be applied in clinical sites and commercialized step by step. On the other hand, additional developments are needed to understand mechanisms and transmit light beyond a skull efficiently. Optogenetic neurostimulations to treat stroke were actively studied at a preclinical in vivo stage for a purpose of understanding a treatment mechanism and verifying effectiveness. The optogenetic stroke treatment showed potential for application as a therapeutic technique for stroke in parallel with conventional drugs. A limitation to be solved for clinical applications is that the safety and stability of optogenetic probes to express neurons must be secured.

5. Conclusions

In this article, optical modalities for research, diagnosis, and treatment of stroke and the consequent brain damage were described. The optical imaging techniques employed for studying cerebrovascular strokes, such as fluorescence imaging, fNIRS imaging, which is based on light irradiations with multiple near-infrared wavelengths for noninvasive acquisition of hemoglobin information; OCT imaging, which is used to obtain information of label-free brain tissue and blood vessels; and photoacoustic imaging employed for studying cerebral blood vessels and to gather functionalized information were explored. Each of these techniques is extensively employed for research and in diagnostic areas. In particular, fNIRS has been employed as a medical device for a diagnosis of acute cerebral hemorrhage and consequent brain damage at emergency sites. Stroke treatments through optical neurostimulation are extensively researched in preclinical studies using animal models and comparative exploration clinical studies. When the results and prospects of these studies are identified, we expect that practical applications and commercialization would be phased out through technological advancements and large-scale clinical verifications. The establishment of stroke animal model, which is based on photothrombosis, is well understood. Moreover, uniform stroke inducement in terms of optical system investigation and upgrade have been identified in previous studies. We are sure that quantification and verification modalities during the establishment of a stroke animal model and stroke-derived brain damages are essential for preclinical studies focused on diagnostic and therapeutic techniques of stroke. Moreover, these optical techniques could be extensively utilized in developments of diagnostic and therapeutic modalities for other brain diseases such as Alzheimer’s disease and certain psychiatric disorders.