STROKE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/850,685, filed May 21, 2019, which is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

[0002] The present disclosure generally relates to near-infrared light (NIR) treatment, including NIR treatment of ischemic stroke.

BACKGROUND

[0003] Stroke is one of the leading causes of death and disability in the Western world and accounts for approximately 1 in 20 deaths in the United States. Ischemic stroke occurs due to occlusion of an intracranial artery generally resulting in rapid and cytotoxic reductions in blood flow to the brain parenchyma. To date, approved treatments for ischemic stroke include rapid restoration of blood flow (i.e., reperfusion or reperfusion phase) either by pharmacological or surgical modalities. While patient outcomes have improved due to these interventions, a substantial amount of tissue damage may occur during the reperfusion phase. As the ischemic brain is re-oxygenated, reactive oxygen species (ROS) are quickly generated, beginning at the early stages of reflow which further induces cell death by way of complex, interacting molecular pathways. While ROS have been implicated to play a key role, to date, pharmacological strategies targeting ROS have generally not proven effective.

[0004] Thus, there is a need to improve treatment for stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 illustrates exemplary blood flow during stroke ischemia and following reperfusion;

[0006] Figure 2 illustrates an exemplary analysis of cerebral injury in an acute reperfusion phase;

[0007] Figure 3 illustrates an exemplary infarct in a chronic phase of post-stroke reperfusion injury;

[0008] Figure 4 illustrates exemplary relative cerebral blood flow during ischemia and following approximately 4-hour NIR treatment;

[0009] Figure 5 illustrates exemplary infarct volumes in the acute phase of stroke with NIR treatment for approximately 4 hours; and

[0010] Figure 6 illustrates exemplary T2 weighted images (T2WI) of exemplary infarct volumes during the chronic phase of stroke.

DETAILED DESCRIPTION

[0011] The present invention provides a method for treating ischemia-reperfusion injury associated with stroke (e.g., focal stroke). Ischemic stroke is typically caused by occlusion of an intracranial artery. Unlike many other ischemia-reperfusion injuries (e.g., post-arrest global brain ischemia), ischemic stroke is often associated with a longer ischemic period before the occlusion in a patient is removed by medical intervention. The longer ischemic period results in more severe tissue damages during the reperfusion stage, which increases the difficulty of achieving substantial protection by treatment. The present invention identifies a noninvasive method of treating ischemic stroke by applying infrared light (IRL) to affected tissue for an extended duration. It has been demonstrated that a prolonged treatment with IRL, for example, at least four hours, can significantly reduce infarct volume in a subject (for example, by at least 50%, 75%, or 100%), relative to that achieved with a shorter period of treatment time with IRL. A non-exhaustive list of examples of the present invention is provided in the following examples listed 1 to 26 below.

[0012] Example 1 : A method of reducing ischemia-reperfusion injury in a subject having a stroke, the method comprising applying light to tissue subject to ischemia-reperfusion injury for at least 3 hours (e.g., at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, or at least 60 hours) contemporaneous with and/or after the onset of ischemia-reperfusion injury thereby to reduce the extent of ischemia-reperfusion injury, wherein the light applied comprises light having wavelengths in each of the ranges of 730-770 nm and 930-970 nm.

[0013] Example 2: The method of example 1, wherein the light is applied to the tissue for 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours contemporaneous with and/or after the onset of ischemia-reperfusion injury.

[0014] Example 3: The method of example 1 or 2, wherein the injury occurs during the acute phase of stroke.

[0015] Example 4: The method of any one of examples 1-3, wherein the injury occurs during the chronic phase of stroke.

[0016] Example 5: The method of any one of examples 1-4, wherein the illumination reduces the volume of tissue exhibiting an infarct by at least 30%, 40%, 50%, or 60% relative to a subject that has not been subject to the illumination.

[0017] Example 6: The method of any one of examples 1-5, wherein the light application has wavelengths of about 750 nm and about 950 nm. As used in this example, the term“about” indicates deviations of up to 1% above and up to 1% below a given value.

[0018] Example 7: The method of any one of examples 1-5, wherein the light application has wavelengths of about 750 nm and about 940 nm. As used in this example, the term“about” indicates deviations of up to 1% above and up to 1% below a given value.

[0019] Example 8: The method of any one of examples 1-7, wherein the light is substantially free of a wavelength of 810 nm and/or 808 nm. As used in this example, the term“substantially free” indicates that the intensity of the light at the specified wavelength is no greater than 10% of the greater of the maximum intensity of in the range of 730-770 nm or that in the range of 930- 970 nm. In a further example, the term“substantially free” indicates that the intensity of the light at the specified wavelength is no greater than 5% of the greater of the maximum intensity of in the range of 730-770 nm or that in the range of 930-970 nm.

[0020] Example 9: The method of any one of examples 1-8, wherein the light is applied prior to the onset of the reperfusion injury.

[0021] Example 10: The method of any one of examples 1-9, wherein the light is applied in multiple, separate time periods.

[0022] Example 11 : The method of example 10, wherein the light is applied in at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) time periods.

[0023] Example 12: The method of example 11, wherein the light is applied in 2, 3, 4, 5, 6, 7, 8, 9, or 10 time periods.

[0024] Example 13 : The method of example 11 or 12, wherein at least two of the time periods have the same time duration.

[0025] Example 14: The method of any one of examples 10-13, wherein all the time periods have the same time duration.

[0026] Example 15: The method of any one of examples 10-14, wherein the total duration of the light applied to the tissue is at least 3 hours (e.g., at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, or at least 60 hours).

[0027] Example 16: The method of any one of examples 10-15, wherein the total duration of the light applied to the tissue is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours. [0028] Example 17: The method of any one of examples 1-16, wherein the light is generated by one or more light emitting diodes.

[0029] Example 18: The method of any one of examples 1-17, wherein the light is generated by one or more laser diodes.

[0030] Example 19: The method of any one of examples 1-18, wherein the light has a fluence in the range of 5 mW to 80 mW (e.g., 0.1-50 mW, 0.1-20 mW, 0.1-10 mW, 0.1-5 mW, 0.1-1 mW, 1-50 mW, 1-20 mW, 1-10 mW, or 1-5 mW).

[0031] Example 20: The method of any of examples 1-19, wherein the light has an irradiance in the range of 2 mW/cm ² to 32 W/cm ² (e.g., 50 mW/cm ² to 2 W/cm ² , 100 mW/cm ² to 1 W/cm ² , 500 mW/cm ² -5 W/cm ² , or 200-800 mW/cm ² ).

[0032] Example 21 : The method of any of examples 1-20, wherein the light has an irradiance lower than or equal to 5 W/cm ² (e.g., lower than or equal to 2 W/cm ² , or lower than or equal to 1 W/cm ² ).

[0033] Example 22: The method of any of examples 1-21, wherein the light has an irradiance of about 10 mW/cm ² , 20 mW/cm ² , 50 mW/cm ² , 100 mW/cm ² , 200 mW/cm ² , 400 mW/cm ² , or 800 mW/cm ² . As used in this example, the term“about” indicates deviations of up to 10% above and up to 10% below a given value.

[0034] Example 23 : The method of any of examples 1-22, wherein the light has a power density in the range of 0.01-0.1 mW/cm ² , 0.1-1 mW/cm ² , 0.5-5 mW/cm ² , or 3-5 mW/cm ² at the tissue.

[0035] Example 24: The method of any one of examples 1-23, wherein the stroke is focal stroke.

[0036] Example 25: The method of any one of examples 1-24, wherein the stroke is ischemic stroke.

[0037] Example 26: The method of example 25, wherein the duration of ischemia of the stroke is at least 30 minutes, at least 1 hour, or at least 2 hours.

[0038] Novel photoreceptive properties of cyclooxygenase (COX), the terminal enzyme in the mitochondrial electron transport chain (ETC), have been identified. Further, it has been determined that the enzymatic activity of COX can be regulated in a wavelength-specific manner using near-infrared (NIR) light. For example, it has been demonstrated that COX activity can be stimulated by applying NIR wavelengths (e.g., 670 nm and 808 nm), thus increasing mitochondrial respiration. Indeed, this stimulatory effect has been utilized to enhance mitochondrial function during permanent ischemic stroke in animal models, but has generally failed to improve outcomes in a clinical stroke study. The aforementioned strategy of increasing COX, and thus mitochondrial activity during early reperfusion, may exacerbate (rather than reduce) injury. ETC complexes are generally rendered hyperactive due to cellular stress during ischemia exposure, resulting from changes in their regulatory modifications. This mitochondrial stress state drives post-ischemic ROS generation in the early phase of reperfusion.

[0039] It has been determined, however, that COX activity can be reduced by applying specific inhibitory NIR wavelengths (e.g., 750 nm, 950 nm, and the combination thereof). Reduction of COX activity by inhibitory NIR leads to transient, reversible reductions in both mitochondrial respiration, and the mitochondrial membrane potential, and subsequent attenuation of superoxide production. In neuronal culture models, irradiation with inhibitory NIR wavelengths salvage neurons following oxygen glucose deprivation or glutamate exposure. As shown herein, the effect of inhibitory NIR wavelengths in the setting of acute ischemic stroke, which differs in the pathological evolution from global brain ischemia, has been investigated.

[0040] Accordingly, as shown herein, the efficacy of NIR treatment at limiting brain injury progression after acute ischemic stroke using the rat middle cerebral artery occlusion (MCAO) model has been evaluated. To increase translational relevance, experiments were conducted in the classic comorbid model displaying spontaneous hypertension. It was hypothesized that dual wavelength inhibitory NIR, applied immediately following resolution of ischemia, would provide neuroprotection measured by multiple, clinically validated MRI modalities that are becoming the gold standard for identifying potentially salvageable brain tissue. The window of treatment opportunity was chosen in order to target the initial ROS burst that occurs early during reperfusion. In a clinical setting, the onset of reperfusion generally coincides with tissue plasminogen activator (tPA) administration or mechanical thrombectomy (i.e., pharmaceutical or mechanical/surgical). It was also hypothesized that NIR therapy would directly target salvageable tissue that succumbs to injury following reperfusion. Brain injury was quantified by delineating infarct volume using diffusion weighted imaging (DWI) during early reperfusion and T2 weighted image (T2WI) at late reperfusion. Further, to increase rigor, incorporation of the ‘area at risk’ of infarction (i.e., volume of brain rendered ischemic during MCAO, derived from perfusion weighted images (PWI)) was introduced as a covariate in the analysis. A goal of these studies was to establish whether NIR treatment limits infarct size expansion (reperfusion injury) in the early acute phase following stroke (e.g., 24 hours following restoration of blood flow). Additionally, another goal was to determine whether the positive effects of NIR treatment would persist as neuroprotection in late chronic phases of reperfusion (e.g., 7 and 14 days after ischemia).

[0041] Chemicals and reagents were obtained from Sigma-Aldrich unless otherwise stated. Light emitting diodes (LEDs) were obtained from Roithner Lasertechnik. For animal experiments LED array 60 chips were used (e.g., 750 nm, LED750-66-60; 950 nm, LED950-66- 60). Diodes were mounted on heat sinks (e.g., black aluminum, 47x20 for LED array 60 chips) together with a fan (e.g., Evercool) operated in reverse mode. Diodes were calibrated with an optical power meter (e.g., 842-PE) and operated with an energy density of approximately 200 mW/cm ² .

[0042] Animal use protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University and all experiments were performed in accordance with the Guide for the care and use of Laboratory Animals and conform to the ARRIVE guidelines for reporting of In vivo experiments. Animals underwent approximately 90 minutes of transient right Middle cerebral artery occlusion (MCAO) as described below. Approximately one hour into ischemia, PWI was assessed using arterial spin labeling (ASL). After approximately 90 minutes of focal (acute) brain ischemia, reintroduction of blood flow (reperfusion) was established, and treatment was initiated and maintained for approximately 120 or 240 minutes (Protocols 1 and 2, respectively). Following treatment, animals were imaged with PWI and DWI. Approximately twenty-four hours following reperfusion, DWI scans were obtained. On days 7 and 14 the animals were imaged with T2WI.

[0043] Focal brain ischemia was induced in male spontaneously hypertensive rats (SHR) (223-336 g, Envigo) with the MCAO method as previously described. SHR animals were utilized to introduce a clinically relevant comorbidity to the model as well as to reduce variation in infarct size. 20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to constrict cerebral arteries and is synthesized more abundantly in the vasculature of SHR animals. This study reported that inhibiting 20-HETE reduced superoxide formation to the same levels as normotensive rats, suggesting that 20-HETE can generate oxidative stress and vascular dysfunction. Furthermore, male animals were utilized in this model to provide a reproducible infarct, omitting changes in estrogen levels in females as another variable. Animals were anesthetized with isoflurane (e.g., 5% induction, 2% maintenance in a mix of 70% nitrous oxide, 30% oxygen). Surgery was conducted at approximately the same time of day for each animal. Temperature was maintained at approximately 37°C using a homeothermic blanket (e.g., Harvard Apparatus). A neck incision was made, and the external carotid was ligated. A silicon coated tip monofilament suture (e.g., Doccol Corporation) was inserted in the external carotid and advanced into the internal carotid artery until it occluded the MCA. The filament was secured in place and the rats were placed in a temperature- and humidity-controlled chamber (e.g., Tecniplast). Following ischemia, animals were re-anesthetized, the filament withdrawn, and NIR treatment was initiated. Animals were given subcutaneous buprenorphine (e.g., 0.015 mg/kg) and recovered in a temperature/humidity-controlled environment. Weight loss exceeding 10% was addressed with enhanced nutritional support (e.g., DietGel) and/or subcutaneous administration of approximately 5% dextrose in normal saline.

[0044] Rats were randomly enrolled in NIR-treatment or untreated groups during MCAO, prior to reperfusion. NIR was administered with combined COX-inhibitory wavelengths of approximately 750 nm and approximately 950 nm. Briefly, LEDs at approximately 200 mW/cm ² were placed approximately 1.5 cm from the shaved scalp upon filament withdrawal and continued for approximately 120 minutes (Protocol 1) or approximately 240 minutes (Protocol 2). That is, a method of reducing an ischemic reperfusion injury due to an acute stroke included applying near-infrared (NIR) light to a brain tissue subject to acute ischemic reperfusion injury for two (2) hours (Protocol 1) and four hours (protocol 2) to reduce the ischemic reperfusion injury.

[0045] During treatment the rats were maintained at approximately 37 ±0.5°C under isoflurane anesthesia (e.g., approximately 1.5%). Untreated animals underwent the same procedures (i.e., MCAO surgery, anesthesia dose, and duration) but were not treated with NIR. In optimizing this treatment regime, it has been demonstrated that irradiation with the approximate wavelengths of 750 nm and 950 nm at approximately 200 mW/cm ² generally does not alter brain, rectal, or skin surface temperature in rats.

[0046] MRI protocols were performed on a 7.0-Tesla, 20-cm bore superconducting magnet (e.g., ClinScan; Bruker, Karlsruhe, Germany) with a Siemens console. Animals were anesthetized with isoflurane. Pulsed ASL (PWI) images were acquired according to the following exemplary sequence parameters: Relaxation time (TR) of 3500 ms; and echo time (TE) of 16 ms; field-of-view (FoV) read 35.0 mm; FoV phase 81.3%; distance factor 25%; slice thickness 2.0 mm; 4 slices. DWI sequence was conducted according to the following exemplary parameters: TR 10000 ms; TE 50 ms; FoV read 32.0 mm; FoV phase 100.0%; distance factor 0%; slice thickness 0.5 mm; 32 slices. T2WI was acquired according to the following exemplary parameters: TR 3530 ms; TE 38 ms; FoV read 32 mm; FoV phase 100.0%; distance factor 0%; slice thickness 1.0 mm; 24 slices. Relative cerebral blood flow in the MCA territory during ischemia and reperfusion was calculated as previously described. Briefly, voxel intensity in ipsilateral and contralateral hemispheres was averaged across coronal slices of the MCA territory using ImageJ, giving the average relative cerebral blood flow (relCBF) voxel intensity value. The boundary between hypoperfused and physiologically perfused brain tissue was identifiable on the relCBF. Then, using kinetic model equations, CBF was calculated for both hemispheres giving cerebral blood flow rates in mL/lOOg/min. Brains were traced from PWI of each animal during ischemia and following treatment (or matched time-points in untreated controls). Infarct volumes were computed using the semi-automated segmentation tool in Analyze 11.0 (e.g., Biomedical Imaging Resource, Mayo Clinic). Seed points were set in the middle of the hyperintense MCA territory on a single slice and thresholds based on voxel intensity were set by the software until the injured region was outlined. The accuracy of the software generated outline was confirmed by a blinded investigator, then the software applied the exemplary threshold parameters to all slices within a sequence. This process provided an automated and unbiased calculation of the area within each slice. All the slices in the brain were summated then multiplied by the area thickness to generate infarct volume in mm ³ .

[0047] Acquisition and analysis of MRI images were conducted in a blinded manner, without knowledge of the treatment group. Group sizes were determined by power analysis (e.g., GPower3.1). Statistical analysis was performed using GraphPad Prism 7. Brain injury volume in the acute phase, chronic phase, and relative cerebral blood flow was compared using two- factor ANOVA (for group and time) with replication, with Neuman-Keuls multiple comparisons test for post hoc analysis with significance at p<0.05. Slopes and intercepts of the DWI/PWI linear regression lines were compared using analysis of covariance (ANCOVA) with significance at p<0.05.

[0048] Figure 1 illustrates exemplary relative blood flow during cerebral ischemia and following reperfusion. In this regard, an experimental protocol 100 for pulsed arterial spin labeling (PWI) imaging is shown. Further, exemplary PWI images 102 during ischemia and from both groups are shown following reperfusion indicating reduction of blood flow in the ipsilateral hemisphere during ischemia and restoration of blood flow during reperfusion. As quantified in the bar diagram 104, averaged regional cerebral blood flow in the ipsilateral MCA territory and contralateral hemisphere both during ischemia and following reperfusion (n = 7- 8/group) are shown.

[0049] During ischemia (prior to randomization), all animals showed reductions in blood flow below established relCBF thresholds in the ipsilateral hemisphere (see, e.g., Figure 1). With regard to Protocol 1, following the 2-hour NIR treatment window, restoration of blood flow to the ipsilateral hemisphere was confirmed and relCBF was statistically equivalent to the contralateral hemisphere. There was no significant difference in relCBF within the ipsilateral hemispheres between control and NIR-treated animals during ischemia or reperfusion.

[0050] With reference now to Figure 2, an exemplary analysis of cerebral injury in the acute reperfusion phase I is shown. In this regard, an exemplary experimental design 200 for measuring early phase outcomes is shown. Exemplary diffusion weighted imaging (DWI) images 202 were obtained and no significant difference in infarct volume was seen between NIR treated and control animals after 2 hours of treatment. At 24 hours, compared with untreated controls, NIR treated MCAO rats showed an exemplary 21% reduction of infarct volume. An exemplary at risk area analyzed (see, e.g., Figure 2, 202) by PWI at 1-hour ischemia (x-axis) and DWI hyperintensity volume (y-axis) 2 hours and 24 hours following reperfusion is illustrated. This indicates that the regression relationship between infarct volume and area at risk for NIR- treated animals was below the relationship observed in controls.

[0051] The effect of NIR on cerebral injury in the acute post-ischemia phase (i.e., less than 24 hours after reperfusion - see, e.g., Figure 2, 200) was assessed using DWI, which is generally considered to be the effective imaging modality for quantification of early cytotoxic edema and prediction of final infarct size. Figure 2, 202 generally illustrates that DWI hyperintensity volumes following treatment (e.g., 2 hours of reperfusion) did not significantly differ between NIR-treated and control groups (210 vs 256 mm ³ , respectively, p>0.05). At 24 hours following reperfusion, NIR-treated animals showed a significant reduction of approximately 21% in DWI hyperintensity volume when compared with untreated controls: 317 vs. 399 mm ³ , p<0.05 (see, e.g., Figure 2, 202). Repeated measures ANOVA revealed an overall, significant increase in DWI-hyperintensity at 24 vs. 2 hours of reperfusion and a significant difference between NIR- treated and untreated groups at 24 hours of reperfusion. These data suggest that the infarct continues to expand between 2- and 24-hours post-reperfusion and NIR-treatment limits this expansion.

[0052] These concepts are supported by ANCOVA, incorporating‘area at risk’ (volume of brain rendered ischemic during MCAO, delineated by the volume of flow deficit identified by PWI) as a covariate in the analysis of infarct size. Following treatment at 2 hours reperfusion, the volume of DWI hyperintensity occupied only approximately 50% of the area at risk in both groups, and ANCOVA revealed no difference in the regression relationship between DWI hyperintensity volume and area at risk between cohorts (p>0.05). This suggests that no measurable treatment effect at this early timepoint. In contrast, after 24 hours of reperfusion, the volume of at-risk brain that displayed DWI hyperintensity increased to approximately 85% in the control cohort and approximately 73% in the NIR-treated group, and the regression relationship between infarct volume and area at risk for NIR-treated group fell below the relationship observed in controls (*p<0.05; see, e.g., Figure 2, 204).

[0053] With reference now to Figure 3, an exemplary cerebral infarct in chronic phase of post-stroke reperfusion injury is shown. An exemplary experimental design 300 at 7- and 14- days following reperfusion is represented. Exemplary T2-weighted imaging (T2WI)-images 302 of MCAO vs. MCAO with NIR treatment are shown. Compared with untreated controls, NIR treated MCAO rats showed an exemplary 25% reduction in infarct volume. Significant reduction in infarct volume persisted at 14 days post stroke. Accordingly, not only are ischemic reperfusion injury reductions observable via an imaging modality (e.g., magnetic resonance) at 7 days, but also at 14 days. As represented in the graphs 304, regression relationship of PWI (area at risk) vs. infarct volume in MCAO vs. MCAO treated with NIR is illustrated. Accordingly, the exemplary regression relationship is shown between infarct volume and risk region for NIR- treated animals. This indicates that the regression relationship was below the relationship observed in controls.

[0054] Assessment of candidate therapies in rat models of stroke traditionally utilize early infarct analysis at 24 hours following MCAO. However, multiple studies have demonstrated early reductions in infarct volume are often not predictive of final brain injury at later stages of injury progression and thus early signs of efficacy may not indicate meaningful, lasting neuroprotection. Accordingly, to address this issue, animals were maintained for an additional 2 weeks and the therapeutic benefits of NIR were also evaluated with T2WI at 7 and 14 days following MCAO-reperfusion (see, e.g., Figure 3, 300). Seven days following MCAO, NIR- treated animals maintained a significant reduction of approximately 25% in infarct size when compared to untreated controls (271 vs 363 mm ³ , *p<0.05) that persisted at 14 days after MCAO (241 vs. 317 mm ³ , *p<0.05, Figure 3, 302), with no significant change over time. That is, infarction had generally fully evolved by 7 days of reperfusion and neuroprotection persisted at the final time-point of 14 days post-stroke. The volume of hyperintensity (infarct), expressed as a percentage of the at-risk volume of brain, averaged 64% vs. 73% in the respective NIR-treated vs. control groups at 7 days, and approximately 57% vs. 69%, respectively, at 14 days post reperfusion. Accordingly reductions in infarct size or ischemic reperfusion injury size are observable via an imaging modality (e.g., magnetic resonance) at 7 days and 14 days. Observable, in general may mean, for example, by the“naked-eye” or indirectly via the imaging modality. These conclusions were again, supported by ANCOVA at both time-points, the regression relationship between infarct volume and risk region for NIR-treated rats fell below the relationship observed in controls (*p<0.05; see, e.g., Figure 3, 304).

[0055] Moving on to Figure 4, exemplary relative cerebral blood flow during ischemia and following an exemplary 4-hour NIR treatment is shown. An exemplary experimental design 400 is represented, as well as exemplary PWI images 402 during ischemia and from both groups following reperfusion, which indicate restoration of blood flow. Exemplary averaged regional relative cerebral blood flow in the ipsilateral and contralateral MCA territories during ischemia and following approximately 4 hours of reperfusion (n = 6-7/group) is quantified in the exemplary bar diagram 404.

[0056] During ischemia (before randomization), mean relCBF in the ischemic hemisphere was reduced to approximately 10.94 mL/100 g/min while the contralateral hemisphere maintained physiological flow rates of approximately 50.78 mL/100 g/min. Following resolution of ischemia, blood flow significantly increased with no significant difference between animals receiving NIR treatment versus untreated controls during ischemia or reperfusion (see, e.g., Figure. 4, 402, *p<0.05).

[0057] With regard to Protocol 2, the efficacy of extended 4-hour treatment was assessed using the analytical approach described in Protocol 1. As represented in Figure 5, exemplary infarct volumes in the acute phase of stroke with NIR for four hours is shown. An exemplary experimental design 500 is represented, as well as the exemplary images 502 of DWI four hours following reperfusion in NIR treated vs. control, indicating a significant reduction in infarct size of the NIR-treated animals compared to the controls at the end of the 4-hour treatment (which was maintained at 24 hours post-treatment). An exemplary at-risk area during ischemia (PWI) vs. area of infarction (DWI) at four hours following reperfusion is quantified in the graphs 504 of Figure 5. [0058] Early indices of brain injury were quantified with DWI after the 4-hour treatment period (4-hours of reperfusion) and at 24 hours after reperfusion (see, e.g., Figure 5, 500). In contrast to Protocol 1, there is a difference in infarct size between control and NIR-treated groups at the end of treatment (e.g., 228 vs. 138 mm ³ ; see, e.g., Figure 5B, p<0.05), that was maintained at 24 hours post-treatment despite the expected, temporal expansion of the infarcts (e.g., 351 vs. 266 mm ³ , *p<0.05). These data were supported by ANCOVA (see, e.g., Figure 5, 504) and, taken together, suggest that even in the early stage of brain damage (e.g., 4 and 24 hours of reperfusion) NIR therapy limited the progression of brain injury.

[0059] Similar to Protocol 1, it was sought to determine whether the 4-hour NIR treatment paradigm evoked sustained neuroprotection in the chronic stage of reperfusion injury. Figure 6 represents this determination, showing T2WI of infarct volumes during the chronic phase of stroke. An exemplary experimental design 600 is represented, along with exemplary images 602. The images 602 indicate that the NIR treated showed an exemplary 52% reduction in infarct volume at 7- and 14-days following ischemia vs. untreated controls. At risk area vs area of infarction at 7- and 14-days post reperfusion show significant reduction with NIR-treatment, as represented in the graphs 604 of Figure 6. Indeed, as shown, there are reductions in infarct size or ischemic reperfusion injury size that are observable via an imaging modality (e.g., magnetic resonance) at 7 days and 14 days. Observable, in general may mean, for example, by the“naked eye” or indirectly via the imaging modality.

[0060] With continued reference to Figure 6, using T2WI, control and NIR treated groups were imaged 7 days following reperfusion (see, e.g., Figure 6, 602). Mean T2 hyperintensities were reduced by approximately 52% at both 7- and 14-days post-reperfusion as exemplified in Figure 6, 602. Accordingly, this suggests that infarction is fully evolved by 7 days of reperfusion and a positive treatment effect remains at the final time-point of 14 days post reperfusion. The relationship between cerebral infarction measured with T2WI with tissue at- risk for infarction revealed a difference (ANCOVA) in regression line elevation between control and NIR-treated groups. Further, ANOVA of the DWI/PWI ratio found a reduction in this ratio between control and NIR-treated groups at both 7 and 14 days of reperfusion as exemplified in Figure 6, 604. [0061] Ischemic stroke generally accounts for about approximately 9% of deaths globally and is generally the second leading cause of death following heart disease. Those who survive this debilitating disease often face a lifetime of neurological deficits and hence a drastic reduction in quality of life, including cognitive, emotional and functional impairments. The gold standard for stroke treatment is generally considered to be the rapid restoration of blood flow. However, timely reperfusion is, paradoxically, associated with a perpetuation of brain injury, due in part to the production of ROS during early reoxygenation. Oxidative stress is a common pathological mechanism in many disease states and arises from an imbalance between ROS production and ROS scavenging. Once produced, ROS damage cells either directly, or through diverse and complex cell signaling cascades.

[0062] As described herein, the effectiveness of targeting ROS at their source is presented: directly in the mitochondrial ETC during early reperfusion using NIR therapy. It has been determined that irradiation of the brain with NIR immediately upon reperfusion reduces infarct size both acutely and chronically following stroke.

[0063] The data indicate that cerebral blood flow is significantly reduced below ischemic thresholds in the ipsilateral hemisphere and returns to normal levels following reperfusion. Interestingly, infarct volumes were not generally different between NIR-treated and control animals immediately following the approximate 120-minute NIR-treatment. However, animals treated for approximately 240 minutes showed immediate reductions in infarct volume when compared to untreated controls. Nonetheless, reductions in infarct volume were manifest in both Protocols 1 and 2 at approximately 24 hours following reperfusion.

[0064] When infarct volumes against area at risk are plotted, it is shown that for all groups in both Protocol 1 and Protocol 2 that the relationship between area at risk and area of infarction is linear. That is, infarct size is proportional to the area at risk. In addition, there was a downward shift in the regression relationship (with generally no difference in slope) for the NIR-treated cohorts versus controls. As such, it is determined that, over the full range of risk regions, the penumbra was salvaged with NIR treatment, resulting in smaller infarct volumes.

[0065] Benefit of NIR are also extended into the chronic phase of stroke. The data show that infarct volumes remained smaller at 7- and 14-days post stroke with NIR in both Protocols - outcomes that were confirmed by ANCOVA. Interesting, in Protocol 2, the paradigm of prolonged NIR-treatment was associated with an approximately 50% reduction in infarct volume. As such, this indicates that the extended treatment duration improved neurological outcome. The additional targeting of late-stage ROS production caused by excitotoxicity, inhibition of inflammatory cell conversion through ROS signaling as well as a possible, and longer-than-predicted window of mitochondrial stress may potentially explain how the longer treatment duration had such profound reductions in infarct volume.

[0066] Accordingly, it is illustrated and described herein that non-invasive partial inhibition of mitochondrial COX by dual -wavelength NIR treatment following acute (focal) stroke is accompanied by profound and sustained reductions in cerebral infarction. While the dual wavelength NIR treatment included exemplary wavelengths of 750 nm and 950 nm, other NIR wavelengths may be employed. For example, one wavelength may be selected from the range of 730-770 nm and the other wavelength may be selected from the range of 930-970 nm.

[0067] It is noted that the reductions in infarct volume were measured using clinically relevant MRI modalities, which allowed assessment of stroke evolution over time to be monitored in the intact animal. As such, it is demonstrated that such treatment reduces the progression of reperfusion injury following stroke. Furthermore, it is shown that NIR can target more than the initial burst of ROS following reperfusion. That is, extending treatment duration has profound beneficial effects on infarct volume. As a result, this data has shifted the paradigm on the role that NIR can play at later stages of the reperfusion injury process.

[0068] Methods discussed herein include removing a cerebral occlusion (such as an occlusion causing an acute stroke) either pharmacologically or mechanically. After or during removal, providing dual -wavelength near-infrared (NIR) light to mitochondrial electron transport chains in brain tissue to reduce reperfusion injury resulting from a stroke is begun. The dual -wavelength NIR light may be provided for at least two hours, four hours, or more.

[0069] The dual wavelength NIR light may, for example, be selected from a range of 730-770 nm and a range of 930-970 nm. Further, the application of the applying the dual -wavelength NIR light may begin prior to an onset of reperfusion, during an onset of reperfusion, or after an onset of reperfusion. As one example, applying the NIR light may occur during the removal of the cerebral occlusion.

[0070] Such techniques or methods at least minimize post-stroke reperfusion injury (chronic phase).

[0071] When introducing elements of various embodiments of the disclosed materials, the articles“a,”“an,”“the,” and“said” are intended to mean that there are one or more of the elements. The terms“comprising,”“including,” and“having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Furthermore, any numerical examples in the following discussion are intended to be non-limiting, and thus additional numerical values, ranges, and percentages are within the scope of the disclosed embodiments.

[0072] While the preceding discussion is generally provided in the context of medical imaging, it should be appreciated that the present techniques are not limited to such medical contexts. The provision of examples and explanations in such a medical context is to facilitate explanation by providing instances of implementations and applications. The disclosed approaches may also be utilized in other contexts, such as the non-destructive inspection of manufactured parts or goods (i.e., quality control or quality review applications), and/or the non- invasive inspection or imaging techniques.

[0073] While the disclosed materials have been described in detail in connection with only a limited number of embodiments, it should be readily understood that the embodiments are not limited to such disclosed embodiments. Rather, that disclosed can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the disclosed materials. Additionally, while various embodiments have been described, it is to be understood that disclosed aspects may include only some of the described embodiments. Accordingly, that disclosed is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.