Low-Level Light in Combination with Metabolic Modulators for Effective Therapy of Injured Brain

Abstract

Vascular damage occurs frequently at the injured brain causing hypoxia and is associated with poor outcomes in the clinics. We found high levels of glycolysis, reduced adenosine triphosphate generation, and increased formation of reactive oxygen species and apoptosis in neurons under hypoxia. Strikingly, these adverse events were reversed significantly by noninvasive exposure of injured brain to low-level light (LLL). Low-level light illumination sustained the mitochondrial membrane potential, constrained cytochrome c leakage in hypoxic cells, and protected them from apoptosis, underscoring a unique property of LLL. The effect of LLL was further bolstered by combination with metabolic substrates such as pyruvate or lactate both in vivo and in vitro. The combinational treatment retained memory and learning activities of injured mice to a normal level, whereas other treatment displayed partial or severe deficiency in these cognitive functions. In accordance with well-protected learning and memory function, the hippocampal region primarily responsible for learning and memory was completely protected by combination treatment, in marked contrast to the severe loss of hippocampal tissue because of secondary damage in control mice. These data clearly suggest that energy metabolic modulators can additively or synergistically enhance the therapeutic effect of LLL in energy-producing insufficient tissue–like injured brain.

Keywords

hypoxia lactate/pyruvate low-level light mitochondrial function TBI

INTRODUCTION

Traumatic brain injury (TBI) is one of the most common causes of death and long-term disability in the developed world. In the United States, estimated 1.5 million patients had head injuries per year and 5.3 million currently live with TBI-related disabilities.¹ The World Health Organization predicts that road traffic accidents will be the third greatest cause of the global burden of the disease and disablement by 2020. Traumatic brain injury results in cerebral structural damage and functional deficits because of both primary and secondary injury. Although primary injury is caused instantly by blast during the incident, secondary brain injury evolves over time, conferring a gold opportunity for prevention and treatment. It is believed that secondary brain injury results from a cascade of metabolic, cellular, and molecular events in association with inflammation, oxidative stress, perturbation of cellular calcium homeostasis, increased vascular permeability, mitochondrial dysfunction, glutamate excitotoxicity, and apoptosis.^2,3 Hypoxia and its adverse effects on mitochondrial function may take central part in initiation of the cascade and can be great targets for therapy or intervention.

Despite representing only 2% of total body mass, the brain consumes ~20% of the oxygen and 25% of the glucose that a human body intakes and it is one of the most energy-consuming organs. A cell's energy provision, in the form of adenosine triphosphate (ATP), is mostly generated through oxidative phosphorylation in mitochondria. Adequate function of mitochondria is pivotal for the survival and activity of all brain cells, and even brief periods of oxygen or glucose deprivation could shut down brain functions within seconds, damaging neurons within minutes.⁴ Abundant data from retrospective studies and prospective clinical trials have shown brain hypoxia to be an early predictor of adverse outcomes after TBI,^5–7 because efficient ATP production by the mitochondrial respiratory chain relies on continuous oxygen supply.

Low-level light (LLL) refers to red or near-infrared lights with a wavelength of 600 to 1,100 nm and an output power of 1 to 500mW at energy densities of 0.04 to 50 J/cm², relatively low compared with other forms of laser therapy such as ablation, cutting, thermal coagulation, etc. Accumulating evidence suggests that LLL can enhance the activity of mitochondrial respiratory chain and ATP synthesis.⁸ By using mitochondria purified from rat livers, Yu et al⁹ found that LLL elevated the oxygen consumption, phosphate potential, and energy charge of mitochondria. Moreover, via coupling with inhibitors specific for individual protein complexes in the mitochondrial respiratory chain, LLL was confirmed to enhance activities of protein complexes I, III, and IV, but not II or V (ATP synthase) in the mitochondrial respiratory chain.^5,10 Clinically, LLL has been used for over a decade to modulate pain and accelerate wound healing, due in large part to the ability of LLL to stimulate ATP production under various conditions of stress. Because neurons contain a great amount of mitochondria,¹¹ LLL has been shown to be beneficial for treating TBI,¹² stroke,¹³ neurodegenerative diseases,¹⁴ and spinal cord injuries.¹⁵ Our previous investigation showed that mice lacking immediate early responsive gene X-1 (IEX-1) were more susceptible to secondary brain injury than their wild-type littermates, because the null mutation of IEX-1 adversely affected mitochondrial functions and therefore diminished ATP formation in the injured brain.^16,17 Interestingly, noninvasive LLL illumination robustly enhanced ATP generation in injured brains of IEX-1 knockout mice. The enhancement was correlated positively and proportionally with LLL-mediated protection against secondary brain injury in the mice, suggesting a key role for mitochondria in both the pathogenesis of secondary brain injury and the effectiveness of LLL.

The current investigation further reveals that hypoxia accelerates secondary brain damage of injured brains, in agreement with an importance of oxidative phosphorylation in protection against secondary brain injury. Noninvasive LLL illumination improved mitochondrial function, suppressed apoptosis of hypoxic cells, and prevented secondary brain injury. The ability of LLL to improve mitochondrial function in hypoxic cells is unique and may be one of the mechanisms underlying its benefits to TBI. Moreover, LLL and energy metabolic modulators could additively or synergistically enhance the therapeutic effect of LLL on injured brain. The finding is of highly clinical significance, because the combination treatment fully protects hippocampal neutrons from secondary brain injury.

MATERIALS AND METHODS

Cells and Treatments

Human neuroblastoma SH-SY5Y (ATCC, Manassas, VA, USA) cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM; Gibco, Life Technologies, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum in a humidified atmosphere with 5% CO₂. The cells were plated at a density of ~ 1×10⁴ cells per well in a 96-well plate, or 5 × 10⁴ cells per well in a 24-well plate. The cells were subjected to hypoxia by inclusion of 150 μ? cobalt chloride (CoCl₂) in the medium, a well-known hypoxia–mimetic agent,¹⁸ to which 25 mmol/L glucose, 10 mmol/L pyruvate, or 10 mmol/L lactate was added. The concentration of CoCl₂ led to ~ 50% cell death and was thus selected for the study. The hypoxic cultures were then illuminated, at indicated times, by sham light or a continuous wave (CW) near-infrared light–emitting diode array at 830 nm (PhotoMedex, Horsham, PA, USA). A light dose of 0.1, 0.5, 1, 3, or 10 J/cm² was used for illumination, respectively. The effects of LLL, CoCl₂, and metabolic substrates on cell survival were assessed 48 hours after CoCl₂ treatment.

Cell Metabolic Activity

Lactate accumulation was measured in a cell culture medium by an L-lactate assay kit (Eton Bioscience, San Diego, CA, USA). The cells were also collected for ATP quantification using an ATP detection kit per the manufacturer's instructions (Promega, Madison, WI, USA). Levels of reactive oxygen species (ROS) in the cells were analyzed by the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate, which is converted to the highly fluorescent 2′,7′-dichlorofluorescein on interaction with ROS (Molecular Probes, Life Technologies, Grand Island, NY, USA). Both ATP and ROS levels were normalized to protein concentrations that were quantified by the Bradford method (BioRad protein assay kit, Hercules, CA, USA). The mitochondrial membrane potential (ΔΨm) was assayed by flow cytometry using tetraethylbenzimidazolocarbocyanine iodide (JC-1, Life Technologies) dye, a cationic dye that exhibits potential-dependent accumulation in mitochondria. Briefly, cells were stained with 5 μg/mL of JC-1 in serum-free media for 20 minutes at 37°C, after which the cells were washed twice with phosphate-buffered saline containing 2% fetal bovine serum and examined by flow cytometry (BD FACSAria, BD Bioscience, San Jose, CA, USA). Mitochondrial membrane potential was presented as a ratio of JC aggregates (red) to its monomer (green).

Annexin V Analysis of Apoptosis

To corroborate that LLL suppressed apoptosis by sustaining mitochondrial membrane integrity, two apoptosis inducers were evaluated for the effect of LLL. PAC-1, also known as the first procaspase-activating compound, primarily targets a downstream apoptosis executor named procaspase-3, in the mitochondria-dependent pathway, while ABT-737 is an inhibitor of B-cell lymphoma 2 (Bcl-2) family proteins that function to protect the integrity of mitochondrial membrane. SH-SY5Y cells were treated with 10 μM PAC-1 or 2 μM ABT-737 for 2 hours, followed by LLL or sham light treatment. Apoptosis was measured 24 hours later by flow cytometry after staining with allophycocyanin annexin V (BioLegend, San Diego, CA, USA, 1:100, V:V) and propidium iodide (BioLegend, 0.5 mg/mL). The results were analyzed by FlowJo (FlowJo, LLC, Ashland, OR, USA).

Immunofluorescence Assay

Immunofluorescence was performed on paraformaldehyde-fixed cells or acetone-fixed tissue. The fixed cells and tissues were incubated with a blocking buffer (3% bovine serum albumin, 10% goat serum, and 0.4% Triton X-100 (Sigma, St Louis, MO, USA) in phosphate-buffered saline) for 1 hour at room temperature, followed by primary antibody diluted in the blocking buffer at 4°C overnight. After reaction with a secondary antibody for 2 hours at room temperature and washing, the slides were mounted with DAPI (4′, 6′-diamidino-2-phenylindole) containing mounting medium (Invitrogen, Life Technologies, Grand Island, NY, USA). The primary antibodies used were mouse anticytochrome c antibody at a 1:300 dilution (Cell Signaling Technology, Danvers, MA, USA), rabbit antiactive caspase-3 antibody at 1:200 (Abcam, Cambridge, MA, USA), and rabbit anti-α-actin antibody at 1:200 (Abcam). Mitochondria were marked by the MitoTracker probe (Invitrogen); hypoxia was measured by the Cyto-ID hypoxia detection kit (Enzo Life Sciences, Farmingdale, NY, USA) and cell images were captured using a confocal microscope (Olympus FV1000, Olympus, Tokyo, Japan).

Animals

Eight-week-old C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and maintained in a 12-hour light/ dark cycle. All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital and performed according to ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for the care and use of laboratory animals.

Traumatic Brain Injury Induction

Mice were subjected to closed head TBI by a standard controlled cortical impact on the left lateral side with closed skull and scalp as previously described.¹⁷ In brief, the mice were anesthetized with isoflurane and placed on the mobile plate with hair removed from the head. A flat face 2-mm diameter tip of the pneumatic impact device (AMS 201, AmScien Instruments, Richmond, VA, USA) was positioned on the left hemisphere center, lowered gradually down to touch the scalp, and recorded as zero depth (sham control). The punch depth was then set 2.5mm using a screw-mounted adjustment. A 4.9 ± 0.2 m/s velocity and 80 ms contact time were specified by setting 150 pounds per square inch (p.s.i.) for a high pressure and 30 p.s.i. for a low pressure. These parameters were selected to yield a trauma giving a neurologic severity score of 5 to 6 at 1 hour after TBI. After recovery from anesthesia, the mice were returned to cages with postoperative care.

Morris Water Maze

The mice started Morris water maze testing 2 weeks after TBI for assessment of cognitive deficits. The maze was a large circular pool of 150 cm in diameter and 50 cm in height, to which water was filled up to 30 cm at room temperature (22°C). The water was made opaque with powdered milk and the pool was divided arbitrarily into four equal quadrants. A platform was centered in one of the quadrants and submerged 1 cm below the water surface. The position of the platform was kept unaltered throughout the training session. The mouse to be tested was gently placed in the water between two quadrants, facing the wall of the pool with a variable order each day during each trial. The mice were given one trial session each day for five consecutive days. The time taken to find the hidden platform, also named escape latency, was recorded in each trial. If the mouse failed to find the platform within 120 seconds, it was guided gently onto the platform and allowed to remain there for 20 seconds. A significant decrease in escape latency from that of first session was considered as a successful learning. During all the trials, the experimenter always stood at the same position. Care was taken not to disturb the relative location of water maze about other objects in the laboratory serving as prominent visual cues. All the trials were completed between 1300 to 1600 PM in a sound-attenuated laboratory.

Adverse Effects of Hypoxia on Secondary Brain Damage

Mice were subjected to TBI by a standard controlled cortical impact on the cranial window under anesthesia. The cranial window was created by an incision in the scalp to expose the skull and the area of interest was identified under a dissection microscope. The skull cranial window was made into a 3-mm diameter circle by a dental drill on the left hemisphere center. To trigger a rapid oxygen decline inducing hypoxia in the injured brain, Oxyrase (Oxyrase, Mansfield, OH, USA), an enzyme that consumes O₂,¹⁹ was added to the cranial window at a dilution of 1:100 or a final concentration of 0.3 U/mL with 20 mmol/L sodium lactate. After incubation with the Cyto-ID hypoxia probe (Enzo Life Sciences), the cranial window was covered with a round cover glass and closed with glue. The hypoxia probe was converted from nonfluorescent to red fluorescent by nitroreductase activity presented in hypoxic tissue, recorded by an Olympus Fluoview1000 multiphoton imaging system (Olympus) with a spectra-Physics MaiTai HP DeepSee femosecond Ti:Sa laser (Spectra-Physics, Santa Clara, CA, USA). To determine whether LLL could reduce the adverse effects of hypoxia, the mice were treated with LLL at 1 hour after TBI using a near-infrared diode laser of 810 nm at 3 J/cm² (Aculaser, PhotoThera, Carlsbad, CA, USA). The treated mice were killed for histologic examination on the same day or 1, 3, and 7 days after procedure.

Treatment of Traumatic Brain Injury by Low-Level Light Alone or in Combination with Lactate or Pyruvate

Low-level light was performed at 4 hours after TBI using an infrared diode laser of 810 nm (Aculaser) as described.¹⁷ Briefly, the mouse was positioned on a plate and covered by aluminum sheet with a 1-cm diameter hole to expose the contusion site on the head. The laser's pulse frequency was 10 Hz, pulse duration 50 ms, average irradiance 150mW/cm², a total exposure duration time 4 minutes, and energy density 36 J/cm². Owing to the skull and scalp acting as barriers over the brain tissue, the transmitted laser energy to the cortex was ~5.9% ± 0.98% of total input irradiance,²⁰ and the fluence that reached the cortex tissue was ~1.8 to 2.5 J/cm². In some experiments, lactate or pyruvate was freshly prepared at a concentration of 50 mg/mL before scheduled injections and administered intraperitoneally at a dose of 1,000 mg/kg at 1 hour after TBI or 3 hours before the mice were treated with LLL. Control mice received a same volume of saline similarly.

Histologic Examination

Mice were anesthetized and fixed by cardiac perfusion with cold phosphate-buffered saline followed by 10% formalin. Brains were carefully removed, fixed overnight in 10% formalin, and subjected to histopathologic processing and analysis. Hematoxylin and eosin–stained sections of 5-μm thick were scanned by Nanozoomer slide scanner (Olympus America, Center Valley, PA, USA).

Statistical Analysis

The data were presented as mean ± s.e.m. and statistical significance was assessed with one-way analysis of variance or two-way analysis of variance using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant.

RESULTS

Low-Level Light Helps Neurons to Survive Hypoxia

Because of a frequent association of hypoxia to poor outcomes in TBI in the clinics, we investigated a role for hypoxia in secondary brain damage and potential of LLL in protection against the damage based on our previous observations.¹⁷ We first subjected neuronal SH-SY5Y cells to hypoxia by inclusion of CoCl₂ in the medium. This hypoxic mimetic compound–induced hypoxia and the expression of hypoxia-inducible factor 1α significantly (data not shown).¹⁸ Treatment of the cells with 150 μM CoCl₂ for 48 hours caused almost half of the cells to die (Figure 1A). However, the deaths were diminished significantly when the cells were exposed to LLL at energy densities ranging from 3 to 10 J/cm² after 2 hours of CoCl₂ incubation. Low-level light was not effective at lower energy densities (Figure 1A). Stronger protection was then attained when the cells were illuminated with 3 J/cm² LLL twice, one at 0 hours and the other at 2 hours after CoCl₂ addition, or three times at 0, 2, and 18 hours after CoCl₂ addition, respectively (Figure 1A). Low-level light displayed little influence on cell survival in normoxic cultures (control). The ability of LLL to protect the cells from hypoxia-induced cell death appeared to associate with improved mitochondrial functions. As can be seen in Figure 1B, cells in hypoxic cultures produced lactate at levels much greater than in controls, in agreement with a metabolic shift from oxidative phosphorylation to glycolysis. Interestingly, lactate production was reduced to or below the control level when the cells were exposed to LLL at the 0-, 2-, 18-, and 24-hour marks after CoCl₂ addition, with a strongest effect at 2 hours and a weakest one at 24 hours after CoCl₂ incubation (Figure 1B). Owing to the metabolic switch, the hypoxic cells had severely low levels of ATP generation (Figure 1C) and robust increases in ROS production (Figure 1D). The compromised mitochondrial function caused by a decrease in oxygen was partially alleviated by LLL treatment, as evidenced by increasing ATP levels in a light dose–dependent manner (Figure 1C) and suppressing ROS generation (Figure 1D) in these hypoxic cells. Strikingly, although LLL sustained mitochondrial activity and mitochondrial membrane potential (ΔΨm; Figure 1E), it did not alter the level of hypoxia-inducible factor 1α expression (data not shown), confirming mitochondria to be the direct target of LLL. It is noteworthy that LLL only improved mitochondrial functions under hypoxia, and it was without effects in normoxic cultures, in agreement with protective roles of LLL against cellular stress.

Figure 1.

Low-level light (LLL) retains mitochondrial functions under hypoxia. (A) Prevention of hypoxia-induced cell death by LLL. SH-SY5Y cells were treated with CoCl₂ for 2 hours followed by LLL illumination at indicated doses: twice, treatment with LLL at 3 J/cm² in 0 and 2 hours after CoCl₂ incubation and thrice, LLL treatment at 0, 2, and 18 hours after CoCl₂ incubation. (B) Lactate production. The cells were treated with or without CoCl₂ and then illuminated with 3 J/cm² LLL at indicated times after CoCl₂ administration, and lactate accumulation was measured 48 hours after CoCl₂ administration. (C) Effects of CoCl₂ and LLL on adenosine triphosphate (ATP) production. The cells were subject to CoCl₂ treatment, followed by an indicated dose of LLL treatment in 2 hours. Adenosine triphosphate levels were measured 1 hour after LLL illumination. Reactive oxygen species (ROS) production (D) and mitochondrial membrane potential (E) were measured 4 hours after CoCl₂ addition or 2 hours after LLL treatment. The ratio of JC-1 aggregates (red) to monomer (green) fluorescence was calculated as measurement of mitochondrial membrane potential. All results were expressed as means ± s.e.m. n=8; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and NS, nonsignificance.

Low-Level Light Suppresses Apoptosis Induced by Hypoxia

Cells undergoing apoptosis activated caspase-3 and the activation was much greater in the presence of CoCl₂ than its absence, but diminished significantly after LLL exposure (Figure 2A), in agreement with better survival of the cells after LLL treatment (Figure 1A). We postulated that LLL sustained mitochondrial membrane potential (Figure 1E) and prevented cytochrome c leakage, thus reducing caspase-3 activation or apoptosis. In support, cytochrome c and mitochondrial staining revealed their colocalization in control cells, reflected by an orange color merging of green (cytochrome c) and red (MitoTracker) fluorescences (Figure 2B).²¹ The orange color was diminished after CoCl₂ incubation, concomitant with largely nonover-lapping green and red colors, suggesting cytochrome c release from mitochondria (Figure 2B). As expected, LLL illumination blunted cytochrome c leakage and retained cytochrome c in mitochondria in these hypoxic cells (Figure 2B). To further clarify the specificity of LLL in prevention of cytochrome c release from mitochondria, the neurons were treated with two apoptosis-inducing drugs: ABT-737 and PAC-1 that act upstream and downstream of cytochrome c release, respectively. Annexin V staining showed that ABT-737 induced ~ 35% death in the cells after 24 hours of treatment, but the death was reduced substantially after LLL treatment (Figures 2C and 2D). In contrast, LLL did not exhibit any effect on apoptosis induced by PAC-1 (Figure 2E). The results are consistent with the ability of LLL to sustain mitochondrial membrane potential and control apoptosis through prevention of cytochrome c leakage.

Figure 2.

Low-level light (LLL) constrains cytochrome c release in hypoxic cells. Representative images of active caspase-3 (A) and colocalization of cytochrome c and mitochondria (B). SH-SY5Y cells were cultured in complete medium (control) or in the medium containing CoCl₂ for 2 hours, after which the cells were illuminated with LLL at 3 J/cm² as above. Caspase-3 activation and cytochrome c were identified by specific antibodies (green), cell nuclei were marked by DAPI (4′, 6′-diamidino-2-phenylindole, blue), and mitochondria were labeled by MitoTracker (red). Mitochondrial colocalization of cytochrome c is indicated by a merged orange color, arising from green and red in B. Low-level light suppresses apoptosis induced by ABT-737 (C and D), but not by procaspase-activating compound 1 (PAC-1) (E). SH-SY5Y cells were treated either with ABT-737 alone or along with LLL (3 J/cm²) illumination (C and D). The cells were also treated similarly with PAC-1 alone or along with LLL illumination (E). The treated cells were stained with annexin V and propidium iodide (PI) in 24 hours and analyzed by flow cytometry. Representative flow cytometry profiles are given in C. Annexin V-positive and PI-negative cells are early apoptotic cells. Mean percentages ± s.e.m. of early apoptotic cells are summarized in D and E. n=8; ^***P < 0.001 and NS, nonsignificance.

Hypoxia Accelerates but Low-Level Light Protects Secondary Brain Injury

Damage to blood vessels including capillaries in the brain causes an immediate and dramatic decrease in cerebral blood flow that could last for days.^22,23 We found partial or complete damage of many small blood vessels in the hippocampal region after 3 hours of the injury (Figure 3A). On average, one-third of the small blood vessels displayed varying degrees of damage, whereas no such damaged vessels were found in the control hippocampus (Figure 3A). Conceivably, a decrease in cerebral blood flow would disrupt oxygen supply and result in cerebral hypoxia. Although this pathologic scenario is widely viewed, experimental evidence is somewhat lacking. We thus introduced hypoxia to the injured brain and compared resultant histologic changes. To select an inducer for hypoxia, SH-SY5Y cells were treated with CoCl₂ or Oxyrase, and hypoxia was measured by a hypoxia probe containing a nitro (NO₂) moiety that can be reduced in hypoxic cells leading to a release of the fluorescence probe. Although Oxyrase consumed oxygen rapidly and effectively inside and outside the cells, CoCl₂ must enter cells to be effective, which proved to be difficult and insufficient in tissues, in marked contrast to cells, and thus Oxyrase was selected for subsequent studies.¹⁹ As shown in Figure 3B, hypoxia was uniformly and specifically detected in hypoxic cultures as early as 20 minutes of Oxyrase incubation. For in vivo study, Oxyrase was applied topically to the cortex at the injured site of the brain by dropping artificial cerebrospinal fluid comprised of Oxyrase. After 20 minutes of Oxyrase dropping, a hypoxia probe was added to the injured site. The cranial window was closed by a glass cover and monitored by two-photon confocal microscopy. Severe hypoxia was observed at the site of Oxyrase application, not at the control site, as shown by bright red fluorescence over the tissue (Figure 3C). The exogenous hypoxia resulted in expansion and deepening of the lesion from day 1 and throughout the entire experimental period (Figure 3D).

Figure 3.

Low-level light (LLL) protects secondary brain injury induced by hypoxia. (A) Traumatic brain injury (TBI) damages small blood vessels in the hippocampus. Blood vessels in the hippocampus were identified by α-actin antibody, a specific marker for smooth muscle in blood vessels. 4′, 6′-Diamidino-2-phenylindole (DAPI) stained cell nuclei in the vessel wall and arrows indicated the damage in the vessel. Shown in A are two representative small blood vessels from each group. Induction of hypoxia in SH-SY5Y cells (B) and injured brain (C). SH-SY5Y cells were incubated with Oxyrase for 20 minutes and stained with a hypoxia-sensitive probe. The resultant red fluorescence in the cytoplasm confirmed hypoxia in almost all the cells in the presence of Oxyrase but not in the control (B). Likewise, strong hypoxia probe was identified in coronal sections of the Oxyrase-treated brain, but not in the untreated brain (C). The area highlighted by a dashed white square in the left panel was enlarged in the right panel. (D) Gross morphology of the impact of brain over 7 days of the experiment. The detrimental effects of hypoxia and beneficial effects of LLL on the injured brain were analyzed on the day of injury (day 0), and days 1, 3, and 7 after injury. (E) Histologic examination of injured brain at indicated days after TBI. The hippocampus was outlined by the dashed black line: CA, cornu ammonis; DG, dentate gyrus. The hippocampus ratios of the lesion side to the opposite side in the same mice in E were determined by ImageJ and expressed as means ± s.e.m. in F. ^**P < 0.01 and ^***P < 0.001.

Histologically, the lesion with the hypoxic inducer aggressively spread from the cortex into the hippocampus and eventually led to an almost complete loss of the hippocampal dentate gyrus in seven days (Figure 3E). In the controls, free of Oxyrase, however, the lesion was constrained mainly in the cortex and only a small surface portion of the hippocampus was affected, leaving the majority of the hippocampal dentate gyrus intact (Figure 3E, upper). Low-level light treatment restrained spreading of the lesion and completely protected not only the dentate gyrus but also the whole hippocampus from injury whether Oxyrase was applied, with more prominent effects in the presence of Oxyrase. After LLL illumination, the lesions progressed much slower than non-LLL-treated controls both in the presence and absence of Oxyrase (Figures 3D and 3E). To quantify the severity of hippocampal lesions, we compared the size of hippocampus in the traumatic side with that in the opposite side. The volume of the hippocampus diminished considerably on day 3 and tissue loss reached a level as high as 50% on day 7 when compared with the uninjured site, with a more profound loss in the presence of Oxyrase (Figure 3F). Strikingly, LLL treatment completely prevented the loss of hippocampal tissues both in the presence and absence of Oxyrase. Notably, there was a slight increase in the size of hippocampus on day 1 in the injured brain after LLL treatment, because of edema. Edema also occurred in other three groups and was embedded in the measurement owing to a loss of the hippocampal tissue in the groups. In other words, the loss of the hippocampal tissues was underestimated in these groups. The results verify the detrimental effects of hypoxia on the pathogenesis of secondary brain injury and the ability of LLL to protect against secondary brain injury induced by hypoxia and other causes.

Mitochondrial Functions are Additively Improved by the Combination of Low-Level Light with Lactate or Pyruvate

To augment therapeutic efficacy of LLL, we combined LLL with other mitochondria-improving metabolic agents like glucose, lactate, and pyruvate that are all substrates of the mitochondrial tricarboxylic acid cycle. These substrates may be able to further augment oxidative energy metabolism under hypoxic conditions when combined with LLL. As shown in Figure 4A, none of the three substrates increased ATP production in normoxic cultures, but they exhibited variable influences in hypoxic cultures, with a more predominant effect of lactate on ATP production in the culture. However, when combined with LLL, glucose or pyruvate increased oxidative phosphorylation in hypoxic cultures more than lactate (Figure 4A). Moreover, a combination of LLL with glucose-, pyruvate-, or lactate-protected cells from hypoxia-induced death significantly better than any single modality. Notably, the effect of LLL on cell survival was stronger in the presence of pyruvate than in the presence of glucose or lactate (Figure 4B). These data suggest that LLL and energy metabolic modulators together could maximize ATP production and cell survival under a condition of hypoxia.

Figure 4.

Combination of low-level light (LLL) and lactate (Lac), pyruvate (Pyr), or glucose (Glu) improves mitochondrial functions of hypoxic cells. SH-SY5Y cells were cultured in the medium alone (none) or supplemented with glucose Glu, pyruvate Pyr, or lactate Lac. To some of these cultures CoCl₂ was added, along with or without LLL illumination after 2 hours of CoCl₂ incubation. Adenosine triphosphate (ATP) was measured 1 hour after LLL treatment and expressed as obituary luminescence units normalized by protein concentrations (A). Viable cells were determined 48 hours later and expressed as the percentage of control cells cultured in the medium alone (B). Note: CoCl₂ suppressed cell growth by >50% in the medium alone, control. ^{*, **}, and ^*** indicate P < 0.05, <0.01, and <0.001 compared with CoCl₂ without treatment group, #, ##, and ###, which indicate P < 0.05, <0.01, and <0.001, respectively, in the presence or absence of LLL, n = 8.

Low-Level Light and Lactate or Pyruvate Together Fully Protect the Hippocampal Tissue and its Function

We showed that lactate or pyruvate was more effective than glucose in augmentation of LLL-mediated ATP production in vitro (Figure 4). These two substrates were further evaluated in vivo in the injured brain. With severe tissue loss the injured site produced 75% less ATP than the healthy brain and the impairment was only modestly restored by LLL, lactate, or pyruvate alone (Figure 5A). However, a combination of LLL and lactate or pyruvate synergistically or additively increased ATP formation, bringing it to a healthy level in the injured brain. Concurrent with reduced ATP production, the injured brain displayed a robust increase in ROS production, which was, however, effectively suppressed by LLL illumination (Figure 5B). Unlike the effects on ATP production, a combination of LLL with either pyruvate or lactate (Figure 5B) did not further suppress ROS production. We next compared the morphologic changes after single or combinational treatments. Histologic examination revealed severe lesion concomitant with substantial brain tissue loss after TBI (Figure 5C, arrows). The lesion was repaired modestly by treatment with LLL, lactate, or pyruvate alone as suggested by a less severe brain tissue loss measured by gross morphology (Figure 5C) or lesion size (Figure 5D). On the contrary, treatments of LLL and lactate or pyruvate resulted in a faster recovery and less brain tissue loss than any noncombinational treatment (Figures 5C and 5D). Traumatic brain injury mice treated with LLL plus lactate or pyruvate recovered fully in 7 days after TBI, whereas severe cortical lesion remained apparent in other groups of mice. Moreover, TBI-induced cortical lesion and inflammation aggressively spread from the cortex to the hippocampus over 3 days after TBI, causing severe neuron death (white arrow) and inflammation (black arrow) in the hippocampus in untreated control mice (Figure 5E). Under similar conditions, the lesion progression into the hippocampus was completely blocked by treatment with LLL, along with either lactate or pyruvate (Figure 5E), in marked contrast to only modest alleviation mediated by LLL, lactate, or pyruvate alone (Figure 5E).

Figure 5.

Low-level light (LLL) in combination with lactate (Lac) or pyruvate (Pyr) fully protects the hippocampus from secondary damage. Adenosine triphosphate (ATP, A) and reactive oxygen species (ROS, B) production in injured cortex. Cortical ATP and ROS were measured 5 hours after traumatic brain injury (TBI) with indicated treatments. Combinational treatments, as opposed to single treatment, sufficiently elevated the level of cortical ATP production in the injured brain to a normal level (A). Reactive oxygen species production was suppressed robustly by LLL alone, which was not furthered by combination with any substrate (B). (C) Histologic examination of injured brains. The mice were intraperitoneally administered Lac or Pyr 1hour after TBI or exposed to LLL 4 hours after injury, or treated with both protocols. Coronal views showed a loss of brain tissues around the injured site (arrow) over time after TBI, but the loss was effectively prevented by combinational therapy (Lac/LLL and Pyr/LLL), not by single therapy (LLL, Lac, or Pyr) when compared with untreated controls (TBI, C). Quantitative analysis of the lesion sizes was performed using ImageJ and expressed as mean percentages ± s.e.m. relative to the whole brain section (D). The region of the hippocampal dentate gyrus was analyzed on a high magnification (E). The black arrows indicate one of the infiltrated leukocytes and the white arrows indicate one of the necrotic cells in each field. (F) Combinational therapy but not single therapy protects the memory and learning functions of injured mice. Mice were subject to TBI, followed with indicated treatments as in C. After 2 weeks of the initial brain injury, these TBI-experienced mice were evaluated for their learning and memory by Morris water maze test. Latency to reach the platform over 6 test days was reduced in the mice after combinational treatment but not single treatment or no treatment. Data are expressed as mean ± s.e.m. n = 8 for A and B, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with TBI group; or n = 9 for C–F, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; and NS, nonsignificance.

The hippocampal region of the brain is considered to be essential for memory and spatial navigation. These activities were assessed by Morris water maze tests after 2 weeks of the initial brain injury. During the 6-day tests, it took a long escape time for untreated mice to find the hidden platform, and there was no significant improvement over the days of training in the mice, suggesting poor memory and learning (Figure 5F), in agreement with a severe loss of the hippocampal tissue in the mice (Figure 5E). Low-level light alone did not significantly shorten the escape latency in most of the TBI mice. In contrast, the combinational treatment fully restored cognitive ability of TBI mice to a normal level (Figure 5F). The ability of LLL and a metabolic substrate in restoration of the normal cognitive behaviors of the mice confirms full protection of hippocampal neurons from brain injury.

DISCUSSION

Despite promising results from preclinical studies, potential TBI treatments have not yet translated into successful outcomes in clinical trials. Further studies are urgently needed to elucidate the mechanisms underlying secondary brain damage and the specificity of individual treatments. Our previous investigations showed that mice bearing inadequate activity of mitochondria as a sequel of IEX-1 null mutation were prone to secondary brain injury.¹⁷ The observation stresses an importance of mitochondrial activity in protection against secondary brain damage, which is highly anticipated, because neurons are one of the most oxygen-sensitive cells. Damage of blood vessels, in particular, capillary, occurs frequently at the site of injured brain, which disrupts oxygen supply and reduces cerebral blood flow drastically. Severe and prolonged reductions in cerebral blood flow lead to deprivations of oxygen and glucose causing cerebral hypoxia and inadequate mitochondrial functions. Data from retrospective studies and prospective clinical trials have revealed that a high level of brain hypoxia always links to adverse outcomes for TBI. However, heterogeneous pathophysiologic alterations after TBI make it difficult to determine the effect of hypoxia on secondary brain damage. Introduction of systemic hypoxia to TBI mice aggravated neuronal death and lesion,^24,25 but it was not known whether local cerebral hypoxia that simulated brain injury in the clinics was also detrimental to TBI. We therefore dropped Oxyrase directly over the injured brain to induce local cerebral hypoxia. Our data corroborate that cerebral hypoxia accelerates secondary brain damage and brain tissue loss because of hypoxia-induced apoptosis. This new TBI model provides direct experimental evidence about adverse effects of hypoxia on secondary brain injury.

Confirmation of the adverse effect of hypoxia on secondary brain damage reinforces the importance of mitochondrial functions in the protection of injured brain from secondary damage. Our previous study, in line with others, have shown that LLL illumination can sustain mitochondrial functions in cells under various conditions of stress, including enhancement of ATP synthesis, suppression of ROS production, and reduction of apoptosis.^26–28 The ability of sustaining mitochondrial functions in the injured brain may account for the benefit of LLL in TBI management showed recently.^12,17,28 The current investigations further validate that LLL stimulates ATP production and suppresses ROS generation in hypoxic cells and brain tissue. Hypoxia increases cytochrome c release from mitochondria. On release from mitochondria, cytochrome c activates apoptotic protease activating factor-1, caspase-9, and caspase-3, executing apoptosis via the intrinsic mitochondria-dependent pathway. By induction of apoptosis with two different agents, we found that LLL could protect cells from apoptosis induced by ABT-737 that abrogates the antiapoptotic function of Bcl-2 family in the upstream of cytochrome c release, but not PAC-1 that activates caspase-3 directly, a downstream target of cytochrome c release. The finding is consistent with the ability of LLL to sustain mitochondrial membrane potential. These investigations uncover the mechanism of LLL in protecting hypoxic neurons from apoptosis and in prevention of brain tissue loss. Hence, noninvasive LLL illumination can greatly benefit TBI patients, particularly in the patients with severe cerebral hypoxia.

The primary energy source for brain cells is glucose and it is transported from the blood into the brain and metabolized to lactate or pyruvate. Lactate and pyruvate could enter mitochondria and serve as substrates of the tricarboxylic acid cycle and oxidative phosphorylation.²⁹ For many years, lactate has traditionally been thought as a useless ‘dead end’ product of anaerobic metabolism and harmful sometimes. Its elevation in the brain signals cerebral ischemic damage.³⁰ However, lactate and pyruvate can readily cross the blood–brain barrier and enter the tricarboxylic acid cycle,^31,32 being preferential oxidative energy substrates over glucose for neurons.^33,34 The beneficial effect of exogenous glucose, lactate, and pyruvate has been shown in some TBI models,^35–37 but these substrates only modestly reduce the lesion size, with no apparent functional protection in our model. However, when lactate or pyruvate is combined with LLL illumination, mitochondrial functions are improved either additively or synergistically, giving rise to a faster recovery and less brain tissue loss compared with LLL, pyruvate, or lactate alone. Most importantly, the hippocampal region is fully protected from the injury by combinational treatments, whereas in the controls the lesion in the cerebral cortex spreads into the hippocampus underneath. It is well known that the hippocampus takes a central role in the consolidation of information relating to short-term and long-term memory, as well as spatial navigation.³⁸ Although there are some neural stem cells in the adult mammals' hippocampus with the capacity to differentiate into neurons, astrocytes, and oligodendrocytes, differentiation of the cells hardly leads to fully functional recovery of the hippocampus as shown by several investigations.³⁹ On damage of the hippocampus, a severe loss of memory and difficulty in establishing new memories manifest. Therefore, ability of shielding the hippocampus from secondary damage remains the most satisfactory therapeutic outcome of the current approach. Traumatic brain injury is a complicated disease and the current investigation suggests that combinational treatment may yield a better outcome than single treatment. Use of LLL to treat TBI is currently under clinical trials and addition of lactate or pyruvate to the trial may confer better outcomes, in particular, in patients with inadequate mitochondrial functions, which will be the next step of the investigation. Apart from TBI, it is well known that mitochondrial dysfunction is involved in many nervous diseases, such as Alzheimer, Parkinson, amyotrophic lateral sclerosis, schizophrenia, et al. It may be worthwhile to test this novel modality in the treatment or prevention of these neurodegenerative disorders in the future as well.

AUTHOR CONTRIBUTIONS

TD designed and performed the research, analyzed data, and wrote the manuscript. QZ participated in the experimental design, analysis of data, and manuscript writing. MRH participated in the experimental design and manuscript writing. MXW designed and supervised the research and wrote the manuscript.

DISCLOSURE/CONFLICT OF INTEREST

The authors declare no conflict of interest.

Footnotes

ACKNOWLEDGMENTS

The authors thank members of the Photopathology Core at Wellman Center for experimental assistance with histopathology, flow cytometry, and microscopy services. The authors also thank Jeffrey H Wu for editing and members of Wu's Lab for stimulating discussion.

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