Use of the traditional Chinese medicine "compound healthy ear agent" to protect against age-related hearing loss in mice: A proteomics study.

Background: Previous studies have shown that the traditional Chinese medicine (TCM) called "compound healthy ear agent" (CHEA) had anti-apoptosis effects in cochlear hair cells and spiral ganglion neurons, and could protect mice hearing against presbycusis or age-related hearing loss (AHL), as well as aminoglycoside antibiotic-induced ototoxicity. Because its mechanisms of action are still unclear, we investigated the mechanism of action of CHEA against AHL in mice using proteomics techniques. Methods: Eighteen C57BL/6J mice at 1 month of age were randomly divided into three groups: (A) drinking water until 2 months of age, K2M); (B) drinking water until 7 months of age to induce AHL, K7M; (C) drinking water containing CHEA daily until 7 months of age as treatment group, Z7M. At 2 or 7 months mice were sacrificed and their cochleae were removed for proteomics analysis. Results: The numbers of proteins with a false discovery rate (FDR) < 1% were respectively 5873 for qualitative and 5492 for quantitative statistics. The numbers of proteins with differential enrichment at least 1.5-fold (p < 0.05) were respectively 351 for K7M vs K2M groups, 52 for Z7M vs K7M groups, 264 for Z7M vs K2M groups. The differentially expressed proteins in the Z7M group were involved in synaptic molecular transmission, energy metabolism, immune response, antioxidant defenses, and anti-apoptosis. Conclusion: The TCM CHEA played a protective role against AHL in mice by regulating the expression of specific proteins and genes in cochlear hair cells and spiral ganglion neurons. Besides the pathways expected to be involved (antioxidant and anti-apoptosis), proteins related to immune response is a new finding of the present study.

Phenylalanine-Arginine-ß-Naphthylamide Enhances the Photobactericidal Effect of Methylene Blue on Pseudomonas aeruginosa.

Objective: To investigate the effectiveness, dosing sequence, concentration, and mechanism of antimicrobial photodynamic inactivation (aPDI) using methylene blue (MB) plus phenylalanine-arginine-ß-naphthylamide (PAßN) against Pseudomonas aeruginosa. Methods: P. aeruginosa bacterial suspension was incubated with MB for different times (5-240 min), and then, 10 J/cm2 red light was irradiated. The efflux pump inhibitor (EPI) PAßN (10-100 µg/mL) was combined with MB (1-20 µM) in different sequences (PAßN-first, PAßN+MB, PAßN-after). Colony-forming units were then determined by serial dilution. Results: Using MB 10 µM plus 10 J/cm2, the killing effect of MB-aPDI on P. aeruginosa increased first and then decreased with longer incubation time. The killing effect of MB+PAßN-aPDI on P. aeruginosa was better than that of MB-aPDI (p < 0.05) by up to 2 logs. PAßN-first had the best killing effect, whereas PAßN-after had the worst killing effect. The killing effect increased with PAßN concentration and at 100 µg/mL reached 5.1 logs. Conclusions: The EPI PAßN enhanced the bactericidal effect of MB-aPDI on P. aeruginosa, especially when added before MB. It is proposed that MB is a substrate of the resistance-nodulation-division family efflux pump.

Antimicrobial photodynamic therapy for oral Candida infection in adult AIDS patients: A pilot clinical trial.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) using methylene blue (MB) plus potassium iodide (KI) has been shown to be effective in killing Candida albicans in many in vitro and in vivo studies, however, there are limited reports of clinical investigations. This study aimed to explore the clinical application of aPDT with MB plus KI for the treatment of oral infection caused by C. albicans in adult acquired immune deficiency syndrome (AIDS) patients. METHODS: A total of 21 adult AIDS patients with C. albicans oral candidiasis were divided into two groups according to MB concentration and received two consecutive aPDT treatments. Immediately before and after the aPDT treatments, C. albicans yeast isolates were recovered to measure the colony-forming units per mL (CFU/mL), biofilm formation, and to analyze the 25S rDNA genotype. Patients were assessed for the clinical recovery of oral lesions and improvement of symptoms. RESULTS: The Log10 CFU/mL of C. albicans decreased significantly after the second aPDT but not the first aPDT. There was no significant difference between the two MB concentrations. Both aPDT protocols decreased the oral lesions and clinical symptoms with no significant difference after 2-fraction aPDT. The biofilm formation of C. albicans isolates did not change before and after aPDT. The killing efficiency of 2-fraction-aPDT was not associated with either biofilm formation or 25S rDNA genotype. CONCLUSIONS: Two-fraction-aPDT with MB plus KI could reduce the number of viable C. albicans fungal cells and improve the clinical symptoms of oral candidiasis in adult AIDS patients, regardless of the biofilm formation or 25S rDNA genotype of infected C. albicans isolates.

Some Ototoxic Drugs Destroy Cochlear Support Cells Before Damaging Sensory Hair Cells.

A wide variety of ototoxic drugs are capable of damaging the sensory hair cells in the mammalian cochlea resulting in permanent hearing loss. However, the toxic properties of these drugs suggest that some could potentially damage cochlear support cells as well. To test the hypothesis, we treated postnatal day three rat cochlear cultures with toxic doses of gentamicin, cisplatin, mefloquine, and cadmium. Gentamicin primarily destroyed the hair cells and disrupted the intercellular connection with the surrounding support cells. Gentamicin-induced hair cell death was initiated through the caspase-9 intrinsic apoptotic pathway followed by activation of downstream executioner caspase-3. In contrast, cisplatin, mefloquine, and cadmium initially damaged the support cells and only later damaged the hair cells. Support cell death was initiated through the caspase-8 extrinsic apoptotic pathway followed later by downstream activation of caspase-3. Cisplatin, mefloquine, and cadmium significantly reduced the expression of actin and laminin, in the extracellular matrix, leading to significant disarray of the sensory epithelium.

Exploration of Copper-Cysteamine Nanoparticles as a New Type of Agents for Antimicrobial Photodynamic Inactivation.

Copper-cysteamine (Cu-Cy) nanoparticles (NPs) are a new type of sensitizers that can be activated by UV light, X-rays, microwaves and ultrasound to produce reactive oxygen species for cancer treatment. Here, for the first time, we explored Cu-Cy NPs for bacteria inactivation by treating gram-positive bacteria (methicillin-resistant Staphylococcus aureus and Enterococcus faecalis) and gram-negative bacteria (Escherichia coli and Acinetobacter baumannii), respectively. The results show that Cu-Cy NPs are very effective in killing gram-positive bacteria but are quite limited in killing gram-negative bacteria yet. The major killing mechanism is cell damage by singlet oxygen and Cu-Cy NPs are potential agents for bacteria inactivation.

Amphiphilic tetracationic porphyrins are exceptionally active antimicrobial photosensitizers: In vitro and in vivo studies with the free-base and Pd-chelate.

Antimicrobial photodynamic inactivation (aPDI) employs the combination of nontoxic photosensitizing dyes and visible light to kill pathogenic microorganisms regardless of drug-resistance, and can be used to treat localized infections. A meso-substituted tetra-methylpyridinium porphyrin with one methyl group replaced by a C12 alkyl chain (FS111) and its Pd-derivative (FS111-Pd) were synthesized and tested as broad-spectrum antimicrobial photosensitizers when excited by blue light (5 or 10 J/cm2 ). Both compounds showed unprecedented activity, with the superior FS111-Pd giving 3 logs of killing at 1 nM, and eradication at 10 nM for Gram-positive methicillin-resistant Staphylococcus aureus. For the Gram-negative Escherichia coli, both compounds produced eradication at 100 nM, while against the fungal yeast Candida albicans, both compounds produced eradication at 500 nM. Both compounds could be categorized as generators of singlet oxygen (ΦΔ = 0.62 for FS111 and 0.71 for FS111-Pd). An in vivo study was carried out using a mouse model of localized infection in a partial thickness skin abrasion caused by bioluminescent Gram-negative uropathogenic E. coli. Both compounds were effective in reducing bioluminescent signal in a dose-dependent manner when excited by blue light (405 nm), but aPDI with FS111-Pd was somewhat superior both during light and in preventing recurrence during the 6 days following PDT.

Comparison of thiocyanate and selenocyanate for potentiation of antimicrobial photodynamic therapy.

We have previously shown that antimicrobial photodynamic therapy (aPDT) mediated by different photosensitizers (PS) can be potentiated by a variety of inorganic salts. Potassium thiocyanate (KSCN) potentiated aPDT mediated by methylene blue (MB), while potassium selenocyanate (KSeCN) potentiated aPDT mediated by MB, Rose Bengal and the anionic porphyrin 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin dihydrochloride. However, the mechanisms of action that were proposed were fundamentally different. In the present study, we compare these two salts (KSCN and KSeCN) with different light-activated PS and different oxidative reactions for killing gram-positive and gram-negative bacteria. Overall KSeCN was more powerful than KSCN, and worked with a wider range of PS, while KSCN only worked with phenothiazinium salts. KSeCN produced killing when cells were added after light suggesting production of a semistable species called selenocyanogen (SeCN)2 . We tested three different oxidative reactions that can all potentially kill bacteria: lead tetraacetate (Pb[OAc]4 ); Fenton reagent (hydrogen peroxide [H2 O2 ] and ferrous sulfate) H2 O2 and horseradish peroxidase (HRP). In every case, KSeCN was substantially more effective (several logs) than KSCN in potentiating the bacterial killing. We conclude that (SeCN)2 is the mediator for aPDT using KSeCN, while sulfur trioxide radical anion is the mediator for KSCN using phenothiaziums. For H2 O2 /HRP with KSCN, hypothiocyanite is proposed to be the antibacterial agent in the literature, while hyposelenocyanite is said not to exist. Pb[OAc]4 is known to produce (SeCN)2 from KSeCN as well as the analogous (SCN)2 from KSCN. The mediators from Fenton reaction are unclear (pseudohalogen radical ions?) Both KSCN (which occurs naturally in the human body) and KSeCN may be clinically applicable.

Antimicrobial Photodynamic Inactivation Mediated by Tetracyclines in Vitro and in Vivo: Photochemical Mechanisms and Potentiation by Potassium Iodide.

Tetracyclines (including demeclocycline, DMCT, or doxycycline, DOTC) represent a class of dual-action antibacterial compounds, which can act as antibiotics in the dark, and also as photosensitizers under illumination with blue or UVA light. It is known that tetracyclines are taken up inside bacterial cells where they bind to ribosomes. In the present study, we investigated the photochemical mechanism: Type 1 (hydroxyl radicals); Type 2 (singlet oxygen); or Type 3 (oxygen independent). Moreover, we asked whether addition of potassium iodide (KI) could potentiate the aPDI activity of tetracyclines. High concentrations of KI (200-400 mM) strongly potentiated (up to 5 logs of extra killing) light-mediated killing of Gram-negative Escherichia coli or Gram-positive MRSA (although the latter was somewhat less susceptible). KI potentiation was still apparent after a washing step showing that the iodide could penetrate the E. coli cells where the tetracycline had bound. When cells were added to the tetracycline + KI mixture after light, killing was observed in the case of E. coli showing formation of free molecular iodine. Addition of azide quenched the formation of iodine but not hydrogen peroxide. DMCT but not DOTC iodinated tyrosine. Both E. coli and MRSA could be killed by tetracyclines plus light in the absence of oxygen and this killing was not quenched by azide. A mouse model of a superficial wound infection caused by bioluminescent E. coli could be treated by topical application of DMCT and blue light and bacterial regrowth did not occur owing to the continued anti biotic activity of the tetracycline.

Comparison of two functionalized fullerenes for antimicrobial photodynamic inactivation: Potentiation by potassium iodide and photochemical mechanisms.

A new fullerene (BB4-PPBA) functionalized with a tertiary amine and carboxylic acid was prepared and compared with BB4 (cationic quaternary group) for antimicrobial photodynamic inactivation (aPDI). BB4 was highly active against Gram-positive methicillin resistant Staphylococcus aureus (MRSA) and BB4-PPBA was moderately active when activated by blue light. Neither compound showed much activity against Gram-negative Escherichia coli or fungus Candida albicans. Therefore, we examined potentiation by addition of potassium iodide. Both compounds were highly potentiated by KI (1-6 extra logs of killing). BB4-PPBA was potentiated more than BB4 against MRSA and E. coli, while for C. albicans the reverse was the case. Addition of azide potentiated aPDI mediated by BB4 against MRSA, but abolished the potentiation caused by KI with both compounds. The killing ability after light decayed after 24 h in the case of BB4, implying a contribution from hypoiodite as well as free iodine. Tyrosine was readily iodinated with BB4-PPBA plus KI, but less so with BB4. We conclude that the photochemical mechanisms of these two fullerenes are different. BB4-PPBA is more Type 2 (singlet oxygen) while BB4 is more Type 1 (electron transfer). There is also a possibility of direct bacterial killing by electron transfer, but this will require more study to prove.

Antimicrobial photodynamic inactivation is potentiated by the addition of selenocyanate: Possible involvement of selenocyanogen?

We previously showed that antimicrobial photodynamic inactivation (aPDI) of Gram-positive and Gram-negative bacteria mediated by the phenothiazinium dye, methylene blue (MB), was potentiated by the addition of potassium thiocyanate (10 mM). The mechanism was suggested to involve a singlet oxygen-mediated reaction with SCN to form sulfite and cyanide and then to produce sulfur trioxide radical anion. We now report that potassium selenocyanate (concentrations up to 100 mM) can also potentiate (up to 6 logs of killing) aPDI mediated by a number of different photosensitizers (PS): MB, rose bengal and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin dihydrochloride (as low as 200 nM). When a mixture of selenocyanate with these PS in solution was illuminated and then bacteria were added after the light, there was up to 6 logs of killing (Gram-negative > Gram-positive) but the antibacterial species decayed rapidly (by 20 minutes). Our hypothesis to explain this antibacterial activity is the formation of selenocyanogen (SeCN)2 by reaction with singlet oxygen (1 O2 ) as shown by quenching of 1 O2 by SeCN and increased photoconsumption of oxygen. The fact that lead tetraacetate reacted with SeCN (literature preparation of (SeCN)2 ) also produced a short-lived antibacterial species supports this hypothesis.

A traditional Chinese medicine compound (Jian Er) for presbycusis in a mouse model: Reduction of apoptosis and protection of cochlear sensorineural cells and hearing.

Age-related hearing loss (AHL) or presbycusis is steadily increasing due to the overall aging of the Chinese population. Traditional Chinese medicine (TCM) has long been used to prevent and treat deafness, but its effectiveness and mechanism of action are still uncertain. The present study tested a TCM preparation called "Jian Er" in a mouse model of prebycusis.

Potentiation by potassium iodide reveals that the anionic porphyrin TPPS4 is a surprisingly effective photosensitizer for antimicrobial photodynamic inactivation.

We recently reported that addition of the non-toxic salt, potassium iodide can potentiate antimicrobial photodynamic inactivation of a broad-spectrum of microorganisms, producing many extra logs of killing. If the photosensitizer (PS) can bind to the microbial cells, then delivering light in the presence of KI produces short-lived reactive iodine species, while if the cells are added after light the killing is caused by molecular iodine produced as a result of singlet oxygen-mediated oxidation of iodide. In an attempt to show the importance of PS-bacterial binding, we compared two charged porphyrins, TPPS4 (thought to be anionic and not able to bind to Gram-negative bacteria) and TMPyP4 (considered cationic and well able to bind to bacteria). As expected TPPS4+light did not kill Gram-negative Escherichia coli, but surprisingly when 100mM KI was added, it was highly effective (eradication at 200nM+10J/cm2 of 415nm light). TPPS4 was more effective than TMPyP4 in eradicating the Gram-positive bacteria, methicillin-resistant Staphylococcus aureus and the fungal yeast Candida albicans (regardless of KI). TPPS4 was also highly active against E. coli after a centrifugation step when KI was added, suggesting that the supposedly anionic porphyrin bound to bacteria and Candida. This was confirmed by uptake experiments. We compared the phthalocyanine tetrasulfonate derivative (ClAlPCS4), which did not bind to bacteria or allow KI-mediated killing of E. coli after a spin, suggesting it was truly anionic. We conclude that TPPS4 behaves as if it has some cationic character in the presence of bacteria, which may be related to its delivery from suppliers in the form of a dihydrochloride salt.

Repeated transcranial low-level laser therapy for traumatic brain injury in mice: biphasic dose response and long-term treatment outcome.

We previously showed that near-infrared laser photobiomodulation (PBM) (810 nm, CW, 18 J/cm2 , 25 mW/cm2 ) delivered to the mouse daily for 3-days after a controlled cortical impact traumatic brain injury (TBI) gave a significant improvement in neurological/cognitive function. However the same parameters delivered 14X daily gave significantly less benefit. This biphasic dose response intrigued us, and we decided to follow the mice that received 3X or 14X laser treatments out to 56-days post-TBI. We found the 14X group showed worse neurological function than the no-treatment TBI group at 2-weeks, but started to improve steadily during the next 6-weeks, and by 56-days were significantly better than the no-treatment TBI mice, but still worse than the 3X mice. A marker of activated glial cells (GFAP) was significantly increased in the brain regions (compared to both untreated TBI and 3X groups) at 4-weeks in the 14X group, but the GFAP had fallen to low levels in both 3X and 14X groups by 8-weeks. We conclude that an excessive number of laser-treatments delivered to mice can temporarily inhibit the process of brain repair stimulated by tPBM, but then the inhibitory effect ceases, and brain repair can resume. The mechanism may be temporary induction of reactive gliosis.

[Protecting Effects of Jian'erji on Age-related Hearing Loss of C57BL/6J Mice].

Objective To observe decreased hearing in aged C57BL/6J mice, and to study pro- tective effects of Jian' erji ( JEJ ) for age-related hearing loss (AHL) and its possible mechanism. Methods Totally 36 C57BL/6J mice were randomly divided into four groups, i.e., the normal control group (n =6) , the AHL control group (n =12) , the high dose JEJ group (n =12) , the low dose JEJ group (n =6). Mice in the normal control group drank tap water from ablactation till 2 months old. Mice in the AHL control group drank tap water from ablactation till 7 months old. Mice in high and low dose JEJ groups drank JEJ at the daily dose of 3. 65 g/kg and 0. 91 g/kg respectively from ablactation till 7 months old. Six mice were selected from each group for auditory brainstem response (ABR) using brainstem evoked potentiometer on the day of ending the test. The cochlear tissue, auditory cortex, and liver were immediately collected from 6 mice of the high dose JEJ group and 6 of the AHL control group at the same ages. Contents of malondialdehyde (MDA) , end product of lipid peroxidation were detected by UV spectrophotometer using MDA coomassie blue kit. Results ABR thresholds evoked by short-pure tone from 4 to 48 KHz were in the normal range of 2 months old mice in the normal control group. Compared with 2 months old mice in the normal control group, ABR thresholds were significantly elevated in 7 months old mice of the AHL control group (P <0. 05). Significant differences also existed in ABR thresholds from 8 to 48 KHz in the high dose JEJ group (P <0. 05). Compared with 7 months old mice of the AHL control group, MDA contents in cochlear tissue, auditory cortex, and liver were obviously reduced in the high dose JEJ group (P <0. 01). Conclusions C57BL/6J mice showed significant symptoms of AHL in high frequency range at 7 months old. Daily drinking of high dose JEJ could significantly delay the occurrence and progress of AHL. Its protection might be related to antioxidant effects JEJ contained.

Low-level laser therapy for traumatic brain injury in mice increases brain derived neurotrophic factor (BDNF) and synaptogenesis.

Transcranial low-level laser (light) therapy (LLLT) is a new non-invasive approach to treating a range of brain disorders including traumatic brain injury (TBI). We (and others) have shown that applying near-infrared light to the head of animals that have suffered TBI produces improvement in neurological functioning, lessens the size of the brain lesion, reduces neuroinflammation, and stimulates the formation of new neurons. In the present study we used a controlled cortical impact TBI in mice and treated the mice either once (4 h post-TBI, 1-laser), or three daily applications (3-laser) with 810 nm CW laser 36 J/cm(2) at 50 mW/cm(2). Similar to previous studies, the neurological severity score improved in laser-treated mice compared to untreated TBI mice at day 14 and continued to further improve at days 21 and 28 with 3-laser being better than 1-laser. Mice were sacrificed at days 7 and 28 and brains removed for immunofluorescence analysis. Brain-derived neurotrophic factor (BDNF) was significantly upregulated by laser treatment in the dentate gyrus of the hippocampus (DG) and the subventricular zone (SVZ) but not in the perilesional cortex (lesion) at day 7 but not at day 28. Synapsin-1 (a marker for synaptogenesis, the formation of new connections between existing neurons) was significantly upregulated in lesion and SVZ but not DG, at 28 days but not 7 days. The data suggest that the benefit of LLLT to the brain is partly mediated by stimulation of BDNF production, which may in turn encourage synaptogenesis. Moreover the pleiotropic benefits of BDNF in the brain suggest LLLT may have wider applications to neurodegenerative and psychiatric disorders. Neurological Severity Score (NSS) for TBI mice.

Transcranial low-level laser therapy enhances learning, memory, and neuroprogenitor cells after traumatic brain injury in mice.

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 expression was lower in the lesion region at 4 days post-TBI. Double-stained BrdU-NeuN (neuroprogenitor cells) was increased in the dentate gyrus and SVZ. Increases in double-cortin (DCX) and TUJ-1 were also seen. Our study results suggest that tLLLT may improve TBI both by reducing cell death in the lesion and by stimulating neurogenesis.

Transcranial low-level laser therapy improves neurological performance in traumatic brain injury in mice: effect of treatment repetition regimen.

Low-level laser (light) therapy (LLLT) has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI). We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a) sham-TBI sham-treatment, (b) real-TBI sham-treatment, and (c) real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm(2), 18 J/cm(2), spot diameter 1 cm) while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU) at days 21-27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx) had significant improvements in neurological severity score (NSS), and wire-grip and motion test (WGMT). However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups) and less Fluoro-Jade staining for degenerating neurons (at 14 days) than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.

Transcranial low level laser (light) therapy for traumatic brain injury.

We review the use of transcranial low-level laser (light) therapy (LLLT) as a possible treatment for traumatic-brain injury (TBI). The basic mechanisms of LLLT at the cellular and molecular level and its effects on the brain are outlined. Many interacting processes may contribute to the beneficial effects in TBI including neuroprotection, reduction of inflammation and stimulation of neurogenesis. Animal studies and clinical trials of transcranial-LLLT for ischemic stroke are summarized. Several laboratories have shown that LLLT is effective in increasing neurological performance and memory and learning in mouse models of TBI. There have been case report papers that show beneficial effects of transcranial-LLLT in a total of three patients with chronic TBI. Our laboratory has conducted three studies on LLLT and TBI in mice. One looked at pulsed-vs-continuous wave laser-irradiation and found 10 Hz to be superior. The second looked at four different laser-wavelengths (660, 730, 810, and 980 nm); only 660 and 810 nm were effective. The last looked at different treatment repetition regimens (1, 3 and 14-daily laser-treatments).

Low-level laser therapy for closed-head traumatic brain injury in mice: effect of different wavelengths.

BACKGROUND AND OBJECTIVES: Traumatic brain injury (TBI) affects millions worldwide and is without effective treatment. One area that is attracting growing interest is the use of transcranial low-level laser therapy (LLLT) to treat TBI. The fact that near-infrared light can penetrate into the brain would allow non-invasive treatment to be carried out with a low likelihood of treatment-related adverse events. LLLT may treat TBI by increasing respiration in the mitochondria, causing activation of transcription factors, reducing inflammatory mediators and oxidative stress, and inhibiting apoptosis. STUDY DESIGN/MATERIALS AND METHODS: We tested LLLT in a mouse model of closed-head TBI produced by a controlled weight drop onto the skull. Mice received a single treatment with continuous-wave 665, 730, 810, or 980 nm lasers (36 J/cm(2) delivered at 150 mW/cm(2)) 4-hour post-TBI and were followed up by neurological performance testing for 4 weeks. RESULTS: Mice with moderate-to-severe TBI treated with 665 and 810 nm laser (but not with 730 or 980 nm) had a significant improvement in Neurological Severity Score that increased over the course of the follow-up compared to sham-treated controls. Morphometry of brain sections showed a reduction in small deficits in 665 and 810 nm laser treated mouse brains at 28 days. CONCLUSIONS: The effectiveness of 810 nm agrees with previous publications, and together with the effectiveness of 660 nm and non-effectiveness of 730 and 980 nm can be explained by the absorption spectrum of cytochrome oxidase, the candidate mitochondrial chromophore in transcranial LLLT.

Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice.

BACKGROUND AND OBJECTIVE: Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI. STUDY DESIGN/MATERIALS AND METHODS: TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm(2) for 12-minutes giving a fluence of 36-J/cm(2). Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test. RESULTS: The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests. CONCLUSION: The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.