Weijun Xuan

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/cm2 for 12-minutes giving a fluence of 36-J/cm2. 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.

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/cm2, 18 J/cm2, 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.

Low-Level Laser Therapy for Closed-Head Traumatic Brain Injury in Mice: Effect of Different Wavelengths

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.

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

Abstract. 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 (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).

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.

Low-level light therapy aids traumatic brain injury

Traumatic brain injury (TBI) caused by falls, motor vehicle accidents, and violence leads to skull fractures, intracranial hemorrhages, elevated intracranial pressure, and cerebral contusion. Severe and moderate TBI, accidental or inflicted, is a major health and socio-economic problem throughout theworld, especially in children and young adults. Despite promising preclinical data, most therapeutic trials for TBI performed in recent years have not demonstrated any significant improvement in outcomes.1 Because of this disappointing state of affairs, a plethora of experimental therapies that are not based on standard pharmaceutical agents have been investigated,2 including several physical treatments.3 Low-level laser therapy (LLLT), also known as photobiomodulation, is an emerging therapeutic approach in which cells or tissues are exposed to low-levels of red and near-IR light. Its experimental applications have broadened to include serious diseases such as heart attack,4 stroke,5 and spinal cord injury.6 LLLT may have beneficial effects in the acute treatment of TBI by increasing mitochondrial respiration, activating transcription factors, reducing key inflammatory mediators, inhibiting apoptosis (programmed cell death), stimulating angiogenesis, and increasing neurogenesis7 (see Figure 1). We studied the effect of an 810nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. We found that at low fluences (0.3–3Jcm2/ mitochondrial respiration was stimulated, as shown by the increase in adenosine triphosphate (ATP), Ca2C; and mitochondrial membrane potential. This, in turn, generated low amounts of reactive oxygen species (ROS) and nitric oxide (NO) that activated signaling pathways and gene transcription without causing cytotoxicity (see Figure 2). At 10J/cm2; the stimulation of these parameters was reduced because instead of activating mitochondrial respiration, they damaged Figure 1. Possible mechanisms of transcranial low-level laser therapy (LLLT) for traumatic brain injury (TBI). Mitochondrial signaling causes increased neuronal survival; lowered edema, inflammation and excitotoxicity; and increased angiogenesis, neurotrophins, and neural progenitor cells. ROS: Reactive oxygen species. NO: Nitric oxide. NGF: Nerve growth factor. BDNF: Brain-derived neurotrophic factor. NT-3: Neurotrophin-3.

Transcranial low-level light therapy produces neuroprotection, neurogenesis and BDNF after TBI in mice.

We have previously shown that transcranial low level light therapy (LLLT) can ameliorate brain damage in mice subjected to traumatic brain injury and improve neurological function. We used a 810-nm laser and delivered 18 J/cm2 at an irradiance 25 mW/cm2. LLLT was either delivered once at 4 hours after controlled cortical impact TBI, once a day for 3 days, or once a day for 14 days. One and 3 applications of LLLT had beneficial effects on the mice, with 3 being better than 1, but 14 applications had no beneficial effect. We now report immunofluorescence studies in mouse brain sections that offer some explanation for this intriguing finding. Mice were injected with BrdU for 1 week before sacrifice (a marker for proliferating cells) and antibodies to double cortin (DCX-1,a marker of migrating neurons), Tuj-1 ( a marker of neuroprogenitor cells), BDNF (brain derived neurotrophic factor) and synapsin-1 ( a marker for newly formed synaptic connections between existing neurons). We found increased BrdU incorporation indicating proliferating cells in the dentate gyrus of the hippocampus, the subventricular layer of the lateral ventricle, as well as the brain tissue surrounding the cortical lesion. Interestingly these cells were more abundant at 7 days than at 28 days post TBI. Co-labeling of BrdU with Neu-N was performed indicating that the proliferating cells were in fact neuronal in nature. Mice with 3 laser treatments had much more BrdU incorporation than mice with 14. Upregulation of BDNF was seen at 7 days, and increased expression of DCX-1 and Tuj-1 was seen at 28 days in the lesion region, indication that neuroprogenitor cells may have migrated there from sites of neurogenesis. Increased syapsin-1 was seen in the cortex at 28 days indicating that neural plasticity may be stimulated by LLLT. Taken together these data suggest that transcranial LLLT may have applications beyond TBI in areas such as neurodegenerative disease and psychiatric disorders.

In vivo studies of low level laser (light) therapy for traumatic brain injury

Low-level laser (or light) therapy (LLLT) is attracting growing interest to treat both stroke and traumatic brain injury (TBI). The fact that near-infrared light can penetrate into the brain allows non-invasive treatment to be carried out with a low likelihood of treatment-related adverse events. It is proposed that red and NIR light is absorbed by chromophores in the mitochondria of cells leading to changes in gene transcription and upregulation of proteins involved in cell survival, antioxidant production, collagen synthesis, reduction of chronic inflammation and cell migration and proliferation. We developed a mouse model of controlled cortical impact (CCI) TBI and examined the effect of 0, 1, 3, and 14 daily 810-nm CW laser treatments in the CCI model as measured by neurological severity score and wire grip and motion test. 1 laser Tx gave a significant improvement while 3 laser Tx was even better. Surprisingly 14 laser Tx was no better than no treatment. Histological studies at necropsy suggested that the neurodegeneration was reduced at 14 days and that the cortical lesion was repaired by BrdU+ve neural progenitor (stem) cells at 28 days. Transcranial laser therapy is a promising treatment for acute (and chronic TBI) and the lack of side-effects and paucity of alternative treatments encourages early clinical trials.