Uri Oron

Infrared Laser Therapy for Ischemic Stroke: A New Treatment Strategy Results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1)

Background and Purpose— The NeuroThera Effectiveness and Safety Trial–1 (NEST-1) study evaluated the safety and preliminary effectiveness of the NeuroThera Laser System in the ability to improve 90-day outcomes in ischemic stroke patients treated within 24 hours from stroke onset. The NeuroThera Laser System therapeutic approach involves use of infrared laser technology and has shown significant and sustained beneficial effects in animal models of ischemic stroke. Methods— This was a prospective, intention-to-treat, multicenter, international, double-blind, trial involving 120 ischemic stroke patients treated, randomized 2:1 ratio, with 79 patients in the active treatment group and 41 in the sham (placebo) control group. Only patients with baseline stroke severity measured by National Institutes of Health Stroke Scale (NIHSS) scores of 7 to 22 were included. Patients who received tissue plasminogen activator were excluded. Outcome measures were the patients’ scores on the NIHSS, modified Rankin Scale (mRS), Barthel Index, and Glasgow Outcome Scale at 90 days after treatment. The primary outcome measure, prospectively identified, was successful treatment, documented by NIHSS. This was defined as a complete recovery at day 90 (NIHSS 0 to 1), or a decrease in NIHSS score of at least 9 points (day 90 versus baseline), and was tested as a binary measure (bNIH). Secondary outcome measures included mRS, Barthel Index, and Glasgow Outcome Scale. Primary statistical analyses were performed with the Cochran-Mantel-Haenszel rank test, stratified by baseline NIHSS score or by time to treatment for the bNIH and mRS. Logistic regression analyses were conducted to confirm the results. Results— Mean time to treatment was >16 hours (median time to treatment 18 hours for active and 17 hours for control). Time to treatment ranged from 2 to 24 hours. More patients (70%) in the active treatment group had successful outcomes than did controls (51%), as measured prospectively on the bNIH (P=0.035 stratified by severity and time to treatment; P=0.048 stratified only by severity). Similarly, more patients (59%) had successful outcomes than did controls (44%) as measured at 90 days as a binary mRS score of 0 to 2 (P=0.034 stratified by severity and time to treatment; P=0.043 stratified only by severity). Also, more patients in the active treatment group had successful outcomes than controls as measured by the change in mean NIHSS score from baseline to 90 days (P=0.021 stratified by time to treatment) and the full mRS (“shift in Rankin”) score (P=0.020 stratified by severity and time to treatment; P=0.026 stratified only by severity). The prevalence odds ratio for bNIH was 1.40 (95% CI, 1.01 to 1.93) and for binary mRS was 1.38 (95% CI, 1.03 to 1.83), controlling for baseline severity. Similar results held for the Barthel Index and Glasgow Outcome Scale. Mortality rates and serious adverse events (SAEs) did not differ significantly (8.9% and 25.3% for active 9.8% and 36.6% for control, respectively, for mortality and SAEs). Conclusion— The NEST-1 study indicates that infrared laser therapy has shown initial safety and effectiveness for the treatment of ischemic stroke in humans when initiated within 24 hours of stroke onset. A larger confirmatory trial to demonstrate safety and effectiveness is warranted.

Low-Level Laser Therapy Applied Transcranially to Rats After Induction of Stroke Significantly Reduces Long-Term Neurological Deficits

Background and Purpose— Low-level laser therapy (LLLT) modulates various biological processes. In the present study, we assessed the hypothesis that LLLT after induction of stroke may have a beneficial effect on ischemic brain tissue. Methods— Two sets of experiments were performed. Stroke was induced in rats by (1) permanent occlusion of the middle cerebral artery through a craniotomy or (2) insertion of a filament. After induction of stroke, a battery of neurological and functional tests (neurological score, adhesive removal) was performed. Four and 24 hours poststroke, a Ga-As diode laser was used transcranially to illuminate the hemisphere contralateral to the stroke at a power density of 7.5 mW/cm2. Results— In both models of stroke, LLLT significantly reduced neurological deficits when applied 24 hours poststroke. Application of the laser at 4 hours poststroke did not affect the neurological outcome of the stroke-induced rats as compared with controls. There was no statistically significant difference in the stroke lesion area between control and laser-irradiated rats. The number of newly formed neuronal cells, assessed by double immunoreactivity to bromodeoxyuridine and tubulin isotype III as well as migrating cells (doublecortin immunoactivity), was significantly elevated in the subventricular zone of the hemisphere ipsilateral to the induction of stroke when treated by LLLT. Conclusions— Our data suggest that a noninvasive intervention of LLLT issued 24 hours after acute stroke may provide a significant functional benefit with an underlying mechanism possibly being induction of neurogenesis.

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rats age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

Skeletal muscle cell activation by low-energy laser irradiation: A role for the MAPK/ERK pathway

Low‐energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum‐starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum‐starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI‐ and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N‐terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3‐sec irradiation induced ERK1/2 phosphorylation, a 12‐sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis α (TNF‐α) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation.

Abstract. The effect of low energy laser (He-Ne) irradiation on bone repair in the cortical part of the tibia of the rat was investigated using biochemical and radioactive labeling methods. A fixed round hole was created in the lateral aspect of the tibia and the newly formed tissue was collected from the gap in the cortical bone. Alkaline phosphatase (ALP) and calcium progressively accumulated at the site of injury, peaking at 9 and 13 days postinjury, respectively. Direct irradiation of the hole injury with He-Ne laser on days 5 and 6 postinjury altered osteoblastic activity at the injured site as reflected by alkaline phosphatase activity. The laser irradiation also caused a significant increase (∼2-fold) in calcium accumulation at the site of injury for 9–18 days postinjury. The rate of calcium deposition, measured by radioactive calcium, was significantly higher (∼2-fold) in the laser-irradiated rats as compared with controls. It is concluded that the process of bone repair in a hole created in the rat tibia is markedly enhanced by direct He-Ne laser irradiation of the injured site at the optimal energy level and time postinjury.

Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts.

Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.

Induction of autologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart

The adult mammalian heart is known to have a very limited regenerative capacity following acute ischemia. In this study we investigated the hypothesis that photobiostimulation of autologous bone‐marrow‐derived mesenchymal stem cells (MSCs) by low‐level laser therapy (LLLT) applied to the bone marrow (BM), may migrate to the infarcted area and thus attenuate the scarring processes following myocardial infarction (MI).

Promotion of Angiogenesis by Low Energy Laser Irradiation

The effect of low energy laser (He-Ne) irradiation (LELI) on the process of angiogenesis in the infarcted rat heart and in the chick chorioallantoic membrane (CAM), as well as the proliferation of endothelial cells in tissue culture, was investigated. Formation of new blood vessels in the infarcted rat heart was monitored by counting proliferating endothelial cells in blood vessels. In the CAM model, defined areas were laser-irradiated or nonirradiated and blood vessel density was recorded in each site in the CAM at various time intervals. Laser irradiation caused a 3.1-fold significant increase in newly formed blood vessels 6 days post infarction, as compared with nonirradiated rats. In the CAM model, a slight inhibition of angiogenesis up to 2 days post irradiation and a significant enhancement of angiogenesis in the laser-irradiated foci as compared with control nonirradiated spots were evident. The LELI caused a 1.8-fold significant increase in the rate of proliferation in endothelial cells in culture over nonirradiated cells. It is concluded that LELI can promote the proliferation of endothelial cells in culture, which may partially explain the augmentation of angiogenesis in the CAM model and in the infarcted heart. These results may have clinical significance by offering therapeutic options to ameliorate angiogenesis in ischemic conditions.

Detailed endocardial mapping accurately predicts the transmural extent of myocardial infarction

OBJECTIVES This study delineates between infarcts varying in transmurality by using endocardial electrophysiologic information obtained during catheter-based mapping. BACKGROUND The degree of infarct transmurality extent has previously been linked to patient prognosis and may have significant impact on therapeutic strategies. Catheter-based endocardial mapping may accurately delineate between infarcts differing in the transmural extent of necrotic tissue. METHODS Electromechanical mapping was performed in 13 dogs four weeks after left anterior descending coronary artery ligation, enabling three-dimensional reconstruction of the left ventricular chamber. A concomitant reduction in bipolar electrogram amplitude (BEA) and local shortening indicated the infarcted region. In addition, impedance, unipolar electrogram amplitude (UEA) and slew rate (SR) were quantified. Subsequently, the hearts were excised, stained with 2,3,5-triphenyltetrazolium chloride and sliced transversely. The mean transmurality of the necrotic tissue in each slice was determined, and infarcts were divided into <30%, 31% to 60% and 61% to 100% transmurality subtypes to be correlated with the corresponding electrical data. RESULTS From the three-dimensional reconstructions, a total of 263 endocardial points were entered for correlation with the degree of transmurality (4.6 +/- 2.4 points from each section). All four indices delineated infarcted tissue. However, BEA (1.9 +/- 0.7 mV, 1.4 +/- 0.7 mV, 0.8 +/- 0.4 mV in the three groups respectively, p < 0.05 between each group) proved superior to SR, which could not differentiate between the second (31% to 60%) and third (61% to 100%) transmurality subgroups, and to UEA and impedance, which could not differentiate between the first (<30%) and second transmurality subgroups. CONCLUSIONS The degree of infarct transmurality extent can be derived from the electrical properties of the endocardium obtained via detailed catheter-based mapping in this animal model.

Near Infrared Transcranial Laser Therapy Applied at Various Modes to Mice following Traumatic Brain Injury Significantly Reduces Long-Term Neurological Deficits

Near-infrared transcranial laser therapy (TLT) has been found to modulate various biological processes including traumatic brain injury (TBI). Following TBI in mice, in this study we assessed the possibility of various near-infrared TLT modes (pulsed versus continuous) in producing a beneficial effect on the long-term neurobehavioral outcome and brain lesions of these mice. TBI was induced by a weight-drop device, and neurobehavioral function was assessed from 1 h to 56 days post-trauma using the Neurological Severity Score (NSS). The extent of recovery is expressed as the difference in NSS (dNSS), the difference between the initial score and that at any other later time point. An 808-nm Ga-Al-As diode laser was employed transcranially 4, 6, or 8 h post-trauma to illuminate the entire cortex of the brain. Mice were divided into several groups of 6-8 mice: one control group that received a sham treatment and experimental groups that received either TLT continuous wave (CW) or pulsed wave (PW) mode transcranially. MRI was taken prior to sacrifice at 56 days post-injury. From 5-28 days post-TBI, the NSS of the laser-treated mice were significantly lower (p<0.05) than those of the non-laser-treated control mice. The percentage of surviving mice that demonstrated full recovery at 56 days post-CHI (NSS=0, as in intact mice) was the highest (63%) in the group that had received TLT in the PW mode at 100 Hz. In addition, magnetic resonance imaging (MRI) analysis demonstrated significantly smaller infarct lesion volumes in laser-treated mice compared to controls. Our data suggest that non-invasive TLT of mice post-TBI provides a significant long-term functional neurological benefit, and that the pulsed laser mode at 100 Hz is the preferred mode for such treatment.