Stroke

Zh Nevrol Psikhiatr Im S S Korsakova. 2014;114(7):43-48.

Effect of intravenous laser irradiation on some blood biochemical indicators in the acute stage of lacunar infarcts.

[Article in Russian]
Nechipurenko NI1, Anatskaia LN, Matusevich LI, Pashkovskaia ID, Shcherbina NI.

Author information

  • 1Respublikanski? nauchno-prakticheski? tsentr nevrologii i ne?rokhirurgii Ministerstva zdravookhraneniia Respubliki Belarus', Minsk.

Abstract

Objective. To investigate the dynamics of lipid metabolism, C-reactive protein (CRP), lipid peroxidation and antioxidant system in lacunar infarction (LI) in chronic cerebral ischemia.

Material and methods. Two groups of patients were studied. The main group included 31 patients who received intravenous laser irradiation of blood (ILIB) with semiconductor laser (wavelength – 0.67 microns, the power output – 3-2 mW) in addition to standard treatment. Patients of the control group (n=22) received only standard treatment.

Results. A statistically significant decrease in total cholesterol levels to normal values due to the significant reduction of the content of antiatherogenic fraction of cholesterol (high-density lipoprotein and atherogenic low-density lipoprotein cholesterol) was found in the main group after treatment. The reduction in atherogenic cholesterol fractions in both groups was associated with the decrease in apolipoprotein B level. The level of CRP was higher than normal in the main and control groups of patients before and after treatment, which indicated the risk of vascular diseases in patients with LI. After treatment, superoxide dismutase activity returned to normal values. In patients of the main group, the catalase activity increased while the level of reduced glutathione did not change and lipid peroxidation products remained on the high level.

Conclusion. Additional antioxidant therapy is needed for these patients.

 
Rev Neurosci.  2013;24(2):205-26. doi: 10.1515/revneuro-2012-0086.

Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders.

Fitzgerald M1, Hodgetts S, Van Den Heuvel C, Natoli R, Hart NS, Valter K, Harvey AR, Vink R, Provis J, Dunlop SA.
  • 1School of Animal Biology, The University of Western Australia, Crawley, Australia. lindy.fitzgerald@uwa.edu.au

Abstract

Irradiation in the red/near-infrared spectrum (R/NIR, 630-1000 nm) has been used to treat a wide range of clinical conditions, including disorders of the central nervous system (CNS), with several clinical trials currently underway for stroke and macular degeneration. However, R/NIR irradiation therapy (R/NIR-IT) has not been widely adopted in clinical practice for CNS injury or disease for a number of reasons, which include the following. The mechanism/s of action and implications of penetration have not been thoroughly addressed. The large range of treatment intensities, wavelengths and devices that have been assessed make comparisons difficult, and a consensus paradigm for treatment has not yet emerged. Furthermore, the lack of consistent positive outcomes in randomised controlled trials, perhaps due to sub-optimal treatment regimens, has contributed to scepticism. This review provides a balanced précis of outcomes described in the literature regarding treatment modalities and efficacy of R/NIR-IT for injury and disease in the CNS. We have addressed the important issues of specification of treatment parameters, penetration of R/NIR irradiation to CNS tissues and mechanism/s, and provided the necessary detail to demonstrate the potential of R/NIR-IT for the treatment of retinal degeneration, damage to white matter tracts of the CNS, stroke and Parkinson's disease.

Evid Based Complement Alternat Med. 2013; 2013: 594906.

Published online 2013 December 2.

Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats

Chiung-Chyi Shen, 1 , 2 , 3 , 4 Yi-Chin Yang, 1 Ming-Tsang Chiao, 1 Shiuh-Chuan Chan, 5 and Bai-Shuan Liu 6 ,*
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Copyright © 2013 Chiung-Chyi Shen et al.
 
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

This study investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs in neuronal cells. MTT assays indicated no significant difference between the amount of cells with (LS+) and without (LS?) laser treatment (P > 0.05). However, immunofluorescent staining and western blot analysis results indicated a significant increase in the neural stem-cell marker, nestin, following exposure to low-level laser irradiation (P < 0.05). Furthermore, stem cell implantation was applied to treat rats suffering from stroke. At 28 days posttreatment, the motor functions of the rats treated using i-ADSCs (LS+) did not differ greatly from those in the sham group and HE-stained brain tissue samples exhibited near-complete recovery with nearly no brain tissue damage. However, the motor functions of the rats treated using i-ADSCs (LS?) remained somewhat dysfunctional and tissue displayed necrotic scarring and voids. The western blot analysis also revealed significant expression of oligo-2 in the rats treated using i-ADSCs (LS+) as well as in the sham group (P < 0.05). The results demonstrated that low-level laser irradiation exerts a positive effect on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.

1. Introduction

Stroke has become a common disease and has been shown to be associated with the consumption of high amounts of oil and salt. These dietary habits cause blood vessels to narrow and become prone to occlusion, which can lead to stroke. Strokes can be broadly classified into 2 categories: ischemic and hemorrhagic strokes. Differing treatment methods are used according to stroke type and lesion location. Therefore, at stroke onset, computed tomography or magnetic resonance imaging is often used for diagnosis to provide physicians with a basis of treatment [1]. The treatment methods used for the 2 types of stroke are different. (1) Ischemic stroke is caused by thrombosis or blood clots in the brain vessels, which prevent blood flow into the brain tissues. Anticoagulants or antiplatelet medications must be administered to patients as soon as possible. (2) Hemorrhagic stroke is caused by the rupture or hemorrhaging of brain vessels caused by peripheral brain trauma (subarachnoid hemorrhage). The majority of cases of hemorrhagic stroke are caused by brain trauma (e.g., car and workplace accidents). Treating these patients often requires neurosurgical interventions [2]. Ischemic strokes constitute the majority of stroke cases. When patients suffer from ischemic stroke, the brain tissues necrotize gradually because of the lack of nutrients if anticoagulants or antiplatelet medications are not administered within 3 h of the stroke. After necrosis occurs, necrotic brain tissue cannot regenerate or recover its functions even if blood-vessel reperfusion occurs [3]. In recent years, stem cell treatment has become an innovative therapy for brain tissues that cannot regenerate.

Body fat extraction (e.g., abdomen, thigh, or buttock fat) has been used for body shaping procedures in the past, and the extracted fat was discarded as medical waste. Presently, medical experts have confirmed that adipose tissues contain large amounts of mesenchymal stem cells that exhibit in vitro proliferation and multiple differentiation ability, characteristics that facilitate the repair and regeneration of damaged tissues or organs [4]. In addition, adipose-derived stem cells (ADSCs) possess several characteristics: (1) they can be easily harvested in abundant quantities without invasiveness, (2) they can proliferate in in vitro cultures, and (3) they can be applied to a wide range of body tissue types because these cells migrate to lesion sites automatically to repair damage. Studies have shown that ADSCs can differentiate into many different cell types, such as adipose, bone, cartilage, smooth muscle, cardiac muscle, endothelial, blood, liver, and even neuronal cells [5]. Because of these characteristics, ADSCs will likely be one of the major sources of autologous stem cells in the future.

The effectiveness of laser therapy on biological bodies has been confirmed, and laser therapy has been successfully applied in new technological applications such as microsurgery. Lasers can be classified as high or low power, depending on the energy levels used. High-power lasers involve using high energy levels to provoke blood coagulation, stop bleeding, cut tissues, and even damage cells, whereas low-level lasers involve using electromagnetic or photochemical processes to achieve therapeutic effects [6]. A low-level laser is defined as a laser with extremely low power and energy too low to destroy the molecular bonding capacity (e.g., hydrogen bonds and van der Waals forces) of tissues. Therefore, low-level lasers do not cause molecular structural change, protein denaturation, or cell death. Irradiation from low-level lasers on tissue does not cause an obvious temperature increase at the treatment site (less than 0.1 to 0.5°C); thus, any physiological responses of the tissues are produced by the stimulation of the laser itself, and this mechanism is described by the theory of laser biostimulation [7]. In addition, by using an appropriate energy level, low-level lasers are primarily used to stimulate biological cells to induce or strengthen physiological responses for facilitating local blood circulation, regulating cell functions, promoting immunological functions, and facilitating cell metabolism and proliferation. Using lasers to generate these physiological changes enables treatment goals such as anti-inflammation and wound-healing promotion to be met [89]. Many studies have shown that low-level laser irradiation exerts beneficial biological effects on bone, neuronal, and skin healing [1012]. However, the type, wavelength, power, and energy level of lasers used in previous studies have varied, and various effects have been observed in different cells when differing levels of laser energy were applied. Previous studies have shown that using a low-level laser with an 820–830 nm wavelength can reduce neural damage, facilitate neuronal healing, and accelerate neural recovery after an osteotomy [1314]. Using a low-level laser with a 660 nm wavelength has been demonstrated to exert healing effects on musculoskeletal injuries and inflammation [15]. In addition, many studies have indicated that a low-level laser with a 660 nm wavelength can effectively promote neural regeneration and accelerate the reinnervation of muscle fibers to promote the recovery of motor functions [1618]. Recent evidence has suggested that protein aggregates such as ?-amyloid- (A?) associated neurotoxicity and dendrite atrophy might be a consequence of brain-derived neurotrophic-factor (BDNF) deficiency. Meng et al. observed that the upregulation of BDNF caused by using low-level laser therapy (LLLT) to activate the extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) pathway can ameliorate A?-induced neuron loss and dendritic atrophy, thus identifying a novel pathway through which LLLT protects against A?-induced neurotoxicity [19].

Currently, most low-level laser therapies in practical clinical applications emphasize treatment courses covering an extensive tissue area in a relatively short period. Multichannel-laser hair treatment, which is currently available for the physical treatment of alopecia, is one such example. Therefore, we used a large-area LLLT that differs from the irradiation methods previously reported in the literature (such as single-spot low-energy laser exposure or the scanning method) to increase the local area of exposure [18]. In this study, stem cells were extracted from adipose tissues, and neural-stem-cell- (NSC-) differentiating agents were used to culture these ADSCs to transform them into induced adipose-derived stem cells (i-ADSCs), which were used as experimental cells. First, we investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs into neuronal cells. We then investigated whether i-ADSCs treated with laser irradiation and injected via an intravenous route could integrate and survive in various locations in rat brains. We investigated if this treatment could improve the neurological dysfunctions caused by ischemic brain damage in rats and if rats could produce BDNF, using this treatment. Studies using intravenous injection to transplant i-ADSC for the treatment of ischemic stroke with protocols similar to that used in the current study are rare. In this study, we further combined the biostimulation theory from large-area LLLT with transplantation of i-ADSCs to induce neural differentiation to investigate the effectiveness of ischemic stroke treatment. In a previous study [20], we established a transient ischemia-reperfusion stroke rodent model by using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. The effectiveness of the treatment was assessed by comparing HE-staining and western blot analysis, as well as the evaluation of motor skill indices by using the rotarod and grip-strength tests. Our protocol has the potential to be developed for application in the clinical treatment of patients with ischemic stroke.

2. Materials and Methods

2.1. Isolation and Culture of ADSCs

A flowchart illustrating the experimental design of the study is shown in Figure 1. Eight-week-old male Sprague-Dawley (SD) rats were used for isolating rat ADSCs. The ADSCs were harvested from the rats' subcutaneous anterior abdominal wall. Inguinal fat pads were excised, washed sequentially in serial dilutions of betadine, and finely minced as tissues in phosphate-buffered saline (PBS). The tissues were digested with 0.3% of Type I collagenase (Sigma) at 37°C for 60 min. The digested tissue/cell suspension was filtered through a 100-mesh filter to remove the debris, and the filtrate was centrifuged at 1000 rpm for 10 min. The cellular pellet was resuspended using DMEM/F12 (10% FBS, 1% P/S) and then cultured for 24 h at 37°C in 5% CO2. Unattached cells and debris were then removed and the adherent cells were cultured using fresh medium. The cells were cultured to 80% confluency before being released with 0.05% trypsin and then subcultured.

Figure 1

A flowchart illustrating the experimental design. Detailed procedures are described in Section 2.

2.2. ADSC Neuronal Predifferentiation

In this study, i-ADSCs obtained from culturing ADSCs by adding NSC-differentiating agents were used as experimental cells. ADSCs within 3–5 passages were detached and induced using NSC media supplementation. ADSCs were resuspended in a serum-free DMEM/F12 medium supplemented with an N2 supplement (Sigma), 20 ng/mL of epidermal growth factor (Gibco, NY, USA), and fibroblast growth factor (Gibco, NY, USA).

2.3. Setup of a Low-Level Laser Application Method

The probe of the laser irradiation device was fixed vertically on a clean, open experimental bench. The distance between the probe and the cell culture dish was 30 cm. Laser irradiation was applied in a 25°C environment by using an AlGaInP-diode laser (Konftec Co., Taipei, Taiwan) with a wavelength of 660 nm at an output power of 50 mW and frequency of 50 Hz. In the control group, the cells that did not receive laser irradiation treatment, i-ADSCs (LS?) (n = 10), were compared with the experimental cells, i-ADSCs (LS+) (n = 10), which were subjected to a laser irradiation treatment of 10 min. The cells in the i-ADSCs (LS?) group were cultured for 7 days, whereas the cells in the i-ADSCs (LS+) group were treated using low-level lasers on the following day for 10 min and then cultured for 6 days. The cells receiving laser irradiation were collected at various times for analysis according to the purposes of the experimental protocols. After the completion of the cultures, an optical microscope was used for observing cell morphology.

2.4. MTT Assay

The principle of the MTT (3-[4,5-dimethylthiazol-2-y1]-2,4-diphenyltetrazolium bromide) assay is that the mitochondria of living cells can transform the yellow chemical substance MTT tetrazolium into the purple non-water-soluble substance MTT formazan through the effect of succinate dehydrogenase. DMSO can be used to dissolve the purple-colored products. No such response occurs in dead cells. An optical absorbance of 570 nm was measured using an enzyme immunoassay analyzer. A higher absorbance value indicates a larger amount of cells. In this study, ADSCs neuronal predifferentiation was first distributed in a 96-well plate with approximately 10000 cells per well. The cells in the i-ADSCs (LS?) group were then cultured in a 37°C environment and a 5% CO2 environment for 5 days and 7 days, respectively. The cells in the i-ADSCs (LS+) group were cultured in a 37°C environment and a 5% CO2 environment for 4 days and 6 days, respectively. After the completion of the cultures, the medium was removed by several rinses with PBS. An MTT solution of 100 ?L was added to each well of a 96-well plate in the dark (1 mL of MTT reagent was added to 9 mL of phenol-red-free, serum-free medium) and incubated in a 37°C, 5% CO2 environment for 2 h. The MTT solution was then removed and the cells were dissolved using DMSO. An optical absorbance of 570 nm was measured using an enzyme immunoassay analyzer to compare the values between the different groups.

2.5. Immunocytochemistry of i-ADSCs

After 7 days in culture, the subcultured neurospheres were washed using 0.1 M PBS 3 times and fixed with 4% paraformaldehyde for 1 h. Following the fixation, the cells were permeated with 0.1% of Triton X-100 for 10 min and then blocked with 5% nonfat milk for 30 min. The phenotypic expression of these neurospheres was examined by implementing immunocytochemical staining accompanied by antibodies against glial fibrillary acidic proteins (GFAPs) for astrocytes, mouse monoclonal antinestins for NSCs, and doublecortin (DCX), which has recently been used as a marker for neurogenesis. Briefly, the fixed cells were washed 3 times in cold PBS. After washing with PBS, the aforementioned primary antibodies were added and the slides were maintained at room temperature overnight. In the following day, the primary antibodies were removed by washing 3 times with PBS and the secondary antibodies were added before incubating the cells for 1 h. After washing off the secondary antibodies, the cells were incubated with tertiary antibodies tagged with peroxidase-antiperoxidase for 1 h. The tertiary antibodies were washed off using PBS. The cells were incubated with DAPI (Sigma, St. Louis, MO, USA) diluted with the cell culture medium for 10 min. Finally, the cells were mounted with 90% glycerol and examined using fluorescent microscopy (Olympus IX-71, Inc., Trenton, NJ, USA).

2.6. Animals and Induction of the MCAO Model

In a previous study [20], we established a transient ischemia-reperfusion stroke rodent model, using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan. Thirty-two adult male SD rats were randomly allocated to 3 groups: the i-ADSCs (LS?) therapy group (n = 12), the i-ADSCs (LS+) therapy group (n = 12), and a sham group (n = 8). The rats in all 3 groups were euthanized on the 28th day after MCAO was performed. For MCAO procedures, anesthesia was induced using 4% isoflurane (Baxter, USA) and maintained using 2% isoflurane. A midline cervical incision was made to isolate the right bilateral common carotid artery. A 25 mm-long 3-0 nylon surgical thread was then inserted into the right carotid bifurcation. In this study, 2 rounds were used to provide a more complete blockage of blood flow in the artery. When the blunted distal end met resistance, the proximal end of the thread was tightened at the carotid bifurcation. The right common, internal, and external carotid arteries were carefully separated from the adjacent vagal nerve, and the distal portions of the external and common carotid arteries were ligated. A small incision was subsequently made at the proximal portion of the external carotid artery, and a 3-0 nylon monofilament suture was gently inserted (approximately 18 mm) into the internal carotid artery. After 60 min of MCAO, the nylon surgical thread was removed to allow complete reperfusion of the ischemic area. During ischemia, rectal temperature was monitored and maintained at approximately 37°C by using a heating pad and an overhead lamp. The anesthetized rats intravenously received i-ADSCs at a concentration of 2 × 107 mL?1 via their femoral veins. The rats in the sham group underwent the same surgical procedures except that the right-sided middle cerebral artery was not occluded.

2.7. Rotarod Test

An accelerating rotarod test was performed for each rat before and on the 7th, 14th, 21st, and 28th day after cerebral ischemia-reperfusion was induced. Before the ischemia-reperfusion experiment was conducted, the animals were subjected to 3 training sessions per day for 3 days on the accelerating rotarod to obtain stable duration on the rotarod spindle. The diameter of the rotarod spindle was 7 cm. The surface of the rotarod spindle was made of knurled Perspex to provide an adequate grip, which prevented animals from slipping off the spindle. The speed of the spindle was increased from 4 to 40 rpm over a period of 5 min and the duration that the animal stayed on the device was recorded. The rats that were capable of staying on the rotarod longer than 150 s after 3 training sessions were selected for the experiments. On the testing days, the animals were tested twice, and the longest durations on the rotarod were recorded.

2.8. Grip Strength Test

Each rat was supported in a horizontal position approximately parallel to a grip bar (Model DPS-5R: range 0–5 kgf, Japan). The researcher set the rat's forepaws on the grip bar and pulled the animal horizontally away from the bar by the base of its tail until the rat released its grip. The pulling motion was smooth and continuous. The researcher supported the rat by the abdomen when the grip was released. The reading on the strain gauge remained constant at the point of maximal value, which was recorded as the measure of forepaw grip strength. The researcher supported the rat body by both the chest and the base of the tail at an angle of ?45° down the tail. The rat was facing away from the grip bar. The rat was encouraged to grasp the bar by moving its hind paw to the bar. When the rat grasped the bar with both hind paws, establishing a “full” grip, the upper body of the rat was lowered so that the rat was in a nearly horizontal position. The rat was pulled horizontally by the base of the tail until it released its grip and was supported as previously described. The reading on the strain gauge remained constant at the point of maximal value (force was measured in grams), which was recorded as the measurement of forepaw grip strength. Three values were obtained in succession, and the median value was used as the daily score. The data were expressed as the percentage of the baseline (preischemic) value.

2.9. Hematoxylin-Eosin Staining of the Cerebellum

The SD rats were anesthetized using 10% chloral hydrate (4 ?L/kg), administered intraperitoneally, and were euthanized on the 28th day after the MCAO operation and sham treatment. For each rat, the left cerebellum was rapidly removed and postfixed in formalin for 24 h. The postfixed tissues were embedded in paraffin wax and 6-?m-thick serial coronal sections were obtained and mounted on poly-L-lysine-coated glass slices. To assess the histological changes in the MCAO and sham groups, the paraffin-embedded left cerebellum sections were stained using hematoxylin-eosin (HE), according to standard protocol before the assay was performed.

2.10. Western Blot Analysis

Proteins were extracted from the rat brains by using a cold lysis buffer (10 mM of tetra sodium pyrophosphate, 20 mM of Hepes, 1% Triton X-100, 100 mM NaCl, 2 ?g/mL of aprotinin, 2 ?g/mL of leupeptin, and 100 ?g/mL of phenylmethylsulfonyl fluoride). The protein concentrations from tissue extracts or ADSC-conditioned medium were determined using the Bradford protein assay. Equal amounts of protein were placed in a 2× sample buffer (0.125 M Tris-HCl, pH 6.8, 2% glycerol, 0.2 mg/mL of bromophenol blue dye, 2% SDS, and 10% ?-mercaptoethanol) and electrophoresed through 10% SDS-polyacrylamide gel. The proteins were then transferred onto a nitrocellulose membrane by using electroblotting. The membranes were blocked for 1 h at room temperature in a Tris-buffered saline with Tween-20 (TBST) and 5% nonfat milk. The primary antibodies (1 : 1000) with appropriate dilutions were incubated for 1 h at room temperature in TBST and 5% nonfat milk. The blots were then washed and incubated with a peroxidase-conjugated secondary antibody (1 : 2000) for 1 h in TBST. The chemiluminescent substrate for the secondary antibody was developed using the ECL detection system (Amersham, UK). The blots were exposed to film for 3–5 min and then developed.

2.11. Statistical Analysis

The data were expressed as the mean value ± standard error of the mean. The statistical significance of the differences between the groups was determined using a one-way analysis of variance followed by Tukey's test. An alpha level of less than 0.05 (P < 0.05) was considered statistically significant.

3. Results

3.1. Effects of the Low-Level Laser on Cell Morphology

The ADSCs were passaged 3–5 times after the initial plating of the primary culture. Rat ADSCs appeared to be a monolayer of large and flat cells (Figure 2(a)). Many cells in the i-ADSCs (LS?) and i-ADSCs (LS+) groups induced a neuronal phenotype and exhibited, among one another, bipolar and multipolar elongations of neuronally induced cell-forming networks. The results show that the stem cells in both i-ADSCs (LS?) and i-ADSCs (LS+) groups developed tentacles, indicating that ADSCs were facilitating the induction of differentiation into neuronal cells. Comparative optical micrographs revealed that some attached cells exhibited a spread-out shape with a spindle-like and fibroblastic phenotype in the i-ADSCs (LS?) group (Figure 2(b)). However, most of i-ADSCs-expressing neurites extended radially, connecting like bridges with those from adjacent cells in the i-ADSCs (LS+) group (Figure 2(c)).

Figure 2

The morphology of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells after low-level laser irradiation. (a) Undifferentiated ADSCs; (b) i-ADSCs (LS?); (c) i-ADSCs (LS+). The arrow denotes the neuronal-like cells. 

3.2. Effects of the Low-Level Laser on Cell Proliferation and Differentiation

In this study, MTT assays were performed on Day 5 and Day 7 to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell proliferation. After analyzing the optical absorbance values, the results showed that, on Day 5, cell activity was slightly higher in the i-ADSCs (LS+) group compared with that in the i-ADSCs (LS?) group. However, the difference was not statistically significant (P > 0.05). On Day 7, the cell amounts in both the i-ADSCs (LS+) and i-ADSCs (LS?) groups were larger than the amounts on Day 5. However, the cell proliferation rates were similar on Day 5 without major differences (P > 0.05) (Figure 3).

Figure 3

The cell activity of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells in both the i-ADSCs (LS?) and i-ADSCs (LS+) groups on Days 5 and 7 after culture.

In this study, immunofluorescent staining and western blots were used to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell differentiation. Immunofluorescent staining was performed for the NSC marker, nestin, glial cell marker, GFAP antibody, and neuronal precursor-cell-marker protein, DCX. After the staining was completed, fluorescent microscopy was used to observe the amount of fluorescence expression of each antibody. The results showed that the fluorescence expression of the nestin was higher in the cells in the i-ADSCs (LS+) group than that in the cells in the i-ADSCs (LS?) group. These results indicated that ADSC differentiation into neuronal cells was facilitated after large-area low-level laser irradiation (Figure 4(a)). For GFAP, no difference was observed in the amount of fluorescence expression of GFAP between the i-ADSCs (LS?) and i-ADSCs (LS+) groups (Figure 4(b)). Furthermore, for DCX, no difference was observed between the 2 groups because the cells in both groups still exhibited stem cell morphology (Figure 4(c)).

Figure 4

The immunofluorescent staining for (a) nestin; (b) GFAP; and (c) DCX of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells in both the i-ADSCs (LS?) (left) and i-ADSCs (LS+) (right) groups. The scale bar represents 

Western blot is used to quantify the expression of marker proteins. Therefore, western blot analysis was used to compare the amount of nestin expression between the i-ADSCs (LS?) and i-ADSCs (LS+) groups in this study. The results showed that cells in the i-ADSCs (LS+) group exhibited a substantially higher nestin expression compared with the cells in the i-ADSCs (LS?) group (P < 0.05) (Figure 5). Regarding the results of GFAP and DCX, no difference was observed in the fluorescence expressions of the i-ADSCs (LS?) and i-ADSCs (LS+) groups. Therefore, western blot analyses were not shown for GFAP and DCX. This result is consistent with the findings obtained using immunofluorescent staining.

Figure 5

The cell growth of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells with or without laser irradiation. (a) The amount of nestin expression. (b) The figure showing the quantification. GAPDH served as the internal reference. 

3.3. Evaluation of Behavior Recovery after Stroke in the Animals

In this study, treadmill and forepaw-grip tests were used to evaluate motor function recovery after stem-cell transplantation treatment in rats with ischemic stroke. The treadmill test was performed on Day 7 after stem-cell transplantation was performed on the rats with stroke. The rats from either the i-ADSCs (LS+) group or the i-ADSCs (LS?) group were unable to run as quickly as the rats in the sham group. From Day 14, the rats in the i-ADSCs (LS+) group gradually recovered the ability to run. By contrast, it was observed that the rats in the i-ADSCs (LS?) group recovered slightly; however, the degree of recovery was lower than that in the i-ADSCs (LS+) group. On Day 21 after stem-cell transplantation, the recovery of running function was still more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the motor function of the rats in the i-ADSCs (LS+) group was approaching the level of the rats in the sham group, whereas the performance of the rats in the i-ADSCs (LS?) group was still considerably weaker than that of the sham group (Figure 6).

Figure 6

The treadmill test for evaluating the recovery of the motor function of running in rats with ischemic stroke with i-ADSCs (LS?) and i-ADSCs (LS+) transplantation.

Grip-strength tests were performed on Day 7 after stem-cell transplantation. The findings were similar to the treadmill test results; the grip behavior of the rats in both the i-ADSCs (LS+) group and the i-ADSCs (LS?) group was worse than that of the rats in the sham group. On Day 14, the grip strength of the rats in the i-ADSCs (LS+) group was considerably recovered. By contrast, although the rats in the i-ADSCs (LS?) group showed some progress, the improvement was minimal. On Day 21, the rats in the i-ADSCs (LS+) group continued to recover, whereas it was observed that the animals in the i-ADSCs (LS?) group were not recovering as quickly. On Day 28, grip strength recovery in the i-ADSCs (LS+) group approached that of the sham group, whereas grip strength in the i-ADSCs (LS?) group was still lower (Figure 7).

Figure 7

The grip test for evaluating the recovery of grip strength in rats with ischemic stroke treated with i-ADSCs (LS?) and i-ADSCs (LS+) transplantation.

3.4. Repair of Brain Tissues after Treatment in Animals

After euthanizing the rats, the brain specimens were treated with paraffin and then sliced. The HE-immunostaining method was used to observe the repair of brain tissues after stem-cell transplantation was performed. An upright microscope (10x) was used to macroscopically observe the brain tissue, and the results showed that brain tissue was completely repaired in the rats in the i-ADSCs (LS+) group with nearly no necrotic brain tissue. By contrast, obvious necrotic scars were observed at the ischemic sites of the brain tissue from the rats in the i-ADSCs (LS?) group (Figure 8(a)). Observed under a microscope and magnified 200 times, the results showed that the brain tissue from the rats in the i-ADSCs (LS+) group was as dense as normal brain tissues, whereas numerous cavities were observed in the brain tissue from rats in the i-ADSCs (LS?) group (Figure 8(b)).

Figure 8

The observation of brain tissue necrosis in rats with ischemic stroke treated with i-ADSCs (LS?) (left) and i-ADSCs (LS+) (right) transplantation: (a) 10x; (b) 200x.

Oligodendrocytes, which are glial cells found in normal brains, form myelin in the central nervous system. Oligodendrocytes substantially decrease after brain cells are damaged, leading to myelin collapse and the loss of neural-signal conduction. Therefore, a western blot was used to analyze the amount of expression of the oligodendrocyte cell protein, oligo-2, to confirm the repair of brain tissues after stem-cell transplantation was performed in rats with stroke. The results showed that the amount of oligo-2 protein response in the brain tissues of the i-ADSCs (LS+) group was as high as that in the sham group. By contrast, the oligo-2 protein response in the i-ADSCs (LS?) group was substantially lower (P < 0.05). These results indicated that stem-cell transplantation treatments can repair brain tissues damaged by ischemia (Figure 9).

Figure 9

The observation of brain tissue repair in rats with ischemic stroke treated with i-ADSCs (LS?) and i-ADSCs (LS+) transplantation. (a) The amount oligo-2 expression. (b) Quantification. GAPDH served as the internal reference. *Significance (P < 

 

4. Discussion

Stem cells possess the ability to proliferate, regenerate, differentiate, and secrete cytokines. Previous studies have proven that stem-cell therapy substantially improves the damage caused by stroke. Stem cells are derived from various sites. Most previous studies have used bone marrow mesenchymal stem cells; however, we used adipose stem cells in the present study, which are easily accessible, and abundant and exhibit high differentiation and proliferation activity. Moreover, adipose stem cells do not trigger strong immune reactions (resulting in low exclusion) and rarely form teratomas. In the literature, it has been demonstrated that adipose stem cells are essential adult stem cells that differentiate into various mesoderm tissues similarly to bone marrow mesenchymal stem cells [2123]. Furthermore, adipose stem cells are more easily accessible than mesenchymal stem cells; therefore, adipose stem cells can be used as a substitute for bone marrow mesenchymal stem cells for repairing damaged tissues in the future. Adipose stem cells are generally more practical than bone marrow mesenchymal stem cells for use in research [2326].

In this study, we used large-area low-level laser irradiation to induce ADSCs to differentiate into neuronal cells. The MTT assay analysis showed that the cell activity of the i-ADSCs increased on Days 5 and 7 of culture after large-area low-level laser irradiation [2728]. However, although cell activity increased on both Days 5 and 7, the activity was also increased for the group that did not receive laser irradiation treatment. No significant difference was observed between the groups with or without laser treatment (P > 0.05). Previous studies have shown that, depending on irradiation parameters, various types of cell respond differently to laser irradiation [29]. Although the mechanism underlying this phenomenon remains obscure, several hypotheses have been proposed to explain the mechanism of laser action [30]. We speculate that the lack of significant effects might be attributable to the short duration of low-level laser irradiation, which might not provide sufficient energy to the ADSCs for them to facilitate proliferation. Alternatively, using low-level lasers on the ADSCs might not sufficiently enhance cell proliferation. Therefore, future studies should investigate the appropriate duration of low-level laser irradiation and determine how much energy is required to accelerate cell proliferation [31,32].

To understand if using large-area low-level lasers exerted a positive effect on cell differentiation, we used immunofluorescent staining and western blot analysis to evaluate whether these lasers were capable of accelerating the induction of cell differentiation [33]. The immunofluorescent staining results showed that the nestin level in the group with i-ADSCs treated using large-area low-level laser irradiation increased substantially compared with that of the group that did not receive laser treatment. This result indicated that large-area low-level laser can accelerate the differentiation of ADSCs into neuronal cells [3435]. LLLT has been demonstrated to regulate neuronal function both in vitro and in vivo. Previous studies have reported that laser treatments accelerated nerve cell sprouting and cell migration, which begin within 24 h of seeding. During the first week of cultivation, irradiated cultures contain a high number of neurons exhibiting large perikaria and branched neuronal fibers, which interconnect to form networks [36]. The possible mechanism of LLLT at the cellular level has been attributed to the acceleration of electron transfer reactions, resulting in the increase of reactive oxygen species and Ca2+as versatile second messengers [37]. Previous studies have shown that applying LLLT could influence cellular processes by altering DNA synthesis and protein expression [38], biomodulating cytoskeletal organization [39], and stimulating cellular proliferation [38]. Such properties suggest that LLLT, or interventions with similar neurobiological effects, can be used to treat neurodegeneration, a phenomenon that underlies debilitating clinical conditions.

However, no obvious differences were observed for GFAP and DCX in the test results. This can be explained by the differentiation agent used in this study, which exerts its effects primarily by inducing ADSCs to differentiate into NSCs. Therefore, although the ADSCs were treated using a low-level laser, they still did not differentiate into neural glia cells. The DCX protein can only be discovered after neurons have been formed from NSCs, which demonstrates that DCX is a late-stage protein that cannot be expressed when cells still exhibit the morphology of NSCs. Therefore, the amount of DCX expression is not affected by low-level laser exposure. Western blot analysis was used to determine the amount of nestin expression after i-ADSCs were exposed to large-area low-level laser irradiation and culture for 7 days. The results showed that the amount of nestin was higher with laser treatment. This is similar to the findings from the immunofluorescent-staining method, indicating that laser irradiation can accelerate the differentiation of ADSCs into neuronal cells. These results indicated that ADSCs can be induced to differentiate into neuronal cells after treatment by large-area laser irradiation for 10 min. Future studies should establish the precise duration of large-area LLLT required to achieve improved results.

In this study, treadmill and forepaws grip tests were used to evaluate motor function recovery after stem cell transplantation treatments in rats with stroke. The treadmill test results showed that the running function was weaker for the rats in the sham group on Day 7 after treatment. We speculate that this might have been because the brain-tissue lesion area from ischemia was too large; therefore, the transplanted stem cells did not have sufficient time to noticeably repair brain tissue. Therefore, the motor functions of the rats remained impaired on Day 7. When tested on Day 14, recoveries in motor functions were observed in both the i-ADSCs (LS?) and i-ADSCs (LS+) groups, with superior recovery in the i-ADSCs (LS+) group, indicating that the damaged brain tissue was repaired. On Day 21, the recovery of the rats' running function was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group, indicating that the brain tissue repair capability was superior to that in the i-ADSCs (LS+) group. When tested on Day 28, the running function of the rats in the i-ADSCs (LS+) group was close to that of the rats in the sham group. However, the motor function of the i-ADSCs (LS?) group remained impaired, indicating that hind-paw motor function recovery was accelerated after i-ADSCs (LS+) treatment.

The grip test results were similar to the treadmill test results. On Day 7 after stem-cell transplantation treatment of the rats with stroke, grip strength was low for both the i-ADSCs (LS+) group and i-ADSCs (LS?) groups because the damaged brain tissues were just about to be repaired; therefore, the forepaws of the rats remained weak. When tested on Day 14, no major improvement was observed in the grip strength of the rats in the i-ADSCs (LS?) group. By contrast, the grip strength improved considerably in the i-ADSCs (LS+) group, indicating that the brain tissue repair capability was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Days 21 and 28, the tests showed that grip strength recovery was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the grip strength in the i-ADSCs (LS+) group recovered to a level close to that of the sham group, indicating that, after i-ADSCs (LS+) treatment, the damaged brain tissues of the rats with stroke were repaired quickly, enabling the recovery of forepaw-grip strength. Based on these results, we concluded that damaged brain tissues can be repaired faster and motor function can be recovered efficiently in rats with stroke after i-ADSCs (LS+) treatment.

The i-ADSCs could differentiate into neuronal cells after transplantation into the brain. As a result, they moved and repaired damaged cerebral tissue selectively and improved cerebral functions by enhancing angiogenesis, renewal of neurons, and proliferation of nerve cells [4041]. In this study, we used the HE-immunostaining method and western blot analysis to evaluate brain tissue repair on Day 28 after stem cell transplantation treatment was performed in rats with stroke. The results of immunostaining were observed using a microscope. The structures were magnified 200 times, which showed that the brain tissue in the stroke lesion was dense and similar to that of normal brain tissue in the i-ADSCs (LS+) group. By contrast, numerous cavities were observed in the ischemic lesions of the brain tissues of the rats in the i-ADSCs (LS?) group. These results indicated that necrotizing brain tissue after ischemia was quickly repaired when i-ADSCs (LS+) was used to treat the rats with stroke. We speculate that using i-ADSCs (LS+) treatment can accelerate the induction of ADSCs to differentiate into neuronal cells. Therefore, although the same number of stem cells was transplanted for treatment, a greater number of NSCs were observed in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. Furthermore, because NSCs can protect damaged brain tissues from continuous deterioration, they can also help brain tissues to repair. Therefore, transplanting a greater amount of NSCs would likely assist in repairing brain tissues more effectively.

Regarding the results of the western blot analysis, the oligo-2 amounts after stem-cell transplantation treatment were analyzed. Because oligodendrocytes form myelin in the central nervous system, myelin levels collapse when oligodendrocytes die. Nerve conduction is delayed or interrupted after the death of oligodendrocytes, leading to limb disabilities. Therefore, the evaluation of oligo-2 could be a crucial reference in assessing the degree of brain tissue recovery after stem-cell transplantation treatments in rats with stroke. In this study, using western blot analysis showed that the amount of expression of oligo-2 in brain tissues treated with i-ADSCs (LS+) was similar to that in normal brain tissues. By contrast, brain tissues treated with i-ADSCs (LS?) exhibited a lower oligo-2 expression, indicating that using i-ADSCs (LS+) treatment in rats with stroke can repair myelin in the central nervous system, leading to the recovery of neural-signal conduction and motor function. Based on these experimental results, we concluded that using i-ADSCs (LS+) treatment in rats with stroke cannot only accelerate the repair of damaged brain tissues for the partial recovery of motor functions, but also enable the central nervous system to recover the velocity of neural-signal conduction. These results confirm that the transplantation of i-ADSCs (LS+) can accelerate repairs in rats with ischemic stroke because i-ADSCs (LS+) can more efficiently differentiate into NSCs.

5. Conclusion

In this study, we used i-ADSCs treated with large-area low-level laser irradiation to evaluate the effects of a low-level laser on cell proliferation and differentiation. The results showed that although a low-level laser cannot facilitate cell proliferation, it can accelerate the induction of ADSCs differentiating into NSCs. In this study, we successfully created large-area cell and tissue damage in rat brains by using an embolic stroke animal model. Stem-cell transplantation with either i-ADSCs (LS+) or i-ADSCs (LS?) was performed to evaluate the degree of repair after stroke in the animals. Because large-area low-level laser irradiation can accelerate the differentiation of ADSCs into NSCs, and NSCs can protect damaged brain tissues to prevent continuous deterioration from damage and to help with repair, the motor function recovery was thus superior in the rats treated using i-ADSCs (LS+) compared with that in the rats treated using i-ADSCs (LS?). From the brain tissue slices from each group of rats, we discovered that i-ADSCs (LS+) treatment more effectively repaired necrotizing brain tissues after ischemia in rat brains. Furthermore, the western blot analysis also showed that the amount of oligo-2 increased in i-ADSCs (LS+)-treated rats with stroke, confirming the repair of myelin in cerebral neurons to further assist in the recovery of neural-signal conduction in the central nervous system.

Therefore, in the present study we demonstrated that using large-area low-level lasers exerts positive effects on inducing ADSCs differentiation, and it effectively treated ischemic stroke in rats, regarding motor function recovery. In future studies, the effects of large-area low-level laser irradiation time and the appropriate dosage for the proliferation and differentiation of ADSCs should be evaluated. If the optimal irradiation time and dosage for ADSC proliferation and differentiation can be discovered in animal experiments similar to those in this study, we believe that superior experimental results can be obtained. Furthermore, if primate or canine experimental animals can be used to conduct the experimental protocols described herein, the concerns associated with individual animal differences and errors associated with motor function assessments can be minimized to obtain more reliable experimental data. Therefore, the findings of this study contribute to the development of cell therapy, which can benefit patients with stroke.

Conflict of Interests

There is no conflict of interests.

Acknowledgments

This work was supported by grants from the Taichung Veterans General Hospital and Central Taiwan University of Science and Technology (Grant. TCVGH-CTUST 1027701) and the National Science Council (Grant. NSC 102-2314-B-075A-019-MY2), Taiwan.

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Brain Res. 2010 Jan 8;1306:100-5. Epub 2009 Oct 23.

Transcranial near infrared laser treatment (NILT) increases cortical adenosine-5'-triphosphate (ATP) content following embolic strokes in rabbits.

Lapchak PA, De Taboada L.

University of California San Diego, Department of Neuroscience, 9500 Gilman Drive MTF316, La Jolla, CA 92093-0624, USA. plapchak@ucsd.edu

Erratum in:

  • Brain Res. 2010 Mar 19;1321:182.

Abstract

Transcranial near infrared laser therapy (NILT) improves behavioral outcome following embolic strokes in embolized rabbits and clinical rating scores in acute ischemic stroke (AIS) patients; however, the cellular mechanism(s) involved in NILT neuroprotection have not been elucidated. It has been proposed that mitochondrial energy production may underlie a response to NILT, but this has not been demonstrated using an in vivo embolic stroke model. Thus, we evaluated the effect of NILT on cortical ATP content using the rabbit small clot embolic stroke model (RSCEM), the model originally used to demonstrate NILT efficacy and initiate the NEST-1 clinical trial. Five minutes following embolization, rabbits were exposed to 2 min of NILT using an 808 nm laser source, which was driven to output either continuous wave (CW), or pulsed wave modes (PW). Three hours after embolization, the cerebral cortex was excised and processed for the measurement of ATP content using a standard luciferin-luciferase assay. NILT-treated rabbits were directly compared to sham-treated embolized rabbits and naïve control rabbits. Embolization decreased cortical ATP content in ischemic cortex by 45% compared to naive rabbits, a decrease that was attenuated by CW NILT which resulted in a 41% increase in cortical ATP content compared to the sham embolized group (p>0.05). The absolute increase in ATP content was 22.5% compared to naive rabbits. Following PW NILT, which delivered 5 (PW1) and 35 (PW2) times more energy than CW, we measured a 157% (PW1 p=0.0032) and 221% (PW2 p=0.0001) increase in cortical ATP content, respectively, compared to the sham embolized group. That represented a 41% and 77% increase in ATP content compared to naive control rabbits. This is the first demonstration that embolization can decrease ATP content in rabbit cortex and that NILT significantly increases cortical ATP content in embolized rabbits, an effect that is correlated with cortical fluence and the mode of NILT delivery. The data provide new insight into the molecular mechanisms associated with clinical improvement following NILT.

Stroke. 2009 Published online before print February 20, 2009, doi: 10.1161/STROKEAHA.109.547547

Submitted on January 12, 2009
Revised on January 26, 2009
Accepted on January 27, 2009

Effectiveness and Safety of Transcranial Laser Therapy for Acute Ischemic Stroke

Justin A. Zivin MD, PhD*; Gregory W. Albers MD; Natan Bornstein MD; Thomas Chippendale MD, PhD; Bjorn Dahlof MD, PhD; Thomas Devlin MD, PhD; Marc Fisher MD; Werner Hacke MD, PhD; William Holt DO; Sanja Ilic MD; Scott Kasner MD; Robert Lew PhD; Marshall Nash MD; Julio Perez MD; Marilyn Rymer MD; Peter Schellinger MD, PhD; Dietmar Schneider MD; Stefan Schwab MD; Roland Veltkamp MD; Michael Walker PhD; Jackson Streeter MD; for the NEST-2 Investigators

From the Department of Neurosciences (J.Z.), University of California San Diego, La Jolla, Calif; Stanford Stroke Center (G.A.), Stanford University Medical Center, Palo Alto, Calif; Tel Aviv Medical Center (N.B.), Tel Aviv, Israel; Scripps Hospital (T.C.), Encinitas, Calif; Sahlgrenska University Hospital (B.D.), Gothenburg, Sweden; Erlanger Health System (T.D.), Chattanooga, Tenn; University of Massachusetts Medical School (M.F.), Worcester, Mass; Department of Neurology (W.H.), Universität Heidelberg, Heidelberg, Germany; Fawcett Memorial Hospital (W.A.H.), Port Charlotte, Fla; Triage Wireless, Inc (S.I.), San Diego, Calif; the Department of Neurology (S.E.K.), University of Pennsylvania School of Medicine, Philadelphia, Pa; Boston University (R.L.), Boston, Mass; DeKalb Neurology Associates (M.N.), Decatur, Ga; Hospital Nacional Dos de Mayo (J.P.), Lima, Peru; St. Luke’s Health System (M.R.), Kansas City, Mo; Universitätsklinikum Erlangen (P.S.), Erlangen, Germany; the Department of Neurology (D.S.), Universität Leipzig, Leipzig, Germany; Universitätsklinikum Erlangen (S.S.), Erlangen, Germany; Department of Neurology (R.V.), Universität Heidelberg, Heidelberg, Germany; Stanford Center for Biomedical Informatics Research (M.W.), Stanford School of Medicine, Palo Alto, Calif; and PhotoThera, Inc (J.S.), Carlsbad, Calif.

* To whom correspondence should be addressed. E-mail: jzivin@ucsd.edu.

Background and Purpose—We hypothesized that transcraniallaser therapy (TLT) can use near-infrared laser technology totreat acute ischemic stroke. The NeuroThera Effectiveness andSafety Trial–2 (NEST-2) tested the safety and efficacyof TLT in acute ischemic stroke.

Methods—This double-blind,randomized study compared TLT treatment to sham control. Patientsreceiving tissue plasminogen activator and patients with evidenceof hemorrhagic infarct were excluded. The primary efficacy endpoint was a favorable 90-day score of 0 to 2 assessed by themodified Rankin Scale. Other 90-day end points included theoverall shift in modified Rankin Scale and assessments of changein the National Institutes of Health Stroke Scale score.

Results—Werandomized 660 patients: 331 received TLT and 327 received sham;120 (36.3%) in the TLT group achieved favorable outcome versus101 (30.9%), in the sham group (P=0.094), odds ratio 1.38 (95%CI, 0.95 to 2.00). Comparable results were seen for the otheroutcome measures. Although no prespecified test achieved significance,a post hoc analysis of patients with a baseline National Institutesof Health Stroke Scale score of <16 showed a favorable outcomeat 90 days on the primary end point (P<0.044). Mortalityrates and serious adverse events did not differ between groupswith 17.5% and 17.4% mortality, 37.8% and 41.8% serious adverseevents for TLT and sham, respectively.

Conclusions—TLTwithin 24 hours from stroke onset demonstrated safety but didnot meet formal statistical significance for efficacy. However,all predefined analyses showed a favorable trend, consistentwith the previous clinical trial (NEST-1). Both studies indicatethat mortality and adverse event rates were not adversely affectedby TLT. A definitive trial with refined baseline National Institutesof Health Stroke Scale exclusion criteria is planned.

J Photochem Photobiol B.J Photochem Photobiol B. 2009 Dec 2;97(3):145-51. Epub 2009 Sep 11.

Effect of phototherapy with low intensity laser on local and systemic immodulation following focal brain damage in rat.

Moreira MS, Velasco IT, Ferreira LS, Ariga SK, Barbeiro DF, Meneguzzo DT, Abatepaulo F, Marques MM.LIM-51, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

Brain injury is responsible for significant morbidity and mortality in trauma patients, but controversy still exists over therapeutic management for these patients. The objective of this study was to analyze the effect of phototherapy with low intensity lasers on local and systemic immunomodulation following cryogenic brain injury. Laser phototherapy was applied (or not-controls) immediately after cryogenic brain injury performed in 51 adult male Wistar rats. The animals were irradiated twice (3 h interval), with continuous diode laser (gallium-aluminum-arsenide (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP), 660 nm) in two points and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). The experimental groups were: Control (non-irradiated), RL3 (visible red laser/ 3 J/cm(2)), RL5 (visible red laser/5 J/cm(2)), IRL3 (infrared laser/3 J/cm(2)), IRL5 (infrared laser/5 J/cm(2)). The production of interleukin-1IL-1beta (IL-1beta), interleukin6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) was analyzed by enzyme immunoassay technique (ELISA) test in brain and blood samples. The IL-1beta concentration in brain of the control group was significantly reduced in 24 h (p<0.01). This reduction was also observed in the RL5 and IRL3 groups. The TNF-alpha and IL-6 concentrations increased significantly (p<0.01 and p<0.05, respectively) in the blood of all groups, except by the IRL3 group. The IL-6 levels in RL3 group were significantly smaller than in control group in both experimental times. IL-10 concentration was maintained stable in all groups in brain and blood. Under the conditions of this study, it is possible to conclude that the laser phototherapy can affect TNF-alpha, IL-1beta and IL-6 levels in the brain and in circulation in the first 24 h following cryogenic brain injury.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Nov-Dec;(6):3-11.

 

Many-level polysensory stimulation of brain functions by physical therapeutic agents.

[Article in Russian]

 

Tyshkevich TG, Ponomarenko GN.

A combination of physiotherapeutic methods for neurorehabilitative treatment has been developed and applied to the treatment of 576 patients with neurosurgical problems including the loss of brain functions as a sequel to nervous system lesions of traumatic, vascular, and other origin. Methodologically, this complex is adapted to the level and extent of the lesion and the character of regeneration of the nervous tissues. It implies many-level stimulation of neuroregeneration by syndromically and pathogenetically substantiated application of physical factors in the early post-injury and postoperative periods. The proposed approach allows the brain function to be completely restored by virtue of persistent compensatory changes in the nervous system. A combination of many-level magnetic, electrical, and laser stimulation is recommended to manage lesions in the speech, motor, and visual analyzers. Combined laser and differential electrostimulation may be prescribed to patients with nerve lesions, extremely high frequency therapy to those with epileptic syndrome, combined microwave therapy to cases with impairment of consciousness, and a variant of systemic UV irradiation with underwater shower-massaging for the treatment of vegetative and asthenic disturbances. Selected physiological aspects of the action of the above physical factors are specified. This physiotherapeutic system is protected by 20 RF patents of invention.

Stroke. 2008 Nov;39(11):3073-8. Epub 2008 Aug 7.

Safety profile of transcranial near-infrared laser therapy administered in combination with thrombolytic therapy to embolized rabbits.

Lapchak PA, Han MK, Salgado KF, Streeter J, Zivin JA.

Department of Neuroscience, University of California San Diego, La Jolla, CA 92093-0624, USA. plapchak@ucsd.edu

Abstract

BACKGROUND AND PURPOSE: Transcranial near-infrared laser therapy (TLT) is currently under investigation in a pivotal clinical trial that excludes thrombolytic therapy. To determine if combining tissue plasminogen activator (tPA; Alteplase) and TLT is safe, this study assessed the safety profile of TLT administered alone and in combination with Alteplase. The purpose for this study is to determine if the combination of TLT and thrombolysis should be investigated further in a human clinical trial.

METHODS: We determined whether postembolization treatment with TLT in the absence or presence of tPA would affect measures of hemorrhage or survival after large clot embolism-induced strokes in New Zealand white rabbits.

RESULTS: TLT did not significantly alter hemorrhage incidence after embolization, but there was a trend for a modest reduction of hemorrhage volume (by 65%) in the TLT-treated group compared with controls. Intravenous administration of tPA, using an optimized dosing regimen, significantly increased hemorrhage incidence by 160%. The tPA-induced increase in hemorrhage incidence was not significantly affected by TLT, although there was a 30% decrease in hemorrhage incidence in combination-treated rabbits. There was no effect of TLT on hemorrhage volume measured in tPA-treated rabbits and no effect of any treatment on 24-hour survival rate.

CONCLUSIONS: In the embolism model, TLT administration did not affect the tPA-induced increase in hemorrhage incidence. TLT may be administered safely either alone or in combination with tPA because neither treatment affected hemorrhage incidence or volume. Our results support the study of TLT in combination with Alteplase in patients with stroke.

Int J Stroke. 2008 May;3(2):88-91.

Laser therapy in acute stroke treatment.

Yip S, Zivin J.

Department of Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0624, USA.

Abstract

Recent development of near infrared light therapy (NILT) as an acute stroke treatment is promising. In various preclinical animal stroke models, NILT has been shown to be effective in improving long-term stroke outcome. More importantly, NILT has a long postischemic therapeutic window that has not been previously observed in other treatment modalities. The preliminary efficacy and safety of NILT in acute stroke patients were demonstrated in the recently published phase II NeuroThera Effectiveness and Safety Trial (NEST-1). If confirmed by the NEST-II trial, NILT will revolutionize acute stroke management as ut has a long time window (possible 24 hr) for therapy. Moreover, understanding the mechanisms of action of NILT will provide a new therapeutic target for future drug or device development.

Neuroscience. 2007 Sep 21;148(4):907-14. Epub 2007 Jul 12

Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: an extended therapeutic window study using continuous and pulse frequency delivery modes.

Lapchak PA, Salgado KF, Chao CH, Zivin JA.

University of California San Diego, Department of Neuroscience, MTF 316, 9500 Gilman Drive, La Jolla, CA 92093-0624, USA. plapchak@ucsd.edu

Photon or near-infrared light therapy (NILT) may be an effective neuroprotective method to reduce behavioral dysfunction following an acute ischemic stroke. We evaluated the effects of continuous wave (CW) or pulse wave (P) NILT administered transcranially either 6 or 12 h following embolization, on behavioral outcome. For the studies, we used the rabbit small clot embolic stroke model (RSCEM) using three different treatment regimens: 1) CW power density of 7.5 mW/cm(2); 2) P1 using a frequency of 300 mus pulse at 1 kHz or 3) P2 using a frequency of 2 ms pulse at 100 Hz. Behavioral analysis was conducted 48 h after embolization, allowing for the determination of the effective stroke dose (P(50)) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the P(50) compared with the control group. Quantal dose-response analysis showed that the control group P(50) value was 1.01+/-0.25 mg (n=31). NILT initiated 6 h following embolization resulted in the following P(50) values: (CW) 2.06+/-0.59 mg (n=29, P=0.099); (P1) 1.89+/-0.29 mg (n=25, P=0.0248) and (P2) 1.92+/-0.15 mg (n=33, P=0.0024). NILT started 12 h following embolization resulted in the following P(50) values: (CW) 2.89+/-1.76 mg (n=29, P=0.279); (P1) 2.40+/-0.99 mg (n=24, P=0.134). At the 6-h post-embolization treatment time, there was a statistically significant increase in P(50) values compared with control for both pulse P1 and P2 modes, but not the CW mode. At the 12-h post-embolization treatment time, neither the CW nor the P1 regimens resulted in statistically significant effect, although there was a trend for an improvement. The results show that P mode NILT can result in significant clinical improvement when administered 6 h following embolic strokes in rabbits and should be considered for clinical development.

Expert Rev Neurother.2007 Aug;7(8):961-5.

Laser treatment for stroke.

Lampl Y.

Edith Wolfson Medical Center, Department of Neurology, Holon, Israel. y_lampl@hotmail.com

Low-level laser therapy is an irradiation technique that has the ability to induce biological processes using photon energy. There are studies showing proliferation and angiogenesis after irradiation in skeletal muscle post-myocardial infarction tissue cells. Most evidence of efficacy is based on the increase in energy state and the activation of mitochondrial pathways. In the brain, there is similar evidence of cellular activity with laser irradiation. In vivo studies reinforced the efficacy of this technique for a better neurological and functional outcome post-stroke. The evidence is based on in vivo animal studies of various models and one human clinical study. Although the data is very promising, some fundamental questions remain to be answered, such as the exact mechanism along the cascade of post-stroke interconnective molecular disturbance, the optimal technique and time of treatment, and the long-term safety aspects. The answers to these questions are expected to evolve within the next few years.

Stroke. 2007 Jun;38(6):1843-9. Epub 2007 Apr 26.

Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1).

Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B, Borenstein P, Andersson B, Perez J, Caparo C, Ilic S, Oron U.

Wolfson Medical Center, Department of Neurology, Holon, Israel.

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).

CONCLUSIONS: 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.

J Neurotrauma. 2007 Apr;24(4):651-6.

Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits.

Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler V, Shohami E.

Department of Orthopedics, Assaf Harofeh Medical Center, Zerifin, Israel. amiroronmd@gmail.com

Low-level laser therapy (LLLT) has been evaluated in this study as a potential therapy for traumatic brain injury (TBI). LLLT has been found to modulate various biological processes. Following TBI in mice, we assessed the hypothesis that LLLT might have a beneficial effect on their neurobehavioral and histological outcome. TBI was induced by a weight-drop device, and motor function was assessed 1 h post-trauma using a neurological severity score (NSS). Mice were then divided into three groups of eight mice each: one control group that received a sham LLLT procedure and was not irradiated; and two groups that received LLLT at two different doses (10 and 20 mW/cm(2) ) transcranially. An 808-nm Ga-As diode laser was employed transcranially 4 h post-trauma to illuminate the entire cortex of the brain. Motor function was assessed up to 4 weeks, and lesion volume was measured. There were no significant changes in NSS at 24 and 48 h between the laser-treated and non-treated mice. Yet, from 5 days and up to 28 days, the NSS of the laser-treated mice were significantly lower (p < 0.05) than the traumatized control mice that were not treated with the laser. The lesion volume of the laser treated mice was significantly lower (1.4%) than the non-treated group (12.1%). Our data suggest that a non-invasive transcranial application of LLLT given 4 h following TBI provides a significant long-term functional neurological benefit. Further confirmatory trials are warranted.

Photomed Laser Surg. 2006 Aug;24(4):458-66

Effects of power densities, continuous and pulse frequencies, and number of sessions of low-level laser therapy on intact rat brain.

Ilic S, Leichliter S, Streeter J, Oron A, DeTaboada L, Oron U.

Photothera Inc., Carlsbad, California, USA.

OBJECTIVE: The aim of the present study was to investigate the possible short- and long-term adverse neurological effects of low-level laser therapy (LLLT) given at different power densities, frequencies, and modalities on the intact rat brain. BACKGROUND DATA: LLLT has been shown to modulate biological processes depending on power density, wavelength, and frequency. To date, few well-controlled safety studies on LLLT are available. METHODS: One hundred and eighteen rats were used in the study. Diode laser (808 nm, wavelength) was used to deliver power densities of 7.5, 75, and 750 mW/cm2 transcranially to the brain cortex of mature rats, in either continuous wave (CW) or pulse (Pu) modes. Multiple doses of 7.5 mW/cm2 were also applied. Standard neurological examination of the rats was performed during the follow-up periods after laser irradiation. Histology was performed at light and electron microscopy levels. RESULTS: Both the scores from standard neurological tests and the histopathological examination indicated that there was no long-term difference between laser-treated and control groups up to 70 days post-treatment. The only rats showing an adverse neurological effect were those in the 750 mW/cm2 (about 100-fold optimal dose), CW mode group. In Pu mode, there was much less heating, and no tissue damage was noted. CONCLUSION: Long-term safety tests lasting 30 and 70 days at optimal 10x and 100x doses, as well as at multiple doses at the same power densities, indicate that the tested laser energy doses are safe under this treatment regime. Neurological deficits and histopathological damage to 750 mW/cm2 CW laser irradiation are attributed to thermal damage and not due to tissue-photon interactions.

Lasers Surg Med. 2006 Jan;38(1):70-3

Transcranial application of low-energy laser irradiation improves neurological deficits in rats following acute stroke.

Detaboada L, Ilic S, Leichliter-Martha S, Oron U, Oron A, Streeter J.

Photothera, Inc., 2260 Rutherford Road, Carlsbad, California 92008, USA.

BACKGROUND AND OBJECTIVES: Low-level laser therapy (LLLT) has been shown to have beneficial effects on ischemic skeletal and heart muscles tissues. The aim of the present study was to approve the effectiveness of LLLT treatment at different locations on the brain in acute stroked rats. STUDY DESIGN/MATERIALS AND METHODS: Stroke was induced in 169 rats that were divided into four groups: control non-laser and three laser-treated groups where laser was employed ipsilateral, contralateral, and both to the side of the induced stroke. Rats were tested for neurological function. RESULTS: In all three laser-treated groups, a marked and significant improvement in neurological deficits was evident at 14, 21, and 28 days post stroke relative to the non-treated group. CONCLUSIONS: These observations suggest that LLLT applied at different locations in the skull and in a rather delayed-phase post stroke effectively improves neurological function after acute stroke in rats.

Progress in Laser Therapy

Jackson Streeter
PhotoThera, Carlsbad, CA;

This paper appears in: Lasers and Electro-Optics Society, 2006. LEOS 2006. 19th Annual Meeting of the IEEE
Publication Date: Oct. 2006
On page(s): 665-666
ISBN: 0-7803-9555-7
INSPEC Accession Number: 9364378
Digital Object Identifier: 10.1109/LEOS.2006.278888
Current Version Published: 2007-01-15

 
 
 
Abstract
The presentation covers fundamental operating principles of some of the most widely used methods of low-level laser therapy (LLLT). It includes also recently developed LLLT technologies and medical devices such as LLLT cardiovascular and brain therapy, tissue regeneration and pain relive. The mechanism of LLLT involving interaction with mitochondria. The effects of LLLT are wavelength specific upon a known mitochondrial receptor (cytochrome C oxidase). Targeting of this receptor results in formation of adenosine triphosphate (ATP), enhanced mitochondrial survival and maintenance of cytochrome C oxidase activity

Lik Sprava. 2006 Apr-May;(3):51-4.

Effect of magnet-laser therapy on the central nervous system functional state in patients with ischemic stroke.

 

[Article in Russian]

Datsenko IV.

Twenty three patients aged from 41 to 75, which have had ischemic stroke in the carotid basin (up to 2 years after an acute period of the stroke), have been examined. The course of magneto-laser therapy lasted 15 days. The author carried out neurological examinations, determined the state of psychoemotional activity, cerebral hemodynamics and frequency-amplitude indices of the brain to assess the mechanisms of MLT effect on the CNS functional state in patients being in a rehabilitative period after ischemic stroke. The course of MLT administration improves cerebral hemodynamics, increases the level of the bioelectrical activity of the brain. We can recommend based on obtained results MLT in the system of rehabilitation of patients which had had ischemic stroke.

Zhong Xi Yi Jie He Xue Bao. 2005 Mar;3(2):128-31.  

 

Protective effect of low-level irradiation on acupuncture points combined with iontophoresis against focal cerebral ischemia-reperfusion injury in rats.

[Article in Chinese]

Dai JY, Ge LB, Zhou YL, Wang L.

Acupuncture Clinic, Institute of Qigong, Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China. djysh2002@yahoo.com.cn

OBJECTIVE: To investigate the effects of low-level laser irradiation on acupuncture points combined with iontophoresis against brain damage after middle cerebral artery occlusion (MCAO) in rats.

METHODS: Sixty-nine SD rats were randomly divided into five groups, including normal group, sham operation group, model group, electro-acupuncture group and low-level laser irradiation on acupuncture points combined with iontophoresis group (LLLI group). The cerebral ischemia-reperfusion (I/R) model was established by thread embolism of middle cerebral artery. The rats in the LLLI group, as well as the electro-acupuncture group were given treatment as soon as the occlusion finished (0 hour) and 12, 24 hours after the occlusion. We observed the changes of neurological deficit scores and the body weight of the rats at different time. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ratos brain tissue were tested.

RESULTS: The neurological deficit score of the LLLI group was significantly lower than that of the model group. The body weight and the activity of SOD of the rats decreased slightly, and the content of MDA decreased significantly after the treatment.

CONCLUSION: The low-level laser irradiation on acupuncture points combined with iontophoresis can prevent focal cerebral ischemia-reperfusion injury. One of its mechanisms may be increasing the activity of SOD and decreasing the damage of the oxidation products to the body.

Stroke. 2004 Aug;35(8):1985-8. Epub 2004 May 20

Transcranial infrared laser therapy improves clinical rating scores after embolic strokes in rabbits.

Lapchak PA, Wei J, Zivin JA.

Department of Neuroscience, University of California San Diego, MTF 316, 9500 Gilman Drive, La Jolla, CA 92093-0624, USA. plapchak@ucsd.edu

BACKGROUND AND PURPOSE: Because photon energy delivered using a low-energy infrared laser may be useful to treat stroke, we determined whether transcranial laser therapy would improve behavioral deficits in a rabbit small clot embolic stroke model (RSCEM).

METHODS: In this study, the behavioral and physiological effects of laser treatment were measured. The RSCEM was used to assess whether low-energy laser treatment (7.5 or 25 mW/cm2) altered clinical rating scores (behavior) when given to rabbits beginning 1 to 24 hours postembolization. Behavioral analysis was conducted from 24 hours to 21 days after embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the P50 compared with the control group.

RESULTS: In the present study, the P50 value for controls were 0.97+/-0.19 mg to 1.10+/-0.17 mg; this was increased by 100% to 195% (P50=2.02+/-0.46 to 2.98+/-0.65 mg) if laser treatment was initiated up to 6 hours, but not 24 hours, postembolization (P50=1.23+/-0.15 mg). Laser treatment also produced a durable effect that was measurable 21 days after embolization. Laser treatment (25 mW/cm2) did not affect the physiological variables that were measured.

CONCLUSIONS: This study shows that laser treatment improved behavioral performance if initiated within 6 hours of an embolic stroke and the effect of laser treatment is durable. Therefore, transcranial laser treatment may be useful to treat human stroke patients and should be further developed.

Vopr Kurortol Fizioter Lech Fiz Kult. 2003 Mar-Apr;(2):19-20.  

Magnetic and laser therapy of acute ischemic stroke

[Article in Russian]

Samosiuk NI.

The paper presents the technique of frequency-modulated magnetolaser therapy (FMMLT) used in combined treatment of 121 patients with ischemic stroke in acute period. The results were compared with those in the control group of 30 patients who received conventional drug treatment. The results of the comparison allowed the author to recommend FMMLT in ischemic stroke especially in the period of "therapeutic window".

Lasers Surg Med 2002  31:283-8

Treatment of experimentally induced transient cerebral ischemia with low energy laser inhibits nitric oxide synthase activity and up-regulates the expression of transforming growth factor-beta 1.

Leung MC, Lo SC, Siu FK, So KF

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) has been shown to be neurotoxic while transforming growth factor-beta 1 (TGF-beta1) is neuroprotective in the stroke model. The present study investigates the effects of low energy laser on nitric oxide synthase (NOS) and TGF-beta1 activities after cerebral ischemia and reperfusion injury.

STUDY DESIGN/MATERIALS AND METHODS: Cerebral ischemia was induced for 1 hour in male adult Sprague-Dawley (S.D.) rats with unilateral occlusion of middle cerebral artery (MCAO). Low energy laser irradiation was then applied to the cerebrum at different durations (1, 5, or 10 minutes). The activity of NOS and the expression of TGF-beta1 were evaluated in groups with different durations of laser irradiation.

RESULTS: After ischemia, the activity of NOS was gradually increased from day 3, became significantly higher from day 4 to 6 (P < 0.001), but returned to the normal level after day 7. The activity and expression of the three isoforms of NOS were significantly suppressed (P < 0.001) to different extents after laser irradiation. In addition, laser irradiation was shown to trigger the expression of TGF-beta1 (P < 0.001).

CONCLUSIONS: Low energy laser could suppress the activity of NOS and up-regulate the expression of TGF-beta1 after stroke in rats.

Vopr Kurortol Fizioter Lech Fiz Kult. 2000 May-Jun;(3):17-21.  

The optimization of an early rehabilitation program for cerebral stroke patients: the use of different methods of magnet0- and laser therapy.

[Article in Russian]

Kochetkov AV, Gorbunov FE, Minenkov AA, Strel'tsova EN, Filina TF, Krupennikov AI.

Magnetotherapy and laser therapy were used in complex and complex-combined regimens in 75 patients after cerebral ischemic or hemorrhagic stroke starting on the poststroke week 4-5. Clinico-neurologic, neurophysiological and cerebrohemodynamic findings evidence for the highest effectiveness of neurorehabilitation including complex magneto-laser therapy in hemispheric ischemic and hemorrhagic stroke of subcortical location in the absence of marked clinico-tomographic signs of dyscirculatory encephalopathy. Complex-combined magneto-laser therapy is more effective for correction of spastic dystonia. Mutual potentiation of magnetotherapy and laser therapy results in maximal development of collateral circulation and cerebral hemodynamic reserve (84% of the patients). Complex effects manifest in arteriodilating and venotonic effects. Complex magneto-laser therapy is accompanied by reduction of hyperthrombocythemia and hyperfibrinogenemia.

Lik Sprava. 1996 Jul-Sep;(7-9):142-5.  

The treatment of patients with chronic cerebral circulatory failure by using laser puncture and the microclimate of the biotron.

[Article in Ukrainian]

Macheret IeL, D'iachenko OIe, Korkushko OO.

A mode is proposed of treatment of chronic cerebrovascular disorders, such as initial manifestations of cerebral blood supply insufficiency (IMBSI) and dyscirculatory encephalopathy (DE) stage I-II in hypertensive disease, involving the use of laser puncture and microclimate of biotron. All patients (n = 162) were exposed to laser puncture (10-12 procedures). Laser puncture treatments were devised according to classical approaches of reflexotherapy, using determinants of electropuncture diagnostic method by Riodoraku. The treatments were carried out with the aid of infrared portable laser "Biomed-001". IMBSI patients presenting with vegetovascular dystonia and about 70% of IMBSI patients presenting with hypertensive disease derived benefit from a course of laser puncture, as evidenced by REG, EEG, acupuncture diagnosis, iridodiagnosis according. In DE stage I-II patients and about 30% IMBSI patients presenting with hypertensive disease good therapeutic effect occurred after treatment in a ward with a stable microclimate of biotron. The proposed method can be used for treating chronic cerebrovascular disorders and administering stroke prophylaxis.

Zhongguo Zhong Xi Yi Jie He Za Zhi. 2000 Apr;20(4):264-6.  

Effect of intravascular laser irradiation of blood and traditional Chinese medical therapy on immune function in senile cerebral infarction patients of kidney deficiency type

[Article in Chinese]

Xiao X, Chu X, Ni J.

Shenzhen Municipal People's Hospital, Guangdong (518020).

OBJECTIVE: To observe the effect of intravascular laser irradiation of blood (ILIB) therapy on cellular immunity, change of T-lymphocyte subsets and humoral immunity in senile cerebral infarction patients of Kidney deficiency type. METHODS: Seventy-five patients were divided randomly into the ILIB group and the control group treated by conventional medicine (CM). Serum CD3, CD4, CD8, IgG, IgA, IgM, C3 and C4 levels of patients were determined before and after treatment for self-control and comparing between various groups and that of normal control. RESULTS: Before treatment, in patients of both groups, the levels of CD3, CD4, CD4/CD8, C3 were all lower than normal levels significantly, C4 and IgM higher than normal (P < 0.05, P < 0.01), the level of IgG lowered in patients inclined to Kidney-Yang deficiency and raised in those inclined to Kidney-Yin deficiency (P < 0.01). After treatment, in the ILIB group, CD3, CD4 and CD4/CD8 raised significantly (P < 0.05, P < 0.01), IgG and C3 varied towards normal control (P < 0.01, P < 0.05), and C4 lowered but without significance. In the control group, the indexes changed also toward normal but without significance except the change of IgG (P < 0.05). As for IgA and IgM, marked changes were not found in both groups in comparison between before and after treatment. CONCLUSION: ILIB therapy could bi-directionally regulate cellular and humoral immunity in senile cerebral infarction patients of Kidney deficiency type, which was similar to the function in supplementing Qi and invigorating Kidney of Chinese herbal medicine.

INTRAVASCULAR LASER THERAPY ON THE CEREBRAL CIRCULATION ISCHEMIC DISTURBANCES

T.O. Makhovskaya, V.V. Skupchenko

Far Eastern State Medical University (Khabarovsk), State Medical University (Samara), Russia

The dynamics of clinical and pathophysiological alterations on the various forms of cerebral circulation ischemic disturbances (CCID) was investigated in the course of helium-neon laser therapy (HNL). There were treated 600 patients. Clinical, vegetative, and neurophysiological pattern indices were examined. Results of the complex investigation reliably testified that vegetative indices play the important role in CCID pathogenesis, accompanied by pathologic neuro-dynamic disbalance formation. Patients with phase somatovegetative hyperactivity prevailed. Clinical effect of HNL correlated with system vegetative dynamic, its effectiveness was higher in the patients with initial sympathicotonia. HNL was not effective on cholinergic influences. After HNL positive neuro-physiological changes were registered in patients with initial adrenergic activity, there were no changes at cholinergic intensity or slight modulate effect was observed. HNL improved blood circulation, blood filling was increased in the affected vascular basin, the increased cerebral arteries tone decreased, pulse blood filling increased, venous circulation was improved. Therefore, HNL has neurodynamic effect, relaxes sympathicotonic influences and has vagotrope regulatory effect. Photoneurodynamic HNL influence renders trophotroimages action, preventing or reducing cerebral tissue ischemization at all stages of cerebro-vascular diseases with sympatic pattern and is not expedient on neurodynamic disbalance in the form of parasympathicotonia. HNL allows to receive stable therapeutic effect in patients with initial cerebral blood supply insufficiency, transient disturbances of cerebral blood circulation, slight insult, ischemic insult in the acute phase, discirculatory encephalopathy at the first stage.

Laser Acupuncture to Treat Paralysis in Stroke Patients, CT Scan Lesion Site Study

Margaret Naeser, Ph.D., Lic.Ac., Dipl.Ac. (NCCAOM)

Department of Neurology, Boston University School of Medicine and Neuroimaging/Aphasia Research, V.A. Boston Healthcare System, Boston, MA. mnaeser@bu.edu

Purpose:

1) To study the effectiveness of low-level laser stimulation of acupuncture points to treat paralysis in stroke patients; 2) To examine the relationship between neuroanatomical lesion sites on CT scan and potential for improvement following laser acupuncture treatments. We have conducted previous research with needle stimulation of acupuncture points in the treatment of paralysis in stroke patients (1-3).

Subjects:

Seven stroke patients participated (ages 48-71 years when entering the study; 5 men, 2 women). Five cases had single left hemisphere stroke; two cases, single right hemisphere stroke. Five patients were treated for residual arm/leg paralysis; they had greatly reduced arm and leg power (and severely reduced or no voluntary isolated finger movement). Two cases were treated only for hand paresis; they had good arm and leg power, but they had mildly reduced isolated finger movement. CT scans were obtained on all patients after at least 3 months poststroke.

Six patients began receiving the laser acupuncture treatments during the chronic phase poststroke (10 months to 6.5 years). These times are beyond the spontaneous recovery period of up to 6 months poststroke (4, 5). One hand paresis case began receiving treatments during the acute phase poststroke (1 month poststroke). Because all patients were beyond the spontaneous recovery period except for one, each patient served as his/her own control. No sham laser treatments were administered. None of the stroke patients was receiving physical therapy or occupational therapy treatments during the course of the laser acupuncture treatments.

Method:

A 20 mW Gallium Aluminum Arsenide (780 nm) near-infrared, diode laser (Uni-laser, Denmark) with 1 mm diameter aperture, was used for 20-40 seconds (51-103 J/cm2) on each acupuncture point. The laser was used for 20 seconds on shallow points (hands and face), and 40 seconds on deeper acupuncture points (arms and legs). The points used on the paralyzed arm included: LI 4 (Hegu), LI 11 (Quchi), LI 15 (Jianyu), TW 5 (Waiguan), TW 9 (Sidu), and three distal Baxie points in the web-spaces between the fingers. The points used on the paralyzed leg included: ST 31 (Biguan), ST 36 (Zusanli), GB 34 (Yanglingquan), GB 39 (Xuanzhong), and LIV 3 (Taichong). Points used on the non-paralyzed side included LI 4 (Hegu) and ST 36 (Zusanli). These points include some of those used in our previous research where needle acupuncture was used to treat paralysis in stroke patients (1-3). If facial paralysis was present, the following points on the paralyzed side were used: ST 4 (Dicang), ST 6 (Jiache), ST 7 (Xiaguan), LI 20 (Yingxiang), and SI 18 Quanliao).

The patients were tested a few days prior to the first laser acupuncture treatment, and within a few days after completing the 20th, 40th and/or 60th laser acupuncture treatment. P.T. and O.T. testers were blinded; testers were part of a needle acupuncture study with real or sham or no acupuncture. Some patients received only 20 or 40 treatments. The number of treatments a patient received (20, 40 or 60) was based solely on patient availability and transportation issues. All patients were offered a maximum of 60 laser treatments. The patients were treated 2 – 3 times per week, for 3 – 4 months.

For patients with arm/leg paralysis, improvement was defined as a minimum increase of at least 10% isolated active range of motion, on at least one arm/leg test, following 20, 40 or 60 laser acupuncture treatments. For the patients treated for hand paresis, improvement was defined as an increase of at least 1 lb., on at least one hand strength test, following 20, 40 or 60 laser acupuncture treatments.

Results:

Overall, 5/7 patients (71.4%) treated with laser acupuncture showed improvement. Four of the six chronic stroke patients (66%) showed improvement. The single acute stroke patient (hand paresis case) also showed improvement.

Three of the five arm/leg cases showed a minimum of at least 10% improvement in isolated active range of motion on knee flexion; knee extension and/or shoulder abduction (range, +11 to +28%; mean, +15.8%, S.D., 7.08).

The two cases with hand paresis each showed improvement in hand strength. For the chronic hand paresis case (33 months poststroke), grip strength, pre- treatment, 62.7 lbs., post- 20 treatments, 68.4 lbs; strength in first 2 fingers opposing thumb (3-Jaw Chuck), pre- 12, post- 18 lbs.; strength in index finger opposing thumb (Tip Pinch), pre- 8, post- 11 lbs; and strength in thumb opposing the lateral surface of index finger (Lateral Pinch) pre- 12, post- 14 lbs. For the acute hand paresis case (starting at 1 month poststroke), grip strength, pre- 32.2, post- 20 Tx.'s, 47.7 lbs.; 3-Jaw Chuck, pre- 0, post- 11.3 lbs.; Tip Pinch, pre- 0, post- 10.7 lbs; Lateral Pinch, pre- 3.7, post- 14.7 lbs.

The five cases who showed improvement following the laser acupuncture treatments

had either no lesion in, or lesion in less than half of the motor pathway areas, including the periventricular white matter (PVWM) area on CT scan. The PVWM area is located adjacent to the body of the lateral ventricle, superior to the posterior limb, internal capsule. The two arm/leg cases who showed no improvement following the laser acupuncture treatments had lesion in more than half of the motor pathway areas, including the PVWM area. These behavioral and neuroanatomical findings are similar to our previous research using needle acupuncture to treat paralysis in stroke patients.

The PVWM area appears to be the most important area to examine on CT scan or MRI scan, in understanding whether a stroke patient is likely to benefit from needle or laser acupuncture to help reduce the severity of paralysis. This area contains many important intra- and inter-hemispheric pathways including, in part: 1) The descending pyramidal fibers from motor cortex, where the pathways for the leg are more medial. 2) The body of the caudate nucleus. 3) The mid-callosal pathways. 4) The medial subcallosal fasciculus containing connections to caudate from supplementary motor area and cingulate gyrus. 5) The occipito-frontal fasciculus. 6) The superior lateral thalamic peduncle which includes projections from dorsomedial nucleus and anterior nucleus to cingulate and projections from the ventrolateral nucleus to motor cortex.

Thus, even within this small PVWM region there are numerous motor systems that might, if incompletely damaged, respond to needle or laser acupuncture. These systems include dorsal striatum, supplementary motor area, or the frontal-striatal-ventrolateral thalamic-frontal loop, as well as the descending pyramidal system.

One patient with severe arm/leg paralysis did have improvement in her facial paralysis with good control of food and liquids in the left side of her mouth for the first time poststroke (4 years poststroke). She also improved in walking, with a “loosening” of the left Achilles tendon.

The author has observed that red-beam laser stimulation (4.59 J/cm2) on the Jing-Well points on the fingers (LU 11, Shaoshang; LI 1, Shangyang; PC 9, Zhongchong; TW 1, Guanchong; HRT 9, Shaochong; SI 1, Shaoze), in combination with the use of a microamps TENS device (MicroStim 100 TENS, Tamarac, FL) placed on the hand (HRT 8, Shaofu; and TW 5; Waiguan), is helpful in treating hand paresis and reducing hand spasticity in stroke patients (6, p. 40, Naeser Laser HAND Treatment Program). This method is also helpful in the prevention/ reduction of contractures of the hand, in patients with severe hand paralysis (personal observation).

Discussion:

The use of low-level laser for long-term treatment is especially desirable for chronic stroke patients with hand paresis. The patient can be trained to treat him/herself at home, using an inexpensive 5mW red-beam diode, laser pointer and a microamps TENS device (MicroStim 100, Tamarac, FL). See Websites listed below.

Acupuncture studies using needle acupuncture have observed the best outcome levels when acupuncture treatments were initiated at less than 3 months poststroke (7, 8), and especially when the acupuncture treatments were initiated at less than 24 hours and 36 hours poststroke (9, 10).

This is the first study to examine the effect of low-level laser therapy on acupuncture points to treat paralysis in stroke patients where lesion location was known for each patient. Results suggest that low-level laser therapy on acupuncture points is effective to help reduce the severity of paralysis in stroke patients, especially those with mild-moderate paralysis. The treatments should be initiated as soon as possible poststroke, even within 24 hours poststroke. A comprehensive rehabilitation program of P.T., O.T. plus needle and/or laser acupuncture is recommended.

References

1.       Naeser MA, Alexander MP, Stiassny-Eder D, Galler V, Hobbs J, Bachman D: Real versus sham acupuncture in the treatment of paralysis in acute stroke patients: A CT scan lesion site study. Journal of Neurologic Rehabilitation 1992;6:163-173.

2.       Naeser MA, Alexander MP, Stiassny-Eder D, Lannin LN, Bachman D: Acupuncture in the treatment of hand paresis in chronic and acute stroke patients – Improvement observed in all cases. Clinical Rehabilitation 1994; 8:127-141.

3.       Naeser MA, Alexander MP, Stiassny-Eder D, Galler V, Hobbs J, Bachman D: Acupuncture in the treatment of paralysis in chronic and acute stroke patients – Improvement correlated with specific CT scan lesion sites. Acupuncture & Electrotherapeutics

4.       Bard G, Hirschberg GG: Recovery of voluntary motion in upper extremity following hemiplegia. Arch Phys Med Rehabil 1965; 46:567-572.

5.       Sunderland A, Tinson D, Bradley L, Hewer R: Arm function after stroke: An evaluation of grip strength as a measure of recovery and a prognostic indicator. J Neurol, Neurosurg, and Psych 1989; 52:1267-1272.

6.       Naeser MA, Wei XB: Laser Acupuncture, An Introductory Textbook for Treatment of Pain, Paralysis, Spasticity and Other Disorders. Boston, Boston Chinese Medicine, 1994, p. 40.

7.       Zhang WX, Li SC, Chen GB, Zhang QM, Wang YX, Fang YA. Acupuncture treatment of apoplectic hemiplegia. Journal of Traditional Chinese Medicine 1987;7:157-160.

8.       Johansson K, Lindgren I, Widner H, Wiklung I, Johansson BB: Can sensory stimulation improve the functional outcome in stroke patients? Neurol 1993; 43:2189-2192.

9.       Li DM, Li WD, Wei LH, Zhao YL, Lu HZ. Clinical observation on acupuncture therapy for cerebral hemorrhage. J. of Traditional Chinese Medicine 1989;9(1):9-13.

10.    Hu HH, Chung C, Liu TJ, Chen RC, Chen CH, Chou P, Huang WS, Lin JCT, Tsuei JJ. A randomized controlled trial on the treatment for acute partial ischemic stroke with acupuncture. Neuroepidemiology 1993;12:106-113.

Additional Information:

www.Acupuncture.com/Acup/Naeser.htm and www.Acupuncture.com/Acup/laser.htm

See also: Naeser MA: Neurological Rehabilitation: Acupuncture and Laser Acupuncture to Treat Paralysis in Stroke and Other Paralytic Conditions and Pain in Carpal Tunnel Syndrome. Chapter in National Institutes of Health Consensus Development Conference on Acupuncture sponsored by the Office of Alternative Medicine and the Office of Medical Applications of Research. Bethesda, MD, November 3-5, 1997. pp. 93-109

Abstract based on: Naeser MA, Alexander MP, Stiassny-Eder D, Galler V, Hobbs J, Bachman D, Lannin L: Laser Acupuncture in the Treatment of Paralysis in Stroke Patients: A CT Scan Lesion Site Study. Am J of Acupuncture, 23(1):13-28, 1995:

Zhong Xi Yi Jie He Xue Bao. 2005 Mar;3(2):128-31.  

Protective effect of low-level irradiation on acupuncture points combined with iontophoresis against focal cerebral ischemia-reperfusion injury in rats.

[Article in Chinese]

Dai JY, Ge LB, Zhou YL, Wang L.

Acupuncture Clinic, Institute of Qigong, Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China. djysh2002@yahoo.com.cn

OBJECTIVE: To investigate the effects of low-level laser irradiation on acupuncture points combined with iontophoresis against brain damage after middle cerebral artery occlusion (MCAO) in rats.

METHODS: Sixty-nine SD rats were randomly divided into five groups, including normal group, sham operation group, model group, electro-acupuncture group and low-level laser irradiation on acupuncture points combined with iontophoresis group (LLLI group). The cerebral ischemia-reperfusion (I/R) model was established by thread embolism of middle cerebral artery. The rats in the LLLI group, as well as the electro-acupuncture group were given treatment as soon as the occlusion finished (0 hour) and 12, 24 hours after the occlusion. We observed the changes of neurological deficit scores and the body weight of the rats at different time. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ratos brain tissue were tested. RESULTS: The neurological deficit score of the LLLI group was significantly lower than that of the model group. The body weight and the activity of SOD of the rats decreased slightly, and the content of MDA decreased significantly after the treatment. CONCLUSION: The low-level laser irradiation on acupuncture points combined with iontophoresis can prevent focal cerebral ischemia-reperfusion injury. One of its mechanisms may be increasing the activity of SOD and decreasing the damage of the oxidation products to the body.