Photobiomodulation Therapy to Promote Angiogenesis in Diabetic Mice with Hindlimb Ischemia.

Objective: To assess whether photobiomodulation therapy (PBMT) induces angiogenesis in diabetic mice with hindlimb ischemia (HLI). Background: Patients with diabetes mellitus (DM) are at high risk of developing peripheral arterial disease (PAD) in the lower extremities. PBMT has been shown to promote angiogenesis both in vitro and in vivo and could be a treatment for DM patients with PAD. Methods: Femoral artery ligation/excision in mice was performed to induce HLI as an animal model of PAD. PBMT at a dose of 660 nm and 1.91 J/cm2 was delivered for 10 min on 5 consecutive days after the HLI surgery. Control mice received HLI only. Mice in the DM group were injected with streptozocin to induce diabetes before HLI surgery. Mice in the laser and DM+ laser groups received both HLI and PBMT, and the latter group had induced DM. After the laser treatment, lower limb blood flow was evaluated by laser Doppler. The capillary density and CD31 were analyzed by immunofluorescence staining, and protein levels of vascular endothelial growth factor (VEGF)-A, hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinases (ERK) were measured by Western blotting of tissue samples. Results: Compared with the control and DM mice, the laser and DM+ laser groups had more than double the capillary density and blood perfusion rate. Levels of CD31 and VEGF-A proteins in groups that received laser were increased by 1.9- to 3.2-fold compared with groups that did not undergo laser treatment. Animals treated with PBMT exhibited significantly increased HIF-1α expression and ERK phosphorylation compared with animals that did not receive this treatment, and the amount of phospho-eNOS and iNOS increased and decreased, respectively. Conclusions: PBMT can induce therapeutic angiogenesis, indicating that low intensity laser could be a novel treatment for PAD patients.

Effects of prenatal photobiomodulation treatment on neonatal hypoxic ischemia in rat offspring.

Neonatal hypoxic-ischemic (HI) injury is a severe complication often leading to neonatal death and long-term neurobehavioral deficits in children. Currently, the only treatment option available for neonatal HI injury is therapeutic hypothermia. However, the necessary specialized equipment, possible adverse side effects, and limited effectiveness of this therapy creates an urgent need for the development of new HI treatment methods. Photobiomodulation (PBM) has been shown to be neuroprotective against multiple brain disorders in animal models, as well as limited human studies. However, the effects of PBM treatment on neonatal HI injury remain unclear. Methods: Two-minutes PBM (808 nm continuous wave laser, 8 mW/cm2 on neonatal brain) was applied three times weekly on the abdomen of pregnant rats from gestation day 1 (GD1) to GD21. After neonatal right common carotid artery ligation, cortex- and hippocampus-related behavioral deficits due to HI insult were measured using a battery of behavioral tests. The effects of HI insult and PBM pretreatment on infarct size; synaptic, dendritic, and white matter damage; neuronal degeneration; apoptosis; mitochondrial function; mitochondrial fragmentation; oxidative stress; and gliosis were then assessed. Results: Prenatal PBM treatment significantly improved the survival rate of neonatal rats and decreased infarct size after HI insult. Behavioral tests revealed that prenatal PBM treatment significantly alleviated cortex-related motor deficits and hippocampus-related memory and learning dysfunction. In addition, mitochondrial function and integrity were protected in HI animals treated with PBM. Additional studies revealed that prenatal PBM treatment significantly alleviated HI-induced neuroinflammation, oxidative stress, and myeloid cell/astrocyte activation. Conclusion: Prenatal PBM treatment exerts neuroprotective effects on neonatal HI rats. Underlying mechanisms for this neuroprotection may include preservation of mitochondrial function, reduction of inflammation, and decreased oxidative stress. Our findings support the possible use of PBM treatment in high-risk pregnancies to alleviate or prevent HI-induced brain injury in the perinatal period.

Effects of Photobiomodulation on Degranulation and Number of Mast Cells and Wound Strength in Skin Wound Healing of Streptozotocin-Induced Diabetic Rats.

BACKGROUND: A lack of effective treatments still exists for patients suffering from diabetes mellitus. Photobiomodulation is proved as a beneficial therapeutic modality for wounds. OBJECTIVE: The aim of this study is to examine the effect of degranulation of mast cells and total number of mast cells in the remodeling step of an ischemic model of wound healing under the influence of photobiomodulation and conditioned medium (CM) from human bone marrow-derived mesenchymal stem cells (hBM-MSCs-CM), or CM, administered alone and or in combination. MATERIALS AND METHODS: Initially, type 1 diabetes mellitus was induced in 72 male adult rats. Then, after a month, one incision was made on the back of each rat. Subsequently, the rats were divided into four groups. The first group was considered as the control (placebo) group, the second group received CM, the third group received photobiomodulation, and the fourth group received photobiomodulation+CM. On days 4, 7, and 15, samples were extracted from the wound for histological and tensiometric examinations. The total number of mast cells, including the three types of mast cells, was counted by the stereological methods. The tensiometric properties of the repairing tissue were examined. RESULTS: The administration of photobiomodulation and CM, alone or in combination, significantly increased the tensiometric properties within the healing wounds. Histologically, photobiomodulation+CM, CM, and photobiomodulation groups showed a significant decrease in the three types of mast cells and in the total number of mast cells compared with the control group on day 15. CONCLUSIONS: We conclude that photobiomodulation and CM alone and or in combination significantly accelerated the healing process in a rat with a diabetic and ischemic wound, and significantly decreased the total number of mast cells and degranulation of mast cells. We suggest that the increased number of type 2 mast cells in the control group adversely affected the tensiometric properties of wounds in this group.

Effect of 810 nm Near-Infrared Laser on Revascularization of Ischemic Flaps in Rats.

OBJECTIVE: To investigate the effect of 810 nm near-infrared (NIR) laser on the revascularization of ischemic flaps. BACKGROUND: It has long been proved that photobiomodulation therapy (PBMT) improves the blood supply of flaps. NIR laser improves the treatment of hypodermis-located lesions and of flap survival, but basic research on the use of 810 nm NIR laser for ischemic flap revascularization is still lacking. MATERIALS AND METHODS: We prepared two symmetrical long random-pattern flaps on the backs of 60 rats. Each flap was 6 cm long, 1 cm wide, and 1 cm to the middle line. The flaps were divided into an irradiated flap group and an internal control group. The irradiated flaps underwent postoperative 810 nm laser therapy with the energy density of 11.30 J/cm2 daily. The control flaps were covered by stainless steel to avoid laser irradiation. We observed the viability of the flaps. The flaps underwent Hematoxylin and Eosin (H&E) staining for the observation of histomorphology, immunohistochemical staining of factor VIII for the capillary count, α-smooth muscle actin for the small arterial count, and vascular endothelial growth factor for the integrated optical density (OD) of the positive stained color. RESULTS: The irradiated flaps showed significantly better flap survival than the control flaps. H&E staining showed that the irradiated flaps had clear tissue structure and little inflammatory cell infiltration. The control flaps demonstrated comparatively worse results. Vascular endothelial growth factor staining showed that the difference in integrated OD between the irradiated flaps and the control flaps was not statistically significant. α-smooth muscle actin and factor VIII staining showed significantly greater numbers of arterioles and capillaries in the irradiated flaps than the control flaps after 4 days of irradiation. CONCLUSIONS: PBMT with 810 nm NIR laser could enhance ischemic flap revascularization and increase flap viability.

Enhanced survival of ischemic skin flap by combined treatment with bone marrow-derived stem cells and low-level light irradiation.

The aim of this study is to examine the enhanced survival effect of ischemic skin flap by combined treatment with bone marrow-derived stem cells (BMSCs) and low-level light irradiation (LLLI). The neovasculogenic effect of BMSCs induced by LLLI was detected using a wound healing and tube formation assay. ICR mice were divided into four groups: control group, LLLI group, BMSCs group, and combine-treated group. The percentage of skin flap necrosis area was calculated on the seventh post-operative day. Specimens were harvested for histologic analyses. LLLI promoted BMSC migration and tube formation. The flap survival rate of combined treated group was significantly higher than that of the control group. Histologic results demonstrated a significant increase in neovascularization in the combined treatment group. This study demonstrates that combination treatment of BMSCs and LLLI could enhance the survival of ischemic skin flap in a mouse model.

Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia.

Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.

Low level light therapy by LED of different wavelength induces angiogenesis and improves ischemic wound healing.

BACKGROUND AND OBJECTIVE: Low-level light therapy (LLLT) has been revealed as a potential means to improve wound healing. So far, most studies are being performed with irradiation in the red to near-infrared spectra. Recently, we showed that blue light (470 nm) can significantly influence biological systems such as nitric oxide (NO) metabolism and is able to release NO from nitrosyl-hemoglobin or mitochondrial protein complexes. Therefore, the aim of this study was to evaluate and compare the therapeutic value of blue or red light emitting diodes (LEDs) on wound healing in an ischemia disturbed rodent flap model. STUDY DESIGN/MATERIALS AND METHODS: An abdominal flap was rendered ischemic by ligation of one epigastric bundle and subjected to LED illumination with a wavelength of 470 nm (blue, n = 8) or 629 nm (red, n = 8) each at 50 mW/cm(2) and compared to a non-treated control group (n = 8). Illumination was performed for 10 minutes on five consecutive days. RESULTS: LED therapy with both wavelengths significantly increased angiogenesis in the sub-epidermal layer and intramuscularly (panniculus carnosus muscle) which was associated with significantly improved tissue perfusion 7 days after the ischemic insult. Accordingly, tissue necrosis was significantly reduced and shrinkage significantly less pronounced in the LED-treated groups of both wavelengths. CONCLUSIONS: LED treatment of ischemia challenged tissue improved early wound healing by enhancing angiogenesis irrespective of the wavelength thus delineating this noninvasive means as a potential, cost effective tool in complicated wounds.

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2.

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm(2) on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm(2) and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm(2). The random skin flap was performed measuring 10×4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner.

Experimental model for low level laser therapy on ischemic random skin flap in rats

OBJETIVO: Propor o desenvolvimento de um modelo experimental para verificar o efeito da laserterapia de baixa intensidade na viabilidade do retalho cutâneo randômico em ratos. MÉTODOS: A amostra constituiu-se de 24 ratos, da linhagem Wistar-EPM. O retalho cutâneo randômico foi realizado com dimensões de 10x4 cm e uma barreira plástica foi interposta entre o mesmo e o leito doador. O Grupo 1 (controle), foi submetido a uma simulação de tratamento com a irradiação laser de diodo (830 nm). O Grupo 2 foi submetido à irradiação laser de diodo (830 nm). Os animais foram submetidos a terapia a laser com densidade de energia de 36 J/cm2 (72 segundos) imediatamente após a operação e nos outros quatros dias subseqüentes. A caneta do laser foi posicionada a 90 graus em contato com o retalho cutâneo em um ponto a 2,5 cm da base cranial do retalho. No sétimo dia pós-operatório foram calculadas as porcentagens da área de necrose. RESULTADOS: O Grupo 1 apresentou média da área de necrose de 48,86% e o Grupo 2 – 23,14%. Após a análise estatística, os resultados mostraram que o Grupo 2 foi mais eficaz, quando comparado ao controle (p<0,001).CONCLUSÃO: O modelo experimental mostrou-se factível para estudos dos efeitos da terapia a laser de baixa intensidade em retalho cutâneo randômico em ratos.

Experimental model for low level laser therapy on ischemic random skin flap in rats.

PURPOSE: To develop an experimental model to be used in the study of low level Laser therapy on viability of random skin flap in rats. METHODS: The sample was 24 Wistar-EPM rats. The random skin flap measured 10 x 4 cm and a plastic sheet was interposed between the flap and donor site. Group 1 (control) underwent sham irradiation with diode laser (830 nm). Group 2 was submitted to laser irradiation with diode laser (830 nm). The animals were submitted to Laser therapy with 36 J/cm(2) energy density (72 seconds) immediately after the surgery and on the four subsequent days. The probe was usually held in contact with the skin flap surface on a point at 2.5 cm cranial from the flap base. On the seventh postoperative day, the percentage of necrotic area was measured and calculated. RESULTS: Group 1 reached an average necrotic area of 48.86%, Group 2 - 23.14%. After the statistic analysis, compared with the control group, Group 2 showed a statistically significant increase in survival area (p<0.001). CONCLUSION: The experimental model proved to be reliable to be used in the study of effects of low level laser therapy in random skin flap in rats.

Effects of diode laser therapy on blood flow in axial pattern flaps in the rat model.

Axial pattern skin flaps are a very important reparative tool for the plastic and reconstructive surgeon in the reconstruction of tissue defects. From whatever unfortunate reason, part or all of such flaps occasionally suffers from irreversible ischaemia with loss of the flap. Infrared diode laser therapy has been shown to improve local and systemic circulation. The present study was designed to assess the effect of an 830 nm diode laser (power density, 18.5 W/cm(2), energy density 185 J/cm(2)) on the blood flow of axial pattern flaps in the rat model and their survival, compared with unirradiated controls. The flaps were raised in all animals ( n=40), and blood flow assessed with laser speckle flowmetry (LSF). In the experimental groups (3 groups, n=10 per group), the flaps were irradiated either directly over the dominant feeder vessel (iliolumbar artery), at the proximal end or at the distal end of the flap itself and blood flow assessed during irradiation. Flowmetry was performed again in all animals at 5 and 10 min postirradiation, and the flaps sutured back in position. The unirradiated controls were handled in exactly the same way, but the laser was not activated. The survival rate of the flaps was assessed on the fifth postoperative day. LSF demonstrated significant increased blood flow in the flaps at 5 and 10 min postirradiation in all experimental groups compared with the control animals. At five days postirradiation, there was significantly better survival of the flaps in all the experimental groups compared with the controls ( p<0.01), but no significant difference was seen between any of the experimental groups. We conclude that laser therapy increases the blood flow and perfusion of transferred flaps, and that this has significant effects on the survival of the flaps. One possible mechanism of modulation of the autonomic nervous system is discussed.

[The effect of low-intensity laser radiation on the regional hemodynamics in strangulated intestinal obstruction]. / Vliianie nizkointensivnogo lazernogo izlucheniia na regionarnuiu gemodinamiku pri stranguliatsionnoi neprokhodimosti kishechnika.

In the experiment on 214 white rats, the effect of different types of low-intensive laser irradiation and existing methods of treatment on regional hemodynamics in strangulated ileus was studied. Combined use of ultraviolet and helium-neon lasers has to be proved to be the most effective. Ultraviolet laser irradiation contributed to increase in elasticity of the arterial wall and cupping off the vascular spasm, helium-neon irradiation had a stimulating effect on the tissues of the intestinal wall.