830-nm irradiation increases the wound tensile strength in a diabetic murine model.

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the effects of low-power laser irradiation on wound healing in genetic diabetes. STUDY DESIGN/MATERIALS AND METHODS: Female C57BL/Ksj/db/db mice received 2 dorsal 1 cm full-thickness incisions and laser irradiation (830 nm, 79 mW/cm(2), 5.0 J/cm(2)/wound). Daily low-level laser therapy (LLLT) occurred over 0-4 days, 3-7 days, or nonirradiated. On sacrifice at 11 or 23 days, wounds were excised, and tensile strengths were measured and standardized. RESULTS: Nontreated diabetic wound tensile strength was 0.77 +/- 0.22 g/mm(2) and 1.51 +/- 0.13 g/mm(2) at 11 and 23 days. After LLLT, over 0-4 days tensile strength was 1.15 +/- 0.14 g/mm(2) and 2.45 +/- 0.29 g/mm(2) (P = 0.0019). Higher tensile strength at 23 days occurred in the 3- to 7-day group (2.72 +/- 0.56 g/mm(2) LLLT vs. 1.51 +/- 0.13 g/mm(2) nontreated; P < or = 0.01). CONCLUSION: Low-power laser irradiation at 830 nm significantly enhances cutaneous wound tensile strength in a murine diabetic model. Further investigation of the mechanism of LLLT in primary wound healing is warranted.

In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes.

BACKGROUND AND OBJECTIVE: The effects of low-level laser light irradiation are still highly contested, and the mechanisms of its action still unclear. This study was conducted to test the effects of low-level laser irradiation at 660 nm on human lymphocytes and to investigate the possible mechanisms by which these effects are produced. STUDY DESIGN/MATERIALS AND METHODS: Whole blood obtained by phlebotomy was irradiated at 660 nm by using energy fluences between 0 and 5.0 J/cm(2). The lymphocytes were isolated after irradiation of the whole blood. For the control experiment, the lymphocytes were first isolated and then irradiated at the same wavelength and energy fluence for comparison. The proliferation of lymphocytes and the formation of free radicals and lipid peroxides were monitored. Hemoglobin was also irradiated in a cell-free environment to test for the production of lipid peroxides. RESULTS: Lymphocyte proliferation was significantly higher (P<0.05) as expressed by a Stimulation Index in samples irradiated in the presence of whole blood compared with lymphocytes irradiated after isolation from whole blood. Free radical and lipid peroxide production also increased significantly when samples were irradiated in the presence of red blood cells. CONCLUSION: The present study supports the hypothesis that one mechanism for the photobiostimulation effect after irradiation at 660 nm is the reaction of light with hemoglobin, resulting in oxygen radical production.

Widespread susceptibility among inbred mouse strains to the induction of lupus autoantibodies by pristane.

Unlike other agents associated with drug-induced lupus, the isoprenoid alkane pristane induces autoantibodies pathognomonic of lupus, including anti-Sm, anti-dsDNA, and anti-ribosomal P in BALB/c and SJL/J mice. The susceptibility of other strains of mice to pristane-induced lupus is unknown and is the focus of the present study. Anti-nRNP/Sm, anti-Su, and anti-ribosomal P autoantibodies were produced by most strains of mice surveyed within several months of pristane treatment, although there was marked interstrain variability in their frequencies, levels, and times of onset. In sharp contrast, the production of autoantibodies against the double-stranded RNA binding proteins NF45/NF90/p110 was restricted to B6 and B10.S mice. We conclude that pristane selectively induces lupus-specific autoantibodies in virtually any strain of mouse regardless of its genetic background. However, H-2-linked as well as non-H2 genes influenced the expression of individual autoantibody markers. The widespread susceptibility of pristane-treated mice to lupus autoantibody production and the relatively small effect of MHC are unique features of this chemically induced lupus syndrome, with potential implications for understanding the pathogenesis of autoantibodies in idiopathic human systemic lupus erythematosus.

Induction of hypergammaglobulinemia and macrophage activation by silicone gels and oils in female A.SW mice.

Although most published epidemiological studies have found little evidence of systemic autoimmune disease associated with silicone breast implants, there still remains a question of whether silicones can cause local and/or systemic immune dysfunction. This study further investigates the effects of silicones on autoantibody and immunoglobulin production and macrophage activation in female A.SW mice. Sixty mice were divided among four treatment groups receiving a 0.5-ml intraperitoneal injection of either phosphate-buffered saline (PBS), pristane, silicone gel, or silicone oil. Test bleeds were taken periodically for 6 months. In contrast to pristane, neither silicone gel nor silicone oil induced lupus-associated antinuclear autoantibodies (immunoglobulin G [IgG] anti-nRNP/Sm, Su, and ribosomal P) or lupus nephritis. However, serum IgM became elevated persistently within 1 month of silicone gel or silicone oil administration. Also, the level of IgG3 was clearly elevated in silicone oil-treated mice. In contrast, IgG1, IgG2a, and IgG2b levels were not affected greatly by either silicone gel or oil. Furthermore, peritoneal macrophages from silicone- and pristane-treated mice produced higher levels of interleukin-1beta (IL-1beta) and IL-6 than those from PBS-treated mice after lipopolysaccharide stimulation. These results suggest that silicone gels and oils are capable of inducing hypergammaglobulinemia and activating macrophages in female A.SW mice.

Mechanisms of adjuvancy: I--Metal oxides as adjuvants.

The exact mechanism of how immune adjuvants function still remains largely unknown, despite their long history of use. This work reports the properties of alum and the related compounds Al(OH)3 or Al2O3. Experiments were performed in rats to determine the relative adjuvancy of silica, talc, ground glass, Al2O3, SnO2, ZrO2, hematite and magnetite. Antibody response and cell-mediated immunity (CMI) to ovalbumin (OVA) were determined and were found to be significantly enhanced by silica and talc. Antibody response to OVA was moderately enhanced by Al2O3, hematite, and magnetite, while CMI to OVA was not affected, SnO2, ZrO2, and ground glass only gave a slight adjuvant effect. The magnitude of adjuvancy appeared to correlate with the magnitude of the inflammatory response produced by each metal oxide and also correlated with their surface area. No correlation could be drawn between the hydrophilicity or hydrophobicity of the metal oxides and the magnitude of their adjuvancy.

Effects of photostimulation on wound healing in diabetic mice.

BACKGROUND AND OBJECTIVE: Low-level laser irradiation at certain fluences and wavelengths can enhance the release of growth factors from fibroblasts and stimulate cell proliferation in vitro. We evaluated whether low-level laser irradiation can improve wound healing in diabetes mellitus. STUDY DESIGN/MATERIALS AND METHODS: Genetically diabetic mice (C57BL/Ksj/db/db) were used as the animal model for this wound healing study. The experimental animals were divided among four groups: negative control, positive control (topical basic fibroblast growth factor [bFGF] on wound), laser therapy group; and a combination group of laser therapy and topical bFGF. An argon dye laser (Lexel Auora Model 600) at a wavelength of 630 nm and an output of 20 m W/cm2 was used as the light source. The speed of wound closure and histological evaluation were used to analyze the experimental results. RESULTS: Laser irradiation enhanced the percentage of wound closure over time as compared to the negative control group (58.4 +/- 2.6 vs. 40.8 +/- 3.4 at day 10 and 95.7 +/- 2 vs. 82.3 +/- 3.6 at day 20, P < .01). Histological evaluation showed that laser irradiation improved wound epithelialization, cellular content, granulation tissue formation, and collagen deposition in laser-treated wounds as compared to the negative control group (6.4 +/- 0.16 vs. 3.8 +/- 0.13 at day 10 and 12 +/- 0.21 vs. 8.2 +/- 0.31, P < .01). CONCLUSION: This study of laser biostimulation on wound healing in diabetic mice suggests that such therapy may be of great benefit in the treatment of chronic wounds that occur as a complication of diabetes mellitus.

Improvement of host response to sepsis by photobiomodulation.

BACKGROUND AND OBJECTIVE: Late sepsis causes immunosuppression and is associated with energy depletion in lymphocytes. Adjuvant treatment with ATP-MgCL2 appears to improve cellular energetics and decrease mortality. Laser irradiation can promote cell proliferation and increase cellular ATP synthesis, which may improve the host immune response in sepsis. The purpose of this study was to determine whether laser irradiation (LI) has a stimulatory effect on the immune response in sepsis using an animal model. STUDY DESIGN/MATERIALS AND METHODS: The cecal ligation and puncture (CLP) rat model was used. Thirty-six SD rats were divided equally among four groups: control (nonoperative), sham operation, CLP treated with laser irradiation, and CLP without laser irradiation. The peritoneal cavity of each animal in CLP/laser group was irradiated immediately after CLP using an Argon-dye laser at a wavelength of 630 nm and at a fluence of 5 J/cm2. Some animals were euthanized 24 hr following CLP and were used to evaluate the immune response (lymphocyte proliferation). In a separate experiment, the survival of septic rats was observed for 60 days. Lymphocytes isolated from normal rat spleens were used to observe for biostimulatory effects in vitro. RESULTS: LI significantly improved ex-vivo lymphocyte proliferation of cells from septic rats (179.7 +/- 17.2 vs. 129.5 +/- 7.8; P < 0.01) and enhanced survival in septic rats (79% vs. 42%; P < 0.001). LI significantly stimulated lymphocyte proliferation in the presence of mitogenic stimuli and enhanced lymphocyte ATP synthesis (P < 0.05). CONCLUSION: LI improves the host immune response and survival rate in sepsis in an animal model. Our studies suggest that LI may be useful as an adjuvant therapy for sepsis.

Adjuvancy effect of different types of silicone gel.

Women with silicone gel-filled breast implants (SBIs) are likely to be at a slightly higher risk of developing an autoimmune-like syndrome. This risk, although small, may be associated with the immunological adjuvancy property of the silicone gel. However, not all silicone gels are chemically formulated exactly the same and their adjuvancy behavior may vary. This study compared, in rats, the adjuvant effect of three different lots of silicone gel using ovalbumin (OVA) as the test antigen. Test bleeds were taken at 21, 48, 62, and 84 days post immunization and the rat sera were analyzed for anti-OVA antibodies by enzyme linked immunosorbent assay (ELISA). A delayed type hypersensitivity (DTH) test was performed on all the treated rats beginning at 14 post-immunization days. The results showed that silicone gel #3 (McGhan lot #S0400488) produced the highest mean anti-OVA antibody titer followed by silicone gel #1 (DC lot #HH019581) and silicone gel #2 (McGhan lot #DP9339). The DTH results showed that rats treated with silicone gel #1 and #3 had a clear positive response, whereas silicone gel #2 caused only a minimal response. These results demonstrate the immunological adjuvancy difference among three types of silicone gel. The chemical composition of each of these silicone gels, that would help explain these results, is yet to be determined.

Photomodulation of oxidative metabolism and electron chain enzymes in rat liver mitochondria.

Low-level laser irradiation has been applied in a variety of laboratory studies and clinical trials for photobiostimulation over the last three decades. Considerable skepticism exists regarding the concept of photostimulation within the medical community. One of the major difficulties with photoirradiation research is that it lacks experimentally supportable mechanisms for the alleged photobiostimulatory effects. This study was undertaken to determine whether oxidative metabolism and electron chain enzymes in rat liver mitochondria can be modulated by photoirradiation. Oxygen consumption, phosphate potential, and energy charge of rat liver mitochondria were determined following photoirradiation. Activities of mitochondrial enzymes were analyzed to assess the specific enzymes that are directly involved with the photostimulatory process. An argon-dye laser at a wave-length of 660 nm and at a power density of 10 mW/cm2 was used as a photon source. Photoirradiation significantly increased oxygen consumption (0.6 J/cm2 and 1.2 J/cm2, P < 0.05), phosphate potential, and the energy charge (1.8 J/cm2 and 2.4 J/cm2, P < 0.05) of rat liver mitochondria and enhanced the activities of NADH: ubiquinone oxidoreductase, ubiquinol: ferricytochrome C oxidoreductase and ferrocytochrome C: oxygen oxidoreductase (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2 and 4.8 J/cm2, P < 0.05). The activities of succinate ubiquinone oxidoreductase, ATPase, and lactate dehydrogenase were not affected by photoirradiation.

Effects of photodynamic therapy combined with methotrexate on C6 rat glioma cells: a preliminary study.

This study was undertaken to determine if methotrexate (MTX) is effective against tumor cells surviving photodynamic therapy (PDT). C6 rat glioma cells were exposed to Photofrin and irradiated at 630 nm using power densities of 1.2 or 4.8 J/cm2 to simulate the conditions for cells in solid tumors which survive PDT. Cells were then treated with MTX at 5.0, 0.5, or 0.05 microM concentrations. MTT assay of cell proliferation was performed at 24, 48, 72, and 96 h postirradiation. During the first 48 h of incubation, MTX alone was more effective than PDT and MTX. After 48 h, the combination treatment was more effective. Further studies of combined PDT and chemotherapy are warranted.

Induction of type II collagen arthritis in the DA rat using silicone gel as adjuvant.

The Dark Agouti (DA) rat has been shown recently to have a high susceptibility for developing arthritis when challenged with either heterologous or homologous collagen II mixed with mineral oil, or with mineral oil challenge alone. This study determined the arthritogenic potential of silicone gel by either mixing it with bovine collagen II (BII) or by injecting silicone gel alone in DA rats. The incidence of collagen induced arthritis was as follows: PBS group- 0/10, silicone gel group- 4/10, and IFA group- 8/9. Anti-BII antibodies were formed in most of the rats treated with either silicone gel or IFA and these groups of rats showed a positive DTH reaction. The PBS treated rats were negative for both anti-BII antibodies and DTH reaction. The incidence of arthritis formation in rats injected with silicone gel alone was 0/10, while the IFA injected rats showed an incidence of 8/10. Silicone gel taken from a commercial breast implant thus is capable of mediating collagen induced arthritis in the DA rat. However, silicone gel alone does not appear to be arthritogenic.

Induction of type II collagen arthritis in the DA rat using silicone gels and oils as adjuvant.

The relative safety (or otherwise) of silicone gel filled breast implants remains a controversial issue. The Dark Agouti (DA) rat has been shown recently to have a high susceptibility for developing arthritis. This study determined the arthritogenic potential of silicone gel, silicone oil, and the low molecular weight octamethylcyclotetrasiloxane (D4), by either mixing it with bovine collagen II (BII) or by injecting silicone gel alone in DA rats. Three separate experiments were performed using 110 female DA rats with 10 rats per treatment group. The incidence of collagen induced arthritis was as follows: Experiment I (6 micrograms BII)- PBS = 0/10, silicone gel = 4/10, and IFA = 8/9; Experiment II (125 micrograms BII)- PBS = 0/10, silicone gel = 7/10, IFA = 10/10, 1,000 cs silicone oil = 3/10, D4 = 0/10, and 1% D4 in 1,000 cs silicone oil = 1/10; Experiment III (adjuvant alone)-IFA = 8/10, silicone gel = 0/10. Anti-BII antibodies were formed in most of the rats treated with either silicone gel or IFA mixed with BII and these groups of rats showed a positive DTH reaction. The PBS treated rats were negative for both anti-BII antibodies and DTH reaction. Silicone gel taken from a commercial breast implant thus is capable of mediating collagen induced arthritis in the DA rat. However, silicone gel alone does not appear to be arthritogenic.

The effect of molecular weight and gel preparation on humoral adjuvancy of silicone oils and silicone gels.

Silicone gels from commercial breast implants have been shown previously by our laboratory to be potent humoral adjuvants, while the low molecular size 20 centistoke (cs) silicone oil (M.W. 1900) possesses no measurable adjuvant properties. It became necessary to shear the silicone gel during our previous experiments in order to facilitate injection through a syringe and needle, it is conceivable that shearing may reduce the molecular weight of the silicone gel used. This investigation was undertaken to determine whether humoral adjuvancy of silicone oils is dependent on molecular weight and whether the method of shearing the silicone gel affects its adjuvancy. Four Dow Corning 360 Medical silicone oils (100 cs, M.W. approximately 5,000; 350 cs, M.W. approximately 10,000; 1000 cs, M.W. approximately 16,500 and 12,500 cs, M.W. approximately 60,000) and Dow Corning octamethylcyclotetrasiloxane (D4, M.W. 296) were tested for their humoral adjuvancy by immunizing 64 Sprague Dawley rats with 50 micrograms of bovine serum albumin (BSA) mixed with each oil. The rats were periodically bled and the sera were analyzed for anti-BSA antibodies by ELISA. In a separate experiment, three silicone gel preparations with reproducible characteristics were prepared by using a tissue homogenizer and varying the applied shear force. Each of these preparations was tested for its humoral adjuvancy as previously described for silicone oils. Rats immunized with BSA mixed with the highest molecular size silicone oil tested (M.W. approximately 60,000) showed a significant increase in anti-BSA antibodies as compared to the lower molecular size oils. The three silicone gel preparations showed no difference in their adjuvancy effect. Thus, the humoral adjuvancy of silicone oil appears to be dependent on molecular weight. Differential shearing of the silicone gel does not alter its humoral adjuvancy.

The effect of silicone-gel on the immune response.

Silicone materials have been used in medical applications for at least 30 years. Despite this long history of use the question whether silicones can mediate an immunological reaction that may be detrimental to the host remains unanswered. Most studies on the biocompatability of silicones conclude that silicones are chemically stable compounds, which however are often capable of eliciting a benign chronic inflammatory response. Recently, our laboratory has conducted a series of animal experiments aimed at determining the immunological adjuvancy potential of silicone-gel taken from commercial breast implants. Our previous studies have indicated that silicone-gel is a potent humoral (antibody) adjuvant. Our present studies have found that silicone-gel is capable of eliciting auto-antibodies to rat thyroglobulin and bovine collagen II. However this immune response did not produce any histological evidence of thyroiditis or arthritis. Theories to explain why silicone-gel behaves as an adjuvant are discussed along with discussion of the hypothesis on the desirability of replacing silicone-gel with a more hydrophilic material in bioimplants.

Preliminary assessment of the Con-Bio laser in vivo.

A variety of lasers are available for the treatment of pigmented and vascular lesions. This paper presents our preliminary evaluation of the Q-switched 532 nm Con-Bio laser (Continuum Biomedical, Livermore, CA) in a murine model. Mice were anesthetized with intraperitoneal pentobarbital. Laser impacts (10 nsec, 150 mJ max.) were created on the ears at fluences of 1.5, 2, 3, 4, 5, and 6 J/cm 2. Acute study animals were sacrificed 15 min postinjury. Tissues were fixed in formalin and examined via light microscopy. Chronic study animals were allowed to heal for 3 weeks postoperatively. Parameters analyzed included the presence or absence of blistering, hemorrhage, visual assessment of thermal injury, microscopic evidence of vascular coagulation, or disruption and cosmesis. Acute vascular disruption and coagulation were present in all samples. Healing and cosmesis were good at all fluences tested. Tests of the laser in a prefocused mode produced a tissue cavitation effect with a depth of coagulation of 0.73 +/- 0.44 mm and hemorrhage of 0.68 +/- 0.41 mm. A chronic study of the vascular effect on larger vessels (1-2 mm) was conducted on a rabbit ear. Acute disruption was observed, however, all vessels were recanalized when examined grossly and histologically at 3 weeks. These preliminary results suggest that this new laser may be useful for the treatment of cutaneous vascular lesions. Further studies and clinical trials are warranted.

The effect of laser irradiation on the release of bFGF from 3T3 fibroblasts.

Studies have shown that low-level laser irradiation increases the proliferation of fibroblasts in cell culture. The mechanism of action is unknown. Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that has been detected in most tissues and which supports cell proliferation and differentiation. The purpose of this study was to determine whether laser irradiation (660 nm) can stimulate production of bFGF from fibroblast cells in cell culture. Our study showed that fibroblasts irradiated with laser energy at 2.16 J/cm2 demonstrated increased cell proliferation and enhanced production of bFGF, whereas fibroblasts irradiated with laser energy at 3.24 J/cm2 neither demonstrated increased cell proliferation or an enhanced release of bFGF as compared to the control group. These results provide direct evidence that the proliferation of fibroblasts as a result of stimulation by low level laser irradiation may be associated with the autocrine production of bFGF from fibroblasts.