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Abstracts and Articles re:NASA Light Emitting Diode Medical Applications
Harry T. Whelan1a,5,7, Ellen V. Buchmann1a,
Noel T. Whelan1a,7, Scott G. Turner1a,
Vita Cevenini7, Helen Stinson7, Ron Ignatius2, Todd Martin2,
Joan Cwiklinski1a, Glenn A. Meyer1c, Brian Hodgson3,4, Lisa Gould1b,
Mary Kane1b, Gina Chen1b, James Caviness6
1aDepartments of Neurology, 1bPlastic Surgery, 1cNeurosurgery,
Medical College of Wisconsin, Milwaukee, WI 53226, (414) 456-4090
2Quantum Devices, Inc Barneveld, WI 53507 (608) 924-3000
3Childrens Hospital of Wisconsin, Milwaukee, WI 53201 (414) 266-2044
44th Dental Battalion, 4th Force Service Support Group, USMCR, Marietta, GA
5Naval Special Warfare Group TWO, Norfolk, VA 23521, (757) 462-7759
6Submarine Squadron ELEVEN, San Diego, CA 92106, (619)553-8719
7NASA-Marshall Space Flight Center, AL 35812, (256) 544-2121
We have all heard how space technology can benefit us all here on earth; well this is no exception when we look at LED therapy. While the researchers in the field were fine-tuning their devices for pain relief, NASA needed a means to produce light without the added heat produced by incandescent light bulbs for space missions and their plant experiments. NASA settled on (LEDs) because of their ability to produce a scattered light of various wavelengths that were of benefit to plants in the confinements of a space vehicle in space flight, while producing no significant increase in thermal heat. They worked, and NASA took the next step. Could LEDs help in healing injuries to astronauts while in space flight. One of the major dilemmas for NASA regarding long-term space flight is the well-documented effect of muscle and bone atrophy that occurs to astronauts while in space. In addition it has been shown that injuries that occur while in space tend not to heal until the astronaut is back within the earths gravity. The LEDs that produced near-infrared light used in NASAs research were shown to stimulate the basic energy processes by activating color sensitive chemicals within the cells. DNA synthesis in fibroblasts and muscle cells had been quintupled. The light absorbed by the cells stimulated the metabolism in muscle and bone as well as skin and subcutaneous tissue. What people and animals had felt through utilizing this technology in real life, NASA was proving to be true in the laboratory.
LED-ENHANCEMENT OF CELL GROWTH
Studies on cells
exposed to microgravity and hypergravity indicate that human cells need
gravity to stimulate growth. As the gravitational force increases or
decreases, the cell function responds in a linear fashion. This poses
significant health risks for astronauts in long-term space flight. The
application of light therapy with the use of NASA LEDs will significantly
improve the medical care that is available to astronauts on long-term
space missions. NASA LEDs stimulate the basic energy processes in the
mitochondria (energy compartments) of each cell, particularly when near-infrared
light is used to activate the color sensitive chemicals (chromophores,
cytochrome systems) inside. Optimal LED wavelengths include 680, 730
and 880 nm and their laboratory has improved the healing of wounds in
laboratory animals by using both LED light and hyperbaric oxygen. Furthermore,
DNA synthesis in fibroblasts and muscle cells has been quintupled using
NASA LED light alone, in a single application combining 680, 730 and
880 nm each at 4 Joules per centimeter squared.
AN ISCHEMIA ANIMAL MODEL SYSTEM PROVIDE PRE-CLINICAL DATA RELEVANT TO
HUMAN HEALING PROBLEMS, CHRONIC NON-HEALING WOUNDS.
LED-Wound Healing in Rats
An ischemic wound is a wound in which there is a lack of oxygen to the wound bed due to an obstruction of arterial blood flow. Tissue ischemia is a significant cause of impaired wound healing which renders the wound more susceptible to infection, leading to chronic, non-healing wounds. Despite progress in wound healing research, we still have very little understanding of what constitutes a chronic wound, particularly at the molecular level, and have minimal scientific rationale for treatment.
In order to study the effects of NASA LED technology and hyperbaric oxygen therapy (HBO), we developed a model of an ischemic wound in normal Sprague Dawley rats. Two parallel 11-cm incisions were made 2.5 cm apart on the dorsum of the rats leaving the cranial and caudal ends intact. The skin was elevated along the length of the flap and two punch biopsies created the wounds in the center of the flap. A sheet of silicone was placed between the skin and the underlying muscle to act as a barrier to vascular growth, thus increasing the ischemic insult to the wounds. The four groups, each consisting of 15 rats, in this study include: the control (no LED or HBO), HBO only, LED (880 nm) only, and LED and HBO in combination. The HBO was supplied at 2.4 atm for 90 minutes, and the LED was delivered at a fluence of 4J/cm2 for fourteen consecutive days. A future study will incorporate the combination of three wavelengths (670nm, 728nm, and 880nm) in the treatment groups.
The wounds were traced manually on days 4, 7, 10, and 14. These tracings were subsequently scanned into a computer and the size of the wounds was tracked using SigmaScan Pro software. Figure 1 depicts the change in wound size over the course of the 14-day experiment. The combination of HBO and LED (880 nm) proves to have the greatest effect in wound healing in terms of this qualitative assessment of wound area. At day 7, wounds of the HBO and LED (880nm) group are 36% smaller than those of the control group. That size discrepancy remains even by day 10. The LED (880nm) alone also showed to speed wound closure. On day 7, the LED (880 nm) treated wounds are 20% smaller than the control wounds. By day 10, the difference between these two groups has dropped to 12%. This is due to the fact that there is a point when the wounds from all of the groups will be closed. Hence, the early differences are the most important in terms of determining the optimal effects of a given treatment. This can be seen in Figure 1 at day 14 when the points are converging due to the fact that the wounds are healing.
Analysis of the biochemical makeup of the wounds at days 4, 7, and 14 is currently underway. The day 0 time point was determined by evaluating the punch biopsy samples from the original surgery. The levels of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were determined using ELISA (enzyme linked immunosorbent assay). The changes in the VEGF concentration throughout the 14-day experiment can be seen in Figure 2. The LED (880 nm) group experiences a VEGF peak at day 4 much like the control group. In contrast, the hyperoxic effect of the HBO suppresses the day 4 peak, and instead, the HBO groups peak at day 7. The synergistic effect of the HBO and LED (880 nm) can be seen at day 4. The VEGF level for the group receiving both treatments is markedly higher at day 4 than the HBO only group. The HBO and LED (880 nm) treated group also experiences the day 7 peak characterized by the HBO treatment. Hence, there is a more uniform rise and fall to the VEGF level in the combined treatment group as opposed to the sudden increases seen in the control, LED only, and HBO only groups. By day 14, the HBO treated groups have dropped closer to the normal level than the LED (880 nm) only or control groups.
The synergistic effects of HBO and LED (880 nm) can be seen easily in Figure 3. The pattern of the changes in basic fibroblast growth factor (FGF-2) concentration is similar to that of the VEGF data. It is clear that the LED (880 nm) day 4 peak is higher than the day 4 peak of the control group. These peaks can be attributed to the hypoxic effect of the tissue ischemia created in the surgery. The hyperoxia of the HBO therapy has a greater effect on suppressing the FGF-2 concentration at day 4 than the VEGF concentration at the same time point. The synergy of the two treatments is evident when looking at the HBO and LED (880 nm) treated group. The concentration of FGF-2 at day 4 is significantly enhanced by the LED (880 nm) treatment. Whereas, the level would normally drop off by day 7 for a LED-only treated wound, the HBO effect seizes control causing the concentration of FGF-2 to plateau. Hence, an elevated FGF-2 concentration is achieved throughout the greater part of the 14 day treatment with both HBO and LED (880) therapies. Further analysis of the excised wounds will include matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) determination by ELISA, histological examination, and RNA extraction.
Figure 1. Change in wound size (%) in rat ischemic wound model.
LED-WOUND HEALING IN HUMAN SUBJECTS
Preclinical and clinical LED-Wound Healing studies were reported previously (Whelan et al., 1999, 2000); and additional human trials are summarized below:
Submarine atmospheres are low in oxygen and high in carbon dioxide, which compounds the absence of crew exposure to sunlight, making wound healing slower than on the surface. An LED array with 3 wavelengths combined in a single unit (670, 720, 880 nm) was delivered to Naval Special Warfare Group-2 in Norfolk and a data collection system has been implemented for musculoskeletal training injuries treated with NASA LEDs. Data collection instruments now include injury diagnosis, day from injury, range of motion measured with goniometer, pain intensity scales reported on scale 1-10, girth-circumferential measurements in cm, percent changes over time in all of the aforementioned parameters, and number of LED-treatments required for the subject to be fit-for-full-duty (FFD). Data have also been received from Naval Special Warfare Command (Norfolk & San Diego) where 18-20 patients per day are being treated with NASA-LEDs and results indicate >40% improvement in musculoskeletal training injuries. Data has also been received from the USS Salt Lake City (submarine SSN 716 on Pacific deployment) reporting 50% faster (7 day) healing of lacerations in crew members compared to untreated control healing (approximately 14 days).
FIGURE 3. Change in basic fibroblast growth factor (FGF-2) concentration (mg/mg Protein) vs. Time (Day) in rat ischemic wound model.
In order to better understand the effects of LEDs on cell growth and proliferation, we have measured radiolabeled thymidine incorporation in vitro in several cell lines treated with LEDs at various wavelengths and energy levels. As previously reported (Whelan, 2000), 3T3 fibroblasts (mouse derived skin cells) responded extremely well to LED exposure. Cell growth increased 150-200% over untreated controls. Additionally, we have treated osteoblasts (rat derived bone cells), and L6 rat skeletal muscle cells with LEDs and have found that both fibroblasts and particularly osteoblasts demonstrated a growth-phase specificity to LED treatment, responding only when cells are in the growth phase. In these experiments, fibroblasts and osteoblasts at a concentration of 1x104 cells/well were seeded in 24 well plates with a well diameter of 2 square centimeters. DNA synthesis was determined on the second, third and fourth days in culture for both fibroblasts (figure 1) and osteoblasts (figure 2). Exposure to LED irradiation accelerated the growth rate of fibroblasts and osteoblasts in culture for 2 to 3 days (growing phase), but showed no significant change in growth rate for cells in culture at 4 days (stationary phase). These data are important demonstrations of cell-cell contact inhibition, which occurs in vitro once cell cultures approach confluence. This is analogous in vivo to a healthy organism, which will regenerate healing tissue, but stop further growth when healing is complete. It is important to demonstrate that LED treatment accelerates this normal healing.