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The NASA Light-Emitting Diode Medical Program – Progress in Space Flight and Terrestrial Applications

Harry T. Whelan, M. D. 1a, 2,3, John M Houle, B. S. 1a,
Noel T. Whelan1a, 3, Deborah L. Donohoe, A. S., L. A. T. G. 1a,
Joan Cwiklinski, M. S. N., C. P. N. P. 1a, Meic H. Schmidt, M. D. 1c,
Lisa Gould, M. D., PhD. 1b, David Larson, M. D. 1b,
Glenn A. Meyer, M. D. 1a, Vita Cevenini3, Helen Stinson, B. S. 3

1a Departments of Neurology, 1bPlastic Surgery and 1cNeurosurgery,
Medical College of Wisconsin, Milwaukee, WI 53226, (414) 456-4090
2Naval Special Warfare Group TWO, Norfolk, VA 23521, (757) 462-7759
3NASA-Marshall Space Flight Center, AL 35812, (256) 544-2121

Abstract. This work is supported and managed through the NASA Marshall Space Flight Center – SBIR Program. Studies on cells exposed to
microgravity and hypergravity indicate that human cells need gravity to stimulate cell 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. LED-technology developed for
NASA plant grown experiments in space shows promise for delivering light deep into tissues of the body to promote wound healing and human
tissue growth. This LED-technology is also biologically optimal for photodynamic therapy of cancer.

The application of light therapy with the use of NASA LED's will significantly improve the medical care that is available to astronauts on
long-term space missions. NASA LED's 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. The depth of near-infrared light penetration into human tissue has been
measured spectroscopically (Chance, et al 1988). Spectra taken from the wrist flexor muscles in the forearm and muscles in the calf of
the leg demonstrate that most of the light photons at wavelengths between 630-800 nm travel 23 cm through the surface tissue and
muscle between input and exit at the photon detector. Our laboratory has improved the healing of wounds in laboratory animals by
using NASA 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.

Muscle and bone atrophy are well documented in astronauts, and various minor injuries occurring in space have been reported not to
heal until landing on Earth. Long term space flight, with its many inherent risks, also raises the possibility of astronauts being injured
performing their required tasks. The fact that the normal healing process is negatively affected by microgravity requires novel
approaches to improve wound healing and tissue growth in space. NASA LED arrays have already flown on Space Shuttle missions for
studies of plant growth. The U. S. Food and Drug Administration (FDA) has approved human trials. The use of light therapy with LED's
is an approach to help increase the rate of wound healing in the microgravity environment, reducing the risk of treatable injuries
becoming mission catastrophes.

Wounds heal less effectively in space than here on Earth. Improved wound healing may have multiple applications which benefit civilian
medical care, military situations and long-term space flight. Laser light and hyperbaric oxygen have been widely acclaimed to speed
wound healing in ischemic, hypoxic wounds. An excellent review of recent human experience with near-infrared light therapy for
wound healing was published by Conlan, et al in 1996. Lasers provide low energy stimulation of tissues which results in increased
cellular activity during wound healing (Beauvoit, 1989, 1995; Eggert, 1993; Karu, 1989; Lubart, 1992, 1997; Salansky, 1998; Whelan, 1999;
Yu, 1997). Some of these activities include increased fibroblast proliferation, growth factor syntheses, collagen production and
angiogenesis. Lasers, however, have some inherent characteristics, which make their use in a clinical setting problematic, including
limitations in wavelengths and beam width. The combined wavelengths of light optimal for wound healing cannot be efficiently
produced, and the size of wounds which may be treated by lasers is limited. Light-emitting diodes (LED's) offer an effective alternative
to lasers. These diodes can be made to produce multiple wavelengths, and can be arranged in large, flat arrays allowing treatment of
large wounds. Our experiments suggest potential for using LED light therapy at 680, 730 and 880 nm simultaneously, alone and in
combination with hyperbaric oxygen therapy, both alone and in combination, to accelerate the healing process in Space Station
Missions, where prolonged exposure to microgravity may otherwise retard healing. NASA LED's have proven to stimulate wound
healing at near-infrared wavelengths of 680, 730 and 880 nm in laboratory animals, and have been approved by the U. S. Food and Drug
Administration (FDA) for human trials. Furthermore, near-infrared LED light has quintupled the growth of fibroblasts and muscle cells
in tissue culture. The NASA LED arrays are light enough and mobile enough to have already flown on the Space Shuttle numerous
times. LED arrays may prove to be useful for improving wound healing and treating problem wounds, as well as speeding the return of
deconditioned personnel to full duty performance. Potential benefits to NASA, military, and civilian populations include treatment of
serious burns, crush injuries, non-healing fractures, muscle and bone atrophy, traumatic ischemic wounds, radiation tissue damage,
compromised skin grafts, and tissue regeneration.

Photodynamic therapy (PDT) is a cancer treatment modality that recently has been applied as adjuvant therapy for brain tumors. PDT
consists of intravenously injecting a photosensitizer, which preferentially accumulates in tumor cells, into a patient and then activating
the photosensitizer with a light source. This results in free radical generation followed by cell death. The development of more
effective light sources for PDT for brain tumors has been facilitated by applications of space light-emitting diode array technology; thus
permitting deeper tumor penetration of light and use of better photosensitizers. Lutetium Texaphyrin (Lutex) and Benzoporphyrin
Derivative (BPD) are new, second generation photosensitizers that can potentially improve PDT for brain tumors. Lutex and BPD have
major absorption peaks at 730 nm and 680 nm respectively, which gives them two distinct advantages. First, longer wavelengths of light
penetrate brain tissue easily so that larger tumors could be treated; and second, the major absorption peaks mean that more of the
drug is activated upon exposure to light. Tumorcidal effects of Lutex and BPD have been studied in vitro using canine glioma and
human glioblastoma cell cultures. Using light-emitting diedes (LED) with peak emissions of 728 nm and 680 nm as a light source, a
greater than 50 percent cell kill was measured in both cell lines by tumor DNA synthesis reduction. The effectiveness of Lutex and
BPD against tumor cells in vitro thus established, we have taken the first step toward determining their in vivo efficacy by performing
experiments to determine the largest doses of both Lutex, or BPD, and light that can be administered to dogs before toxicity is seen,
i. e. the maximum tolerated dose (MTD). Using this dose allows us to effect maximum tumor cell destruction during in vivo studies.

For longer wavelengths of light, the improved NASA LED-technology is required. LED's are an effective alternative to lasers for PDT.
Laser conversion to near-infrared wavelengths is inherently costly and inefficient, using an argon ion or KTP/ YAG laser beam that is
converted by a dye module, usually to 630 nm. LED's have been frequently used to emit longer wavelength broad spectrum near-infrared
light of 25-30 nm bandwidths. LED lamps traditionally consist of an array of semiconducting LED chips. In recent years,
improvements in semiconductor technology have substantially increased the light output of LED chips. A novel type of LED chip is
based on the semiconductor Aluminum Gallium Arsenide (AlGaAs). These LED chips have been manufactured to emit light with peak
wavelengths of 680 and 730 nm, which are optimal wavelengths for the absorption spectrum of the new photosensitizers used for
cancer PDT.

Human trials have begun at the Medical College of Wisconsin, Naval Special Warfare Command and NASA-Marshall Space Flight

Photodynamic Therapy with NASA LED Human Subjects
Preclinical studies of LED-photodynamic therapy were reported previously (Whelan, 1993, 1999; Schmidt, 1996, 1999)
The first patient treated with the NASA LED probe in our Photodynamic Therapy Phase II study, is a 20 year old female who
underwent PDT on May 4, 1999 for a brain tumor (anaplastic ependynoma). She had had two previous PDT treatments with laser light
in another area of the brain. The most recent tumor recurrence formed in a different location of the brain than the previous two sites
and is thought to be the result of the laser light not being able to penetrate the tissue deep enough to prevent recurrence. LED and
photofrin together allow for deeper penetration of tissue, thereby exposing surrounding tissue which may contain stray cancer cells to
the LED light.

Post surgically, she experienced some edema as is expected from brain surgery, and was treated successfully, and discharged on May 14,
1999. Follow-up MRI scans do not demonstrate tumor growth. A subsequent 21 year-old male with brain tumor (glioblastoma) was
treated August 25, 1999, with LED-photodynamic therapy, he tolerated the procedure well and shows no evidence residual tumor on
subsequent neuro imaging.

Preclinical LED-Wound Healing studies were reported previously (Whelan, 1999)

LED-Diabetic Mice
Type II, Diabetic Mice with excisional skin wounds were treated with LED's at 680, 730, and 880 nm, 4J/ cm2 of energy.
A repeated measures analysis was conducted using a General Linear Model with SqrtArea as the dependent variable and Treat as the
independent variable. The interaction effect Day* Treat is significant (p-value=. 0095), indicating there there is a significant difference
between treatments on some days. This test is of primary interest in this situation, because it shows that the treatments are effective
for some part of the treatment period. This analysis was carried out using the SAS statistical software package, published by the SAS
Institute, Inc.

LED-Human Subjects
Pt #1 is a 31 year-old white female insulin dependent diabetic (type 1) with end-stage renal disease status post renal transplant failure
and bilateral below knee amputations. Her left amputation site has poor healing compared to the right. She was receiving Hyperbaric
Oxygen therapy because of bilateral arterial insufficiency ulcers on her hands. In January of 1999 she had a hangnail on her right third
finger and sores developed at the tip. Multiple fissures and sores on both hands further occurred. Sores covered with black eschar
developed and her hands are dry with very limited range of motion. She has similar processes on both hands and had similar processes
developing on her feet prior to undergoing bilateral below the knee amputations. At that time she complained of cold-induced
cyanosis of the fingers and has been developing fissuring of the skin on her hands and some small spots on her fingers. She is a
nonsmoker and has been on dialysis for her renal failure. The renal failure and the arterial insufficiency ulcers of her extremities have
been felt to be due to vasculitis She does not use alcohol and has no drug allergies. She had renal transplant for renal failure in 1996
with removal of the transplanted kidney in 1998. She has received Prednisone and Cytoxan for her vasculitis and she also takes
Synthroid for hypothyroidism. She has had problems in the past with gastroparesis and had a temporary percutaneous endoscopic
gastrostomy tube in place, but has never had a diagnosis of gastroesophageal dysmotility. She has no history of calcinosis and she is
anuric because of renal failure. A renal biopsy performed December 5, 1997 of her transplanted kidney showed "focal necrotizing
glomerular nephritis, no evidence of acute rejection, no evidence of recurrent diabetic nephopathy". She received 20 treatments of 8
J/ cm2 NASA LED light therapy to all surfaces of both hands. Prior to starting treatment her hands were cold and dusky, lacking
sensation. They had many eschar ulcerations. After light treatment we noted an improvement in color and hands were warm to the
touch. Her complaints of pain and itching indicated a return of sensation.

PT #2 is a 76 year-old white mail, borderline diabetic with two open sores on his right medial malleolus. These wounds were
traumatic, caused when he was wounded by mortar fire during World War II in the Battle of the Bulge. He has had open sores in that
area since that time. These wounds have required two split thickness grafts. One was performed in 1945 and another in 1965. Both
grafts have been of questionable success. Mr. Marek has been treated with 52 Hyperbaric Oxygen Treatments to date and has received
19 treatments of 8 J/ cm2 of NASA LED therapy to his ankle wound. At the start of NASA LED treatment the area had opened into
one large 2cm X 3cm wound draining sanguinous fluid. Since treatment we have seen an increase in tissue granulation from the inside
towards the outer edges of the wound. Area has decreased to 1.5cm X 2.8cm and there is a significant decrease in drainage.
Transcutaneous Oxygen Measurement (T. C. pO2) readings have shown an improvement in vasculature to the area.

Further In Vitro LED Cell Growth Studies
In-vitro studies continue with the 3T3 Fibroblasts, and L6 Rat Skeletal Muscle cell line. Stimulation with LED's clearly shows
proliferation of both cell lines with the DNA synthesis assay. The studies indicate the 3T3 Fibroblasts with stimulation of 4 J/ cm2, 8
J/ cm2 and 12 J/ cm2 of energy show proliferation of cells within 3 hours after exposure to LED. Studies on the L6 Skeletal Muscle
depict growth over a period of 48 hours after exposure to 4 J/ cm2 of energy. This data demonstrates immediate benefit of LED
exposure in-vitro, as in the 3T3 Fibroblasts, but also over a period of time, over 48 hours with the L6 Muscle Cells.

We wish to thank Karen Zeqiri for assistance in manuscript preparation. The LED arrays were provided by Quantum Devices, Inc.,
Barneveld, WI. We also gratefully acknowledge the Department of Defense, Air Force Material Command, Armstrong Laboratories,
Davis Hyperbaric Laboratory, Brooks Air Force Base, TX for providing the hyperbaric chamber used in this research. The hyperbaric
oxygen treatments of our human subjects were performed by Estelle Woodard, C. R. T., C. H. T. This work was supported by the
National Aeronautics and Space Administration, Marshall Space Flight Center SBIR grants: NAS8-99015 and NAS8-97277, Children's
Hospital Foundation, the MACC Fund and Quantum Devices, Inc.

Beauvoit B., Evans S. M., Jenkins T. W., Miller E. E., Chance B., "Contribution of the Mitochondrial Compartment to the Optical Properties of the Rat
Liver: A Theoretical and Practical Approach," Analytical Biochemistry 226, 167-174 (1995).
Beauvoit B., Kitai T., Chance B., "Correlation between the Light Scattering and the Mitochondrial Content of Normal Tissues and Transplantable
Rodent Tumors," Biophysical Journal 67, 2501-25 10 (1994).
Chance B., Nioka S., Kent J., McCully K., Fountain M., Greenfield R., Holtom G., "Time-Resolved Spectroscopy of Hemoglobin and Myoglobin in
Resting and Ischemic Muscle," Analytical Biochemistry 174, 698-707 (1988)
Conlan M. J., Rapley J. W., Cobb C. M., "Biostimulation of wound healing by low-energy laser irradiation," J. Clin. Periodont. 23, 492-496 (1996).
Eggert H. R., Blazek V., "Optical Properties of Normal Human Brain Tissues In The Spectral Range of 400 to 2500 nm," Advances in Experimental
Medicine & Biology 333, 47-55 (1993).
Karu T., "Photochemical Effects Upon the Cornea, Skin and Other Tissues (Photobiology Of Low-Power Laser Effects," Hlth Physics 56, 69 1-704
Lubart R., Friedman H., Sinyakov M., Cohen N., Breitbart H., "Changes in Calcium Transport in Mammalian Sperm Mitochondria and Plasma
Membranes Caused by 780 nm Irradiation," Lasers in Surg & Med 21, 493-499 (1997).
Lubart R., Wollman Y., Friedman H., Rochkind S. Laulicht L., "Effects of visible and near-infrared lasers on cell cultures," Journal of Photochemistry
& Photobiology 12( 3), 305-3 10 (1992).
Salansky N., "Low energy photon therapy for wound healing." Intnl Med Instr, Canadian Defense Ministry, Personal Communication. (1998).
Schmidt M. H., Bajic D. M., Reichert K. W. II, Martin T. S., Meyer G. A., Whelan H. T., "Light –emitting diodes as a light source for intra-operative
photodynamic therapy." Neurosurg 38( 3), 552-556 (1996).
Schmidt M. H., Reichert K. W. II, Ozker K., Meyer G. A., Donohoe D. L., Bajic D. M., Whelan N. T., Whelan H. T., "Preclinical Evaluation of
Benzoporphyrin Derivative Combined with a Light-Emitting Diode Array for Photodynamic Therapy of Brain Tumors." Pediatr Neurosurg 30, 225-
231 (1999).
Whelan H. T., Schmidt M. H., Segura A. D., McAuliffe T. L., Bajic D. M., Murray K. J., Moulder J. E., Strother D. R., Thomas J. P., Meyer G. A., "The role of
photodynamic therapy in posterior fossa brain tumors: A pre-clinical study in a canine glioma model." Journal of Neurosurgery 79( 4), 562-8
Whelan H. T., Houle J. M., Donohoe D. L., Bajic D. M., Schmidt M. H., Reichert K. W., Weyenberg G. T., Larson D. L., Meyer G. A., Caviness J. A., "Medical
Applications of Space Light-Emitting Diode Technology— Space Station and Beyond." Space Tech. & App Int'l Forum 458, 3-15 (1999).
Yu W., Naim J. O., Lanzafame R. J., "The Effect Of Laser Irradiation On The Release Of bFGF From 3T3 Fibroblasts." Photochemistry & Photobiology
59, 167-70 (1994).


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