Wavelength equivalence of red light between 595-nm and 695-nm for laser-induced hair regrowth

Transcription

1 Wavelength equivalence of red light between 595-nm and 695-nm for laser-induced hair regrowth Michael R Hamblin, PhD, Associate Professor, Department of Dermatology, Harvard Medical School Wellman Center for Photomedicine at Massachusetts General Hospital, Boston MA, Hamblin@helix.mgh.harvard.edu Phone: Abstract. Low-level laser or light therapy is used for hair regrowth in cases of male pattern baldness or androgenetic alopecia. Red light delivered by a laser or LED is absorbed in the mitochondria of cells in the hair follicle and activates transcription factors leading to stimulation of the anagen phase of the hair cycle. This review examines the work that has been published on comparing the relative effectiveness of different wavelengths of red light (between 595 and 695-nm) on biological endpoints relevant to stimulation of hair regrowth.

2 1. Introduction. Male androgenetic alopecia (AGA) is the most frequent type of thinning or loss of hair in males. The condition, also known as male pattern baldness, causes hair loss as early as late adolescence. The therapies that have been developed for AGA include minoxidil, propecia and laser therapy. 2. Low -level laser (light) therapy (LLLT). In 1967 a few years after the first working laser was invented, Endre Mester in Semmelweis University, Budapest, Hungary decided to test if laser radiation might cause cancer in mice [1]. He shaved the hair off their backs, divided them into two groups and gave a laser treatment with a low powered ruby laser (694-nm) to one group. They did not get cancer and to his surprise the hair on the treated group grew back more quickly than the untreated group. This was the first demonstration of "laser biostimulation". Since then the use of red light whether delivered by a laser or by a noncoherent light source such as light-emitting diodes has become widely used in medicine and physical therapy. There are many approved and generally accepted applications for indications such as wound healing, relief of pain and inflammation, and stimulation of tissue repair. 3. LLLT for hair regrowth A Spanish group has reported [2, 3] on the use of HeNe laser for both androgenic alopecia and alopecia areata. A report from Finland [4] compared three different light sources used for male-pattern baldness (HeNe laser, InGaAl diode laser at 670-nm and non-coherent 635-nm LED and measured blood flow in the scalp. 4. Molecular basis for LLLT. The first law of photobiology states that for low power visible light to have any effect on a living biological system, the photons must be absorbed by electronic absorption bands belonging to some molecular chromophore or photoacceptor [5]. One approach to finding the identity of this chromophore is to carry out action spectra. This is a graph representing biological photoresponse as a function of wavelength, wave number, frequency, or photon energy and should resemble the absorption spectrum of the photoacceptor molecule. The existence of a structured action spectrum is strong evidence that the phenomenon under study is a photobiological one (i.e., cellular photoacceptors and signaling pathways exist). The second important consideration involves the optical properties of tissue. Both the absorption and scattering of light in tissue are wavelength dependent (both much higher in the blue region of the spectrum than the red) and the principle tissue chromophores (hemoglobin and melanin) have high absorption bands at wavelengths shorter than 600-nm. Water begins to absorb significantly at wavelengths greater than 1150-nm. For these reasons there is a so-called optical window in tissue covering the red and near-infrared wavelengths, where the effective tissue penetration of light is maximized (Figure 1). Therefore although blue, green and yellow light may have significant effects on cells growing in optically transparent culture medium, the use of LLLT in animals and patients almost exclusively involves red and near-infrared light ( nm).

3 It was suggested in 1989 that the mechanism of LLLT at the cellular level was based on the absorption of monochromatic visible and NIR radiation by components of the cellular respiratory chain [6]. Respiration occurs in subcellular organelles called mitochondria. The inner mitochondrial membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (Complex III), cytochrome c oxidase (Complex IV), ATP synthase (Complex V) and two freely-diffusible molecules ubiquinone and cytochrome c that shuttle electrons from one complex to the next. In 1995, an analysis of five action spectra suggested that the primary photoacceptor for the red-nir range in mammalian cells is cytochrome c oxidase [7]. It is remarkable that the action spectra that were analyzed had very close (within the confidence limits) peak positions in spite of the fact that these are seemingly different processes. The enzyme contains two iron centers, haem a and haem a 3 (also referred to as cytochromes a and a 3 ), and two copper centers, Cu A and Cu B [8]. Fully oxidized cytochrome c oxidase has both iron atoms in the Fe(III) oxidation state and both copper atoms in the Cu(II) oxidation state, while fully reduced cytochrome c oxidase has the iron in Fe(II) and copper in Cu(I) oxidation states. All the many individual oxidation states of the enzyme have different absorption spectra [9], thus probably accounting for slight differences in action spectra of LLLT that have been reported. 5. Wavelength equivalence between nm. There have been a few studies that have compared the relative effectiveness of different wavelengths in the red region of the visible spectrum on some photobiomodulation endpoint. These studies have almost all been carried out in vitro. The reason for this preponderance of in vitro experiments is the following. The amount of work needed to compare different wavelengths in vivo in an animal study (or even more so if it were done in a clinical study in human patients) is overwhelming. Moreover the task of comparing the relative effectiveness of different wavelengths is much more complicated than it appears at first sight. This is because of the biphasic nature of the dose response curve. Actually there is not even clear agreement of what measurements should be used in constructing the dose response curve in the first place. Although delivered fluence or energy density (J/cm 2 ) is the most widely employed measurement of light dose, some investigators will use irradiance or power density (measured in mw/cm 2 ). These latter workers would maintain that LLLT should be delivered at a low irradiance and that this value is more important than the total energy density delivered (in other words the length of time light is on for). It has been realized for many years since the birth of LLLT that there exists and optimum dose of light for any particular application. In other words doses smaller or more importantly lager than this optimum value will have less effect. Doses very much larger than this optimum may not only have lost all positive biological effects, but may actually have an overall negative effect. This observation is sometimes termed the Arndt-Schulz law, in which low doses are said to be stimulatory while large doses are said to be inhibitory.

4 Figure 1. Graphical representation of biphasic dose response known as the Arndt-Schultz curve (taken from [10]). A graphical representation of the Arndt-Schulz dose response curve is shown in Figure 1 [10]. It must be said however that it would seem unlikely that the peak should be so sharp. It is almost a certainty that the exact position of the maximum effect on the Arndt-Schulz dose curve will be different for different wavelengths and for different biological applications. Therefore we can deduce from these comments that a rigorous investigation of the relative effectiveness of different wavelengths should include construction of a dose response curve broad enough to determine the position of the maximum effect. Not surprisingly this level of experimental detail has seldom if ever been performed. The studies that have been reported have basically taken a single light dose and compared the biological effects for a range of wavelengths. Although many practitioners of LLLT maintain that a laser is better than a non-coherent light source for producing biostimulation effects, it is also difficult to compare many wavelengths using laser light simply because a range of different lasers with different wavelengths is not available in many laboratories. There are two basic approaches to generating the different wavelengths of light that have been reported. Karu s group in Russia has used a broad band lamp and a monochromator that can be adjusted to produce band pass filtering effects (typically the light beam will have a full width half maximum of about 14-nm. It also possible to use a tunable pumped dye laser that can produce monochromatic light with a moderate degree of coherence such as the study reported by Moore et al [11].

5 Moore et al carried out an in vitro study that looked at the proliferation of two primary cultures of mouse cells (skin fibroblasts and aortic endothelial cells) when illuminated with laser light at an energy density of 10 J/cm2.delivered at an irradiance of 5 mw/cm2 over a time period of 30 minutes. The results shown in Figure 2 show a positive growth stimulating effect for both cells at all tested wavelengths in the red (although interestingly not at the NIR wavewlength of 810-nm). 675, 665, were the best 655-nm and 645-nm somewhat less good and 635 and 625-nm were least effective but still showed positive stimulation effects. It should be emphasized that this order of effectiveness of these wavelengths only applies at this specific fluence value and the order of effectiveness may be completely different at another fluence value (for instance at the popular fluence of 4J/cm 2 ). Karu has published many papers containing comparisons of different wavelengths [12-19]. The light was generated by a monochromator and the fluence was 0.01 J/cm 2 delivered at an irradidance of 1 mw/cm 2 for an exposure time of 10 seconds. and the biological endpoints were different and included proliferation, DNA synthesis, RNA synthesis, and adhesion to a glass substrate all measured in the human cervical carcinoma cell line known as HeLa cells. In some cases comparisons were made between actively proliferating (log-phase) and confluent (plateau phase) cultures [20]. Karu also investigated the Gram-negative bacterium Escherichia coli, in order to demostrate the broad applicability of her findings [21]. Figure 3 shows the action spectra obtained. Karu s analysis of the data [14] gave the following wavelength ranges for four peaks in the LLLT action spectrum: 1) nm, 2) nm, 3) nm, 4) nm.

6 Figure 3. Action spectra in the region of nm carried out on HeLa cells for (A) stimulation of DNA synthesis rate in log-phase and (B) plateau-phase cultures; (C) stimulation of RNA synthesis rate in logphase and (D) plateau-phase cultures; (E) increase of cell attachment to a glass matrix. Experimental curves adapted from [11], curve fittings (solid line), and Lorentzian fittings (dashed line) are shown. Dose 100 J/m 2 (A-D) or 52 J/m 2 (E). 6 Conclusion The studies summarized in this report taken together suggest that all tested wavelengths of light between 595-nm and 695-nm are capable of having a biological

7 stimulating effect in vitro when delivered at the appropriate fluence. Since it has been well established that certain red wavelengths can stimulate the growth of hair in men suffering from AGA, the logical conclusion is that all the red wavelengths would be capable of stimulating hair growth in men with AGA when the fluence, irradiance and treatment repetition schedule for each particular wavelength has been optimized.

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