In our recent studies, we employed *in vivo* phage display technique and intended to identify peptides that will specifically home to radiation or drug treatment responsive tumors (Han et al., 2008; Passarella et al., 2009). During the studies, we first treated tumors in mice with radiation and tyrosine kinase inhibitors. Then a peptide phage library was injected from the tail vein of tumor bearing mice for tumor binding screening. After several rounds of *in vivo* screening and enrichment of phages isolated from the treated tumors (Fig. 2), one phage clone, encoding HVGGSSV peptides, was identified preferentially target to treatment responsive tumors. The binding preference of those phages were confirmed by fluorescence labeled phage or peptide imaging (Han et al., 2008; Passarella et al., 2009).

#### **5.2 HVGGSSV peptide as imaging/targeting probe for radiation responsive tumors**

To explore HVGGSSV peptide's clinical application in noninvasive imaging of tumor response to treatment, fluorescent labeled HVGGSSV peptide were used to target human tumors in several mouse models. Optical imaging studies indicated that the signal intensities of peptide binding within tumors correlate to the overall efficacy of treatment regimens on tumor growth control in multiple tumor models that had been treated with a variety of RTK inhibitors with or without combination of radiation (Han et al., 2008). SPECT/CT provides high spatial resolution and sensitivity in functional imaging. We Phage-Displayed Recombinant Peptides for Non-Invasive Imaging Assessment of Tumor Responsiveness to Ionizing Radiation and Tyrosine Kinase Inhibitors 503

502 Current Topics in Ionizing Radiation Research

been used for magnetic resonance imaging (MRI) (Frangioni, 2003; Reubi and Maecke, 2008). Peptides can also be conjugated to other tumor targeted polymers or nanoparticles and dramatically increase their tumor targeted selectivity and efficiency (Hariri et al., 2010;

Fig. 3. HVGGSSV peptide labeled with near-infrared (NIR) fluorescent dyes specifically located to radiation-treated tumors.  HVGGSSV peptide as one imaging probes to detect tumor response to**

In our recent studies, we employed *in vivo* phage display technique and intended to identify peptides that will specifically home to radiation or drug treatment responsive tumors (Han et al., 2008; Passarella et al., 2009). During the studies, we first treated tumors in mice with radiation and tyrosine kinase inhibitors. Then a peptide phage library was injected from the tail vein of tumor bearing mice for tumor binding screening. After several rounds of *in vivo* screening and enrichment of phages isolated from the treated tumors (Fig. 2), one phage clone, encoding HVGGSSV peptides, was identified preferentially target to treatment responsive tumors. The binding preference of those phages were confirmed by fluorescence

**5.2 HVGGSSV peptide as imaging/targeting probe for radiation responsive tumors**  To explore HVGGSSV peptide's clinical application in noninvasive imaging of tumor response to treatment, fluorescent labeled HVGGSSV peptide were used to target human tumors in several mouse models. Optical imaging studies indicated that the signal intensities of peptide binding within tumors correlate to the overall efficacy of treatment regimens on tumor growth control in multiple tumor models that had been treated with a variety of RTK inhibitors with or without combination of radiation (Han et al., 2008). SPECT/CT provides high spatial resolution and sensitivity in functional imaging.  (Adapted from Han et al., 2008).

**5.1 Discovery of HVGGSSV peptide**

Fig. 4. SPECT/CT imaging of the HVGGSSV peptide within LLC tumors after treatment. The biotinylated HVGGSSV peptide was complexed with iodine-125-labeled streptavidin. The implanted tumor was treated once with radiation (5 Gy) alone (**A**), or combination of Sunitinib (40 mg/Kg) and radiation (5 Gy) (**B**) before intravenous administration of the imaging probe. Shown are 3D virtual rendering (3D-VR) images (far left) and hybrid SPECT/CT fusion images in the coronal, sagittal, and transaxial planes (the second to the fouth from the left, respectively) acquired 4 hours after the imaging probe administration. The LLC tumors was pointed with arrows, high resolution images enable spatially localizing the radiation-responding cells within the peripheral and central regions of the tumors.

Phage-Displayed Recombinant Peptides for Non-Invasive Imaging

peptides will play a major role in the future molecular imaging.

**6. Perspectives**

**7. References**

Assessment of Tumor Responsiveness to Ionizing Radiation and Tyrosine Kinase Inhibitors 505

The treatment-induced TIP-1 expression on the cell surface is detectable in the first few hours after the treatment and before the onset of treatment associated apoptosis or cell death. Majority of the cells expressing TIP-1 on the cell surface are the live albeit such cells are less potent in proliferation and more susceptible to subsequent radiation treatment (Wang et al., 2010). The increased susceptibility to the subsequent irradiation might explain why the peptide binding is predictive in assessing tumor overall responsiveness in the early stage of treatment course. The treatment-inducible TIP-1 translocation before the onset of cell apoptosis or death further suggests potentials of the HVGGSSV peptide in non-invasive

The development of imaging technologies revolutionizes medial diagnosis and clinical management. Functional molecular imaging becomes one critical part of personalized medicine. Peptide, with its small sizes and versatile structures, is increasingly recognized as a promising imaging probe to predict the outcomes of radiotherapy and other medical treatments. Although some disadvantages associated with peptides, such as its degradation inside human body and its low affinity with its targets, with chemical modification to improve its stability and association with nanoparticles to increase its binding affinity,

Allal, A. S., Slosman, D. O., Kebdani, T., Allaoua, M., Lehmann, W., and Dulguerov, P.

Aloj, L., and Morelli, G. (2004). Design, synthesis and preclinical evaluation of radiolabeled peptides for diagnosis and therapy. Current pharmaceutical design *10*, 3009-3031. Arap, W., Kolonin, M. G., Trepel, M., Lahdenranta, J., Cardo-Vila, M., Giordano, R. J., Mintz,

Brown, J. M. (1975). Selective radiosensitization of the hypoxic cells of mouse tumors with the nitroimidazoles metronidazole and Ro 7-0582. Radiation research *64*, 633-647. Cai, W., Niu, G., and Chen, X. (2008). Imaging of integrins as biomarkers for tumor

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imaging properties of an (111)In-labeled galectin-3 binding peptide in prostate

imaging assessment of tumor response to radiation and tyrosine kinase inhibitors.

employed this imaging modality to detect tumor response to radiation by using the HVGGSSV peptide. The mice were treated with radiation alone or combination of radiation and one TKI - Sunitinib (40 mg/Kg,). After the treatment, the HVGGSSV peptide complexed with 125I-labeled streptavidin was selectively targeted to the tumors treated with radiation or radiation combined with Sunitinib. High resolution SPECT/CT images (Fig. 4) also showed that majority of the imaging probes were located in the peripheral area of the tumors that were treated with radiation alone. However, treatment with radiation and Sunitinib extended the imaging probe binding to both the peripheral and central parts of the subcutaneous tumors. This data might reflect the radiosensitization effect of Sunitinib.

The tumor targeting potential of HVGGSSV peptide has been further explored in several drug delivery studies. In these studies, HVGGSSV peptide has been conjugated to different nanoparticles, such as liposome, FePt, and nanoparticle albumin bound (nab) (Hariri et al., 2011; Hariri et al., 2010; Lowery et al., 2011), and selectively targeted those nanoparticles to irradiated tumors. One study also showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel-treated groups and significantly increase tumor growth delay as compared with controls (Hariri et al., 2010).

## **5.3 The biological basis of the HVGGSSV peptide imaging**

#### **5.3.1 Peptide receptor identification**

To understand the physiology underlines peptides binding, we need to identify the molecular targets of peptides. However, peptides are usually unstable. Their surface charges and structures will change dramatically in different environment.  Therefore, traditional affinity purification methods are of little use because of high background of nonspecific binding. To date, there are very few identified receptors for peptides in contrast to the great number of discovered cancer targeting peptides (Sugahara et al., 2009). New strategies are needed for identifying peptide's receptors.

### **5.3.2 TIP-1 as a molecular target of HVGGSSV peptide**

In our recent studies of one peptide (HVGGSSV), we utilized a phage cDNA library screening to search for peptide's receptors. Because several rounds of phage display screening can significantly enrich the low-affinity or low-abundance proteins, we successfully identified a PDZ protein - TIP-1 as the target of HVGGSSV peptide (Wang et al., 2010). Through the PDZ domain, TIP-1 binds to the classic C-terminal PDZ motif within the HVGGSSV peptide. One TIP-1-specific antibody that inhibited the in vitro interaction between TIP-1 and the HVGGSSV peptide attenuated the peptide's accumulation within irradiated tumors. Imaging with TIP-1-specific antibody recapitulated the pattern of peptide imaging in tumor-bearing mice. Mutation in the classic PDZ binding motif of the HVGGSSV peptide destroyed the specific binding within irradiated tumors. These results also demonstrated the potentials of screening phage-displayed cDNA library in discovery of molecular targets of the peptides with a simple structure and low affinity.

#### **5.3.3 The biological relevance of TIP-1 relocation onto tumor cell surface to the radiation response of tumor cells**

With a TIP-1 specific antibody, it was further identified that radiation induced translocation of the basically intracellular TIP-1 protein onto the cell surface in a dose-dependent manner. The treatment-induced TIP-1 expression on the cell surface is detectable in the first few hours after the treatment and before the onset of treatment associated apoptosis or cell death. Majority of the cells expressing TIP-1 on the cell surface are the live albeit such cells are less potent in proliferation and more susceptible to subsequent radiation treatment (Wang et al., 2010). The increased susceptibility to the subsequent irradiation might explain why the peptide binding is predictive in assessing tumor overall responsiveness in the early stage of treatment course. The treatment-inducible TIP-1 translocation before the onset of cell apoptosis or death further suggests potentials of the HVGGSSV peptide in non-invasive imaging assessment of tumor response to radiation and tyrosine kinase inhibitors.

## **6. Perspectives**

504 Current Topics in Ionizing Radiation Research

employed this imaging modality to detect tumor response to radiation by using the HVGGSSV peptide. The mice were treated with radiation alone or combination of radiation and one TKI - Sunitinib (40 mg/Kg,). After the treatment, the HVGGSSV peptide complexed with 125I-labeled streptavidin was selectively targeted to the tumors treated with radiation or radiation combined with Sunitinib. High resolution SPECT/CT images (Fig. 4) also showed that majority of the imaging probes were located in the peripheral area of the tumors that were treated with radiation alone. However, treatment with radiation and Sunitinib extended the imaging probe binding to both the peripheral and central parts of the subcutaneous tumors. This data might reflect the radiosensitization effect of Sunitinib. The tumor targeting potential of HVGGSSV peptide has been further explored in several drug delivery studies. In these studies, HVGGSSV peptide has been conjugated to different nanoparticles, such as liposome, FePt, and nanoparticle albumin bound (nab) (Hariri et al., 2011; Hariri et al., 2010; Lowery et al., 2011), and selectively targeted those nanoparticles to irradiated tumors. One study also showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel-treated groups and significantly increase

To understand the physiology underlines peptides binding, we need to identify the molecular targets of peptides. However, peptides are usually unstable. Their surface charges and structures will change dramatically in different environment.  Therefore, traditional affinity purification methods are of little use because of high background of nonspecific binding. To date, there are very few identified receptors for peptides in contrast to the great number of discovered cancer targeting peptides (Sugahara et al., 2009). New

In our recent studies of one peptide (HVGGSSV), we utilized a phage cDNA library screening to search for peptide's receptors. Because several rounds of phage display screening can significantly enrich the low-affinity or low-abundance proteins, we successfully identified a PDZ protein - TIP-1 as the target of HVGGSSV peptide (Wang et al., 2010). Through the PDZ domain, TIP-1 binds to the classic C-terminal PDZ motif within the HVGGSSV peptide. One TIP-1-specific antibody that inhibited the in vitro interaction between TIP-1 and the HVGGSSV peptide attenuated the peptide's accumulation within irradiated tumors. Imaging with TIP-1-specific antibody recapitulated the pattern of peptide imaging in tumor-bearing mice. Mutation in the classic PDZ binding motif of the HVGGSSV peptide destroyed the specific binding within irradiated tumors. These results also demonstrated the potentials of screening phage-displayed cDNA library in discovery of

tumor growth delay as compared with controls (Hariri et al., 2010).

**5.3 The biological basis of the HVGGSSV peptide imaging**

strategies are needed for identifying peptide's receptors.

**5.3.2 TIP-1 as a molecular target of HVGGSSV peptide**

molecular targets of the peptides with a simple structure and low affinity.

**5.3.3 The biological relevance of TIP-1 relocation onto tumor cell surface to the**

With a TIP-1 specific antibody, it was further identified that radiation induced translocation of the basically intracellular TIP-1 protein onto the cell surface in a dose-dependent manner.

**5.3.1 Peptide receptor identification**

**radiation response of tumor cells**

The development of imaging technologies revolutionizes medial diagnosis and clinical management. Functional molecular imaging becomes one critical part of personalized medicine. Peptide, with its small sizes and versatile structures, is increasingly recognized as a promising imaging probe to predict the outcomes of radiotherapy and other medical treatments. Although some disadvantages associated with peptides, such as its degradation inside human body and its low affinity with its targets, with chemical modification to improve its stability and association with nanoparticles to increase its binding affinity, peptides will play a major role in the future molecular imaging.

## **7. References**

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**24**

*Argentina*

**Biosafety in the Use of Radiation:**

**Infrared and Ultraviolet Lamps in an**

Rodolfo Esteban Avila, Maria Elena Samar, Gustavo Juri,

*Department of Cell Biology, Histology and Embryology, Faculty of Medical Sciences, National University of Cordoba,*

Juan Carlos Ferrero and Hugo Juri

**Biological Effects Comparison Between**

**Laser Radiation, Intense Pulsed Light and**

**Experimental Model in Chicken Embryos**

Physics is the science that studies the interaction between matter and energy. This discipline studies the general properties of bodies, the forces that modify the transfer of energy and the interaction between particles. Physics now has many branches, and one of them is the

Biomedical Physics in Medicine applies the principles and methods of physics. This will generate new knowledge and progress towards new horizons in the management of certain diseases. Thus the study of radiation led to progress in the field of diagnosis and therapy of

Radiation it's called all energy that propagates as a wave through space. Radiation can be classified as ionizing radiation (cosmic rays, gamma rays, x-rays) and non-ionizing radiation. The concept of non-ionizing radiation includes ultraviolet (UV), infrared (IR) and

Non-ionizing radiations are those that radiation interaction with matter does not generate

Non-ionizing radiation (laser, ultraviolet and infrared) are commonly used in medicine for diagnosis and therapy. However, they have a deleterious effect on organogenesis (organ

UV radiation is part of the natural light. According to its wavelength is recognized groups A

UV radiation sources are natural (the sun) and artificial (hospitals, industries, cosmetics, etc.). UVC radiation does not reach the surface because it is retained by the layer of ozone in the

**1. Introduction**

Biomedical Physics.

some diseases.

others lasers.

formation).

stratosphere.

**1.1 Ultraviolet radiation (UV)**

ions due to its energy content is relatively low.

(400 - 320 nm), B (320 - 290 nm) and C (290 - 200 nm).

## **Biosafety in the Use of Radiation: Biological Effects Comparison Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in an Experimental Model in Chicken Embryos**

Rodolfo Esteban Avila, Maria Elena Samar, Gustavo Juri, Juan Carlos Ferrero and Hugo Juri *Department of Cell Biology, Histology and Embryology, Faculty of Medical Sciences, National University of Cordoba, Argentina*

## **1. Introduction**

510 Current Topics in Ionizing Radiation Research

Valadon, P., Garnett, J. D., Testa, J. E., Bauerle, M., Oh, P., and Schnitzer, J. E. (2006).

Vallerga, A. K., Zarling, D. A., and Kinsella, T. J. (2004). New radiosensitizing regimens, drugs, prodrugs, and candidates. Clin Adv Hematol Oncol *2*, 793-805. Wang, H., Yan, H., Fu, A., Han, M., Hallahan, D., and Han, Z. (2010). TIP-1 translocation

Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M. (2000). Selection of

Williams, K. J., Telfer, B. A., Brave, S., Kendrew, J., Whittaker, L., Stratford, I. J., and Wedge,

Zhivotosky, B., and Orrenius, S. (2001). Assessment of apoptosis and necrosis by DNA

vivo. Proc Natl Acad Sci U S A *103*, 407-412.

antitumor activity. Clin Cancer Res *10*, 8587-8593.

ionizing radiation. PloS one *5*, e12051.

Nature *405*, 665-668.

18 13.

Screening phage display libraries for organ-specific vascular immunotargeting in

onto the cell plasma membrane is a molecular biomarker of tumor response to

peptides with semiconductor binding specificity for directed nanocrystal assembly.

S. R. (2004). ZD6474, a potent inhibitor of vascular endothelial growth factor signaling, combined with radiotherapy: schedule-dependent enhancement of

fragmentation and morphological criteria. Curr Protoc Cell Biol *Chapter 18*, Unit

Physics is the science that studies the interaction between matter and energy. This discipline studies the general properties of bodies, the forces that modify the transfer of energy and the interaction between particles. Physics now has many branches, and one of them is the Biomedical Physics.

Biomedical Physics in Medicine applies the principles and methods of physics. This will generate new knowledge and progress towards new horizons in the management of certain diseases. Thus the study of radiation led to progress in the field of diagnosis and therapy of some diseases.

Radiation it's called all energy that propagates as a wave through space. Radiation can be classified as ionizing radiation (cosmic rays, gamma rays, x-rays) and non-ionizing radiation. The concept of non-ionizing radiation includes ultraviolet (UV), infrared (IR) and others lasers.

Non-ionizing radiations are those that radiation interaction with matter does not generate ions due to its energy content is relatively low.

Non-ionizing radiation (laser, ultraviolet and infrared) are commonly used in medicine for diagnosis and therapy. However, they have a deleterious effect on organogenesis (organ formation).

## **1.1 Ultraviolet radiation (UV)**

UV radiation is part of the natural light. According to its wavelength is recognized groups A (400 - 320 nm), B (320 - 290 nm) and C (290 - 200 nm).

UV radiation sources are natural (the sun) and artificial (hospitals, industries, cosmetics, etc.). UVC radiation does not reach the surface because it is retained by the layer of ozone in the stratosphere.

Biosafety in the Use of Radiation: Biological Effects Comparison

directional light.

Lasers and biomedical use:

pacemakers or pregnant women.

**1.4 Intense Pulsed Light (IPL)**

et al., 1993, Avila et al., 1994 , Samar et al., 1995)

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 513

The best known property is the emission of beams of highly monochromatic, coherent and

There are different laser for medical use, classified as high-power-like carbon dioxide (CO2),

The term laser means in English Light Amplification by Stimulated Emission of Radiation. The laser is a device or devices that produce a very special light (visible or invisible

Einstein laid the theoretical basis for his work on the Quantum Theory of Radiation in 1917,

As light is reflected, the laser is absorbed, burning, and changes its direction through

The use of laser energy is not without several problems as the high price of the apparatus and the need for prior learning to use. In addition there is one type of laser that fits all

From the medical point of view the use of laser can be used to remove tumors, seal blood vessels to reduce blood loss, sealed lymph vessels to reduce swelling and decrease the

Aesthetic laser therapy treatments can be used to remove warts and moles and to remove tattoos. It is also used to treat stretch marks, cellulite, sagging skin, acne sequels.It is based on the fundamental physiological properties of the laser, anti-inflammatory, spasmolytic

Solid lasers: the most used is Neodymium-Yag laser. In ophthalmology it is used to

 Gas lasers: they are the most widely used therapeutic and amongst these the heliumneon laser, used in beauty treatments, therapies reductive, etc. (red), the argon laser used in dermatology (bluish green) and the laser CO2 used in surgery as a scalpel. The Helium-Neon laser is used to treat various conditions "satisfactory" mainly

Among the lasers used in medical practice are the 308nm excimer. This type of laser emits

Contraindications: The laser should not be used in patients affected by neoplasia (cancer). Nor can it be used in the presence of acute infections and in patients treated with photosensitizing drugs or remedies. Not recommended laser surgery in people with

In a previous study, we irradiated chick embryos and new born chickens with He-Ne laser, infrared and ultraviolet radiation, finding post irradiation histopathologic changes. (Samar

In addition to the laser, since 1995 it also has been available a device that emits at 308 nm, called intense pulsed light (IPL) which is basically a XeCl lamp that has proved to be a useful tool for the treatment of the changes of the skin. The IPL provides high energy pulsed excitation consisting of 85% of ultraviolet radiations UV B and 15% of radiations UV A. This technology is also known as "Photoderm". Although the active medium of this lamp is also XeCl, the emission is polychromatic but also non-coherent; therefore is not a laser. However,

applications, but each type has it sitting indications, many of which are unique to it.

for surgical use, and low power-such as Helium-Neon (He-Ne) for clinical use.

depending on their wavelength), created by man and acts like solid matter.

by which the energy (light) can be converted into mass and mass into energy.

spread of tumor cells and nerve endings sealed to reduce postoperative pain.

and antiphlogistic effects and bio-stimulants. (Andreu & Valiente Zaldivar 1996).

coagulate tissue. It is also used to treat hyperpigmentation of the skin

osteomioarticular (rehabilitation), skin disorders and wound healing.

85% of ultraviolet (UV) B and 15% of UV radiation type A.

different lenses. As solid mass you can cut, melt, burn, and transmit.

The natural radiation that reaches us is UVA and UVB. The UVC is the most dangerous to health because of its higher energy.

As for the benefits of using ultraviolet (UV), is recognized for use in phototherapy of patients with psoriasis, vitiligo and other skin diseases. In addition, ultraviolet rays have a bactericidal action, allowing its use in pressure ulcers.

However, involvement of UV radiation in different pathologies has led to the approach of concepts such as photo-aging, accelerated aging process due to modification by UV radiation, DNA (deoxyribonucleic acid) and lipids membrane and induction of programmed cell death (apoptosis) of epithelial cells and the activation of enzymes degrading the collagen in the skin.

While studying the effects of ultraviolet radiation on the apoptotic induction and angiogenesis the results are mixed for use in the therapy of skin diseases including cancer and photocarcinogenesis, understood as the mechanism of DNA mutation due to the alteration of repair when damaged by UV radiation.

Ultrviolet radiation is involved in the alterations of protein synthesis, the immunosuppressive properties and Its relationship to skin cancer and change in the synthesis of melanin.

Ultraviolet radiation B are related to the induction and progression of cutaneous melanoma in mice, so its use helps to know the specific immune processes committed to these cancers, and thus to develop new treatments for melanoma. This type of radiation has also been used experimentally to elucidate the mechanisms of cellular radioresistance in ovarian cell lines.

## **1.2 Infrared radiation (IR)**

Other natural rays are infrared (IR) or infrared radiation (the prefix infra means below, correspond to an emission of energy in the form of electromagnetic waves in the spectrum located immediately after the red zone). These rays have wavelengths between 800 and 0.25 nm and cause heating of the exposed regions, being the less penetrating wavelength formerly called them "calorific rays."

As for the infrared rays have a wavelength between 800 and 0.25 nm, cause heating of the exposed areas and those are more penetrating radiation of shorter wavelength. Special lamps are used commercially to produce 14,000 to infrared.

Its heating effect allows blood vessels to dilate and increase blood flow.

They are also used in conjunction with hyperthermia and photodynamic therapy to treat tumor in the ablation of organs, and therapy of processes related to inflammation of nerves, muscles and joints.

It has been postulated that IR produce a higher activity of phagocytosis and metabolic reactivity in infectious and has been used in phototherapy of patients with certain skin tumors.

However, the intracellular level, has shown that infrared radiation acting microtubule disrupting the centrosome (organoids) in mammalian cells in vitro.

#### **1.3 Laser radiation**

The laser light (artificial) could be defined as an amplification process that culminates in the production of a light. In turn, electricity is the band of the electromagnetic spectrum including UV radiation, visible light and IR radiation. The laser light is not normally found in nature and is of extraordinary intensity.

The best known property is the emission of beams of highly monochromatic, coherent and directional light.

There are different laser for medical use, classified as high-power-like carbon dioxide (CO2), for surgical use, and low power-such as Helium-Neon (He-Ne) for clinical use.

The term laser means in English Light Amplification by Stimulated Emission of Radiation.

The laser is a device or devices that produce a very special light (visible or invisible depending on their wavelength), created by man and acts like solid matter.

Einstein laid the theoretical basis for his work on the Quantum Theory of Radiation in 1917, by which the energy (light) can be converted into mass and mass into energy.

As light is reflected, the laser is absorbed, burning, and changes its direction through different lenses. As solid mass you can cut, melt, burn, and transmit.

The use of laser energy is not without several problems as the high price of the apparatus and the need for prior learning to use. In addition there is one type of laser that fits all applications, but each type has it sitting indications, many of which are unique to it.

From the medical point of view the use of laser can be used to remove tumors, seal blood vessels to reduce blood loss, sealed lymph vessels to reduce swelling and decrease the spread of tumor cells and nerve endings sealed to reduce postoperative pain.

Aesthetic laser therapy treatments can be used to remove warts and moles and to remove tattoos. It is also used to treat stretch marks, cellulite, sagging skin, acne sequels.It is based on the fundamental physiological properties of the laser, anti-inflammatory, spasmolytic and antiphlogistic effects and bio-stimulants. (Andreu & Valiente Zaldivar 1996).

Lasers and biomedical use:

512 Current Topics in Ionizing Radiation Research

The natural radiation that reaches us is UVA and UVB. The UVC is the most dangerous to

As for the benefits of using ultraviolet (UV), is recognized for use in phototherapy of patients with psoriasis, vitiligo and other skin diseases. In addition, ultraviolet rays have a

However, involvement of UV radiation in different pathologies has led to the approach of concepts such as photo-aging, accelerated aging process due to modification by UV radiation, DNA (deoxyribonucleic acid) and lipids membrane and induction of programmed cell death (apoptosis) of epithelial cells and the activation of enzymes

While studying the effects of ultraviolet radiation on the apoptotic induction and angiogenesis the results are mixed for use in the therapy of skin diseases including cancer and photocarcinogenesis, understood as the mechanism of DNA mutation due to the

Ultrviolet radiation is involved in the alterations of protein synthesis, the immunosuppressive properties and Its relationship to skin cancer and change in the

Ultraviolet radiation B are related to the induction and progression of cutaneous melanoma in mice, so its use helps to know the specific immune processes committed to these cancers, and thus to develop new treatments for melanoma. This type of radiation has also been used experimentally to elucidate the mechanisms of cellular radioresistance in ovarian cell lines.

Other natural rays are infrared (IR) or infrared radiation (the prefix infra means below, correspond to an emission of energy in the form of electromagnetic waves in the spectrum located immediately after the red zone). These rays have wavelengths between 800 and 0.25 nm and cause heating of the exposed regions, being the less penetrating wavelength

As for the infrared rays have a wavelength between 800 and 0.25 nm, cause heating of the exposed areas and those are more penetrating radiation of shorter wavelength. Special

They are also used in conjunction with hyperthermia and photodynamic therapy to treat tumor in the ablation of organs, and therapy of processes related to inflammation of nerves,

It has been postulated that IR produce a higher activity of phagocytosis and metabolic reactivity in infectious and has been used in phototherapy of patients with certain skin

However, the intracellular level, has shown that infrared radiation acting microtubule

The laser light (artificial) could be defined as an amplification process that culminates in the production of a light. In turn, electricity is the band of the electromagnetic spectrum including UV radiation, visible light and IR radiation. The laser light is not normally found

health because of its higher energy.

degrading the collagen in the skin.

synthesis of melanin.

**1.2 Infrared radiation (IR)**

muscles and joints.

**1.3 Laser radiation**

in nature and is of extraordinary intensity.

tumors.

formerly called them "calorific rays."

lamps are used commercially to produce 14,000 to infrared.

Its heating effect allows blood vessels to dilate and increase blood flow.

disrupting the centrosome (organoids) in mammalian cells in vitro.

bactericidal action, allowing its use in pressure ulcers.

alteration of repair when damaged by UV radiation.

The Helium-Neon laser is used to treat various conditions "satisfactory" mainly osteomioarticular (rehabilitation), skin disorders and wound healing.

Among the lasers used in medical practice are the 308nm excimer. This type of laser emits 85% of ultraviolet (UV) B and 15% of UV radiation type A.

Contraindications: The laser should not be used in patients affected by neoplasia (cancer). Nor can it be used in the presence of acute infections and in patients treated with photosensitizing drugs or remedies. Not recommended laser surgery in people with pacemakers or pregnant women.

In a previous study, we irradiated chick embryos and new born chickens with He-Ne laser, infrared and ultraviolet radiation, finding post irradiation histopathologic changes. (Samar et al., 1993, Avila et al., 1994 , Samar et al., 1995)

### **1.4 Intense Pulsed Light (IPL)**

In addition to the laser, since 1995 it also has been available a device that emits at 308 nm, called intense pulsed light (IPL) which is basically a XeCl lamp that has proved to be a useful tool for the treatment of the changes of the skin. The IPL provides high energy pulsed excitation consisting of 85% of ultraviolet radiations UV B and 15% of radiations UV A. This technology is also known as "Photoderm". Although the active medium of this lamp is also XeCl, the emission is polychromatic but also non-coherent; therefore is not a laser. However,

Biosafety in the Use of Radiation: Biological Effects Comparison

basophilic and metachromatic substances, alcohol-resistant.

Sample (egg) with thermic sensor (ts ). Egg holder (eh). Window in the egg shell (we).

Fig. 1. The system adaptable to the experimental requirements.

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 515

The samples taken was fixed in Bouin and were processed by routine histological technique and stained with: hematoxylin and eosin; conventional Histochemistry: PAS for the demonstration of glycoproteins, Alcian Blue at pH 2.5 and 1.0 for the demonstration of glycosaminoglycans sulfated and sulfated; Toluidine blue at pH 3.8 to demonstrate

the use in medicine and the risks associated with it are comparable to the medical lasers of the high energy (class 3b and 4) and therefore their use should be subject to the same safety guidelines. (Spencer & Hadi, 2004). (Town, et al. 2007).

The IPL is used in a similar way to the excimer laser in the treatment of various pathologies of the skin: psoriasis, vitiligo, etc. Clinically it is also used to stimulate the regeneration of the cartilage in degenerative processes. It has been postulated that its action is based on the activation of the cell division, collagenous and elastic fibers formation, regeneration of blood vessels, cicatrisation of bone tissue and reepithelization of damaged tissue. ( Schoenewolf et al. 2011)

Nevertheless, there are some discrepancies in results obtained with animal experimentation on the medical use of not-ionizing radiations. (Chan et al. 2007 )

However, the biological effect of the 308 nm IPL has not been investigated using the chick embryos as sensor.

## **1.5 Objective**

Different studies led us to establish preliminary results concerning the action of He-Ne laser, ultraviolet, infrared, LIP on the different tissues of chick embryos and in salivary glands and tongue of newly hatched chicks.

We also carry out the observation of effects that these types of radiation have on the chorioallantoic membrane of chick embryos, particularly on the formation of new blood vessels.

Moreover, the exposure time and the amount of non-ionizing radiation doses used in animal experiments and in medical practice are not yet fully known. Thus, our experimental design established in embryonated eggs and newly hatched chicks will contribute to the quantitative analysis (exposure and dose) of radiation mentioned above.

The purpose of this chapter describes the biological effects comparison between laser radiation, intense pulsed light and infrared and ultraviolet lamps in an experimental model in chicken embryos.

The chick embryo is a good model to evaluate direct effects of ionizing radiation because of easy handling and availability ITS.

Describe the most important results with different sources that emit non-ionizing radiation.

## **2. Materials and methods**

## **2.1 Experimental model of chicken embryo**

We used the chick embryo model as a mechanism for measuring biological effects of radiation on tissues, and this is an interesting method to be easily replicable.

Also to compare the results obtained by applying the radiation we have established in previous studies, the sequence of morphological changes, biochemical and histochemical occurring during differentiation and growth of the tongue, stomach, and ovaries both mesonephros "in ovo" as "in vitro" from 7 to 21 days post-birth immediately.

The problem groups were irradiated for 5 minutes through a window opened in the egg shell, and the eggs were maintained aseptically for 24hs in an incubator.We used the following radiations: intense pulsed light (excimer laser wavelength 308 nm) He-Ne laser (power 5mV, wavelength 632.8 nm), UV germicidal lamp and IR lamp OSRAM infraphil.

The control group were irradiated and only opened a window in the eggshell. All controls and aseptically problems remained in an incubator at 37 ° C for 24 hours post-irradiation.

the use in medicine and the risks associated with it are comparable to the medical lasers of the high energy (class 3b and 4) and therefore their use should be subject to the same safety

The IPL is used in a similar way to the excimer laser in the treatment of various pathologies of the skin: psoriasis, vitiligo, etc. Clinically it is also used to stimulate the regeneration of the cartilage in degenerative processes. It has been postulated that its action is based on the activation of the cell division, collagenous and elastic fibers formation, regeneration of blood vessels, cicatrisation of bone tissue and reepithelization of damaged tissue. ( Schoenewolf et

Nevertheless, there are some discrepancies in results obtained with animal experimentation

However, the biological effect of the 308 nm IPL has not been investigated using the chick

Different studies led us to establish preliminary results concerning the action of He-Ne laser, ultraviolet, infrared, LIP on the different tissues of chick embryos and in salivary glands and

We also carry out the observation of effects that these types of radiation have on the chorioallantoic membrane of chick embryos, particularly on the formation of new blood

Moreover, the exposure time and the amount of non-ionizing radiation doses used in animal experiments and in medical practice are not yet fully known. Thus, our experimental design established in embryonated eggs and newly hatched chicks will contribute to the

The purpose of this chapter describes the biological effects comparison between laser radiation, intense pulsed light and infrared and ultraviolet lamps in an experimental model

The chick embryo is a good model to evaluate direct effects of ionizing radiation because of

Describe the most important results with different sources that emit non-ionizing radiation.

We used the chick embryo model as a mechanism for measuring biological effects of

Also to compare the results obtained by applying the radiation we have established in previous studies, the sequence of morphological changes, biochemical and histochemical occurring during differentiation and growth of the tongue, stomach, and ovaries both

The problem groups were irradiated for 5 minutes through a window opened in the egg shell, and the eggs were maintained aseptically for 24hs in an incubator.We used the following radiations: intense pulsed light (excimer laser wavelength 308 nm) He-Ne laser (power 5mV, wavelength 632.8 nm), UV germicidal lamp and IR lamp OSRAM infraphil. The control group were irradiated and only opened a window in the eggshell. All controls and aseptically problems remained in an incubator at 37 ° C for 24 hours post-irradiation.

quantitative analysis (exposure and dose) of radiation mentioned above.

radiation on tissues, and this is an interesting method to be easily replicable.

mesonephros "in ovo" as "in vitro" from 7 to 21 days post-birth immediately.

guidelines. (Spencer & Hadi, 2004). (Town, et al. 2007).

on the medical use of not-ionizing radiations. (Chan et al. 2007 )

al. 2011)

embryos as sensor.

in chicken embryos.

tongue of newly hatched chicks.

easy handling and availability ITS.

**2.1 Experimental model of chicken embryo**

**2. Materials and methods**

**1.5 Objective**

vessels.

The samples taken was fixed in Bouin and were processed by routine histological technique and stained with: hematoxylin and eosin; conventional Histochemistry: PAS for the demonstration of glycoproteins, Alcian Blue at pH 2.5 and 1.0 for the demonstration of glycosaminoglycans sulfated and sulfated; Toluidine blue at pH 3.8 to demonstrate basophilic and metachromatic substances, alcohol-resistant.

Sample (egg) with thermic sensor (ts ). Egg holder (eh). Window in the egg shell (we). Fig. 1. The system adaptable to the experimental requirements.

Biosafety in the Use of Radiation: Biological Effects Comparison

E1

source (308 nm). (Avila et al, 2009)

Fig. 2. Scheme of the experimental setup

Notebook

E2

Filter Exposure

F7: Clear

F8:Yellow

F9: Blue

F10: Orange

F11:Green

time /s

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 517

LPI 300 nm

S1

E1: Spectrometer Ocean Optics HR4000, E2: Energy meter, E3: Energy sensor, S1: sample (egg), LPI: IPL

Optical fiber

In Table 1 we shown the light intensity transmitted in the different units by the IPL with the different filters used of acrylic (Ac) and cellophane (Cel) with the exposure time that are normally given for skin treatment. The selection criteria used for the filters to work with were those which showed a transmittance grater than 30%. This transmittance percentage

> Transmitted Fluence (mJ/cm2)

Filter absorbance

Filter

E3

Filter transmittance (%)

guarantees the possibility to observe the effects of radiation on tissues.

Transmitted Intensity (mW)

None 20 18.1 147.50 0.00 100 F1: Green acetate 20 0.46 3.74 1.59 2.5 F2: Blue acetate 20 0.00 0.00 2.00 0.0 F3: Clear acetate 20 0.00 0.00 2.00 0.0 F4: Yellow acetate 20 0.00 0.00 2.00 0.0 F5: Orange acetate 20 6.70 54.60 0.43 37.0 F6: Red Cellophane 10 3.29 13.40 1.04 9.1

Cellophane 10 18.0 73.34 0.30 49.7

Cellophane 10 2.2 8.96 1.21 6.07

Cellophane 10 0.48 1.95 1.87 1.32

Cellophane 10 9.2 37.50 0.60 25.0

Cellophane 20 5.25 42.8 0.53 30.0

Table 1. Intensity of the light from the XeCl lamp (IPL) transmitted through the different

filters, measured at 30 cm from the lamp. (Avila et al. 2009).

## **2.2 Infrared radiations**

In 1996 we published design of a thermic detection system applied to chick embryonated eggs irradiated with infrared rays (Avila et al. 1996).

Infrared radiations are widely used in medical therapeutics. It has been argued that doses and periods of time employed in experimental animals are higher than those used in clinic. Thus, we considered of interest to analyse aspects of dosimetry and thermic effects of infrared rays with current methods in medical practice, using in ovo chick embryo as a model of easy control.

To this end we designed a system to measure temperatures and their acquisitions and software for its handling. The system consists of: a) thermic points: thermocouples or termistores adaptable to the experimental requirements and calibrated with a greater precision within a range of ten degrees around the incubation temperature; b) acquirer circuit of thermic data (hardware): it generates a time base that varies with the thermic sensor. Software: the PC XT or AT detects changes in the time base by means of a programs' in a Turbo Pascal; c) storage and analysis of data allows, through a menu the scale selection, time of program data to be acquired, storage and recovery of the diskette information and graphic impression; d) chick's embryonated eggs.

This system allows to measure temperature distribution in small physical spaces with little disturbance of the system to be measured in irradiated bodies, to analyse variations of the temperatures in time and to secure a greater confidence and automatism to obtain the required data. Figure 1.

### **2.3 Experimental Intense Pulsed Light (IPL) system with and without different filters (Avila et al. 2009)**

To carry out the studies of the effects of the radiation on the different tissues in embryos of chicken we designed the experimental plan that is shown in the Figure 2.

The system consists of an Intense Pulsed Light (IPL) radiation source of 308 nm from DEKA model: Excilite (XeCl Excimer Light) wave length of 308 nm, capable to emit a radiation density of 4,5 J/cm2. The emission spectrum of this lamp was characterized in this work and is shown in the Figure 2.

Besides we used a filter set:

Acetate: Colorless, Orange, Green, Blue and Yellow.

Cellophane: Colorless, Orange, Green, Blue, Yellow and Red

A spectrometer (E1) Ocean Optics model HR4000 was used to characterize the emission of the IPL and the filters used in this work. The measurements of energy were carried out with an energy meter (E2) Scientech model 756 and a sensor (E3) model 362 with complete range (200nm to 1m). With this system we measured the total emitted energy per surface unit, mJ/cm2, on each sample with the different filters used and also the variation of the emission spectrum of the IPL after passing through the filter.

Optical characteristics of the light emitted by the XeCl lamp were determined, Intense Pulsed Light (IPL), using the experimental plan shown in Figure 2. The typical emission spectrum of this lamp was obtained by means of this system We determined that 85% of UVB radiation emitted by the IPL corresponds to 307.94 nm and only 15% to UVA centered in 367,5 nm. This implies that the effects seen on the tissue samples were due mainly by only one emission with very similar characteristics to the effects that could be caused by a laser radiation of this wave length.

E1: Spectrometer Ocean Optics HR4000, E2: Energy meter, E3: Energy sensor, S1: sample (egg), LPI: IPL source (308 nm). (Avila et al, 2009)

#### Fig. 2. Scheme of the experimental setup

516 Current Topics in Ionizing Radiation Research

In 1996 we published design of a thermic detection system applied to chick embryonated

Infrared radiations are widely used in medical therapeutics. It has been argued that doses and periods of time employed in experimental animals are higher than those used in clinic. Thus, we considered of interest to analyse aspects of dosimetry and thermic effects of infrared rays with current methods in medical practice, using in ovo chick embryo as a

To this end we designed a system to measure temperatures and their acquisitions and software for its handling. The system consists of: a) thermic points: thermocouples or termistores adaptable to the experimental requirements and calibrated with a greater precision within a range of ten degrees around the incubation temperature; b) acquirer circuit of thermic data (hardware): it generates a time base that varies with the thermic sensor. Software: the PC XT or AT detects changes in the time base by means of a programs' in a Turbo Pascal; c) storage and analysis of data allows, through a menu the scale selection, time of program data to be acquired, storage and recovery of the diskette information and

This system allows to measure temperature distribution in small physical spaces with little disturbance of the system to be measured in irradiated bodies, to analyse variations of the temperatures in time and to secure a greater confidence and automatism to obtain the

**2.3 Experimental Intense Pulsed Light (IPL) system with and without different filters**

chicken we designed the experimental plan that is shown in the Figure 2.

To carry out the studies of the effects of the radiation on the different tissues in embryos of

The system consists of an Intense Pulsed Light (IPL) radiation source of 308 nm from DEKA model: Excilite (XeCl Excimer Light) wave length of 308 nm, capable to emit a radiation density of 4,5 J/cm2. The emission spectrum of this lamp was characterized in this work and

A spectrometer (E1) Ocean Optics model HR4000 was used to characterize the emission of the IPL and the filters used in this work. The measurements of energy were carried out with an energy meter (E2) Scientech model 756 and a sensor (E3) model 362 with complete range (200nm to 1m). With this system we measured the total emitted energy per surface unit, mJ/cm2, on each sample with the different filters used and also the variation of the emission

Optical characteristics of the light emitted by the XeCl lamp were determined, Intense Pulsed Light (IPL), using the experimental plan shown in Figure 2. The typical emission spectrum of this lamp was obtained by means of this system We determined that 85% of UVB radiation emitted by the IPL corresponds to 307.94 nm and only 15% to UVA centered in 367,5 nm. This implies that the effects seen on the tissue samples were due mainly by only one emission with very similar characteristics to the effects that could be caused by a laser

**2.2 Infrared radiations**

model of easy control.

required data. Figure 1.

is shown in the Figure 2. Besides we used a filter set:

radiation of this wave length.

**(Avila et al. 2009)**

eggs irradiated with infrared rays (Avila et al. 1996).

graphic impression; d) chick's embryonated eggs.

Acetate: Colorless, Orange, Green, Blue and Yellow.

spectrum of the IPL after passing through the filter.

Cellophane: Colorless, Orange, Green, Blue, Yellow and Red

In Table 1 we shown the light intensity transmitted in the different units by the IPL with the different filters used of acrylic (Ac) and cellophane (Cel) with the exposure time that are normally given for skin treatment. The selection criteria used for the filters to work with were those which showed a transmittance grater than 30%. This transmittance percentage guarantees the possibility to observe the effects of radiation on tissues.

Table 1. Intensity of the light from the XeCl lamp (IPL) transmitted through the different filters, measured at 30 cm from the lamp. (Avila et al. 2009).

Biosafety in the Use of Radiation: Biological Effects Comparison

associated to the plasma membrane and basement membrane.

constitute reliable experimental models for this type of studies.

which might appear once laser therapy has been installed.

**<IP_ADDRESS> Biological effects on the lingual cartilage of the chicken embryo**

cartilage of the tongue chick embryo using various filters. (Avila et al. 2009)

security measures and understand the possible effects on an experimental model.

**chicken irradiated with He-Ne laser (Avila et al. 1997)**

epithelial cells.

He-Ne laser.

**3.1.3 Newborn chicken**

**3.2 Intense Pulsed Light (IPL) 3.2.1 Tongue of the chicken embryo**

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 519

The embryos were irradiated for 5 minutes through a window opened in the eggshell and aseptically maintained in incubator for 24 hours. The gonads were dissected out and

Controls: The ovaries were formed by a germinative or superficial epithelium, with germ and epithelial cells, and by primary sex cords compressed between them, although separated by a reduced stroma. The cords contained germ cells The surface coat of the germinative epithelium presented a thin layer of PAS positive, alcianophilic at pH 2.5 and orthochromatic material. Basement membranes and intercord extracellular substance were also PAS positive. Problems: Disorganization of the tissue structure was well manifest in irradiated gonads, accompanied by negativization of the histochemical reactions. A lymphocytic infiltration was also found. No structural alterations were observed in germ or

It is concluded that laser radiations would act producing decrease of the mucosubstances

They would also provoke the appearance of an inflammatory mononuclear infiltration.

**<IP_ADDRESS> Structural and cytochemical modifications in the lingual glands of the newborn**

Despite the increasing and successful use of laser in Medicine and Odontology, the possible iatrogenic and otherwise deleterious side effects of this radiation remain mostly unknown. In previous studies, it was shown that both the embryonic and the post-hatched chicken

Hence, the purpose of the present work was to analyze the structural and cytochemical alterations of the lingual glands of the newborn chicken irradiated with low energy

This laser produced regressive structural changes of the glands towards the embryonic stage as well as hyperplasia of the reserve glandular basal cells. Furthermore, a decrease in the

These results corroborate the pathogenic effects of the He-Ne- laser on the experimental model employed and, at the same time, emphasize the importance of considering, regarding clinical applications, possible previous neoplastic alterations as well as adverse reactions

In 2009 we described the biological effects produced by intense pulsed light (Xe-Cl) on the

The Laser used correctly in the medical practice offers clear advantages compared with traditional therapies. The improvement and even the elimination of many significant skin lesions can be achieved with reduced risks to patients. However, it is important to keep

The chick embryo is a good model to evaluate the direct effects of non-ionizing radiation for its easy handling and availability. The purpose of this communication is to show our

glycoprotein content and a rise in the sulfated glycosaminoglycans were also found.

processed for the following techniques: H/E, PAS, Alcian blue, and Toluidine blue.

Once we characterized the IPL emission and the filter absorption we proceeded to radiate the chicken embryos by previously removing their white membrane (below the shell of the egg). We first carried out a set of measurements without using any filters and later using the filters that were previously selected.

Before starting the radiation process in the different samples we determined the absorption spectrum of the albumen of the egg and of the white membrane. From these results we determined that the white membrane should be removed before the radiation of each sample since it has a very large absorption coefficient to the study wave length and would not permit to determine the effects of the radiation in the embryo tissues.

The acetate filters (transparent and orange) and cellophane filters (transparent orange and green) allow to the passage of the radiations producing biological effects in the organs of the chicken embryo, for this reason it is not recommended to be used as a filter.

The acetate filters (green, blue and yellow) and cellophane filters (red, yellow and blue) do not allow to the passage of radiation, without showing any biological effects in the chicken embryo.

## **3. Results**

## **3.1 He-Ne laser irradiation**

### **3.1.1 Chick embryo mesonephros**

A study on the effects of He-Ne laser irradiation on glomeruli and renal tubules of the chick embryo mesonephros at 7 days of in ovo development was made. (Avila et al, 1992)

To this purpose, He-Ne laser irradiation (potency: 5 mW, wavelength: 632.8 nm) was beamed for 5 minutes through a window opened in the egg shell, and the eggs were maintained aseptically for 24 h in an incubator.

Mesonephros were dissected out and processed for hematoxylin/eosin, periodic acid-Schiff, and Alcian blue at pH 2.5 and 1.0, toluidine Blue at pH 3.8.

In controls, the glomeruli were formed by coiled capillaries with a homogeneous basement membrane, the proximal tubules presented a high cubic epithelium with acidophilic cytoplasm and a developed brush border. Distal and collecting segments were lined by cubic epithelium. An alcianophilic and PAS-positive reaction stood out at the membrane coats of the proximal tubules and tubular and glomerular basement membranes. No glomerular alterations were observed during the experiment. However, there was a marked enlargement of the tubular interstitium, with edema and lymphohistiocytic inflammatory infiltration. Some tubular cells desquamated to the lumen. Other cells presented a raveled apical surface. Some nuclei were dispersed out, and mixing with chromatin, formed diminutive granules. Pyknotic nuclei were seen occasionally. Epithelial necrosis and cytoplasmic debris in the lumen were also noted. For mucins, some zones showed brush border coats of the proximal tubules as discontinuous.

#### **3.1.2 Chick embryo ovary**

We published in 1992 the structural changes induced by He-Ne laser on the chick embryo ovary. (Avila et al. 1992)

The morpho-histochemical alterations that occur in the chicken ovary at 7 days of incubation after irradiation with He-Ne laser of a potency of 5 mw and at a wavelength of 632.8 nm were studied.

The embryos were irradiated for 5 minutes through a window opened in the eggshell and aseptically maintained in incubator for 24 hours. The gonads were dissected out and processed for the following techniques: H/E, PAS, Alcian blue, and Toluidine blue.

Controls: The ovaries were formed by a germinative or superficial epithelium, with germ and epithelial cells, and by primary sex cords compressed between them, although separated by a reduced stroma. The cords contained germ cells The surface coat of the germinative epithelium presented a thin layer of PAS positive, alcianophilic at pH 2.5 and orthochromatic material. Basement membranes and intercord extracellular substance were also PAS positive. Problems: Disorganization of the tissue structure was well manifest in irradiated gonads, accompanied by negativization of the histochemical reactions. A lymphocytic infiltration was also found. No structural alterations were observed in germ or epithelial cells.

It is concluded that laser radiations would act producing decrease of the mucosubstances associated to the plasma membrane and basement membrane.

They would also provoke the appearance of an inflammatory mononuclear infiltration.

## **3.1.3 Newborn chicken**

518 Current Topics in Ionizing Radiation Research

Once we characterized the IPL emission and the filter absorption we proceeded to radiate the chicken embryos by previously removing their white membrane (below the shell of the egg). We first carried out a set of measurements without using any filters and later using the

Before starting the radiation process in the different samples we determined the absorption spectrum of the albumen of the egg and of the white membrane. From these results we determined that the white membrane should be removed before the radiation of each sample since it has a very large absorption coefficient to the study wave length and would

The acetate filters (transparent and orange) and cellophane filters (transparent orange and green) allow to the passage of the radiations producing biological effects in the organs of the

The acetate filters (green, blue and yellow) and cellophane filters (red, yellow and blue) do not allow to the passage of radiation, without showing any biological effects in the chicken

A study on the effects of He-Ne laser irradiation on glomeruli and renal tubules of the chick

To this purpose, He-Ne laser irradiation (potency: 5 mW, wavelength: 632.8 nm) was beamed for 5 minutes through a window opened in the egg shell, and the eggs were

Mesonephros were dissected out and processed for hematoxylin/eosin, periodic acid-Schiff,

In controls, the glomeruli were formed by coiled capillaries with a homogeneous basement membrane, the proximal tubules presented a high cubic epithelium with acidophilic cytoplasm and a developed brush border. Distal and collecting segments were lined by cubic epithelium. An alcianophilic and PAS-positive reaction stood out at the membrane coats of the proximal tubules and tubular and glomerular basement membranes. No glomerular alterations were observed during the experiment. However, there was a marked enlargement of the tubular interstitium, with edema and lymphohistiocytic inflammatory infiltration. Some tubular cells desquamated to the lumen. Other cells presented a raveled apical surface. Some nuclei were dispersed out, and mixing with chromatin, formed diminutive granules. Pyknotic nuclei were seen occasionally. Epithelial necrosis and cytoplasmic debris in the lumen were also noted. For mucins, some zones showed brush

We published in 1992 the structural changes induced by He-Ne laser on the chick embryo

The morpho-histochemical alterations that occur in the chicken ovary at 7 days of incubation after irradiation with He-Ne laser of a potency of 5 mw and at a wavelength of 632.8 nm

embryo mesonephros at 7 days of in ovo development was made. (Avila et al, 1992)

not permit to determine the effects of the radiation in the embryo tissues.

chicken embryo, for this reason it is not recommended to be used as a filter.

filters that were previously selected.

embryo.

**3. Results**

**3.1 He-Ne laser irradiation**

**3.1.2 Chick embryo ovary**

ovary. (Avila et al. 1992)

were studied.

**3.1.1 Chick embryo mesonephros**

maintained aseptically for 24 h in an incubator.

and Alcian blue at pH 2.5 and 1.0, toluidine Blue at pH 3.8.

border coats of the proximal tubules as discontinuous.

#### **<IP_ADDRESS> Structural and cytochemical modifications in the lingual glands of the newborn chicken irradiated with He-Ne laser (Avila et al. 1997)**

Despite the increasing and successful use of laser in Medicine and Odontology, the possible iatrogenic and otherwise deleterious side effects of this radiation remain mostly unknown. In previous studies, it was shown that both the embryonic and the post-hatched chicken constitute reliable experimental models for this type of studies.

Hence, the purpose of the present work was to analyze the structural and cytochemical alterations of the lingual glands of the newborn chicken irradiated with low energy He-Ne laser.

This laser produced regressive structural changes of the glands towards the embryonic stage as well as hyperplasia of the reserve glandular basal cells. Furthermore, a decrease in the glycoprotein content and a rise in the sulfated glycosaminoglycans were also found.

These results corroborate the pathogenic effects of the He-Ne- laser on the experimental model employed and, at the same time, emphasize the importance of considering, regarding clinical applications, possible previous neoplastic alterations as well as adverse reactions which might appear once laser therapy has been installed.

## **3.2 Intense Pulsed Light (IPL)**

## **3.2.1 Tongue of the chicken embryo**

## **<IP_ADDRESS> Biological effects on the lingual cartilage of the chicken embryo**

In 2009 we described the biological effects produced by intense pulsed light (Xe-Cl) on the cartilage of the tongue chick embryo using various filters. (Avila et al. 2009)

The Laser used correctly in the medical practice offers clear advantages compared with traditional therapies. The improvement and even the elimination of many significant skin lesions can be achieved with reduced risks to patients. However, it is important to keep security measures and understand the possible effects on an experimental model.

The chick embryo is a good model to evaluate the direct effects of non-ionizing radiation for its easy handling and availability. The purpose of this communication is to show our

Biosafety in the Use of Radiation: Biological Effects Comparison

availability.

radiation.

without color and a green.

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 521

measures and know the possible effects on an experimental model. The chick embryo is a good model to evaluate the direct effects of ionizing radiation is its easy handling and

The purpose of this communication is to show our histopathological findings in chick embryo wings with and without barrier protection when subjected to intense pulsed light

We used intense pulsed light of 308 nm wavelength Excilite model DEKA brand, Luz Excimer (XeCl) Wavelength 308 nm. Embryos were irradiated through an open window in the eggshell without barriers (controls) or barriers (problems). The eggs were maintained aseptically for 24 hours in an incubator. The barrier used was transparent cellophane

The most outstanding results obtained without a barrier, with clear cellophane and green were epithelial haematic fibrinous exudate and epithelial hyperplasia. In the stroma are varying degrees of vasocongestion, erythrocyte extravasation, and focal hemorrhage and edema, mononuclear infiltrate. It is concluded that tissue changes observed are consistent with possible changes produced by these collateral photothermal radiation, which warns of

We believe it is important to study the means to ensure the safety of operators and patients.

1. Control. 10 X H / E. 2. Problem:epithelial hyperplasia. 40 X. H / E. 3. Problem: Extravasation erythrocytes. 40 X H / E. 4. Problem: Fibrinous exudate 40 X. H / E. 5. Problem: epithelial hyperplasia, edema and mononuclear infiltrate (3), which migrate into the epithelium sectors 40 XH / E. 6. Issue:

Fig. 4. Epithelium and stroma of the chick embryo wing 15 days irradiated with (problem)

Stromal with vessels congestion and epithelial hyperplasia. 10 X. H / E

or without (control) intense pulsed light of 308 nm.

possible adverse effects when these are applied indiscriminately. Figure 4.

histological findings in organs of the chick embryo with and without protective barrier to be subjected to radiation excimer.

We used the following emitter: intense pulsed light (excimer Xe-Cl laser of 308 nm wavelength). It was irradiated embryos through an open window on eggshells. Aseptically the eggs were kept for 24 hours in an incubator. The protective barriers were used with and without colored glass, latex, cellophane, paper, polycarbonate of different colors and thicknesses.

The tissue changes observed are consistent with possible side effects of these fotothermical radiations we warned about possible side effects when they are applied indiscriminately. We believe it is important to explore different means to safeguard the safety of operators and patients. Figure 3.

Fig. 3. Tongue of chicken 15 days of incubation in "ovo" irradiated with Intense Pulsed Light (Xe-Cl) 308 nm wavelength. Stain: hematoxylin and eosin. 5 x . The most striking findings consisted degenerative changes at the level of cartilage necrosis following groups glandular mucosa nature, accompanied by leukocyte infiltration, vascular wall thickening at the expense of the tunica media, perivascular edema.

## **3.2.2 Chick embryo wing**

### **<IP_ADDRESS> Histopathologic findings in epithelium and stroma of the chick embryo wing irradiated with intense pulsed light of 308 nm**

Intense Pulsed Light (IPL) is used in medical practice enabling improved significantly even elimination of many skin lesions of patients. However, it is important to keep safety

histological findings in organs of the chick embryo with and without protective barrier to be

We used the following emitter: intense pulsed light (excimer Xe-Cl laser of 308 nm wavelength). It was irradiated embryos through an open window on eggshells. Aseptically the eggs were kept for 24 hours in an incubator. The protective barriers were used with and without colored glass, latex, cellophane, paper, polycarbonate of different colors and

The tissue changes observed are consistent with possible side effects of these fotothermical radiations we warned about possible side effects when they are applied indiscriminately. We believe it is important to explore different means to safeguard the safety of operators

Fig. 3. Tongue of chicken 15 days of incubation in "ovo" irradiated with Intense Pulsed Light (Xe-Cl) 308 nm wavelength. Stain: hematoxylin and eosin. 5 x . The most striking findings consisted degenerative changes at the level of cartilage necrosis following groups glandular mucosa nature, accompanied by leukocyte infiltration, vascular wall thickening at the

**<IP_ADDRESS> Histopathologic findings in epithelium and stroma of the chick embryo wing**

Intense Pulsed Light (IPL) is used in medical practice enabling improved significantly even elimination of many skin lesions of patients. However, it is important to keep safety

expense of the tunica media, perivascular edema.

**irradiated with intense pulsed light of 308 nm**

**3.2.2 Chick embryo wing**

subjected to radiation excimer.

thicknesses.

and patients. Figure 3.

measures and know the possible effects on an experimental model. The chick embryo is a good model to evaluate the direct effects of ionizing radiation is its easy handling and availability.

The purpose of this communication is to show our histopathological findings in chick embryo wings with and without barrier protection when subjected to intense pulsed light radiation.

We used intense pulsed light of 308 nm wavelength Excilite model DEKA brand, Luz Excimer (XeCl) Wavelength 308 nm. Embryos were irradiated through an open window in the eggshell without barriers (controls) or barriers (problems). The eggs were maintained aseptically for 24 hours in an incubator. The barrier used was transparent cellophane without color and a green.

The most outstanding results obtained without a barrier, with clear cellophane and green were epithelial haematic fibrinous exudate and epithelial hyperplasia. In the stroma are varying degrees of vasocongestion, erythrocyte extravasation, and focal hemorrhage and edema, mononuclear infiltrate. It is concluded that tissue changes observed are consistent with possible changes produced by these collateral photothermal radiation, which warns of possible adverse effects when these are applied indiscriminately. Figure 4.

We believe it is important to study the means to ensure the safety of operators and patients.

1. Control. 10 X H / E. 2. Problem:epithelial hyperplasia. 40 X. H / E. 3. Problem: Extravasation erythrocytes. 40 X H / E. 4. Problem: Fibrinous exudate 40 X. H / E. 5. Problem: epithelial hyperplasia, edema and mononuclear infiltrate (3), which migrate into the epithelium sectors 40 XH / E. 6. Issue: Stromal with vessels congestion and epithelial hyperplasia. 10 X. H / E

Fig. 4. Epithelium and stroma of the chick embryo wing 15 days irradiated with (problem) or without (control) intense pulsed light of 308 nm.

Biosafety in the Use of Radiation: Biological Effects Comparison

**4. Discussion and conclusion**

handling and availability.

on organs of chicken embryos.

effects on organs of chicken embryos.

personnel that uses this type of IPL.

Zaldivar 1996).

preventive end treating purposes. (Veterany et al. 2004)

(Samar et al., 1993, Avila et al., 1994 , Samar et al., 1995)

the rate was produced (28%) of synthesis of the matrix.

how a protection against the detrimental effects of IRA can be achieved.

Between Laser Radiation, Intense Pulsed Light and Infrared and Ultraviolet Lamps in... 523

The chick embryo is a good model to evaluate direct effects of ionizing radiation is its easy

We irradiated chick embryos and newly hatched chicks with He-Neon laser and infrared radiation (IR) and ultraviolet (UV) radiation found histopathological changes post. On the other hand, in the databases searched found no experimental studies on the biological effects of radiation from pulsed light of 308 nm (XeCl Excimer Light) in chick embryo organs. So also did experiments with intense pulsed light corroborating deleterious effects

On the other hand, in the databases searched found no experimental studies on the biological effects of radiation from pulsed light of 308 nm (XeCl Excimer Light) in chick embryo organs. So also did experiments with intense pulsed light corroborating deleterious

Authors studied the Influence of UV-B radiation on embryonic development of chickens Hampshire breed and considered that it is possible to state that short-lasting UV radiation appealing can have positive influence on organisms, which can be used in medicine for

Some authors ( Schroeder et al. 2008) imply that IRA-radiation is capable of altering gene expression which brings forward a pro-aging phenotype of the skin. Apart from minimising exposure to natural IRA and responsible use of artificial IRA sources, the questions arises

The XeCl (IPL) lamp used in these experiments present a very monochromatic spectrum of emission of 307,94 nm and its effects could be very similar to those corresponding to a XeCl laser. The IPL is normally used for skin treatment for which the spectroscopic characterization carried out lets us determine the effects of this wave length on the tissues. (Nahavandi et al., 2008) The filters used basically acted only as intensity attenuators and they let us determine which optic material is more convenient for the protection of the

It is because of this that we investigated the histopathological changes produced on an organ of the oral cavity of the chicken, the tongue and especially those centered in the cartilage, being similar to those described in prior publications produced in different organs of the embryo of chicken radiated with ultraviolet radiation, infrared and HeNe Lasers.

Clinically it is used as cartilage stimulus for its regeneration in the degenerative processes. It's been postulated that its action is based on the cell multiplication, the formation of collagen and elastic fibers, the vessel regeneration, the scar formation of bony tissues and the re-epithelization of the damaged tissue. (Baumanna et al., 2006 and Andreu &

Others authors. (Baumanna et al., 2006) studied the influence of laser radiation in human osteoartritic condrocites using different wave lengths (laser diode of 690nm and laser Nd: YAG of 1064 nm), power densities and exposition times and they observed that uaing a specific set of parameters (2 W; 16W/cm2; 60 s; 120 J) they observed an increase in the synthesis of the matrix (material of cartilage originating from 36 patients). They also describe that, using a very high power density, but a constant energy density, a reduction of

## **3.2.3 Heart of the chick embryo**

## **<IP_ADDRESS> Biological effects on the heart of the chick embryo**

The 308 nm excimer laser is a new application in cardiology for the treatment of congenital heart malformations, vascular and ischemic cardiomyopathy. (Spencer & Hadi, S. M. 2004). This type of laser emits 85% of ultraviolet (UV) type B and 15% of UV radiation type A. However, the literature does not describe the changes that occur in myocardial cells and surrounding embryonic tissue when exposed to this type of laser.

Moreover, the intense pulsed light therapy with high-energy UVB spectrum is used in a manner similar to the excimer laser in different conditions. Intense Pulsed Light (IPL), is based on generating a polychromatic light source, high intensity incoherent.

A. Control: cardiomyocytes. Coloration hematoxylin / eosin. B. Problem: disorganized tissue with interstitial edema, vasocongestion, exudate. Pericardium with leukocyte exudate. Coloration hematoxylin / eosin. C. Problem: Interstitium with blood vessels. Cardiomyocytes. Masson's Trichrome Staining. D. Problem: Interstitium and cardiomyocytes. Reduction of glycoproteins. PAS Staining

Fig. 5. Heart of the chick embryo 15 days days irradiated with (problem) or without (control) intense pulsed light of 308 nm.

## **4. Discussion and conclusion**

522 Current Topics in Ionizing Radiation Research

The 308 nm excimer laser is a new application in cardiology for the treatment of congenital heart malformations, vascular and ischemic cardiomyopathy. (Spencer & Hadi, S. M. 2004). This type of laser emits 85% of ultraviolet (UV) type B and 15% of UV radiation type A. However, the literature does not describe the changes that occur in myocardial cells and

Moreover, the intense pulsed light therapy with high-energy UVB spectrum is used in a manner similar to the excimer laser in different conditions. Intense Pulsed Light (IPL), is

A. Control: cardiomyocytes. Coloration hematoxylin / eosin. B. Problem: disorganized tissue with interstitial edema, vasocongestion, exudate. Pericardium with leukocyte exudate. Coloration hematoxylin / eosin. C. Problem: Interstitium with blood vessels. Cardiomyocytes. Masson's Trichrome Staining. D. Problem: Interstitium and cardiomyocytes. Reduction of glycoproteins. PAS Staining Fig. 5. Heart of the chick embryo 15 days days irradiated with (problem) or without

(control) intense pulsed light of 308 nm.

**3.2.3 Heart of the chick embryo**

**<IP_ADDRESS> Biological effects on the heart of the chick embryo**

surrounding embryonic tissue when exposed to this type of laser.

based on generating a polychromatic light source, high intensity incoherent.

The chick embryo is a good model to evaluate direct effects of ionizing radiation is its easy handling and availability.

We irradiated chick embryos and newly hatched chicks with He-Neon laser and infrared radiation (IR) and ultraviolet (UV) radiation found histopathological changes post. On the other hand, in the databases searched found no experimental studies on the biological effects of radiation from pulsed light of 308 nm (XeCl Excimer Light) in chick embryo organs. So also did experiments with intense pulsed light corroborating deleterious effects on organs of chicken embryos.

On the other hand, in the databases searched found no experimental studies on the biological effects of radiation from pulsed light of 308 nm (XeCl Excimer Light) in chick embryo organs. So also did experiments with intense pulsed light corroborating deleterious effects on organs of chicken embryos.

Authors studied the Influence of UV-B radiation on embryonic development of chickens Hampshire breed and considered that it is possible to state that short-lasting UV radiation appealing can have positive influence on organisms, which can be used in medicine for preventive end treating purposes. (Veterany et al. 2004)

Some authors ( Schroeder et al. 2008) imply that IRA-radiation is capable of altering gene expression which brings forward a pro-aging phenotype of the skin. Apart from minimising exposure to natural IRA and responsible use of artificial IRA sources, the questions arises how a protection against the detrimental effects of IRA can be achieved.

The XeCl (IPL) lamp used in these experiments present a very monochromatic spectrum of emission of 307,94 nm and its effects could be very similar to those corresponding to a XeCl laser. The IPL is normally used for skin treatment for which the spectroscopic characterization carried out lets us determine the effects of this wave length on the tissues. (Nahavandi et al., 2008) The filters used basically acted only as intensity attenuators and they let us determine which optic material is more convenient for the protection of the personnel that uses this type of IPL.

It is because of this that we investigated the histopathological changes produced on an organ of the oral cavity of the chicken, the tongue and especially those centered in the cartilage, being similar to those described in prior publications produced in different organs of the embryo of chicken radiated with ultraviolet radiation, infrared and HeNe Lasers. (Samar et al., 1993, Avila et al., 1994 , Samar et al., 1995)

Clinically it is used as cartilage stimulus for its regeneration in the degenerative processes. It's been postulated that its action is based on the cell multiplication, the formation of collagen and elastic fibers, the vessel regeneration, the scar formation of bony tissues and the re-epithelization of the damaged tissue. (Baumanna et al., 2006 and Andreu & Zaldivar 1996).

Others authors. (Baumanna et al., 2006) studied the influence of laser radiation in human osteoartritic condrocites using different wave lengths (laser diode of 690nm and laser Nd: YAG of 1064 nm), power densities and exposition times and they observed that uaing a specific set of parameters (2 W; 16W/cm2; 60 s; 120 J) they observed an increase in the synthesis of the matrix (material of cartilage originating from 36 patients). They also describe that, using a very high power density, but a constant energy density, a reduction of the rate was produced (28%) of synthesis of the matrix.

Biosafety in the Use of Radiation: Biological Effects Comparison

287-290.

pp. 8-13.

1799.

87-89.

2, pp. 215-20.

522-525.

*Morphol., 27(4)*:1003-1008.

No. 202(9), pp. 523-532.

*Laser Med Surg*, No. 11, pp. 87-89.

*Dermatol*. No. 42, pp. 166-172.

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Avila, R. E., Samar, M.E., Juri, H. & Fabro, S. P. de. (1992). Effects of He Ne laser

Avila, R. E.; Samar, M. E.; Juri, H.; Femopase, G. A.; Hidalgo, M.; Ferrero, J. C.; Rinaldi, C.;

Baumanna, M., Bjorn, J., Rohdec E., Bindigd, U., Mullerc, G. & Schellera, E. (2006). Influence

cultures – An in-vitro investigation. *Med Laser Appl*, No. 21, pp. 191–198. Chan, H. H., Yang, C. H., Leung, J. C., Wei, W. I. & Lai, K. N. (2007). An animal study of

Moseley, H. (1994). Ultraviolet and laser radiation safety. *Phys. Med. Biol.,* No. 39, pp. 1765-

Nahavandi, H., Neumann, R., Holzer, G. & Knobler, R. (2008). Evaluation of safety and

Parker, S. (2007). Laser regulation and safety in general dental practice. *Br Dent J.* Vol. 12,

Samar, M. E., Avila, R. E., de Ferrais, M. E., Ferraris, R. V. & de Fabro, S. P. (1993).

Samar, M. E., Avila, R. E., Juri, H., Centurión, C. & Fabro, S. P. de. (1993). Histologycal

Samar, M. E., Avila, R. E., Juri, H., Plivelic, T. & Fabro, S. P. de. (1995). Histopathological

Samar, M. E.; Avila, R. E. & Juri, H. (1993). Histological changes produced by He-Ne

Schroeder, P., Haendeler, J. & Krutmann. (2008). The role of near infrared radiation in

Schoenewolf, N. L., Barysch, M. J. & Dummer, R. (2011). Intense pulsed light. *Curr Probl*

Sliney D. H. (2006) Risks of occupational exposure to optical radiation. *Med Lav, Vol.* 97, No.

Spencer, J. M. & Hadi, S. M. (2004). The excimer lasers. *J. Drugs Dermatol.,* No. 5, pp.

Town, G., Ash, C., Eadie, E. & Moseley, H. (2007). Measuring key parameters of intense pulsed light (IPL) devices. *J. Cosmet. Laser Ther.,* Vol. 9, No. 3, pp. 148-160.

photoaging of the skinJ*. Exp Gerontol*, Vol. 43, No. 7, pp. 629-632.

*J. Eur. Acad. Dermatol. Venereol.,* Vol. 22, No. 3, pp. 311-315.

study. *Acta Odontol Latinoam*, Vol. 7, No. 2, pp. 23-32.

chicken. *J Clin Laser Med Surg*, Vol. 13, No. 4, pp. 267-272.

irradiation on chick embryo mesonephros*. J Clin Laser Med Surg*, No. 10, pp.

Fonseca, I. & Juri, G. (2009). Biological Effects Produced by Intense Pulsed Light (Xe-Cl) on the Cartilage of the Tongue Chick Embryo Using Various Filters. *Int. J.*

of wavelength, power density and exposure time of laser radiation on chondrocyte

the effects on p16 and PCNA expression of repeated treatment with highenergy laser and intense pulsed light exposure. *Lasers Surg. Med.,* Vol. 39, No. 1,

efficacy of variable pulsed light in the treatment of unwanted hair in 77 volunteers.

Embryogeny of human labial glands: a structural, ultrastructural and cytochemical

changes produced by He-Ne laser on different tissues from chick embryo. *J Clin*

alterations induced by He-Ne laser in the salivary glands of the posthatched

laser on different tissues from chick embryo. *J. Clin. Laser Med. Surg., No. 11, pp.*

By using only the filters with a transmittance grater than 30%, we could guarantee the possibility to observe the biological effects in tissue radiation.

Many countries have published safety guidelines for the use of lasers and most of them are harmonized with the international standards of the International Electrotecnic Commission (IEC). The maximum limit permitted of the exposure with the (MPE) laser used in international safety standards of the IEC are based on guidelines of the International Commission in the protection against the not-ionizing radiation (ICNIRP). (Moseley 1994; Sliney 2006; Parker,2007).

The CIS 825-1 norm (1993) is set for the manufacturers; However, it also offers some limitations oriented on the safety for users. All commercial lasers must exhibit the risk classification indicated.

We repeat that the use of IPL in spite of having similar biological effects than UV Lasers, do not have an equal standard safety classification.

The study of different types of radiation in our experimental model allowed to obtain information regarding the behavior of cells and tissues. We believe it is important to study the various means to ensure the safety of patients and operators. Also allow those responsible for advising on the lamps to regulate medical and aesthetic use.

We conclude that these tissue changes are compatible with photothermal side effects of this radiation that warn us about possible adverse effects of dose and time used experimentally.

## **5. References**

By using only the filters with a transmittance grater than 30%, we could guarantee the

Many countries have published safety guidelines for the use of lasers and most of them are harmonized with the international standards of the International Electrotecnic Commission (IEC). The maximum limit permitted of the exposure with the (MPE) laser used in international safety standards of the IEC are based on guidelines of the International Commission in the protection against the not-ionizing radiation (ICNIRP). (Moseley 1994;

The CIS 825-1 norm (1993) is set for the manufacturers; However, it also offers some limitations oriented on the safety for users. All commercial lasers must exhibit the risk

We repeat that the use of IPL in spite of having similar biological effects than UV Lasers, do

The study of different types of radiation in our experimental model allowed to obtain information regarding the behavior of cells and tissues. We believe it is important to study the various means to ensure the safety of patients and operators. Also allow those

We conclude that these tissue changes are compatible with photothermal side effects of this radiation that warn us about possible adverse effects of dose and time used

Andreu, M. I. & Valiente Zaldivar, C. (1996). Efectos biológicos de la radiación láser de

Avila, R. E., Ferraris, R. & Samar, M. E. (2001). Efectos biológicos inmediatos de las

Avila, R. E., Plivelic, T., Samar, M. E. & Benavidez, E. (1996). Diseño de un sistema de

Avila, R. E., Samar, M. E., De Fabro, S. P., Leguina, M. & Juri, H. O. (1994). Histologic

Avila, R. E., Samar, M. E., de Fabro, S. P. & Plivelic, T. S. (1997). Structural and cytochemical

Avila, R. E., Samar, M. E., Fabro, S. P. de Juri, H. & Leguina, M. (1994) Alteraciones

Avila, R. E., Samar, M. E., Juri, H., Fabro, S. P. de, Centurión, C. & Sánchez Mazzaferro, R.

infrarrojos. *Rev Fac Cienc Med Univ Nac Cordoba*, No. 54, pp. 13-18.

*Med. Univ. Nac. Cordoba,* Vol 52 No.1, pp. 27-30.

*Univ Nac Cordoba*, Vol. 52, No. 1, pp. 27-30.

*Fac Cienc Med Univ NacCordoba*, No. 50, pp. 7-10.

laser. *Acta Odontol Latinoam*, Vol. 10, No. 2, pp. 81-88.

baja potencia en la reparación hística. *Rev Cubana Estomatol*, Vol. 33, No. 2, pp. 60-

radiaciones ultravioletas en el mesonefros del embrión de pollo. *Rev Med Cordoba*,

detectores térmicos aplicado en huevos embrionados de pollo, irradiados con rayos

changes produced by non ionizing radiation in the chick embryo. *Rev. Fac. Cien.*

modifications in the lingual glands of the newborn chicken irradiated with He-Ne

histológicas por radiaciones no ionizantes en el embrión de pollo. *Rev FacCienc Med*

(1992). Structural changes induced by He-Ne laser on the chick embryo ovary. *Rev*

responsible for advising on the lamps to regulate medical and aesthetic use.

possibility to observe the biological effects in tissue radiation.

Sliney 2006; Parker,2007).

classification indicated.

experimentally.

**5. References**

63. 9.

No. 89, pp. 9-11.

not have an equal standard safety classification.

**25**

*Australia*

**Alternatives to Radiation**

Shazia Naser-Ud-Din

**Investigations in Orthodontics**

*School of Dentistry, University of Queensland, Brisbane,*

There has been a massive paradigm shift in investigatory tools at present. A greater surge of interest in soft tissue analysis, real time imaging and functional recordings to enhance our understanding of the complexity of the biological system is underway; with high emphasis on non-invasiveness and cost effectiveness. Current studies are looking at the possibilities of using non-ionizing alternatives to derive the required parameters to diagnose, treatment plan and evaluate the orthodontic case. This is in stark contrast to some decades ago where

This chapter will explore the different investigations traditionally used for Orthodontic treatment planning and evaluation of treatment objectives. Since the introduction of lateral cephalometrics in 1931 by Broadbent and Hoffrath this 2 dimensional investigation has been considered the ultimate. However, it has several key limitations – radiation exposure particularly for growing children, 2 dimensional representation of a 3 dimensional living body with static image output. The need to provide a more holistic view to the case assessment with new investigatory tools like 3D imaging, ultrasonography, video recordings and EMG are perhaps the way for the future to assess growing living functioning

At the time of writing there is a wide array of imaging technology with innovative developments occurring at a phenomenal pace. Nearly every time one searches the world wide web something new is presented. The new hype- 3D video softwares are considered to be the next big thing 1. The imaging industry finds wide application in defence, architecture, resource industries and general entertainment. In the medical field, it has immense application for diagnosis and potential to generate understanding not previously possible

In orthodontics, the focus is more based on the external features and soft tissues and 3D reproductions are invaluable not only for diagnosis in general medical scenarios, but more so for treatment evaluation and long-term growth changes. However, the majority of published work tends to come from Japan followed by European and USA researchers 7-9. In this chapter a brief historical background of the different imaging techniques namely Lateral cephalometrics, sEMG, Ultrasonography & 3D imaging as used in contemporary investigations along with important findings from recent studies from author's research

static hard tissue investigations were considered gold standard.

with 2D investigations. The classic examples are MRI and CT scans 2-6.

involving such technology are discussed.

**1. Introduction**

body.

Veterány, L., Hluchý, S. & Veterányová, A. (2004). The Influence of Ultra-Violet Radiation on Chicken Hatching. *J Environ Sci Health A Tox Hazard Subst Environ Eng*. Vol. 39, No. 9, pp. 2333-2339.

## **Alternatives to Radiation Investigations in Orthodontics**

Shazia Naser-Ud-Din

*School of Dentistry, University of Queensland, Brisbane, Australia*

## **1. Introduction**

526 Current Topics in Ionizing Radiation Research

Veterány, L., Hluchý, S. & Veterányová, A. (2004). The Influence of Ultra-Violet Radiation

No. 9, pp. 2333-2339.

on Chicken Hatching. *J Environ Sci Health A Tox Hazard Subst Environ Eng*. Vol. 39,

There has been a massive paradigm shift in investigatory tools at present. A greater surge of interest in soft tissue analysis, real time imaging and functional recordings to enhance our understanding of the complexity of the biological system is underway; with high emphasis on non-invasiveness and cost effectiveness. Current studies are looking at the possibilities of using non-ionizing alternatives to derive the required parameters to diagnose, treatment plan and evaluate the orthodontic case. This is in stark contrast to some decades ago where static hard tissue investigations were considered gold standard.

This chapter will explore the different investigations traditionally used for Orthodontic treatment planning and evaluation of treatment objectives. Since the introduction of lateral cephalometrics in 1931 by Broadbent and Hoffrath this 2 dimensional investigation has been considered the ultimate. However, it has several key limitations – radiation exposure particularly for growing children, 2 dimensional representation of a 3 dimensional living body with static image output. The need to provide a more holistic view to the case assessment with new investigatory tools like 3D imaging, ultrasonography, video recordings and EMG are perhaps the way for the future to assess growing living functioning body.

At the time of writing there is a wide array of imaging technology with innovative developments occurring at a phenomenal pace. Nearly every time one searches the world wide web something new is presented. The new hype- 3D video softwares are considered to be the next big thing 1. The imaging industry finds wide application in defence, architecture, resource industries and general entertainment. In the medical field, it has immense application for diagnosis and potential to generate understanding not previously possible with 2D investigations. The classic examples are MRI and CT scans 2-6.

In orthodontics, the focus is more based on the external features and soft tissues and 3D reproductions are invaluable not only for diagnosis in general medical scenarios, but more so for treatment evaluation and long-term growth changes. However, the majority of published work tends to come from Japan followed by European and USA researchers 7-9.

In this chapter a brief historical background of the different imaging techniques namely Lateral cephalometrics, sEMG, Ultrasonography & 3D imaging as used in contemporary investigations along with important findings from recent studies from author's research involving such technology are discussed.

Alternatives to Radiation Investigations in Orthodontics 529

Fig. 1. **Lateral Cephaolmetric with Linear (Blue)-** 9 linear ( Co-Go, Ar-Go, Go-Me, Masseter Length, R1-R2, R3-R4, N-Me, N-Gn, AGoNotch)**.Angular ( Green)** -6 angular ( GoAngle, UGoAngle, LGoAngle, Max-Man, Frankfort Horizontal to Mandibular plane, ODI) **& Proportional (Red) analysis-**5 proportional (NGn-ArGo, LAFH/TAFH,LPFH/TPFH, ArGo-

GoMe, Jarabak Ratio)17

## **2. Lateral cephalometrics**

Lateral cephalometric techniques have dominated in mainstream orthodontic, orthognathic and dentofacial growth studies for over seven decades. It is considered a versatile tool and has served the profession well since it was first introduced in 1931 by Broadbent 10. Cephalograms have improved our understanding of growth through seminal research 11,12. It is an important diagnostic tool utilized extensively in orthodontics. Recent literature search brought out 28,200 items related to "lateral cephalometrics in Orthodontics" while a similar search on PubMed found 808 documents on related topics. This highlights the volume of work done in the field.

There are two major limitations with cephalometrics; primarily, the additive radiation dosage of progressive films and, secondly, it is a two dimensional representation of the three dimensional craniofacial region. Moreover, the validity of cephalometric analyses has been questioned13 and there have been concerns about the application of such simple analyses in diagnosis and treatment planning. Errors generally are attributed to orientation, geometry and association. A meta-analysis was conducted on six studies for random and systematic error along with repeatability and reproducibility 14 with good repeatability found for limited landmarks namely menton (Me) posterior nasal spine (PNS) anterior nasal spine (ANS) sella (S) pterygomaxillary fissure(Ptm) point A deepest point on the anterior maxillary margin (A) and point B deepest point on the anterior symphysis region of the mandible(B).

A number of computer software sites such as the Dolphin® (Los Angeles CA, USA), Mona Lisa® (Tidbinbilla, Canberra, Australia) (Fig.2) etc. are available to analyse the lateral cephalogram with multiple analyses at a click or a mouse scroll. However, the computer analyses have also been criticized for error through loss of contrast, related to pixel size and poor calibration. Overall, the trained eye can effectively trace and resolve finer details up to 0.1mm or smaller 15 as compared to 2.48mm accuracy recorded from Active Appearance Models (AAM) which is a computer program designed to locate landmarks 16. Such technology needs refinement with mathematical model in order to provide a higher level of accuracy and reproducibility with work in progress by most of the softwares.

Furthermore, the lateral cephalogram has been supplemented with other radiographic conventional images such as panoramic radiography, periapicals, and hand wrist films. The later is now being replaced by the cervical maturation index (CMI) which can be derived from standard lateral cephalograms reinforcing the trend towards minimizing radiation exposure in growing individuals.

There is a galaxy of analyses to choose from. One needs to be mindful of the analyses being a composite of several unique dimensions often with a signature eponym. So it is imperative to choose specific dimensions of relevance to the study at hand. Prudent selection of analysis proves to be time efficient. Figure 1 depicts the basic linear, angular and proportional analysis for majority of diagnostic features commonly required for treatment planning in Orthodontics.

The common range and standard deviations for the different variables from a recent study are given in Table 1. These reflect the general spread for the variables elicited in Fig.1( part of the research project of Dr S Naser-ud-Din for the fulfillment of PhD University of Adelaide 2009)18.

Lateral cephalometric techniques have dominated in mainstream orthodontic, orthognathic and dentofacial growth studies for over seven decades. It is considered a versatile tool and has served the profession well since it was first introduced in 1931 by Broadbent 10. Cephalograms have improved our understanding of growth through seminal research 11,12. It is an important diagnostic tool utilized extensively in orthodontics. Recent literature search brought out 28,200 items related to "lateral cephalometrics in Orthodontics" while a similar search on PubMed found 808 documents on related topics. This highlights the

There are two major limitations with cephalometrics; primarily, the additive radiation dosage of progressive films and, secondly, it is a two dimensional representation of the three dimensional craniofacial region. Moreover, the validity of cephalometric analyses has been questioned13 and there have been concerns about the application of such simple analyses in diagnosis and treatment planning. Errors generally are attributed to orientation, geometry and association. A meta-analysis was conducted on six studies for random and systematic error along with repeatability and reproducibility 14 with good repeatability found for limited landmarks namely menton (Me) posterior nasal spine (PNS) anterior nasal spine (ANS) sella (S) pterygomaxillary fissure(Ptm) point A deepest point on the anterior maxillary margin (A) and point B deepest point on the anterior symphysis region of the

A number of computer software sites such as the Dolphin® (Los Angeles CA, USA), Mona Lisa® (Tidbinbilla, Canberra, Australia) (Fig.2) etc. are available to analyse the lateral cephalogram with multiple analyses at a click or a mouse scroll. However, the computer analyses have also been criticized for error through loss of contrast, related to pixel size and poor calibration. Overall, the trained eye can effectively trace and resolve finer details up to 0.1mm or smaller 15 as compared to 2.48mm accuracy recorded from Active Appearance Models (AAM) which is a computer program designed to locate landmarks 16. Such technology needs refinement with mathematical model in order to provide a higher level of

Furthermore, the lateral cephalogram has been supplemented with other radiographic conventional images such as panoramic radiography, periapicals, and hand wrist films. The later is now being replaced by the cervical maturation index (CMI) which can be derived from standard lateral cephalograms reinforcing the trend towards minimizing radiation

There is a galaxy of analyses to choose from. One needs to be mindful of the analyses being a composite of several unique dimensions often with a signature eponym. So it is imperative to choose specific dimensions of relevance to the study at hand. Prudent selection of analysis proves to be time efficient. Figure 1 depicts the basic linear, angular and proportional analysis for majority of diagnostic features commonly required for treatment planning in

The common range and standard deviations for the different variables from a recent study are given in Table 1. These reflect the general spread for the variables elicited in Fig.1( part of the research project of Dr S Naser-ud-Din for the fulfillment of PhD University of

accuracy and reproducibility with work in progress by most of the softwares.

**2. Lateral cephalometrics**

volume of work done in the field.

exposure in growing individuals.

mandible(B).

Orthodontics.

Adelaide 2009)18.

Fig. 1. **Lateral Cephaolmetric with Linear (Blue)-** 9 linear ( Co-Go, Ar-Go, Go-Me, Masseter Length, R1-R2, R3-R4, N-Me, N-Gn, AGoNotch)**.Angular ( Green)** -6 angular ( GoAngle, UGoAngle, LGoAngle, Max-Man, Frankfort Horizontal to Mandibular plane, ODI) **& Proportional (Red) analysis-**5 proportional (NGn-ArGo, LAFH/TAFH,LPFH/TPFH, ArGo-GoMe, Jarabak Ratio)17

Alternatives to Radiation Investigations in Orthodontics 531

Fig. 2. MonaLisa ® Program for custom made cephalometric analysis. For efficient and

Our understanding of the neurophysiology of the muscles of mastication has increased substantially in the past 60 years. With the introduction of electrophysiological tools in the 1950's 19, an array of experimental protocols have become available around the globe. Although, the majority of studies have used animal models due to the invasiveness of the procedures, results from such studies still provide valuable data that is extrapolated into humans. The earliest EMG studies reported on muscles of mastication during basic mandibular movements were from Carlsöö(1952), Göpfert & Göpfert (1955) in Moller( Moller 196620. These preliminary studies improved our fundamental understanding of the complex masticatory system. Other work of historical interest is from Perry & Ahlgren21,22. EMG registers signals of muscle contractility through action potentials delivered by the motoneurones. Highly refined bipolar surface electrodes are sensitive to these electrical

In the human model, indirect methods are employed and are preferably, non-invasive. Surface electromyography (sEMG) is a tool that is used extensively to explore the neural circuitry. Although, it has its limitations (discussed later) is overall considered user-friendly,

standardized analysis computer based software packages are widely utilized.

**3. sEMG (surface Electro Myo Graphy)**

signals and, once amplified, are visible as the EMG recordings.

non-invasive and has extensive applications in neurophysiology.

Table 1. Common distribution for the variables from cephalometrics.  Common distribution for the variables from cephalometrics. ( Part of the doctorate

project Dr S Naser-ud-Din University of Adelaide Australia, 2009)

Fig. 2. MonaLisa ® Program for custom made cephalometric analysis. For efficient and standardized analysis computer based software packages are widely utilized.

## **3. sEMG (surface Electro Myo Graphy)**

Our understanding of the neurophysiology of the muscles of mastication has increased substantially in the past 60 years. With the introduction of electrophysiological tools in the 1950's 19, an array of experimental protocols have become available around the globe. Although, the majority of studies have used animal models due to the invasiveness of the procedures, results from such studies still provide valuable data that is extrapolated into humans. The earliest EMG studies reported on muscles of mastication during basic mandibular movements were from Carlsöö(1952), Göpfert & Göpfert (1955) in Moller( Moller 196620. These preliminary studies improved our fundamental understanding of the complex masticatory system. Other work of historical interest is from Perry & Ahlgren21,22.

EMG registers signals of muscle contractility through action potentials delivered by the motoneurones. Highly refined bipolar surface electrodes are sensitive to these electrical signals and, once amplified, are visible as the EMG recordings.

In the human model, indirect methods are employed and are preferably, non-invasive. Surface electromyography (sEMG) is a tool that is used extensively to explore the neural circuitry. Although, it has its limitations (discussed later) is overall considered user-friendly, non-invasive and has extensive applications in neurophysiology.

Alternatives to Radiation Investigations in Orthodontics 533

Muscle spindle reflexes are stronger in short face height individuals17,34 which may explain the phenomenon with bruxism and brachyfacial tendency.  The authors believe their system can help diagnose different types of bruxism and is aptly termed the innovative bruxism analysing system. Once again, the literature supports the use of sEMG as a useful mode of investigation.

EMG has provided insight into the neuromuscular system but there are inherent limitations which must be minimized in order to derive meaningful data. This has been reviewed thoroughly 36. EMG accuracy may be enhanced by the use of intramuscular electrodes compared with sEMG electrodes. However, sEMG electrodes with built-in amplifiers can assist in reducing cross-talk and movement artifact. Passive surface electrodes require skin preparation and monitoring of resistance below 10Ω for clear data recording. Contemporary sEMG involves the use of bipolar electrodes, which suppress noise. The amplification should counter distortion, preferably 10 x higher than the electrode-to-skin electrical resistance. The filter level is dependent upon skin thickness and the frequencies recorded. It should not eliminate any of the frequencies within the range of recording and the choice lies

translate into better designs for splints and Orthodontic appliances.

between upper (high-cut or low pass) and lower (low-cut or high pass).

Fig. 3. Subject seated comfortably throughout the experiment session. sEMG recorded bilaterally from the masseter and the digastric muscles: (a) biting on a custom made impression; (b) left central incisor mechanically stimulated by an orthogonal probe;(c) local anaesthetic was infiltrated to eliminate periodontal mechanoreceptor input during the

(a)

(b)

(c)

spindle trials. An ear clip provided the electrical ground.

**3.3 Limitations & methodological issues**

## **3.1 Development of sEMG devices**

The first chewing apparatus was described by Moller in 1966 20. It was a landmark achievement in which bipolar recordings could be saved on photographic oscilloscopes rather than printed by inkwriters. Therefore, several experiments were designed to enhance our understanding of muscle activity during chewing, postural activities, full effort clenching, swallowing patterns and even facial morphology. This innovative apparatus only required an EMG recorder with electrodes. The electrical activity was recorded on three channels. Although, an advanced methodology for its time, Moller's apparatus had the serious limitation of un-standardized positioning of the subject. This produced a lack of reproducibility, albeit the reproducibility factor poses a challenge even with contemporary advancements. Other methodological issues arose from interference from neighbouring potentials, rejection of common voltage, distortion of amplitude and uneven distribution of electrical activity 20. Over time, several global studies have been conducted which have improved designs and chewing devices 23,24 leading to an enhanced understanding of the reflexes in the oro-facial regions.

The periodontal-masseteric reflex was first described by Goldberg 1971 25 highlighting the importance of not only PMR but also the role of the masseter muscle and central connections, due to the short latency. This study concluded that there was a central connection located in the mesencephalic nucleus that was responsible for the excitatory masseteric reflex evoked by PMR and gingival receptors. One can appreciate that, even with a very simplistic methodology and manual tapping of the teeth, Goldberg was able to deduce these important findings.

## **3.2 EMG studies and significance in orthodontics**

Work in this field has resulted in few conclusive findings. A canonical correlation analysis between facial morphology, age, gender and EMG during rest and contraction has not found a statistically significant correlation 26. EMG and bite force have been studied extensively20,27,28 with consistent findings of reduced force levels in dolichofacial patterns.

Different vertical facial types, both in adults and children, produce differences in EMG responses recorded over the course of a day 29. Masseter muscle EMG activity was found to be consistently longer in short vertical dimension facial types as compared to high mandibular plane angle individuals. The variables of bite force, muscle efficiency and mechanical advantage in children with vertical growth patterns 30 were negatively correlated with muscle efficiency and vertical proportions.

Morimitsu *et al.* documented recordings of EMG from masseter muscles which showed positive correlations with linear cephalometric measurements; particularly, the muscle activity was significantly related to mandibular dimensions such as (Co-Go) and body length (Go- Me) 31. In fact, the muscle activity increased with decrease in vertical proportions such as mandibular plane angle. Furthermore, they compared the muscle activities with anterior-posterior skeletal base relationships indicated by Sella Nasion Point A (SNA) and Sella Nasion Point B (SNB) variables. Morimitsu *et al.* strongly advocated the use of EMG for examining masticatory activity, but one could argue that the reproducibility with electrode placement at subsequent visits may prove to be a challenge 32.

A comprehensive study by Tuxen *et al.* 33 compared masseter muscle fibre types along with function from EMG and facial morphology. They concluded that even with intensive analysis of different dimensions, linear regression analyses failed to show any significant association highlighting, once again, the complexity of the craniofacial region.