II.4. TATTOOS
II.4.1. Tattoos: Definition. Evolution.
Tattoos are permanent marks on the skin obtained by injecting pigment in the dermis. The process of tattooing dates approximately 8000 years back, and has been reported in most known civilizations. Nowadays, professional tattoos are performed with a device based on a 1891 patent created after Thomas Edison’s engraving machine. The modern tattoo process has been adapted to minimize the health risk of the clients by using sterile mixtures, single-use components and aseptic techniques. Often, patients ask surgeons to preserve the tattoo as much as possible, if it interferes with the operating site61.
II.4.2. Physiology and histology of tattoos.
Tattoos are created by creating pores in the skin in which pigment is placed. Usually, a fast oscillating needle (50-3000 Hz, usually ~80-150 Hz) injects pigment through the epidermis at various depths, usually, in the papillary dermis. The pigment is fixated in the skin due to the inflammatory reaction caused by needle and pigment, as presented in Figure 630.
Figure 6. Left. The needle passage inside the skin. The basement membrane seals back in time. Right: Depth of pigment placement is paramount to the final aspect of the tattoo. The deeper the pigment is placed inside the dermis, the more attenuated the initial color will be.
https://s-media-cache-ak0.pinimg.com/736x/80/c5/3e/80c53ea0bb0931e12256161429c2378d–stretch-mark-remedies-skin-needling.jpg
http://www.cosmetictattoo.org/admin/images/articles/pigment-depth.jpg
Figure 7 displays how the pigment is placed both in the dermis and the epidermis, but the latter is quickly removed by the fast shedding pace of the epidermis. The ink is stored in the extrafibrillar collagen matrix30, but also inside the activated macrophages and granulation tissue following skin trauma. Since the pigment cannot be broken down, the tattoo is thus fixated, usually after 2-3 months.
Figure 7. Tattoo fixation. Initially, ink is dispersed in both epidermis and papillary dermis. After 1 month, the epidermis is completely regenerated and only minimal ink loss occurs until the basement membrane heals (2-3 months).
http://archives.evergreen.edu/webpages/curricular/1999-2000/humanbio/TattooInk.htm
Tattoo final color is influenced by skin thickness alteration: increased in UV light exposure (sun or tanning salons), inflammatory conditions (dermatitis, psoriasis, allergies), systemic diseases (diabetes mellitus, scleroderma, hypothyroidism) and decreased with age, chronic sun exposure, local corticoid therapy, menopause, frequent skin exfoliation. In time, tattoos are advancing and dispersing toward the reticular dermis, thus fading in color and acquiring a blue tint, losing the initial clarity of the image.
Figure 8.Tattoo final color is determined both by the pigment used and the skin color. The initial aspect will fade as the tattoo heals (up to 3 months).
http://www.cosmetictattoo.org/article/why-do-cosmetic-tattoos-change-colour-part-2.html
The ideal pigment has a big molecular size, is not immunogenic and not soluble or degrading under the action of inflammatory molecules and cell enzymes, thus maintaining its location and structure over a longer period of time38,48,65.
II.4.3. Tattoo usage in medical sciences and medical practice
Tattoos have been previously used in medical practice or training. Tattooed pig skin is used for training model in dermatology specialists and residents41. Endoscopic marking of colon for oncological limit resection have been reported3. Tattoo migrated in lymph nodes can mimic malignancy aspect in PET-CT scans32. Certain red pigments can induce irritation, photosensitivity and even cancer50. Tattoos have been previously used in research to mark wound margins, but no validation of the method was provided48.
II.5. RAT MODELS FOR BURNS RESEARCH
II.5.1. Definition
“An animal model is a living organism in which normative biology or behavior can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animal21.”
Rats (Wistar Rats, Rattus norvegicus) are a good model for wound healing, especially due to their availability, low maintenance, reduced size and fast regeneration, being preferred over other mammals that are less versatile to work with19,53,55,67. Rats and humans have common characteristics that are documented in literature and recommend them for use in skin healing models. Nonetheless, the use of rodent models in burn research is still debated, due to little translational value between rat models and human pathophysiology. A humanized, genetically modified rat model has also been developed in 2015.18 Furthermore, rats are loose skinned mammals, and thus heal primarily by wound contraction rather than epithelialization, through the contraction of panniculus carnosus muscle (Figure 9). Another main difference is the presence of L-gluconolactone enzyme helps synthesize vitamin C and accelerates collagen synthesis44,51.Rat hair follicles have anagen and telogen cycles that influence burn healing (the former heal faster than the latter)1,9.
Figure 9.Histological slide of rat skin section presenting the location of panniculus carnosus14, located in the subcutaneous tissue, deep to panniculus adiposus.
II.5.2. Classification by burn mechanism
Burn models are more difficult to create than other acute injury models due to high variability of the parameters. Burn agent material, temperature and contact time need precise standardization and have often inter-operator variability. Several models have been described in the literature, but most are not reproducible, do not provide sufficient method details and none offer a perfect solution of a standardized animal model45.
Contact
The most common way to induce burns by skin contact is using metal objects (bar, plate, rod, block, weights) that are pressed against the skin with constant pressure for a standardized amount of time, resulting in deep partial or full thickness (second or third degree) burns49. Usually, the rods are heated by immersion in hot water, but other methods such as flame or radiation are not uncommon. Temperature can be standardized by measuring the temperature of the water or of the brass rod, but such practice is not always reported9. The pressure is dependent on rod weight and can be measured with pressure sensors embedded in the rod. Conductive metals are desired, but for achieving a deeper burn, higher temperatures are needed (100-400°C) and frequent reheating is necessary. Also, contact burns can cause the skin to stick to the infliction device, thus aggravating the injury in a non-standardized manner22,37,57,58. Other contact burns models, though less popular, have been inflicted by direct electric current, electric coil, hot cloth, hot sponge, molten wax1.
Scalding / immersion
Scalding injuries can be simulated by steam for controlled depth or by immersion in boiling water (Walker and Mason method is the most popular technique). Ethanol baths have also been reported. The rat under anesthesia is placed in a cylindrical tube with a variable opening that corresponds to burn site and size. The animal needs to be heavily sedated during the procedure because the trauma is much greater than a series of small injuries1,2,66.
Chemical
Chemical burns can be inflicted through standardized protocols of precise dosage of various substances. The downside lies in the difficulty to control the chemical reaction between the compound and the skin, possible resulting burn variability2.
Radiation
Accurate burns can be induced by laser. Tissue damage depends on energy input and wavelength. Computer-aided beam allows more control over the outcome in terms of constant contact time, depth and burn location8,17,46.
II.6. MEDICAL IMAGE PROCESSING
II.6.1. Image processing. Definition, uses.
Image processing represents a sum of mathematical operations that provide an output in form of quantitative results or parameters regarding data encoded in an image format (static image or image batches, such as a video), in order to extract targeted information. The basic steps are: 1) importing the image (or converting an analog image into a digital image), 2) manipulation the image and analyzing parameters, 3) output the results in a useful format. The main directions of image processing are: 1) visualization (obtaining parts of image that are not clearly visible), 2) image restoration (enhancing an image for better visualization), 3) image retrieval (obtain an image from a dataset), 4) measure parameters (histograms, shapes, sizes, objects), 5) automatic image recognition (artificial intelligence algorithms that identify objects). Image processing is becoming increasingly used in medicine for image recognition, big data analysis, sorting algorithms and even diagnostics. In burn healing research, image processing is used to recognize burn injury (in both animal models and human patients), estimate burn area, compute burn area evolution in time and predict healing and burn depth based on wound color. While some techniques are difficult, inexact and expensive, other methods can be applied with ease and have a shorter learning curve. Hence, the current paper aims to describe a simple, easy to learn method that is applicable for measuring burn area in prospective studies in animal models27.
II.6.2. ImageJ software
ImageJ is open source software develop in Java ® platform that is adapted for medical images processing. It has dedicated modules for various function and has a visual interface that allows both beginners and experienced users to perform a multitude of tasks35.