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The effect of long-term black tattoo on the skin of adult male guinea pigs and the probable role of imiquimod cream on tattoo removal: a histological and immunohistochemical study

Mohamed, Enas T.; Mahmoud, Somaya H.; Shaalan, Aly A.E.; Mohamed, Abeer Abdel Khalik

The Egyptian Journal of Histology: December 2011 - Volume 34 - Issue 4 - p 751–761
doi: 10.1097/01.EHX.0000407038.46912.43
Original articles

Background Tattoo is related to many skin problems, one of them being the necessity to remove it. Imiquimod is a novel synthetic compound with immunomodulatory action, and its role as a safe method to remove short-term tattoo within 7 days after tattooing was confirmed.

Objective The present study was conducted to investigate the effect of long-term (after 3 weeks) black tattoo on the skin of guinea pigs and the role of imiquimod cream in removing it.

Methods This study was carried out on 32 adult male guinea pigs. The animals were randomized and divided equally into four groups: group I (control group); group II (imiquimod cream group): animals in this group received topical application of imiquimod 5% cream once every 6 h for 7 days. Group III (tattoo group): animals in this group were tattooed once with black tattoo pigment. Group IV (tattoo/imiquimod cream group): animals in this group were tattooed with black tattoo pigment, followed, after a gap of 3 weeks, by a topical application of imiquimod 5% cream on the tattooed skin once every 6 h for 7 days. At the end of the experiment, the animals were sacrificed, and specimens were taken from the skin of each animal. The specimens were subjected to different histological techniques and immunohistochemical stains for detecting macrophage Ab-1Mac 387. For statistical analysis, the slides were examined to assess the epidermal thickness, optical density of collagen fibers, and macrophage expression in the epidermis and the dermis.

Results The epidermis of the tattoo/imiquimod cream, the tattoo, and the imiquimod cream groups showed a significant increase in thickness compared with the control group. Keratinocytes showed cellular atypia, vaculation, pyknosis, and chromatin margination. The tattoo and the tattoo/imiquimod cream groups showed a significant increase in collagen fibers, whereas the cream group showed a significant decrease. Localization of tattoo pigments was seen in the tattoo/imiquimod cream group. Macrophage Ab-1Mac 387 expression in the skin of the tattoo/imiquimod cream and the imiquimod cream groups showed a significant increase compared with the tattoo group.

Conclusion Tattoo causes skin damage. Although imiquimod 5% cream alone caused localization of tattoo pigments and activation of macrophages, it has a hazardous side effect on the skin. Further studies should be conducted to find other ways to remove long-term tattoo with minimal side effects.

Department of Histology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence to Abeer Abdel Khalik Mohamed, Department of Histology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt Tel: +20 966 535 928 225/+20 966 569 518 143; e-mail: abeeeeer2000@yahoo.com

Received May 28, 2011

Accepted July 21, 2011

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Introduction

Tattoos are a form of body art that have been practiced throughout history by various cultures. In addition to religious considerations, permanent tattoos are commonly applied for cosmetic purposes on the eyebrows, lips, eyes, and even moles [1]. Tattooing is a process of marking the skin with drops of insoluble ink by a needle into the dermis [2]. Tattoo ink consists of a carrier and a pigment; the carrier acts as a solvent for the pigment. The pigment originated from many sources as the original mineral pigments, modern industrial organic pigments, and vegetable-based pigments. Pigments have a variety of colors, the most common and widely used one being black [3].

Tattoo causes many skin problems such as acute inflammatory reactions, eczematous hypersensitivity reactions, photoaggravated reactions, granulomatous reactions, lichenoid reactions, and pseudolymphomatous reactions [4]. One of the common problems related to tattoos is the necessity to remove it. Tattoo removal process is difficult, painful, and the degree of success depends on the materials and regime used [5].

Imiquimod is a novel synthetic compound with an immunomodulatory role; it is a member of the imidazoquinolone family [6]. The use of imiquimod was confirmed clinically and histopathologically as a safe and nonsurgical method of acute-phase tattoo removal (from 6 h to 7 days after tattoo) [7]. However, the effect of imiquimod cream on the removal of chronic phase tattoo (more than 7 days after tattoo) is not evident till now. Hence, the present study was conducted to investigate the effect of long-term black tattoo on the skin of guinea pigs and the role of imiquimod cream in removing it.

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Materials and methods

This study was carried out on 32 male adult guinea pigs weighing 200–250 g each. Animals were acclimatized to their place for 1 week before the start of the experiment, and housed in their cages with free access to food and tap water.

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Experimental protocols

Animals were randomized and divided into four groups (eight animals per group). Group I (control group): animals in this group were not exposed to any tattoo or treatment. Group II (imiquimod cream group): animals in this group received topical application of imiquimod 5% cream once every 6 h for 7 days [7]. Its trade name is Aldara cream (produced by 3M Health Care Limited, Loughborough LE11 1EP England UK). Group III (tattoo group): animals in this group were tattooed once with black tattoo pigment (Black-Mei-Chai-Na Company, Ltd. Taiwan Export,labvision Lab Vision in Fremont, California, USA which contains purified water 100%, iron oxide black 32%, and veegum 10%). Group IV (tattoo/imiquimod cream group): animals in this group were tattooed with black tattoo pigment, followed, after a gap of 3 weeks, by a topical application of imiquimod 5% cream on the tattooed skin once every 6 h for 7 days [7].

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Animal preparation

Hair on the back of the animals was shaved in a specific area (1 × 1 cm) with ordinary hair clippers [8].

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Tattoo application

With the help of an expert tattooist, black tattoo was applied once in a 1 × 1 cm skin area, using a tattoo machine, after subjecting animals to light ether anesthesia. The hygiene of the skin of all animals was taken care of, to prevent any contamination or infection.

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Imiquimod 5% cream application

A one-fifth portion of the cream sachet (the sachet=amount of cream covers nearly 5 × 5 cm of skin) of 5% imiquimod cream was applied to the 1 × 1 cm skin area once every 6 h for 7 days [7].

At the end of the experiment, animals in each group were sacrificed by decapitation under light ether anesthesia. One specimen was taken from each animal. The specimens were fixed in 10% neutral buffered formalin solution and processed, and paraffin blocks of 5-μm-thick sections were prepared. Sections were stained with H&E stain for assessment of the general skin architecture and Masson’s trichrome for collagen fibers. Immunohistochemical staining was carried out for detection of macrophages, using the avidin–biotin complex method (Lab Vision) with the monoclonal antibody Macrophage/L1 Protein/Calprotectin Ab-1 (Clone MAC 387) [9,10]. For immunohistochemical staining, deparaffinization, and rehydration, sections were incubated in 0.006% hydrogen peroxide for 60 min, to block the endogenous peroxidase, and washed with PBS. Sections were incubated for 2 h in the primary antibody [Macrophage/L1 Protein/Calprotectin Ab-1 (Clone MAC 387)]. Negative controls were run routinely in parallel by omitting of the primary antibody. The binding of the primary antibody was visualized using the commercial avidin–biotin–peroxidase detection kit (Dako, Carpinteria, California, USA) according to the manufacturer’s instructions. Staining was completed with DAB Chromogenic for 1–2 min and slides were counterstained with Harris’s hematoxylin, dehydrated, and then coverslipped. A brown cytoplasmic reaction was observed.

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Data analysis

Using the Heidi Soft image analysis system, the following quantitative measurements were performed:

  1. (1) Epidermal thickness in H&E-stained sections.
  2. (2) Optical density of collagen fibers in Masson's trichrome-stained sections.
  3. (3) Optical density of macrophage expression in the epidermis and dermis in the immunohistochemical stained sections.
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Statistical analysis

All statistical analyses were performed using the statistical software package SPSS 15.0 for Windows (SPSS Inc., Chicago, Illinois, USA). The data obtained were tabulated and statistically analyzed using the Student t-test for statistical significance to compare between the two different groups. P < 0.05 was considered statistically significant.

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Results

Animals were active, with no clinical abnormalities during the period of the experiment, except some visible irritability among the imiquimod group animals at the time of cream application. There were no deaths among the animals.

In brief, no changes were observed in the skin of animals in the control group, whereas the skin of animals in the imiquimod group (group II) appeared erythematous 2 days after the cream application and this increased in severity until the end of the experiment. The skin of animals in the tattoo group (group III) showed the black tattoo pigment (in surface area 1 × 1 cm) with mild erythema in the skin surrounding the tattooed area, which appeared immediately after tattoo application and then started to disappear 1 week later. However, the skin of the tattoo/imiquimod cream group (group IV) showed a decrease in the black tattooed surface area. Skin erythema was not obvious because of the presence of the tattoo pigment. By the end of cream application, the tattoo pigment neither disappeared nor faded.

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Histological and immunohistochemical results

Group I

H&E-stained sections of the control group showed normal structure of the epidermis, the dermis, and the hypodermis (Figs 1 and 2). The mean epidermal thickness in this group was 7.34 ± 1.4 (Table 1). Using Masson’s trichrome-stained sections, the dermis of animals in this group showed greenish fine collagen fibers in the papillary dermis and coarse irregular interwoven fibers in the reticular dermis and around the hair follicles (Fig. 3). The mean optical density of collagen fibers in this group was 0.24 ± 0.04 (Table 2). Using immunohistochemical techniques, macrophage Ab-1Mac387 expression in the skin of this group showed a mild reaction in the form of a brown color in Langerhan’s cells in the epidermis and in the connective tissue surrounding the sebaceous glands and hair follicles (Fig. 4). The mean optical densities of macrophage Ab-1Mac387 in the epidermis and the dermis of this group were 0.33 ± 0.02 and 0.31 ± 0.02 sequentially (Table 3).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

Table 1

Table 1

Table 2

Table 2

Table 3

Table 3

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Group II

H&E-stained sections of the imiquimod group showed acanthosis, represented by a significant increase in the epidermal thickness compared with the control group (Figs 5 and 6). The mean epidermal thickness in this group was 15.4 ± 1.4 (Table 1). Keratinocytes showed hyperplasia and apparent atypia in all epidermal layers. The atypia were in the form of deeply stained nuclei of variable sizes and shapes, with prominent nucleoli. Necrosis in the form of vacuolated cytoplasm with pyknotic nuclei was shown in some keratinocytes (Fig. 6). A few nuclei with chromatin margination were also observed (Fig. 6). Stratum corneum showed the loss of a basket wave appearance (compact layers) (Fig. 6). The sections showed partial dermo-epidermal separation in some areas (Figs 5 and 6). Apparent mononuclear cellular infiltration was observed in the dermis of all sections (Fig. 6). Masson’s trichrome-stained sections in this group revealed a significant decrease in collagen fibers represented by fragmented strands of collagen fibers in both the papillary and the reticular dermis and around the hair follicles. The mean optical density of collagen fibers in this group was 0.13 ± 0.03 (Fig. 7) (Table 2). Using immunohistochemical-stained sections of this group, the expression of macrophage Ab-1Mac387 showed a significant increase in reaction in the epidermis and the dermis compared with the control group. The mean optical density of macrophage Ab-1Mac387 in the epidermis of this group was 0.59 ± 0.06 and in the dermis was 0.44 ± 0.01 (Fig. 8) (Table 3).

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

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Group III

H&E-stained sections in the tattoo group revealed (acanthosis) a significant increase in the epidermal thickness compared with the control group, and its mean was 13.1 ± 2.5 (Table 1). Diffuse black tattoo pigment in both the papillary and the reticular dermis was also detected (Figs 9 and 10). The sections showed apparent keratinocytic atypia and necrosis in the form of vacuolated cytoplasm with pyknotic nuclei (Fig. 10). The sections showed apparent hyperplasia of keratinocytes in the stratum spinosum and thickness and spaces between keratinocytes. A few nuclei with chromatin margination were also observed (Fig. 10). Stratum corneum showed the loss of a basket wave appearance (Fig. 10). Mononuclear cellular infiltration was observed in the dermis and around the hair follicles in all sections (Fig. 10). Masson’s trichrome-stained sections in this group showed a moderate increase in the amount and the density of collagen fibers in both the reticular and the papillary layers and around the hair follicles compared with the control group (Fig. 11). The mean optical density of collagen fibers in this group was 0.37 ± 0.02; there was a statistically significant increase compared with the control group (Table 2). Using immunohistochemical techniques, macrophage Ab-1Mac387 expression in the skin of this group showed a significant decrease in reaction in the epidermis and the dermis compared with the control group (Fig. 12). The mean optical density of macrophage Ab-1Mac387 in the epidermis of this group was 0.10 ± 0.01 and in the dermis was 0.19 ± 0.04 (Table 3).

Figure 9

Figure 9

Figure 10

Figure 10

Figure 11

Figure 11

Figure 12

Figure 12

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Group IV

H&E-stained sections in the tattoo/imiquimod cream group showed (acanthosis) a significant increase in the epidermal thickness compared with the control group. The mean epidermal thickness in this group was 14.5 ± 2.1 (Table 1). A slightly localized black tattoo pigment was observed in the dermis (Fig. 13). Keratinocytes showed atypia and marked necrosis of keratinocytes in the form of a vacuolated cytoplasm with pyknotic nuclei (Fig. 14). Chromatin margination in the stratum spinosum layer was also detected in a few cells (Fig. 14). The stratum corneum showed the loss of a basket wave appearance. Partial dermo-epidermal separation was also seen in some areas (Fig. 14). Mononuclear cellular infiltration was observed in the dermis and around the hair follicles in all sections in this group. Masson’s trichrome-stained sections in this group showed a mild increase in the amount and density of collagen fibers in both the reticular and the papillary layers and around the hair follicles compared with the control group (Fig. 15). The mean optical density of collagen fibers of this group was 0.30 ± 0.04; there was a statistically significant increase compared with the control group (Table 2). Expression of macrophage Ab-1Mac387 in sections of this group showed a significant increase in reaction in the epidermis and the dermis compared with the control group (Fig. 16). The mean optical density of macrophage Ab-1Mac387 in the epidermis of this group was 0.52 ± 0.08 and in the dermis was 0.37 ± 0.09 (Table 3).

Figure 13

Figure 13

Figure 14

Figure 14

Figure 15

Figure 15

Figure 16

Figure 16

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Discussion

The present study was conducted to investigate the effect of long-term black tattoo on the skin of guinea pigs and the role of imiquimod cream in removing it. The epidermis of the tattoo group showed a significant increase in thickness, keratinocytic atypia, and necrosis of keratinocytes. These findings were previously documented in many studies on humans and experimental animals [11–16]. The increase in epidermal thickness and keratinocytic atypia noticed in the tattooed skin is speculated to reflect the compensatory hypertrophy of the tattooed skin, resulting from the inflammatory reaction that occurred within the keratinocytes. This inflammation is caused by dysregulation in their growth through the production of soluble mediators with proinflammatory properties such as IL-1α and growth activation by TGFα [16]. Necrosis of keratinocytes, which was detected in the present study, is consistent with some studies that reported different grades of necrosis of keratinocytes with tattoo [14,17]. Some investigators did not detect necrosis of keratinocytes [16,18]. This difference may be explained by their use of red tattoo dyes instead of the black ones used in the present study. These two dyes are different in their chemical composition [3]. Necrosis may be attributed to keratinocyte and dermal fibroblast activation, which results in the secretion of different cytokines such as TNFα and IL-1α [13,19]. These cytokines lead to the upregulation of nitric oxide (NO), which causes epidermal cells necrosis. NO is produced under the effect of inducible NO-synthase found in Langerhan’s cells [20,21].

Dermal changes in the tattoo group in the present study were evident by a significant increase in the density of collagen fibers. The previous findings in the dermis are similar to those of Cui et al. [15], who reported variable dermal fibrosis with thick collagen bundles. This can be explained by the secretion of IL-1 by the activated keratinocytes as an inflammatory response to the tattoo pigments, and this IL-1 induces an increased fibroblast synthesis of collagen types I and III [21]. An apparent mononuclear cellular infiltration was observed in the dermis and around the hair follicles in this group. This is similar to what was detected by many studies [13,14]. This infiltration can be attributed to the inflammatory role of macrophages in response to the tattoo pigment [16]. Macrophages and keratinocytes secrete the cytokines TNF and chemokines together with NO. Passing through a cascade of immune responses, leukocytes, mainly monocytes and neutrophils, rapidly accumulate at the site of inflammation [11]. Tattoo pigments were found in the papillary and reticular dermis of the skin of this group, and this was also detected by Engel et al. [22] who found that the ink itself was initially dispersed as fine granules in the upper dermis. This can be explained by the penetration of the tattoo machine into the skin by about a millimeter or less, which deposits a drop of insoluble ink with each puncture [21].

The macrophage Ab-1Mac 387 is a macrophage antibody that reacts with the calprotectin inside the macrophages wherever they are found and this also includes Langerhan’s cells. In the normal skin, the expression of this antibody is in the Langerhan’s cells in the epidermis, because they contain calprotectin, and in a few macrophages in the dermis [23]. The expression of macrophage Ab-1Mac 387 in the tattoo group in the present study showed a significant decrease in reaction in the epidermis and the dermis. No studies were conducted before on macrophage Ab-1Mac 387 detection in tattooed skin, but the idea of using macrophage recruitment to remove the tattoo pigment from the tattooed skin was applied in the study of Cohen (1995), who used macrophages after laser treatment to engulf the altered pigment and they recruited them using the macrophage colony-stimulating factor [23]. The mechanism by which the tattoo induced the loss of macrophage Ab-1Mac 387 reaction in the epidermis and the dermis in this group may be attributed to the presence of tattoo ink particles inside the macrophages; these particles were sufficiently large to inhibit the activity of macrophages and resulted in an appearance of macrophage ‘freezing’ [24,25].

In the present study, the epidermis of the imiquimod cream group showed a significant increase in thickness, keratinocytic atypia, and necrosis of keratinocytes. The increased epidermal thickness is consistent with the results of a study conducted by Rajan and Langtry [25], which showed that the use of imiquimod 5% cream for the treatment of superficial basal cell carcinomas caused generalized exacerbation of psoriasis and increase in epidermal thickness. The keratinocytic necrosis found in this study was also found in some studies conducted using imiquimod 5% cream in the treatment of different diseases: for example, Berman (2003), who used this cream for the keloid management [26], and Berman et al. [27], who studied the effect of imiquimod 5% cream on basal cell carcinomas. The increased epidermal thickness and the necrosis of keratinocytes found in the epidermis of this group can be attributed to the mechanism of action of imiquimod 5% cream, which is an immune system modulation and stimulation through binding to the toll receptor 7 present on dendritic cells, macrophages, and monocytes. Subsequent activation of these cells leads to the release of proinflammatory mediators IFNα, TNFα, IL-16, IL-8, IL-10, and IL-12. These mediators lead to hypertrophy in the keratinocytes, which lead to the subsequent increase in the epidermal thickness [26–28].

Dermal changes in the imiquimod 5% cream group in the present study were principally manifested by a significant decrease in the density of collagen fibers, dermo-epidermal separation, and mononuclear cellular infiltration. This can be explained by the fact that activated macrophages, dendritic cells, and monocytes under the effect of the cream secrete fibroblast-inhibiting cytokines IFNα, IFNγ, and TNFα, which induces a decreased fibroblast synthesis of collagen types I and III [29,30]. The partial dermo-epidermal separation seen in some skin areas in this group may be because of the edema that occurred in the skin as an inflammatory response to the cream. The mononuclear cellular infiltration that was observed in the dermis and around the hair follicles in the skin of the imiquimod cream group is consistent with the study of Michalopoulos et al. [30], who found infiltration of macrophages and lymphocyte markers such as CD68+ macrophages, CD3+ T cells, and CD8+ T cells during the use of imiquimod cream for the treatment of lentigo maligna in humans. The interpretation of this finding is also through its mechanism of action as an immune-modulator drug that activates the immune system to send many inflammatory cells to the site of imiquimod cream application [31].

There was a significant increase in the expression of macrophage Ab-1Mac 387 in the epidermis and the dermis of the imiquimod cream group in the present study. Many immunohistochemical studies were conducted to assess the function of imiquimod 5% cream as an immunomodulator, but none of them studied macrophage Ab-1Mac 387 in specific. One of these is the study conducted by Somani et al. [32] on atypical nevi treated by imiquimod 5%, who found an increase in the expression of other macrophage markers such as CD4+ and CD8+ cells. Another one is the study conducted by Ooi et al. [33] on actinic keratosis, who found a statistically significant increase in the biomarkers’ level for CD4+ CD8+. Somani et al. [32] also reported an increase in the granulocyte–macrophage colony-stimulating factor and the granulocyte colony-stimulating factor together with the macrophage inflammatory protein1-α using imiquimod 5% cream. The increased expression of macrophage Ab-1Mac 387 in the epidermis of this group confirms the mechanism of action of imiquimod 5% cream on the immune system modulation and macrophage activation [28]. There are not many studies conducted on imiquimod 5% cream as a way of tattoo removal. Most of them studied the role of imiquimod as an adjuvant to laser removal of tattoos. Limited studies have been conducted to assess the role of imiquimod cream alone on tattooed skin in the removal of either short-term [7] or long-term tattoo.

The epidermis of the tattoo/imiquimod cream group in the present study showed a significant increase in thickness, necrosis of keratinocytes, keratinocytic atypia, and mononuclear cellular infiltration. This may be attributed to the combined inflammatory effects of the tattoo and the imiquimod cream and the mechanical irritation of the tattoo machine on keratinocytes. Dermal changes were principally manifested by the significant increase in the density of collagen fibers. Solis et al. [7] documented the presence of fibrosis in their study, in which they used imiquimod cream to remove the acute-phase tattoo (6 h after tattoo application) from the skin of guinea pigs. This finding may be explained by the inflammatory reaction induced by the tattoo, which leads to the secretion of different fibroblast-activating cytokines such as IL-1. These cytokines oppose the fibroblast-inhibiting cytokines secreted under the effect of the imiquimod cream, and the net result is an activated fibroblast and therefore increased synthesis of collagen [7]. Tattoo pigments could still be seen in this group and localized in both the papillary and the reticular dermis, with no change in the density of the color. This finding is consistent with Ehrlich [34], who used this cream alone to remove the tattoo in a trial performed on one of his patients who had a tattoo for many years. He found that, after 6 weeks of cream application, the tattoo did not clear significantly. However, Solis et al. [7] found, in their study on guinea pigs, that the application of imiquimod cream 6 h after tattooing (short-term tattoo) completely removed the tattoo clinically and histopathologically. The discrepancy between this study and what Solis et al. [7] found in their study may be because they started the cream application directly 6 h after tattoo application (short-term tattoo); during this period, the tattoo pigments are still dispersed as fine granules in the epidermis and not inside the macrophages, and so the macrophages are not yet frozen; therefore, with more active macrophages recruited by the cream, the tattoo pigments could be removed easily.

The expression of macrophage Ab-1Mac 387 in the epidermis of the tattoo/imiquimod cream group in the present study showed a significant increase that in turn confirms the mechanism of action of imiquimod 5% cream in activating the immune system even in the presence of an immune system deactivator such as the tattoo ink. Hence, in a long-term tattoo, the tattoo pigment may be taken up by macrophages and causes their freezing. According to the present study, the cream could not activate the frozen macrophages and the new macrophages recruited could not remove the tattoo as well; therefore, the tattoo pigments could not be removed and were still seen in the dermis. Hence, in accordance with the previous results, tattoo causes apparent histopathological changes in the skin. However, imiquimod 5% cream alone is not an effective method for removal of long-term tattoo; in addition, imiquimod 5% cream leads to inflammatory side effects on normal and tattooed skin. Further studies should be conducted to find other methods for the removal of long-term tattoos with minimal side effects.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

Ab-1Mac 387 expression; histology; imiquimod cream; keratinocytes; macrophage and immunohistochemistry; melanocytes; skin; tattoo

© 2011 The Egyptian Journal of Histology