Evaluation of EMLA Cream for Preventing Pain during Tattooing of Rabbits: Changes in Physiological, Behavioural and Facial Expression Responses

Background

Ear tattooing is a routine procedure performed on laboratory, commercial and companion rabbits for the purpose of identification. Although this procedure is potentially painful, it is usually performed without the provision of analgesia, so compromising animal welfare. Furthermore, current means to assess pain in rabbits are poor and more reliable methods are required. The objectives of this study were to assess the physiological and behavioural effects of ear tattooing on rabbits, evaluate the analgesic efficacy of topical local anaesthetic cream application prior to this procedure, and to develop a scale to assess pain in rabbits based on changes in facial expression.

Methodology/Principal Findings

In a crossover study, eight New Zealand White rabbits each underwent four different treatments of actual or sham ear tattooing, with and without prior application of a topical local anaesthetic (lidocaine/prilocaine). Changes in immediate behaviour, heart rate, arterial blood pressure, serum corticosterone concentrations, facial expression and home pen behaviours were assessed. Changes in facial expression were examined to develop the Rabbit Grimace Scale in order to assess acute pain. Tattooing without EMLA cream resulted in significantly greater struggling behaviour and vocalisation, greater facial expression scores of pain, higher peak heart rate, as well as higher systolic and mean arterial blood pressure compared to all other treatments. Physiological and behavioural changes following tattooing with EMLA cream were similar to those in animals receiving sham tattoos with or without EMLA cream. Behavioural changes 1 hour post-treatment were minimal with no pain behaviours identifiable in any group. Serum corticosterone responses did not differ between sham and tattoo treatments.

Conclusions

Ear tattooing causes transient and potentially severe pain in rabbits, which is almost completely prevented by prior application of local anaesthetic cream. The Rabbit Grimace Scale developed appears to be a reliable and accurate way to assess acute pain in rabbits.     

医学文眉深浅探讨

本文认为医学文眉是古代文身术与近代医学的结合,是永久性画眉。详细地介绍了医学文眉的材料,方法与适应证。首次将文眉分为追求自然型与润色修饰型。正常求美者和眉型不足者都适合于文眉。鉴于目前除皮肤切除术外,尚无彻底消除文眉痕迹的方法,本文还分析了文眉术后的近期、远期效果,及文眉效果不满意时的补救措施,强调高水平的文眉。讨论了选色深浅、针刺深浅及皮肤深浅与文眉的关系,各深浅的优缺点,并提议文眉时针刺的深度以浅为妙。文中所介绍和讨论的方法。同样适用于文身术。     

The impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice

Genetic material for polymerase chain reaction (PCR) and Southern blot analysis on transgenic mice is normally obtained by tail biopsy. Additionally, it may be necessary to tattoo the mice, as it is essential to have a good and permanent identification. The aim of this study was to evaluate the effects of amputating the tip of the tail to obtain a biopsy for genetic analysis and of ink tattooing on welfare in C57BL/6J mice, a strain often used as genetic background for transgenes. The behaviour of the animals, fluctuating asymmetry (FA, a measure of developmental instability) and the level of restitution in the remaining part of the tail were evaluated and used for an assessment of the impact of these procedures on the welfare of the animals. One group of mice was marked by tail tattooing at various ages. Another group of mice were tail amputated at 12 or 20 days of age. Body weight and FA were followed, and at the end of the experiment, the level of fear/anxiety was assessed using a light-dark box. In the group of tail-amputated animals observation of climbing behaviour and a beam walking test for balance was performed. Seven weeks after tail amputation, the animals were euthanized. The remaining part of the tail was evaluated histopathologically. Body weight, behaviour in the light-dark box and balance test results were not influenced by tail amputation or tattooing. FA was only transiently increased by tattooing. Climbing behaviour was reduced just after tail amputation at 20 days of age. No signs of neuromas were found in the amputated tails, but seven weeks after amputation a significant number of mice did not have fully regenerated glandular tissue and hair follicles in the tail. It is concluded that both tail amputation and tail tattooing seem to have minor short-term negative effects on welfare and that the tissues on the tail probably do not regenerate fully after amputation.     

玉米抗镰刀菌穗腐病接种方法及抗病资源筛选研究

对玉米抗穗腐病性鉴定中采用的牙签法与花丝通道注射法进行比较研究表明,牙签法接种果穗的发病程度重于花丝通道注射法接种。牙签法接种病菌于果穗子粒与穗轴之间,有利于病原菌的发育扩展,是一种比较理想的、容易操作的接种方法。采用牙签法接种串珠镰刀菌,对178份玉米自交系和15份杂交种进行抗穗腐病鉴定,筛选出高抗(HR)自交系1份、抗病(R)玉米自交系34份、抗病(R)玉米杂交种12份。     

Identification of newborn rats by tattooing

Pups were identified by toe clipping or tattooing the plantar surface of the paws on day 4 after delivery. Their growth, maturation, and reproductive capability were not affected by either identification method. In the toe clipping group, however, the duration until fall in the suspension test was significantly shortened, indicating that this identification method may not be suitable for some behavioural tests. The clipping also disturbs the skeletal investigation of toes and is not recommended from the view point of animal welfare. Palm tattooing, on the other hand, satisfies the fundamental requirements for long-term identification of rats.     

One size fits all: a surgical technique for the correction of all types of prominent ears

Patient satisfaction after surgery to correct the prominent ear relates to the degree of retroposition of the ear. Angular breaks in the cartilage seen with other cartilage-transecting procedures (and even after conchal resections), which are noticed by patients, led to the development of this technique. It is an easy operative technique that may be modified to suit all prominent ears, including the constricted cup ear, and it does not leave any sharp, visible ridges in the region of the anti-helix or concha. The procedure combines and modifies a number of techniques that have been described previously, which allows more flexibility in the treatment of this deformity.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian, and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis, and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL.p53^-/- mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^-/- mice show chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfb/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfb/Smad pathway.     

Complications of tattooing

Pathologic consequence of tattooing is relatively rare, but in light of the great number of persons in military service, where tattooing seems to be popular, consideration of the complications seems worth while.Four unusual cases of patients with cutaneous lesions in tattoos are reported. One patient had Boeck's sarcoid, in which the cutaneous eruption was limited solely to the tattooed areas on the body and involved all the various colors of the tattoo. The cutaneous eruption was only a single manifestation of generalized sarcoidosis. Another patient had secondary syphilis with the cutaneous lesions involving multi-colored tattoos, including the red areas. These tattoos were applied in Hawaii, and presumably the red dye was not the usual cinnabar (mercuric sulfide). As a general rule, the cutaneous lesions of secondary syphilis do not involve the red areas because of the treponomicidal action of mercuric sulfide.In the third case the characteristic eruption of discoid lupus erythematosus was present. It began over the red areas of a tattoo on the patient's forearm. It has been stated that mercuric sulfide is a photo-sensitizing agent, and it is believed that this explains the localization of the eruption in this case. The fourth patient had eruption, caused by mercury sensitivity, in an area of tattoo. The eruption was noted soon after the tattooing was done, and it persisted for seven years.     

COMPLICATIONS OF TATTOOING

Pathologic consequence of tattooing is relatively rare, but in light of the great number of persons in military service, where tattooing seems to be popular, consideration of the complications seems worth while.Four unusual cases of patients with cutaneous lesions in tattoos are reported. One patient had Boeck''s sarcoid, in which the cutaneous eruption was limited solely to the tattooed areas on the body and involved all the various colors of the tattoo. The cutaneous eruption was only a single manifestation of generalized sarcoidosis.Another patient had secondary syphilis with the cutaneous lesions involving multi-colored tattoos, including the red areas. These tattoos were applied in Hawaii, and presumably the red dye was not the usual cinnabar (mercuric sulfide). As a general rule, the cutaneous lesions of secondary syphilis do not involve the red areas because of the treponomicidal action of mercuric sulfide.In the third case the characteristic eruption of discoid lupus erythematosus was present. It began over the red areas of a tattoo on the patient''s forearm. It has been stated that mercuric sulfide is a photo-sensitizing agent, and it is believed that this explains the localization of the eruption in this case.The fourth patient had eruption, caused by mercury sensitivity, in an area of tattoo. The eruption was noted soon after the tattooing was done, and it persisted for seven years.     

Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.     

Convenient methods for marking newborn rats

Newborn rats were marked directly by tattooing or branding on their body except for the cervical and dorsal area within 24 hours after birth. The results obtained were as follows. Tattoos (less than 2 x 2 mm) and brands (circular, 3-4 mm in diameter) on the skin were clearly recognizable during 21 days observation, and followed no deaths. The marking methods resulted in no differences in body weight gain or external differentiation when compared with untreated animals. Normal behavior was observed in the inclined plane test at day 7 or 21 after tattooing or branding. Newborn rats marked with tattoos or brands grew even under the care of foster mothers without any troubles.     

玉米自交系间杂交种F1农艺性状的主成分分析

用Hótlling倡导的主成分分析法,(Principal comporient analysis),分析了玉米32个自交系间80个杂交组合的F_1世代.分析性状有株高、穗位高、雄穗分枝数、单株有效穗数、穗长、秃尖长、穗行数、行粒数、穗重、百粒重和单株产量.结果表明,前4个主成分贡献率占了总变异的大部分,因此就前4个主成分得分值对80个杂交组合进行了选择,为玉米组合的选择提供了直接依据.     

Tattooing in reconstruction of the nipple and areola: a new method

In order to produce the characteristic color of the nipple-areola complex and in unilateral reconstructions to match the other side, a modified tattooing method has been introduced. The quite different requirements between reconstruction and ornamental studio tattooing can only be satisfied by different techniques. For the first time, pigment particles have been suspended in a gel rather than adsorbed on an opaque mineral. Our porcine study has shown how the pigment is distributed in the dermis without penetration of the gel particles. The first 16 of the authors' 29 patients have been reviewed with an analysis of their appearance. Using this new method, a third achieved a completely natural appearance and two-thirds were judged to be intermediate, compared with an unnatural, although sometimes acceptable, result in all the earlier conventional tattoos.     

Intradermal tattoo as an adjunct to nipple-areola reconstruction

Improving results in breast reconstruction have encouraged more authentic restoration of the lost nipple-areola. While recreation of nipple shape can be reliably achieved, appropriate color has remained elusive, except when a composite nipple graft has been harvested from the normal breast, often at a significant aesthetic, emotional, and oncologic cost. Extensive experience with intradermal tattooing of the nipple-areola in over 100 patients over a 5-year period has shown this technique to be exceptionally safe and effective. Both medical-grade and commercial machines are available at varying prices, and medical-grade pigments may be obtained in a variety of authentic flesh tones derived from titanium or iron oxides. In nearly every case, tattooing has helped with either nipple-areola color, size, shape, or position, without any significant complications. Some degree of tattoo fading is common, requiring occasional late touch-ups and, more rarely, complete retattooing.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M “arrest,” showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53^−/− mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^−/− mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.Key words: mouse, regeneration, p53, p21, MRL, ear-hole, Tgfβ     

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.     

IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22(-/-) mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A(-/-) mice produced little ear swelling (p < 0.001, versus IL-23-injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23-induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23-induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23-mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.     

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.