C-type natriuretic peptide (CNP) inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor growth by modulating inflammation in Swiss albino mice.

C-type natriuretic peptide (CNP) exhibits anti-inflammatory activity besides its natriuretic and diuretic functions. The present study aimed to determine the anticancer and synergistic therapeutic activity of CNP against a 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor mouse model. CNP (2.5 µg/kg body weight) was injected either alone and/or in combination with Cisplatin (CDDP) (2 mg/kg body weight) for 4 weeks. The dorsal skin tumor incidences/growth and mortality rate were recorded during the experimental period of 16 weeks. The serum C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels, infiltrating mast cells, and AgNORs proliferating cells count were analyzed in control and experimental mice. Further, the expression profile of marker genes of proliferation, inflammation, and progression molecules were analyzed using Reverse transcriptase-polymerase chain reaction (RT-PCR)/quantitative PCR (qPCR), western blot, and immunohistochemistry. The DMBA/Croton oil-induced mice exhibited 100% tumor incidence. Whereas, CNP alone, CDDP alone, and CNP+CDDP combination-treated mice exhibited 58%, 46%, and 24% tumor incidence, respectively. Also, a marked reduction in the levels of serum CRP and LDH, the number of infiltrating mast cells count and AgNORs proliferating cells count were noticed in the mice skin sections. Further, a significant reduction in both mRNA and protein expression levels of proliferation, inflammation, and progression markers were noticed in CNP (p < 0.01), CDDP (p < 0.01), and CNP+CDDP combination (p < 0.001) treated mice, respectively. The results of the present study suggest that CNP has anticancer activity. Further, the CNP+CDDP treatment has more promising anticancer activity as compared with CNP or CDDP alone treatment, probably due to the synergistic antiproliferative and anti-inflammatory activities of CNP and CDDP.

Depth Map for Face and Neck Deep Chemical Peel Resurfacing.

BACKGROUND: Chemical peels are applied to the face and neck to improve rhytides and the photoaged appearance of the skin. Peels can be applied to different skin depths depending on the types of chemicals, the volume of solution, and the amount of pressure or friction applied. If a peel is applied too superficially, rhytides will not be removed. If a peel is applied too deeply, scarring or hypopigmentation could occur. OBJECTIVE: To create face and neck depth maps for chemical peeling, which can guide safety when removing rhytides and improving the skin's appearance. MATERIALS AND METHODS: A multicenter retrospective review of records was conducted of patients who underwent phenol-croton oil peeling, from January 1, 2018, to December 31, 2018. Information was collected on facial and neck cosmetic units peeled, peel formula and strength used, outcomes, and complications. RESULTS: A total of 410 patients received deep peels. Two depth maps were created that corresponded to the most common patterns of deep chemical peel applications. CONCLUSION: Different areas of the face and neck are treated with different chemical peel application depths to safely improve rhytides and appearance. Depth maps are created to balance safety and efficacy.

4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis.

Leukocyte transendothelial migration (TEM) is an essential component of the inflammatory response. In vitro studies with human cells have demonstrated that platelet/endothelial cell adhesion molecule (PECAM) functions upstream of CD99 during TEM; however, results in vivo with mice have been apparently contradictory. In this study we use four-dimensional (4D) intravital microscopy to demonstrate that the site and order of function of PECAM and CD99 in vivo are dependent on the strain of mice. In FVB/n mice, PECAM functions upstream of CD99, as in human cells in vitro, and blocking antibodies against either molecule arrest neutrophils before they traverse the endothelium. However, in C57BL/6 mice, PECAM and CD99 appear to function at a different step, as the same antibodies arrest leukocyte migration through the endothelial basement membrane. These results are the first direct comparison of PECAM and CD99 function in different murine strains as well as the first demonstration of the sequential function of PECAM and CD99 in vivo.

Anti-inflammatory effects of anthocyanins-rich extract from bilberry (Vaccinium myrtillus L.) on croton oil-induced ear edema and Propionibacterium acnes plus LPS-induced liver damage in mice.

Bilberry (Vaccinium myrtillus L.) has been known to play a protective role in human health due to its high anthocyanin content. This study investigated the anti-inflammatory effects of bilberry extract (BE, containing 42.04% anthocyanin) on Propionibacterium acnes (P. acnes) plus lipopolysaccharide (LPS) induced liver injury and croton oil-induced ear edema in mice. Results showed that BE could effectively inhibit croton oil-induced ear edema and liver inflammation provoked by P. acnes plus LPS, as reflected by the reduced plasma alanine aminotransferase and aspartate aminotransferase activities. These findings were confirmed by hepatic pathological examination. Moreover, BE administration markedly suppressed the increase of liver mRNA levels of iNOS, TNF-α, IL-1ß and IL-6, and the protein levels of iNOS, TNF-α and NF-κB. In addition, liver malondialdehyde and NO contents were significantly reduced by BE treatment. These results indicated that BE has potent protective effects on acute and immunological inflammation, which might contribute to the study of the anti-inflammatory effects of natural products and healthy food.

P2X7 receptor is required for neutrophil accumulation in a mouse model of irritant contact dermatitis.

Irritant contact dermatitis (ICD) is an inflammatory reaction caused by chemical toxicity on the skin. The P2X7 receptor (P2X7R) is a key mediator of cytokine release, which recruits immune cells to sites of inflammation. We investigated the role of P2X7R in croton oil (CrO)-induced ICD using in vitro and in vivo approaches. ICD was induced in vivo by CrO application on the mouse ear and in vitro by incubation of murine macrophages and dendritic cells (DCs) with CrO and ATP. Infiltrating cells were identified by flow cytometry, histology and myeloperoxidase (MPO) determination. Effects of the ATP scavenger apyrase were assessed to investigate further the role of P2X7R in ICD. Animals were also treated with N-1330, a caspase-1 inhibitor, or with clodronate, which induces macrophage apoptosis. CrO application induced severe inflammatory Gr1(+) cell infiltration and increased MPO levels in the mouse ear. Selective P2X7R antagonism with A438079 or genetic P2X7R deletion reduced the neutrophil infiltration. Clodronate administration significantly reduced Gr1(+) cell infiltration and local IL-1ß levels. In vitro experiments confirmed that A438079 or apyrase treatment prevented the increase in IL-1ß that was evoked by macrophage and DC incubation with CrO and ATP. These data support a key role for P2X7 in ICD-mediated inflammation via modulation of inflammatory cells. It is tempting to suggest that P2X7R inhibition might be an alternative ICD treatment.

Chemopreventive effect of Lagenaria siceraria in two stages DMBA plus croton oil induced skin papillomagenesis.

Cancer chemoprevention is a dietary or therapeutic strategy to prevent, suppress, or delay carcinogenesis either at initiation or progression level with nontoxic agents. Use of natural dietary compounds has been a major chemopreventive approach to modulate tumorigenic pathways. In the present study, we have evaluated Lagenaria siceraria (bottle gourd), a common vegetable of Indian household for its chemomodulatory potential. The fruit has been used in traditional medicine for a very long time for health benefits and to cure pain, ulcers, fever, cough, asthma, and other bronchial disorders. However, despite its reported beneficial effect the chemo modulatory potential of this plant has not been reported. Therefore chemopreventive effect of bottle gourd juice (BGJ) was studied against 7,12-dimethylbenz(a)anthracene (DMBA) plus croton oil induced skin papillomagenesis in Swiss albino mice. The effect was studied both at antiinitiation and antiinitiation/promotion level followed by histopathological study. A dose of 2.5% and 5% given in drinking water showed significant decrease in papilloma number, papilloma incidence, papilloma multiplicity, papilloma latency, papilloma volume, and papilloma size in different size range. Histopathological study showed chemopreventive effect by minimizing loss of stratification, a decrease in number of epithelial layers, reducing dermal infiltration and protection for various cytoplasmic changes. Higher dose of BGJ was found to be more effective than lower dose and the chemopreventive effect was maximum for antiinitiation/promotion treatment. Altogether, this study reports the chemopreventive effect of Lagenaria siceraria on skin papillomagenesis for the first time and suggests that its consumption may help in suppression of skin cancer.

Extraction optimization of Tinospora cordifolia and assessment of the anticancer activity of its alkaloid palmatine.

OBJECTIVE: To optimize the conditions for the extraction of alkaloid palmatine from Tinospora cordifolia by using response surface methodology (RSM) and study its anticancerous property against 7,12-dimethylbenz(a)anthracene (DMBA) induced skin carcinogenesis in Swiss albino mice. METHODS: The effect of three independent variables, namely, extraction temperature, time, and cycles was investigated by using central composite design. A single topical application of DMBA (100 µg/100 µL of acetone), followed 2 weeks later by repeated application of croton oil (1% in acetone three times a week) for 16 weeks, exhibited 100 percent tumor incidence (Group 2). RESULTS: The highest yield of alkaloid from Tinospora cordifolia could be achieved at 16 hours of extraction time under 40°C with 4 extraction cycles. Alkaloid administration significantly decreases tumor size, number, and the activity of serum enzyme when compared with the control (Group 2). In addition, depleted levels of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase and increased DNA damage were restored in palmatine treated groups. CONCLUSION: The data of the present study clearly indicate the anticancer potential of palmatine alkaloid in DMBA induced skin cancer model in mice.

Oral Intake of Combined Natural Immunostimulants Suppresses the 7,12-DMBA/ Croton Oil Induced Two-step Skin Carcinogenesis in Swiss Albino Mice.

BACKGROUND: The immune system is critical in fighting cancer, so is it possible that the natural stimulation of this system can slow down or stop the evolution of cancer? Our in vivo study aimed to evaluate the protective effect of the combination of five types of immunostimulants, which are Beta-glucan and Arabinogalactan as polysaccharides and three mushroom extracts (Reishi, Maitake, and Shiitake), on 7,12-Dimethyl Benz[a]anthracene (DMBA)/ Croton oil-induced papilloma in Swiss albino mice. METHODOLOGY: We used blood count analyses to estimate broadly the immunological reaction and biochemical techniques to determine the oxidative stress variations in the enzymatic activity of Superoxide dismutase (SOD), Catalase (CAT), and Glutathion peroxidase (GPx), which could have a preventive function against cancer development. RESULTS: The cutaneous application of the DMBA/Croton oil caused precancerous hyperplasia in squamous cells (papilloma) on the back of the mice. Tumor development was accompanied by a decrease in SOD and GPx activities. The treatment with the immunostimulants led to the total disappearance of the incidence of skin papillomas and also showed a nearly back to normal SOD activity but not CAT and GPx activities. The increase in the level of immune cells (lymphocytes, monocytes, and white blood cells) reflected a clear enhancement of the immune system activity. DISCUSSION: The healthy epidermis observed with treated mice simultaneously subjected to the cancerogenosis protocol suggests the inhibition of spinous cell proliferation leading to the total suppression of the hyperplasia. Moreover, the increase in the level of immune cells in this batch reflects an inflammatory reaction. Indeed, previous studies reported that immunostimulants, including Betaglucan involve a release of some inflammatory mediators who would be at the origin of its anticancer activity. Cancerogenesis has clearly disrupted the activities of the antioxidant enzymes, but the relationship between the two process is often complex. Bibliographic data led us to suggest that low catalytic activities of CAT and GPx observed in treated mice simultaneously subjected to the cancerogenesis protocol, would have induce an accumulation of H2O2 which has often been described as an inducer of cancer cells apoptosis. CONCLUSION: Immunostimulants used in our study could have an effective protective effect against skin carcinogenesis via the enhancement of the global function of the immune system and modulation of the antioxidant defense. KEYWORDS: Immunostimulants, Beta-glucan, Arabinogalactan, Reishi, Maitake, Shiitake, DMBA, Croton oil, Oxidative stress, Carcinogenesis. ABBREVIATIONS: C, control group; Dc, drug control group; Pc, positive control group; St, sick treated group;DMBA, 7,12 Dimethyl Benz[a]anthracene; NK, natural killer; CAT, catalase; SOD, superoxide dismutase, GPx, glutathione peroxidase; IS, immunostimulants; WBC, White blood cells; LY, Lymphocytes; MO, Monocytes; ROS, Reactive oxygen species; ONAB, Office national des aliments de bétail.

Mechanisms of olive leaf extract-ameliorated rat arthritis caused by kaolin and carrageenan.

Olive leaf extract (OLE) has antioxidant and antiinflammatory actions. However, the role of OLE in mechanical inflammatory arthritis (osteoarthritis, OA) is unclear. This study investigated the effect of OLE on the development of kaolin and carrageenan-induced arthritis, a murine model of OA. Administration of OLE significantly ameliorated paw swelling, the paw Evans blue content and the histopathological scores. In the human monocyte cell line, THP-1, the OLE reduced the LPS-induced TNF-α production and was dose dependent. Croton oil-induced ear edema in mice also revealed that treatment with OLE suppressed ear edema, myeloperoxidase (MPO) production and was dose dependent. These results indicated that OLE is an effective antiarthritis agent through an antiinflammation mechanism. Also OLE may be beneficial for the treatment of OA in humans.

Antiinflammatory activity of coumarins from Ligusticum lucidum Mill. subsp. cuneifolium (Guss.) Tammaro (Apiaceae).

Four coumarin derivatives [selidinin 1, (+)-praeruptorin A 2, visnadin 3 and (R)-(+)-7-(2',3'-epoxy-3'-methylbutoxy)-coumarin 4] were isolated from the aerial parts of Ligusticum lucidum Mill. subsp. cuneifolium (Guss.) Tammaro (Apiaceae). This is the first report on the identification of these compounds in the Ligusticum genus. Their topical antiinflammatory activity was evaluated as the inhibition of croton oil-induced ear dermatitis in mice. Each compound (0.3 µmol/cm(2) ) induced a significant oedema reduction and compound 4 exerted an effect similar to that of the equimolar dose of the reference drug indomethacin.

Histological case-control study of peeling-induced skin changes by different peeling agents in surgically subcutaneous undermined skin flaps in facelift patients.

A histological evaluation of peeling-induced skin changes in subcutaneous undermined preauricular facial skin flaps of nine patients was performed. There were three treatment groups: Trichloroacetic acid (TCA) 25%, TCA 40% and phenol/croton oil; one group served as control. Two independent evaluators determined the epidermal and dermal thickness and the depth of necrosis (micrometre). The percentual tissue damage due to the peeling was calculated, and a one-sample t-test for statistical significance was performed. On the basis of the histomorphological changes, peeling depth was classified as superficial, superficial-partial, deep-partial and full thickness chemical burn. The histological results revealed a progression of wound depth for different peeling agents without full thickness necrosis. TCA peels of up to 40% can be safely applied on subcutaneous undermined facial skin flaps without impairing the vascular patency, producing a predictable chemical burn, whereas deep peels such as phenol/croton oil peels should not be applied on subcutaneous undermined skin so as to not produce skin slough or necrosis by impairing vascular patency.

Ethnopharmacological study and topical anti-inflammatory activity of crude extract from Poikilacanthus glandulosus (Nees) Ariza leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies are important tools as records and documentation of the empirical uses of medicinal plants in traditional communities with the purpose of generating useful knowledge to lead to the development of new medicines, biodiversity conservation and enhancement of knowledge and local culture. Poikilacanthus glandulosus is widely used by the population of City of Santiago, in Brazil, nevertheless, it does not have any validation regarding its use and its medicinal effects. AIM: The objective of this study was to perform one ethnopharmacological survey about P. glandulosus in the City of Santiago and determine the anti-inflammatory activity in order to prove its uses in popular medicine. METHODS: Personal and ethnopharmacological data were collected through a prepared questionnaire. The phytochemical analysis was performed observing the individual methodology for each reaction and by HPLC-UV. The antiedematogenic and anti-inflammatory (cell infiltration and histological procedure) activities of the P. glandulosus (0.01-1000µg/ear) were evaluated in the ear edema model induced by topical application of croton oil. RESULTS: P. glandulosus is known in City of Santiago as "Gaiana" and its macerated leaves and branches are prepared with alcohol or sugarcane liquor especially for insect bites, cicatrization and inflammation. HPLC analysis revealed the presence of maslinic acid (2.024±0.10mg/g), uvaol (0.124±0.02mg/g) and sitosterol (0.502±0.05mg/g). The topical application of crude extract of P. glandulosus reduced in a dose-dependent manner the croton oil-induced ear edema and myeloperoxidase activity (neutrophils infiltration marker) with maximum inhibition of 87±2% and 64±12%, respectively at 1000µg/ear. Dexamethasone (100µg/ear), used as a positive control, inhibited croton oil-induced ear edema in 89±3% and decreased myeloperoxidase activity in 50±3%. Both P. glandulosus as dexamethasone reduced cell infiltration when evaluated by histological procedure CONCLUSION: This work allowed us to understand the specie P. glandulosus through ethnopharmacological study and it showed that the crude extract presented antiedematogenic and anti-inflammatory actions, proving their traditional use as anti-inflammatory.

Chemomodulatory Potential of Flaxseed Oil Against DMBA/Croton Oil-Induced Skin Carcinogenesis in Mice.

The present study was conducted to evaluate the potential of flaxseed oil to prevent chemically induced skin cancer in mice. Cancer was induced on 2-stage skin carcinogenesis model by single topical application of 7,12 dimethylbenz [a]anthracene (DMBA), as, initiator, and two weeks later it was promoted by croton oil treatment thrice a week on the dorsal surface of mice for 16 weeks. Flaxseed oil (FSO; 100µL/animal/d) was orally administered 1 week before and 1 week after DMBA application (Peri-initiation stage). The animals of the FSO-administered group showed a significant reduction in tumor incidence (76.67%), cumulative number of tumors (37), tumor yield (3.7), and tumor burden (4.81) when compared with the carcinogen-treated control animals. Biochemical parameters in skin and liver tissue such as LPO and phase I enzymes were significantly (P < .01) reduced in the FSO-treated experimental group, whereas the phase II enzymes (GST, DT-diaphorase) and antioxidant parameters (GSH, GPx, SOD, catalase, and vitamin C) exhibited a significant (P < .01) elevation when compared with the animals of the carcinogen-treated control group. Histopathological alterations in the carcinogen-treated control animals were also observed in the form of epidermal hyperplasia, keratinized pearl formation, and acanthosis in skin and tumors, whereas these were found to be reduced after FSO administration. The results of the present study demonstrate that the oral administration of FSO has the potential to modulate the levels of LPO, antioxidants, and detoxification enzymes in the DMBA-croton oil-induced skin carcinogenesis in mice.

Anti-inflammatory properties of an oxidized sterol.

A polar photoproduct of cholesterol oxidation, 7-ketocholesterol, was able to inhibit in a dose-dependent manner the mouse ear-swelling response to irritants such as croton oil or cantharidin. Its anti-inflammatory properties were much less than equivalent concentrations of hydrocortisone, but the oxidized sterol did not induce any systemic effects (as measured by thymolytic activity), as did topical hydrocortisone. It is concluded that 7-ketocholesterol has weak anti-inflammatory activity, and its mode of action may be different from that of glucocorticoids.

Cutaneous chemical carcinogenesis: past, present, and future.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin.

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

Topical treatment of alopecia areata.

It has been shown previously that alopecia areata can be treated with dinitrochlorobenzene (DNCB) and other contact allergens. Whether these agents work by inducing immunologic stimulation or simply a nonspecific inflammatory reaction has not been definitively demonstrated. To test the relative importance of these two mechanisms, we have randomly studied 22 patients with alopecia areata to whom either DNCB or croton oil was applied topically. Sixty-three percent of patients without spontaneous regrowth of hair regrew hair after DNCB application. None of those treated with croton oil regrew hair when treated later with DNCB. Therefore, a proved contact allergen was shown to be required for therapeutic success. Patient acceptance of the induced contact dermatitis was excellent. In light of recent data on the mutagenicity of DNCB to bacteria, other contact allergens for topical immunotherapy are being sought.

An examination of the phenol-croton oil peel: part IV. Face peel results with different concentrations of phenol and croton oil.

In Part IV of this examination of the phenol-croton oil peel, the author presents peeling solutions using phenol in concentrations between 16% and 50% as the carrier for croton oil. Previously, in Part I, the author showed that phenol alone in concentrations of less than 50% has no significant peeling effect on the skin in the absence of taping. All of these formulas are dependent on the addition of croton oil for their peeling action. A topographic map of the face is presented that divides the face into the zones that the author believes are best treated with different strengths of croton oil. Five patients peeled between late 1992 and late 1995 were chosen as examples to illustrate the effect of different strengths of croton oil between 0.25% and 2.78%. The author has documented their immediate postoperative course photographically to show the effect of the different concentrations. It is clinically apparent that peels using croton oil between 0.25% and 0.5% generally heal within 7 days; peels between 0.6% and 1.0% usually heal within 9 or 10 days, and peels using concentrations higher than 1% heal later and have some risk of pigmentation loss. Peels using croton oil concentrations at 2% and above almost always have pigmentation loss and have healing delays in areas other than the thick skin of the lower nose and perioral area. The practical clinical formulas distributed at the time of the presentation of this article at the 1996 Annual Meeting of the American Society for Aesthetic Plastic Surgery in Orlando, Florida, entitled "Heresy Phenol Formulas--1996," are provided here. These have been used in both the United States and Europe over the past few years. A metric standard for drop size is suggested at 0.04 ml. This relates to the drop size used clinically over the years to measure croton oil. The adoption of this unit will make formulas around the world easier to calculate and compare. The author has produced a metric formula using the suggested standard size drop for croton oil. This uses 35% phenol as the carrier and provides the same range of treatment dilutions as the 1996 "Heresy Phenol Formulas." The need for research into "carriers" and solvents for croton oil is pointed out. Despite what is not known about how it works, the combination of croton seed extract and phenol has been a success story in providing facial rejuvenation from the 1920s to the present. The croton oil-phenol peel in its many formulas still sets the standard for facial rejuvenation.