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Infrared thermal measurement method to evaluate the skin cooling effect of topical products and the impact of microstructure of creams
Journal of Drug Delivery Science and Technology 39 (2017) 296e299
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Infrared thermal measurement method to evaluate the skin cooling effect of topical products and the impact of microstructure of creams Reena Murthy a, S. Rangappa b, Michael A. Repka b, K. Vanaja a, H.N. Shivakumar a, S. Narasimha Murthy a, b, * a b
Institute for Drug Delivery & Biomedical Research (IDBR), Bangalore, Karnataka, India Department of Pharmaceutics and Drug Delivery, University of Mississippi, University, MS 38677, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2016 Received in revised form 6 April 2017 Accepted 8 April 2017 Available online 9 April 2017
The sensorial characteristic of topical products determines the patient acceptability of the topical products. The primary focus of the present study was to assess the feasibility of using Infrared (IR) thermal imaging technique to measure the skin cooling effect of topical creams. The secondary objective was to investigate the effect of microstructure of topical creams on the skin cooling effect of topical cream products that are identical in composition. The results showed that IR thermal measurement technique was found to be a sensitive and reliable for quantitative evaluation of skin cooling effect of topical products. The globule size of the creams was found to influence the skin cooling effect caused by creams despite their sameness in composition. © 2017 Published by Elsevier B.V.
Keywords: Skin cooling effect IR thermal imaging Topical creams Globule size distribution Microstructure of creams
1. Introduction Sensory characteristics are an important attribute of topical pharmaceutical and cosmetic products. The sensorial characteristic of topical products determines the patient acceptability of the products [1]. Topical products induces a cooling perception or skin cooling effect immediately after application on the skin. The skin cooling is regarded as a desirable characteristic as long as the cooling is above physiologically acceptable threshold. The coolness perception holds great significance in pediatric products and could improve patient adherence to therapy. Therefore, formulators intend to develop products that are capable of generating significant cooling effect when applied on the skin. Evaluation of sensorial characteristics has been performed based on scoring or grading method by a panel of human volunteers. The method often fails to discriminate marginal differences in performance between the products. Moreover, a large number of human volunteers/experts would be required in this conventional scoring method of evaluation. Thus, the development of
* Corresponding author. Pharmaceutics and Drug Delivery, School of Pharmacy, University Of Mississippi, University, MS 38677, United States. E-mail address: [email protected] (S.N. Murthy). http://dx.doi.org/10.1016/j.jddst.2017.04.015 1773-2247/© 2017 Published by Elsevier B.V.
instrumental methods that could quantify the sensorial performance of products would be preferable over the conventional method of scoring and grading. IR thermal technique has gained greater importance in assessing the numerous pathological conditions such as diabetic foot syndrome associated with extremities diabetic neuropathy and lower extremity peripheral artery disease [2e6]. Also, this method was utilized in the estimation of respiration rate and tidal volume and in certain nasal septal defects [7,8]. Recently, the effect of triamcinolone acetonide topical aerosol on the skin cooling was studied using IR camera [9]. Thus far, no systematic studies have been performed to evaluate the coolness perception of topical creams. The major emphasis of the present study was to investigate the skin cooling effect of topical products with the use of IR thermal imaging technique. The surface temperature was estimated with the aid of a thermographic camera which detects/analyze infrared radiation emitted from the skin surface. Further, the second main objective was to understand the effect of microstructural differences in products that were compositionally identical on the skin cooling effect. Therefore, different o/w formulations that were identical in composition but different in their globule size distribution were prepared by implementing different homogenizing conditions and the products were subjected to evaluation of skin cooling effect in human volunteers.
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
2. Materials & methods
297
Table 2 Globule size in different o/w cream formulations in relation to their homogenization protocol.
2.1. Equipment All Thermal images were recorded using IR camera FLIR Ax5 Series (Flir Systems, Danderyd, Sweden) equipped with focal plane array microbolometer thermal detector, 160 128 IR resolution, spectral range 7.5e13 mm. Thermal sensitivity was 0.05 C. 2.2. Screening of commercial products The preliminary study involved determination of skin cooling effect of eight commercially available skin care products by IR thermal technique. The objective was to identify a product that leads to relatively higher drop in skin temperature so that it could serve as a positive control or a reference standard in the next experiment involving custom made creams. The study was performed upon approval and as per the regulations of the Institutional Review board; The University of Mississippi (IRB protocol No.16e043). Six healthy human volunteers of either sex aged between 18 and 45years were selected for the study. To ensure thermal equilibrium, tests were performed in a room with controlled ambient temperature (23O C) and relative humidity (70%) and reasonable light conditions. In each session, one product was evaluated by each individual. A circular area of 30 mm was marked on the dorsal surface of the left hand. The skin surface temperature was recorded at five predetermined spots using IR thermal camera prior to the application of the product. A premeasured quantity (30 mg) of the marketed product was applied within the circular area. The product was removed with a soft plastic spatula after 20 min and the surface temperature was measured at five spots. The average temperature drop in the applied region was noted. 2.3. Preparation of custom made cream products Seven o/w cream formulations with same quantitative and qualitative characteristics were prepared after initial pilot studies as per the composition mentioned in Table 1. Nile red was dissolved in oil phase to visualize the globule size. The oil and the aqueous phase components were melted in a thermostated glass vessel maintained at 80 C separately. Later, oil phase was gradually added to the aqueous phase while maintaining the temperature at 80 C. A different homogenization protocol was followed for each cream as mentioned in Table 2. After homogenization, controlled cooling was done for all the products, except for the 7th formulation, until the temperature of formulation reaches 25 C. The 7th formulation was cooled at room temperature. The globule size of the products was determined by confocal microscopy. 2.4. Confocal microscopy of creams 10 mg of cream was applied on glass slide. The cream was spread using a film applicator alongside the applicator blade (Gardco® Table 1 Composition of custom made o/w creams. Ingredient
Quantity (% w/w)
Cetostearyl alcohol Mineral oil Cremophore A25 Cremophore A6 Propylene glycol Water purified
7 12 1.5 1.5 8 70
Formulation
Average Globule Size (mm)
Homogenization Protocol
F1 F2 F3 F4 F5 F6 F7
11.37 ± 7.03 7.41 ± 2.19 2.98 ± 1.25 1.71 ± 0.41 4.30 ± 1.33 4.36 ± 0.88 4.25 ± 0.99
500 rpm -20 min 1000 rpm -20 min 3000 rpm -20 min 5000 rpm -20 min 3000 rpm -10 min 3000 rpm -40 min 3000 rpm - gradual cooling
Microm II applicator blade, Japan) with 10 mm clearance between slide and tip of the blade. The slide was observed under confocal microscope (Zeiss LSM 510) at 40X objective. The excitation wavelength used for Nile red was 515 nm and emission wavelength was 525e605 nm. 2.5. Evaluation of skin cooling effect of custom made cream products All the custom made cream products were evaluated in the same six volunteers, altering the order of application as explained above. The marketed product which showed maximum cooling effect was used as positive control. Later, the individuals were blind folded and were asked to score the coolness perception with reference to the positive control on a scale of 0e10, with 10 being equivalent to the positive control and 0 being least or nil coolness perception. 2.6. Statistical analysis The obtained data was subjected to paired student's t-test using graph pad prism5 (version 5.02, GraphPad Software, San Diego, CA). The data are expressed as mean ± SEM and the level of significance was established at p < 0.05. 3. Results & discussion The IR thermal camera is a highly sensitive instrument with a high resolution. It can measure small differences in temperature across a small distance between the points of measurement on the skin surface. The images make it clear to identify the area of application for temperature measurement. The marked area on the skin shows a clear difference in color when the drop in skin surface temperature is significant (Fig. 1). The commercial creams were selected with different bases. In general, o/w creams (P2, P3 and P8) generated relatively more drop in skin temperature compared to coco butter (P4) or hydrocarbon bases (P7). The cream that resulted in highest drop in skin temperature (P8) was considered as a positive control for evaluating custom made creams. (Fig. 2). The custom made creams were prepared at different homogenization conditions. The globule size of the products was determined based on homogenization speed. The globule size decreased with increase in homogenization speed. At 500 rpm homogenization speed, the globules were irregular and did not appear to be uniformly distributed. The skin cooling effect was different for different products indicating that microstructural differences could have a significant impact on the sensorial attributes of the topical products (Fig. 3). The results clearly shows the need for a systematic investigation into the impact of microstructure on the sensorial characteristics, as it could influence the patient acceptability of the product, patient
298
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
Fig. 1. Representative pictures of IR thermal imaging of the skin area before and after application of the creams. The circle indicates the applied area. The temperature was measured on give points (four sides and center of the circle in each case).
Fig. 2. Skin cooling effect of eight different commercial topical products (n ¼ 6 ± s.d).
Fig. 4. Correlation between the IR measurements and human subject scoring of skin cooling effect in custom made creams that were identical in composition and different in microstructure. The Pearson's correlation coefficient was 0.825 suggesting a reasonable correlation.
Fig. 3. Skin cooling effect of different custom made o/w creams (n ¼ 6 ± s.d).
compliance and market potential as well. The scoring method was also used in this case to assess the validity of the thermal measurement method. There was reasonable correlation (R2 ¼ 0.825) between the human scoring method and the IR thermal measurement technique (Fig. 4). The negative slope of the linear graph suggested an inverse trend because the scoring method ranked the products in the order of least to the most preferred products in terms of skin cooling effect. Fig. 5 represents the relationship between the average globule size and skin cooling effect of custom made creams that were identical in composition. The correlation coefficient was not strong enough to arrive at a mathematical expression. However one can appreciate the inverse trend suggesting that, as the globule size increases in o/w creams, the skin cooling efficacy decreases. The F1 was treated as an outlier due to ineffective homogenization of the dispersed phase in the continuous phase. The reason for decrease in skin cooling effect with increase in globule size could only be speculated at this stage. When equal amount of different products is spread uniformly across unit surface area, the ratio of dispersed
Fig. 5. Influence of Globule size on the Skin cooling effect of different custom made creams that were identical in composition.
phase and external phase remains constant across all products. In case of a product with larger globules, the external phase will be relatively more continuous as compared to product with smaller globules. The skin cooling effect is generally due to evaporation of water from the skin surface. The evaporation would be relatively rapid from a less continuous external phase than from a more continuous external phase. This in turn is likely to lead to relatively higher drop in skin temperature in case of products with smaller globules than the product with larger globules, owing to efficient utilization of skin heat in the former as compared to the later.
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
4. Conclusions The skin cooling effect is one of the important characteristics of topical products that determine the patient compliance, product acceptability and market potential. IR thermal technique would be a useful and reliable tool in determining the skin cooling effect. It is a sensitive technique that can discriminate the products quantitatively unlike the conventional method of scoring by a group of volunteers. Despite the sameness in composition, the products could elicit different skin cooling effect as a function of microstructure of the topical products. References [1] Chandler J. Mark, The importance of sensory in topical pharmaceutical product, Chim. Oggi e Chem. Today. 3 (2013) 46e48. [2] N. Papanas, K. Papatheodorou, D. Papazoglou, S. Kotsiou, E. Maltezos, Association between foot temperature and sudomotor dysfunction in type 2 diabetes,
299
J Diabetes Sci Tech. (2010) 4803e4807. [3] E.J. Boyko, J.H. Ahroni, V. Stensel, R.C. Forsberg, D.R. Davignon, D.G. Smith, A prospective study of risk factors for diabetic foot ulcer, Seattle Diabet. Foot Study. Diabetes Care 22 (1999) 1036e1042. [4] P.C. Sun, H.D. Lin, S.H.E. Jao, Y.C. Ku, R.C. Chan, C.K. Cheng, Relationship of skin temperature to sympathetic dysfunction in diabetic at-risk feet, Diabetes Res Clin. Pract. 73 (2006) 41e46. [5] M. Bharara, J. Cobb, D. Claremont, Thermography and thermometry in the assessment of diabetic neuropathic foot: a case for furthering the role of thermal techniques, Int J Low. Extrem Wounds 54 (2006) 250e260. [6] C.L. Huang, Y.W. Wu, C.L. Hwang, Y.S. Jong, C.L. Chao, W.J. Chen, et al., The application of infrared thermography in evaluation of patients at high risk for lower extremity peripheral arterial disease, J. Vas. Surg. 54 (2011) 1074e1080. [7] J. Lindemann, K. Wiesmiller, T. Keck, K. Kastl, Dynamic nasal infrared thermography in patients with nasal septal perforations, Am. J. Allerg. Rheno. 23 (2009) 471e474. [8] D. Mitchell, C.H. Wyndham, T. Hodgson, Emissivity and transmittance of excised human skin in its thermal emission wave band, J. Appl. Physiol. 23 (1967) 390e394. [9] R.V. Linkner, B.A. Andrew Sohn, K. Goldenberg, M. Lebwohl, Infrared camera evaluation of the cooling effect of triamcinolone acetonide aerosol, J. Clin. Aest Dermatol. 6 (2013) 28e31.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Infrared thermal measurement method to evaluate the skin cooling effect of topical products and the impact of microstructure of creams Reena Murthy a, S. Rangappa b, Michael A. Repka b, K. Vanaja a, H.N. Shivakumar a, S. Narasimha Murthy a, b, * a b
Institute for Drug Delivery & Biomedical Research (IDBR), Bangalore, Karnataka, India Department of Pharmaceutics and Drug Delivery, University of Mississippi, University, MS 38677, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2016 Received in revised form 6 April 2017 Accepted 8 April 2017 Available online 9 April 2017
The sensorial characteristic of topical products determines the patient acceptability of the topical products. The primary focus of the present study was to assess the feasibility of using Infrared (IR) thermal imaging technique to measure the skin cooling effect of topical creams. The secondary objective was to investigate the effect of microstructure of topical creams on the skin cooling effect of topical cream products that are identical in composition. The results showed that IR thermal measurement technique was found to be a sensitive and reliable for quantitative evaluation of skin cooling effect of topical products. The globule size of the creams was found to influence the skin cooling effect caused by creams despite their sameness in composition. © 2017 Published by Elsevier B.V.
Keywords: Skin cooling effect IR thermal imaging Topical creams Globule size distribution Microstructure of creams
1. Introduction Sensory characteristics are an important attribute of topical pharmaceutical and cosmetic products. The sensorial characteristic of topical products determines the patient acceptability of the products [1]. Topical products induces a cooling perception or skin cooling effect immediately after application on the skin. The skin cooling is regarded as a desirable characteristic as long as the cooling is above physiologically acceptable threshold. The coolness perception holds great significance in pediatric products and could improve patient adherence to therapy. Therefore, formulators intend to develop products that are capable of generating significant cooling effect when applied on the skin. Evaluation of sensorial characteristics has been performed based on scoring or grading method by a panel of human volunteers. The method often fails to discriminate marginal differences in performance between the products. Moreover, a large number of human volunteers/experts would be required in this conventional scoring method of evaluation. Thus, the development of
* Corresponding author. Pharmaceutics and Drug Delivery, School of Pharmacy, University Of Mississippi, University, MS 38677, United States. E-mail address: [email protected] (S.N. Murthy). http://dx.doi.org/10.1016/j.jddst.2017.04.015 1773-2247/© 2017 Published by Elsevier B.V.
instrumental methods that could quantify the sensorial performance of products would be preferable over the conventional method of scoring and grading. IR thermal technique has gained greater importance in assessing the numerous pathological conditions such as diabetic foot syndrome associated with extremities diabetic neuropathy and lower extremity peripheral artery disease [2e6]. Also, this method was utilized in the estimation of respiration rate and tidal volume and in certain nasal septal defects [7,8]. Recently, the effect of triamcinolone acetonide topical aerosol on the skin cooling was studied using IR camera [9]. Thus far, no systematic studies have been performed to evaluate the coolness perception of topical creams. The major emphasis of the present study was to investigate the skin cooling effect of topical products with the use of IR thermal imaging technique. The surface temperature was estimated with the aid of a thermographic camera which detects/analyze infrared radiation emitted from the skin surface. Further, the second main objective was to understand the effect of microstructural differences in products that were compositionally identical on the skin cooling effect. Therefore, different o/w formulations that were identical in composition but different in their globule size distribution were prepared by implementing different homogenizing conditions and the products were subjected to evaluation of skin cooling effect in human volunteers.
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
2. Materials & methods
297
Table 2 Globule size in different o/w cream formulations in relation to their homogenization protocol.
2.1. Equipment All Thermal images were recorded using IR camera FLIR Ax5 Series (Flir Systems, Danderyd, Sweden) equipped with focal plane array microbolometer thermal detector, 160 128 IR resolution, spectral range 7.5e13 mm. Thermal sensitivity was 0.05 C. 2.2. Screening of commercial products The preliminary study involved determination of skin cooling effect of eight commercially available skin care products by IR thermal technique. The objective was to identify a product that leads to relatively higher drop in skin temperature so that it could serve as a positive control or a reference standard in the next experiment involving custom made creams. The study was performed upon approval and as per the regulations of the Institutional Review board; The University of Mississippi (IRB protocol No.16e043). Six healthy human volunteers of either sex aged between 18 and 45years were selected for the study. To ensure thermal equilibrium, tests were performed in a room with controlled ambient temperature (23O C) and relative humidity (70%) and reasonable light conditions. In each session, one product was evaluated by each individual. A circular area of 30 mm was marked on the dorsal surface of the left hand. The skin surface temperature was recorded at five predetermined spots using IR thermal camera prior to the application of the product. A premeasured quantity (30 mg) of the marketed product was applied within the circular area. The product was removed with a soft plastic spatula after 20 min and the surface temperature was measured at five spots. The average temperature drop in the applied region was noted. 2.3. Preparation of custom made cream products Seven o/w cream formulations with same quantitative and qualitative characteristics were prepared after initial pilot studies as per the composition mentioned in Table 1. Nile red was dissolved in oil phase to visualize the globule size. The oil and the aqueous phase components were melted in a thermostated glass vessel maintained at 80 C separately. Later, oil phase was gradually added to the aqueous phase while maintaining the temperature at 80 C. A different homogenization protocol was followed for each cream as mentioned in Table 2. After homogenization, controlled cooling was done for all the products, except for the 7th formulation, until the temperature of formulation reaches 25 C. The 7th formulation was cooled at room temperature. The globule size of the products was determined by confocal microscopy. 2.4. Confocal microscopy of creams 10 mg of cream was applied on glass slide. The cream was spread using a film applicator alongside the applicator blade (Gardco® Table 1 Composition of custom made o/w creams. Ingredient
Quantity (% w/w)
Cetostearyl alcohol Mineral oil Cremophore A25 Cremophore A6 Propylene glycol Water purified
7 12 1.5 1.5 8 70
Formulation
Average Globule Size (mm)
Homogenization Protocol
F1 F2 F3 F4 F5 F6 F7
11.37 ± 7.03 7.41 ± 2.19 2.98 ± 1.25 1.71 ± 0.41 4.30 ± 1.33 4.36 ± 0.88 4.25 ± 0.99
500 rpm -20 min 1000 rpm -20 min 3000 rpm -20 min 5000 rpm -20 min 3000 rpm -10 min 3000 rpm -40 min 3000 rpm - gradual cooling
Microm II applicator blade, Japan) with 10 mm clearance between slide and tip of the blade. The slide was observed under confocal microscope (Zeiss LSM 510) at 40X objective. The excitation wavelength used for Nile red was 515 nm and emission wavelength was 525e605 nm. 2.5. Evaluation of skin cooling effect of custom made cream products All the custom made cream products were evaluated in the same six volunteers, altering the order of application as explained above. The marketed product which showed maximum cooling effect was used as positive control. Later, the individuals were blind folded and were asked to score the coolness perception with reference to the positive control on a scale of 0e10, with 10 being equivalent to the positive control and 0 being least or nil coolness perception. 2.6. Statistical analysis The obtained data was subjected to paired student's t-test using graph pad prism5 (version 5.02, GraphPad Software, San Diego, CA). The data are expressed as mean ± SEM and the level of significance was established at p < 0.05. 3. Results & discussion The IR thermal camera is a highly sensitive instrument with a high resolution. It can measure small differences in temperature across a small distance between the points of measurement on the skin surface. The images make it clear to identify the area of application for temperature measurement. The marked area on the skin shows a clear difference in color when the drop in skin surface temperature is significant (Fig. 1). The commercial creams were selected with different bases. In general, o/w creams (P2, P3 and P8) generated relatively more drop in skin temperature compared to coco butter (P4) or hydrocarbon bases (P7). The cream that resulted in highest drop in skin temperature (P8) was considered as a positive control for evaluating custom made creams. (Fig. 2). The custom made creams were prepared at different homogenization conditions. The globule size of the products was determined based on homogenization speed. The globule size decreased with increase in homogenization speed. At 500 rpm homogenization speed, the globules were irregular and did not appear to be uniformly distributed. The skin cooling effect was different for different products indicating that microstructural differences could have a significant impact on the sensorial attributes of the topical products (Fig. 3). The results clearly shows the need for a systematic investigation into the impact of microstructure on the sensorial characteristics, as it could influence the patient acceptability of the product, patient
298
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
Fig. 1. Representative pictures of IR thermal imaging of the skin area before and after application of the creams. The circle indicates the applied area. The temperature was measured on give points (four sides and center of the circle in each case).
Fig. 2. Skin cooling effect of eight different commercial topical products (n ¼ 6 ± s.d).
Fig. 4. Correlation between the IR measurements and human subject scoring of skin cooling effect in custom made creams that were identical in composition and different in microstructure. The Pearson's correlation coefficient was 0.825 suggesting a reasonable correlation.
Fig. 3. Skin cooling effect of different custom made o/w creams (n ¼ 6 ± s.d).
compliance and market potential as well. The scoring method was also used in this case to assess the validity of the thermal measurement method. There was reasonable correlation (R2 ¼ 0.825) between the human scoring method and the IR thermal measurement technique (Fig. 4). The negative slope of the linear graph suggested an inverse trend because the scoring method ranked the products in the order of least to the most preferred products in terms of skin cooling effect. Fig. 5 represents the relationship between the average globule size and skin cooling effect of custom made creams that were identical in composition. The correlation coefficient was not strong enough to arrive at a mathematical expression. However one can appreciate the inverse trend suggesting that, as the globule size increases in o/w creams, the skin cooling efficacy decreases. The F1 was treated as an outlier due to ineffective homogenization of the dispersed phase in the continuous phase. The reason for decrease in skin cooling effect with increase in globule size could only be speculated at this stage. When equal amount of different products is spread uniformly across unit surface area, the ratio of dispersed
Fig. 5. Influence of Globule size on the Skin cooling effect of different custom made creams that were identical in composition.
phase and external phase remains constant across all products. In case of a product with larger globules, the external phase will be relatively more continuous as compared to product with smaller globules. The skin cooling effect is generally due to evaporation of water from the skin surface. The evaporation would be relatively rapid from a less continuous external phase than from a more continuous external phase. This in turn is likely to lead to relatively higher drop in skin temperature in case of products with smaller globules than the product with larger globules, owing to efficient utilization of skin heat in the former as compared to the later.
R. Murthy et al. / Journal of Drug Delivery Science and Technology 39 (2017) 296e299
4. Conclusions The skin cooling effect is one of the important characteristics of topical products that determine the patient compliance, product acceptability and market potential. IR thermal technique would be a useful and reliable tool in determining the skin cooling effect. It is a sensitive technique that can discriminate the products quantitatively unlike the conventional method of scoring by a group of volunteers. Despite the sameness in composition, the products could elicit different skin cooling effect as a function of microstructure of the topical products. References [1] Chandler J. Mark, The importance of sensory in topical pharmaceutical product, Chim. Oggi e Chem. Today. 3 (2013) 46e48. [2] N. Papanas, K. Papatheodorou, D. Papazoglou, S. Kotsiou, E. Maltezos, Association between foot temperature and sudomotor dysfunction in type 2 diabetes,
299
J Diabetes Sci Tech. (2010) 4803e4807. [3] E.J. Boyko, J.H. Ahroni, V. Stensel, R.C. Forsberg, D.R. Davignon, D.G. Smith, A prospective study of risk factors for diabetic foot ulcer, Seattle Diabet. Foot Study. Diabetes Care 22 (1999) 1036e1042. [4] P.C. Sun, H.D. Lin, S.H.E. Jao, Y.C. Ku, R.C. Chan, C.K. Cheng, Relationship of skin temperature to sympathetic dysfunction in diabetic at-risk feet, Diabetes Res Clin. Pract. 73 (2006) 41e46. [5] M. Bharara, J. Cobb, D. Claremont, Thermography and thermometry in the assessment of diabetic neuropathic foot: a case for furthering the role of thermal techniques, Int J Low. Extrem Wounds 54 (2006) 250e260. [6] C.L. Huang, Y.W. Wu, C.L. Hwang, Y.S. Jong, C.L. Chao, W.J. Chen, et al., The application of infrared thermography in evaluation of patients at high risk for lower extremity peripheral arterial disease, J. Vas. Surg. 54 (2011) 1074e1080. [7] J. Lindemann, K. Wiesmiller, T. Keck, K. Kastl, Dynamic nasal infrared thermography in patients with nasal septal perforations, Am. J. Allerg. Rheno. 23 (2009) 471e474. [8] D. Mitchell, C.H. Wyndham, T. Hodgson, Emissivity and transmittance of excised human skin in its thermal emission wave band, J. Appl. Physiol. 23 (1967) 390e394. [9] R.V. Linkner, B.A. Andrew Sohn, K. Goldenberg, M. Lebwohl, Infrared camera evaluation of the cooling effect of triamcinolone acetonide aerosol, J. Clin. Aest Dermatol. 6 (2013) 28e31.