Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants.
Background
DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants.Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes.
Results
We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes.Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.
Conclusion
This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

Background Molecular biological studies of plants require high-quality DNA. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method [ 1 ] and its modifications [ 2 , 3 ]. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Most methods require the use of liquid nitrogen [ 4 ] or freeze-drying (lyophilization) [ 5 , 6 ] of tissue for the initial grinding, and these processes are unavailable in many regions of the world. After grinding the tissues in various extraction buffers, DNA is extracted with phenol-chloroform, or the extract is dialyzed against EDTA and a buffered Tris-HCl solution [ 7 ]. After extraction, the aqueous phase is concentrated, either by ethanol or isopropanol precipitation [ 8 , 9 ], or with microconcentrators ( e.g ., the Wizard genomic DNA purification system; Promega, USA). However, these methods are not time efficient for consistently obtaining PCR-quality DNA from calluses and plants, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. In our laboratory, we investigate the stability of transgenes expressed in calluses or plants transformed by nuclear or chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Southern blot analysis to confirm homologous recombination in chloroplast transformation [ 10 ]. For our purposes, we desire a simple and fast procedure for obtaining plant genomic DNA for PCR, and good-quality DNA for complete enzyme digestion for Southern blot analysis. Therefore, we present a protocol for extracting genomic DNA from fresh calluses and plant leaves that is applicable to a variety of organisms, regardless of the complexity of their genomes. In addition, we present a rapid and reliable procedure for extracting genomic DNA for PCR or Southern blot analysis from a small amount (~0.5 cm 2 ) of leaf tissue.
Background
Molecular biological studies of plants require high-quality DNA. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method [ 1 ] and its modifications [ 2 , 3 ]. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed.Most methods require the use of liquid nitrogen [ 4 ] or freeze-drying (lyophilization) [ 5 , 6 ] of tissue for the initial grinding, and these processes are unavailable in many regions of the world. After grinding the tissues in various extraction buffers, DNA is extracted with phenol-chloroform, or the extract is dialyzed against EDTA and a buffered Tris-HCl solution [ 7 ]. After extraction, the aqueous phase is concentrated, either by ethanol or isopropanol precipitation [ 8 , 9 ], or with microconcentrators ( e.g ., the Wizard genomic DNA purification system; Promega, USA). However, these methods are not time efficient for consistently obtaining PCR-quality DNA from calluses and plants, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc.e.gIn our laboratory, we investigate the stability of transgenes expressed in calluses or plants transformed by nuclear or chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Southern blot analysis to confirm homologous recombination in chloroplast transformation [ 10 ]. For our purposes, we desire a simple and fast procedure for obtaining plant genomic DNA for PCR, and good-quality DNA for complete enzyme digestion for Southern blot analysis. Therefore, we present a protocol for extracting genomic DNA from fresh calluses and plant leaves that is applicable to a variety of organisms, regardless of the complexity of their genomes. In addition, we present a rapid and reliable procedure for extracting genomic DNA for PCR or Southern blot analysis from a small amount (~0.5 cm 2 ) of leaf tissue.Results and discussion We describe a simple and reproducible procedure for RAPD or PCR amplification of transgenes from various plant sources. Three different variations of the genomic DNA extraction protocol for RAPD analysis were compared. After simple plant leaf and callus tissue homogenization with DNA extraction buffer using a hand-operated homogenizer, the leaf and callus cells were lysed with 20% SDS. Then, genomic DNA was extracted with the same volume of phenol/chloroform/isoamyl alcohol (25:24:1). An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 μl of the diluted supernatant as a template (Figure  1 , lane 1). Alternatively, after phenol/chloroform/isoamyl alcohol (25:24:1) extraction, the supernatant was transferred to a fresh tube for a second phenol/chloroform/isoamyl alcohol (25:24:1) extraction followed by a chloroform extraction. An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 μl of the diluted supernatant as the DNA template (Figure  1 , lane 2). In the third variation, after chloroform extraction the supernatant was transferred to a fresh tube and precipitated with two volumes of ethanol. After washing the DNA pellet with 70% ethanol, the DNA pellet was dissolved in 50 μl of sterile dH 2 O containing 20 μg ml -1  DNase-free RNase A. For PCR, 50 ng of the DNA were used as the template (Figure  1 , lane 3). DNA samples prepared using the three different extraction procedures (lanes 1, 2, and 3 in Figure  1 ) were subjected to PCR amplification using two 10-mer random primers: RAPD-1 and RAPD-2 (Genotech, Korea) (Figure  1 ). All the genomic DNA samples produced a clear, sharp, and reproducible PCR product when primer RAPD-1 was used for PCR amplification (Figure  1A ). Although three variations of the DNA extraction procedure were used, there was little difference between lanes 1, 2, and 3. Only a difference in the intensity of the band was observed, which may be due to the different template concentrations used for the PCR reaction. This result suggests that the supernatant after the first phenol treatment (protocol 1) was sufficiently pure to be used as the DNA template for PCR amplification. Therefore, PCR amplification with another random primer, RAPD-2, was performed using the DNA template extracted using the simplest protocol (Figure  1B ). The PCR amplification was successful, and the same banding pattern was seen when we repeated the PCR amplification. Therefore, we confirmed that the DNA template extracted using the simplest method was sufficient for RAPD, and it was used as the DNA template to amplify specific DNA or transgenes from transgenic calluses or plants. To examine the presence of  bar  [ 11 , 12 ] or the LTB gene [ 13 ] at a directed site in the chloroplast DNA after homologous recombination in transplastomic tobacco plants, putative transformants were screened by PCR analysis (Figure  2 ). PCR amplification using primer combinations Bar-F/Bar-R, 1-F/1-R, and LTB-F/LTB-R resulted in 550-, 1700-, and 380-bp fragments, respectively. Primers 2-F/2-R produced 2200- or 1900-bp fragments containing  bar  and LTB, respectively, which confirmed the site-specific integration in the chloroplast genome (Table  1 ). No detectable product was produced using genomic DNA from wild-type plants (Figure  2B , lane 1), demonstrating the specificity of these primers and genomic DNA extracts. Therefore, we concluded that chloroplast DNA was also amplified, although we did not use liquid nitrogen, but simply used a hand-operated homogenizer with a plastic tip. We also successfully amplified specific foreign genes from transgenic tobacco plants transformed using the nuclear transformation method, including the α-interferon (550 bp) [ 14 ], the core epitope of the PEDV gene (420 bp) [ 15 , 16 ], the LTB gene (380 bp) [ 17 ], and the A plus B subunit of the  Helicobacter pylori  urease gene (2450 bp) [ 18 ] (Figure  3 ). Specific PCR amplification was also conducted using transgenic calluses as well as transgenic plants. In transgenic calluses derived from Siberian ginseng plants, α-interferon was successfully amplified, showing a 580-bp fragment in 1% agarose gels. Using the third protocol, the DNA concentrations obtained were between 20 and 30 μg/0.5 cm 2  plant leaf, and the absorbance ratios (A 260 /A 280 ) were between 1.7 and 2.0. However, the DNA concentrations from rice, maize, and poplar were relatively low (< 3 μg). This may be because homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electrophoresed on 1% agarose gels, and high-molecular-weight DNA was obtained (Figure  4A ). When the genomic DNA was digested with  Eco RI and  Hin dIII, the DNA was completely digested, and could be used for Southern blot analysis. Therefore, we concluded that the purity and quality of the genomic DNA was sufficient for enzyme digestion. There are many advantages in using our genomic DNA extraction method to obtain template for PCR amplification. Many different plants could be amplified using the same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA repeatedly from all eight plant sources examined. Our procedure is not only very simple, but is also time and cost effective. After homogenization in DNA extraction buffer using a hand-operated homogenizer, the template DNA for PCR could be extracted by phenol/chloroform/isopropyl alcohol treatment. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. The time required for our DNA extraction method is less than 30 min, which is extraordinary compared with other genomic DNA extraction methods. With our procedure, leaf tissue (~0.5 cm 2 ) is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction. There is no sample waste with our method, whereas much larger samples are required when plant samples are ground in a mortar and pestle with liquid nitrogen and transferred to a tube. Previously reported techniques require several steps [ 19 ], use of expensive enzymes such as proteinase K [ 20 ], or beads and shakers [ 21 ]. Although the protocol for one-step plant DNA isolation was developed by Burr et al. [ 22 ], if plant material more than 1 mm 2  was used in the extraction, co-extracts (e.g., chlorophyll) were extracted alongside the DNA and inhibited the PCR. On the contrary, our protocol does not require appropriate sample size to extract DNA. Warner et al. [ 23 ] also reported a rapid DNA extraction method in barley, which requires NaOH. However, the extracted DNA samples were easily degraded. The DNA samples extracted by our protocol were very stable and could be stored for a long time without degradation. We find the new method very useful in our laboratory, since limited transgenic plant tissue or callus is sometimes available in a culture bottle. Therefore, the simplicity, efficiency, speed, and lack of a requirement for expensive facilities make our method an attractive alternative to existing methods of genomic DNA extraction.
Results and discussion
We describe a simple and reproducible procedure for RAPD or PCR amplification of transgenes from various plant sources. Three different variations of the genomic DNA extraction protocol for RAPD analysis were compared. After simple plant leaf and callus tissue homogenization with DNA extraction buffer using a hand-operated homogenizer, the leaf and callus cells were lysed with 20% SDS. Then, genomic DNA was extracted with the same volume of phenol/chloroform/isoamyl alcohol (25:24:1). An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 μl of the diluted supernatant as a template (Figure  1 , lane 1). Alternatively, after phenol/chloroform/isoamyl alcohol (25:24:1) extraction, the supernatant was transferred to a fresh tube for a second phenol/chloroform/isoamyl alcohol (25:24:1) extraction followed by a chloroform extraction. An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH 2 O, and PCR was performed using 1 μl of the diluted supernatant as the DNA template (Figure  1 , lane 2). In the third variation, after chloroform extraction the supernatant was transferred to a fresh tube and precipitated with two volumes of ethanol. After washing the DNA pellet with 70% ethanol, the DNA pellet was dissolved in 50 μl of sterile dH 2 O containing 20 μg ml -1  DNase-free RNase A. For PCR, 50 ng of the DNA were used as the template (Figure  1 , lane 3).DNA samples prepared using the three different extraction procedures (lanes 1, 2, and 3 in Figure  1 ) were subjected to PCR amplification using two 10-mer random primers: RAPD-1 and RAPD-2 (Genotech, Korea) (Figure  1 ). All the genomic DNA samples produced a clear, sharp, and reproducible PCR product when primer RAPD-1 was used for PCR amplification (Figure  1A ). Although three variations of the DNA extraction procedure were used, there was little difference between lanes 1, 2, and 3. Only a difference in the intensity of the band was observed, which may be due to the different template concentrations used for the PCR reaction. This result suggests that the supernatant after the first phenol treatment (protocol 1) was sufficiently pure to be used as the DNA template for PCR amplification. Therefore, PCR amplification with another random primer, RAPD-2, was performed using the DNA template extracted using the simplest protocol (Figure  1B ). The PCR amplification was successful, and the same banding pattern was seen when we repeated the PCR amplification. Therefore, we confirmed that the DNA template extracted using the simplest method was sufficient for RAPD, and it was used as the DNA template to amplify specific DNA or transgenes from transgenic calluses or plants.To examine the presence of  bar  [ 11 , 12 ] or the LTB gene [ 13 ] at a directed site in the chloroplast DNA after homologous recombination in transplastomic tobacco plants, putative transformants were screened by PCR analysis (Figure  2 ). PCR amplification using primer combinations Bar-F/Bar-R, 1-F/1-R, and LTB-F/LTB-R resulted in 550-, 1700-, and 380-bp fragments, respectively. Primers 2-F/2-R produced 2200- or 1900-bp fragments containing  bar  and LTB, respectively, which confirmed the site-specific integration in the chloroplast genome (Table  1 ). No detectable product was produced using genomic DNA from wild-type plants (Figure  2B , lane 1), demonstrating the specificity of these primers and genomic DNA extracts. Therefore, we concluded that chloroplast DNA was also amplified, although we did not use liquid nitrogen, but simply used a hand-operated homogenizer with a plastic tip. We also successfully amplified specific foreign genes from transgenic tobacco plants transformed using the nuclear transformation method, including the α-interferon (550 bp) [ 14 ], the core epitope of the PEDV gene (420 bp) [ 15 , 16 ], the LTB gene (380 bp) [ 17 ], and the A plus B subunit of the  Helicobacter pylori  urease gene (2450 bp) [ 18 ] (Figure  3 ). Specific PCR amplification was also conducted using transgenic calluses as well as transgenic plants. In transgenic calluses derived from Siberian ginseng plants, α-interferon was successfully amplified, showing a 580-bp fragment in 1% agarose gels.barbarHelicobacter pyloriUsing the third protocol, the DNA concentrations obtained were between 20 and 30 μg/0.5 cm 2  plant leaf, and the absorbance ratios (A 260 /A 280 ) were between 1.7 and 2.0. However, the DNA concentrations from rice, maize, and poplar were relatively low (< 3 μg). This may be because homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electrophoresed on 1% agarose gels, and high-molecular-weight DNA was obtained (Figure  4A ). When the genomic DNA was digested with  Eco RI and  Hin dIII, the DNA was completely digested, and could be used for Southern blot analysis. Therefore, we concluded that the purity and quality of the genomic DNA was sufficient for enzyme digestion.EcoHinThere are many advantages in using our genomic DNA extraction method to obtain template for PCR amplification. Many different plants could be amplified using the same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA repeatedly from all eight plant sources examined. Our procedure is not only very simple, but is also time and cost effective. After homogenization in DNA extraction buffer using a hand-operated homogenizer, the template DNA for PCR could be extracted by phenol/chloroform/isopropyl alcohol treatment. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. The time required for our DNA extraction method is less than 30 min, which is extraordinary compared with other genomic DNA extraction methods. With our procedure, leaf tissue (~0.5 cm 2 ) is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction. There is no sample waste with our method, whereas much larger samples are required when plant samples are ground in a mortar and pestle with liquid nitrogen and transferred to a tube. Previously reported techniques require several steps [ 19 ], use of expensive enzymes such as proteinase K [ 20 ], or beads and shakers [ 21 ]. Although the protocol for one-step plant DNA isolation was developed by Burr et al. [ 22 ], if plant material more than 1 mm 2  was used in the extraction, co-extracts (e.g., chlorophyll) were extracted alongside the DNA and inhibited the PCR. On the contrary, our protocol does not require appropriate sample size to extract DNA. Warner et al. [ 23 ] also reported a rapid DNA extraction method in barley, which requires NaOH. However, the extracted DNA samples were easily degraded. The DNA samples extracted by our protocol were very stable and could be stored for a long time without degradation.We find the new method very useful in our laboratory, since limited transgenic plant tissue or callus is sometimes available in a culture bottle. Therefore, the simplicity, efficiency, speed, and lack of a requirement for expensive facilities make our method an attractive alternative to existing methods of genomic DNA extraction.Conclusions Our objective was to extract genomic DNA with a simple and fast procedure for PCR and enzyme digestion. The present protocol is for extracting genomic DNA from fresh calluses or plant leaf tissues that is applicable to a variety of organisms, regardless of the complexity of their genomes. Our procedure is not only very simple, but is also time and cost effective. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. In addition, a very small sample is required for DNA extraction.
Conclusions
Our objective was to extract genomic DNA with a simple and fast procedure for PCR and enzyme digestion. The present protocol is for extracting genomic DNA from fresh calluses or plant leaf tissues that is applicable to a variety of organisms, regardless of the complexity of their genomes. Our procedure is not only very simple, but is also time and cost effective. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. In addition, a very small sample is required for DNA extraction.Methods Plant material We examined plant material from plant collections commonly used for foreign gene expression: tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), cabbage ( Brassica oleracea ), rice ( Oryza sativa ), lettuce ( Lactuca sativa ), maize ( Zea mays ), poplar ( Populus nigra ), and Siberian ginseng ( Eleutherococcus senticosus ). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l -1  MS salts [ 24 ], 30 g l -1  sucrose, and 7.5 g l -1  bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification. DNA extraction (Figure  5 ) We tested three different variations of the genomic DNA extraction procedure. About 0.5 cm 2  of culture room- or greenhouse-grown plant leaves were put in a 1.5-ml microfuge tube. The leaf tissue was homogenized in 50 μl DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl pH 7.5, and 50 mM EDTA pH 7.5), using a hand-operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 μl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 μl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH 2 O containing 20 μg/ml DNase-free RNase A. The concentration and purity were determined from the A 260 /A 280  ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII (Life Technologies, USA) in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. Analysis of DNA and PCR amplifications Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400 (Biorad, USA) in a total volume of 25 μl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table  1 ), 50 ng template DNA from plants, and 0.25 U Ex-Taq DNA polymerase (Takara, Japan) using the following profile: a 3-min denaturation at 94°C and 40 cycles of 1-min denaturation at 94°C, 1-min annealing at 37°C for RAPD or 55°C for specific transgene amplification, and a 2-min extension at 72°C, followed by a final extension at 72°C for 7 min. The PCR products were resolved by electrophoresis in 1.0% agarose gels.
Methods
Plant material We examined plant material from plant collections commonly used for foreign gene expression: tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), cabbage ( Brassica oleracea ), rice ( Oryza sativa ), lettuce ( Lactuca sativa ), maize ( Zea mays ), poplar ( Populus nigra ), and Siberian ginseng ( Eleutherococcus senticosus ). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l -1  MS salts [ 24 ], 30 g l -1  sucrose, and 7.5 g l -1  bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification.
Plant material
We examined plant material from plant collections commonly used for foreign gene expression: tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), cabbage ( Brassica oleracea ), rice ( Oryza sativa ), lettuce ( Lactuca sativa ), maize ( Zea mays ), poplar ( Populus nigra ), and Siberian ginseng ( Eleutherococcus senticosus ). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l -1  MS salts [ 24 ], 30 g l -1  sucrose, and 7.5 g l -1  bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification.Nicotiana tabacumSolanum tuberosumBrassica oleraceaOryza sativaLactuca sativaZea maysPopulus nigraEleutherococcus senticosusDNA extraction (Figure  5 ) We tested three different variations of the genomic DNA extraction procedure. About 0.5 cm 2  of culture room- or greenhouse-grown plant leaves were put in a 1.5-ml microfuge tube. The leaf tissue was homogenized in 50 μl DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl pH 7.5, and 50 mM EDTA pH 7.5), using a hand-operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 μl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 μl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH 2 O containing 20 μg/ml DNase-free RNase A. The concentration and purity were determined from the A 260 /A 280  ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII (Life Technologies, USA) in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture.
DNA extraction (Figure  5 )
We tested three different variations of the genomic DNA extraction procedure. About 0.5 cm 2  of culture room- or greenhouse-grown plant leaves were put in a 1.5-ml microfuge tube. The leaf tissue was homogenized in 50 μl DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl pH 7.5, and 50 mM EDTA pH 7.5), using a hand-operated homogenizer (Sigma, Z35997-1) with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 μl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 μl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH 2 O containing 20 μg/ml DNase-free RNase A. The concentration and purity were determined from the A 260 /A 280  ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII (Life Technologies, USA) in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture.EcoHinAnalysis of DNA and PCR amplifications Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400 (Biorad, USA) in a total volume of 25 μl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table  1 ), 50 ng template DNA from plants, and 0.25 U Ex-Taq DNA polymerase (Takara, Japan) using the following profile: a 3-min denaturation at 94°C and 40 cycles of 1-min denaturation at 94°C, 1-min annealing at 37°C for RAPD or 55°C for specific transgene amplification, and a 2-min extension at 72°C, followed by a final extension at 72°C for 7 min. The PCR products were resolved by electrophoresis in 1.0% agarose gels.
Analysis of DNA and PCR amplifications
Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of  Eco RI and  Hin dIII in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400 (Biorad, USA) in a total volume of 25 μl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table  1 ), 50 ng template DNA from plants, and 0.25 U Ex-Taq DNA polymerase (Takara, Japan) using the following profile: a 3-min denaturation at 94°C and 40 cycles of 1-min denaturation at 94°C, 1-min annealing at 37°C for RAPD or 55°C for specific transgene amplification, and a 2-min extension at 72°C, followed by a final extension at 72°C for 7 min. The PCR products were resolved by electrophoresis in 1.0% agarose gels.EcoHinAuthors' contributions TJK developed the method and performed majority of the experiments. MSY provided technical assistance, funding and supervision for the work. All authors have read and approved the final manuscript.
Authors' contributions
TJK developed the method and performed majority of the experiments. MSY provided technical assistance, funding and supervision for the work. All authors have read and approved the final manuscript.


Background Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood.
Background
Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood.Methods Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF).
Methods
Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF).Results TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] – no pain group, n = 8, 0.69 [0–1.27]; pain group, n = 10, 2.15 [0.77–4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 [ 1 - 3 ], p = 0.008; TRPV4, median [range] – no pain group, n = 6, [0–1]; pain group, n = 11, 1 [0.5–2], p = 0.014).
Results
TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] – no pain group, n = 8, 0.69 [0–1.27]; pain group, n = 10, 2.15 [0.77–4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 [ 1 - 3 ], p = 0.008; TRPV4, median [range] – no pain group, n = 6, [0–1]; pain group, n = 11, 1 [0.5–2], p = 0.014).Conclusion Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.
Conclusion
Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.

Background Breast pain is a common problem, which can affect up to 70% of women [ 1 ]. Breast pain or mastalgia can be cyclical or non-cyclical. The cyclical type of breast pain has been attributed to sex hormonal changes through the menstrual cycle that may increase the size of the breast tissue, which stretches the internal structures and causes pain or soreness. Numerous studies have demonstrated variation in pain perception during the menstrual cycle [ 2 - 5 ]. Heat sensitivity is increased in the luteal (17–22) phase of the menstrual cycle [ 6 ] and lowest in the periovulatory phase (day 12–16), but other studies have shown variation at other times in the cycle. Non-cyclical breast pain can be caused by hormonal influences particularly oestrogen, and other causes such as macromastia, local infection or inflammation; rarely, breast cancer can present as breast pain. Macromastia may cause areas of numbness in the breast and problems with nipple erectile function, which is thought to be related to the stretching of the nerve supply with increase in breast size [ 7 ]. Post-surgical breast pain is also a significant entity, with about 50% of women who undergo mastectomy suffering from chronic pain one year after their operation [ 8 , 9 ]. The mechanisms of breast pain in the majority of women are not well understood at the cellular or molecular level. We hypothesized a relationship between clinical breast pain, nerve growth factor (NGF) and its regulated ion channels or receptors expressed by nociceptor fibres. Estrogens upregulate NGF receptor mRNA in sensory neurons [ 10 ], and enhance the proliferative effects of NGF [ 11 , 12 ]. As NGF is a key molecule that determines the sensitivity of nociceptors in humans [ 13 ] and animal models [ 14 ], sex hormonal influences could be responsible for altered NGF activity during the menstrual cycle, leading to cyclical breast soreness or pain. NGF expression is also increased by inflammation, and this is responsible for the collateral nerve fibre sprouting and hypersensitivity of nociceptor fibres associated with inflammation. The hypersensitivity is, in part, mediated via the capsaicin or vanilloid receptor 1 (TRPV1), which is required for thermal hyperalgesia in rodents [ 15 , 16 ], and is activated by heat pain. Thermal hyperalgesia can occur during the menstrual cycle and it is well known that the core body temperature alters during the cycle (this is a qualitative test for ovulation), and thus heat conductance and perception and tolerance of heat alters during the cycle [ 2 , 6 ]. The TRPV1 receptor is activated also by the products of inflammation. We have therefore studied TRPV1-expressing nerve fibres and NGF in skin from women with and without breast pain and tenderness. The recently discovered vanilloid thermoreceptors TRPV3 and TRPV4, which are also expressed by sensory fibres and activated by warmth, were also studied [ 17 , 18 ].
Background
Breast pain is a common problem, which can affect up to 70% of women [ 1 ]. Breast pain or mastalgia can be cyclical or non-cyclical. The cyclical type of breast pain has been attributed to sex hormonal changes through the menstrual cycle that may increase the size of the breast tissue, which stretches the internal structures and causes pain or soreness. Numerous studies have demonstrated variation in pain perception during the menstrual cycle [ 2 - 5 ]. Heat sensitivity is increased in the luteal (17–22) phase of the menstrual cycle [ 6 ] and lowest in the periovulatory phase (day 12–16), but other studies have shown variation at other times in the cycle. Non-cyclical breast pain can be caused by hormonal influences particularly oestrogen, and other causes such as macromastia, local infection or inflammation; rarely, breast cancer can present as breast pain. Macromastia may cause areas of numbness in the breast and problems with nipple erectile function, which is thought to be related to the stretching of the nerve supply with increase in breast size [ 7 ]. Post-surgical breast pain is also a significant entity, with about 50% of women who undergo mastectomy suffering from chronic pain one year after their operation [ 8 , 9 ].The mechanisms of breast pain in the majority of women are not well understood at the cellular or molecular level. We hypothesized a relationship between clinical breast pain, nerve growth factor (NGF) and its regulated ion channels or receptors expressed by nociceptor fibres. Estrogens upregulate NGF receptor mRNA in sensory neurons [ 10 ], and enhance the proliferative effects of NGF [ 11 , 12 ]. As NGF is a key molecule that determines the sensitivity of nociceptors in humans [ 13 ] and animal models [ 14 ], sex hormonal influences could be responsible for altered NGF activity during the menstrual cycle, leading to cyclical breast soreness or pain. NGF expression is also increased by inflammation, and this is responsible for the collateral nerve fibre sprouting and hypersensitivity of nociceptor fibres associated with inflammation. The hypersensitivity is, in part, mediated via the capsaicin or vanilloid receptor 1 (TRPV1), which is required for thermal hyperalgesia in rodents [ 15 , 16 ], and is activated by heat pain. Thermal hyperalgesia can occur during the menstrual cycle and it is well known that the core body temperature alters during the cycle (this is a qualitative test for ovulation), and thus heat conductance and perception and tolerance of heat alters during the cycle [ 2 , 6 ]. The TRPV1 receptor is activated also by the products of inflammation. We have therefore studied TRPV1-expressing nerve fibres and NGF in skin from women with and without breast pain and tenderness. The recently discovered vanilloid thermoreceptors TRPV3 and TRPV4, which are also expressed by sensory fibres and activated by warmth, were also studied [ 17 , 18 ].Methods Patients Eighteen patients were recruited (n = 12 breast reduction for macromastia; n = 6 breast reconstruction) at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration. Clinical pain assessment Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire [ 19 ] were produced, along with a 10 cm unmarked visual analogue scale (VAS). The presence of breast pain was defined from the results of the Breast Pain Questionnaire using the total Pain Rating Index (PRI (total)) [ 19 ] and VAS scores marked by the patient in centimetres being more than zero to identify those patients with breast pain. Only two patients were taking simple analgesia for breast pain. Immunohistochemistry Full thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, USA). Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline (PBS; 0.1 M phosphate; 0.9% w/v saline; pH 7.3). After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 (polyclonal rabbit anti-TRPV1; GlaxoSmithKline, Harlow, UK; 1/5000; 1/10,000), TRPV3 (polyclonal rabbit anti-TRPV3; GlaxoSmithKline, Harlow, UK; 1/1000), TRPV4 (polyclonal rabbit anti-TRPV4; GlaxoSmithKline, Harlow, UK; 1/250; 1/1000), recombinant human NGF (polyclonal rabbit anti-NGF; Genentech, San Francisco, USA; 1/4000) or marker of large and some small calibre nerve fibres (mixed mouse monoclonal antibodies to neurofilaments 200 kD, 70 kD and 57 kD; DAKO cytomation Cambs., UK, 1/50,000; Novocastra, Newcastle upon Tyne, UK, 1/500). Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications [ 18 , 20 ]. Sites of antibody attachment were revealed using biotinylated goat anti- rabbit or biotinylated horse anti-mouse IgG (Vector Laboratories, High Wycombe, Bucks., U.K.) and nickel-enhanced, immunoperoxidase (avidin-biotin complex – ABC elite; Vector Laboratories, High Wycombe, Bucks., U.K.). Nuclei were counterstained with 0.1% w/v aqueous neutral red. The intensity of NGF immunostaining was graded on a scale 0 – 3 where 0 = negative or no immunoproduct, 1 = weak immunoproduct, 2 = intermediate intensity immunoproduct and 3 = intense immunoproduct. Intra-epidermal and sub-epidermal TRPV1-, TRPV4 or neurofilament – positive fibres were counted and the length and thickness of the epidermis was measured using a calibrated microscope, eyepiece graticule. Similarly, fibres that extended through the epidermis were counted, along with arborising "clusters" of fibres. The two observers who performed the histological studies were blinded with regard to clinical pain scores. Statistical analyses A non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™).
Methods
Patients Eighteen patients were recruited (n = 12 breast reduction for macromastia; n = 6 breast reconstruction) at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration. Clinical pain assessment Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire [ 19 ] were produced, along with a 10 cm unmarked visual analogue scale (VAS). The presence of breast pain was defined from the results of the Breast Pain Questionnaire using the total Pain Rating Index (PRI (total)) [ 19 ] and VAS scores marked by the patient in centimetres being more than zero to identify those patients with breast pain. Only two patients were taking simple analgesia for breast pain.
Patients
Eighteen patients were recruited (n = 12 breast reduction for macromastia; n = 6 breast reconstruction) at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration.Clinical pain assessment Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire [ 19 ] were produced, along with a 10 cm unmarked visual analogue scale (VAS). The presence of breast pain was defined from the results of the Breast Pain Questionnaire using the total Pain Rating Index (PRI (total)) [ 19 ] and VAS scores marked by the patient in centimetres being more than zero to identify those patients with breast pain. Only two patients were taking simple analgesia for breast pain.
Clinical pain assessment
Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire [ 19 ] were produced, along with a 10 cm unmarked visual analogue scale (VAS). The presence of breast pain was defined from the results of the Breast Pain Questionnaire using the total Pain Rating Index (PRI (total)) [ 19 ] and VAS scores marked by the patient in centimetres being more than zero to identify those patients with breast pain. Only two patients were taking simple analgesia for breast pain.Immunohistochemistry Full thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, USA). Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline (PBS; 0.1 M phosphate; 0.9% w/v saline; pH 7.3). After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 (polyclonal rabbit anti-TRPV1; GlaxoSmithKline, Harlow, UK; 1/5000; 1/10,000), TRPV3 (polyclonal rabbit anti-TRPV3; GlaxoSmithKline, Harlow, UK; 1/1000), TRPV4 (polyclonal rabbit anti-TRPV4; GlaxoSmithKline, Harlow, UK; 1/250; 1/1000), recombinant human NGF (polyclonal rabbit anti-NGF; Genentech, San Francisco, USA; 1/4000) or marker of large and some small calibre nerve fibres (mixed mouse monoclonal antibodies to neurofilaments 200 kD, 70 kD and 57 kD; DAKO cytomation Cambs., UK, 1/50,000; Novocastra, Newcastle upon Tyne, UK, 1/500). Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications [ 18 , 20 ]. Sites of antibody attachment were revealed using biotinylated goat anti- rabbit or biotinylated horse anti-mouse IgG (Vector Laboratories, High Wycombe, Bucks., U.K.) and nickel-enhanced, immunoperoxidase (avidin-biotin complex – ABC elite; Vector Laboratories, High Wycombe, Bucks., U.K.). Nuclei were counterstained with 0.1% w/v aqueous neutral red. The intensity of NGF immunostaining was graded on a scale 0 – 3 where 0 = negative or no immunoproduct, 1 = weak immunoproduct, 2 = intermediate intensity immunoproduct and 3 = intense immunoproduct. Intra-epidermal and sub-epidermal TRPV1-, TRPV4 or neurofilament – positive fibres were counted and the length and thickness of the epidermis was measured using a calibrated microscope, eyepiece graticule. Similarly, fibres that extended through the epidermis were counted, along with arborising "clusters" of fibres. The two observers who performed the histological studies were blinded with regard to clinical pain scores.
Immunohistochemistry
Full thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, USA). Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline (PBS; 0.1 M phosphate; 0.9% w/v saline; pH 7.3). After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 (polyclonal rabbit anti-TRPV1; GlaxoSmithKline, Harlow, UK; 1/5000; 1/10,000), TRPV3 (polyclonal rabbit anti-TRPV3; GlaxoSmithKline, Harlow, UK; 1/1000), TRPV4 (polyclonal rabbit anti-TRPV4; GlaxoSmithKline, Harlow, UK; 1/250; 1/1000), recombinant human NGF (polyclonal rabbit anti-NGF; Genentech, San Francisco, USA; 1/4000) or marker of large and some small calibre nerve fibres (mixed mouse monoclonal antibodies to neurofilaments 200 kD, 70 kD and 57 kD; DAKO cytomation Cambs., UK, 1/50,000; Novocastra, Newcastle upon Tyne, UK, 1/500). Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications [ 18 , 20 ]. Sites of antibody attachment were revealed using biotinylated goat anti- rabbit or biotinylated horse anti-mouse IgG (Vector Laboratories, High Wycombe, Bucks., U.K.) and nickel-enhanced, immunoperoxidase (avidin-biotin complex – ABC elite; Vector Laboratories, High Wycombe, Bucks., U.K.). Nuclei were counterstained with 0.1% w/v aqueous neutral red. The intensity of NGF immunostaining was graded on a scale 0 – 3 where 0 = negative or no immunoproduct, 1 = weak immunoproduct, 2 = intermediate intensity immunoproduct and 3 = intense immunoproduct. Intra-epidermal and sub-epidermal TRPV1-, TRPV4 or neurofilament – positive fibres were counted and the length and thickness of the epidermis was measured using a calibrated microscope, eyepiece graticule. Similarly, fibres that extended through the epidermis were counted, along with arborising "clusters" of fibres. The two observers who performed the histological studies were blinded with regard to clinical pain scores.Statistical analyses A non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™).
Statistical analyses
A non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™).Results Clinical pain assessment Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4–30); Mean (SEM) 13.44(3.07) and VAS – Median (range) 5(3.7–6.7); Mean (SEM) 5.14(0.31)]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea. Immunostaining TRPV1 TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin (Fig.  1A ). In breast pain, TRPV1-immunoreactive fibres appeared to be more abundant in the epidermis and frequently seen to pass along the junction of the stratum corneum (intra-epidermal fibres – IEF; Fig.  1B, Ci, ii ) often with multiple fibres (fibre "clusters"; Fig.  1Di, ii - arrows). Intra-epidermal fibres and fibre "cluster" counts/millimetre of epithelium were significantly higher in patients with breast pain (Table  1 ; Fig.  2 ). This significance was maintained despite exclusion of patients with previous breast surgery (i.e. the breast reconstruction patients; Pain, n = 7; No Pain, n = 5; p = 0.0303). While some specimens from pain patients showed thinning of the epidermis, this was not so overall for the pain group (Table  1 ). Figure 1 TRPV1-immunoreactive nerve fibres in breast skin . ( A ) Normal, control skin : TRPV1-immunoreactive nerve fibres (small arrows) in the sub-epidermis. ( B ) Painful skin (macromastia patient): intra-epidermal, TRPV1-immunoreactive nerve fibre (arrow) deriving from a large, sub-epidermal fascicle and extending to the stratum corneum. ( Ci ) Painful skin (breast reconstruction patient): TRPV1-immunoreactive intra-epidermal fibres passing along the junction between the epidermis and stratum corneum ( Cii -enlarged area from  Ci ). ( Di ) Painful skin (macromastia patient): multiple branching, TRPV1-immunoreactive intra-epidermal nerve fibres (arrows) extending to the stratum corneum ( Dii -enlarged area from  Di ). Large double arrows indicate relative epidermal thickness. Scale bars: A, B, C(i), D(i) = 50 μm; C(ii), D(ii) = 10 μm. Table 1 Histology results Patients with no breast pain (N = 8) Patients with breast pain (N = 10) P-Mann Whitney Test Intra-epidermal Median (range) Median (range) TRPV1 intra-epidermal fibres 0.69 (0.00 – 1.27) 2.15 (0.77 – 4.38) 0.0009** TRPVR1 fibre "clusters" 0.15 (0.00 – 0.27) 0.37 (0.00–0.85) 0.0085* Sub-epidermal TRPV1 fibres 2.98 (1.59–4.04) 3.79 (0.63 – 5.92) 0.1457 Neurofilament fibres 3.38 (0.83 – 5.92) 2.95 (0.94 – 4.95) 0.7618 NGF staining 2.5 (1.0–3.0) 2.38 (1.0–3.0) 0.5726 Epidermal Thickness (mm) 0.04 (0.02 – 0.12) 0.04 (0.03 – 0.05) 0.9654 Figure 2 Quantification of TRPV1-immunoreactive, intra-epidermal fibres and breast pain . Scattergrams show TRPV1-immunoreactivity in ( A ) intra-epidermal fibres and ( B ) intra-epidermal fibre "clusters" in patients with and without breast pain. TRPV3 TRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain (Fig.  3 ). TRPV3-immunoreactivity was not detected in skin nerve fibres in this study. Figure 3 TRPV3-immunoreactivity in breast skin . TOP PANELS: TRPV3-immunoreactive keratinocytes mostly in basal layer and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm BOTTOM PANEL: Scattergram showing grading assessment and a significant increase (*P < 0.05) of TRPV3 immunoreactivity in patients with pain. TRPV4 Basal keratinocytes also displayed TRPV4 immunoreactivity in groups of cells which were particularly strong at the apex of dermal papillae, where immunoreactivity appeared most strong at the cell membrane junction (Fig  4 ). Quantification of immunostaining showed a significant (p < 0.03) increase with breast pain (Fig.  4 ). TRPV4 immunoreactivity was also detected in fine nerve fibres scattered through the sub-epidermis, and showed a trend (p = 0.402) for increased frequency in painful subjects (Fig  4 ). Figure 4 TRPV4-immunoreactivity in breast skin . TOP FOUR PANELS: TRPV4-immunoreactivity in keratinocytes mostly at the cell membrane and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm MIDDLE PANEL: Fine, sub-epidermal, TRPV4-immunoreactive fibres (arrows). Scale bar = 100 μm BOTTOM PANELS: Scattergrams showing grading assessment of sub-epidermal fibres (left panel) and keratinocytes (right panel) significantly increased (*P < 0.05) in patients with pain. NGF NGF immunoreactivity was present in basal keratinocytes in all samples (Fig  5Ai, ii ) with little difference in intensity between pain and no pain specimens (Table  1 ). In some pain specimens the epidermis appeared to be thinner, and there was evidence of NGF expression in suprabasal as well as basal keratinocytes (Fig  5Bi  and  5ii , – arrows), which correlated with the presence of TRPV1positive fibres (seen in a serial section from the same case shown in Fig  1B ). Figure 5 NGF-immunoreactivity in breast skin . ( Ai ) Normal, control skin: NGF-immunoreactive basal keratinocytes. ( Aii )-enlarged area from Ai showing NGF confined to single layer of keratinocytes. ( Bi ) Painful skin (macromastia patient): NGF-immunoreactive basal and supra-basal ( Bii -arrows) keratinocytes in skin with thin epidermis. Nerve marker (neurofilaments) Neurofilaments showed nerve fibres in sub-epidermal and dermal regions only but no significant changes were detected between groups (Table  1 ).
Results
Clinical pain assessment Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4–30); Mean (SEM) 13.44(3.07) and VAS – Median (range) 5(3.7–6.7); Mean (SEM) 5.14(0.31)]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea.
Clinical pain assessment
Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4–30); Mean (SEM) 13.44(3.07) and VAS – Median (range) 5(3.7–6.7); Mean (SEM) 5.14(0.31)]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors (burning, hot), while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea.Immunostaining TRPV1 TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin (Fig.  1A ). In breast pain, TRPV1-immunoreactive fibres appeared to be more abundant in the epidermis and frequently seen to pass along the junction of the stratum corneum (intra-epidermal fibres – IEF; Fig.  1B, Ci, ii ) often with multiple fibres (fibre "clusters"; Fig.  1Di, ii - arrows). Intra-epidermal fibres and fibre "cluster" counts/millimetre of epithelium were significantly higher in patients with breast pain (Table  1 ; Fig.  2 ). This significance was maintained despite exclusion of patients with previous breast surgery (i.e. the breast reconstruction patients; Pain, n = 7; No Pain, n = 5; p = 0.0303). While some specimens from pain patients showed thinning of the epidermis, this was not so overall for the pain group (Table  1 ). Figure 1 TRPV1-immunoreactive nerve fibres in breast skin . ( A ) Normal, control skin : TRPV1-immunoreactive nerve fibres (small arrows) in the sub-epidermis. ( B ) Painful skin (macromastia patient): intra-epidermal, TRPV1-immunoreactive nerve fibre (arrow) deriving from a large, sub-epidermal fascicle and extending to the stratum corneum. ( Ci ) Painful skin (breast reconstruction patient): TRPV1-immunoreactive intra-epidermal fibres passing along the junction between the epidermis and stratum corneum ( Cii -enlarged area from  Ci ). ( Di ) Painful skin (macromastia patient): multiple branching, TRPV1-immunoreactive intra-epidermal nerve fibres (arrows) extending to the stratum corneum ( Dii -enlarged area from  Di ). Large double arrows indicate relative epidermal thickness. Scale bars: A, B, C(i), D(i) = 50 μm; C(ii), D(ii) = 10 μm. Table 1 Histology results Patients with no breast pain (N = 8) Patients with breast pain (N = 10) P-Mann Whitney Test Intra-epidermal Median (range) Median (range) TRPV1 intra-epidermal fibres 0.69 (0.00 – 1.27) 2.15 (0.77 – 4.38) 0.0009** TRPVR1 fibre "clusters" 0.15 (0.00 – 0.27) 0.37 (0.00–0.85) 0.0085* Sub-epidermal TRPV1 fibres 2.98 (1.59–4.04) 3.79 (0.63 – 5.92) 0.1457 Neurofilament fibres 3.38 (0.83 – 5.92) 2.95 (0.94 – 4.95) 0.7618 NGF staining 2.5 (1.0–3.0) 2.38 (1.0–3.0) 0.5726 Epidermal Thickness (mm) 0.04 (0.02 – 0.12) 0.04 (0.03 – 0.05) 0.9654 Figure 2 Quantification of TRPV1-immunoreactive, intra-epidermal fibres and breast pain . Scattergrams show TRPV1-immunoreactivity in ( A ) intra-epidermal fibres and ( B ) intra-epidermal fibre "clusters" in patients with and without breast pain. TRPV3 TRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain (Fig.  3 ). TRPV3-immunoreactivity was not detected in skin nerve fibres in this study. Figure 3 TRPV3-immunoreactivity in breast skin . TOP PANELS: TRPV3-immunoreactive keratinocytes mostly in basal layer and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm BOTTOM PANEL: Scattergram showing grading assessment and a significant increase (*P < 0.05) of TRPV3 immunoreactivity in patients with pain. TRPV4 Basal keratinocytes also displayed TRPV4 immunoreactivity in groups of cells which were particularly strong at the apex of dermal papillae, where immunoreactivity appeared most strong at the cell membrane junction (Fig  4 ). Quantification of immunostaining showed a significant (p < 0.03) increase with breast pain (Fig.  4 ). TRPV4 immunoreactivity was also detected in fine nerve fibres scattered through the sub-epidermis, and showed a trend (p = 0.402) for increased frequency in painful subjects (Fig  4 ). Figure 4 TRPV4-immunoreactivity in breast skin . TOP FOUR PANELS: TRPV4-immunoreactivity in keratinocytes mostly at the cell membrane and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm MIDDLE PANEL: Fine, sub-epidermal, TRPV4-immunoreactive fibres (arrows). Scale bar = 100 μm BOTTOM PANELS: Scattergrams showing grading assessment of sub-epidermal fibres (left panel) and keratinocytes (right panel) significantly increased (*P < 0.05) in patients with pain. NGF NGF immunoreactivity was present in basal keratinocytes in all samples (Fig  5Ai, ii ) with little difference in intensity between pain and no pain specimens (Table  1 ). In some pain specimens the epidermis appeared to be thinner, and there was evidence of NGF expression in suprabasal as well as basal keratinocytes (Fig  5Bi  and  5ii , – arrows), which correlated with the presence of TRPV1positive fibres (seen in a serial section from the same case shown in Fig  1B ). Figure 5 NGF-immunoreactivity in breast skin . ( Ai ) Normal, control skin: NGF-immunoreactive basal keratinocytes. ( Aii )-enlarged area from Ai showing NGF confined to single layer of keratinocytes. ( Bi ) Painful skin (macromastia patient): NGF-immunoreactive basal and supra-basal ( Bii -arrows) keratinocytes in skin with thin epidermis. Nerve marker (neurofilaments) Neurofilaments showed nerve fibres in sub-epidermal and dermal regions only but no significant changes were detected between groups (Table  1 ).
Immunostaining
TRPV1 TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin (Fig.  1A ). In breast pain, TRPV1-immunoreactive fibres appeared to be more abundant in the epidermis and frequently seen to pass along the junction of the stratum corneum (intra-epidermal fibres – IEF; Fig.  1B, Ci, ii ) often with multiple fibres (fibre "clusters"; Fig.  1Di, ii - arrows). Intra-epidermal fibres and fibre "cluster" counts/millimetre of epithelium were significantly higher in patients with breast pain (Table  1 ; Fig.  2 ). This significance was maintained despite exclusion of patients with previous breast surgery (i.e. the breast reconstruction patients; Pain, n = 7; No Pain, n = 5; p = 0.0303). While some specimens from pain patients showed thinning of the epidermis, this was not so overall for the pain group (Table  1 ). Figure 1 TRPV1-immunoreactive nerve fibres in breast skin . ( A ) Normal, control skin : TRPV1-immunoreactive nerve fibres (small arrows) in the sub-epidermis. ( B ) Painful skin (macromastia patient): intra-epidermal, TRPV1-immunoreactive nerve fibre (arrow) deriving from a large, sub-epidermal fascicle and extending to the stratum corneum. ( Ci ) Painful skin (breast reconstruction patient): TRPV1-immunoreactive intra-epidermal fibres passing along the junction between the epidermis and stratum corneum ( Cii -enlarged area from  Ci ). ( Di ) Painful skin (macromastia patient): multiple branching, TRPV1-immunoreactive intra-epidermal nerve fibres (arrows) extending to the stratum corneum ( Dii -enlarged area from  Di ). Large double arrows indicate relative epidermal thickness. Scale bars: A, B, C(i), D(i) = 50 μm; C(ii), D(ii) = 10 μm. Table 1 Histology results Patients with no breast pain (N = 8) Patients with breast pain (N = 10) P-Mann Whitney Test Intra-epidermal Median (range) Median (range) TRPV1 intra-epidermal fibres 0.69 (0.00 – 1.27) 2.15 (0.77 – 4.38) 0.0009** TRPVR1 fibre "clusters" 0.15 (0.00 – 0.27) 0.37 (0.00–0.85) 0.0085* Sub-epidermal TRPV1 fibres 2.98 (1.59–4.04) 3.79 (0.63 – 5.92) 0.1457 Neurofilament fibres 3.38 (0.83 – 5.92) 2.95 (0.94 – 4.95) 0.7618 NGF staining 2.5 (1.0–3.0) 2.38 (1.0–3.0) 0.5726 Epidermal Thickness (mm) 0.04 (0.02 – 0.12) 0.04 (0.03 – 0.05) 0.9654 Figure 2 Quantification of TRPV1-immunoreactive, intra-epidermal fibres and breast pain . Scattergrams show TRPV1-immunoreactivity in ( A ) intra-epidermal fibres and ( B ) intra-epidermal fibre "clusters" in patients with and without breast pain.
TRPV1
TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin (Fig.  1A ). In breast pain, TRPV1-immunoreactive fibres appeared to be more abundant in the epidermis and frequently seen to pass along the junction of the stratum corneum (intra-epidermal fibres – IEF; Fig.  1B, Ci, ii ) often with multiple fibres (fibre "clusters"; Fig.  1Di, ii - arrows). Intra-epidermal fibres and fibre "cluster" counts/millimetre of epithelium were significantly higher in patients with breast pain (Table  1 ; Fig.  2 ). This significance was maintained despite exclusion of patients with previous breast surgery (i.e. the breast reconstruction patients; Pain, n = 7; No Pain, n = 5; p = 0.0303). While some specimens from pain patients showed thinning of the epidermis, this was not so overall for the pain group (Table  1 ).Histology resultsPatients with no breast pain (N = 8)Patients with breast pain (N = 10)P-Mann Whitney TestIntra-epidermalMedian (range)Median (range)TRPV3 TRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain (Fig.  3 ). TRPV3-immunoreactivity was not detected in skin nerve fibres in this study. Figure 3 TRPV3-immunoreactivity in breast skin . TOP PANELS: TRPV3-immunoreactive keratinocytes mostly in basal layer and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm BOTTOM PANEL: Scattergram showing grading assessment and a significant increase (*P < 0.05) of TRPV3 immunoreactivity in patients with pain.
TRPV3
TRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain (Fig.  3 ). TRPV3-immunoreactivity was not detected in skin nerve fibres in this study.TRPV4 Basal keratinocytes also displayed TRPV4 immunoreactivity in groups of cells which were particularly strong at the apex of dermal papillae, where immunoreactivity appeared most strong at the cell membrane junction (Fig  4 ). Quantification of immunostaining showed a significant (p < 0.03) increase with breast pain (Fig.  4 ). TRPV4 immunoreactivity was also detected in fine nerve fibres scattered through the sub-epidermis, and showed a trend (p = 0.402) for increased frequency in painful subjects (Fig  4 ). Figure 4 TRPV4-immunoreactivity in breast skin . TOP FOUR PANELS: TRPV4-immunoreactivity in keratinocytes mostly at the cell membrane and graded from grade 0/negative (top left) to grade 3/strong staining (bottom right). Scale bar = 100 μm MIDDLE PANEL: Fine, sub-epidermal, TRPV4-immunoreactive fibres (arrows). Scale bar = 100 μm BOTTOM PANELS: Scattergrams showing grading assessment of sub-epidermal fibres (left panel) and keratinocytes (right panel) significantly increased (*P < 0.05) in patients with pain.
TRPV4
Basal keratinocytes also displayed TRPV4 immunoreactivity in groups of cells which were particularly strong at the apex of dermal papillae, where immunoreactivity appeared most strong at the cell membrane junction (Fig  4 ). Quantification of immunostaining showed a significant (p < 0.03) increase with breast pain (Fig.  4 ). TRPV4 immunoreactivity was also detected in fine nerve fibres scattered through the sub-epidermis, and showed a trend (p = 0.402) for increased frequency in painful subjects (Fig  4 ).NGF NGF immunoreactivity was present in basal keratinocytes in all samples (Fig  5Ai, ii ) with little difference in intensity between pain and no pain specimens (Table  1 ). In some pain specimens the epidermis appeared to be thinner, and there was evidence of NGF expression in suprabasal as well as basal keratinocytes (Fig  5Bi  and  5ii , – arrows), which correlated with the presence of TRPV1positive fibres (seen in a serial section from the same case shown in Fig  1B ). Figure 5 NGF-immunoreactivity in breast skin . ( Ai ) Normal, control skin: NGF-immunoreactive basal keratinocytes. ( Aii )-enlarged area from Ai showing NGF confined to single layer of keratinocytes. ( Bi ) Painful skin (macromastia patient): NGF-immunoreactive basal and supra-basal ( Bii -arrows) keratinocytes in skin with thin epidermis.
NGF
NGF immunoreactivity was present in basal keratinocytes in all samples (Fig  5Ai, ii ) with little difference in intensity between pain and no pain specimens (Table  1 ). In some pain specimens the epidermis appeared to be thinner, and there was evidence of NGF expression in suprabasal as well as basal keratinocytes (Fig  5Bi  and  5ii , – arrows), which correlated with the presence of TRPV1positive fibres (seen in a serial section from the same case shown in Fig  1B ).Nerve marker (neurofilaments) Neurofilaments showed nerve fibres in sub-epidermal and dermal regions only but no significant changes were detected between groups (Table  1 ).
Nerve marker (neurofilaments)
Neurofilaments showed nerve fibres in sub-epidermal and dermal regions only but no significant changes were detected between groups (Table  1 ).Discussion While breast pain and tenderness is a common problem, in the majority of women the mechanisms underlying breast pain are poorly understood. Our study focused on Nerve Growth Factor (NGF) and the expression of the capsaicin receptor 1 (TRPV1) in nociceptor fibres, as these are key molecules in pain and hypersensitivity. Our finding, that TRPV1-positive intra-epidermal fibres were significantly increased in patients with breast pain and tenderness, is both novel and important. The increased and abnormal "clusters" of intra-epidermal fibres were shown in patients who had no previous breast surgery, and no known episodes of mastitis. Hence this may be a surrogate marker for "idiopathic" or macromastia related breast pain in some patients. Studies are in progress to correlate changes in TRPV1 nerve fibres with quantitative sensory perception thresholds. The cause of the increased intra-epidermal fibres is not known. Given the trend to thinning of the epidermis, normally associated with denervation [ 21 ], in the biopsies from some patients with breast pain, the increased intra-epidermal fibres here may represent nerve fibre sprouts following cutaneous terminal damage. The intra-epidermal fibre morphology and "clusters" would be in keeping with this explanation, as fibres ran in unusual patterns. There was no overall change in the sub-epidermal fibre counts for TRPV1 or the structural nerve marker neurofilament. NGF-immunostaining intensity was similar in the different groups. However, in some patients, there appeared to be staining for NGF in the supra-basal epidermis in addition to the basal cells, which is usually associated with inflammation or denervation; increased NGF is known to cause collateral sprouting [ 22 ]. Further studies, using quantitative NGF assays and in situ hybridisation, need to be performed to address this issue (these studies would require more substantial skin biopsies). The menstrual cycle influences on NGF levels are also difficult to determine with the current sample size. Our recent studies have demonstrated increased TRPV1-immunoreactive nerve fibres in inflammatory bowel disease [ 23 ], and in the mucosal and sub-mucosal layers of patients with rectal hypersensitivity, where they correlated with thermal and mechanical hypersensitivity, suggesting increase of polymodal nociceptors [ 20 ]. We proposed that topical capsaicin or resiniferatoxin treatment, which reduces the numbers of TRPV1 positive fibres, may be a useful therapeutic approach in rectal hypersensitivity. Topical capsaicin has been reported to be useful for treatment for post-mastectomy chronic pain [ 24 ], but it is uncertain if it could substantially help breast pain or tenderness in macromastia, as some of the symptoms are likely to arise from deeper structures. Oral selective TRPV1 antagonists thus deserve consideration: both mechanical and thermal hyperalgesia may be reversed by capsazepine, a TRPV1 antagonist [ 25 ], again suggesting an effect on polymodal nociceptors. Thus patients may be helped with respect to mechanical symptoms, which predominate in comparison with thermal descriptors. Little is known of the roles of TRPV3 and TRPV4 in human pain pathophysiology and keratinocyte function. While we have previously demonstrated TRPV3 in human sensory neurons [ 18 ], no TRPV3 staining was observed in skin innervation in this study, presumably as the levels in the periphery were below the detection limit of our method. It may be speculated that increased TRPV3 and TRPV4 in observed in keratinocytes may alter keratinocyte expression of NGF and other molecules, which in turn may sensitise nociceptors.
Discussion
While breast pain and tenderness is a common problem, in the majority of women the mechanisms underlying breast pain are poorly understood. Our study focused on Nerve Growth Factor (NGF) and the expression of the capsaicin receptor 1 (TRPV1) in nociceptor fibres, as these are key molecules in pain and hypersensitivity. Our finding, that TRPV1-positive intra-epidermal fibres were significantly increased in patients with breast pain and tenderness, is both novel and important. The increased and abnormal "clusters" of intra-epidermal fibres were shown in patients who had no previous breast surgery, and no known episodes of mastitis. Hence this may be a surrogate marker for "idiopathic" or macromastia related breast pain in some patients. Studies are in progress to correlate changes in TRPV1 nerve fibres with quantitative sensory perception thresholds.The cause of the increased intra-epidermal fibres is not known. Given the trend to thinning of the epidermis, normally associated with denervation [ 21 ], in the biopsies from some patients with breast pain, the increased intra-epidermal fibres here may represent nerve fibre sprouts following cutaneous terminal damage. The intra-epidermal fibre morphology and "clusters" would be in keeping with this explanation, as fibres ran in unusual patterns. There was no overall change in the sub-epidermal fibre counts for TRPV1 or the structural nerve marker neurofilament. NGF-immunostaining intensity was similar in the different groups. However, in some patients, there appeared to be staining for NGF in the supra-basal epidermis in addition to the basal cells, which is usually associated with inflammation or denervation; increased NGF is known to cause collateral sprouting [ 22 ]. Further studies, using quantitative NGF assays and in situ hybridisation, need to be performed to address this issue (these studies would require more substantial skin biopsies). The menstrual cycle influences on NGF levels are also difficult to determine with the current sample size.Our recent studies have demonstrated increased TRPV1-immunoreactive nerve fibres in inflammatory bowel disease [ 23 ], and in the mucosal and sub-mucosal layers of patients with rectal hypersensitivity, where they correlated with thermal and mechanical hypersensitivity, suggesting increase of polymodal nociceptors [ 20 ]. We proposed that topical capsaicin or resiniferatoxin treatment, which reduces the numbers of TRPV1 positive fibres, may be a useful therapeutic approach in rectal hypersensitivity. Topical capsaicin has been reported to be useful for treatment for post-mastectomy chronic pain [ 24 ], but it is uncertain if it could substantially help breast pain or tenderness in macromastia, as some of the symptoms are likely to arise from deeper structures. Oral selective TRPV1 antagonists thus deserve consideration: both mechanical and thermal hyperalgesia may be reversed by capsazepine, a TRPV1 antagonist [ 25 ], again suggesting an effect on polymodal nociceptors. Thus patients may be helped with respect to mechanical symptoms, which predominate in comparison with thermal descriptors.Little is known of the roles of TRPV3 and TRPV4 in human pain pathophysiology and keratinocyte function. While we have previously demonstrated TRPV3 in human sensory neurons [ 18 ], no TRPV3 staining was observed in skin innervation in this study, presumably as the levels in the periphery were below the detection limit of our method. It may be speculated that increased TRPV3 and TRPV4 in observed in keratinocytes may alter keratinocyte expression of NGF and other molecules, which in turn may sensitise nociceptors.Conclusion Breast pain and tenderness appears to be associated with abnormal intra-epidermal innervation. This may reflect re-innervation of skin following nerve stretch damage, and/or collateral sprouting. While further studies are necessary to establish functional links between the TRPV1, TRPV3 and TRPV4 immunohistological changes and breast pain, our findings indicate a path for increasing understanding and treatment of breast pain.
Conclusion
Breast pain and tenderness appears to be associated with abnormal intra-epidermal innervation. This may reflect re-innervation of skin following nerve stretch damage, and/or collateral sprouting. While further studies are necessary to establish functional links between the TRPV1, TRPV3 and TRPV4 immunohistological changes and breast pain, our findings indicate a path for increasing understanding and treatment of breast pain.List of abbreviations TRPV = transient receptor potential vanilloid; NGF = Nerve Growth Factor; VAS = Visual Analogue Score; PRI = Pain Rating Index
List of abbreviations
TRPV = transient receptor potential vanilloid; NGF = Nerve Growth Factor; VAS = Visual Analogue Score; PRI = Pain Rating IndexCompeting interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions PG and EW recruited patients, collected biopsies and participated in immunohistology studies. PF participated in immunohistology and coordination of the study. AH participated in design of the study and recruitment of patients. JD, GS and CB provided antibodies and helped draft the manuscript. PA conceived the original study, its design and coordination, and helped with the manuscript. All authors read and approved the final manuscript.
Authors' contributions
PG and EW recruited patients, collected biopsies and participated in immunohistology studies. PF participated in immunohistology and coordination of the study. AH participated in design of the study and recruitment of patients. JD, GS and CB provided antibodies and helped draft the manuscript. PA conceived the original study, its design and coordination, and helped with the manuscript. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders

It is indeed regrettable that a distinguished and thoughtful scientist such as Elizabeth Blackburn should have been dismissed from the President's Council on Bioethics. Scientific perspectives such as hers are surely needed on this committee.Her dismissal was apparently the consequence of her disagreement with some of the text of the Council's report, “Beyond Therapy: Biotechnology and the Pursuit of Happiness” (2003). The thrust of this report is that some of the directions of current biological research will, if carried to fulfillment, result in major changes in the nature of human life—changes that the report regards with foreboding.In their essay, Drs.  Blackburn and Rowley (2004)  try to bypass these concerns with the argument that we really are not able to accomplish any of these changes yet and, indeed, some may never be possible.I would suggest that as scientists we should face these issues forthrightly. We should not seek refuge in presentday uncertainties. The authors of the report are not naïve nor ignorant. Yes, if these lines of research are successful, their outcome will change the nature of human life.As an example, consider current research into the causes of aging. Clearly, we do not at present know how to achieve major increases in the human life span (although we are able to do so in lower life forms). But it is plausible that we will learn how to do so. And surely a, say, doubling of the human life span would change the nature of human life.Likewise, if we learn to modify the human gene pool so as to produce exceptional individuals or to alter human capabilities, or if powerful drugs are developed that may commandeer the human psyche, the nature of human life will be altered.But so be it. The nature of human life has changed repeatedly and profoundly in the past—with the invention of agriculture, with the invention of writing, with the development of machines and mechanical power, with the advent of modern science and medicine. The nature of human life is different in 2004 a.d. from what it was in 1000 a.d. or 46 b.c. or 5000 b.c. or 10,000 b.c., and it will change again in the future.The concerns expressed in the report are earnest, and they should be confronted in earnest.