Scandinavian Journal of Plastic and Reconstructive Surgery
ISSN: 0036-5556 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iphs18
65
Absorption, Retention and Excretion of Zn in Rats 65 with Wounds Treated with Zn-Labelled Zinc Tape Göran Hallmans, Ann-Marie Nilsson & Rolf Sjöström To cite this article: Göran Hallmans, Ann-Marie Nilsson & Rolf Sjöström (1978) Absorption, 65
65
Retention and Excretion of Zn in Rats with Wounds Treated with Zn-Labelled Zinc Tape, Scandinavian Journal of Plastic and Reconstructive Surgery, 12:2, 111-117, DOI: 10.3109/02844317809012981 To link to this article: http://dx.doi.org/10.3109/02844317809012981
Published online: 08 Jul 2009.
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Date: 15 March 2016, At: 02:56
Scand J Plast Reconstr Surg 12: 1 11-1 17, 1978
ABSORPTION, RETENTION AND EXCRETION OF 65ZnIN RATS WITH WOUNDS TREATED WITH 65Zn-LABELLED ZINC TAPE’ Goran Hallmans, Ann-Marie Nilsson and Rolf Sjostrom From the Department of Pathology I (Head F . Bergman) and the Biophysical Laboratory (Head B . Bergman), University of Umeii, Umeii, Sweden
Downloaded by [] at 02:56 15 March 2016
(Submitted for publication April 14, 1978)
Abstract. Absorption, retention and excretion of “Zn in rats from excisional wounds treated with “Zn-labelled zinc tape were studied. The absorption of 65Znwas marked and indicates that the absorption of zinc from the tape is more than sufficient to account for the increases in zinc concentrations seen in several tissues. The distribution, retention and excretion of 65Znfrom the zinc tape followed a pattern similar to that of 65Zn injected into non-operated and operated rats. This indicates that the zinc from the tape is present in the body not as zinc oxide, as it is in the adhesive substance of the tape, but as zinc in those forms which are originally found in the body and that zinc oxide is ionized before absorption. In tissues with a rapid uptake of zinc more than 50% of the total concentration of zinc derived from the zinc tape after eight days of treatment. In operated animals which were not treated with zinc tape a decline was seen in the serum zinc concentration and excretion of zinc with a concomitant increase in the liver concentration of zinc.
Zinc appears to be essential for the normal process of wound healing in both humans (Hallbook & Lanner, 1972) and animals (Sandstead, Lanier, Shepard & Gillespie, 1970). Zinc is an integral part of several metalloenzymes (Riordan & Vallee, 1976), takes part at multipe sites in nuclei acid metabolism (Kirchgessner, Roth & Weigand 1976) and seems to be closely involved in the homeostasis of various inflammatory cells (Chvapil, Zukoski, Hattler, Stankova, Montgomery, Carlson & Ludwig 1976). It has previously been shown that zinc concentrations in serum and several tissues in rats increase significantly when excisional wounds are treated with various zinc compounds (Hallmans 1 9 7 7 ~ ; 1978) and that serum zinc increases in humans when burns are treated with zinc tape (Hallmans, 19776). The present experiment in rats was desiged in order to determine firstly whether or not the increases in zinc concentrations are derived from zinc absorbed from the zinc tape, secondly whether or not the zinc from the tape is freely exchangeable with the body
zinc and thirdly how much of the zinc present in selected tissue derives from the zinc tape. For this purpose a 65Zn-labelled zinc tape was prepared and the absorption, retention and excretion of ‘j5Zn in rats with excisional wounds treated with this tape was compared to that in rats with and without excisional wounds administered g5ZnC12subcutaneously. MATERIAL AND METHODS Animals Thirty-two male, albino rats of the Sprague-Dawley strain weighing 110-175 g were used. The rats were housed in individual metabolism cages of acrylic resin and stainless steel which permitted collection of urine and faeces. The animals were fed a pellet diet containing 100 pg zinclg and de-ionized water ad libitum. Radioisotopes A zinc tape containing 65Znwas prepared for the present experiment. Zinc oxide powder was placed in a quartz glass tube and irradiated with thermal neutrons at aflux of approximately 2.2x IOls neutrons. cm-2. sec-’ for 72 hours (ABAtomenergi, Studsvik, Sweden). The irradiated zinc oxide was mixed mechanically for three hours with an adhesive substance composed of gum, resin and trichloroethylene. The final concentration of zinc was 17% of the total dry weight. The mixture was then spread as evenly as possible on cotton gauze and dried. The range of activity of “Zn was 5-10 pCi/cm2 zinc tape. The 65Zn-labelled zinc tape was stored for one month in order to practically eliminate activity of other zinc radioisotopes before being used in the present experiment. The =Zn injection solution was prepared by diluting a stock solution of 65ZnClz(camer-free) in 0.1 mol/l HCI with physiologic saline to a final concentration of 40 pCi/ml. A single subcutaneous injection of 0.5 or 1.0 ml of this solution was administered in the neck of animals which were not treated with adhesive B6Zn-labelled zinc tape.
Supported by grants from the Swedish Medical Research Council (Project No. 4518) and “Forenade Liv” Mutual Group Life Insurance Company, Stockholm, Sweden Scand J Plast Reconstr Surg I 2
112
G . Hallmans et al.
after 4 days. At sacrifice, samples were taken of serum, coagulated blood, hair, cortical bone (tibia diaphysis c-c crugvlum without bone marrow), testis, liver, pancreas, kidney and H serum granulation tissue (from the small, plastic foil treated wounds) for gamma scintillation and atomic absorption spectrophotometric measurements. In series II, 8 rats weighing 110-125 g were used for an autoradiographic study. Four rats were assigned to group A and two each to groups B and C. The operations and treatment procedure were in principle the same as in series I except that the two wounds were equal in size and approximately 4 cm in diameter. In group A both wounds were treated with 65Zn-labelled zinc tape. Forty-eight hours prior to operation each animal in groups B and C was injected with 40 pCi "Zn subcutaneously. After 4 and 8 days two animals from group A and one animal from each of groups B and C were killed and prepared for autoradiography. Duplicate samples were taken of blood from the tails of V the rats treated with 65Zn-labelled zinc tape (group A) of I z 3 4 5 6 ; a both series using 50 p1 micropipettes at 6, 12, 24, 48 and days dler opantion Fig. 1 . Concentration of @Zn (nCi/ml) in serum and 72 hours after operation. For animals killed after 8 days coagulum during 8 days after operation in animals treated (5 animals) samples were also taken at the same intervals after redressing. The blood samples were allowed to with BJZn-labelledzinc tape (series I and 11). The wounds coagulate, they were then centrifuged and the microwere redressed with BsZn-labelled zinc tape on day 4. The pipettes were divided at the margin between the coagulum results are presented as the mean+_S.E.M. and serum after which the lengths of the two portions were measured. The activity of 65Zn in serum and coagExperimental design, operations, ulum was then measured. Faeces and urine were collected from all animals for wound treatment procedure Two series of experiments were performed. In series I, 24 analysis. In animals from series I, except those killed after rats weighing 125-175 g were used. Eight rats were as- 24 hours, faeces were collected at 4 day intervals. In signed to each of the following groups: group A: operated animals from series II, faeces were collected every day. animals, BKZn-labelledzinc tape treated; group B: operated The faeces were dried at 110°C for 3 days, weighed, ashed animals, plastic foil treated, injected with 65Znsubcutane- overnight at 550"C, weighed again, dissolved in 3 mol/l ously; group C: unoperated animals, injected with B5Zn HCI for 24 hours after which the 65Znactivity was measured. After further dilution with 0.6 mol/l HCI the zinc subcutaneously. In groups A and B two circular wounds were made on concentration was determined by atomic absorption the dorsolateral area of the skin on either side of the spine spectrophotometry. The content of zinc and B5Znin urine between the mid-thoracic and the mid-lumbar regions. were analyzed in a similar manner. Before wounding, the hair on the back and sides of the Gamma scintillation and atomic absorption animals was removed using an electric clipper. The wound spectrophotometric measurements sites were marked with two inked metal tubes and the The distribution of 65Znin selected tissues in the bodies of wounds made through the panniculus carnosus to the animals of series I was measured by gamma scintillation fascia using curved scissors. After wound retraction the and the total zinc concentrations of these tissue samples diameter of the wound located on the left side of the spine determined using atomic absorption spectrophotometry. was approximately 5 cm and that of the wound on the right The sample preparation procedure has previously been described by Bergman &Wing (1974). The tissue samples side approximately 2 cm. In group A the larger wound was treated with "Znwere removed with stainless steel instruments in such a labelled zinc tape, the smaller wound with a sterile, plastic way as to avoid contamination from the zinc tape. Instrufoil. In group B the plastic foil was placed on the sur- ments which had been used for opening the bandage and face of both wounds. To protect the wounds from damage removing zinc contaminated granulation tissue were never and avoid contamination to the cages from the 6nZn-la- used for other purposes. Each sample was placed in belled zinc tape, a bandage of a gauze sponge surrounded a pre-weighed and acid washed borosilicate tube and by a plastic coated tape was applied around the bodies of weighed. The dry weights of the samples were taken after the animals. Forty-eight hours prior to the operations each 3 days at llO'C, by which time the weights had become of the animals in groups B and C was injected with 20 pCi constant. The dried samples were ashed at 500°C (serum) 65Znsubcutaneously. or 550°C (other tissues and 65Zn-labelled zinc tape) for 24 hours. The resultant ash was dissolved in 0.5 rnl 3 moll1 At 1 , 4 and 8 days post-operatively two or three animals from each group were killed by decapitation under ether HCI overnight and then diluted with 2 ml distilled water. The activity of 65Zn was measured in a 7.6X7.6 cm NaI anesthesia. For those animals from groups A and B which were to survive 8 days, the wound dressings were changed (Tl) well scintillation detector with a single channel an-
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Scand J Plasr Reconstr Surg I2
Absorption, retention and excretion of 65Zn
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4-
J l
-
- - - - -
113
-
Fig. 2 . Activity of 65Znin selected tissues of ssZn-labelled zinc tape treated animals exprested as nCi/g wet weight and nCi/pg Zn (series I). The arrow indicates the level of the E5Zn/Znratio of the zinc tape before treatment. 1
8
8
days ailw oparaion
alyzer (AE 3207, AB Atomenergi, Studsvik, Sweden) calibrated to count at the 1.116 MeV y-peak of "Zn. A reference sample was prepared by diluting a known amount of the injection solution to the same volume a s the samples, 2.5 ml. The background counting was recorded on each occasion and subtracted from the sample and reference counting rates. Samples of the ashed 65Znlabelled zinc tape were further diluted before measurement of "Zn activity. After the gamma scintillation measurements the samples were further diluted as necessary with 0.6 mol/l HCI for zinc determinations by atomic absorption spectrophotometry (Bergman, Sjostrom & Wing, 1974). The analysis was performed on a Varian AAdDB spectrophotometer at 213.9 nm using reference samples of zinc in 0.6 mol/l HCI.
In each sample from animals injected with the 65Zn solution the relative activity (R. A,) was calculated: R.A.=
65Znactivitylg wet weight of the sample "Zn activity injected/gram body weight
The specific activity (S.A.) in each sample was calculated by dividing the R.A. by the zinc concentration (mg Zn/g wet weight; for faeces, mg Zn/g dry weight). In samples taken from "Zn-labelled zinc tape treated animals, the activity of "Zn was expressed as nCi/g wet or dry weight and nCi/pg Zn or nCi/mg Zn. Autoradiography
The distribution of 85Zn in the rats of series If was studied by the autoradiographic technique presented by Bergman
Fig.3. Relative (R. A.) and specific
activity (S. A,) in selected tissues of animals injected with "Zn (series I). The means for the operated and non-operated animals combined are presented.
S c u d J PIust Reconsir Surg 12
114
G. Hallmans et al.
0
COntlOl
m
plastic toll zinc tape
t
Fig. 4 . Zinc concentrations (pglg
Downloaded by [] at 02:56 15 March 2016
wet weight) in selected tissues of non-operated (control), plastic foil treated and zinc tape treated animals (series I). Concentrations in samples taken at different time intervals ( I , 4 and 8 days) are combined and presented as the mean f S.E.M. Levels of significance related to non-operated control animals are indicated in the figure. *p
ISSN: 0036-5556 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iphs18
65
Absorption, Retention and Excretion of Zn in Rats 65 with Wounds Treated with Zn-Labelled Zinc Tape Göran Hallmans, Ann-Marie Nilsson & Rolf Sjöström To cite this article: Göran Hallmans, Ann-Marie Nilsson & Rolf Sjöström (1978) Absorption, 65
65
Retention and Excretion of Zn in Rats with Wounds Treated with Zn-Labelled Zinc Tape, Scandinavian Journal of Plastic and Reconstructive Surgery, 12:2, 111-117, DOI: 10.3109/02844317809012981 To link to this article: http://dx.doi.org/10.3109/02844317809012981
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphs18 Download by: [137.189.171.235]
Date: 15 March 2016, At: 02:56
Scand J Plast Reconstr Surg 12: 1 11-1 17, 1978
ABSORPTION, RETENTION AND EXCRETION OF 65ZnIN RATS WITH WOUNDS TREATED WITH 65Zn-LABELLED ZINC TAPE’ Goran Hallmans, Ann-Marie Nilsson and Rolf Sjostrom From the Department of Pathology I (Head F . Bergman) and the Biophysical Laboratory (Head B . Bergman), University of Umeii, Umeii, Sweden
Downloaded by [] at 02:56 15 March 2016
(Submitted for publication April 14, 1978)
Abstract. Absorption, retention and excretion of “Zn in rats from excisional wounds treated with “Zn-labelled zinc tape were studied. The absorption of 65Znwas marked and indicates that the absorption of zinc from the tape is more than sufficient to account for the increases in zinc concentrations seen in several tissues. The distribution, retention and excretion of 65Znfrom the zinc tape followed a pattern similar to that of 65Zn injected into non-operated and operated rats. This indicates that the zinc from the tape is present in the body not as zinc oxide, as it is in the adhesive substance of the tape, but as zinc in those forms which are originally found in the body and that zinc oxide is ionized before absorption. In tissues with a rapid uptake of zinc more than 50% of the total concentration of zinc derived from the zinc tape after eight days of treatment. In operated animals which were not treated with zinc tape a decline was seen in the serum zinc concentration and excretion of zinc with a concomitant increase in the liver concentration of zinc.
Zinc appears to be essential for the normal process of wound healing in both humans (Hallbook & Lanner, 1972) and animals (Sandstead, Lanier, Shepard & Gillespie, 1970). Zinc is an integral part of several metalloenzymes (Riordan & Vallee, 1976), takes part at multipe sites in nuclei acid metabolism (Kirchgessner, Roth & Weigand 1976) and seems to be closely involved in the homeostasis of various inflammatory cells (Chvapil, Zukoski, Hattler, Stankova, Montgomery, Carlson & Ludwig 1976). It has previously been shown that zinc concentrations in serum and several tissues in rats increase significantly when excisional wounds are treated with various zinc compounds (Hallmans 1 9 7 7 ~ ; 1978) and that serum zinc increases in humans when burns are treated with zinc tape (Hallmans, 19776). The present experiment in rats was desiged in order to determine firstly whether or not the increases in zinc concentrations are derived from zinc absorbed from the zinc tape, secondly whether or not the zinc from the tape is freely exchangeable with the body
zinc and thirdly how much of the zinc present in selected tissue derives from the zinc tape. For this purpose a 65Zn-labelled zinc tape was prepared and the absorption, retention and excretion of ‘j5Zn in rats with excisional wounds treated with this tape was compared to that in rats with and without excisional wounds administered g5ZnC12subcutaneously. MATERIAL AND METHODS Animals Thirty-two male, albino rats of the Sprague-Dawley strain weighing 110-175 g were used. The rats were housed in individual metabolism cages of acrylic resin and stainless steel which permitted collection of urine and faeces. The animals were fed a pellet diet containing 100 pg zinclg and de-ionized water ad libitum. Radioisotopes A zinc tape containing 65Znwas prepared for the present experiment. Zinc oxide powder was placed in a quartz glass tube and irradiated with thermal neutrons at aflux of approximately 2.2x IOls neutrons. cm-2. sec-’ for 72 hours (ABAtomenergi, Studsvik, Sweden). The irradiated zinc oxide was mixed mechanically for three hours with an adhesive substance composed of gum, resin and trichloroethylene. The final concentration of zinc was 17% of the total dry weight. The mixture was then spread as evenly as possible on cotton gauze and dried. The range of activity of “Zn was 5-10 pCi/cm2 zinc tape. The 65Zn-labelled zinc tape was stored for one month in order to practically eliminate activity of other zinc radioisotopes before being used in the present experiment. The =Zn injection solution was prepared by diluting a stock solution of 65ZnClz(camer-free) in 0.1 mol/l HCI with physiologic saline to a final concentration of 40 pCi/ml. A single subcutaneous injection of 0.5 or 1.0 ml of this solution was administered in the neck of animals which were not treated with adhesive B6Zn-labelled zinc tape.
Supported by grants from the Swedish Medical Research Council (Project No. 4518) and “Forenade Liv” Mutual Group Life Insurance Company, Stockholm, Sweden Scand J Plast Reconstr Surg I 2
112
G . Hallmans et al.
after 4 days. At sacrifice, samples were taken of serum, coagulated blood, hair, cortical bone (tibia diaphysis c-c crugvlum without bone marrow), testis, liver, pancreas, kidney and H serum granulation tissue (from the small, plastic foil treated wounds) for gamma scintillation and atomic absorption spectrophotometric measurements. In series II, 8 rats weighing 110-125 g were used for an autoradiographic study. Four rats were assigned to group A and two each to groups B and C. The operations and treatment procedure were in principle the same as in series I except that the two wounds were equal in size and approximately 4 cm in diameter. In group A both wounds were treated with 65Zn-labelled zinc tape. Forty-eight hours prior to operation each animal in groups B and C was injected with 40 pCi "Zn subcutaneously. After 4 and 8 days two animals from group A and one animal from each of groups B and C were killed and prepared for autoradiography. Duplicate samples were taken of blood from the tails of V the rats treated with 65Zn-labelled zinc tape (group A) of I z 3 4 5 6 ; a both series using 50 p1 micropipettes at 6, 12, 24, 48 and days dler opantion Fig. 1 . Concentration of @Zn (nCi/ml) in serum and 72 hours after operation. For animals killed after 8 days coagulum during 8 days after operation in animals treated (5 animals) samples were also taken at the same intervals after redressing. The blood samples were allowed to with BJZn-labelledzinc tape (series I and 11). The wounds coagulate, they were then centrifuged and the microwere redressed with BsZn-labelled zinc tape on day 4. The pipettes were divided at the margin between the coagulum results are presented as the mean+_S.E.M. and serum after which the lengths of the two portions were measured. The activity of 65Zn in serum and coagExperimental design, operations, ulum was then measured. Faeces and urine were collected from all animals for wound treatment procedure Two series of experiments were performed. In series I, 24 analysis. In animals from series I, except those killed after rats weighing 125-175 g were used. Eight rats were as- 24 hours, faeces were collected at 4 day intervals. In signed to each of the following groups: group A: operated animals from series II, faeces were collected every day. animals, BKZn-labelledzinc tape treated; group B: operated The faeces were dried at 110°C for 3 days, weighed, ashed animals, plastic foil treated, injected with 65Znsubcutane- overnight at 550"C, weighed again, dissolved in 3 mol/l ously; group C: unoperated animals, injected with B5Zn HCI for 24 hours after which the 65Znactivity was measured. After further dilution with 0.6 mol/l HCI the zinc subcutaneously. In groups A and B two circular wounds were made on concentration was determined by atomic absorption the dorsolateral area of the skin on either side of the spine spectrophotometry. The content of zinc and B5Znin urine between the mid-thoracic and the mid-lumbar regions. were analyzed in a similar manner. Before wounding, the hair on the back and sides of the Gamma scintillation and atomic absorption animals was removed using an electric clipper. The wound spectrophotometric measurements sites were marked with two inked metal tubes and the The distribution of 65Znin selected tissues in the bodies of wounds made through the panniculus carnosus to the animals of series I was measured by gamma scintillation fascia using curved scissors. After wound retraction the and the total zinc concentrations of these tissue samples diameter of the wound located on the left side of the spine determined using atomic absorption spectrophotometry. was approximately 5 cm and that of the wound on the right The sample preparation procedure has previously been described by Bergman &Wing (1974). The tissue samples side approximately 2 cm. In group A the larger wound was treated with "Znwere removed with stainless steel instruments in such a labelled zinc tape, the smaller wound with a sterile, plastic way as to avoid contamination from the zinc tape. Instrufoil. In group B the plastic foil was placed on the sur- ments which had been used for opening the bandage and face of both wounds. To protect the wounds from damage removing zinc contaminated granulation tissue were never and avoid contamination to the cages from the 6nZn-la- used for other purposes. Each sample was placed in belled zinc tape, a bandage of a gauze sponge surrounded a pre-weighed and acid washed borosilicate tube and by a plastic coated tape was applied around the bodies of weighed. The dry weights of the samples were taken after the animals. Forty-eight hours prior to the operations each 3 days at llO'C, by which time the weights had become of the animals in groups B and C was injected with 20 pCi constant. The dried samples were ashed at 500°C (serum) 65Znsubcutaneously. or 550°C (other tissues and 65Zn-labelled zinc tape) for 24 hours. The resultant ash was dissolved in 0.5 rnl 3 moll1 At 1 , 4 and 8 days post-operatively two or three animals from each group were killed by decapitation under ether HCI overnight and then diluted with 2 ml distilled water. The activity of 65Zn was measured in a 7.6X7.6 cm NaI anesthesia. For those animals from groups A and B which were to survive 8 days, the wound dressings were changed (Tl) well scintillation detector with a single channel an-
Downloaded by [] at 02:56 15 March 2016
T
Scand J Plasr Reconstr Surg I2
Absorption, retention and excretion of 65Zn
Downloaded by [] at 02:56 15 March 2016
4-
J l
-
- - - - -
113
-
Fig. 2 . Activity of 65Znin selected tissues of ssZn-labelled zinc tape treated animals exprested as nCi/g wet weight and nCi/pg Zn (series I). The arrow indicates the level of the E5Zn/Znratio of the zinc tape before treatment. 1
8
8
days ailw oparaion
alyzer (AE 3207, AB Atomenergi, Studsvik, Sweden) calibrated to count at the 1.116 MeV y-peak of "Zn. A reference sample was prepared by diluting a known amount of the injection solution to the same volume a s the samples, 2.5 ml. The background counting was recorded on each occasion and subtracted from the sample and reference counting rates. Samples of the ashed 65Znlabelled zinc tape were further diluted before measurement of "Zn activity. After the gamma scintillation measurements the samples were further diluted as necessary with 0.6 mol/l HCI for zinc determinations by atomic absorption spectrophotometry (Bergman, Sjostrom & Wing, 1974). The analysis was performed on a Varian AAdDB spectrophotometer at 213.9 nm using reference samples of zinc in 0.6 mol/l HCI.
In each sample from animals injected with the 65Zn solution the relative activity (R. A,) was calculated: R.A.=
65Znactivitylg wet weight of the sample "Zn activity injected/gram body weight
The specific activity (S.A.) in each sample was calculated by dividing the R.A. by the zinc concentration (mg Zn/g wet weight; for faeces, mg Zn/g dry weight). In samples taken from "Zn-labelled zinc tape treated animals, the activity of "Zn was expressed as nCi/g wet or dry weight and nCi/pg Zn or nCi/mg Zn. Autoradiography
The distribution of 85Zn in the rats of series If was studied by the autoradiographic technique presented by Bergman
Fig.3. Relative (R. A.) and specific
activity (S. A,) in selected tissues of animals injected with "Zn (series I). The means for the operated and non-operated animals combined are presented.
S c u d J PIust Reconsir Surg 12
114
G. Hallmans et al.
0
COntlOl
m
plastic toll zinc tape
t
Fig. 4 . Zinc concentrations (pglg
Downloaded by [] at 02:56 15 March 2016
wet weight) in selected tissues of non-operated (control), plastic foil treated and zinc tape treated animals (series I). Concentrations in samples taken at different time intervals ( I , 4 and 8 days) are combined and presented as the mean f S.E.M. Levels of significance related to non-operated control animals are indicated in the figure. *p
