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Histomorphometrical Analysis of Porcine Islets of Langerhans
Histomorphometrical Analysis of Porcine Islets of Langerhans J. Kinasiewicz, M. Antosiak-Iwan´ska, and M. Sabat ABSTRACT Xenotransplantation of pig islets of Langerhans may be an efficient method for treatment of type I diabetic patients. Considering the shortage of human pancreas donors, pigs offer readily available islet source. Unfortunately, their morphologic characteristics makes isolation of pig islets of Langerhans extremely difficult. The selection of a suitable donor may favorably affect the success of isolation. Therefore, we examined pig breeds and weights for their influence histomorphometric features of Langerhans islets. Pancreatic tissue biopsy specimens were obtained from Polish Large White pigs (n ⫽ 5), Polish Landrace pigs (n ⫽ 8), Pietrain pigs (n ⫽ 4), and marked-weight pigs of 100 kg (n ⫽ 8) or more than 200 kg (n ⫽ 10). Biopsy specimens were fixed and stored in 4% formaldehyde. Tissue sections were stained with polyclonal guinea pig anti-insulin antibody. The number of islets per 1 cm2 was counted in each section. Mean diameters and areas of the islets were measured to calculate the percentage volume density. Differences were observed among the groups of pigs weighing 200 kg and 100 kg in islet mean diameter and area. The number of islets/cm2 was higher among Pietrain than Polish Landrace pigs (425.45 ⫾ 111.7 versus 315.33 ⫾ 36.59 islet/cm2, P ⬍ .05). Furthermore, Pietrain pigs showed the largest volume density (1.44%). Histological analysis revealed that Pietrain pigs of 100 kg and market-weight pigs of 200 kg were the best donors for islets of Langerhans. TRUCTURAL similarity of human and porcine insulin as well as their blood glucose could promote pigs as alternative islet donors. The main difficulty in isolation of large numbers of viable islets is the lack of connective tissue surrounding the islets which disposes them to fragmentation during the pancreatic digestion process.1,2 Success in isolation may be assured by histological screening of each organ before isolation and selection of a pancreas containing predominantly large islets.3,4 Several studies have suggested genetic or nutritional donor parameters to impact islet isolation.3,5 The discussion about advantages of adult over younger pigs is still open.6,7 In this connection, the aim of our work was to assess the influence of pig breed and weight on histomorphometric images of islets of Langerhans.
S
MATERIALS AND METHODS Pancreatic biopsy specimens were obtained from 100-kg pigs: Polish Large White (PLW, n ⫽ 5); Polish Landrace (PL, n ⫽ 8); Pietrain (P, n ⫽ 4) as well as market-weight pigs of 100 kg (MW, n ⫽ 8) or more than 200 kg (MW, n ⫽ 10). Biopsy specimens were fixed in 4% formaldehyde. For histomorphological analysis of the islets, the biopsy specimens were dehydrated using alcohol gradients, embedded in paraffin, sectioned, deparaffinized, and stained
using primary antibodies against insulin (DAKO, Glostrup, Denmark), and secondary antibodies conjugated with horseradish peroxidase (DAKO). Subsequent to immunohistochemical staining, we analyzed samples using an Olympus microscope (IX71, Olympus Corporation, Tokyo, Japan) equipped with a CCD (charge coupled device) camera (DP70, Olympus) and a software system for image capture. Stained islets were measured, calculated, and analyzed using Cell P software (Olympus). We calculated the percentage volume density of islet to exocrine tissues according to the Principle of Delesse8: Volume density
共%兲 ⫽ area density 共%兲 ⫽ 共关⌸ 兺 r2兴 ⁄ section ⫻ 100, (r ⫺ radius)
area兲
Statistical analysis was performed using Student t-test. From the Nałe˛cz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences (J.K., M.A.-I.), Warsaw, Poland and the Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw (M.S.), Warsaw, Poland. Address reprint requests to Joanna Kinasiewicz, the Nałe˛cz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Trojdena 4, 02-109 Warsaw, Poland. E-mail: [email protected]
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.08.022
Transplantation Proceedings, 43, 3105–3106 (2011)
3105
KINASIEWICZ, ANTOSIAK-IWAN´SKA, AND SABAT
3106
Table 1. Results of Histological Analysis of Pancreatic Biopsy Specimens From Different Pig Breeds (All Weighted 100 kg) and Market-weight Pigs of 100 kg and 200 kg Pig Groups
Number of islets/cm2 pancreatic tissue (Mean ⫾ SD) Islet area [m2] (mean ⫾ SD) Islet size [m] (mean ⫾ SD) Volume density (%)
PLW 100 kg n⫽5
PL 100 kg n⫽8
P 100 kg n⫽4
MW 100 kg n⫽8
MW 200 kg, n ⫽ 10
338.8 ⫾ 101.8
315.3 ⫾ 36.6
425.5 ⫾ 111.7
394.8 ⫾ 149.2
322.7 ⫾ 103.52
2687.0 ⫾ 465.5 56.7 ⫾ 4.6 0.91 ⫾ 0.3
3562.5 ⫾ 1072.2 64.7 ⫾ 9.1 1.11 ⫾ 0.3
3429.3 ⫾ 880.6 63.3 ⫾ 8.8 1.44 ⫾ 0.5
2674.1 ⫾ 820.5 56.9 ⫾ 6.9 1.08 ⫾ 0.6
3719.9 ⫾ 1448.5 65.7 ⫾ 12.4 1.20 ⫾ 0.6
Abbreviations: PLW, Polish Large White pigs; PL, Polish Landrace pigs; P, Pietrain pigs; MW, market-weight pigs.
RESULTS
The results of histological analysis are summarized in Table 1. We showed different islet mean diameters and areas among the pig strains. A slightly larger area of islets was observed among MW pigs of 200 kg compared with 100 kg: 3719.9 m2 ⫾ 1448.5 versus 2674.1 m2 ⫾ 820.5 (P ⫽ ns). The total islet area and size increased with greater pig weight. The P pigs showed a greater number of islets/cm2 than PL pigs: 425.45 ⫾ 111.7 versus 315.33 ⫾ 36.59 islet/cm2, (P ⬍ .05). In addition, the number of islets/cm2 was higher among 100-kg MW pigs than 200-kg MW pigs. The islet volume density calculated on the basis of morphological measurements exhibited the largest value among 100-kg P (1.44%) as well as 200-kg MW pigs (1.2%). DISCUSSION
The problem of the human islet shortage may be resolved by using pig islets. Morphological islet parameters may allow selection of proper donors. We observed that the number of islets/cm2 decreased as the weight of the pig increased, showing limited capability of islets to divide during the maturation process. In contrast, the islet area increased proportionate to pig weight. Our results corresponded to those of Ulrichs et al who reported that the individual islets may grow but do not divide. Adult pigs are regarded as the best islet donors because they exhibit the higher islet area; however, the islet volume density which is recognized as predictor of isolation outcome,3 showed the
highest value among 100-kg P pig demonstrating that not only weight (age), but also breed-dependent factors influence successful isolations. In conclusion, we used histological analyses to identity Pietrain pigs as well as 200-kg market-weight pigs as the best donors of islets of Langerhans. REFERENCES 1. Toso, C, Brandhorst D, Oberholzer J, et al: Isolation of adult porcine islets of Langerhans. Cell Transplant 9:297, 2000 2. Socci C, Ricordi C, Davalli AM, et al: Selection of donors significantly improves pig islet isolation yield. Horm Metab Res 25(suppl):32, 1990 3. Ulrichs K, Bosse M, Wacker HH, et al: Histologic analysis of the porcine pancreas to improve islet yield and integrity after collagenase digestion. Transplant Proc 26:610, 1994 4. Krickhahn M, Buhler C, Meyer T, et al: The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs. Cell Transplant 11:827, 2002 5. Kirchhof N, Hering BJ, Geiss V, et al: Evidence for breeddependent differences in porcine islets of Langerhans. Transplant Proc. 26:616, 1994 6. Dufrane D, Goebbels RM, Fdilat I, et al: Impact of porcine islet size on cellular structure and engraftment after transplantation. Pancreas 30:138, 2005 7. O’Neil JJ, Stegemann JP, Nicholson DT, et al: The siolation and function of porcine islets from market weight pigs. Cell Transplant 10:235, 2001 8. Weibel E: Volume density measurement: the principle of Delesse. In Weibel ER (ed): Stereological Methods, Vol: Practical Methods for Biological Morphometry. London: Academic Press; 1979, p. 51
S
MATERIALS AND METHODS Pancreatic biopsy specimens were obtained from 100-kg pigs: Polish Large White (PLW, n ⫽ 5); Polish Landrace (PL, n ⫽ 8); Pietrain (P, n ⫽ 4) as well as market-weight pigs of 100 kg (MW, n ⫽ 8) or more than 200 kg (MW, n ⫽ 10). Biopsy specimens were fixed in 4% formaldehyde. For histomorphological analysis of the islets, the biopsy specimens were dehydrated using alcohol gradients, embedded in paraffin, sectioned, deparaffinized, and stained
using primary antibodies against insulin (DAKO, Glostrup, Denmark), and secondary antibodies conjugated with horseradish peroxidase (DAKO). Subsequent to immunohistochemical staining, we analyzed samples using an Olympus microscope (IX71, Olympus Corporation, Tokyo, Japan) equipped with a CCD (charge coupled device) camera (DP70, Olympus) and a software system for image capture. Stained islets were measured, calculated, and analyzed using Cell P software (Olympus). We calculated the percentage volume density of islet to exocrine tissues according to the Principle of Delesse8: Volume density
共%兲 ⫽ area density 共%兲 ⫽ 共关⌸ 兺 r2兴 ⁄ section ⫻ 100, (r ⫺ radius)
area兲
Statistical analysis was performed using Student t-test. From the Nałe˛cz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences (J.K., M.A.-I.), Warsaw, Poland and the Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw (M.S.), Warsaw, Poland. Address reprint requests to Joanna Kinasiewicz, the Nałe˛cz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Trojdena 4, 02-109 Warsaw, Poland. E-mail: [email protected]
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.08.022
Transplantation Proceedings, 43, 3105–3106 (2011)
3105
KINASIEWICZ, ANTOSIAK-IWAN´SKA, AND SABAT
3106
Table 1. Results of Histological Analysis of Pancreatic Biopsy Specimens From Different Pig Breeds (All Weighted 100 kg) and Market-weight Pigs of 100 kg and 200 kg Pig Groups
Number of islets/cm2 pancreatic tissue (Mean ⫾ SD) Islet area [m2] (mean ⫾ SD) Islet size [m] (mean ⫾ SD) Volume density (%)
PLW 100 kg n⫽5
PL 100 kg n⫽8
P 100 kg n⫽4
MW 100 kg n⫽8
MW 200 kg, n ⫽ 10
338.8 ⫾ 101.8
315.3 ⫾ 36.6
425.5 ⫾ 111.7
394.8 ⫾ 149.2
322.7 ⫾ 103.52
2687.0 ⫾ 465.5 56.7 ⫾ 4.6 0.91 ⫾ 0.3
3562.5 ⫾ 1072.2 64.7 ⫾ 9.1 1.11 ⫾ 0.3
3429.3 ⫾ 880.6 63.3 ⫾ 8.8 1.44 ⫾ 0.5
2674.1 ⫾ 820.5 56.9 ⫾ 6.9 1.08 ⫾ 0.6
3719.9 ⫾ 1448.5 65.7 ⫾ 12.4 1.20 ⫾ 0.6
Abbreviations: PLW, Polish Large White pigs; PL, Polish Landrace pigs; P, Pietrain pigs; MW, market-weight pigs.
RESULTS
The results of histological analysis are summarized in Table 1. We showed different islet mean diameters and areas among the pig strains. A slightly larger area of islets was observed among MW pigs of 200 kg compared with 100 kg: 3719.9 m2 ⫾ 1448.5 versus 2674.1 m2 ⫾ 820.5 (P ⫽ ns). The total islet area and size increased with greater pig weight. The P pigs showed a greater number of islets/cm2 than PL pigs: 425.45 ⫾ 111.7 versus 315.33 ⫾ 36.59 islet/cm2, (P ⬍ .05). In addition, the number of islets/cm2 was higher among 100-kg MW pigs than 200-kg MW pigs. The islet volume density calculated on the basis of morphological measurements exhibited the largest value among 100-kg P (1.44%) as well as 200-kg MW pigs (1.2%). DISCUSSION
The problem of the human islet shortage may be resolved by using pig islets. Morphological islet parameters may allow selection of proper donors. We observed that the number of islets/cm2 decreased as the weight of the pig increased, showing limited capability of islets to divide during the maturation process. In contrast, the islet area increased proportionate to pig weight. Our results corresponded to those of Ulrichs et al who reported that the individual islets may grow but do not divide. Adult pigs are regarded as the best islet donors because they exhibit the higher islet area; however, the islet volume density which is recognized as predictor of isolation outcome,3 showed the
highest value among 100-kg P pig demonstrating that not only weight (age), but also breed-dependent factors influence successful isolations. In conclusion, we used histological analyses to identity Pietrain pigs as well as 200-kg market-weight pigs as the best donors of islets of Langerhans. REFERENCES 1. Toso, C, Brandhorst D, Oberholzer J, et al: Isolation of adult porcine islets of Langerhans. Cell Transplant 9:297, 2000 2. Socci C, Ricordi C, Davalli AM, et al: Selection of donors significantly improves pig islet isolation yield. Horm Metab Res 25(suppl):32, 1990 3. Ulrichs K, Bosse M, Wacker HH, et al: Histologic analysis of the porcine pancreas to improve islet yield and integrity after collagenase digestion. Transplant Proc 26:610, 1994 4. Krickhahn M, Buhler C, Meyer T, et al: The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs. Cell Transplant 11:827, 2002 5. Kirchhof N, Hering BJ, Geiss V, et al: Evidence for breeddependent differences in porcine islets of Langerhans. Transplant Proc. 26:616, 1994 6. Dufrane D, Goebbels RM, Fdilat I, et al: Impact of porcine islet size on cellular structure and engraftment after transplantation. Pancreas 30:138, 2005 7. O’Neil JJ, Stegemann JP, Nicholson DT, et al: The siolation and function of porcine islets from market weight pigs. Cell Transplant 10:235, 2001 8. Weibel E: Volume density measurement: the principle of Delesse. In Weibel ER (ed): Stereological Methods, Vol: Practical Methods for Biological Morphometry. London: Academic Press; 1979, p. 51